International Dairy Journal 12 (2002) 163171

An overview of the functionality of exopolysaccharides produced by lactic acid bacteria

Patricia Ruas-Madiedo, Jeroen Hugenholtz, Pieternela Zoon*
NIZO Food Research, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands Received 4 March 2001; accepted 10 October 2001

Abstract Lactic acid bacteria (LAB) that produce exopolysaccharides (EPSs) play an important role in the dairy industry because of their contribution to the consistency and rheology of fermented milk products. The EPS polymers can be considered as natural biothickeners because they are produced in situ by the LAB-starters that have General Recognised As Safe status (GRAS). The physico-chemical properties of EPSs determine their viscosifying efciency. Hence, the knowledge of the structurefunction relationship of these biopolymers is crucial in order to choose or design polymers for a specic technological application. In addition, health benets have been attributed to some of these EPSs, particularly antitumor and immunomodulating activities. Also a prebiotic role has been suggested. However, almost all studies were performed in vitro and scarce information is available concerning in vivo experiments with oral administration. This overview focuses on the recent information about the functional properties of lactic acid bacterial EPSs, including both technological and health-promoting aspects. r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Exopolysaccharide; Lactic acid bacteria; Functionality; Technology and physiology

1. Introduction Lactic acid bacteria (LAB) have been used around the world to improve the preservation, sensorial characteristics and nutritional value of a large variety of products, like milk, meat and vegetables. The lactic fermentation of milk is by far the best studied and known. A great variety of dairy products with different avour, texture and health-promoting properties can be obtained from milk using different technologies and starter cultures. Important features of the starter are rapid acidication, microbial preservation of the milk, formation of specic avours, texturing capacities and health benets. The fermentation of lactose during bacterial growth causes acidication of the milk and thereby it protects the milk against spoilage microorganisms and proliferation of pathogens. Another effect of acidication is the neutralisation of the negative charges on the milk proteins, resulting in coagulation. The acid also gives fermented products, such as yoghurt and cheese, their pleasant
*Corresponding author. Tel.: +31-318-659577; fax: +31-318650400. E-mail address: zoon@nizo.nl (P. Zoon).

fresh and mild acid taste. Furthermore, the LAB convert sugars, organic acids, proteins or fats into the typical aroma and avour components. Several LAB strains also can contribute to improve the texture and viscosity of fermented products by means of the synthesis of exopolysaccharides (EPSs). LAB, like many other bacteria, are able to produce several types of polysaccharides that are classied according with their location relative to the cell. Those that are excreted outside the cell wall are called exocellular polysaccharides or EPSs. These can form an adherent cohesive layer and are called capsular polysaccharides. The EPSs also can either be loosely attached or be completely excreted into the environment as slime (Cerning, 1994). The bacterial EPSs are not used as energy sources by the producer microorganism. They probably have a protective function in the natural environment, e.g. against desiccation, phagocytosis and predation by protozoa, phage attack, antibiotics or toxic compounds and osmotic stress. EPSs also have a role in cell recognition, in adhesion to surfaces and in formation of biolms facilitating the colonisation of various ecosystems (Looijesteijn, Trapet, de Vries, Abee, & Hugenholtz, 2001; Whiteld & Valvano, 1993). The

0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 6 0 - 1

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ecological function of the EPSs produced by LAB is not clearly dened and it is probably complex, but it seems to be related with cell adhesion and cell protection in different environments. The EPSs produced by Streptococcus salivarius and S. mutans are involved in the bacterial colonisation and formation of dental plaque (Cerning, 1990). A recent publication of Looijesteijn and coworkers (2001) show that the EPS produced by Lactococcus lactis subsp. cremoris NZ4010 protects the bacteria against several antimicrobial factors such as bacteriophages, metal ions, nisin and lysozyme. Apart from these ecological functions, EPSs from LAB have technological signicance in the production of several fermented dairy products. The use of ropy starters containing EPS-forming S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is a common practice in the production of yoghurt to improve texture, avoiding syneresis and increase the viscosity of the yoghurt. In Scandinavian dairy products, like viili, EPS-forming strains of Lactococcus species are used. In addition to this role in texture formation, it has been suggested that the positive physiological effects on human health of fermented milk products might be attributable to several cell wall components of LAB, particularly extracellular polysaccharides (Kitazawa et al., 1998). In the last decade several reviews on EPSs produced by LAB have been published (Cerning, 1990, 1994, 1995; Kleerebezem et al., 1999; Ricciardi & Clementi, 2000; Sikkema & Oba, 1998; De Vuyst & Degeest, 1999). They focus primarily on the classication, chemical composition and structure of EPSs, biosynthesis and genetics, microbial physiology, production and engineering of EPSs and also consider some applications. Since the

EPS-forming LAB have been used to improve the reological characteristics of dairy products, the better understanding of the structurefunction relationship of EPS in the fermented product is crucial to further technological applications of these biopolymers. Furthermore, the growing market of functional foods enhances the interest in the potential benecial effect of EPSs on human health, although studies in this sense are scarce and limited to in vitro experiments. This overview deals with the recent information on technological and physiological (health-promoting) functionality of the EPSs produced by LAB.

2. General considerations The EPSs produced by LAB can be divided into two categories: homopolysaccharides, containing only one type of monosaccharide, and heteropolysaccharides, composed of repeating units, varying in size from dito heptasaccharides, that could also contain non-sugar molecules. The homopolysaccharides can be clustered into four groups: a-d -glucans, b-d -glucans, fructans and others, like polygalactan (Table 1). The strain-specic differences depend on the degree of branching and the different linking sides (De Vuyst & Degeest, 1999). Homopolysaccharides have high molecular weights but some values reported in literature were over-estimating due to the presence of aggregates in aqueous solutions. The average molar mass of non-aggregated molecules of the dextran (a-glucan) produced by Leuconostoc mesenteroides NRRL B-512F varies between 6.2 and 7.1 106 Da. Levans produced by several strains of S.

Table 1 Homopolysaccharides produced by lactic acid bacteria EPS a-d-glucans Dextran Mutan Alternan b-d-glucans Strain Leuconostoc mesenteroides subsp. mesenteroides Leuconostoc mesenteroides subsp. dextranicum Streptococcus mutans Streptococcus sobrinus Leuconostoc mesenteroides Pediococcus spp. Streptococcus spp. Linkagea a-d -Glcpb (1-6) a-d -Glcpb (1-3) a-d -Glcp (1-3)/(1-6) b-d-Glcp (1-3)

Fructans Levans Inulin-like Polygalactan

a b

Streptococcus salivarius Streptococcus mutans L. lactis subsp. lactis H414

b-d-Frup (2-6) b-d-Frup (2-1) a-d -Galp/b-d -Galpc

Glc: glucose; Gal: galactose; Fru: fructose. At least 50% of the respective linkage. c Homopolysaccharide containing a pentameric repeating unit of galactose.

P. Ruas-Madiedo et al. / International Dairy Journal 12 (2002) 163171 Table 2 Overview of recently published structures of heteropolysaccharides produced by lactic acid bacteriaa Repeating units Lactobacillus Lb. delbrueckii subsp. bulgaricus 291 References Faber, Kamerling, and Vliegenthart (2001)

165

Lb. helveticus Lb161

Staaf et al. (2000)

Lb. helveticus K16

Yang et al. (2000)

Lb. rhamnosus C83 Streptococcus S. macedonius Sc136

NAc

Vanhaverbeke et al. (1998)

Vincent et al. (2001)

S. thermophilus SFi 20

NAc

Navarini et al. (2001)

S. thermophilus Rs S. thermophilus Sts

Faber et al. (1998)

S. thermophilus MR-1C

L-Fuc
Ac0.4

Low et al. (1998)

S. thermophilus S3 S. thermophilus 8Sb

Faber, van den Haak, Kamerling, and Vliegenthart (2001)

_ -D-Rib-

Sug-

NAc

Faber (2000)

S. thermophilus EU20

Marshall et al. (2001)

Lactococcus L. lactis subsp. cremoris NIZO B39

van Casteren, Dijkema, Schols, Beldman, and Voragen (2000)

Ac0.5

L. lactis subsp. cremoris NIZO B891

a

van Casteren, de Waard, Dijkema, Schols, and Voragen (2000)

This table does not contain structures already summarised in previous reviews (De Vuys & Degeest, 1999; Ricciardi & Clementi, 2000; Sikkema & Oba, 1998). () b-d -glucose; (&) a-d-glucose; ( ) b-d-galactose; ( ) a-d -galactose; (E) b-l-rhamnose; (B) a-l-rhamnose; (Rib) d -ribose; (Fuc) fucose; (Nac) N-acetyl, (Ac) acetyl; (G) glycerol. b Sug is 6-O-(30 ,90 -dideoxy-d -threo-d -altro-nononic acid-20 -yl)-a-d -glucopyranose.

mutans have a higher molar mass (2.721.6 106 Da) and S. mutans JC2 synthesises a fructane inulin-like of 12.4 106 Da (Cerning, 1990). The production of homopolysaccharides requires the presence of a specic substrate, such as sucrose, and the assembly of monosaccharide units takes place outside the bacterial cell (Cerning, 1990). These biopolymers are produced in large quantities. For example, a strain of Lb. reuteri (Lb. reuteri LB 121) is able to produce nearly 10 g L1 of two types of hompolysaccharides mainly composed of d -glucose or d -fructose (van Geel-Schutten et al., 1999).

The heteropolysaccharides, produced by a great variety of mesophilic and thermophilic LAB, are formed by repeating units that most often contain a combination of d -glucose, d -galactose and l -rhamnose and, in a few cases, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) or glucuronic acid (GlcA). Sometimes, non-carbohydrate substituents such as phosphate, acetyl and glycerol are present (Table 2). Heteropolysaccharide synthesis differs from homopolysaccharide synthesis in that the precursor repeating units are formed intracellularly and isoprenoid glycosyl

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carried lipids are involved in the process (Cerning, 1990). The repeating units are translocated across the membrane and subsequently polymerised extracellularly. Van Kranenburg (1999) has recently proposed a working model for the synthesis route of NIZO B40 EPS. The molar mass of heteropolysaccharides ranges from 4.0 104 to 6.0 106 Da. The amount of EPS produced in milk by different species and strains varies considerably, although this variation could also depend on the EPS isolation method employed. Growth conditions (pH, temperature and incubation time) and medium composition (carbon, nitrogen sources and other nutrients) can affect the polymer yield and the sugar composition (Looijesteijn, van Casteren, Tuinier, Doeswijk-Voragen, & Hugenholtz, 2000; Torino, Sesma, & de Valdez, 2000; Grobben, Boels, Sikkema, Smith, & de Bont, 2000). The EPS quantity reported ranges from 50 to 350 mg L1 for S. thermophilus, from 60 to 150 mg L1 for Lb. delbrueckii subsp. bulgaricus, from 25 to 600 mg L1 for L. lactis subsp. cremoris and from 50 to 60 mg L1 for Lb. casei (Cerning, 1995). These quantities are limited compared to the production of polymers from other bacteria like, for example, xanthan (1025 g L1) produced by Xanthomonas campestris, which is one of the major commercial polymers (Becker, Katzen, Puhler, & Ielpi, 1998). For some strains, the ability to produce EPS is an unstable characteristic that has been attributed to loss of plasmids in mesophilic LAB (Neve, Geis, & Teuber, 1988; Vedamuthu & Neville, 1986), although the mechanism remains unclear in thermophilic strains. For L. lactis subsp. cremoris strains, the ability to produce EPS was shown to be encoded by plasmids (Forde & Fiztgerald, 1999; van Kranenburg, Marugg, van Swam, Willem, & de Vos, 1997). More recently, van Kranenburg, Vos, van Swam, Kleerebezem, and de Vos (1999) found eps-encoding genes in other 15 strains of L. lactis. By transferring the plasmid through conjugation, EPS production could be introduced into other L. lactis strains.

3. Technological functionality The most important textural characteristics of yoghurt are rmness and the ability to retain water. These properties are related with the gel structure and might be inuenced by the type of culture. The rmness and cohesiveness of yoghurts made with ropy strains decrease with the presence of increased amounts of EPS (Hassan, Frank, Schmidt, & Shalabi, 1996; Marshall & Rawson, 1999). The EPS could interfere with the association between casein micelles resulting in a less rm coagulum. Studies of yoghurt microstructure (Hassan, Frank, Farmer, Schmidt, & Shalabi, 1995; van Marle, 1998) show void spaces around EPS-

producing bacteria in confocal scanning laser micrographies that can affect the integrity of the protein matrix. Yoghurts made with ropy cultures exhibited the highest water holding capacity (Hassan et al., 1996) which decrease the susceptibility to syneresis. The contribution of the lactic acid bacterial EPSs to the rheological properties of stirred yoghurt is a common assumption (Hess, Roberts, & Ziegler, 1997; Rawson & Marshall, 1997; van Marle, van den Ende, de Kruif, & Mellema, 1999). However, no clear correlation between the viscosity of fermented milk and EPS concentration was found. Van Marle and Zoon (1995) showed that the non-ropy LL yoghurt culture produced nearly the same amount of polymer (90 mg L1) as the ropy RR culture (101 mg L1). However, there was a great difference in the viscosity of the two types of yoghurt. Based on permeability measurements and confocal scanning laser micrographs, it is suggested that the spatial structure of the protein network inuences the apparent viscosity of the stirred yoghurt, although this it is not the only factor (van Marle, 1998). WacherRodarte et al. (1993) and Sebastiani and Zelger (1998) also reported that viscosity values of milks fermented with thermophilic strains of LAB were not correlated with the amount of EPS present. Only if the production of a given type of EPS is increased the viscosity of the fermented milk also increased, as shown by Sebastiani and Zelger (1998). For a S. thermophilus strain (S. thermophilus 1) they could increase the EPS production by addition of peptone to the milk and a concomitant increase in viscosity of the milk product was observed. Furthermore, it is supposed that differences in viscosity enhancement of various strains are due to differences in the intrinsic viscosity of the EPSs. Recently, Tuinier, Zoon, Cohen-Stuart, Fleer, and de Kruif (1999b) elucidated the physical characteristics of the EPS produced by L. lactis subsp. cremoris B40. The intrinsic viscosity as well as the concentration and shear rate dependence of all random coil biopolymers could be predicted from the molar mass and hydrodynamic radius of the polymer. These characteristics can be measured by light scattering techniques. The radius (a measure of the size of the polymer) depends on the molar mass (length of the polymer) and on the exibility of the polymer chain (Tuinier et al., 1999c). Similar ndings were obtained from studies on the physical properties of aqueous solutions of the EPS produced by L. lactis subsp. cremoris SBT 0495, which has the same repeating units as EPS B40 (Higashimura, MulderBosman, Reich, Iwasaki, & Robijn, 2000; Oba et al., 1999). To obtain a high viscosity, the molar mass should be high and the chain should be relatively stiff. The importance of molar mass and radius of EPS for yoghurt texture is illustrated by the work of Faber, Zoon, Kamerling, and Vliegenthart (1998). Milks fermented with two strains of S. thermophilus (Rs and

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Sts) were different in Posthumus viscosity (39 and 126 s, respectively) although the permeability coefcient of milk yoghurt gels, the amount of EPS produced and also the repeating unit of the two biopolymers was the same. The only difference was the molar mass of the EPSs; EPS molar mass from strain Sts, the more viscous, was higher than those from strain Rs (3.7 106 and 2.6 106 Da, respectively). The role of chain stiffness in viscosifying properties of EPS was shown by van den Berg et al. (1995) and Tuinier et al. (2001). The average molar mass of EPS produced by Lb. sake 01 (6 106 Da) is in the same order of magnitude as that of xanthan gum (between 4 106 and 9 106 Da), but its intrinsic viscosity is higher (van den Berg et al., 1995). Although the relation between polysaccharide chemistry and chain stiffness is complex, some general features can be distinguished, e.g. it is known that monosaccharides connected by b(1-4) bonds result in stiffer chains compared with a(1-4) or b(1-3) bonds (Tuinier, 1999). Also branches and side groups play an important role in stiffness. Tuinier et al. (2001) showed that the removal of terminally linked galactosyl residues (on side chains) of EPSs produced by L. lactis subsp. cremoris B39 and B891 tended to decrease the chain stiffness and thereby the thickening efciency. The removal of the acetyl group present in B891 EPS did not signicantly affect the chain stiffness. The presence of charges, like the negative charge of the phosphate group of EPSs produced by Lb. sake 01 or L. lactis subsp. cremoris B40, causes, depending on the ionic strength, an increase in the intramolecular repulsion forces in the polymer chain. And hence, leads to an increase of the hydrodynamic volume and the intrinsic viscosity. Also the special branching pattern on the structure of the EPS produced by Lb. helveticus K16 could explain the high viscosity of the EPS in aqueous solution (Yang, Staaf, Huttunen, & Widmalm, 2000) and supports the effect of side groups on the viscosifying properties of EPSs. The interaction of EPS with other food components has not been studied yet into great detail. Some electron microscopy work has been done (Skriver, Buchheim, & Qvist, 1995) and Tuinier, ten Grotenhuis, Holt, Timmins, and de Kruif (1999a) observed depletion effects between EPS and casein micelles at natural milk pH (B6.6). More research is needed in this eld to fully understand the physical functionality of EPSs in food products. The studies former refereed make clear that the knowledge of the physico-chemical properties of EPSs and their interactions with the other components of the food products are crucial in order to design polymers for a certain application. Synthesis of desired EPSs may be achieved by control of culture conditions or by metabolic engineering. Looijesteijn et al. (2000) found that the type of substrate limitation inuenced the molar

mass of EPSs produced by L. lactis subsp. cremoris NIZO B40 and NIZO B891. Under glucose limitation, the molar mass was reduced, but no changes in chemical composition of EPS backbone were found. Some success has been reported in EPS overexpression. The epsD gene, encoding a lactococal priming glucosyltransferase and part of the plasmid-located EPS operon, was placed under control of the NICE (nisin-controlled expression) system. After induction with nisin A, the EPS production of a transformed L. lactis strain was higher than that of the wild-type strain (van Kranenburg et al., 1999). Possibilities for production of heterologous or new EPS structures have also been reported. The transfer of a gene cluster encoding EPS produced by S. thermophilus SFi6 into a non-EPS-producing L. lactis MG1363 yielded an EPS with a different structure from the EPS of the native host (Stingele et al., 1999). The Nacetylgalactosamine present in the backbone structure of the wild-type S. thermophilus SFi6 was substituted by galactose in the recombinant EPS of L. lactis and also the galactose side group branched at the 6 position of the glucose residue was absent. Although these ndings offer perspectives for increased production of EPSs and their potential use as thickening ingredients, most LAB strains are not suitable for the protable production of EPS due to the low production levels of EPS. These bacteria seem more appropriate as functional starters. The LAB, which are GRAS microorganisms, can synthesise the biopolymer in situ, producing a natural product with improved rheological properties.

4. Physiological functionality Since the beginning of the 20th century it is clear that some bacteria have a benecial effect on human health. At present, the development of functional foods containing probiotic bacteria is an expanding market with both health and economic benets. Probiotics are dened as live microbial food ingredients that are benecial to health (Salminen et al., 1998). The probiotic bacteria currently used are predominantly lactobacilli and bidobacteria, some of which produce EPSs. In fact, it has been suggested that the health-promoting effect of EPS-producing strains are related to the biological activities of these biopolymers. Exopolysaccharides might contribute to human health (Fig. 1) as prebiotics or due to antitumor, antiulcer, immunomodulating or cholesterol-lowering activities (De Vuyst & Degeest, 1999). Several studies have suggested that LAB and fermented dairy products have anticarcinogenic activity. Kitazawa et al. (1991) found that the intraperitoneal injection of lyophilised L. lactis subsp. cremoris KVS 20 cells resulted in the growth inhibition of Sarcoma-180

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LAB

EPS
Stomach
Antiulcer

Gut

Prebiotic

Blood Immune system

Lymphocyte proliferation Macrophage activation Cytokine production Cholesterol lowering

property of retarding tumor growth when administrated orally. Oral immune enhancement by this EPS is induced probably through T-cell and not through Bcell participation (Zubillaga et al., 2001). Further research is necessary in this topic as prerequisite to employ the EPS or EPS-producing LAB in functional foods. A prebiotic is a non-digestible food ingredient that benecially affects the host by selectively stimulating the growth and/or activity of a limited number of bacteria in the colon, and thus improves host health (Gibson & Roberfroid, 1995). In order to classify a food ingredient as a prebiotic it must: 1. be neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract; 2. be a selective substrate for a limited number of benecial bacteria commensal to the colon, which are stimulated to grow and/or are metabolically activated; 3. consequently, be able to alter the colonic ora in favour of a healthier composition; 4. induce luminal or systemic effects that are benecial to host health. Non-digestible carbohydrates in general, and fructooligosaccharides in particular (e.g., inulin), are the most common prebiotics. There are no reports of the use of EPSs produced by LAB as prebiotics, despite the benecial effects former summarised. The EPS susceptibility to digestion might be an important characteristic for its possible prebiotic role, but only a few studies appear to have been done on this subject. Looijesteijn et al. (2001) found that the presence of EPS did not exert any protection for the gastrointestinal transit of producer strain L. lactis subsp. cremoris NZ4010. The experiment was carried out in vitro, using a model system simulating the process of digestion in humans, in which the strain was incubated at 371C in a solution containing pepsin, a mixture of pancreatic enzymes, and cholic and taurodeoxy acids. The same authors also tested the resistance of EPS produced by L. lactis subsp. cremoris B40 to digestion in vivo using rats, which were fed with an EPS-containing diet during an experimental period of 2 weeks. The recovery of EPS in the faeces was nearly 100%, indicating that during the passage through the gastrointestinal tract no EPS was digested. The low biodegradability of this EPS was also found by Ruijssenaars, Stingele, and Hartmans (2000), who studied the susceptibility of several EPSs produced by LAB to biological breakdown. The biodegradability of EPSs was tested in vitro in a medium inoculated with a faecal slurry ltrate under anaerobic conditions at 371C. EPSs produced by S. thermophilus SFi39 and SFi12 were degraded by gastrointestinal microorganisms, in contrast to EPSs from L. lactis spp. cremoris B40, Lb. sake 01, S. thermophilus SFi20 and

Antitumoral activiy

Fig. 1. Schematic representation of the possible health-promoting properties of exopolysaccharides produced by lactic acid bacteria.

tumors in mice, but the LAB strain did not exhibit cytotoxicity in in vitro studies against S-180 tumor cells. This suggests that the effectiveness of this strain in preventing tumor proliferation was mediated through immune activity. These authors postulate that the slime material produced by L. lactis subsp. cremoris KVS 20 may be the principal component in the antitumoral effect. A later study (Kitazawa, Yamaguchi, & Itoh, 1992) showed a signicant increase of the B-celldependent mitogenic activity induced by the slime material products from L. lactis subsp. cremoris KVS 20. Also Nakajima, Toba, and Toyoda (1995) found that EPS of L. lactis subsp. cremoris SBT 0495 administered intraperitoneally enhanced the production of specic antibodies in mice, indicating that this EPS may act as adjuvant. The yoghurt starter Lb. delbrueckii subsp. bulgaricus OLL 1073R-1, which produces an EPS, has been reported to exert a host-mediated antitumor activity (Kitazawa et al., 1998). The isolated EPS was fractionated into two components: a neutral polysaccharide and an acidic polysaccharide (APS). It was found that the APS-1073 fraction was a phosphopolysaccharide with effective mitogenic activity mainly for murine B-lymphocytes and proved that the phosphate group acted as a trigger of the mitogenic induction of this EPS. Extracellular polysaccharides produced by LAB enhance other immunological functions such as ! n, Heiska, Herva, proliferation of T-lymphocytes (Forse & Arvilommi, 1987), macrophage activation and induction of cytokine (interferon-g and interleukin-1a) production (Kitazawa, Itoh, Tomioka, Mizugaki, & Yamaguchi, 1996). All the studies referred to have been done in vitro or by injecting the EPS material into mice. But very scarce experiments have been done in vivo by oral administration. The water-soluble EPS (KGF-C) from ker grains was shown to have the

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Lb. helveticus Lh59 strains. The authors tried to relate the susceptibility to biological degradation directly to the primary structure of the EPSs. The EPSs SFi39 and SFi12 had a single b-galactosyl residue as side chain whereas EPS B40 and EPS from L. sake 01 contain two residues, one charged (phosphate group) and one uncharged. These substituents may make the EPS molecule less accessible to degradative enzymes. In fact, van Casteren, Dijkema, Schols, Beldman, and Voragen (1998) tested several enzyme preparations against B40 EPS and showed no activity. Only the crude cellulase preparation from Trichoderma viridae had activity after previous removal, under acid conditions, of the galactosylphosphate residue. To employ EPSs produced by LAB as potential prebiotics more studies on degradability, specicity of breakdown by benecial colonic bacteria and healthy effect in human hosts are required.

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5. Concluding remarks LAB produce a great variety of EPSs with different chemical composition and structure, thus providing useful functional properties in food systems. The use of EPS-producer ropy strains is common in the manufacture of fermented milks and yoghurt in order to obtain a proper consistency. The production in situ of these biopolymers is employed as an alternative to the addition of stabiliser from plants or of non-GRAS microbial origin, additives that are not allowed in several countries. Also, starters producing EPS have been employed for the production of low-fat Mozzarella cheese to enhance moisture retention (Bhaskaracharya & Shah, 2000). Due to the low amount of EPS produced by LAB the use of these substances as food-grade additives is still limited. But the selection of strains and optimisation of fermentation conditions, together with molecular biology and metabolic engineering of EPS synthesis, might allow the construction of modied strains producing higher levels of EPS or new biopolymers. Finally, knowledge of the relationship between EPS composition and its physical and health-promoting properties is essential to increase the range of biopolymers with desirable functions.

Acknowledgements The authors are grateful to M. Kanning, R. Tuinier, H. Snel and C. G. de Kruif at NIZO Food Research for critical reading of the manuscript. P. Ruas-Madiedo also acknowledges the Spanish Ministry of Education and Culture and the European Commission for their postdoctoral fellowships.

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