**2. Fructooligosaccharides**

**1. Introduction**

26 Food Production and Industry

and effective [6,7].

Consumers all around the world are increasingly aware and concerned about safety and the quality of food. Besides the push towards replacement of chemical additives by those obtained from natural sources, this awareness has led to a rising demand for enrichment of foods with bioactive compounds that have beneficial effects on human health [1]. Therefore, nowadays, a variety of gluten free and products enriched with dietary fiber, or containing probiotics and/

Oligosaccharides are carbohydrates, composed of up to twenty monosaccharides linked by glycosydic bonds, widely used in food and pharmaceutical industries. These compounds are obtained from natural sources and through chemical or biotechnological processes [3,4].

Among the various functions of non-digestible oligosaccharides, one that has attracted attention is its prebiotic potential. A prebiotic can be defined as "selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gastro‐ intestinal microbiota that confers benefits upon host well-being and health" [5]. An oligosac‐ charide to be regarded as prebiotic must not be hydrolyzed or absorbed in the upper part of the gastrointestinal tract; and must be assimilated selectively by one or by a limited number of beneficial microorganisms in the colon, promoting benefic luminal or systemic effects. To improve colonic function, live microorganisms can be administered in adequate amounts, being known as probiotics; and to be used in food, these organisms must be able to survive passage through the gut; to proliferate and to colonize the digestive tract; and must be safe

The intestinal benefits of prebiotics, such as fructooligosaccharides and inulin as well as their symbiotic association with probiotic bacteria, encompass prevention and treatment of infectious diseases, including viral or bacterial diarrhea, and chronic inflammatory diseases such as ulcerative colitis [8]. The mechanisms of action of probiotics against gastrointestinal pathogens consist mainly on competition for nutrients and sites of access, production of antimicrobial metabolites, changes in environmental conditions, and modulation of the immune response of the host. Other benefits attributed to prebiotics and probiotics include treatment of inflammatory intestinal and irritable bowel syndrome, prevention of cancer, and

Oligosaccharides can be obtained by extraction from natural sources (milk, vegetables, fruits), and by chemical or biotechnological processes [10,11]. Mixtures of oligosaccharides with different degrees of polymerization and glycosidic linkages are usually formed in the enzy‐ matic processes. Chemical structures and composition of these mixtures depend on the type and source of enzymes, and on process conditions, including the initial concentration of substrate [11,12]. Depending on the initial substrate, production of oligosaccharides can involve different steps: hydrolysis of glycosidic bonds giving rise to monomers, followed by generation of disaccharides and other oligomers through the action of transferases [13,14].

modulation of the immune system, mineral absorption and lipid metabolism [8,9].

or prebiotic and functional oligosaccharides are available in the market [2].

Fructans are carbohydrates in which one or more fructosylfructose links constitute the majority of glycosidic bonds [15]. These carbohydrates can be of the inulin-type with β-(2,1)-Dfructofuranosyl units, found in plants and synthesized by fungi. Additionally, there are the levan-type fructans with β-(6,2)-D-fructofuranosyl units, found in plants and synthesized by bacteria [16].

Levan is a polymer with very high molecular weight that can reach 107 Da [17]. In contrast to levan, inulin from chicory consists of a mixture of oligomers and polymers with a degree of polymerization (DP) that varies from two to approximately sixty units (Figure 1; Table 1) [18]. Around 10% of the fructan chains in native chicory inulin have a DP in the range between two and five [5].

**Figure 1.** Structure of inulin, a linear fructosyl polymer linked by β-(2,1) bonds (n=3-65), attached to a terminal gluco‐ syl residue by an α-(1,2) bond.



**Prebiotics Chemical structure Properties Applicability /**

Soluble fibers; Gel formation; Sugar replacement; Moderate sweetness; Stable (depending on

matrix).

Soluble fiber; Water adsorption; Gel formation; Modifier of viscosity, texture, colour and sensory aspects of food formulations;

Replacement for fat and

Low calorimetric value; Moderate sweetness.

Stable in acidic conditions and in higher

temperatures; Soluble;

Cryoprotector activity; Low ability to crystalyze; Incorporated in various functional foods.

sugar;

FOS Fructosyl units linked by β-

28 Food Production and Industry

by α-(1,2) bond

DP=3-10.

Inulin Mixture of linear fructosyl

GOS Mixture of

polymers and oligomers (DP = 3-65) linked by β-(2,1) bonds, attached to a terminal glucosyl residue by α(1—2) bond.

galactopyranosyl oligomers (DP= 3-8) linked mostly by β-(1,4) or β-(1,6) bonds, although low proportions of β (1,2) or β-(1,3) linkages may also be present. Terminal glucosyl residues

(2,1) bonds, attached to a terminal glucosyl residue

(Variants: Inulin type β-1,2 and Levan type β-2,6 linkages between fructosyl units in the main chain)

**Health benefits**

respiratory tract;

intestinal flora;

system;

species;

type 2;

system;

system;

Prevention of cancer.

and triglycerides levels;

Prevention of cancer.

microbial adherence;

Stimuli of probiotic growth; Reorder intestinal flora; Regulation of intestinal immune

Reinforcement of intestinal barrier; Inhibition of adhesion of pathogens; Mimic molecular receptors, inhibit

Stimulation of probiotic growth; Lowering effect on cholesterol LDL

Influence on inflammatory markers and development of gut associated lymphoid tissue (GALT); Regulation of intestinal immune

Enhancement of immune response; Increase of mineral absorption (Calcium, Iron and Magnesium);

metabolism;

Prevention of intestinal infections and extra intestinal infections (e.g.

Inhibition of pathogens, ordering

Regulation of intestinal immune

Enhancement of immune response; Stimulation of probiotic growth of Lactobacilli and Bifidobacteria

Optimization of colonic function and

Production of short chain fatty acids; Increase of mineral absorption; Reduction of food intake and obesity management and control of diabetes

**Reference**

[8,24,25, 61,67,69, 72,170, 176-186]

[8,19,67, 176,187-195]

[8,105, 196- 206]


FOS: Fructooligosaccharides; GOS: Galactooligosaccharides; COS: Chitooligosaccharides;

XOS: Xylooligosaccharides; IMO: Isomaltooligosaccharides; SOS: Soybean oligosaccharides

**Table 1.** Structure and biological activity of prebiotics.

Inulin-type fructooligosaccharides are made up of two or more fructosyl moieties linked by β-(2,1) bonds and united at the non-reducing end to a terminal glucose residue by an α-(1,2) glycosidic bond (Table 1) [19]. The term fructooligosaccharides (FOS) is mainly used for fructose oligomers that contain one glucose unit and from two to four fructose units bound together by β-(2,1) glycosidic linkages [20,21]. Nevertheless, oligofructose and FOS may be regarded as synonyms for the mixture of small inulin oligomers with DP<10 [6,22]; while short chain FOS (sc-FOS) are fructose oligomers mainly composed of 1-kestose (GF2), nystose (GF3), and 1 F-fructofuranosylnystose (GF4) (Figure 2) [23-25].

Fructans have storage and protective functions in many commonly consumed plants, being a typical part of the diet. Some food sources are richer in high molecular weight fructans, such as inulin, while others have higher levels of sc-FOS [26].

Biotechnological Production of Oligosaccharides — Applications in the Food Industry http://dx.doi.org/10.5772/60934 31

**Prebiotics Chemical structure Properties Applicability /**

100°C);

Stable in a large range of pH values (2,5-8,0); Thermal stability (up to

Antioxidant effects; Antifreezing activity; Low cariogenicity; Low calorimetric value; Low glycemic index.

Low sweetness; Low viscosity; Bulking properties; Humectant; Prevention of sucrose crystallization.

Stabilizer properties; Cryoprotectant effect.

Inulin-type fructooligosaccharides are made up of two or more fructosyl moieties linked by β-(2,1) bonds and united at the non-reducing end to a terminal glucose residue by an α-(1,2) glycosidic bond (Table 1) [19]. The term fructooligosaccharides (FOS) is mainly used for fructose oligomers that contain one glucose unit and from two to four fructose units bound together by β-(2,1) glycosidic linkages [20,21]. Nevertheless, oligofructose and FOS may be regarded as synonyms for the mixture of small inulin oligomers with DP<10 [6,22]; while short chain FOS (sc-FOS) are fructose oligomers mainly composed of 1-kestose (GF2), nystose (GF3),

Fructans have storage and protective functions in many commonly consumed plants, being a typical part of the diet. Some food sources are richer in high molecular weight fructans, such

FOS: Fructooligosaccharides; GOS: Galactooligosaccharides; COS: Chitooligosaccharides; XOS: Xylooligosaccharides; IMO: Isomaltooligosaccharides; SOS: Soybean oligosaccharides

F-fructofuranosylnystose (GF4) (Figure 2) [23-25].

as inulin, while others have higher levels of sc-FOS [26].

XOS Xylose oligomers connected by β-(1,4) linkages (DP=3-6).

30 Food Production and Industry

IMO Glucosyl residues linked to

SOS Oligomers composed by

and 1

maltose or isomaltose by α- (1,6) glycosidic bonds.

galactosyl units linked to sucrose by α-(1,6) bonds. Most abundant are raffinose and stachyose.

**Table 1.** Structure and biological activity of prebiotics.

**Health benefits**

metabolism;

products;

Inhibition of pathogens growth, reordering intestinal flora; Stimulation of probiotic growth; Reinforcement of intestinal barrier; Optimization of colonic function and

Obesity management, reduction of

Optimization of colonic function and metabolism, reduces nitrogenated

food intake and weight.

Increase caecum weight; Antidiabetic effects;

Improve lipid metabolism and obesity management.

Prevention of pathogen proliferation. [2, 156, 213, 220,

**Reference**

[159, 162, 179, 214-216]

[2, 217- 219]

221]

**Figure 2.** Structures of typical fructooligosaccharides (FOS), derived from sucrose. FOS consist of a glucosyl residue α- (1,2) linked to two or more β-(1,2) fructosyl units. Synthesis of these FOS is catalyzed by fructosyltransferases, requir‐ ing a second sucrose molecule as a fructosyl residue donor.

FOS are found in low levels in natural sources such as asparagus, sugar beet, garlic, chicory, onion, Jerusalem artichoke, wheat, honey, banana, barley, tomato, and rye [27-29]. Apart from usually occurring in low concentrations, seasonal conditions also limit their large-scale production from these sources [30].

For this reason, enzymatic processes are used for the industrial production of FOS. One route involves the controlled hydrolysis of long chain fructans (Table 2) [31,32], which results in a large amount of FOS mostly without glucose in their structures. The other route is the synthesis from sucrose, which leads to sc-FOS that contain a molecule of glucose in their structures [11, 33]. The present review will focus on the synthesis of FOS from sucrose.

FOS are produced from sucrose by the action of microbial enzymes with high transfuctosy‐ lating activity: β-D-fructosyltransferase (FTase, EC 2.4.1.9) and β-fructofuranosidase (FFase, EC 3.2.1.26) (Table 2) [34]. Since FTase possesses almost only the transfructosylating activity, it is able to cleave the β-1,2 linkage of sucrose, transferring the fructosyl group to an acceptor molecule, with the resulting formation of fructooligosaccharides and release of glucose [35]. This enzyme shows little affinity towards water as an acceptor, therefore the hydrolase activity of FTase is very low [36].

FFase can catalyze both hydrolytic and transfructosylating reactions, nevertheless, transfruc‐ tosylation only takes place when sucrose concentrations are higher than 500 g L-1 [27,34,36-38]. The production of FOS by the action of FFase on sucrose can occur either by reverse hydrolysis or by transfructosylation [36].



**Type of prebiotics**

FOS Enzymatic reactions:

32 Food Production and Industry

Inulin Natural product,

GOS Enzymatic

transgalactosylation reactions, using lactose as substrate; Fermentation process.

extraction from plants

fructosyltransferases using sucrose as a substrate or from inulin using microbial endoinulinases.

**Obtention source Enzyme processing Microbial producer Industrial product**

*B. macerans Z. mobilis L. reutri A. niger A. japonicus A. foetidus A. sydowi A. pullans C. purpurea F. oxysporum P. citrinum P. frequentans P. spinulosum P. rigulosum P. parasitica S. brevicaulis S. cerevisiae K. marxianus*

Not applicable Not apllicable Inulin-S –

β-Galactosidases *Aspergillus sp.*

*Bacillus sp. B. circulans Kluyveromyces sp. B. bifidum*

Fructosyltransferases or β-fructofuranosidases; Levansucrases; Endoinulinases.

**and manufacturer**

Neosugar Actilight NutraFlora P-95 - GTC Nutrition Raftilose P95 - Orafti Group

SigmaAldrich Fibruline - Trades

Fibrex - Danisco Sugar

Frutafit CLR DP8, Fruta- fit HD DP10, Frutafit TEX DP5, Inulin TEX – Sensus

Inulin GR, HP, HPgel, HPX, LS, ST, Raftilin ST, Raftilose P95, Raftiline HP - Orafti Group

Vivinal GOS Syrup - Bolculo Domo or Friesland Foods Domo

S.A.

**References**

[21,29, 170, 176,

[189, 194, 222]

[105,120, 198, 202-205, 223- 227]

222]


**Table 2.** Obtention and industrial production of prebiotics.

FOS are produced at industrial scale from concentrated sucrose solutions using fungal transfructosylating enzymes mainly from strains of *Aspergillus niger, Aspergillus oryzae* and *Aureobasidium pullulans* [27,29,30]. Moreover, production of FTase from bacteria (*Lactobacillus*) and yeasts (*Rhodotorula*, *Candida*, *Cryptococcus* sp) has been reported [39,40]. The main enzymes used for industrial production of FOS generally give rise to a mixture of molecules with the inulin-type structure, 1 F-FOS, whereas those from yeasts usually form levan-type FOS (6 F-FOS) or neoFOS (6 G-FOS) [41].

The enzymes from *Aureobasidium pullulans* and from *Aspergillus niger* are highly regiospecific in the fructosyl transfer reaction, transferring one fructosyl moiety from sucrose to the 1-OH of the furanoside of another fructose molecule or fructooligosaccharide, with high selectivity [27]. This synthesis is a complex process in which several reactions occur simultaneously, both in parallel and in series, because sc-FOS are also potential substrates of FTase [42].

Catalytic and physicochemical properties of the producing enzymes, as well as production conditions and composition of FOS are different, depending on the microbial strain. For instance, fungal FTases have molecular masses ranging between 180,000 and 600,000, and are homopolymers with two to six monomer units [43].

Fructosyltransferase from *Aureobasidium pullulans* was submitted to preparative scale chro‐ matographic separation on a weak anion-exchanger [42]. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. Analysis of the action of FTase on a FOS substrate (Actilight 950P) showed that sucrose was the only donor of fructosyl moiety used in the transfer reaction catalyzed by the enzyme, while the acceptor could be another molecule of fructose or FOS [42].

A transferase isolated and purified from *Aspergillus aculeatus* exhibited pH and temperature optima of 6.0 and 60°C, respectively, remaining stable with no decrease in activity after 5 h under such conditions [44]. The enzyme was monomeric with a molecular mass of 85 kDa. On the other hand, FFase I from *A. pullulans* DSM2404 had a molecular weight of 430,000 [45]. The biocatalyst from *A. aculeatus* showed both transfructosylation and hydrolytic activity, and the transfructosylation ratio increased to 88% at 600 mg mL-1 of sucrose [44]. Conditions such as sucrose concentration (400 mg mL-1), temperature (60°C) and pH (5.6) favored synthesis of high levels of GF3 and GF4. The major products were GF2 after 4 h and GF4 after 8 h of reaction. Prolonged incubation for 16 h resulted in the conversion of GF4 into GF2 due to hydrolase activity.

The theoretical yield of FOS from sucrose is 75% if 1-kestose is the only FOS produced [46]. However, production yields of FOS are typically low (55–60%) due to the hydrolytic activity which gives rise to glucose and fructose as reaction byproducts [27] and/or to the fact that glucose acts as an inhibitor of the enzymes, reducing the reaction efficiency [36,47,48]. To improve FOS production yields, glucose oxidase has been used to remove glucose via transformation to gluconic acid [49] and glucose isomerase has been used to interconvert glucose to fructose [46]. Nevertheless, it is necessary to seek for strains among the microbial diversity with high transfructosylating activity, able to produce high yields of oligosaccharides and low yields of monomeric sugars [35].

**Type of prebiotics**

34 Food Production and Industry

**Table 2.** Obtention and industrial production of prebiotics.

inulin-type structure, 1

G-FOS) [41].

molecule of fructose or FOS [42].

activity.

homopolymers with two to six monomer units [43].

or neoFOS (6

**Obtention source Enzyme processing Microbial producer Industrial product**

FOS are produced at industrial scale from concentrated sucrose solutions using fungal transfructosylating enzymes mainly from strains of *Aspergillus niger, Aspergillus oryzae* and *Aureobasidium pullulans* [27,29,30]. Moreover, production of FTase from bacteria (*Lactobacillus*) and yeasts (*Rhodotorula*, *Candida*, *Cryptococcus* sp) has been reported [39,40]. The main enzymes used for industrial production of FOS generally give rise to a mixture of molecules with the

The enzymes from *Aureobasidium pullulans* and from *Aspergillus niger* are highly regiospecific in the fructosyl transfer reaction, transferring one fructosyl moiety from sucrose to the 1-OH of the furanoside of another fructose molecule or fructooligosaccharide, with high selectivity [27]. This synthesis is a complex process in which several reactions occur simultaneously, both

Catalytic and physicochemical properties of the producing enzymes, as well as production conditions and composition of FOS are different, depending on the microbial strain. For instance, fungal FTases have molecular masses ranging between 180,000 and 600,000, and are

Fructosyltransferase from *Aureobasidium pullulans* was submitted to preparative scale chro‐ matographic separation on a weak anion-exchanger [42]. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. Analysis of the action of FTase on a FOS substrate (Actilight 950P) showed that sucrose was the only donor of fructosyl moiety used in the transfer reaction catalyzed by the enzyme, while the acceptor could be another

A transferase isolated and purified from *Aspergillus aculeatus* exhibited pH and temperature optima of 6.0 and 60°C, respectively, remaining stable with no decrease in activity after 5 h under such conditions [44]. The enzyme was monomeric with a molecular mass of 85 kDa. On the other hand, FFase I from *A. pullulans* DSM2404 had a molecular weight of 430,000 [45]. The biocatalyst from *A. aculeatus* showed both transfructosylation and hydrolytic activity, and the transfructosylation ratio increased to 88% at 600 mg mL-1 of sucrose [44]. Conditions such as sucrose concentration (400 mg mL-1), temperature (60°C) and pH (5.6) favored synthesis of high levels of GF3 and GF4. The major products were GF2 after 4 h and GF4 after 8 h of reaction. Prolonged incubation for 16 h resulted in the conversion of GF4 into GF2 due to hydrolase

in parallel and in series, because sc-FOS are also potential substrates of FTase [42].

*and T. maritima A. carbonarious L. mesenteroides*

F-FOS, whereas those from yeasts usually form levan-type FOS (6

**and manufacturer**

**References**

F-FOS)

In addition, the supply of sc-FOS is limited compared to their increasing demand in the food industry, because enzymes such as fructosyltransferases are not widely commercially available [50]. For this reason, the production of FOS is usually carried out in a two-stage process, in which the first stage consists of the microbial production of the enzyme with transfructosyla‐ tion activity, while the second involves the reaction of the produced enzyme with sucrose (substrate) to generate FOS [29].

A commercial pectinase preparation from *Aspergillus aculeatus*, Pectinase Ultra SP-L, contains FTase [51,52] besides being composed of different pectinolytic and cellulolytic enzymes. The preparation, used in the food industry to reduce the viscosity of fruit juices [42,53], was the only commercially available source of FTase according to [42].

Enzymes from *Aspergillus japonicus*, *Aspergillus aculeatus* (Pectinex Ultra SP-L) and *Aureobasi‐ dium pullulans* were used to determine the reaction conditions required to obtain high yields of sc-FOS [34,51,54]. High concentrations of sucrose (600-850 g L-1), pH (4.5–6.5), temperature (50–60°C), reaction time (3–5 h) and high ratios of transferase and hydrolase activities of the enzyme favored transfructosylation over hydrolysis reaction [44,53].

In a recent study, twenty-five commercial enzyme preparations used in the food industry were screened for transfructosylation activity. Three preparations showed high transfructosylation activity from sucrose, high ratios of transferase over hydrolase activity, selectivity for the synthesis of sc-FOS and did not hydrolyze the produced sc-FOS after a 12 h reaction time [55]. Among these enzymes, a cellulolytic enzyme preparation, Rohapect CM, catalyzed the synthesis of sc-FOS with relatively high production yield (63.8%), under cost-effective conditions of temperature (50°C), sucrose concentration (2.103 M) and enzyme concentration (6.6 TU/mL), which could provide a process with potential application at industrial scale [50].

The synthesis of FOS from sucrose is economically advantageous because sucrose is less expensive than inulin; however, the use of enzymes as catalysts for industrial processes is expensive. Furthermore, the recovery of soluble enzymes for reuse is not economically feasible. In contrast, enzyme immobilization usually confers high storage and long-term operational stability, facilitates the recovery and reuse of the biocatalyst, allowing a cost-efficient use of the enzyme in continuous operation, among other advantages [56,57].

In this context, the commercial enzyme preparation from *Aspergillus aculeatus* (Pectinex Ultra SP-L) has been studied for production of FOS in free and immobilized form. Immobilization of the enzyme onto Eupergit C led to retention of enzyme activity for 20 days of batch 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 reaction [59].

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

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 FOS production from sucrose was 55%.

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 [29,63,64].

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 (32°C and 385 rpm) [62].

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 mixture of FOS (1 F-FOS, 6 F-FOS and 6 G-FOS, including a non-conventional disaccharide (blastose)) with different glycosidic linkages, which could afford certain benefits regarding their bioactivity [41].

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 FOSproducing processes [65,66].

Immobilization of whole cells of *Aspergillus japonicus* ATCC 20236 onto different lignocellulosic materials was also undertaken to produce fructooligosaccharides. Cells immobilized in the different support materials showed FOS production and FFase activity ranging from 128.35 to 138.73 g/L and from 26.83 to 44.81 U/mL, respectively. Corncobs were the best support for immobilization, providing the highest results of microorganism immobilization, FOS and FFase production. In addition, use of immobilized cells led to higher FOS productivity and yield, as well as higher transfructosylation over hydrolysis ratio of FFase than free cells [64].

Several important health benefits are associated with the consumption of FOS as food ingre‐ dients. These include modulation of colonic microflora; improvement of the gastrointestinal physiology; activation of the immune system; enhancement of the bioavailability of minerals; reduction of the levels of serum cholesterol, triglycerides and phospholipids; and prevention of colonic carcinogenesis [34,44,67,68].

Among the different FOS, 1-kestose is considered to have better therapeutic properties than those with higher degree of polymerization [69]. The chain length is an important factor influencing the physiological effect of the oligomer in the host [69] and fermentation by bifidobacteria and lactobacilli species [70].

In this context, fermentation of oligosaccharides was evaluated using pure FOS mixtures containing three FOS species (GF2, GF3 and GF4). Only two oligosaccharides (GF2 and GF3) were consumed by *Lactobacillus* strains. Moreover, none of the investigated strains metabolized the GF4 species, suggesting an intracellular metabolism after the FOS transport [70]. This transfer apparently involves an ATP-dependent transport system with specificity for a limited scope of substrates [71].

Moreover, β-fructofuranosidase activity enables bifidobacteria to degrade FOS. Nevertheless, this property is strain-dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates [72].

FOS can be used as calorie-free and non-carcinogenic sweeteners. 1-Kestose has enhanced sweetening power compared to other sc-FOS, and 1-kestose-rich sc-FOS syrups can be used as sugar for diabetics [27,73].

Other types of FOS, such as the levan-type and the neo-FOS, have very promising properties; however, they are not yet commercially available [53,74,75].
