Exopolysaccharides of lactic acid bacteria: A review

J Food Sci Technol 2009, 46(1), 1–11

Exopolysaccharides of lactic acid bacteria: A review Behare PV*, Rameshwar Singh, Kumar M, Prajapati JB1, Singh RP Dairy Microbiology Division, National Dairy Research Institute, Karnal-132 001, India 1 SMC College of Dairy Science, Anand Agricultural University, Anand-388 110, India *E-mail: [email protected] Lactic acid bacteria (LAB) strains secrete extracellular polysaccharide either associated with bacterial cell wall or liberated into the medium. The term exopolysaccharide (EPS) is used to describe either type of extaracellular polysaccharide. EPS plays an important role in the improvement of physical properties of fermented milks, acting as a stabilizer, viscosifier, emulsifier or gelling agent thereby providing the product with natural thickness. Besides, EPS of LAB are thought to have beneficial effects on human health such as cholesterol-lowering ability, anticanceral, immunomodulating and antitumoral activities and prebiotic effect. Consumer’s demand for natural, healthy and low-calorie food has increased interest in the dairy industries for development and manufacture of low-fat/fat-free fermented milk products. But fat removal has several undesirable effects on technological properties of fermented milks leading to inferior flavour, texture and rheological properties that consequently hamper consumer acceptability. EPS producing LAB cultures are interesting which offer a natural and usually simplest way for making low-fat/fat-free fermented milks with more acceptable flavour and sensory attributes. The present review discusses the work reported on EPS producing LAB, factors affecting EPS production by LAB, types of EPS and their properties. Literature survey on application of EPS producing LAB in dairy product manufacture has also been presented. Keywords: Lactic acid bacteria, Exopolysaccharide, Homopolysaccharide, Heteropolysaccharide, Rheological properties, Fermented milk

Lactic acid bacteria (LAB) are widely used in the dairy and food industry since ages. They play crucial role in food fermentation process (Wood 1997). Apart from production of lactic acid, flavouring compounds and bacteriocin like substances, several strains of LAB secrete extracellular polysaccharide in favourable environment such as milk (Sikkema and Oba 1998, Cerning and Marshall 1999, Ricciardi and Clementi 2000). The term exopolysaccharide (EPS) is used to describe extracellular polysaccharide either attached as capsule with bacterial cell wall or liberated into the medium as ropy polysaccharide (Sutherland 1972). The EPS play an important role in the improvement of physical properties of fermented milks, which act like a food stabilizer, viscosifier, emulsifier or gelling agent providing a product with natural thickness (Ruas-Madiedo and ReyesGavilan 2005). Some of the examples of LAB EPS are dextran, mutan and fructan produced by Leuconostoc mesenteroides, Streptococcus mutans and Strep. salivarius subsp. thermophilus, respectively (Montiville et al 1978, Cerning 1990). The Gram negative bacteria, Xanthomonas campestris, Acetobacter xylinum and Sphingomonas paucimovilis also produce EPS xanthan, acetan and gellan, respectively that are commercially available as

food additives (Harvey and McNeil 1998). However, EPS extracted from Gram negative bacteria, although produced in larger quantities than EPS produced by food grade LAB, may not be preferred, as former is derived from non-food grade non-GRAS (Generally Recognized As Safe) status organism and had high cost involved in their recovery (De Vuyst et al 2001). Moreover, addition of purified EPS into the food product may not have similar effects as EPS produced in situ by LAB during milk fermentation (Doleyres et al 2005). The properties of EPS in purified form differ considerably from the properties of EPS produced in situ (Duboc and Mollet 2001), latter being a more desirable approach. The in situ EPS production may play useful role in the manufacture of a variety of cultured dairy products such as yoghurt, drinking yoghurt, cheese, cultured cream and milkbased dessert (Crescenzi 1995, Cerning 1995, Bouzar et al 1997, Christiansen et al 1999). In addition, certain EPS produced by LAB exhibit beneficial effects on human health such as cholesterol-lowering ability, anticarcinogenic, immunomodulating and antitumoral activities and prebiotic effects (Harris and Ferguson 1993, Cummings and Englyst 1995, Kitazawa et al 1998, Chabot et al 2001, Dal Bello et al 2001, Korakli et al 1

2002, Pigeon et al 2002). It has been speculated that the increased viscosity of EPS containing foods may increase the residence time of ingested fermented milk in the gastrointestinal tract, which helps in transient colonization by probiotic bacteria (German et al 1999). For this reason, the use of EPS producing strains as natural source of food bio-thickeners with added health benefits has received much attention in recent years. Consumers’ demand for natural, healthy and low-calorie food has increased interest in the dairy industries for development and manufacture of low-fat/fatfree fermented milk products. But fat removal has several undesirable effects on physical properties of fermented milks such as inferior flavour, texture and rheological properties that consequently hamper their acceptability. Several attempts were made including increase in milk solids and addition of stabilizers to tackle such problems (Rohm and Schimid 1993). However, these approaches did not address an increasing consumer demand for products with natural, low-cost and with as few food additives as possible. Furthermore, additives particularly, stabilizers are strictly prohibited in some fermented milk products like dahi (similar to yoghurt) in India and yoghurt in Norway by the stringent regulations. In this con-

J Food Sci Technol 2009, 46(1), 1–11 text, there is no alternative to use EPS producing lactic cultures, which offer a natural and usually acceptable way for making low-fat/fat-free fermented milks.

EPS producing LAB A large variety of LAB, with some strains of Bifidobacteria are reported to produce EPS (Abbad-Andaloussi et al 1995, Roberts et al 1995, Hosono et al 1997, Zotta et al 2008). Most of them belong to the genera of Lactococcus, Streptococcus, Lactobacillus, Leuconostoc and Pediococcus (Table 1). The use of polysaccharide producing lactic culture strains in the fermented milk manufacture is not new. The EPS producing LAB have

been traditionally used in the Scandinavian fermented milk products to impart desirable texture and rheological properties (Macura and Townsley 1984). The products made with ropy strains have smooth body, high viscosity and less syneresis than the products made with non-ropy strains (Wacher-Rodarte et al 1993). Often in the literature, to describe different EPS producing phenotypes, the term ropy, mucoid and slime have been interchangeably used. However, not all mucoid or slime producing cultures are ropy. The ropy colonies are able to form strand when touched with an inoculating loop, whereas, mucoid colonies have glis-

Table 1. Exopolysaccharide producing lactic acid bacterial strains L. lactis subsp. cremoris H414 L. lactis subsp. cremoris SBT0495 L. lactis subsp. cremoris B39 Strep. thermophilus CNCMI 733 Strep. thermophilus Sfi6 Strep. thermophilus Sfi12 Strep. thermophilus Sfi20 Strep. thermophilus Sts Strep. thermophilus S3 Strep. thermophilus MR-1C Strep. thermophilus SY 89, SY 102 Strep. macedophilus Sc136 Strep. thermophilus STD, CH101, LY03, ST 111 Lact. paracasei 34-1 Lact. acidophilus LMG9433 Lact. delbrueckii subsp. bulgaricus rr Lact. delbrueckii subsp. bulgaricus 291 Lact. helveticus Lb161 Lact. helveticus K16 Lact. rhamnosus C83 Lact. sakei 0-1 Leuc. mesenteroides MR15 Bifidobacterium longum ATCC 15707 Pediococcus pentosaceus AP-1

References Gruter et al (1992) Nakajima et al (1992) Van Casteren et al (2000) Doco et al (1990) Stingele et al (1996) Lemoine et al (1997) Navarini et al (2001) Faber et al (1998) Faber et al (2001b) Low et al (1998) Marshall et al (2001) Vincent et al (2001) De Vuyst et al (2003) Robijn et al (1996a) Robijn et al (1996b) Gruter et al (1993) Faber et al (2001a) Staaf et al (2000) Yang et al (2000) Vanhaverbeke et al (1998) Robijn et al (1995) Savadogo et al (2004) Abbad-Andaloussi et al (1995) Smitinont et al (1999)

tening and slimy appearance on agar plates and are not able to produce strands by this method (Vescovo et al 1989, Dierksen et al 1997). The EPS producing lactic bacteria are isolated from dairy and non-dairy environment using different media supplemented with one or more type of sugars (Table 2). The media used for isolation of EPS producing cultures are: liquid EPS selection medium (ESM) containing (g/l) 90 skim milk, 3.5 yeast extract, 3.5 peptone and 10 glucose (Van den Berg et al 1993), milk indicator agar and M17 lactose agar (Terzaghi and Sandine 1975), MRS with high concentration of sugars (100/g) (Van Geel-Schutten et al 1998) and milk agar (Mozzi et al 2001). Large numbers of ropy mesophilic lactococci were isolated from Swedish sour milk and Finnish villi on M17 medium (Neve et al 1988). Ropy colonies produced on media could be easily distinguished from nonropy colonies by forming long, ropy filaments with an inoculation loop. Gancel et al (1989) developed ruthenium red indicator agar, in particular to differentiate EPS and non-EPS producing Strep. thermophilus. The EPS producing strains formed white colonies on agar medium due to the presence of surrounding protective layer of EPS that prevents colorization of the cell, whereas, non-EPS producing strains formed pink colonies. The encapsulated strain of Strep. thermophilus was isolated from the commercial yoghurt by Ariga et al (1992). The ropy nature of the yoghurt could be attributed to ropy character of the strain, which shows large capsule surrounding the cell by staining with Indian ink. Micheli et al (1999) isolated a capsular polysaccharide producing strain, LM-17, from kefir grains and identified as a slime

Table 2. Isolation of exopolysaccharide (EPS) producing lactic acid bacteria from different sources Source of isolation Medium EPS producing isolates* Cheeses, dairy products Liquid ESM 30(4.9%) Non dairy fermented foods ESM with 50 g/l glucose 11 Fermented foods MRS with 100 g/l sucrose 60(33%) Thai fermented foods MRS with 20 g/l sucrose 7(6.7%) Nigerian fermented foods Modified ESM 25(16%) Burkino faso fermented milk MRS agar, Rogosa Agar, M17 Agar 13(26%) Dahi and naturally soured milk Milk agar 92(>90%) Human intestine MRS with 0.25% L-cysteine agar 362(17%) *Values in parentheses are the total percent of lactic cultures isolated

2

References Van den Berg et al (1993) Ludbrook et al (1997) Van Geel-Schutten et al (1998) Smitinont et al (1999) Sanni et al (2002) Savadogo et al (2004) Behare et al (2007) Ruas-Madiedo et al (2007)

J Food Sci Technol 2009, 46(1), 1–11 forming, rod shaped Lactobacillus. Thirteen EPS producing lactic isolates were reported from Burkino faso fermented milks (Savadogo et al 2004). Targeting spacer region between 16S and 23S rRNA genes, the isolates were named as Lb. delbrueckii, Lb. acidophilus, Lb. fermentum, Strep. thermophilus, Pediococcus subsp Leuc. mesenteroides subsp mesenteroides. Despite, possessing the technologically important EPS producing property by some LAB strains, the main problem associated with use of such cultures is the gradual loss of EPS producing character. Numerous investigators have reported instability of EPS producing traits in mesophilic and thermophilic LAB (Cerning et al 1992). This may be due to the frequent transfer of culture and prolonged periods of incubation (Macura and Townsley 1984, Cerning 1990, Cerning et al 1992) and spontaneous loss of plasmid encoded genes in mesophilic strains (Vedamuthu and Neville 1986, Von Wright and Tynkkynen 1987, Neve et al 1988, Vescovo et al 1989). However, in case of thermophilic strains, the EPS synthesis genes are chromosomally located and the instability could be due to mobile genetic elements or to a generalized genomic instability, including deletions and rearrangements (Mollet and Delley 1990, Roussel et al 1994, Germond et al 1995, Guedon et al 1995).

EPS types Based on the composition of monosaccharide repeating units, EPS of LAB are classified into 2 groups, viz., homopolysaccharides and heteropolysaccharides. Homopolysaccharides are a group of polysaccharides, which contain one type of monosaccharides fructose or glucose (Barker and Ajongwen 1991, Monsan et al 2001). Dextran, mutan and levan are the examples of homopolysaccharides produced by some Lactobacillus, Leuconostoc and Streptococcus species, of which, Leuc. dextranicum is a well-known dextran producer (Hamada and Slade 1980, Montiville et al 1978, Funane et al 1995, Monchois et al 1998, 1999, Van Geel-Schuten et al 1999). The homopolysaccharides are synthesized by anchored or secreted transglycosylases, which are able to cata-

lyze the transfer of a corresponding glycosyl moiety (Monsan et al 2001). On the other hand heteropolysaccharides are composed of different monosaccharides repeating units, mostly glucose and galactose and also small amounts of rhamnose, mannose, N-acetylglucosamine, Nacetylgalactosamine, glucuronic acid (Cerning 1990, Van den Berg et al 1995, Stingele et al 1996, 1997, Grobben et al 1997). Heteropolysaccharides contain backbone of repeated subunits that are branched (at positions C2, C3, C4 or C6) or unbranched and that consists of three to eight monosaccahrides (De Vuyst et al 2003). Unlike homopolysaccharides, the heteropolysaccahrides are synthesized intracellularly at the cytoplasmic membrane utilizing sugar nucleotide as precursors for the assembly of polysaccharide chain (Cerning 1995). The glycosyl transferases are the key enzymes for the biosynthesis of the EPS repeating unit, since they catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, thereby forming a glycosidic bond. Different types of heteropolysaccharides are secreted by mesophilic and thermophilic lactic bacteria with respect to sugar composition and molecular mass, the latter varying from 1.0 × 104 to 6.0 × 106 (Cerning 1995, De Vuyst and Degeest 1999). Generally all thermophilic LAB produce heteropolysaccharides in larger amounts than mesophilic LAB (Mozzi et al 2001).

EPS production, isolation and characterization EPS production by dairy LAB varies from species to species and ranges between 25 and 9800 mg/l (Table 3). The media developed for EPS production include skim milk, whey based medium, semidefined medium, chemically defined medium, and basal minimum medium. The choice of an adequate EPS producing medium is of great importance for further isolation process and depends on the degree of purity required. For example, some strains are particular for media in EPS production, may produce EPS only in milk (Cerning 1990) and isolation of EPS from coagulated milk is tedious, time consuming, and includes the risk of degrading or modifying of polymer during manipulation (Cerning et al 1992). 3

Factors such as composition of the medium, temperature and pH greatly affect the EPS production (Cerning et al 1992, Gancel and Novel 1994, Mozzi et al 1994). Most of the extracellular polysaccharide producing organisms utilize carbohydrate as the carbon and energy source and either an ammonium salt or amino acid as the source of nitrogen (Manca de Nadra et al 1985, Cerning 1990, Mozzi et al 1995). Macy (1923) studied the effect of carbohydrates and nitrogenous compounds on EPS production by ropy lactic cultures isolated from finnish product. The cultures have shown ropiness in all the aqueous solutions of lactose, maltose and casein or peptone separately or in combinations, but the best result was obtained in casein and lactose combination. Addition of glucose or sucrose to milk and milk ultrafiltrate increased EPS production by ropy strains of L. lactis subsp. lactis, L. lactis subsp. cremoris and L. casei subsp. casei (Cerning et al 1992). De Vuyst et al (2003) observed enhanced growth and EPS production by Strep. thermophilus strains in enriched milk medium supplemented with 1.0% peptone and 0.5% yeast extract than in simple milk medium without nitrogenous source. The carbohydrate added to the medium not only stimulates culture growth but also increases EPS production by LAB strains. However, type of sugar used in the media is largely dependant on the type of strain used and no sugar gives the best result because different organisms use different carbohydrates. The cultural conditions (type of sugar, temperature of incubation) must be optimized for maximal EPS production (Mozzi et al 2003, Torino et al 2005). Regarding the temperature of EPS production by LAB, there are 2 views: First, EPS production is higher when the cultures are incubated at sub optimal temperature of 18-25ºC than at 30ºC for mesophilic lactococci and at 35-37ºC than at 42ºC for thermophilic cultures (Macy 1923, Sutherland 1972, Schellhaass 1984, Macura and Townsley 1984, Manca de Nadra et al 1985, Cerning et al 1992). Second, the EPS production is growth associated; maximum EPS production is achieved when the cell cultures are in exponential phase (Degeest and De Vuyst

J Food Sci Technol 2009, 46(1), 1–11 Table 3. Exopolysaccharide (EPS) yield obtained by some lactic acid bacteria strains growing in several media by using different isolation procedures LAB strains Culture media* Isolation procedure EPS, mg/1 Strep. thermophilus SY WM UF+ ethanol pptn 152 Strep. thermophilus ChH YC-380 Milk Ethanol pptn 600 Strep. thermophilus CRL 804 Milk Ethanol pptn 166 L. lactis subsp. cremoris LC330 CDM Dialysis 25 L. lactis subsp cremoris T5 SM Pronase+UF+Ethanol pptn 600 L. lactis subsp cremoris B891 SM TCA pptn 80 Lact. delbrueckii subsp. Whole milk TCA + Ethanol pptn 540 bulgaricus 2-11 Lact. delbrueckii subsp. SM TCA + Ethanol pptn 170 bulgaricus CNRZ416 Lact. delbrueckii subsp. Milk Ethanol pptn 150 bulgaricus CRL852 Lact. rhamnosus R BMM TCA + Ethanol pptn 495 Lact. rhamnosus RW-9595M WPM TCA + Ethanol pptn 2300 Lact. helveticus ATCC15807 SM Pronase + Ethanol pptn 549 Lact. paracasei Type V BMM Ethanol pptn 85 Lact. reuteri LB121 MRS-sucrose Ethanol pptn 9800 Lact. sakie 0-1 SDM Ethanol pptn 1580 Lact. fermentum MR6 MRS-glucose TCA + Ethanol pptn 713 Ped pentosaceus AP-3 MRS-sucrose Ethanol pptn 2500 * WM: Whey media, CDM: Chemically defined medium, MRS: De Man Rogosa, sharp medium, SDM: milk, BMM: Basal minimal medium, WPM: Whey permeate medium, pptn: Precipitation

1999, Devuyst et al 2003). The amount of EPS produced by LAB are small as compared to xanthan gum (10 to 25 g/l), an EPS produced by non-food-grade microorganisms (Becker et al 1998, De Vuyst and Degeest 1999). Recently, molecular strategies have been used to modify glycosyltransferases that are involved in the assemblage of EPS repeating units to increase the level of EPS production in LAB strains. Levander et al (2002) obtained 2 times increase in EPS production through overexpression of UDP glucose pyrophosphorylase (GalU) in combination with phosphoglucomutase (PGM). Further, Svenson et al (2005) reported altered levels of Leloir enzymes together with PGM and GalU increased the EPS production 3.3 times over that of the parent strain Strep. thermophilus The complexity of the medium used for the isolation and purification of EPS will depend on the composition of the culture medium used for its production (Table 3). The simplest procedure involves dialysis against water of the culture medium after cell removal by centrifugation, followed by lyphilization (Marshall et al 1995). Ethanol precipitation may be used to concentrate in the

EPS, before dialysis for the isolation of EPS from LAB strains (Dal Bello et al 2001, Ricciardi et al 2002). Additional purification steps have become necessary to reduce the protein content and other components in the final EPS, where culture medium has increased complexity. Three approaches are commonly used to obtain EPS from media with higher protein content. First precipitation with variable amounts of TCA (final ranging from 4 to 14%), digestion with protease or a combination of both (Dupont et al 2000, Frengova et al 2000, Bouzar et al 1997, Cerning et al 1992; Mozzi et al 1995). Apart from protein removal and EPS precipitation other procedures that have been used to purify the EPS fraction include membrane filtration technique such as microfiltration, ultrafiltration (UF) and diafiltration (Tuinier et al 1999b; Staaf et al 2000). A wide range of methodologies have been published all of them are different. Rimada and Abraham (2003) compared different EPS isolation procedures reported in the literature for recovery of EPS produced by Lb. kefir in milk and deproteinized whey (DPW). Method that involves ethanol precipitation twice, direct dialysis or a combination of etha4

References Ricciardi et al (2002) Ludbrook et al (1997) Mozzi et al (2006) Marshall et al (1995) Cerning et al (1992) Ruas-Madiedo et al (2002) Frengova et al (2000) Petry et al (2003) Mozzi et al (2006) Pham et al (2000) Bergmaier et al (2003) Torino et al (2001) Dupont et al (2000) Van Geel-Schutten et al (1999) Degeest et al (2001) Savadogo et al (2004) Smitinont et al (1999) Semi-defined medium, SM: Skim

nol precipitation followed by a dialysis step provided the same polysaccharide concentration and is the most suitable procedure for isolation of kefir polysaccharide from milk and DPW. After the isolation steps, a lyophilized powder is obtained its weight being the simplest indication of the EPS yield (Degeest et al 2001, Frengova et al 2000, Van GeelSchutten et al 1999). The EPS production can also be expressed as the equivalent milligrams of dextran per milliliter. However, values obtained by this method strongly depends on degree of purity of the isolated EPS as protein and non-EPS carbohydrates are also quantified. A colorimetric method for the determination of sugar and related compounds is the phenol sulphuric method described by Dubois et al (1956). This method is widely used as an indication of EPS yield obtained by different isolation procedures (Cerning et al 1992, Gruter et al 1992, Gancel and Novel 1994, Marshall et al 1995, Mozzi et al 1995). Another colorimetric procedure used by several authors for the quantification of sugars involves the use of anthrone reagent (Van den Berg et al 1995, Levander et al 2001, Rimada and Abraham 2001).

J Food Sci Technol 2009, 46(1), 1–11 In order to understand the composition of isolated EPS, preliminary depolymerization requires by total or partial hydrolysis with trifluroacetic acid HCl or H2SO4 at 100 to 120°C for 2 to 8 h. These treatments yield mono or oligosaccharides that are frequently derivatized into alditol acetates, trimethyl silyated (-)-2 butylglycosides. The qualitative monosaccharide compostion of an EPS has been analyzed in the past by TLC (Gruter et al 1992, Lemoine et al 1997). However, this method has low discriminatory power and has been largely surpassed by more reliable techniques. The qualitative and quantitative determination of EPS monosaccharide by HPLC involves the separation of monosaccharides by anion exchange columns and detection by RI. Another technique used for the identification and quantification of mono- and oligosaccharides resulting from the partial hydrolysis of EPS is high-performance anion-exchange chromatography pulse amperometric detection (HPAEC-PAD) (Lemoine et al 1997, Levander et al 2001). The most extensively used technique for the analysis of the monomer composition of EPS isolated from LAB is gas chromatography/mass spectrometry (GC/MS). The monomeric composition of several EPS produced by L. lactis subsp. cremoris, Strep. thermophilus, Lb. delbreuckii subsp. bulgaricus, Lb helveticus, Lb reuteri, Lb. rhamnosus and Bifidobacterium longum was determined using the GC/MS method (Nakajima et al 1990, Marshall et al 1995, Bouzar et al 1997, Yang et al 2000, Petry et al 2003). Many past reports indicate that characterization of EPS gives either homopolysaccharide (glucose or fructose) or heteropolysaccharides (glucose, galactose, rhamnose, etc.). Faber et al (2002) used GLC to determine monosaccharide composition of EPS produced by Strep. thermophilus 8S. The analysis revealed that the EPS contained galactose, glucose, ribose and N-acetyl-D-galactosamine in a molar ratio of 2:1:1:1. Savadogo et al (2004) reported glucose and galactose were the dominating sugars with small amounts of rhamnose, mannose, fructose, arabinose and xylose sugar of EPS obtained from different LAB species. A large biodiversity of EPS from LAB exists regarding their composition and structure,

molecular mass, yield, and functionalities (De Vuyst and Degeest 1999, De Vuyst et al 2003).

Functional properties of EPS Bacterial EPS influence the texture and rheology of fermented milk products at extremely low concentration (Duboc and Mollet 2001). Understanding the nature and molecular characteristics of the polymer would help in determining its effect on functional properties (Kleerebezem et al 1999, Tuinier et al 1999 a,b). Most EPS are random coils with no fixed shape; they have randomly fluctuating tertiary structure. In most cases, EPS contributes to the thickening of the final product, which depends on the viscofying ability of the polymer. The viscofying ability of EPS in solution can be determined by some parameters like intrinsic viscosity, the specific volume occupied by the dispersed particle and the concentration of dispersed polymer (Tuinier et al 1999b). The specific volume of EPS in solution is determined by its molecular mass and its “radius of gyration”, which is a measure of the size of the polymer in solution (measured in nm). To obtain higher intrinsic viscosity, the molecular characteristics, either the molar mass (chain length) or the stiffness of the polysaccharide must increase (Laws and Marshall 2001). The lactic strains producing EPS with greater chain length may produce more viscous product. Two strains of Strep. thermophilus Sts and Rs although produced similar amounts of EPS in milk (135 mg/ l for Rs and 127 mg/ l for Sts), exhibited different viscosity values (measured using the Posthumus method, which measures flow rate) 39 and 126, respectively due to differences in molecular mass of the polymers (Faber et al 1998). The molecular mass of an EPS of Rs strain was 2.6×106 and that of strain Sts was 3.7×106 Da; the latter produced more viscous product than the former. The β (1→4) linkages in the backbone of the polymer lead to stiffer chains, whereas β (1→2) or β (1→3) linkages and α linkages lead to more flexible chains (Rees 1977, Bianchi et al 1986). The L. lactis subsp. cremoris B40 strain showed higher intrinsic viscosity and thickening efficiency because of the stiffer chains β (1→4) present in the 5

backbone of EPS (Ruas-Madiedo et al 2002). On the other hand, the EPS of Strep. thermophilus Sfi20 , which has two β sugars and one α sugar with 1→3 linkages in its backbone, suggested that the polymer was flexible (Navarini et al 2001). The EPS with intrinsic viscosity below 1.5 M/ kg will not be able to increase the viscosity of fermented milk (Ruas-Madiedo et al 2002). Ludbrook et al (1997) isolated EPS from LAB of nondairy origin and assessed thickening properties by preparing 0.3% (w/v) EPS solution. The average intrinsic viscosity of the solution was very low (ranged from 1 to 6 cps) and did not contribute to the thickening under specified conditions. Some authors have reported a direct correlation between the concentration of EPS and viscosity of stirred yoghurt, but no clear relation has been demonstrated (Van Marle and Zoon 1995, Dupont et al 2000) except that if a given strain produces more EPS, the viscosity of fermented milk increases (Sebastiani and Zelger 1998, Ruas-Madiedo et al 2002). De Vuyst et al (2003) attempted to interpret the contribution of heteropolysaccharides to structure/function relationship of fermented milk. They observed no clear cut relationship between amount of EPS produced and viscosity of fermented milk. However, they stated that for high intrinsic viscosity, stiffer chains are required. In addition, the complexity of the primary structure (size, monomer composition and side groups α- and β - linkages, branching) influences the viscofying effects of EPS solution (Yang et al 2000).

Application of EPS producing lactic cultures in low-fat dairy products Milk fat contributes to the body, texture and flavour development of dairy products. Fat reduction to satisfy consumers demand leads to textural and functional defects in low-fat yoghurt, cheeses, dahi, and kefir (Mistry 2001, Haque and Ji 2003, Tunick et al 2003, Guven et al 2005). In low-fat yoghurt and dahi, lack of flavour, weak body and poor texture are the major problems. Mechanical breaking particularly in stirred yoghurt, strongly affects the rheology of the coagulum and favour syneresis since the network formed by the gel is broken

J Food Sci Technol 2009, 46(1), 1–11 (Duboc and Mollet 2001). Low-fat cheeses have poor moisture retention ability, which otherwise moisture present in the cheese partially overcomes the problem of firm, rubbery body and texture created by high casein content (Mistry 2001). Generally for cheese manufacture, the casein to fat ratio is 0.68-0.70, which gives desirable textural attributes. Due to fat reduction, the casein to fat ratio is disturbed and casein content is more in the resultant cheese consequently generating some textural defects. Lowfat Mozzarella cheeses have less tendency to melt and inferior baking characteristics (Fife et al 1996, Rudan and Barbano 1998). These defects can be reduced by the use of additives to some extent but may not find wide acceptance to produce wholesome product. The consistent manufacture of good quality products that have good texture, mouthfeel and stability is important to the dairy industry. The manufacturers have used texture promoting or ropy cultures for many years particularly where addition of stabilizer is prohibited (Marshall and Rawson 1999). These cultures may impart higher intensity to flavour to the yoghurt due to the carbohydrate masking the flavour, mouth feel and other attributes (Tamime and Robinson 1999). To reduce the amount of added milk solids, improve yoghurt viscosity, to enhance texture and mouthfeel and to avoid syneresis during fermentation or upon storage of the fermented milk products, EPS producing

functional starters are interesting (De Vuyst et al 2003). The apparent viscosity of skim milk gel made by both ropy cultures increased as compared to that made by non-ropy cultures (Tamime and Robinson 1999). However, combining two ropy cultures for yoghurt manufacture may not have always, additive effect (Faber et al 1998). Marshall and Rawson (1999) found that mixing a non-ropy strain of Strep. thermophilus with a ropy strain of Lact. delbrueckii subsp. bulgaricus had a greater effect on viscosity of stirred yoghurt than combining 2 ropy strains (Table 4). The authors suggested the interaction and cooperative growth that occur in mixed cultures, which also appear to influence EPS production in yoghurt. The presence of EPS in stirred yoghurt makes the product less susceptible to mechanical damage from pumping, blending and filling machines (Robinson 1981, De Vuyst and Degeest 1999). Although, mechanical processing steps do increase the syneresis of the final product, use of EPS cultures can control this defect (Duboc and Mollet 2001). Hassan et al (1996a, b) examined the rheological and textural properties of yoghurt made with strains differentiated as encapsulated non-ropy (in which an EPS capsule is formed), encapsulated ropy (secretes extracellular slime) and nonencapsulated non-ropy. Yoghurt made with ropy cultures exhibited increased viscosity and shear stress values; however, differences attributed to the type of polysac-

charide secretion (capsule or slime) were apparent. The presence of bacterial capsule may enhance some rheological properties such as viscosity, but may weaken the gel structure. This has caused lower shear stress value compared to slime producers, which produce a more stretchable gel structure (Hassan et al 1996a). The types of EPS produced by the yoghurt bacteria have effects on the texture and syneresis. Yoghurt made with encapsulated non-ropy cultures had the lowest firmness and curd tension, but exhibited less syneresis than unencapsulated cultures. The lower firmness in the yoghurt made by slime producing cultures might have been due to the polysaccharide interfering with the casein structure (Hassan et al 1996b). Several microstructural studies have indicated that it is not the amount of EPS, which is important to the rheological properties of fermented milk but the type of EPS and therefore the interaction of the polymer with the bacterial cell and the milk protein is important during the fermentation (Marshall and Rawson 1999). Folkenberg et al (2005) observed 2 types of microstructure in yoghurt made with different EPS producing cultures, one in which EPS is associated with the protein network and another where EPS appeared to be incompatible with the protein. Yoghurt in which the EPS were associated with protein had ropiness, low serum separation and appeared more resistant to stirring than EPS appeared incompatible with protein.

Table 4. Application of EPS producing LAB in reduced fat dairy products LAB Strep. thermophilus (EPS-) and Lact. delbrueckii subsp. bulgaricus (EPS+) Strep. thermophilus (EPS+) and Lact. delbrueckii subsp. bulgaricus (EPS+) Strep. thermophilus (EPS+) and Lact. delbrueckii subsp. bulgaricus (EPS-) Strep. thermophilus MR-1C (EPS+) and Lact. bulgaricus MR-1R (EPS+) Lact. lactis

Product Stirred yoghurt

Effect Increased viscosity

References Marshall and Rawson (1999)

Yoghurt

Hassan et al (1996a,b)

L. lactis subsp. cremoris JFR1

Cheddar cheese

Lact. lactis

Cream cheese

Strep. thermophilus Lact. kefir

Hungarian cultured milk Kefir

Improved rheological and textural properties Ropiness, low serum separation, higher viscosity Retained moisture, better melting properties Increased moisture retention, melt properties and springiness Increased moisture retention, improved sensory, textural and melting properties Better firmness, consistency and flavour Improved rheological properties Improved texture

Yoghurt Mozzarella cheese Cheddar cheese

6

Folkenberg et al (2005) Perry et al (1997) Michael et al (2003) Awad et al (2005)

Nauth and Hayashi (2004) Obert (1984) Micheli et al (1999)

J Food Sci Technol 2009, 46(1), 1–11 Ayala-Hernandez et al (2008) developed an immobilization technique to remove noninteracting material by a washing step thereby facilitating to observe the interactions between EPS and dairy proteins (casein and whey proteins) in fermented milk permeate and buttermilk using scanning electron microscopy. EPS appeared as filament strands attached to the protein aggregates and to the bacterial cells, confirming for the first time that the EPS molecules interact with dairy proteins. The functions of EPS in dairy products have not been completely understood. Hassan (2008) attributed this to 2 main reasons: the major variations among EPS even from the same group of microorganisms, which makes it difficult to apply information from one EPS to others, and the lack of availability of techniques with the ability to observe the microstructure and distribution of the highly hydrated EPS in fermented dairy products. Incorporation of EPS strains in the starter culture retains significant amount of moisture in a variety of low-fat cheeses that positively influence their functionality (Francois et al 2004, Awad et al 2005, Zisu and Shah 2005). Perry et al (1997) investigated the influence of an EPS-producing starter pair Strep. thermophilus MR-1C and Lact. delbrueckii subsp. bulgaricus MR-1R on the moisture and melt properties of low-fat Mozzarella cheese. Cheese manufactured with MR1C and MR-1R contained significantly (p