An Overview of the Recent Developmentson Fructooligosaccharide Production and Applications Ana Luísa Dominguez, Lígia Raquel Rodrigues, Nelson Manuel Lima & José António Teixeira Food and Bioprocess Technology An International Journal ISSN 1935-5130 Food Bioprocess Technol DOI 10.1007/s11947-013-1221-6 1 23 You may further deposit the accepted manuscript version in any repository.Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. The link must be accompanied by the following text: "The final publication is available at link.com”. If you wish to self-archive your article.springer. provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. please use the accepted manuscript version for posting on your own website. 1 23 . This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. Lda.and health-relevant properties. and low glycemic index. IBB—Institute for Biotechnology and Bioengineering. The gut is a body organ with major influence in several body functions. functional food is a newsworthy and stimulating concept.1007/s11947-013-1221-6 REVIEW An Overview of the Recent Developments on Fructooligosaccharide Production and Applications Ana Luísa Dominguez & Lígia Raquel Rodrigues & Nelson Manuel Lima & José António Teixeira Received: 11 September 2013 / Accepted: 25 October 2013 # Springer Science+Business Media New York 2013 Abstract Over the past years. and market data. as much as it is supported by consensual scientific data generated by the development of functional food science aimed at the improvement of dietary guidelines that integrate new information on the food elements and body function interactions (Roberfroid 2000a). low calorimetric value. FOS are produced through a two-stage process that requires an enzyme production and purification step in order to proceed with the chemical reaction itself. Building a path to an optimum nutrition. Avepark-Edif. A narrow analysis of the topic suggests that all food is functional since it provides basic energy and nutrients essential for surviving. knowledge of health is now being used to improve food. Fructosyltransferase . USA). M. therefore. Conventionally. A. Campus de Gualtar. Food science has evolved from solving problems related to nutritional deficiencies to designing foods that have the potential to induce health benefits and reduce the risk of disease (Clydesdale 2004). Portugal progresses in FOS research such as its production. Keywords Fructooligosaccharides . 4710-057 Braga. Transfructosylation . Zona Industrial da Gandra. Rodrigues : N. functional properties. Several studies have been conducted on the production of FOS. play a key role in the improvement of gut microbiota balance and in individual health. Dominguez (*) : L. many researchers have suggested that deficiencies in the diet can lead to disease states and that some diseases can be avoided through an adequate intake of relevant dietary components. Production yield . Due to their nutrition. aiming its optimization toward the development of more efficient production processes and their potential as food ingredients. Portugal e-mail: anadominguezmatos@gmail. which is an aspiring long-term goal. Yet. are generally regarded as safe (generally recognized as safe status—from the Food and Drug Administration. this review focuses on the latest A. we have witnessed an increase of new information concerning the . Spinpark. FOS are generally used as components of functional foods. The improvement of FOS yield and productivity can be achieved by the use of different fermentative methods and different microbial sources of FOSproducing enzymes and the optimization of nutritional and culture parameter. Lima : J. University of Minho. L.com L. thus. and worth about 150€ per kilogram. Prebiotics. a great interest in dietary modulation of the human gut has been registered. low carcinogenicity. Prebiotics . and over the past decade. Teixeira Centre of Biological Engineering. Rodrigues Biotempo—Consultoria em Biotecnologia. R. Howlett 2008). The first systematic exploration of the positive aspects of food functionality was undertaken in Japan where research programs funded by the Japanese government during the 1980s focused on the ability of some foods to influence physiological functions (Clydesdale 2004.Author's personal copy Food Bioprocess Technol DOI 10. such as fructooligosaccharides (FOS). FOS have been increasingly used by the food industry. Fructofuranosidase Introduction While food has long been used to improve health. 4805-017 Guimarães. the term “functional food” in use nowadays implies that such food carries broader health benefits than just basic nutrition. R. Barco. such as moderate sweetness. it became a clear target for the evolution of functional food. Recently. e. Kunz and Rudloff 2006. colon cancer.. Howlett 2008. and to inhibit lesions that are precursors of adenomas and carcinomas. the functional oligosaccharides. Prebiotics According to the FAO. soybean oligosaccharides. xylooligosaccharides (XOS). 2011). 2011). or drug therapy. oligofructose from inulin). probiotics. Thus. Qiang et al. These include their ability to modulate gut function and transit time. Various non-digestible carbohydrates have been tested for their effect on the microbial community... Mussatto and Mancilha 2007). prebiotics may also induce local (in the colon) or systemic effects through bacterial fermentation products that are beneficial to host health (Manning and Gibson 2004. De Preter et al. Apart from their potential to modify the gut microbiota and its metabolic activities in a beneficial way. 2013). behaving as dietary fibers and prebiotics. The gut microbiota ferments a range of substances. among others. Prebiotics can be used as a strategy to improve the balance of intestinal bacteria differing from the probiotic approach in which exogenous strains are included in food and ultimately reach the individual’s intestinal tract. Howlett 2008. This has led to great efforts in the development of strategies aimed at dietary modulation of the microbiota composition and activity. and obesity. Macfarlane et al. Qiang et al. such as disease. Fructooligosaccharides as Prebiotics FOS is a common name for fructose oligomers. especially in the lower part of the colon where the effects are believed to be most favorable. Furthermore. Strategies for developing prebiotic products aim to provide specific fermentable substrates for beneficial bacteria (bifidobacteria and lactobacilli). and therefore their use as food ingredients has increased rapidly (Simmering and Blaut 2001. the potential of prebiotics is too large to be ignored (Ziemer and Gibson 1998. Nevertheless. These may provide adequate amounts and proportions of fermentation products. the appropriate healthpromoting species are not present in the bowel.or disaccharides such as sucrose (FOS) or lactose (GOS. Even as part of a more complex story. i. and maltitol are among the commonly used prebiotics. Although the association with past outbreaks will remain conjectural. oligosaccharides. isomaltooligosaccharides (IMO). mainly provided by the diet. to activate the immune system. antibiotic. In a typical adult.Author's personal copy Food Bioprocess Technol interface between the diet and its possible role on gut microbiota and.g. Crittenden and Playne 1996). to increase the absorption of minerals. Most prebiotics and prebiotic candidates that have been identified are NDO and are obtained either by extraction from plants (e. in health and disease. 2000. 2008). proteins. which cannot be digested by the host in the small intestine and are available for fermentation by the colonic microbiota. Key criteria for a food ingredient to be classified as a prebiotic are (1) it must not be hydrolyzed or absorbed in the upper part of the gastrointestinal tract so that it reaches the colon in significant amounts and (2) it must be a selective substrate for one or more beneficial bacteria that are stimulated to grow. about 100 g of food ingested each day reaches the large intestine and therefore is susceptible to fermentation by the gut microbiota. Simmering and Blaut 2001). consequently. Among them. and amino acids. These compounds selectively stimulate the proliferation and/or activity of beneficial groups of bacteria that exist in the intestinal microbiota. non-digestible oligosaccharides (NDO) that are distinguished from other carbohydrates for being resistant to digestion in the stomach and small intestine (Playne and Crittenden 2004). aging. possibly followed by an enzymatic hydrolysis (e. Howlett 2008. FOS being the most studied and the best implemented oligosaccharides in the European market (Chen et al. De Preter et al. 2009. other NDO including XOS.g. IMO. which are intermediate in nature between simple sugars and polysaccharides. They . and these are usually understood as inulin-type oligosaccharides. However. As a consequence. or by synthesis (by transglycosylation reactions) from mono. it is reasonable to suggest that prebiotic administration may possibly exert very important prophylactic effects against gut pathogens. to increase the production of butyric and other short-chain fatty acids. non-starch polysaccharides (dietary fiber). 2009. present important physicochemical and physiological properties beneficial to the consumer’s health. Shimizu and Hachimura 2011). Piñeiro et al. galactooligosaccharides (GOS). 2008). These include resistant starch. Szajewska 2010. and soybean oligosaccharides have also been evaluated for their prebiotic effect (Gibson and Roberfroid 1995. prebiotics can potentially reduce some of the risk factors involved in the causes of colorectal diseases and reduce the risk of diseases such as cardiovascular disease. 2004. chicory inulin). and a combination of both (synbiotics. FOS. Nobre et al. if for any reason. they can constitute a more practical and efficient way to manipulate the gut microbiota compared to probiotics. many other helpful and useful effects of prebiotics are being investigated. “a prebiotic is a non-viable food component that confers a health benefit to the host associated with the modulation of the microbiota” (Gibson et al. achieved by the use of prebiotics. the prebiotic is unlikely to be effective (Playne and Crittenden 2004. such as calcium and magnesium. These ingredients are normally restricted to certain carbohydrates. The two main types of fermentation that are carried out in the gut are saccharolytic and proteolytic. Lee et al. these compounds have the capacity of promoting a good balance of intestinal microbiota and decrease gastrointestinal infections. and 1F-β. 2009). nystose (GF3. Many studies were performed to demonstrate such activity. left). As FOS cannot be digested by the enzymes of the small intestine. Indeed. These findings showed lower rates of colorectal cancer in Africa compared to industrialized Western countries. and fructofuranosyl nystose (GF4. Lim et al. which is the second most prevalent cancer in humans. propionate can have anti-inflammatory effects on colon cancer cells (Munjal et al. mainly acetate. they are fermented in the large intestine to selectively stimulate the growth of probiotic-like bacteria that are part of the commensal gut microbiota and then produce short-chain fatty acids (SCFAs). supplying small amounts of energy. Protection Against Colon Cancer Immunomodulatory Effect Fig. respectively. 2013). Also. respectively (Manning and Gibson 2004. in which two.Author's personal copy Food Bioprocess Technol constitute a series of homologous oligosaccharides derived from sucrose usually represented by the formula GFn. Mishra and Mishra 2013). There have been several studies on the use of prebiotics in cancer prevention mainly focusing on animal models. 2005a). 1999). FOS have a low sweetness intensity since they are only about one third as sweet as sucrose. FOS Functional Properties Prebiotics have been postulated to be protective against the development of colon cancer. Bugaut and Bentéjac (1993) reported that butyrate is used by the epithelial cells of the colon mucosa as an energy source. Butyrate is an important source of energy and is thought to have antitumor properties. FOS are also calorie-free since they are scarcely hydrolyzed by the digestive enzymes and not utilized as an energy source in the body. as shown in Fig. and butyrate. This property is quite useful in the various kinds of foods in which the use of sucrose is restricted by its high sweetness. right) Functional foods are reported to enhance the immunity of the consumers. It has been estimated that about 10–60 g/day of dietary carbohydrate reaches the colon acting like a growth factor to particular commensal bacteria. approximately 0–3 kcal/g of sugar substitute.fructofuranosyl nystose (GF4). The beneficial physiologic functions that have been reported for FOS are briefly summarized below. which may be more quickly fermented in the saccharolytic proximal bowel (Manning and Gibson 2004). but the “dietary fiber hypothesis” for protection against colorectal cancer was advanced by Burkitt (1969) based on epidemiological evidence supporting a relationship between diet and colon health. the dietary components and their fermentation metabolites are in close contact with the gut-associated lymphoid tissue (GALT). and butyrate (Yun 1996. and recent preclinical studies demonstrated that it might be chemopreventive in carcinogenesis (Scheppach and Weiler 2004). and according to Kim et al. and four fructosyl units are bound at the β-2. It is known that several species of bacteria commonly found in the colon produce carcinogens and tumor promoters from the metabolism of food components. acetate. 2010). Saad et al. As previously mentioned. which are mainly composed of 1-kestose (GF2). such as SCFAs. 2011. The saccharolytic activity is more favorable than the proteolytic one due to the type of metabolic end products that are formed. propionate. Yu Wang et al. nystose (GF3). being also a growth factor.1 position of glucose. thus. which inhibit the adherence and invasion of pathogens in the colonic epithelia by competing for the same glycoconjugates present on the surface of epithelial . propionate. which is the biggest tissue in the immune system comprising 60 % of all lymphocytes in the body (Delgado et al. In addition to butyrate. they are safe for diabetics. prebiotics are known to prevent the colonization of human gut by pathogenic microorganisms because they encourage the growth of beneficial bacteria (Roberfroid 2000b. It is also importance to notice that long-chain FOS may exert a prebiotic effect in more distal colonic regions compared with the lower-molecular-weight FOS. 1 (Yun 1996. butyrate is a protective agent against colon cancer by promoting cell differentiation. center). where traditional diets consisted of high amounts of unrefined fiber and high intakes of refined carbohydrates. three. 1 Structures of 1-kestose (GF2. (1982). it seems that. 2006). Delzenne et al. Schley and Field (2002) have reviewed some evidences of the immune-enhancing effects of dietary fibers and prebiotic and identified for the first time the gene markers associated with the physiological effects of a particular FOS on a host animal. 2002). (2003) stated that inulin and FOS were able to modulate various parameters of the immune system. Nilsson et al. Effects on Serum Lipid and Cholesterol Concentrations Since cardiovascular risk is the major public health concern in many countries and accounts for more deaths than any other disease or group of diseases. thus making it possible to clarify the mechanisms behind the immunomodulatory effects of FOS. the fiber’s fermentation in the intestine may also influence satiety. the food industry is increasingly interested in the development of functional foods that modulate blood lipids. (1999) in animals. favoring the barrier function. Secondly. Furthermore. The main action of FOS is mainly associated with their fermentation by resident microbiota. 2008). Calcium (Ca) intake in many underdeveloped countries is below the recommended daily allowance. Chaudhri et al. Yu Wang et al. these mechanisms are not completely understood. Another way to contribute to the enhanced mineral absorption is the trophic effect of prebiotics on the gut (cell growth and functional enhancement of the absorptive area). Hosono et al. resulting in changes in either TAG accumulation in the liver (steatosis) or serum lipids. after consumption of FOS. (1986) and Yamamoto et al. (1994) and Jackson et al. 2008). Mineral deficiencies remain an important nutritional issue in the World. thus helping in the proper control and management of diabetes mellitus and obesity (Bennett et al. First. improving the mucus production. Ca is critical in achieving optimal peak bone mass and modulating the rate of bone loss associated with aging. 2010). including resistant starch or FOS. Then. (1994) and Fiordaliso et al. In 1997. Another mineral with a low intake is iron (Fe). has been described by Glore et al. Several mechanisms have been proposed to elucidate the possible roles of FOS in improving mineral absorption. such as cholesterol and triglycerides (Manning and Gibson 2004. other scientific data suggest that the decrease in food intake associated with prebiotics feeding in animals might be linked to the modulation of gastrointestinal peptides involved in the regulation of food intake (Druce et al. which is a major cause of anemia. It has been suggested that this is mediated by an increased production of butyrate and/or certain polyamines (Roberfroid 1993. Current recommendations for the management of type II diabetes and obesity include an increase in dietary fiber intake. Marginal Zn deficiency is suspected to be widespread in populations heavily dependent on cereal-based diets containing few animal products (Baba et al. leading to osteoporosis. One hypothesis is that the production of SCFAs influences postprandial glucose response by reducing fat competition for glucose disposal. Many researchers recognized the effect of prebiotics oral administration. which leads to progressive bone loss. if Ca intake is not enough to offset obligatory losses. another hypothesis relies on the idea that fermentation of SCFAs influences gut hormones and gastric motility (Brighenti et al. a major public health problem. On the other hand. Obesity and obesity-related type II diabetes are typical diseases of the modern Western society. 1996. Beyond glycemic control.Author's personal copy Food Bioprocess Technol cells. Many attempts have been made to control serum triacylglycerol (TAG) concentrations through the modification of dietary habits. 2004. (1995) reported a decrease in total serum cholesterol after . Levrat et al. Even though mineral absorption generally occurs in the upper part of the intestine. Qiang et al. Roberfroid et al. a decrease in glycemia could be part of the process since glucose (together with insulin) is a driver of lipogenesis. Among several studies. 2010). (1999) in humans and by Tokunaga et al. Also. Ingredients showing a prebiotic effect are able to modulate hepatic lipid metabolism in rats or hamsters. Pierre and co-workers (Pierre et al. Raschka and Daniel 2005). 2006. producing SCFAs. 1997) reported the development of GALT associated with inulin and FOS intake. acquired skeletal mass cannot be maintained. altering the colonic pH. and inducing cytokine production (Korzenik and Podolskyl 2006). Yu Wang et al. 2010. Magnesium is the second most abundant intracellular cation in vertebrates. the major site of action leading to improved bioavailability of minerals is the large intestine. Obesity and Diabetes Fiber intakes are associated with increased satiety and lower body weight. 2009). The importance of zinc (Zn) nurture for growth and development has been widely documented. The hypotriglyceridemic effect of nondigestible but fermentable carbohydrates. the SCFAs produced through the fermentation process could play a role in the regulation of lipid metabolism (Delzenne and Kok 2001. The decrease in TAG synthesis and the accumulation of dietary prebiotics compounds could be linked to several events. Improving Mineral Adsorption Another interesting feature of FOS is their positive effect on mineral absorption. Dietary fiber’s viscose and fibrous structure can control the release of glucose with time in the blood. However. and its deficiency has been implicated as a risk factor for osteoporosis. SCFAs decrease luminal pH and thus create an acidic environment more favorable for mineral solubility (GudielUrbano and Goñi 2002. This reaction yields FOS. garlic. such as Aspergillus sp. In this reaction. L’Hocine et al. whereas others designate it as fructosyltransferase (FTase. 2000.1) position of sucrose (Belghith et al. but the activity ratios (U t/U h) of their enzymes vary significantly (Sangeetha et al.2. The authors suggested that the decrease in serum cholesterol could reflect a decrease in TAG-rich lipoproteins. 2004). such as asparagus (Shiomi 1982).. Saad et al. and fructofuranosyl nystose (Fig. A high concentration of the starting substrate is required for efficient transfructosylation. 2005a. There is still a dispute in the scientific community regarding the nomenclature of FOS-producing enzymes. 1999.. FOS are produced from the disaccharide sucrose using enzymes with transfructosylation activity. EC 2. are known to produce extracellular and/or intracellular enzymes with transfructosylation activity (Table 1).e. Although these proteins differ in their subunit structure. This is accompanied by a significant decrease in the hepatic cholesterol content. chemical susceptibility. Sangeetha et al. 2001. 1992. FOS are formed. sugar beet. For most of these sources. Bacillus macerans (Park et al. 1997. 2004. Some researchers use the term β-fructofuranosidase (FFase.7 mg/ kgbody weight per day or 806 mg/day has been estimated (Voragen 1998). honey. Crittenden and Playne 1996. and from different microorganisms (fungi and bacteria) such as Aspergillus niger (L’Hocine et al. Asparagus. in which fructosyl units are bound at the β(2. On the other hand. 1). EC. In this process. 2012). Yun et al. 3.1. Mussatto and Mancilha 2007). EC. FOS Occurrence FOS are found in trace amounts as natural components in fruits. and rye are well-known sources of FOS (Yun 1996. 2005a.1. Yoshikawa et al. L’Hocine et al. 1992). 2012). Singh and Singh 2010. Aspergillus japonicus (Hayashi et al. degree of glycosylation. they occur in many higher plants as reserve carbohydrates. Jerusalem artichoke (Koops and Jonker 1994). some FFases have much higher transfructosylating activity (U t) than hydrolyzing activity (U h). wheat. for chicory. Additionally. EC 3. these values are between 5 and 10 %. 2000).4. onion. The same microorganism can even produce several enzymes with transfructosylation activity holding different characteristics (Yoshikawa et al. 2011. Park et al. several fungal strains. but also some by-products such as glucose and small amounts of fructose (Brenda 2005. 1997). They can be produced by the hydrolysis of inulin or by the transfructosylation of sucrose. 2001). 2007). releasing glucose. degree of glycosylation. As a series of fructose oligomers and polymers derived from sucrose. FFase. Nguyen et al. barley.9). nystose.Author's personal copy Food Bioprocess Technol dietary supplementation with inulin (10 %) in mice or rats. and Candida utilis (Chávez et al. 2006). tomato. the oligosaccharide mixture produced from inulin hydrolysis contains longer fructo-oligomer chains than that produced by the sucrose transfructosylation process. Some bacterial strains have been reported to produce such enzymes..26) and sucrase (sucrose fructosyltransferase. chemical . 2005. Although FFase is generally known as an enzyme catalyzing the hydrolysis of sucrose. namely. 2001). FOS Microbial Production—Transfructosylation Enzymes The commercially available FOS are produced through the enzymatic synthesis from sucrose by microbial enzymes with transfructosylation activity. 2013). 2000). kestose. an average daily consumption of FOS of approximately 13.4. Although these proteins differ in their subunit structure.. A wide variety of fungi produce FFases. FOS concentrations range between 0. Aureobasidium pullulans (Yoshikawa et al. chicory. while in Jerusalem artichoke they can reach 20 %. Sheu et al. Jedrzejczak-Krzepkowska et al. These enzymes are invertase (β-fructofuranosidase fructohydrolase. Aureobasidium sp. fructose oligomers.9. Hayashi et al. Jerusalem artichoke.2. onion (Fujishima et al. and substrate specificity.26. Sangeetha et al. FOS Production FOS represent one of the major classes of bifidogenic oligosaccharides in terms of their production volume. although slightly different end products are obtained as not all of the β(1→2)-linked fructosyl chains end with a terminal glucose. 2006). Transfructosylating activity acts on sucrose by cleaving the β(1. Risso et al. Many authors have reported the purification and characterization of FOS-producing enzymes from various sources and by different microorganisms (bacteria and fungi. molecular weight. banana. i. they all display both hydrolytic and transfer activities. The latter denomination is probably due to the fact that FTase activity was originally found from the side action in the course of FFase preparation when acting on a high concentration of sucrose (Straathof et al. Based on the usual consumption of these natural sources.2) linkage and transferring the fructosyl group to an acceptor molecule such as sucrose. Vandáková et al. FTase.1.2. Nguyen et al. vegetables. The FOS mixture obtained by the enzymatic hydrolysis of inulin closely resembles the mixture produced by the transfructosylation process.1. 2005). Fructosyl-transferring enzymes have been purified and characterized from higher plants. and honey. which are mainly derived from fungal and bacterial sources.3 and 6 %. molecular weight. and Penicillium sp. 1986. (2002) reported U t in A. This study also suggested that the expression of FFase I was not repressed by glucose. (2009) Mussatto and Teixeira (2010) Wallis et al. Fungal FTases have molecular masses ranging between 180. (2006) have reported five FFase extracted from the cell wall of A.5. a maximum enzyme production of 910 U mL−1 was achieved. (2004a) Sangettha et al.000 U dm−3 (80 U mL−1) is achieved. They also reported that with an initial sucrose concentration of 200 g L−1.5 to 6.and extracellular FTase Intracellular FTase Periplasmic FFase Extracellular FTase FTase from commercial enzyme preparation—Pectinex Ultra SP-L Extracellular FTase Intra. (2009) Ghazi et al. 2008). (2013) Park et al. japonicus was also reported by Chen (1995). Wang and Zhou (2006) isolated an A. There are several studies reporting the characteristics of transfructosylation enzymes produced by fungi. (2005b) Ganaie et al. Yoshikawa et al.42 U mL−1 at pH 5. (2005a) Ganaie et al.000 and are homopolymers with two to six monomer units.000 and 600. (2000) Extracellular FTase Extracellular FTase Extracellular FTase Extracellular FTase Intra. (2002) Bekers et al. (2004) Lateef et al.Author's personal copy Food Bioprocess Technol Table 1 Microbial sources of enzymes with transfructosylation activity: FOS-producing enzymes Source Microorganism Enzyme Reference Fungal transfructosylation enzyme Aureobasidium pullulans Intra. Maiorano et al.000 U dm−3 (131 U mL−1) released into the cultivation medium. respectively (Madlová et al. (2005) Ghazi et al. FFase activity in A. they all display both hydrolytic and transfer activities (Ghazi et al. but those of FFases II–V were strongly inhibited in the presence of glucose. and substrate specificity. This strain showed very high enzyme productivity and high FTase . (1992) identified a FFase produced by A.and/or extracellular FFase Intra. purified and partially characterized a FTase and a FFase from the crude extract of A. (1997) identified two FFase secreted by A. mainly Aureobasidium sp. 2007). (2008) reported the chromatographic separation of an intracellular FTase from A . japonicus and optimized the cultural condition for the production of the enzyme and the enzymatic reaction conditions for the industrial utilization of the strain to produce FOS. and Aspergillus sp. the U t being higher in the sucrose concentration range tested (10–600 g L−1) and completely dominating at higher sucrose concentrations. whereas FFase IV may function as a FOS-degrading enzyme with its strong hydrolyzing activity. In 2008. (2013) Lim et al. Later on. (2007) Yoshikawa et al. (2012) Nemukula et al. Wallis et al. Antošová et al. an FTase U t of 80.and/or extracellular FFase Extracellular FFase Extracellular FFase Extracellular FFase Intra. Some studies stated the optimum temperature and pH for these enzyme activities between 50 and 60 °C and from 4. was reported. Antošová et al. japonicus strain from soil and investigated the optimal conditions for FFase. a maximum FTase U t of 131. reaching an activity of 55.and extracellular FTase Intra. niger. (2007) Ganaie et al. considering that FFase I played a key role in FOS production by this fungus. pullulans CCY 27-1-1194 in the extracellular medium and in the whole cells. In their study. niger. (1997) Nguyen et al. (2004a) Vandáková et al. (2013) Chen and Liu (1996) Wang and Zhou (2006) Mussatto et al. pullulans with high U t. (2006) Dominguez et al. (2013) Sangeetha et al.5 and 30 °C. after four cultivation days.and extracellular FFase Intra. (1997) Sangeetha et al. (1999) L’Hocine et al. Hayashi et al. (2002) Aspergillus aculeatus Aspergillus flavus Aspergillus japonicus Aspergillus niger Aspergillus oryzae Bacterial transfructosylation enzyme Aspergillus terreus Penicillium citrinum Penicillium islandicum Bacillus macerans Lactobacillus reuteri Zymomonas mobilis susceptibility. (2001) van Hijum et al.and/or extracellular enzyme Extracellular FTase Extracellular transfructosylation enzyme Intra and/or extracellular FTase Extracellular levansucrase Ganaie et al. L’Hocine et al. 2000.and/or extracellular FTase Yun et al. The isolated enzyme exhibited both U h and U t activities.and extracellular FTase Intra. pullulans. This is also the usual process used to produce the FOS that are currently available on the market (Singh and Singh 2010). the enzyme(s) being produced by SmF in the first stage. time of cultivation. (2002) obtained the highest FTase activity using sucrose at 350 g L−1 and found that biomass production was not influenced by sucrose concentration in the range from 50 to 350 g L−1. niger and estimated its molecular mass to be in the range from 120 to 130 kDa. Nguyen et al.3 kDa. There are mainly two methods that can be used to produce transfructosylation enzymes and FOS by fermentation. The effects of inorganic salts were reported by various authors. high sucrose concentrations resulted in high specific activities of the cells. Chen and Liu (1996) evaluated the effect of various carbon and nitrogen sources in enzyme production and found that sucrose at 25 % (w /v) and yeast extract were the best ones. Extracellular FTase activity in Aspergillus oryzae was identified by Sangeetha et al. Most studies on the experimental conditions for enzyme production are mainly based on shake-flask experiments. schematically represented in Fig. Later on. small amounts of amino acids. Later on. Chen and Liu (1996) studied the effect of inorganic salts and found that the addition of MgSO4⋅7H2O and K2HPO4 shifted the morphology of the fungal growth from filamentous to pellet form without affecting FFase production. (2005) purified an intracellular FFase produced by A . (2001) reached a maximum FFase activity of 149 U L−1 h−1. respectively. 2008). The variables generally studied to define the best operational conditions for enzyme production are the carbon and nitrogen sources. Vandáková et al. FOS Production by SmF The most common and studied method to produce FOS is by the transfructosylation of sucrose in a two-stage process. niger. however. Balasubramaniem et al. However. Control of mycelia morphology in fermentations is often a prerequisite for industrial applications. Antošová et al. agitation.Author's personal copy Food Bioprocess Technol activity. and surfactants (Maiorano et al. with a molecular mass of 116. 2. the same authors reported an FFase activity in that same strain in the range of 15 ± 2 U mL−1 min−1 (Sangeetha et al. polymers. 2005a). Other important factors are related with the addition of different mineral salts. 2005c) the work carried out in bioreactors is unusual. and Fig. These two methods will be further discussed in the following sections. (2004b). namely. this enzyme being produced by A. The morphology of fungal growth can range from dispersed mycelia to compact pellets. their concentrations. and aeration rates. 2 Schematic representation of the two-stage FOS production process by SmF (adapted from Sangeetha et al. (2004) also studied the effect of sucrose on FTase production by Aerobasidium pullulans and reported that sucrose content did not influence the total production of FTase. by submerged fermentation (SmF) or by solid state fermentation (SSF). Although . the enzyme used in the crude state also showed hydrolytic activity. 0 % (g FOS/g sucrose) to 57. pullulans in a two-stage process can vary between 44. and lower sterilization costs (Sangeetha et al. Yoshikawa et al. and by-products of coffee and tea processing industries were used as substrates to produce FTase in SSF by A. FOS Production bySSF Despite most industrial enzymes being produced in SmF. and cork oak were tested as carriers for fungus immobilization (Mussatto et al. with optimum activity at 60 °C and pH 6. Production of High-Content FOS Industrial production of FOS carried out with microbial transfructosylation enzymes was found to give a maximum . (2005a. Hölker et al. and vice versa (Maiorano et al. Mg2+ affects the permeability of the cell wall for A.0 % (g FOS/g sucrose. Moreover. rice bran and wheat bran were found to be the substrates that maximize FTase production. 72 % (g FOS/g sucrose). 2005a. production of FOS and FFase by repeated batch fermentation of P. values ranging between 24. as well as a buffering reagent. product concentration. Nguyen et al. sugarcane bagasse.0 % (g FOS/g sucrose.0 % (g FOS/g sucrose. 2004). 2004. 2001). the risk of contamination is reduced. Optimization of the fermentation medium for β-fructofuranosidase production by A. The FOS production yield by A. K2HPO4 is described as a microelement source for cell growth. Coffee silverskin was found to be the one that provided the higher production yield and FFase activity. which have discouraged the use of this technique for industrial enzyme production. b) reported FOS production yield by A. the growth of fungus in the form of pellets is more attractive to many industrial fermentation processes from the perspective of reducing the power input needed for adequate gas dispersion and mixing (Metz and Kossen 1977. 2001). b) and 5 g L−1 (Vandáková et al. On the other hand.5 was found to be the best initial value for FTase production by A.0 % (g FOS/g sucrose. such as increasing FFase activity and FOS production (Balasubramaniem et al. Maldová et al. Its optimal concentration ranges between 4g L−1 (Sangeetha et al. 2004). expansum immobilized on synthetic fiber was studied. Chen and Liu 1996). Shin et al. b) to 2 g L−1 (Balasubramaniem et al. Table 2 presents a comparison between the FOS production yields reported in the literature. grown in the form of mycelia. 1999) and 70. can also be added to the medium with positive effects. 2008). SSF presents an interesting potential for small-scale units.8 % (g FOS/ g sucrose. Several agricultural by-products like cereal bran. moisture. Additionally. A. Sangeetha et al. these values can vary between 55. corn products. coffee silverskin. japonicus JN19 (Wang and Zhou 2006). Generally.0 %. After the optimization procedures.0. and low capital cost and energy consumption. 2005b) to 25 g L−1 (Dhake and Patil 2007). b). polyurethane foam. The maximum FOS production (52. obtained in various production studies by either SmF or SSF. SSF requires low water volume and thus has a large impact on the economy of the process due to smaller fermenter size. Wang and Zhou 2006). 2005c. 2004b) and 62. may have a high specific growth rate. the most efficient culture media and strain to produce enzymes in SSF are not the same as those in SmF.4 % (g FOS/g sucrose). which are difficult to control under limited water availability. 2005a. 1999) can be expected. loofah sponge. 2012). oryzae CFR202 (Sangeetha et al. 2005b. g FOS/ g sucrose) was obtained after 8 h of reaction. pH. Lim et al. 2005b). The main drawbacks are due to the buildup of gradients—of temperature. and the productivity in SSF was about twice that in SmF. oryzae of 53. the medium composition for SSF required twice more sucrose and yeast extract than the medium for SmF. niger. 2008). The effect of the medium pH in FTase production and microorganism growth has been reported. 48. Lim et al. 1997). reaching 70. japonicus using agro-industrial residues as immobilization supports and nutrient sources. reduced downstream processing. or dissolved oxygen—during cultivation. Wang and Zhou 2006) and 61. NaNO3 is the most common source of inorganic nitrogen in transfructosylation enzymes production by fungi and can be employed in concentrations from 2 g L−1 (Lim et al. 2004b).0 % (g FOS/g sucrose. Shin et al. The use of low-cost agricultural and agro-industrial residues as substrates contributes also to the lower capital and operating costs of SSF as compared to SmF. Some advantages of this process are the simplicity of operation. 2004a. japonicas. such as FeSO4 and CuSO4. Corn cobs. These authors conducted a similar study with Penicillum expansum in which the use of low-cost materials including synthetic fiber. there are also some disadvantages associated with the use of SSF. respectively.Author's personal copy Food Bioprocess Technol fungus. high volumetric productivity. and cork oak were used as support and nutrient sources. leading to scale-up engineering problems (Pandey 2003. pH of 5. However. The results showed different compositions of optimized media for SmF and SSF. pullulans (Vandáková et al.3 g L−1 (Sangeetha et al. reduced stirring. Chiang et al. stainless steel sponge. niger NRRL 330 in SSF and SmF was carried out using a fractional factorial design (Balasubramaniem et al. and Penicillium purpurogenum (Dhake and Patil 2007). 2001. For A. Mussatto and Teixeira 2010). whereas for A. substrate concentration. 2005a. were obtained. In this study. and its optimal concentration varies from 0.0 % (g FOS/g sucrose) after 16 h of reaction. oryzae CFR 202 (Sangeetha et al. Other ions. and 44 % (g FOS/g sucrose) in the three initial cycles (36. and 60 h). Mussatto and Teixeira (2010) reported the increase of FOS yield and productivity by SSF with A . Production yields of 87 % (g FOS/g sucrose). .0 (60 h) 42.2 Sangeetha et al.5 Ganaie et al.0 70. This yield cannot be further increased due to the high amounts of glucose coproduced during the fermentation.0 Yoshikawa et al.0 Lateef et al. (2004b) 24+9 h 59.0 (36 h). many authors have studied the impact of the continuous removal of glucose and residual sucrose from the medium during the FOS conversion. (1997) 72+24 h 55. (2013) 120+12 h 54. (2004a) 96+12 h 53. (2005a) 24+48 h 53.0 Madlová et al. second: Mussatto et al.0 Shin et al.Author's personal copy Food Bioprocess Technol Table 2 FOS yields obtained using FTase and/or FFase from various fungal strains Microorganism Production process Reaction time (fungus cultivation+enzymatic reaction) Production yield (%. (2013) 24 h 28+17 h 61.0 Sangeetha et al. (2009) 24 h 16 h 61. (2005c) 36 h 60 h. by increasing the fermentation yields. (2009) Mussatto and Teixeira (2010) Nguyen et al. (2010) First: 87.1 Sangeetha et al.0–26. (2012) 72. three cycles 58.0 Gomes (2009) Chiang et al.0 Mussatto et al. Therefore. which acts as an inhibitor (Yun 1996). 2005c). in order to obtain higher fermentation yields.8 Ganaie et al. (2004b) 24+18 h 57.0 Prata et al.0 Yun and Song (1993) 48+24 h 55. (2004a) 72+24 h 44. Consequently.0 24+72 h 24.4 Dominguez et al.0–64. (2004a) 72+24 h 53. (2004a) 120+8 h 60.0 Shin et al. (2007) 24+24 h 62.9 Sangeetha et al.0 Sangeetha et al. (2012) Ganaie et al. (1999) 92+8 h 70.8 Wang and Zhou (2006) 21 h 66. g FOS/g sucrose) Reference Aureobasidium pullulans Two-stage process using isolated intracellular enzyme produced by SmF Two-stage process using extracellular enzyme produced by SmF Two-stage process using culture broth homogenate produced by SmF Two-stage process using isolated intracellular enzyme produced by SmF Two-stage process with immobilized cell (SmF) Two-stage process with isolated intracellular FTase produced by SmF Two-stage process using crude FFase preparation produced by SmF One-stage process: present study—SmF Two-stage process using culture broth filtrate produced by SmF One-stage process—SmF Two-stage process using immobilized FFase produced by SmF Two-stage process with isolated intracellular FFase produced by SmF One-stage process using corn cobs as the solid support—SSF One-stage process—SmF One-stage process using coffee silverskin as the solid support—SSF Two-stage process with isolated intracellular and extracellular FFase produced by SmF Two-stage process using washed cells produced by SmF Two-stage process using culture broth filtrate produced by SmF Two-stage process using extracellular enzyme produced by SmF Two-stage process using culture broth homogenate produced by SmF Two-stage process FTase produced by SSF using corn germ as the solid support Two-stage process using extracellular enzyme produced by SmF Two-stage process using whole cells produced by SmF One-stage process—SmF One-stage process using synthetic fiber as the solid support: SFF—repeated batch fermentation Two-stage process using culture broth filtrate produced by SmF 96+25 h 58. 2013). third: 44.6 Sangeetha et al.4 Sangeetha et al.1 63. (2004a) 96+12 h 56.0 (48 h). (2013) Aspergillus flavus Aspergillus ibericus Aspergillus japonicus Aspergillus niger Aspergillus oryzae Penicillium expansum Penicillium chrysogenum 48+24 h theoretical yield of 55–60 % based on the initial sucrose concentration (Sangeetha et al.0 Mussatto et al.0 61. (2008) 48 h 120+24 h 64. a purer final product could be obtained (Nobre et al. (1999) 72+24 h 55. lactulose. through which glucose permeated. while oligosaccharides with four or more monosaccharide units were not fermented (Yoon et al. The complex system produced more than 80 % (g FOS/g sucrose) FOS. China Beneo-Orafti. In their study. Goulas et al. The World demand for prebiotics is estimated to be around 167.000 tons and 390 million Euros. However. Among them. FOS percentage of the reaction product was increased to above 90 % (g FOS/ g sucrose). polydextrose. the global market for FOS from sucrose was estimated to be 20. Lin and Lee 2008). Calcium carbonate slurry was used to control the pH to 5. the European market of functional food is dominated by gut health-targeted products. (2008) reported that recombinant FTases also have interesting biocatalytic properties. for example the market volume in Germany. UK. 2003. of which 228 million Euros account for probiotic. The system used mycelia of A . For example. FOS. Also. and sucrose from food-grade oligosaccharide mixtures. niger (genetic engineering) provided a new powerful tool for the efficient preparative synthesis of tailor-made saccharides 1-kestose and 1-nystose type. mobilis. Nobre et al. isomaltooligosaccharides. France. pullulans ATCC 9348 with FFase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. which were completely fermented within 12 h. Germany. Similarly to Z. In 1995.. japonicus CCRC 93007 or A . the total cost of developing a conventional new food product is estimated to be up to 0. with the remaining being 5– 7 % glucose and 8–10 % sucrose on a dry weight basis. de C. Crittenden and Playne (2002) used a different approach. Sucrose solution with an optimum concentration of 30 % (w/ v) was employed as feed for the complex cell system. (2001) achieved higher FOS yields with a simultaneous removal of glucose using a membrane reactor system with a nanofiltration membrane. Functional dairy products have shown an impressive growth. inulin. Korea Actilight® Profeed® Oligo-Sugar GTC Nutrition. The microbial treatment seems to be a good alternative for increasing the percentage of FOS in a mixture through the removal of mono. As previously mentioned. Belgium NutraFlora® Meioligo® Beneshine™ Ptype Orafti® Fibrulose® Sensus. g FOS/g sucrose).. the use of yeast treatment was found to modify the oligosaccharide composition (Sanz et al. and other functional yoghurts and around 89 million Euros for functional drinks. 2008). and gluconic acid in the reaction mixture was precipitated as calcium gluconate.Author's personal copy Food Bioprocess Technol High FOS production yields adding glucose oxidase to the reaction media have been reported. while the development and marketing costs of functional food products may exceed this level by far.75–1... Ltd. Japan Victory Biology Engineering Co. but not sucrose and FOS. and sucrose) from a mixture of sugars. Hernández et al. Saccharomyces cerevisiae also showed the ability to ferment some common mono. from around 4 million Euros in 1995 to 317 million Euros in 2000.and disaccharides (fructose. hence reducing the glucose content in the media (Yun and Song 1993. at an industrial scale. plus a small amount of calcium gluconate. and the Netherlands account for around two thirds of all sales of functional dairy products in Europe. With the same goal. immobilized cells of the bacterium Zymomonas mobilis were used to remove glucose. the use of microbial treatments implies a further step of purification for the removal of biomass and metabolic products formed during the fermentation in order to obtain a FOS product with few contaminants. Zuccaro et al. FOS are produced enzymatically by two different processes Table 3 Commercially available food-grade FOS Substrate Manufacturer Trade name Sucrose Cheil Foods and Chemicals Inc. as novel substrates (substrate engineering). glucose.000 tons (Menrad 2003.5. and highly active recombinant β-fructofuranosidase from A. The combination of sucrose analogues. Nishizawa et al. 2009). a minimal amount of sorbitol also being formed. although these systems can be more efficient in terms of FOS production yield. Belgium Cosucra Groupe Warcoing. and many authors manage to control this effect and increase FOS production yield (up to 90–98 %. Presently.V. prebiotic. Nonetheless. 2013). galactose. Mexico Frutalose® OLIFRUCTINESP® Inulin Beghin-Meiji Industries. 2001. which was much higher than that of the batch reaction product (55–60 %. (2002) reported a complex biocatalyst system with a bioreactor equipped with a microfiltration module. g FOS/g sucrose) by adding glucose oxidase to the reaction media. 2007. fructose. USA Meiji Seika Kaisha Ltd. thus increasing the production cost. it is important to notice that they are also more expensive. 2005. France . Siró et al. which leads to the formation of gluconic acid.and disaccharides. the Netherlands Nutriagaves de Mexico S. Sheu et al. Other systems aiming glucose removal from the media have also been studied.A. FOS Market Data In general. bringing. Sheu et al. and high-content FOS was discharged continuously from a microfiltration module.5 million Euros.. this process being adequate to be used during the enzymatic synthesis of FOS. and resistant starch are considered the most popular. The fermentation end products were ethanol and carbon dioxide. . & Bentéjac. & Liu.. 56. (1996). I. for her PhD grant reference SFRH/BD/ 23083/2005.-H. 38. Mathes. M. Balasubramaniem.. Germany. 18. USA. 217–241. V. Europe nutraceutics market is expected to have a share of over 20% until 2017.Author's personal copy Food Bioprocess Technol (transfructosylation of sucrose or hydrolysis of inulin) which yield slightly different end products. the opportunity exists for the screening and identification of novel strains capable of producing enzymes with transfructosylation activity and for developing improved and less expensive production methods. M. (1995). S... & Mejdouba. C.. Peterson. J. 8.. (1993). Nutrition Research. C. These FOS products are provided with a purity level above 95 % (Singh and Singh 2010. W. Scazzina. Kaminska. References Antošová. individual FOS molecules with purities varying between 80 and 99 % are only available for analytical purposes. H. & Fettman. A. Baba.... Kirk.. F. Production of β-fructofuranosidase by Aspergillus japonicus. 2. 83. 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