Jurnal FOS

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Food and Bioprocess TechnologyAn International Journal ISSN 1935-5130 Food Bioprocess TechnolDOI 10.1007/s11947-013-1221-6

An Overview of the Recent Developmentson Fructooligosaccharide Production andApplications

Ana Lusa Dominguez, Lgia RaquelRodrigues, Nelson Manuel Lima & JosAntnio Teixeira

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REVIEW

An Overview of the Recent Developmentson Fructooligosaccharide Production and Applications

Ana Lusa Dominguez & Lgia Raquel Rodrigues &Nelson Manuel Lima & Jos Antnio Teixeira

Received: 11 September 2013 /Accepted: 25 October 2013# Springer Science+Business Media New York 2013

Abstract Over the past years, many researchers have sug-gested that deficiencies in the diet can lead to disease statesand that some diseases can be avoided through an adequateintake of relevant dietary components. Recently, a great inter-est in dietary modulation of the human gut has been registered.Prebiotics, such as fructooligosaccharides (FOS), play a keyrole in the improvement of gut microbiota balance and inindividual health. FOS are generally used as components offunctional foods, are generally regarded as safe (generallyrecognized as safe statusfrom the Food and Drug Admin-istration, USA), and worth about 150 per kilogram. Due totheir nutrition- and health-relevant properties, such as moder-ate sweetness, low carcinogenicity, low calorimetric value,and low glycemic index, FOS have been increasingly usedby the food industry. Conventionally, FOS are producedthrough a two-stage process that requires an enzyme produc-tion and purification step in order to proceed with the chemicalreaction itself. Several studies have been conducted on theproduction of FOS, aiming its optimization toward the devel-opment of more efficient production processes and their po-tential as food ingredients. The improvement of FOS yield andproductivity can be achieved by the use of different fermen-tative methods and different microbial sources of FOS-producing enzymes and the optimization of nutritional andculture parameter; therefore, this review focuses on the latest

progresses in FOS research such as its production, functionalproperties, and market data.

Keywords Fructooligosaccharides . Prebiotics .

Transfructosylation . Production yield . Fructosyltransferase .

Fructofuranosidase

Introduction

While food has long been used to improve health, knowledgeof health is now being used to improve food. The first sys-tematic exploration of the positive aspects of food functional-ity was undertaken in Japan where research programs fundedby the Japanese government during the 1980s focused on theability of some foods to influence physiological functions(Clydesdale 2004; Howlett 2008).

A narrow analysis of the topic suggests that all food isfunctional since it provides basic energy and nutrients essen-tial for surviving. Yet, the term functional food in usenowadays implies that such food carries broader health bene-fits than just basic nutrition. Food science has evolved fromsolving problems related to nutritional deficiencies to design-ing foods that have the potential to induce health benefits andreduce the risk of disease (Clydesdale 2004).

Building a path to an optimum nutrition, which is anaspiring long-term goal, functional food is a newsworthyand stimulating concept, as much as it is supported by con-sensual scientific data generated by the development of func-tional food science aimed at the improvement of dietaryguidelines that integrate new information on the food elementsand body function interactions (Roberfroid 2000a).

The gut is a body organ with major influence in severalbody functions; thus, it became a clear target for the evolutionof functional food, and over the past decade, we havewitnessed an increase of new information concerning the

A. L. Dominguez (*) : L. R. Rodrigues :N. M. Lima :J. A. TeixeiraCentre of Biological Engineering, IBBInstitute for Biotechnologyand Bioengineering, University of Minho, Campus de Gualtar,4710-057 Braga, Portugale-mail: [email protected]

L. R. RodriguesBiotempoConsultoria em Biotecnologia, Lda, Avepark-Edif.Spinpark, Zona Industrial da Gandra, Barco, 4805-017Guimares, Portugal

Food Bioprocess TechnolDOI 10.1007/s11947-013-1221-6

Author's personal copy

interface between the diet and its possible role on gut micro-biota and, consequently, in health and disease. This has led togreat efforts in the development of strategies aimed at dietarymodulation of the microbiota composition and activity,achieved by the use of prebiotics, probiotics, and a combina-tion of both (synbiotics; Howlett 2008; Szajewska 2010; DePreter et al. 2011).

Various non-digestible carbohydrates have been tested fortheir effect on the microbial community. Among them, thefunctional oligosaccharides, which are intermediate in naturebetween simple sugars and polysaccharides, present importantphysicochemical and physiological properties beneficial to theconsumers health, behaving as dietary fibers and prebiotics,and therefore their use as food ingredients has increasedrapidly (Simmering and Blaut 2001; Kunz and Rudloff2006; Mussatto and Mancilha 2007).

F O S , g a l a c t o o l i g o s a c c h a r i d e s ( G O S ) ,isomaltooligosaccharides (IMO), soybean oligosaccharides,xylooligosaccharides (XOS), and maltitol are among the com-monly used prebiotics, FOS being the most studied and thebest implemented oligosaccharides in the European market(Chen et al. 2000; Qiang et al. 2009; Nobre et al. 2013).

Prebiotics

According to the FAO, a prebiotic is a non-viable foodcomponent that confers a health benefit to the host associatedwith the modulation of the microbiota (Gibson et al. 2004;Pieiro et al. 2008).

Prebiotics can be used as a strategy to improve thebalance of intestinal bacteria differing from the probioticapproach in which exogenous strains are included infood and ultimately reach the individuals intestinaltract. These compounds selectively stimulate the prolif-eration and/or activity of beneficial groups of bacteriathat exist in the intestinal microbiota. As a consequence,they can constitute a more practical and efficient way tomanipulate the gut microbiota compared to probiotics.However, if for any reason, such as disease, aging,antibiotic, or drug therapy, the appropriate health-promoting species are not present in the bowel, the prebi-otic is unlikely to be effective (Playne and Crittenden 2004;Macfarlane et al. 2008).

The gut microbiota ferments a range of substances, mainlyprovided by the diet, which cannot be digested by the host inthe small intestine and are available for fermentation by thecolonic microbiota. These include resistant starch, non-starchpolysaccharides (dietary fiber), oligosaccharides, proteins,and amino acids, among others. In a typical adult, about100 g of food ingested each day reaches the large intestineand therefore is susceptible to fermentation by the gut micro-biota. Key criteria for a food ingredient to be classified as a

prebiotic are (1) it must not be hydrolyzed or absorbed in theupper part of the gastrointestinal tract so that it reaches thecolon in significant amounts and (2) it must be a selectivesubstrate for one or more beneficial bacteria that are stimulat-ed to grow. Furthermore, prebiotics may also induce local (inthe colon) or systemic effects through bacterial fermentationproducts that are beneficial to host health (Manning andGibson 2004; Howlett 2008; De Preter et al. 2011).

These ingredients are normally restricted to certain carbo-hydrates, i.e., non-digestible oligosaccharides (NDO) that aredistinguished from other carbohydrates for being resistant todigestion in the stomach and small intestine (Playne andCrittenden 2004).

Most prebiotics and prebiotic candidates that have beenidentified are NDO and are obtained either by extraction fromplants (e.g., chicory inulin), possibly followed by an enzymat-ic hydrolysis (e.g., oligofructose from inulin), or by synthesis(by transglycosylation reactions) frommono- or disaccharidessuch as sucrose (FOS) or lactose (GOS; Crittenden and Playne1996). Nevertheless, other NDO including XOS, IMO, andsoybean oligosaccharides have also been evaluated for theirprebiotic effect (Gibson and Roberfroid 1995; Simmering andBlaut 2001).

Apart from their potential to modify the gut microbiota andits metabolic activities in a beneficial way, many other helpfuland useful effects of prebiotics are being investigated. Theseinclude their ability to modulate gut function and transit time;to activate the immune system; to increase the production ofbutyric and other short-chain fatty acids; to increase the ab-sorption of minerals, such as calcium and magnesium; and toinhibit lesions that are precursors of adenomas and carcino-mas. Thus, prebiotics can potentially reduce some of the riskfactors involved in the causes of colorectal diseases and re-duce the risk of diseases such as cardiovascular disease, coloncancer, and obesity. Strategies for developing prebiotic prod-ucts aim to provide specific fermentable substrates for bene-ficial bacteria (bifidobacteria and lactobacilli). These mayprovide adequate amounts and proportions of fermentationproducts, especially in the lower part of the colon where theeffects are believed to be most favorable. Although the asso-ciation with past outbreaks will remain conjectural, it is rea-sonable to suggest that prebiotic administration may possiblyexert very important prophylactic effects against gutpathogens. Even as part of a more complex story, thepotential of prebiotics is too large to be ignored (Ziemerand Gibson 1998; Howlett 2008; Qiang et al. 2009; Shimizuand Hachimura 2011).

Fructooligosaccharides as Prebiotics

FOS is a common name for fructose oligomers, and these areusually understood as inulin-type oligosaccharides. They

Food Bioprocess Technol


constitute a series of homologous oligosaccharides derivedfrom sucrose usually represented by the formula GFn, whichare mainly composed of 1-kestose (GF2), nystose (GF3), and1F-- fructofuranosyl nystose (GF4), in which two, three, andfour fructosyl units are bound at the -2,1 position ofglucose, respectively, as shown in Fig. 1 (Yun 1996;Lee et al. 1999).

FOS Functional Properties

FOS have a low sweetness intensity since they are only aboutone third as sweet as sucrose, supplying small amounts ofenergy, approximately 03 kcal/g of sugar substitute. Thisproperty is quite useful in the various kinds of foods in whichthe use of sucrose is restricted by its high sweetness. FOS arealso calorie-free since they are scarcely hydrolyzed by thedigestive enzymes and not utilized as an energy source inthe body; thus, they are safe for diabetics. Also, prebioticsare known to prevent the colonization of human gut by path-ogenic microorganisms because they encourage the growth ofbeneficial bacteria (Roberfroid 2000b; Mishra and Mishra2013). As FOS cannot be digested by the enzymes of thesmall intestine, they are fermented in the large intestine toselectively stimulate the growth of probiotic-like bacteria thatare part of the commensal gut microbiota and then produceshort-chain fatty acids (SCFAs), mainly acetate, propionate,and butyrate (Yun 1996; Yu Wang et al. 2010). It is alsoimportance to notice that long-chain FOS may exert a prebi-otic effect in more distal colonic regions compared with thelower-molecular-weight FOS, which may be more quicklyfermented in the saccharolytic proximal bowel (Manningand Gibson 2004).

As previously mentioned, these compounds have the ca-pacity of promoting a good balance of intestinal microbiotaand decrease gastrointestinal infections. The beneficial phys-iologic functions that have been reported for FOS are brieflysummarized below.

Protection Against Colon Cancer

Prebiotics have been postulated to be protective against thedevelopment of colon cancer, which is the second most prev-alent cancer in humans. It is known that several species ofbacteria commonly found in the colon produce carcinogensand tumor promoters from the metabolism of food compo-nents. There have been several studies on the use of prebioticsin cancer prevention mainly focusing on animal models, butthe dietary fiber hypothesis for protection against colorectalcancer was advanced by Burkitt (1969) based on epidemio-logical evidence supporting a relationship between diet andcolon health. These findings showed lower rates of colorectalcancer in Africa compared to industrializedWestern countries,where traditional diets consisted of high amounts of unrefinedfiber and high intakes of refined carbohydrates, respectively(Manning and Gibson 2004; Lim et al. 2005a).

The two main types of fermentation that are carried out inthe gut are saccharolytic and proteolytic. The saccharolyticactivity is more favorable than the proteolytic one due to thetype of metabolic end products that are formed, such asSCFAs, acetate, propionate, and butyrate. Butyrate is an im-portant source of energy and is thought to have antitumorproperties. Many studies were performed to demonstrate suchactivity, and according to Kim et al. (1982), butyrate is aprotective agent against colon cancer by promoting cell dif-ferentiation. Bugaut and Bentjac (1993) reported that buty-rate is used by the epithelial cells of the colon mucosa as anenergy source, being also a growth factor, and recent preclin-ical studies demonstrated that it might be chemopreventive incarcinogenesis (Scheppach and Weiler 2004). In addition tobutyrate, propionate can have anti-inflammatory effects oncolon cancer cells (Munjal et al. 2009).

Immunomodulatory Effect

Functional foods are reported to enhance the immunity of theconsumers. Indeed, the dietary components and their fermen-tation metabolites are in close contact with the gut-associatedlymphoid tissue (GALT), which is the biggest tissue in theimmune system comprising 60 % of all lymphocytes in thebody (Delgado et al. 2011; Saad et al. 2013). It has beenestimated that about 1060 g/day of dietary carbohydratereaches the colon acting like a growth factor to particularcommensal bacteria, which inhibit the adherence and invasionof pathogens in the colonic epithelia by competing for thesame glycoconjugates present on the surface of epithelial

Fig. 1 Structures of 1-kestose (GF2, left), nystose (GF3, center), andfructofuranosyl nystose (GF4, right)



cells, altering the colonic pH, favoring the barrier function,improving the mucus production, producing SCFAs, and in-ducing cytokine production (Korzenik and Podolskyl 2006).

Many researchers recognized the effect of prebiotics oraladministration. In 1997, Pierre and co-workers (Pierre et al.1997) reported the development of GALT associated withinulin and FOS intake. Furthermore, Hosono et al. (2003)stated that inulin and FOS were able to modulate variousparameters of the immune system. Schley and Field (2002)have reviewed some evidences of the immune-enhancingeffects of dietary fibers and prebiotic and identified for thefirst time the gene markers associated with the physiologicaleffects of a particular FOS on a host animal, thus making itpossible to clarify the mechanisms behind the immunomodu-latory effects of FOS.

Obesity and Diabetes

Fiber intakes are associated with increased satiety and lowerbody weight. Beyond glycemic control, the fibers fermenta-tion in the intestine may also influence satiety. However, thesemechanisms are not completely understood. One hypothesis isthat the production of SCFAs influences postprandial glucoseresponse by reducing fat competition for glucose disposal. Onthe other hand, another hypothesis relies on the idea thatfermentation of SCFAs influences gut hormones and gastricmotility (Brighenti et al. 2006; Nilsson et al. 2008). Also,other scientific data suggest that the decrease in food intakeassociated with prebiotics feeding in animals might be linkedto the modulation of gastrointestinal peptides involvedin the regulation of food intake (Druce et al. 2004; Chaudhriet al. 2008).

Obesity and obesity-related type II diabetes are typicaldiseases of the modern Western society. Current recommen-dations for the management of type II diabetes and obesityinclude an increase in dietary fiber intake. Dietary fibersviscose and fibrous structure can control the release ofglucose with time in the blood, thus helping in the propercontrol and management of diabetes mellitus and obesity(Bennett et al. 2006).

Improving Mineral Adsorption

Another interesting feature of FOS is their positive effect onmineral absorption. Mineral deficiencies remain an importantnutritional issue in the World. Calcium (Ca) intake in manyunderdeveloped countries is below the recommended dailyallowance, which leads to progressive bone loss. Ca is criticalin achieving optimal peak bone mass and modulating the rateof bone loss associated with aging. Then, if Ca intake is notenough to offset obligatory losses, acquired skeletal masscannot be maintained, leading to osteoporosis, a major publichealth problem. Magnesium is the second most abundant

intracellular cation in vertebrates, and its deficiency has beenimplicated as a risk factor for osteoporosis. Another mineralwith a low intake is iron (Fe), which is a major cause ofanemia. The importance of zinc (Zn) nurture for growth anddevelopment has been widely documented. Marginal Zndeficiency is suspected to be widespread in populationsheavily dependent on cereal-based diets containing few ani-mal products (Baba et al. 1996; Roberfroid et al. 2010; YuWang et al. 2010).

Several mechanisms have been proposed to elucidate thepossible roles of FOS in improving mineral absorption. Eventhough mineral absorption generally occurs in the upper partof the intestine, it seems that, after consumption of FOS, themajor site of action leading to improved bioavailability ofminerals is the large intestine. The main action of FOS ismainly associated with their fermentation by resident micro-biota. SCFAs decrease luminal pH and thus create an acidicenvironment more favorable for mineral solubility (Gudiel-Urbano and Goi 2002; YuWang et al. 2010). Another way tocontribute to the enhanced mineral absorption is the trophiceffect of prebiotics on the gut (cell growth and functionalenhancement of the absorptive area). It has been suggestedthat this is mediated by an increased production of butyrateand/or certain polyamines (Roberfroid 1993; Raschka andDaniel 2005).

Effects on Serum Lipid and Cholesterol Concentrations

Since cardiovascular risk is the major public health concern inmany countries and accounts for more deaths than any otherdisease or group of diseases, the food industry is increasinglyinterested in the development of functional foods that modu-late blood lipids, such as cholesterol and triglycerides(Manning and Gibson 2004; Qiang et al. 2009).

Many attempts have been made to control serum triacyl-glycerol (TAG) concentrations through the modification ofdietary habits. The hypotriglyceridemic effect of non-digestible but fermentable carbohydrates, including resistantstarch or FOS, has been described by Glore et al. (1994) andJackson et al. (1999) in humans and by Tokunaga et al. (1986)and Yamamoto et al. (1999) in animals.

Ingredients showing a prebiotic effect are able to modulatehepatic lipid metabolism in rats or hamsters, resulting inchanges in either TAG accumulation in the liver (steatosis)or serum lipids. The decrease in TAG synthesis and theaccumulation of dietary prebiotics compounds could be linkedto several events. First, a decrease in glycemia could be part ofthe process since glucose (together with insulin) is a driver oflipogenesis. Secondly, the SCFAs produced through the fer-mentation process could play a role in the regulation of lipidmetabolism (Delzenne and Kok 2001; Delzenne et al. 2002).

Among several studies, Levrat et al. (1994) and Fiordalisoet al. (1995) reported a decrease in total serum cholesterol after



dietary supplementation with inulin (10 %) in mice or rats.This is accompanied by a significant decrease in the hepaticcholesterol content. The authors suggested that the decrease inserum cholesterol could reflect a decrease in TAG-richlipoproteins.

FOS Occurrence

FOS are found in trace amounts as natural componentsin fruits, vegetables, and honey. As a series of fructoseoligomers and polymers derived from sucrose, they oc-cur in many higher plants as reserve carbohydrates. Aspara-gus, sugar beet, garlic, chicory, onion, Jerusalem artichoke,wheat, honey, banana, barley, tomato, and rye are well-knownsources of FOS (Yun 1996; Sangeetha et al. 2005a; Mussattoand Mancilha 2007).

For most of these sources, FOS concentrations range be-tween 0.3 and 6%; for chicory, these values are between 5 and10 %, while in Jerusalem artichoke they can reach 20 %.Based on the usual consumption of these natural sources, anaverage daily consumption of FOS of approximately 13.7 mg/kgbody weight per day or 806 mg/day has been estimated(Voragen 1998).

FOS Production

FOS represent one of the major classes of bifidogenic oligo-saccharides in terms of their production volume. They can beproduced by the hydrolysis of inulin or by thetransfructosylation of sucrose. The FOS mixture obtained bythe enzymatic hydrolysis of inulin closely resembles the mix-ture produced by the transfructosylation process, althoughslightly different end products are obtained as not all of the(12)-linked fructosyl chains end with a terminal glucose.Additionally, the oligosaccharide mixture produced from inu-lin hydrolysis contains longer fructo-oligomer chainsthan that produced by the sucrose transfructosylationprocess. In this process, FOS are produced from thedisaccharide sucrose using enzymes with transfructosylationactivity. These enzymes are invertase (-fructofuranosidasefructohydrolase, FFase, EC 3.2.1.26) and sucrase (sucrosefructosyltransferase, FTase, EC 2.4.1.9), which aremainly derived from fungal and bacterial sources. Ahigh concentration of the starting substrate is requiredfor efficient transfructosylation. In this reaction, FOS areformed, namely, kestose, nystose, and fructofuranosylnystose (Fig. 1), but also some by-products such asglucose and small amounts of fructose (Brenda 2005;Crittenden and Playne 1996; Singh and Singh 2010;Saad et al. 2013).

FOS Microbial ProductionTransfructosylation Enzymes

The commercially available FOS are produced through theenzymatic synthesis from sucrose by microbial enzymes withtransfructosylation activity. Some bacterial strains have beenreported to produce such enzymes. On the other hand, severalfungal strains, such as Aspergillus sp., Aureobasidium sp.,and Penicillium sp., are known to produce extracellular and/orintracellular enzymes with transfructosylation activity(Table 1).

Many authors have reported the purification and character-ization of FOS-producing enzymes from various sources andby different microorganisms (bacteria and fungi; Hayashiet al. 1992; LHocine et al. 2000; Park et al. 2001; Nguyenet al. 2005; Jedrzejczak-Krzepkowska et al. 2011; Risso et al.2012). Although these proteins differ in their subunit struc-ture, molecular weight, degree of glycosylation, chemicalsusceptibility, and substrate specificity, they all display bothhydrolytic and transfer activities. The same microorganismcan even produce several enzymes with transfructosylationactivity holding different characteristics (Yoshikawa et al.2007).

There is still a dispute in the scientific community regard-ing the nomenclature of FOS-producing enzymes. Some re-searchers use the term -fructofuranosidase (FFase, EC.3.2.1.26; Nguyen et al. 1999; Sheu et al. 2001), whereasothers designate it as fructosyltransferase (FTase, EC.2.4.1.9;Yun et al. 1997; Sangeetha et al. 2004; Vandkov et al. 2004).The latter denomination is probably due to the fact that FTaseactivity was originally found from the side action in the courseof FFase preparation when acting on a high concentration ofsucrose (Straathof et al. 1986; LHocine et al. 2000).

Transfructosylating activity acts on sucrose by cleaving the(1,2) linkage and transferring the fructosyl group to anacceptor molecule such as sucrose, releasing glucose. Thisreaction yields FOS, i.e., fructose oligomers, in whichfructosyl units are bound at the (2,1) position of sucrose(Belghith et al. 2012). Although FFase is generally known asan enzyme catalyzing the hydrolysis of sucrose, some FFaseshave much higher transfructosylating activity (U t) than hy-drolyzing activity (U h). A wide variety of fungi produceFFases, but the activity ratios (U t/Uh) of their enzymes varysignificantly (Sangeetha et al. 2005a; Yoshikawa et al. 2006).Fructosyl-transferring enzymes have been purified and char-acterized from higher plants, such as asparagus (Shiomi1982), onion (Fujishima et al. 2005), Jerusalem artichoke(Koops and Jonker 1994), and from different microorganisms(fungi and bacteria) such as Aspergillus niger (LHocine et al.2000), Aspergillus japonicus (Hayashi et al. 1992),Aureobasidium pullulans (Yoshikawa et al. 2006), Bacillusmacerans (Park et al. 2001), and Candida utilis (Chvez et al.1997). Although these proteins differ in their subunit struc-ture, molecular weight, degree of glycosylation, chemical



susceptibility, and substrate specificity, they all display bothhydrolytic and transfer activities (Ghazi et al. 2007). FungalFTases have molecular masses ranging between 180,000 and600,000 and are homopolymers with two to six monomerunits. Some studies stated the optimum temperature and pHfor these enzyme activities between 50 and 60 C and from 4.5to 6.5, respectively (Madlov et al. 2000; Maiorano et al.2008).

There are several studies reporting the characteristics oftransfructosylation enzymes produced by fungi, mainlyAureobasidium sp. and Aspergillus sp. Antoov et al.(2002) reported U t in A . pullulans CCY 27-1-1194 in theextracellular medium and in the whole cells. In their study, amaximum FTase U t of 131,000 U dm

3 (131 U mL1) re-leased into the cultivation medium, after four cultivation days,was reported. They also reported that with an initial sucroseconcentration of 200 g L1, an FTase U t of 80,000 U dm

3

(80 U mL1) is achieved. In 2008, Antoov et al. (2008)reported the chromatographic separation of an intracellularFTase from A . pullulans . The isolated enzyme exhibited bothU h and U t activities, the U t being higher in the sucroseconcentration range tested (10600 g L1) and completely

dominating at higher sucrose concentrations. Yoshikawaet al. (2006) have reported five FFase extracted from the cellwall of A . pullulans with high U t. This study also suggestedthat the expression of FFase I was not repressed by glucose,but those of FFases IIV were strongly inhibited in the pres-ence of glucose, considering that FFase I played a key role inFOS production by this fungus, whereas FFase IV may func-tion as a FOS-degrading enzyme with its strong hydrolyzingactivity.

Hayashi et al. (1992) identified a FFase produced by A.japonicus and optimized the cultural condition for the pro-duction of the enzyme and the enzymatic reaction conditionsfor the industrial utilization of the strain to produce FOS. Lateron, FFase activity in A . japonicus was also reported by Chen(1995); a maximum enzyme production of 910 U mL1 wasachieved. Wang and Zhou (2006) isolated an A . japonicusstrain from soil and investigated the optimal conditions forFFase, reaching an activity of 55.42 U mL1 at pH 5.5 and30 C. Wallis et al. (1997) identified two FFase secreted by A.niger. LHocine et al. purified and partially characterized aFTase and a FFase from the crude extract of A . niger. Thisstrain showed very high enzyme productivity and high FTase

Table 1 Microbial sources of enzymes with transfructosylation activity: FOS-producing enzymes

Source Microorganism Enzyme Reference

Fungal transfructosylation enzyme Aureobasidium pullulans Intra- and extracellular FTase Yun et al. (1997)

Intra- and extracellular FTase Sangeetha et al. (2004a)

Intra- and extracellular FTase Vandkov et al. (2004)

Intracellular FTase Lateef et al. (2007)

Periplasmic FFase Yoshikawa et al. (2006)

Extracellular FTase Dominguez et al. (2012)

Aspergillus aculeatus FTase from commercial enzymepreparationPectinex Ultra SP-L

Nemukula et al. (2009)

Ghazi et al. (2005)

Ghazi et al. (2007)

Aspergillus flavus Extracellular FTase Ganaie et al. (2013)

Aspergillus japonicus Intra- and/or extracellular FFase Chen and Liu (1996)

Intra- and/or extracellular FFase Wang and Zhou (2006)

Extracellular FFase Mussatto et al. (2009)

Extracellular FFase Mussatto and Teixeira (2010)

Aspergillus niger Extracellular FFase Wallis et al. (1997)

Intra- and extracellular FFase Nguyen et al. (1999)

Intra- and/or extracellular FTase LHocine et al. (2000)

Extracellular FTase Ganaie et al. (2013)

Aspergillus oryzae Extracellular FTase Sangeetha et al. (2004a)

Extracellular FTase Sangettha et al. (2005a)

Aspergillus terreus Extracellular FTase Ganaie et al. (2013)

Penicillium citrinum Intra- and/or extracellular enzyme Lim et al. (2005b)

Penicillium islandicum Extracellular FTase Ganaie et al. (2013)

Bacterial transfructosylation enzyme Bacillus macerans Extracellular transfructosylation enzyme Park et al. (2001)

Lactobacillus reuteri Intra and/or extracellular FTase van Hijum et al. (2002)

Zymomonas mobilis Extracellular levansucrase Bekers et al. (2002)



activity; however, the enzyme used in the crude state alsoshowed hydrolytic activity. Balasubramaniem et al. (2001)reached a maximum FFase activity of 149 U L1 h1, thisenzyme being produced by A . niger. Nguyen et al. (2005)purified an intracellular FFase produced by A . niger andestimated its molecular mass to be in the range from 120 to130 kDa. Extracellular FTase activity in Aspergillus oryzaewas identified by Sangeetha et al. (2004b), with a molecularmass of 116.3 kDa. Later on, the same authors reported anFFase activity in that same strain in the range of 152 U mL1 min1 (Sangeetha et al. 2005a).

There are mainly two methods that can be used to producetransfructosylation enzymes and FOS by fermentation, name-ly, by submerged fermentation (SmF) or by solid state fer-mentation (SSF). These two methods will be further discussedin the following sections.

FOS Production by SmF

The most common and studied method to produce FOS is bythe transfructosylation of sucrose in a two-stage process,schematically represented in Fig. 2, the enzyme(s) being pro-duced by SmF in the first stage. This is also the usual processused to produce the FOS that are currently available on themarket (Singh and Singh 2010).

Most studies on the experimental conditions for enzymeproduction are mainly based on shake-flask experiments, and

the work carried out in bioreactors is unusual. The variablesgenerally studied to define the best operational conditions forenzyme production are the carbon and nitrogen sources, theirconcentrations, time of cultivation, agitation, and aerationrates. Other important factors are related with the addition ofdifferent mineral salts, small amounts of amino acids, poly-mers, and surfactants (Maiorano et al. 2008).

Chen and Liu (1996) evaluated the effect of various carbonand nitrogen sources in enzyme production and found thatsucrose at 25 % (w /v ) and yeast extract were the best ones,respectively. Later on, Antoov et al. (2002) obtained thehighest FTase activity using sucrose at 350 g L1 and foundthat biomass production was not influenced by sucrose con-centration in the range from 50 to 350 g L1. Vandkov et al.(2004) also studied the effect of sucrose on FTase productionby Aerobasidium pullulans and reported that sucrose contentdid not influence the total production of FTase. However, highsucrose concentrations resulted in high specific activities ofthe cells.

The effects of inorganic salts were reported by variousauthors. Chen and Liu (1996) studied the effect of inorganicsalts and found that the addition of MgSO47H2O and K2HPO4shifted the morphology of the fungal growth from filamentousto pellet form without affecting FFase production. The mor-phology of fungal growth can range from dispersed mycelia tocompact pellets. Control of mycelia morphology in fermenta-tions is often a prerequisite for industrial applications. Although

Fig. 2 Schematic representationof the two-stage FOS productionprocess by SmF (adapted fromSangeetha et al. 2005c)



fungus, grown in the form of mycelia, may have a high specificgrowth rate, the growth of fungus in the form of pellets is moreattractive to many industrial fermentation processes from theperspective of reducing the power input needed for adequategas dispersion and mixing (Metz and Kossen 1977; Chen andLiu 1996). K2HPO4 is described as a microelement source forcell growth, as well as a buffering reagent. Its optimal concen-tration ranges between 4g L1 (Sangeetha et al. 2005a, b) and5 g L1 (Vandkov et al. 2004; Shin et al. 2004a; Lim et al.2005b). Mg2+ affects the permeability of the cell wall for A .pullulans (Vandkov et al. 2004), and its optimal concentra-tion varies from 0.3 g L1 (Sangeetha et al. 2005a, b) to 2 g L1

(Balasubramaniem et al. 2001). NaNO3 is the most commonsource of inorganic nitrogen in transfructosylation enzymesproduction by fungi and can be employed in concentrationsfrom 2 g L1 (Lim et al. 2005b) to 25 g L1 (Dhake and Patil2007). Other ions, such as FeSO4 and CuSO4, can also beadded to the medium with positive effects, such as increasingFFase activity and FOS production (Balasubramaniem et al.2001; Lim et al. 2005b; Wang and Zhou 2006).

The effect of the medium pH in FTase production andmicroorganism growth has been reported. pH of 5.5 wasfound to be the best initial value for FTase production by A.oryzae CFR202 (Sangeetha et al. 2005a, b), A. japonicusJN19 (Wang and Zhou 2006), and Penicillium purpurogenum(Dhake and Patil 2007).

The FOS production yield by A . pullulans in a two-stageprocess can vary between 44.0 % (gFOS/g sucrose; Shin et al.2004b) and 62.0 % (gFOS/g sucrose; Yoshikawa et al. 2008). ForA . japonicas , these values can vary between 55.8 % (gFOS/g sucrose; Wang and Zhou 2006) and 61.0 % (gFOS/g sucrose;Chiang et al. 1997), whereas for A. niger, values rangingbetween 24.0 % (gFOS/g sucrose; Nguyen et al. 1999) and70.0 % (gFOS/g sucrose; Maldov et al. 1999) can be expected.On the other hand, Sangeetha et al. (2005a, b) reported FOSproduction yield by A . oryzae of 53.0 % (gFOS/g sucrose) to57.4 % (gFOS/g sucrose).

FOS Production bySSF

Despite most industrial enzymes being produced in SmF, SSFpresents an interesting potential for small-scale units. Someadvantages of this process are the simplicity of operation, highvolumetric productivity, product concentration, and low capitalcost and energy consumption. Moreover, the risk of contami-nation is reduced. SSF requires low water volume and thus hasa large impact on the economy of the process due to smallerfermenter size, reduced downstream processing, reduced stir-ring, and lower sterilization costs (Sangeetha et al. 2005c;Mussatto and Teixeira 2010). However, there are also somedisadvantages associated with the use of SSF, which havediscouraged the use of this technique for industrialenzyme production. The main drawbacks are due to the

buildup of gradientsof temperature, pH, moisture, substrateconcentration, or dissolved oxygenduring cultivation,which are difficult to control under limited water availability,leading to scale-up engineering problems (Pandey 2003;Hlker et al. 2004).

The use of low-cost agricultural and agro-industrial resi-dues as substrates contributes also to the lower capital andoperating costs of SSF as compared to SmF. Several agricul-tural by-products like cereal bran, corn products, sugarcanebagasse, and by-products of coffee and tea processing indus-tries were used as substrates to produce FTase in SSF by A .oryzae CFR 202 (Sangeetha et al. 2004b). In this study, ricebran and wheat bran were found to be the substrates thatmaximize FTase production, with optimum activity at 60 Cand pH 6.0. The maximum FOS production (52.0 %, gFOS/g sucrose) was obtained after 8 h of reaction. Mussatto andTeixeira (2010) reported the increase of FOS yield and pro-ductivity by SSF with A . japonicus using agro-industrialresidues as immobilization supports and nutrient sources.Corn cobs, coffee silverskin, and cork oak were used assupport and nutrient sources. Coffee silverskin was found tobe the one that provided the higher production yield and FFaseactivity, reaching 70.0 % (gFOS/g sucrose) after 16 h of reaction.These authors conducted a similar study with Penicillumexpansum in which the use of low-cost materials includingsynthetic fiber, polyurethane foam, stainless steel sponge,loofah sponge, and cork oak were tested as carriers for fungusimmobilization (Mussatto et al. 2012). Additionally, produc-tion of FOS and FFase by repeated batch fermentation of P.expansum immobilized on synthetic fiber was studied. Pro-duction yields of 87 % (gFOS/g sucrose), 72 % (gFOS/g sucrose),and 44 % (gFOS/g sucrose) in the three initial cycles (36, 48, and60 h), respectively, were obtained.

Generally, the most efficient culture media and strainto produce enzymes in SSF are not the same as those inSmF, and vice versa (Maiorano et al. 2008). Optimization ofthe fermentation medium for -fructofuranosidase productionby A . niger NRRL 330 in SSF and SmFwas carried out usinga fractional factorial design (Balasubramaniem et al.2001). The results showed different compositions ofoptimized media for SmF and SSF. After the optimiza-tion procedures, the medium composition for SSF re-quired twice more sucrose and yeast extract than the medi-um for SmF, and the productivity in SSFwas about twice thatin SmF.

Table 2 presents a comparison between the FOS productionyields reported in the literature, obtained in various productionstudies by either SmF or SSF.

Production of High-Content FOS

Industrial production of FOS carried out with microbialtransfructosylation enzymes was found to give a maximum



theoretical yield of 5560 % based on the initial sucroseconcentration (Sangeetha et al. 2005c). This yield cannot befurther increased due to the high amounts of glucose co-produced during the fermentation, which acts as an inhibitor(Yun 1996).

Therefore, in order to obtain higher fermentation yields, manyauthors have studied the impact of the continuous removal ofglucose and residual sucrose from the medium during the FOSconversion. Consequently, by increasing the fermentation yields,a purer final product could be obtained (Nobre et al. 2013).

Table 2 FOS yields obtained using FTase and/or FFase from various fungal strains

Microorganism Production process Reaction time (funguscultivation+enzymaticreaction)

Production yield(%, gFOS/gsucrose)

Reference

Aureobasidiumpullulans

Two-stage process using isolatedintracellular enzyme produced by SmF

96+25 h 58.0 Yun and Song (1993)

Two-stage process using extracellularenzyme produced by SmF

48+24 h 55.9 Sangeetha et al. (2004a)

Two-stage process using culture brothhomogenate produced by SmF


Two-stage process using isolatedintracellular enzyme produced by SmF

72+24 h 53.0 Shin et al. (2004a)

Two-stage process with immobilized cell(SmF)

72+24 h 44.0 Shin et al. (2004b)

Two-stage process with isolated intracellularFTase produced by SmF

24+9 h 59.0 Lateef et al. (2007)

Two-stage process using crude FFasepreparation produced by SmF

24+24 h 62.0 Yoshikawa et al. (2008)

One-stage process: present studySmF 48 h 64.1 Dominguez et al. (2012)

Aspergillus flavus Two-stage process using culture brothfiltrate produced by SmF

120+24 h 63.4 Ganaie et al. (2013)

Aspergillus ibericus One-stage processSmF 24 h 61.064.0 Gomes (2009)

Aspergillus japonicus Two-stage process using immobilized FFaseproduced by SmF

28+17 h 61.0 Chiang et al. (1997)

Two-stage process with isolated intracellularFFase produced by SmF

72+24 h 55.8 Wang and Zhou (2006)

One-stage process using corn cobs as thesolid supportSSF

21 h 66.0 Mussatto et al. (2009)

One-stage processSmF 24 h 61.0 Mussatto et al. (2009)

One-stage process using coffee silverskin asthe solid supportSSF

16 h 70.0 Mussatto and Teixeira(2010)

Aspergillus niger Two-stage process with isolated intracellularand extracellular FFase produced by SmF

24+72 h 24.026.0 Nguyen et al. (1999)

Two-stage process using washed cellsproduced by SmF

92+8 h 70.0 Madlov et al. (1999)

Two-stage process using culture brothfiltrate produced by SmF

72+24 h 55.8 Ganaie et al. (2013)

Aspergillus oryzae Two-stage process using extracellularenzyme produced by SmF


Two-stage process using culture brothhomogenate produced by SmF


Two-stage process FTase produced by SSFusing corn germ as the solid support

120+8 h 60.0 Sangeetha et al. (2004b)

Two-stage process using extracellularenzyme produced by SmF


Two-stage process using whole cellsproduced by SmF

24+48 h 53.0 Sangeetha et al. (2005c)

Penicillium expansum One-stage processSmF 36 h 58.0 Prata et al. (2010)

One-stage process using synthetic fiber asthe solid support: SFFrepeated batchfermentation

60 h, three cycles First: 87.0 (36 h); second:72.0 (48 h); third: 44.0(60 h)

Mussatto et al. (2012)

Penicilliumchrysogenum

Two-stage process using culture brothfiltrate produced by SmF

48+24 h 42.5 Ganaie et al. (2013)



High FOS production yields adding glucose oxidase to thereaction media have been reported, and many authors manageto control this effect and increase FOS production yield(up to 9098 %, gFOS/g sucrose) by adding glucose oxi-dase to the reaction media, which leads to the formationof gluconic acid, hence reducing the glucose content inthe media (Yun and Song 1993; Sheu et al. 2001; Linand Lee 2008).

Other systems aiming glucose removal from the mediahave also been studied. For example, Sheu et al. (2002)reported a complex biocatalyst system with a bioreactorequipped with a microfiltration module. The system usedmycelia of A . japonicus CCRC 93007 or A . pullulans ATCC9348 with FFase activity and Gluconobacter oxydans ATCC23771 with glucose dehydrogenase activity. Calcium carbon-ate slurry was used to control the pH to 5.5, and gluconic acidin the reaction mixture was precipitated as calcium gluconate.Sucrose solution with an optimum concentration of 30 % (w /v ) was employed as feed for the complex cell system, andhigh-content FOS was discharged continuously from amicrofiltration module. The complex system produced morethan 80 % (gFOS/g sucrose) FOS, with the remaining being 57 % glucose and 810 % sucrose on a dry weight basis, plus asmall amount of calcium gluconate.

Nishizawa et al. (2001) achieved higher FOS yields with asimultaneous removal of glucose using a membrane reactorsystem with a nanofiltration membrane, through which glu-cose permeated, but not sucrose and FOS. FOS percentage ofthe reaction product was increased to above 90 % (gFOS/g sucrose), which was much higher than that of the batch reac-tion product (5560 %, gFOS/g sucrose). Nonetheless, althoughthese systems can be more efficient in terms of FOS produc-tion yield, it is important to notice that they are also moreexpensive.

With the same goal, Crittenden and Playne (2002) used adifferent approach. In their study, immobilized cells of thebacterium Zymomonas mobilis were used to remove glucose,fructose, and sucrose from food-grade oligosaccharide mix-tures, which were completely fermented within 12 h. Thefermentation end products were ethanol and carbon dioxide,a minimal amount of sorbitol also being formed. Similarly toZ . mobilis , Saccharomyces cerevisiae also showed the abilityto ferment some common mono- and disaccharides (fructose,glucose, galactose, and sucrose) from a mixture of sugars,while oligosaccharides with four or more monosaccharideunits were not fermented (Yoon et al. 2003; Goulas et al.2007; Hernndez et al. 2009). The microbial treatment seemsto be a good alternative for increasing the percentage of FOSin a mixture through the removal of mono- and disaccharides,this process being adequate to be used during the enzymaticsynthesis of FOS. However, the use of microbial treatmentsimplies a further step of purification for the removal of bio-mass and metabolic products formed during the fermentation

in order to obtain a FOS product with few contaminants, thusincreasing the production cost. Also, the use of yeast treatmentwas found to modify the oligosaccharide composition (Sanzet al. 2005; Nobre et al. 2013).

Zuccaro et al. (2008) reported that recombinant FTases alsohave interesting biocatalytic properties. The combination ofsucrose analogues, as novel substrates (substrate engineering),and highly active recombinant -fructofuranosidase from A .niger (genetic engineering) provided a new powerful tool forthe efficient preparative synthesis of tailor-made saccharides1-kestose and 1-nystose type.

FOS Market Data

In general, the total cost of developing a conventional newfood product is estimated to be up to 0.751.5 million Euros,while the development and marketing costs of functional foodproducts may exceed this level by far. Presently, the Europeanmarket of functional food is dominated by gut health-targetedproducts. Germany, France, UK, and the Netherlands accountfor around two thirds of all sales of functional dairy productsin Europe. Functional dairy products have shown an impres-sive growth, bringing, for example the market volume inGermany, from around 4 million Euros in 1995 to 317 millionEuros in 2000, of which 228 million Euros account for probi-otic, prebiotic, and other functional yoghurts and around 89million Euros for functional drinks. The World demand forprebiotics is estimated to be around 167,000 tons and 390million Euros. Among them, FOS, inulin, isomalto-oligosaccharides, polydextrose, lactulose, and resistant starchare considered the most popular. In 1995, the global marketfor FOS from sucrose was estimated to be 20,000 tons(Menrad 2003; Sir et al. 2008).

As previously mentioned, at an industrial scale, FOS areproduced enzymatically by two different processes

Table 3 Commercially available food-grade FOS

Substrate Manufacturer Trade name

Sucrose Beghin-Meiji Industries, France Actilight

Profeed

Cheil Foods and Chemicals Inc., Korea Oligo-Sugar

GTC Nutrition, USA NutraFlora

Meiji Seika Kaisha Ltd., Japan Meioligo

Victory Biology Engineering Co., Ltd.,China

Beneshine P-type

Inulin Beneo-Orafti, Belgium Orafti

Cosucra Groupe Warcoing, Belgium Fibrulose

Sensus, the Netherlands Frutalose

Nutriagaves de Mexico S.A. de C.V.,Mexico

OLIFRUCTINE-SP



(transfructosylation of sucrose or hydrolysis of inulin) whichyield slightly different end products. The companies thatcommercially produce FOS from sucrose or inulin and theirtrade names are listed in Table 3. These FOS products areprovided with a purity level above 95 % (Singh and Singh2010; Nobre et al. 2013).

The enzymatic synthesis route to FOS using FTase from A .niger was first developed by Meiji Seika Kaisha Ltd., Japan,resulting in the launch of the commercial product Meioligo.Then, this company also established a joint venture withBeghin-Meiji Industries, France, to produce FOSmarketed as Actilight, and also with GTC Nutrition,USA, to produce FOS under the trade name NutraFlora(Singh and Singh 2010). According to the Meiji HoldingsCo., Ltd. Annual Report (2011), they presented total net salesof almost 21 million Euros and operating incomes of 0.5million Euros.

Additionally, individual FOS molecules with purities vary-ing between 80 and 99 % are only available for analyticalpurposes. The main companies supplying 1-kestose (GF2),nystose (GF3), and 1F-- fructofuranosyl nystose (GF4) areSigma Aldrich, Megazyme, and Wako Chemicals GmbH(Nobre et al. 2013).

Conclusions

Modern consumers are increasingly interested in their personalhealth and expect the foods they eat to bebeyond tasty andattractivealso safe and healthy. Non-digestible carbohydratessuch as dietary fibers, oligosaccharides, and resistant starch havevarious physiologic functions, and the effects of many non-digestible carbohydrates on well-being, better health, and reduc-tion of the risk of diseases have been well evaluated. Amongfunctional oligosaccharides, FOS present important physico-chemical and physiological properties beneficial to the healthof consumers. For this reason, their use as food ingredients hasincreased rapidly, presenting a significant growth on the func-tional food market all over the world. Europe nutraceuticsmarket is expected to have a share of over 20% until 2017. Inview of the great demand for FOS as food ingredients, theopportunity exists for the screening and identification of novelstrains capable of producing enzymes with transfructosylationactivity and for developing improved and less expensive pro-duction methods. In this review, the beneficial effects of FOSand how they can play a key role in the food market have beendiscussed, bearing in mind that more effective less costly pro-duction methods can be a main advantage in the food industry.

Acknowledgments Agncia de Inovao (AdI)Project BIOLIFE ref-erence PRIME 03/347. Ana Dominguez acknowledges Fundao para aCincia e a Tecnologia, Portugal, for her PhD grant reference SFRH/BD/23083/2005.

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An Overview of the Recent Developments on Fructooligosaccharide Production and ApplicationsAbstractIntroductionPrebioticsFructooligosaccharides as PrebioticsFOS Functional PropertiesProtection Against Colon CancerImmunomodulatory EffectObesity and DiabetesImproving Mineral AdsorptionEffects on Serum Lipid and Cholesterol Concentrations

FOS OccurrenceFOS ProductionFOS Microbial ProductionTransfructosylation EnzymesFOS Production by SmFFOS Production bySSFProduction of High-Content FOS

FOS Market DataConclusionsReferences

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