Appl Microbiol Biotechnol (2013) 97:70737080 DOI 10.

1007/s00253-013-5079-3

MINI-REVIEW

Current studies on physiological functions and biological production of lactosucrose

Received: 13 May 2013 / Revised: 21 June 2013 / Accepted: 21 June 2013 / Published online: 5 July 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Lactosucrose (O- - D -galactopyranosyl-(1,4)O--D-glucopyranosyl-(1,2)--D-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by -fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by -galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes. Keywords Lactosucrose . Prebiotic . Transglycosylation . Physiological function . Biotechnological production

Introduction It is well known that lactose (O--D-galactopyranosyl-(1,4)-Dglucopyranose) and sucrose (O--D-glucopyranosyl-(1,2)--Dfructofuranoside) are two of the most common and the cheapest disaccharides widely existing in nature. Several million tons of lactose is produced annually as a cheap by-product of the dairy industry. In practical industry, disposal of excess whey and lactose is an ongoing environmental problem. Sucrose is the most common sweetener used in the food industry; however, excess sucrose consumption easily leads to potential health
Wanmeng Mu and Qiuming Chen have equal contribution to this work. W. Mu (*) : Q. Chen : X. Wang : T. Zhang : B. Jiang State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China e-mail: wmmu@jiangnan.edu.cn

risks, such as tooth decay, obesity, type 2 diabetes, and heart disease. On the other hand, lactose and sucrose are increasingly recognized as important cheap sources to produce many valuable bioactive derivative compounds through biological technology. Functional lactose derivatives include lactulose (Panesar and Kumari 2011), epilactose (Mu et al. 2013), galactooligosaccharide (Fanaro et al. 2005), D-tagatose (Kim 2004; Oh 2007), lactobionic acid (Gutierrez et al. 2012), lactitol (Gee and Johnson 2005), etc. Functional sucrose derivatives include isomaltulose (Lina et al. 2002), trehalulose (Ooshima et al. 1991), isomalt (Gostner et al. 2005), fructooligosaccharides (Roberfroid and Slavin 2000), raffinose (Abou Hachem et al. 2012), etc. In addition, lactose and sucrose have recently been widely used as a resource to produce bio-ethanol by yeast fermentation (Guimaraes et al. 2010; Smith 2008). Using both lactose and sucrose as substrates, biological transglycosylation reaction may produce another bioactive derivative compound, lactosucrose. This rare trisaccharide is a kind of indigestible carbohydrate (Mussatto and Mancilha 2007) and, therefore, is low in calories and suitable for lowcalorie foods. Like many other oligosaccharides, lactosucrose has a prebiotic effect (Ohkusa et al. 1995) and promotes intestinal mineral absorption (Teramoto et al. 2006). It has been approved as an important functional food ingredient for foods for specified health uses (FOSHU) due to its promising properties in Japan. The present article is a review of recent studies on the physiological functions of lactosucrose, as well as its biological production from lactose and sucrose using fructofuranosidase, levansucrase, or -galactosidase.

Lactosucrose Chemical structure and physicochemical properties Lactosucrose (O- - D -galactopyranosyl-(1,4)-O- - D glucopyranosyl-(1,2)--D-fructofuranoside) is also known as

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4G--D-galactosylsucrose and lactosylfrucoside. The molecular formula and molecular weight of lactosucrose (CAS no. 8741956-5) are C 18 H 32 O 16 and 504.4 g/mol, respectively. Lactosucrose is a trisaccharide consisting of D-glucose, D-galactose, and D-fructose (Fig. 1). Lactosucrose has much higher solubility (3,670 g l1 at 25 C) in water than sucrose (2,000 g l1 at 25 C). By contrast to other oligosaccharide sweeteners, lactosucrose has a high quality taste similar to sucrose (Fujita et al. 2009). It has 30 % of relative sweetness compared with sucrose. It is stable for 1 h at pH 4.5 and 120 C. The lactosucrose powder is highly hygroscopic with a relatively high moisture-binding ability (Playne and Critten 2009). Physiological functions Lactosucrose is scarcely hydrolyzed by human digestive enzymes or by acetone powder prepared from rat intestines (Fujita et al. 1991), but it is selectively utilized by intestinal Bifidobacterium (Fujita et al. 2009; Ohkusa et al. 1995; Takei et al. 2006); therefore, it is considered as a kind of indigestible oligosaccharide with prebiotic properties. Many literatures have shown that lactosucrose has a beneficial effect in intestinal microflora maintenance and intestinal protection. The Bifidobacterium multiplication promoted by lactosucrose produces short chain fatty acids, reduces intestinal pH, and inhibits the proliferation of intestinal pathogenic bacteria, especially Clostridium, thus decreasing the formation of toxic products, such as ammonia, phenol, indole, skatol, and ethylphenol (Fujita et al. 1995; Ogata et al. 1993; Terada et al. 1992, 1994; Yoneyama et al. 1992). The administration of lactosucrose significantly stimulates the

growth of Bifidobacterium and improves the intestinal microenvironment in Crohns disease and ulcerative colitis patients (Teramoto et al. 1996). Honda et al. (1999) have reported the protective effect of lactosucrose on intracolonic indomethacin-induced small intestinal ulcers in rats. In addition, lactosucrose has potential treatment effect on inflammatory bowel disease due to its ability to modify the intestinal flora (Kanauchi et al. 2003). Generally, indigestible oligosaccharides, such as fructooligosaccharides (Morohashi et al. 1998), oligofructose (van den Heuvel et al. 1999), difructose anhydride III (Hang et al. 2011), and galactooligosaccharides (Chonan et al. 1995), can enhance intestinal calcium absorption in rats or humans. As a member of indigestible oligosaccharides, lactosucrose also displays effective calcium absorption-promoting activity. Fujita et al. (1999) have reported that feeding calcium and lactosucrose mixture to weanling rats could enhance the ability of the femur and tibial cancellous cartilage to resist breakage. Using 45Ca as a tracer to follow calcium transport and deposition, Kishino et al. (2006) have proven that dietary supplementation with lactosucrose may effectively increase calcium absorption from the intestine in growing rats. Supplementation of lactosucrose in a single (Kikuchi et al. 2003) and 2-week administration (Fujita et al. 2006) may enhance the intestinal calcium absorption in healthy men. In addition, the administration of lactosucrose has a long-term effect that enhances intestinal calcium absorption and reduces bone resorption in healthy young women (Teramoto et al. 2006). Lactosucrose shows potential beneficial effect in inhibiting body fat accumulation and preventing obesity (Kimura et al. 2002). It has been reported that lactosucrose supplementation decreases the serum cholesterol levels in mice (Han et al.

Fig. 1 Chemical structures of lactose, sucrose, and lactosucrose

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1998). Lactosucrose can also inhibit the absorption of 2monoacylglycerol by rat brush border membrane vesicles prepared from the small intestine (Han et al. 1999). Through inhibiting -monoglyceride absorption, lactosucrose reduces intestinal absorption of dietary fat and reduces the parametrial adipose tissue weight of female mice fed with a high-fat diet (Okuda and Han 2001). Mizote et al. (2009) suggest that lactosucrose inhibits intestinal lipid absorption through directly interacting with triglyceride, thus reducing adipose tissue accumulation. Recently, lactosucrose has been found having potential immunoregulatory effect. Lactosucrose supplementation induces the modification of intestinal microflora environment and indirectly enhances the IgA in the gut and suppresses the systemic immune response to the dominant type 2 helper T (Hino et al. 2007). In addition, it has been reported that lactosucrose intake suppresses IgE response induced by intraperitoneal immunization with ovalbumin/alum in a mice model, suggesting that lactosucrose has the potential ability to prevent IgE-mediated allergic diseases (Taniguchi et al. 2007). In addition, lactosucrose is an excellent excipient for spray-dried powders and shows promising protein stabilization of IgG (Schule et al. 2008). It has also been explored as a pet food ingredient due to its prebiotic properties (Hussein et al. 1999). Commercial importance Lactosucrose has been used as a prebiotic ingredient in a range of food products which are certified as FOSHU and are labeled to indicate their effects on intestinal conditions in Japan. By 2009, over 30 food products containing lactosucrose have been approved as FOSHU. As a sweetener as well as a prebiotic ingredient, lactosucrose has been commercially used in diverse food and beverage systems, including confectionaries, desserts, sweets, bakery products, yoghurts, coffee, and tea. However, its use is largely confined to Japan. Annual sales volume of lactosucrose reaches several kilotons in Japan. The two major manufacturers of lactosucrose are Ensuiko Sugar Refining Co. and Hayashibara Shoji Inc., with the product brand name Nyuka-Origo and Newka-Oligo, respectively.

Levansucrases To our best knowledge, enzymatic synthesis of lactosucrose was first described by Avigad (1957) who reported the production of lactosucrose by levansucrase from Aerobacter levanicum, acting on a system comprised of sucrose as transfructosyl donor and lactose as acceptor. Later on, many microorganisms harboring levansucrase activity were screened and characterized to have bioconversion ability from lactose and sucrose into lactosucrose, such as Bacillus natto (Takahama et al. 1991), Bacillus subtilis ATCC 6501 (Hara et al. 1994), B. subtilis KCCM 32835 (Park et al. 2005), Paenibacillus polymyxa IFO 3020 (Choi et al. 2004), Sterigmatomyces elviae ATCC 18894 (Lee et al. 2007a, b), and Pseudomonas aurantiaca (Han et al. 2007). In addition, the recombinant levansucrases from B. subtilis NCIMB 11871 (Seibel et al. 2006) and Zymomonas mobilis (Han et al. 2009) could produce lactosucrose from lactose and sucrose. In enzymology, levansucrase (systematic name: sucrose:2,6--D-fructan 6--D-fructosyltransferase) catalyzes the transfer of the fructosyl residue from sucrose to different acceptors, including water (sucrose hydrolysis), glucose (exchange), sucrose (oligofructoside synthesis), and fructan (polymerization) (Martinez-Fleites et al. 2005). It displays an important role in fructan biosynthesis in bacteria. Seibel et al. (2006) have reported that the levansucrases from B. subtilis NCIMB 11871 have a broad acceptor spectrum for transfructosylation, including various D-monosaccharides, L-monosaccharides, and disaccharides, and produce a series of sucrose analogs with sucrose serving as a glycosyl donor substrate. Also, this bacterial levansucrase shows effective lactosucrose-producing ability at a productivity of 21.9 g l1 h1 with a final conversion rate of 34 %, when lactose and sucrose are used as transfructosyl acceptor and donor, respectively (Seibel et al. 2006). It is suggested that lactosucrose formation is realized by the ping-pong mechanism involving the formation of a transient fructosylenzyme intermediate (shown in Fig. 2) (Fujita et al. 1991). -Fructofuranosidase from Arthrobacter sp. K-1 Lactosucrose synthesis from sucrose and lactose can also be carried out by using -fructofuranosidase from Arthrobacter sp. K-1. -Fructofuranosidase (systematic name: - D fructofuranoside fructohydrolase), also known as saccharase or invertase, widely exists in bacteria, yeasts, fungi, and plants (Roitsch and Gonzalez 2004). It mainly catalyzes the hydrolysis of terminal nonreducing -D-fructofuranoside residues in -D-fructofuranosides, especially sucrose, and is one of the cheapest commercial carbohydrases (Kotwal and Shankar 2009). In addition to its hydrolytic action, fructofuranosidase may catalyze transfructosylation (Win

Enzymes producing lactosucrose As a rare trisaccharide, lactosucrose scarcely exists in nature and is difficult to be chemically synthesized. Lactosucrose can be produced through transfructosylation reaction by fructofuranosidase (EC 3.2.1.26) from Arthrobacter sp. K-1 or a range of levansucrases (EC 2.4.1.10), or transgalactosylation reaction by -galactosidase (EC 3.2.1.23) from Bacillus circulans, with sucrose and lactose as substrates.

7076 Fig. 2 The supposed mechanism of lactosucrose production by B. natto levansucrase (Fujita et al. 1991)

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et al. 2004), and the acceptor specificity and product structure for transfructosylation may vary, depending on the source of the enzymes and on the reaction conditions (Smaali et al. 2012; Win et al. 2004). The transfructosylation of -fructofuranosidase has widely been used to produce short chain fructooligosaccharides (Vega-Paulino and Zuniga-Hansen 2012). Fujita et al. screened a bacterium, Arthrobacter sp. K-1, from soil, which had fructose-transferring enzyme activity, and then purified and characterized -fructofuranosidase (Fujita et al. 1990a). Arthrobacter sp. K-1 -fructofuranosidase catalyzes mostly hydrolysis when incubated with sucrose alone even at high concentration; however, interestingly, in the presence of suitable acceptors, the enzyme preferentially catalyzes the transfructosylation from sucrose to the acceptor molecules (Fujita et al. 1990b, 1991). It has a very broad specificity for transfructosylation acceptor, and lactose ranks in the most effective acceptors, with transfer ratios more than 50 % (Fujita et al. 1990b), and the transfructosylated product was determined as lactosucrose with lactose serving

as transfructosylation acceptor (Fujita et al. 1994; Pilgrim et al. 2001). The enzyme is known to not only catalyze the transfer reaction producing lactosucrose, but also to hydrolyze sucrose and lactosucrose (Fig. 3) (Pilgrim et al. 2001). Although -fructofuranosidase widely occurs in nature, so far, only -fructofuranosidase from Arthrobacter sp. K-1 has been exclusively characterized having conversion ability of lactose and sucrose into lactosucrose. The gene encoding Arthrobacter sp. K-1 -fructofuranosidase has been sequenced (Ito et al. 2002) and shows that it belongs to the glycoside hydrolase family 68 in the CAZy database (http://www.cazy.org) (Cantarel et al. 2009). The enzyme has been cloned and expressed in Escherichia coli, and the recombinant Arthrobacter sp. K-1 -fructofuranosidase has highly similar biochemical properties to the native enzyme, showing optimum pH and temperature at 6.5 and 55 C. In addition, the enzyme is relatively thermostable and remained approximately 70 % of initial activity after incubated at 60 C for 30 min (Ito et al. 2002). The crystal structure of

Fig. 3 Enzymatic reactions by -fructofuranosidase from Arthrobacter sp. K-1 with lactose and sucrose as substrates (Pilgrim et al. 2001)

Appl Microbiol Biotechnol (2013) 97:70737080 Fig. 4 Synthetic scheme for lactosucrose and its analogs by B. circulans -galactosidase with lactose and sucrose as substrates (Li et al. 2009)

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Arthrobacter sp. K-1 -fructofuranosidase has been recently dissolved, and the structural features of the catalytic cleft and the tunnel at the bottom of the cleft show that it is more suitable for lactosucrose synthesis than other -fructofuranosidases with dissolved structure (Tonozuka et al. 2012). -Galactosidase from B. circulans -Galactosidase (systematic name: - D -galactoside galactohydrolase), also known as lactase, is an extremely common glycosyl hydrolase in nature (Grosova et al. 2008). It not only catalyzes lactose hydrolysis, but also catalyzes transgalactosylation reaction. It is widely used for the hydrolysis of milk lactose and the synthesis of galactooligosaccharides

and lactulose from lactose in commerce (Juajun et al. 2011; Panesar and Kumari 2011; Park and Oh 2010). -Galactosidase from B. circulans catalyzes the transfer of galactose units to various glucose and galactose derivatives, forming tri- and tetrasaccharides with -1,4 linkage, including lactosucrose (Farkas et al. 2003). Li et al. (2008, 2009) have recently reported the effect of the enzymatic synthesis of lactosucrose and its analogs from lactose and sucrose by B. circulans galactosidase (Fig. 4). Production of lactosucrose by B. circulans -galactosidase is due to the transfer of the galactosyl residue from the donor lactose to the acceptor sucrose. On the contrary, lactose and sucrose are the glycosyl acceptor and donor, respectively, during the lactosucrose production by transfructosylation of levansucrases and Arthrobacter sp. K-1 -fructofuranosidase.

Table 1 Biotechnical production of lactosucrose using levansucrase and whole cells harboring levansucrase activity Strains Biocatalyst Substrates concentration used 85.5 g l1 sucrose and 85.5 g l1 lactosea 225 g l1 sucrose and 225 g l1 lactose 225 g l1 sucrose and 225 g l1 lactose 205 g l1 sucrose and 410 g l1 lactosea 510 g l1 sucrose and 360 g l1 lactose 180 g l1 sucrose and 180 g l1 lactose 250 g l1 sucrose and 250 g l1 lactose 250 g l1 sucrose and 250 g l1 lactose Lactosucrose produced Productivity (g l1 h1) NR 28 18.3 21.9 NR NR References

B. natto Paenibacillus polymyxa IFO 3020 B. subtilis KCCM 32835 B. subtilis NCIMB 11871 Pseudomonas aurantiaca Z. mobilis

Purified enzyme Free whole cells Free whole cells Crude recombinant enzyme NR Crude recombinant enzyme

31 % of total sugars 170 g l1 183 g l1 68 g l1b 285 g l1 28.5 % of total sugars (by single enzyme); 43.2 % of total sugars (by coupling with glucose oxidase) 140.91 g l1 (basal medium); 183.78 g l1 (optimized medium) 192 g l1

Takahama et al. (1991) Choi et al. (2004) Park et al. (2005) Seibel et al. (2006) Han et al. (2007) Han et al. (2009)

S. elviae mutant

Free whole cells

NR

Lee et al. (2007b)

S. elviae mutant

Immobilized whole cells

NR

Lee et al. (2007a)

NR not reported
a b

The values were converted from the original references after exchanging the unit The lactosucrose yield is 34 % of initial sucrose concentration (Seibel et al. 2006)

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Biological lactosucrose production Park et al. studied the lactosucrose productivity of a range of microorganisms harboring levansucrase activity and found that they all could produce lactosucrose, but the productivity remarkably depended on the microbial strain (Park et al. 2005). So far, there have been many references reporting the lactosucrose production by levansucrase from various microorganisms. The lactosucrose yield and productivity using levansucrase and whole cells harboring levansucrase activity were shown in Table 1. When sucrose and lactose were both used with the concentration of 225 g l1, Paenibacillus polymyxa IFO 3020 (Choi et al. 2004) and B. subtilis KCCM 32835 (Park et al. 2005) could produce 170 and 183 g l1 of lactosucrose, with productivity of 28 and 18.3 g l1 h1, respectively. The yield of lactosucrose formation by Pseudomonas aurantiaca was improved by increasing the ratio of [sucrose]/[lactose], but decreased by extending reaction time. Under optimized conditions, Pseudomonas aurantiaca produced 285 g l1 of lactosucrose (Han et al. 2007). Lee et al. (2007a) carried out the N-methyl-N-nitro-N-nitrosoguanidine mutagenesis of S. elviae ATCC 18894 and obtained a beneficial mutant with 54.3 % higher lactosucrose production than the wild type. Using response surface methodology to optimize the culture medium of S. elviae mutant, the cell mass and lactosucrose production increased to 23.3 and 30.42 %, respectively, and the lactosucrose yield reached 183.78 g l1 (Lee et al. 2007b). Using the same mutant strain, they developed a continuous production of lactosucrose by immobilized whole cells in a packed bed reactor (Lee et al. 2007a). In addition, it was reported that the conversion efficiency of lactosucrose produced by Z. mobilis levansucrase could be remarkably improved by coupling glucose oxidase, which could remove byproduct glucose from the reaction products (Han et al. 2009). The industrial production of lactosucrose by Arthrobacter sp. K-1 -fructofuranosidase has been developed in Ensuiko Sugar Refining Co Ltd, Japan, for two decades (Fujita et al. 1992). Kawase et al. (2001) developed a continuous lactosucrose production process by the -fructofuranosidase using simulated moving bed reactor, and the conversion ratio of sucrose into lactosucrose was improved to over 80 %, which was much higher than that of the batch process (50 % sucrose conversion). In addition, using 300 g l1 sucrose and 300 g l1 lactose, galactosidase from B. circulans produced lactosucrose with the maximum yield of 56 g l1, which was significantly lower than that produced by levansucrases and Arthrobacter sp. K-1 fructofuranosidase (Li et al. 2009).

optimized enzyme for industrial lactosucrose production should be thermostable and have higher transglycosylation specificity for lactosucrose production and higher conversion of sucrose and lactose into lactosucrose. The crystal structures of the lactosucrose-producing enzymes have been identified. Based on the structure information, molecular modification would be deeply researched to obtain the more ideal enzyme for industrial lactosucrose production. The presence of other carbohydrate products and residual amounts of unused lactose and sucrose in the bioprocess is unfavorable to the product application. Therefore, the downstream process researches should be strengthened in the future.
Acknowledgments This work was supported by the NSFC Project (Nos. 31171705 and 21276001), the 973 Project (No. 2012CB720802), the 863 Project (No. 2011AA100904), the Fundamental Research Funds for the Central Universities (No. JUSRP51304A), and the Support Project of Jiangsu Province (Nos. BE2011622, BE2011766, and BE2010626).

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