FEMS MicrobiologyReviews87 ~1990)113-130 Publishedby Elsevier

113

FEMSRE00164

ExoceUular polysaccharides produced by lactic acid bacteria Jutta Cerning Station de RecherchesLaiti~res, CRJ, INRA Jouy-en-Josas,France

Key words: Heteropolysaccharides; Dextrans; Mutans; Thermophilic lactic acid bacteria; Mesophilic lactic acid bact~:~ ~; Mutans streptococci; Leuconostoc mesenteroides

1. SUMMARY

2. INTRODUCTION

The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined. Mutans streptococci, which include Streptococcu~ mutans and S. sobrinus produce soluble and insoluble a-glucans. The latter may contain as much as 90% a - l - 3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque. Dextrans produced by Leuconostoc mesenteroides are high molecular weight a-glucans having 1-6, 1-4 and 1-3 linkages, varying from slightly to highly branched; 1-6 linkages are predominant. Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry. The produced polymers play a key role in the rheological behaviour and the texture of fermented milks. One of the main problems in this field is the transitory nature of the thickening trait. This instability is not yet completely understood. Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases.

The bacterial cell may synthesize a number of polysaccharides which are defined by their location relative to the cell. Some are intracellularly located in the cytosol and used as carbon or energy sources, others are cell wall constituents such as peptidoglycan and teichoic acids and a third group is located outside the cell wall. The latter group occurs in two basic forms, either as a capsule intimately associated with the cell surface, or secreted into the environment. In some cases, both capsular and unattached polysaccharides are produced by the same microbe. Distinguishing between the two forms can be difficult. These polymers are the subject of the present article. Depending on their structural relationship to the bacterial cell, they have been variously named slime, capsular or microcapsular polysaccharides. The name exopolysaccharides (EPS) as proposed by Sutherland [1] provides a general term for all these forms of bacterial polysaccharides found outside the cell wall and will be used in this article. The aim of this review is to summarize information available o n polymers produced by lactic acid bacteria. However, several thousands of papers have been devoted to EPS and for those who wish more detailed information than is pre-. seated in this paper, reference to reviews and monographs is included [1-9]. Most of the examples chosen here are based on those with which the reviewer has experience in her own laboratory

Correspondence to: Jutta Cerning, Station de Recherches

Laiti~resCRJ, INRA 78350, Jouy-en.Josas,France

0168-6445/90/$03.50 © 1990 Federationof European MicrobiologicalSocieties

114 and on those that in her view have a considerable importance in the rheolo~cal behaviour of fermented milks. Essentially three groups of EPS formed by lactic acid bacteria are the focus of this review. Dextrans produced by Leuconostoc mesenteroides and mutans formed by "mutat~" streptococci which are a-giucans, levans produced by Streptococcus salivarius which are fructans and a large .heterogeneous group of EPS produced by mesophilic and thermophilic lactic acid bacteria. Probably no other polymer has been so widely studied as dextrans, clinical research characterizing mutans abounds, while the interest in studying polysaccharides produced by mesophilic and thermophilic lactic acid bacteria lies principally in the role which they play in the rheological behaviour and the texture of fermented milks.

3. PRODUCTION Most mucoid bacteria produce EPS under all cultural conditions, but the growth conditions must be optimized for maximal production. Polysaccharide production is normally highest under aerobic conditions. Therefore, more polymer is usually excreted during growth on solid media than obtained from comparable amounts of cells grown in liquid media. The composition of most EPS found in bacterial slime or capsule appears to be independent of the carbon and energy source provided for growth and polymer synthesis [1]. Synthesis of exocellular homopolysaceharides such as levans, mutans and dextrans requires a specific substrate, usually sucrose, reflecting the involvement of highly specific enzymes acting on oligosaccharide carbon sources. EPS production in defined media can be stimulated by limiting nutrients, such as nitrogen and by providing excess carbohydrate. The limitation of carbon and energy sources results in minimal EPS production. Deficiency of nitrogen, phosphorous or sulphur sources in the presence of carbohydrates leads to increased polymer production in Escherichia coll. A high carbon/nitrogen ratio in the medium favours EPS production in some mucoid strains. Dextrans and mutans which are synthesized at

or outside the cell surface are easy to isolate in the laboratory. Traditionally, dextran production is accomplished by growing cultures of Leuconostoc mesenteroides in sugar solutions fortified with suitable nutrients, growth stimulants, buffer salts and minerals, at or near neutral pH. The dextran is harvested from the fermentation medium by two ethanol precipitations. Although no further process control is required, fermentation parameters such as temperature, pH, and initial sucrose concentration affect the conversion rate and dextran production [8]. The disadvantages of conventional dextran production are the time and energy required to propagate cells, and the synthesis of dextran under constantly changing fermentation conditio- " resulting in less than optimal dextral production, xr~ the 1950s the use of a cell-free enzyme solution permitted dextran synthesis under controlled conditions yielding a polymer of greater purity. Koepsrell and Tsuchiya [10] first established the conditions for production of dextransucrase and these were further described by Jeanes [11]. It is possible to obtain eight times more dextansuerase activity with the improved system. Addition of certain sugars can promote formation of short chain oligosaccharides at the expense of high molecular weight dextrans. This reaction was used to modify dextran synthesis using oligosaccharides and low molecular weight dextrans as starters or primers in cell-free enzyme liquor/sucrose reactions. For making pharmaceutical and industrial dextran products, native dextran is employed as starting material, which is then submitted to acid or enzymatic hydrolysis, solvent fractiunation procedures, or ultrasonic treatment. The latter treatment breaks down the dextran molecule preferentially at its midpoint. There are some significant differences in the quantities and chemical nature of the extracellular glucans synthesized by various serotypes of S. mutans [12]. $. sobrinus (type d) produces higher amounts of glucan than type c and the ratio of insoluble to soluble glucans is higher with the type d strain. The difference is explained by the presence of the higher proportions of a - l - 3 linkages in the type of glucans. Glucans produced by S. mutans (serotype a to g) after incubation with

115 cell-free GTase and sucrose can be separated further into one water-insoluble and three soluble fractions. Each fraction possesses a different content of a - l - 3 glucosidic linkage, a different molecular weight and a different reactivity with concanavalin A or S. mutans cells. The different methods of production (temperature, time) to obtain a maximum of viscosity or slime production by mesophilic and thermophilic lactic acid bacteria are mostly assessed by visual observations. Viscosity, consistency, resistance to flow and formation of stringiness, long ropy strands during fall from a pipette tip are used as test criteria. Colonies are scored for ropiness by picking with an inoculation loop. Ropy colonies (top +) can be easily distinguished from non-ropy colonies (rop-) by the occurrence of long ropy filaments [13]. Only recently have attempts been made to quantify more precisely the polysaccharide production responsible for the physical or visual observations. The instability of the slime-producing trait in thermophilic and mesophilic lactic acid bacteria has been reported by numerous investigators, who observed that growth of certain strains of Lactococcus increased the viscosity of whey but that the culture gradually lost this property when frequently transferred. Also, the organism lost the property of slime production more qu/cHy at high temperatures. This spontaneous loss of slime-producing ability by lactic acid bacteria has been related to the involvement of plasmid-encoded genes. This is true for mesophilic lactic acid bacteria [13,14], but does not seem to be true for EPS-producing thermophilic lactic acid bacteria, which often do not contain plasmids ([13]; M. Veaux, unpublished results). Whether the transitory nature of the ropy character is related to the presence of glycohydrolases capable of degrading the polymer is unclear at this time. Viscosity measurements which are used as an indication of EPS production are difficult to interpret, particularly for non-Newtunian solutions. Viscosity in liquid media may not only be affected by the amount of EPS released, but also by the type of polymer and the effect of other metabolic products which are excreted into the medium. Therefore, an organism might be producing the same quantity of slime but

with a slightly different structure resulting in different rheological characteristics of the medium. Metabolic products, such as lactic acid, may also influence the rheological behaviour of fermented milks and interactions might take place in the culture medium between polysaccharides and other milk constituents, i.e. casein [15]. Bouillanne and Desmazeaud [16] attempted to categorize strains of $. salivarius ssp. thermophilus used in yoghurt manufacture according to their viscosifying characteristics. They concluded that slime-producing strains were not easily categorized, and that the viscous trait could not be linked to acid production, proteolytic ability or acetaldehyde synthesis. The relationship between EPS production and viscosity measurements is often complicated by the decrease of viscosity upon prolonged incubation. Macura and Townsley [17] found a decrease of viscosity after 24 h of cell growth, but the decrease varied with the strain of ropy bacteria. .This suggests that a degrading agent, possibly a glucohydrolase, is destroying slime progressively. Similar observations have been made with strains of S. salivarius ssp. thermophilus [18] and with Lb. hilgardii (M. Pidoux, unpublished results). Slime-producing strains are also used in the manufacture of kefir [19]. Viscous Lb. hilgardii and Leuconostoc sp. have been found in the kefir grains. Slime production by these strains is believed to ~ responsible for embedding the cells into the grains, which is necessary as the grains are recovered, dried and re-used for many successive inoculations. According to ScheUhaass [20], all ropy mesophilic and thermophih'c lactic acid bacteria strains are able to grow and produce slime in milk or milk dialysate enriched with different nutrients, as indicated by an increase in relative viscosity of the media. She concluded that neither growth nor slime production seemed to be linked specifically to the presence of casein or whey proteins. It is difficult to interpret these results, because no quantitative determination of polymer production has been performed. We found in our laboratory that it is indeed possible to obtain EPS production on milk uitrafiltrate enriched with amino acids, but the amounts produced are low.compared to

116

those in milk cultures. The same strain of Lb. delbrueckii ssp. bulgaricus capable of forming 250 mg EPS/1 produced only 50 mg/1 in an ultrafdtrate culture medium. A strain of S. salivarius ssp. thermophilus formed only 20 m g / l EPS on milk ultrafiltrate, while as much as 350 mg/1 had been found in milk [21]. In contrast to non-ropy strains of Lb. delbrueckii ssp. lmlgaricus, ~,,hich produce little if any exocellular polysaccharides, non-ropy strains of $. salivarius ssp. thermophilus produce on average 50 mg of EPS per liter. The amounts of polymer formed by ropy strains of both species vary considerably even under the same experimental conditions. Some strains eventually even lose their repy character but may recover it after several transfers. Addition of 15 C~Lsein to non-fat dry milk is also efficient in some cases, bat *.he stimulation is more pronounced w~th Lb. delbrueckii ssp. bulgaricus than with S. salivarius ssp. thermophilus [21]. When a ropy strain of S. salivarius ssp. thermophilus is grown in association with a nonropy strain of Lb. delbrueckii ssp. bulgaricus in milk, EPS production can reach quantities of almost 800 mg/l. Spoilage lactic acid bacteria of the genu~ Pediococcus found in wine and beer manufacture can produce small amounts of/3-glucan, which is very undesirable and makes the product unsuitable for consumption. The quantity produced varies from 50 to 100 mg/1 when the medium contains glucose, and 5 to 10 mg/1 in the presence of various sugars [22]. It was shown recently [23] that two different homofermentative lactobacilli and a Leuconostoc strain produced slime on vacuum.packed cooked sausage even in the absence of added sugar. Again the endproduct becomes unsuitable for consumption.

4. PROPERTIES

4.1. Isolation Isolation of EPS generally presents few problems when R is secreted as an exocellular slime [1]. The lack of physical attachment between polysac.

charide and cell permits the use of differential centrifugation to be employed. The main problem in such preparations is the high viscosity of the slime solutions which hinders deposition of the cells. Different slime preparations vary in their intrinsic viscosity and there are no general rules for centrifugal speeds necessary to obtain adequate separation. Addition of electrolytes (salts) may be useful in the precipitation by neutralizing charges on the polysaccharide. If the polysaccharide is in capsule form it must be detached from the cells. Again it is difficult to generalize as capsules are much more readily removed from some strains than from others. Gentle stirring or mixing in a homogenizer may suffice, but sometimes more drastic procedures may have to be employed. Depending on the culture medium used or methods employed, the isolation of a polymer contalnin S various contaminations and some degradation may also occur. Dextrans and mutans produced in cell-frce culture medium are usually obtained after centrifugation and repeated ethanol precipitation. The kefir polymer is obtained in the same manner [19,24]. Mechanical isolation and subsequent purification of a polymer material is difficult and tedious, when the slime-producing microorganisms such as thermophilic and mesophilic lactic acid bacteria .are grown on a coagulated milk system. The slime is found together with and surrounding the microorganisms that have produced it and from which it can hardly be entirely separated. Furthermore, it is mixed with different carbohydrates and protein material from the milk itself.

4.2. Purification In general, purification of EPS, i.e. removal of extraneous matter such as proteins and nucleic acids, is a difficult matter owing to the high viscosity of most polymers. The deproteinization technique of Seva8 [25] is still used successfully, provided that dilute (0.5% w/v) solutions of EPS are used. Pretreatment with enzymes, such as deoxyribonuclease, ribonuclease, trypsin, pronase and proteinase K result in isolation and improved purity of the polysaecharide. It is possible to obtain a carbohydrate fraction as pure as 95% by

117 repeat¢.d ethanol precipitation when isolating dextrans, mutans and kefirans from cell-free culture m~ainm. When more complex culture media (milk, milk ultrafiltrate, whey), are employed, isolation and purification of the EPS is much more difficult. The crude preparation of polysaccharides obtained from a whey culture with Lb. helveticus vat. "'yoghurti" "~6] was purified by DEAE ceihdose column chromatography with a 92.5~ yield. Small amounts of proteins contaminating the polymer were removed by a second DEAE step and CM cellulose colunm chromatography. The exopolysaccharide fractions produced by different strains of Lb. delbrueckii ssp. bulgaricus [27] and S. salivarius ssp. thermophilus [28] were purified in a similar manner. Ethanol precipitated slime recovered from a whey protein culture of Lc. lactis ssp. cremoris [17] ~as TCA extracted and yielded a crude preparation containing 47~ protein. Ethanol precipitated polysaccharides from milk culture containing S. salivarius ssp. thermophilus were purified by ion exchange chromatography (Dowex) followed by gel trdtration on Sephacryl S-1000 [28]. 4. 3. Composition and structure 4.3.1. Homopolysaccharides Dextrans and mutans are a-giucans composed of a-D-glycopyranosyl residues. The main dextran synthesizing bacteria belong to the genus Leuconostoc, species mesenteroides and dextranicum. Mutans are produced by "mutans" streptococci, which include S. mutans and S. sobrinus. Although each bacterial strain produces a unique D-giucan, a common feature to all dextrans is the preponderance of a-1-6 linkages with some branch points at position 2, 3 or 4. Mutans differ from dextrans in containing a high percentage of a - l - 3 linkages; often a water-soluble fraction rich in 1-6 linkages and a water-insoluble fraction rich in 1-3 linkages are found. Differences in solubility and physical characteristics are due to the proportions and types of linkages and how these are arranged in each a-giucan molecule. Dextrans are generally identified by a code number of the corresponding bacterial strain and

much of the recent work has been conducted with the strain L. mesenteroides N R R L B-512F. Most dextrans have high molecular weights, but chain lengths show considerable variation. Some values, obtained by light scattering or ultracentrifugation measurements and ranging from 80 to 600 million Da [29,30] were questioned by Ebert [31] who stated that the over-estimating was due to the presen~ of very stable aggregates in aqueous solutions. This was confirmed recently [32,33] in studies on the effects of synthesis temperature on the structure of dextran NRRL B-512F. Light scattering measurements revealed that the average molecular weight of non-associated molecules is almost independent of the synthesis temperature and indicated much smaller values than all those reported earlier in the literature (6.2-7.1 × 106). Furthermore, the work demonstrated that branching increases with synthesis temperature. As a result, confor,3ation of dextran changes from an expanded random coil type if synthesis temperature is reiatively low (5°C), to a compact solid sphere ~ype if synthesized at 30°C, with dextran produced at 25°C showing intermediate characteristics. Seymour and co-workers [34--38] described the structural analysis of a number of dextraus, including those produced by Leuconostoc mesente~ roides B-742. This strain produces two exocellular dextrans, namely, fraction L, which consists of an a-D-(.1-6) backbone with a-D-(1-4) branch-poiLts, and a fraction S, which consists of an a-D-(1-6) backbone with a-D-(1-3) branch-points. According to their results, 13-742 dexu~an fraction consists of a linear chain of a-D-(1-6)-iinked D-glucopyranosyl residues, each bearing a single a-D-giUcopyranosyl group linked to 0-3, to give a comblike structure. Nearly all of the D-glucoSyi residues in the backbone chain could be substituted in this way, with approximately one in 10 or 20, lacking a D-glucosyl group in 0-3. It was assumed that such a dextran would be completely resistant to hydrolysis by most endodextranases. Cbt6 and Robyt [39] found that B-742 S dextran may not always be so highly branched. Native dextrans were hydrolysed enzymatically with various endo- and exodextranases to elucidate more accurately structural differences and

118

their degree of branching [40-42]. Kobayashi and Matsuda [43] demonstrated that the glucoamylase from Rhizopus niveus can be used to remove Dglucose residues from the non-reducing ends of dextran, its action being stopped at the branch points, thus giving information on the side chain length. The maximum percentage of hydrolysis decreased when the temperature of dextran synthesis increased. The percentage of hydrolysis (28.4) obtained with dextran 25 is very close to those reported by several authors (30, 32, 295, respectively) for native dextran NRRL B512F. The percentage of 1-3 bonds was 3.4~ and 4.8~ for dextran 5 and dextran 30, respectively [32]. It is, however, not easy to interpret enzymatic studies. The interpretation of such studies is based on the assumption of a certain type of branching and conclusions from physical measurements (intrinsic viscosity) are not always in agreement with a change in the fimiting percentage of hydrolysis. The question remains as to whether dextrans have a laminated, comb or ramified structure. Since a considerable part of the branches seems to consist of single a-v-glucosylpyranosyl groups, the comblike structure was believed to be the most probable [9]. Recently however Sabatie et al. [33] stated that the comblike structure was too simplistic and believed that there was stronger evidence for a ramified structure. Hamada and Slade [12] and Loesche [44] ,~omprehensively reviewed many aspects of mutans elaborated by Streptococcus species. Water-insoluble gincan from S. mutans strain OMZ176 contains as much as 90~ a - l - 3 linkages. In contrast to the insoluble mutan from S. mutans, gelatinous glucan from S. sanguis strain 84 has equal amounts of a-1-3 and a-1-6 glucosidic linkages. Ample evidence has accumulated supporting the original findings by Guggenheim [45] regarding the chemical structure of water-insoluble mutans from various strains of S. mutans as well as other oral streptococcal species. The large proportions of a - l - 3 glucosidic linkages found in the insoluble glucan explain the insoluble nature of this polymer. Smith degradation and methylation analysis have shown that the consecutive a-1-3 glucosidic linkage form long chains and the b~ckbone of a highly branched insoluble glucan [12]. However,

the insoluble mutan, extracted in an alkaline solution and ethanol precipitated, can be separated into structurally different water-soluble and insoluble fractions. The latter possesses more a - l - 3 linkages than does the former and is more resistant to the enzymic action of a-1-6 glucanase i.e. dextranase. S. salivarius strains (ATCC 9759, ATCC 13419) synthesize fructans of the levan typo with 2-61inked/~-fructofuranoside residues branched in the 1 position (15-175) and of high molecular weight (2.7 × 106-21.6 × 106). S. mutans strain JC2 produces a fructan (12.4 × 106) of the inulin type with 1-2-linked/~-fructofuranosidose residues and some branching in the 6 position (65) [12]. Soluble mutans isolated from S. mutans strains JC2, FA and Inghritt and S. sanguis strmn 903 were predominantly 1-6-1inked and to a lesser extent 1-3-linked (9-125) with branching in the 3 and 6 position (19-235). High (12.4 >: 106) and low molecular weight glucans (129000-136000) were found. It was not established if the a - l - 3 linkages were part of the main chains, or if two types of glucan synthesizing enzymes were present in S. mutans and $. sanguis, one synthesizing a-l-6-linked (soluble) and the other a-l-3-1inked (insoluble) polymer [46-50]. In the last 10 years knowledge of enzymes involved in mutan synthesis has increased and it is clear now that several glucosyltransferases are produced by mutans streptococci. These chemical differences in the various polysaccharides reflect the complexity of exocellular polysaccharide synthesis from sucrose. Many factors, including the type of growth media, incubation time, sucrose concentration and the presence of polysaccharide-degrading enzymes probably influence the molecular weight and structure of these polymers. 4.3.2. HeteropoO,saccharides

Exocellular polysaccharides produced by thermophilic and mesophific l~ctic acid bacteria have been investigated to a much lesser extent, compared to other polymers. The common~ feature of these strains is the low EPS production [27,28], and the transitory nature o f the slime producing trait. Oroux [51] investigated the composition of

119 slime secreted by a strain of Lb. delbrueckii ssp. bulgaricus and reported that galactose was the main constituent of the EPS, with some arabinose, mannose and glucose also present. The polymer had a low specific optical rotation, indicating the existence of t-linkages. Periodate oxidation and Smith degradation also revealed a - l - 6 linkages. Some linkages were resistant to periodate oxidation suggesting 1-3,6 branch points or/]-1-3 linear linkages. The polysaccharide recovered from a yoghurt starter culture was reported as an a-glucan by Tamime [52], as glucose was the only monomer detected by gas chromatography. Oda et al. [26] investigated the chemical composition of EPS produced by Lb. heiveticus, var."joghurti", which was only composed of glucose and galactose monomers in a ratio of 2:1. The polymer had a high molecular weight and showed anti-tumour activity. Manca de Nadra et al. [53] obtained a polymer on synthetic medium produced by Lb. delbrueckii ssp. bulgaricus and composed of glucose and fructose in a ratio of 1 : 1. The polymer recovered by Marshall [54] from yoghu~ contained galactose, glucose, xylose and uronic acid residues. Schellhaass [20] isolated EPS from ropy strains of S. salivarius ssp. thermophilus, Lb. delbrueckii ssp. bulgaricus, and Lc. lactis ssp. c~'emoris, cultured on milk ultrafiltrate to which casamino acids had been added. The polymer material isolated from each of the ropy strains had a similar chemical composition consisting of 2~ moisture, 0.09~ ash, 0.3~ nitrogen and 85~ carbohydrate. Reducing sugar concentrations (reducing equivalent) were different for the slime isolated from each of the strains. This could indicate that the strains produce EPS of differing length. However, different batches of slime produced by the same strain also showed variable reducing equivalents contents. These findings indicate that the size of the EPS produced by a ropy strain can vary. Sutherland [2] postulated that the molecular weight of the EPS produced by bacteria is affected by the growth rate of the culture. Polymers isolated by Schellhaass from mesophilic and thermophilic lactic acid bacteria had similar sugar compositions [20]. They were composed of galactose and glucose in the ratio of 2 : 1, and the existence of a and fl linkages within the

polysaccharide was observed. No uronic acids, deoxy and 3,6-dideoxysugars nor aminosugars were identified. 1-6 Linkages were absent, but the existence of 2 and 1 linkages is suggested. The protein content of the EPS studied was very low and similar for all mesophilic and thennophilic lactic acid bacteria. Macura and Townsley [17], suggested a glycoprotein nature of the slime produced by mesophilic lactic acid bacteria. Ceming et al. [27] showed that ropy strains of Lb. delbrueckii ssp. bulgaricus produce a viscosifying exocellular water-soluble heteropolysaccharide composed of galactose, glucose and rhamnose in an approximate molar ratio of 4 : 1 : 1. The molecular weight of the polymer was about 500000 Da, and the intrinsic viscosity was 4.7 dl/g, indicating that the polymer had remarkable thickening properties. It was shown [18] that ropy s~:alns of S. salivarius ssp. thermophilu.~ grown on skim milk also produced exocellular polysaecharide, essentially composed of galactose and glucose. Small amounts of xylose, arabinose, rhamnose and mannose were also identified. Gel filtration chromatography on CL Sepharose 4B showed two fractions, the first had a molecular weight close to blue dextran (2 × 106), the second, 35000 Da. The latter was possibly a degradation product. Also, the latter fraction was much more resistant to acid hydrolysis than the fraction of high molecular weight. However, the sugar composition, as determined by HPTLC, appeared to be the same, i.e. all monomers identified in the total EPS were present in the two fractions (J. Ceruing, unpublished results). Doco et al. [28] determined structural elements of an EPS produced on skim milk after only 3-4 h of incubation. They obtained a polymer composed of galactose, glucose and N-acetylgalactosamine in a ratio of 2 : 1 : 1 , the molecular weight was 1.106 Da and the intrinsic viscosity 1.54 di/g. There is tittle agreement (Table 1) as to the chemical composition of EPS isolated from thermol~hilic lactic acid bacteria cultures by various authors. However, in most cases, galactose and glucose are the most frequent monomers released after acid hydrolysis. Galactose seems to be the major monomer in most cases, probably because glucose is mainly metabolized after lactose hydro-

120

Table 1 Examples of polysaccharidecomposition Microorganism

Monomersin EPS Gala Gluc

Fru

Lb. delbrueckii

+

+

-

ssp. bu/gar/cus

+ + +

+ + + +

. + .

+

+

-

+

+

.

+

+

.

. .

S. salivarius ssp. thermophilus Lb. delbrueckii ssp. bulgaricus S. salivarius ssp. thermophilus Lc. lactis ssp. .'remori$

Reference

+

+ +

. -

+

+

-

+ +

+ +

.

Rha

Man

-

+

.

. . +

.

.

.

.

.

tr

. .

51 26 53 27 20

-

-

+

-

18 28 20

-

+ -

. . tr

. .

-

.

tr

.

. .

-

.

.

+ -

. .

Neu

.

.

.

+

. .

GalA .

.

tr

.

.

tr .

.

+

.

. .

.

tr b

+

. .

.

Ara

-

. .

. .

Xyl

. .

. .

52 54 18 55 20

= Gal, galactose; Gluc, glucose; Fru. fnmtose; Rha. rhamnose; Man. mannose; Xyl, xylose; Ara, arabinose; GalA; galactosamine; Neu, neuramic acid b Tr, traces

lysis while galactose is excreted into the medium a n d is available for polymer synthesis [54]. M i n o r differences may b e explained by different growth conditions and isolation and purification procedures. The gel-forming polysaccharide of the sugary kefir grains (11.5~ of dry matter) or one taken from a Lb. h i l g a r d i i culture were identified as dextrans with some 1 - 3 glucose linkages in the main chain, with a ratio of b r a n c h e d / t o t a l units of 0.19 a n d 0.14, respectively, instead of 0.07 for the non-gelling polysaccharide [19]. Romaskaya a n d D y m e n t [55] showed that viscous extraceUular polymers formed b y Lc. l a c t i s ssp. l a c t i s , L c . l a c t i s s s p . c r e m o r i s a n d L c . l a c t i s ssp. l a c t i s v a r . d i a c e t y l a c t i s were carbohydrate-protein complexes. The carbohydrate polymer included glucose, galactose and rhamnose. Macura a n d Townsley [17] isolated the viscous nmterial produced b y a strain of Lc. l a c t i s ssp. c r e m o r i s and proposed that it was most likely a glycoprotein. The material isolated contained protein-bound hexose.like components, methyl pentoses a n d sialic acid. N o aminosugars were found and the amino acid profile was very similar to that of deproteinized whey

in which the culture was grown. This may suggest whey protein c o n t a m i n a t i o n of the sample. The slime material isolated from vacuumpacked cooked meat products h a d a molecular weight in the range of 3 0 0 0 0 - 7 0 0 0 0 D a a n d contained glucose a n d galactose in a ratio of 1 0 : 1 1 0 : 2 [23]. Lactic acid bacteria of the genus P e d i o c o c c u s [22] found in beer and wine manufacture produce fl-glucans of high molecular weight (higher than 2 × 10 6) with fl-1-3 a n d fl-1-2 linkages 4.4. F u n c t i o n

and use

4. 4.1. G e n e r a l b a c k g r o u n d

Historically the research involving bacterial EPS has been mainly of medical origin. Most of the earfier studies were those of the immunologist studying haptens a n d antigens present at the bacterial surface a n d their role in pathogenicity. Capsule production per se is not sufficient to ensure virulence in bacterial species. Evidence from early studies proved that m a n y harmless bacteria including those found in soil or the aquatic environment are heavily encapsulated. The role of

EPS has not been clearly established, and it is probably diverse and complex. It has been suggested that EPS may play a role in protecting the cell against desiccating, phagoeytosis and phage attack, providing higher oxygen tension, partieipating in uptake of metal ions, functioning as adhesive agents, interactions between plants and bacteria, and in development systems such as those found in myxobacteria. Exocellular polysaceharides do not appear to function as energy sources, since slime-forming bacteria are usually not capable of catabolizing the polymer which they synthesize. Degradation of EPS produced by $. pyogenes [56] has been observed but it is not certain whether the organism utilizes the decomposed capsule. Unrelated organisms in mixed cultures can however use slime produced by various organisms [57]. The relationship between encapsulation and role in virulence has been studied and it has been proposed that the degree of hydrophobieity of the bacterial surface dictates the susceptibility to become engulfed by neutrophiles. Encapsulated organisms having a more hydrophih'e surface were shown less susceptible to phagoeytosis by human neutrophiles [57]. The same author proposed a similar hyphothesis to explain protection of soil bacteria against predation by amoeba and flagellated soil protozoa. Whether capsular polysaceharides provide protect.ion against physically adverse conditions or not has not been dearly demonstrated [57]. Slime formarion and adhesion to solid surfaces in various aqueous emdronments is well known, but the process of adsorption of bacteria to solid surfaces is a complex process involving many physical and chemical factors and is not completely understood

[2o1. Bacteriophage active on encapsulated strains are generally exopolysaceharide specific, non-encapsulated or non-slime forming producing mutans are resistant to phage. Lindberg [5g] proposed a model to explain the mechanism of adsorption of the phage to the capsule and subsequent injection of its nucleic acids. He suggested that the phage recognize and bind to the exopolysaccharide. The tail moves along the glycan strands supplying an opening for the phage head via endoglycosidase activity which is locate~l in the spikes of the base

plate. The nucleic acid injection is then triggered by another receptor on the host cell wall. This model would require at least two receptors, the enzyme-substrate reaction between the tail spike and the exopolysaccharide and a recognition receptor associated with the wall or membrane. Numerous investigators have shown that capsules are not necessary for cell viability. Capsules or slime can be removed physically or enzymatically without adverse effect on bacterial growth. Further incubation of these cultures led to synthesis of new polysaceharide. Also, mutants unable to form exopolysaccharide are readily isolated. They occur spontaneously or after mutagenesis [59,60]. Although loss of ability to produce polysaccharide may not cause any significant change in the viability in vitro, it could be important for survival under highly competitive conditions in a natural habitat. The functions of EPS in natural environments have been previously reviewed in detail [57,611.

4.4.2. Dental plaque

Mutans and fructans synthesized by S. mutans and S. salivarius--mutans in particular--are considered to be critically important in dental plaque formation and hence in the pathogenesis of dental caries because they are water-insoluble and possess a marked ability to promote adherence when synthesized de novo on various solid surfaces. Various mechanisms for bacterial colonization and plaque formation have been considered, such as acid precipitation and enzyme precipitation ot" salivary glycoproteins, non-selective or selective microbial adherence theories. Of these, the concept of selective adherence via specific ionic, hydrophobic and lectin-like interactions is best able to explain the colonization of the acquired enamel pellicle of teeth by S. sanguis and mutans streptococci [12,44]. In fact, mutan is able to adhere to the inert enamel surface of the tooth and thus attach the bacterial cell to the tooth. Since glycosyltransferase is present in the mutan network, mutan continues to be synthesized, entrapping more bacterial cells of the same or different species and building up the yellowish film known dent~ ~!aque

122

4.4.3. Endocarditis Isolation of a great number of streptococcal strains from patients with subacute endocarditis revealed that $. sanguis (16.4~), $. boris (15.1~) and $. mutans (14.2~) [12] are the most numerous. From 54 serotyped $. mutans strains, serotype c was the most prevalent. Isolates cultured from dental plaque have revealed that serotype c is the most frequently detected serotype irrespective of age, country, sampling site, or isolation and serotyping proee~lures. Serotype c usually comprises about 80~ of the total isolates. The initial event of the pathogenicity of bacterial endocarditis is the attachment of bacteria to heart valves, particularly those with damaged aortic valves possessing a platelet-fibrin thrombus. Cell-bound glucan appears to promote the establishment of S. mutans and other giucan-producing streptococci on the heart valves. Adherence to damaged valves is approximately five times higher than adherence to normal valves. These results may explain the high prevalence of glucan-synthesizing streptococci, including S. mutans as the causative agent of subacute endocarditis. 4.4.4. Uses of dextran The potential uses of native dextran include secondary recovery of petroleum from oil drilling muds, stabilization of soil aggregates, protective coating for seeds, deflocculants in paper products, metal plating processes, surgical sutures and in foods, stabilizing and imparting viscosity to syrups [8]. Low molecular weight dextrans have their biggest outlet in the pharmaceutical industry, where fractions of various molecular weights and molecular distributions have been used as blood plasma extenders and blood flow improvers. The original products proposed for this purpose had a molecular weight of 150000, but they were antigenic and may have caused side effects. Therefore, lower molecular weight dextrans are used, dextran 70 (70000 M w) as a blood volume expander and dextran 40 (40000 Mw) as a blood flow improver. Clinical dextrans of this type usually have to be manufactured from the NRRL 8512 strain of Leuconostoc mesenteroides. Dextran derivatives and activated dextrans find several commercial uses. Another successful application for dextran is its

use in the manufacture of molecular sieves. These are prepared by cross-linking dextran with epichlorhydrin in the presence of sodium hydt'oxide. The degree of cross-linking in these insoluble hydrophilic gels determines the water regain value and pore size and hence the molecular exclusion limit. 4.4.5. Function of EPS produced by thermophilic and mesophilic lactic acid bacteria Traditionally, yoghurt manufacturers try to improve the body and texture of the product by manipulation of one or more of the following factors: composition of the yoghurt mix, heat treatment of the mix prior to inoculation, starter culture and incubation conditions, handling of the ripened yoghurt, and addition of stabilizers. Stabilizers used in the manufacture of cultured milk products are hydrocolloids of animal or plant origin. They are added to improve consistency and viscosity as well as to hinder whey separation and to bind free water. However, the use of stabilizers is prohibited in France and the Netherlands. Over the last 30 years yoghurt has evolved into many variations. The traditional plain "cup-set" yoghurt now occupies a small fraction of the yoghurt market, which is dominated by stirred yoghurt in its various forms such as fruit yoghurt, flavoured yoghurt made with artificial flavours or sugars or drinking yoghurt with lower total solids. Stirred yoghurt loses part of its initial texture after the different mechanical steps of processing [62]. An alternative way to improve yoghurt texture and viscosity is to use slime-producing bacteria in starter cultures. The use of slime-producing strains of thermophilic lactic acid bacteria to increase the viscosity of yoghurt and decrease the susceptibility to syneresis has been advocated by many dairy researchers. In the Scandinavian countries slimeproducing mesophilic lactic acid bacteria are used in the manufacture of different fermented milks (Villi) and the group of Fors6n [59,60,63-65] has investigated this field. Stirred yoghurt, yoghurt drinks and low-fat yoghurts have become very popular within the last few years, and yoghurt without stabilizers has gained popularity in many countries, because of the increased desire of the consumer for "100~ real yoghurt" and for the

123 "natural" product. Furthermore, it has been claimed that exocellu!ar polysaccharides isolated from lactic acid bacteria cultures may have antitumour activity [26,28]. In the production of Finnish fermented milk, "Viili", the existence of a slime-forming variant of Lc. lactis ssp. cremor~s in the dairy starter culture is essential for proper consistency [66]. As it has been mentioned before, the ability to produce slime material is an unstable property. Growth temperature affects consistent slime production and growth at higher temperatures and prolonged storage without transfer enhances the loss of the mucoid character [17]. According to McKay [67], the effect of temperature on the appearance of non-slime-forming variants suggests that plasmid DNA may be associated with slime production. It has since been confirmed by several authors, that the ropy character of mesophilic lactic acid bacteria is plasmid-carried [13,14,68,69]. This does not seem to be true for ropy strains of thermc~ philic lactic acid bacteria, in which plasmids could not be identified [13,21]. 5. BIOSYNTHESIS AND ASSEMBLY Exocellular polysaccharides are synthesized in different growth phases and under a variety of conditions, depending on the organism studied. The process of synthesis involved can be divided into two principal categories based essentially on the site of synthesis and the nature of the precursors, i.e. synthesis outside the cell or at the cell membrane. Homopolysaccharides, such as dextrans and levans produced by Leuconostoc mesenteroides and mutans streptococci are synthesized outside the cell in the presence of a donor molecule, sucrose, and an acceptor: Sucrose + glucan (or fructan) acceptor --* dextran (or fructan, levan) + fructose (or glucose) Heteropolysaccharide synthesis differs from homopolysaccharide synthesis in that they are produced at the cytoplasmic membrane utilizing precursors formed intracellularly. Sugar nucleotides play an essential role in heteropolysaccharide

synthesis due to their role in sugar interconversions as well as sugar activation, which is necessary for monosaccharide polymerization. The involvement of isoprenoid glycosyl carrier fipids in exopolysaccharide formation has been demonstrated [1,70]. The mode of action of dextransucrase has been the subject of controversy for the last 30 years. Different factors including single or multi chain reaction, primer requirement, direction of chain growth, chain termination, acceptor mechanisms and branch linkages have to be considered [8]. In early studies it was suggested that dextran synthesis might be a multi chain reaction, but it was shown later that the single chain reaction mechanism operates in the synthesis of the skeletal dextran chain. Thus dextransucrase is a transglycosidase operating by transferring the glycosyl group of the donor (sucrose) to a suitable acceptor, i.e. the growing dextran chain. It was believed that dextran associated with the enzyme could prime the polymerization reaction, but it was shown later that sucrose can initiate its own polymerization and that primers are not necessary to initiate polymer growth. Theoretically, employing a single chain mechanism of skeletal chain growth, the structural polarity of dextran molecules allows growth to take place by glycosyl transfer to either end of the molecule. Partially purified dextransucrase preparations have been obtained from Leuconostoc, Streptococcus and Lactobaciilus species [71]. The degree of specificity of particular enzymes has not been completely established. Difficulties during purification arise from the tendency for dextransucrase and dextran to aggregate, and instability of the enzyme resulting in possible proteolytic modification. Cellular dextransucrase activity may be a mixture of enzymes, each specific for one of the hydroxyl groups undergoing substitution, a single enzyme with differing affinities for different hydroxyl positions or both [72]. Enzymes isolated from different bacterial strains produce polymers with various proportions of the possible linkages, and existing substituents or degree of polymerization affect the position of further substitution. Some radiochemical evidence indicates that extension occurs at the reducing end of the growing

124 polymer with the enzyme from Leuconostoc. This was concluded from the absence of released D(14C) glucose with exo-l,4-a-D-glucanase after a pulse of label and the isolation of D-(14C) glucitol on reduction and hydrolysis. However, the dimiitished dextran synthesis with dextransucrase from Leuconostoc mesenteroides in the presence of exo-l,4-a-D-glucanase was ascribed to competition between the exo-D-glucanase and dextransucrase for D-glycosyl groups and it was concluded that addition occurred at the non-reducing end of the growing dextran. Under different experimental conditions, the dextransucrase activity from S. mutans added D-glucose to the non-reducing end of isomaltosaccharides of DP-6 (DP-degree Cf polymerization) and formed an a-(1-3)-a-Dbranch linkage if the DP was above 6. Robyt and Walseth [73] investigated the acceptor reaction using (14C)-labelled acceptors. They prepared a dextran charged enzyme by reacting dextransucrase with sucrose and then separated this charged enzyme from fructose and sucrose on a Biogel P6 column. When the charged enzyme was incubated in turn with three acceptors (14C)glucose, (~4C)-fructose and (~4C)-maitc~, each reaction gave two labelled products of high and low molecular weight, which were separated on Biogel P6. Reduction and acid hydrolysis of the products showed that all the acceptors were incorporated into the products at the reducing end. These results showed that contrary to previous views the acceptor reactions occur in the absence of sucrose. Different mechanisms have been proposed to explain the formation of branch linkages in dextran. Early proposals suggested that the branch points were synthesized by means of a secondary branching enzyme believed to be a more thermally stable dextransucrase. The group of Cbt6 and Robyt [39,74,75] has described glucansucrases from different strains of Leuconostoc that are caPable of forming types of a- v- l- 3 linkages at the branch points and in a linear posit,ion. Leuconos. toc mesenteroides strain B 1355 was shown to preduce two separate dextrans ('termed "L" and "S" fractions) with 0Afferent physical characteristics. The "L" fraction is a typical dextran with 95% 1-6 linkages, the "S" fraction contains alternate 1-6 and 1-3 linkages. This fraction has been

named alternan and the enzyme which forms it from sucrose "alternansucrase" [74]. The percentage of a-D-l-3 branch points in fraction S glucan was variable, depending on the conditions under which it was synthesized from sucrose by the exoceHular glucansucrase, a-D-l-3 branch formarion by this enzyme occurs by an acceptor reaction in which a-D-glucosyl groups are transferred from sucrose to OH-3 groups on a-D-l-6 linked I~-glucan chains. Thus, any change in reaction conditions that affect the rate of acceptor reactions relative to chain elongation also affects the degree of branching in the S dextran. The isolated glucansucrase was capable of modifying other dextrans by transferring D-glucosyl groups on OH-3 of D-glUCOSYIresidues in these dextrans as well. Kobayashi and Matsuda [43] found that diminished dextran synthesis with dextransucrase from Leuconostoc mesenteroides in the presence of exo 1-4 a-glucanase was due to competition between the two enzymes for v-glucosyl groups and they concluded that addition occurred at the nonreducing end of growing dextran. Common features exist between dextran and mutan syl~thesis [71], i.e., the exocellular polysaccharide is synthesized by dextransucrase which polymerizes the glucose moiety of sucrose into water-sohible and water-insoluble polyglucans with the liberation of fructose. It should be noted that dextransucrase is also known as glucosyltransferase. It is understood that the mutan polymerization is cataiysed by two types of extracellular glucosyltransferases (GTF), one synthesizing a water-soluble product from sucrose (S) and another synthesizing a water-insoluble product from sucrose (I). These two enzymes, when combined, synthesize a complex highly branched water-insoluble glucan [12,76-78]. Mutan synthesis by GTF I of S. mutans requires the presence of a water-soluble glucan primer, a requirement which can be fulfilled by the product of GTF S. Two types of GTF S were identified [76,79-82], one required a primer dextran and formed highly brancl~ed water-sol.~ble products and the other .had no primer reqmrement and synthesized a water soluble product that was only slightly branched. Other reports of primer-independent GTF S [83,84] gave no indication that two distinct GTF S

125 enzymes might be produced by S. rnutans. It seems clear however, that at least two distinct GTF S are co-produced in S. mutans serotypes a, d and g [79,80,85], but not in other serotypes [82]. Three types of GTF, including a primer-independent GTF capable of forming a slightly branched water-soluble glucan (S) were described by McCabe [85]. The latter did not respond catalytically to the presence of either clinical dextran or the highly branched water-soluble glucan produced by primer-dependent GTF S. It was however capable of binding these polysaccharides at a non-catal~,tic site and responded to the low molecular weight acceptor 1-O-methyl-a-D-glucopyranoside. The water-sohible glucan product of primer-independent GTF S was a superior priming glucan for primer-independent GTF enzymes as compared with the glucan product of primer-dependent GTF S. When primer-independent GTF S was present in reaction mixtures, glucan synthesis was stimulated by primer-dependent GTF S and by GTF synthesizing water-insoluble glucans by at least 10-fold. The presence of similar amounts of primer-dependent GTF S had no effect on synthesis of water-insoluble glucan by GTF. Primer in'dependent GTF seems to be a potent source for l~ming glucan for the primer-dependent GTF em,,.ymes. The possession of a non-catalytic binding'~site for glucan was shown for the GTF of $. muta;ns, and this suggests that it may also serve as a gluten receptor on the S. mutans cell surface. The p~er-independent GTF isolated recently [86] from S.~ mutans strains was a 1,6-a-D-glucansynthetasL~ which produced oligo-isomaltosaccharides. "i~e enzyme is thought to be a donor source for the bra~ching enzv,ne of S. mutans. The cell-asso

Exocellular polysaccharides produced by lactic acid bacteria.

The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structur...
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