Exopolysaccharide-Producing Lactic Acid Bacteria – Health-Promoting Properties And Application In The Dairy Industry

Apart from their basic metabolic activities, bacteria are also capable of synthesizing many biopolymers which differ in structure and chemical properties and thus in their functions in the cells. Considering their localization, biopolymers may be divided into intracellular (a small group with limited applications) and extracellular (a large group with wide applicability) ones. One of the classes of extracellular biopolymers includes exopolysaccharides (EPS). The EPS may be secreted outside a bacterial cell or may be produced as a capsule bound with external cellular membranes [66]. The EPS serve various functions in bacterial cells, like: protecting them against adverse effects of the environment (e.g., high or low temperature, high or low pH, and toxic metal ions) and against biological factors (e.g., phage attack), or helping them to colonize the environment (they are constituents of biofilms). It is presumed that EPS do not serve as a source of energy to bacterial cells, though some probiotic strains of lactic acid bacteria (LAB) were shown to be capable of EPS degradation [75]. Some LAB strains used in the dairy industry to produce fermented milks, e.g., yoghurt, kefir, sour milk and other fermented milk drinks, are able to synthesize EPS (the so-called EPS(+) strains). The application of EPS(+) strains may have highly positive effects on the rheological properties and quality of the manufactured fermented products [7, 12]. The EPS produced by lactic acid bacteria during formation of the casein curd of milk are capable of water retention and thereby inhibit syneresis in fermented drinks. In addition, by reacting with proteins, they may contribute to the reinforcement of the casein network, which improves the rheological properties, quality of the final product, and cheese yield. The use of adjunct EPS(+) starter cultures improves the smoothness, viscosity and stability of a yoghurt gel and of other fermented milks [4, 22, 32]. The character of changes induced in fermented products by the presence of EPS is determined by the chemical composition and structure of these compounds, including e.g., their molecular weight, type of bonds and the presence of side chains. The rheological properties of EPS-containing food products are also affected by the time of their most active production by LAB during food manufacture [21, 22, 67, 75]. The most active LAB strains were shown to produce EPS at even 3 g/l [58]. The EPS display also some health-promoting properties. An increase in the viscosity of EPS-containing products is believed to extend the time of their gastrointestinal passage, which may be beneficial for temporary gut colonization by LAB [15]. In addition, many studies have shown the immunomodulatory, hypocholesterolemic, anti-carcinogenic, and anti-ulcerous activities of EPS [24, 26, 42, 43, 50, 56, 59].

The EPS are high-molecular, long-chain linear biopolymers with side chains, which are constituted by carbohydrate units linked with α- and β-glycosidic bonds. They may be secreted by a cell to the extracellular space and remain bound with its surface thus forming a capsule (CPS – capsular exopolysaccharides). EPS may also be released to the external environment in the form of slime exopolysaccharides. Other types of polysaccharides include, e.g., cell wall polysaccharides (CWPS) linked with ionic or covalent bonds with the peptidoglycan layer on the cell’s surface [88]. Taking into account the EPS structure, they may be divided into homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Molecules of HoPS consist of successively repeated monosaccharides of one type (e.g., D-glucose or D-fructose), and include two major groups: glucans (dextran, mutan, alternan, reuteran, curdlan) and fructans (levan, inulin-type fructans) [63, 67, 88]. In turn, HePS are built of sub-units containing 3 to 8 monosaccharides: D-glucose, D-galactose, L-fructose, L-rhamnose or, alternatively, acids: D-glucuronic, L-glucuronic and D-mannuronic. The HePS may also contain amino sugars, like, e.g., N-acetyl-D-glucosamine or N-acetyl-D-galactosamine [7, 67]. Molecular weights of HePS range from 10⁴ to 6×10⁶ Da [7].

Glucans – being representatives of HoPS – are divided into α-D-glucans and β-D-glucans [63]. The production of α-D-glucans (e.g., dextran, mutan, alternan, reuteran) is assisted by dextransucrase which is an extracellular enzyme synthesized by, among others, bacteria of the Leuconostoc, Streptococcus, and Lactobacillus genera. In turn, β-D-glucans (e.g., curdlan) contain glucose residues linked with β-1,3-glycosidic bonds. The LAB capable of their production include strains from Pediococcus, Oenococcus, and Lactobacillus genera [7]. One of the HoPS α-D-glucans is dextran. The dextran molecule synthesized by Leuconostoc mesenteroides has a linear structure and is built mainly of D-glucose residues (95%) linked with α-1,6-glycosidic bonds. The remaining part is constituted by side α-1,3-glycosidic bonds. Differences in the structure of dextrans isolated from various LAB include mainly: type, number and arrangement of side chains in a molecule. Bacterial strains which produce dextran include strains from Leuconostoc, Streptococcus and Lactobacillus genera. In the pure form, dextran is applied as a component of gel used for filtration (Sephadex) and as a blood substitute [7, 36]. Another example of α-D-glucan is mutan, which is built of D-glucose molecules linked in over 50% with α-1,3-glycosidic bonds. A high activity of the mutan-producing enzyme was reported in Ln. mesenteroides NRRL B-523 and B-1149 strains and in some strains from the genus Streptococcus. Mutan is responsible for the adhesion of oral cavity microflora to teeth surface, which contributes to the formation of dental plaque and calculus [7, 63]. Alternan – which contains alternatively arranged α-1,6 and α-1,3-glycosidic bonds – is produced by an enzyme called alternansucrase. Capability for alternansucrase synthesis has so far been reported for three strains of Ln. mesenteroides: NRRL B-1355, NRRL B-1501, and NRRL B-1498 [63]. In turn, reuteran is an α-D-glucan produced by reuteransucrase enzyme isolated from Lactobacillus reuteri 121 and ATCC 55730 [44, 45]. Curdlan is a neutral, gel-forming β-D-glucan with a straight chain [48]. Curdlan and other polysaccharides belonging to this group were described as anti-carcinogens activating macrophages and leukocytes [7].

One of the most extensively described HePS is kefiran, built of mannose, glucose and galactose in approx. ratio of 1:5:7 [87]. Ability to produce kefiran was reported for: Lactobacillus kefiranofaciens, Lb. kefirgranum, Lb. parakefiri, Lb. kefir, Lb. plantarum, and Lb. delbrüeckii subsp. bulgaricus [3, 84].

The capability to synthesize EPS by LAB varies and depends on many factors, and is species- and strain-specific. EPS production by lactic bacilli (Lb. plantarum) accounts for ca. 0.14 g/l [81], Lb. bulgaricus – 0.06–0.15 g/l [83] and Lb. fermentum – 0.75 g/l [41]. In turn, lactic streptococci (S. thermophilus) are able to produce 0.1 g/l [89]. However, the greatest production of EPS was demonstrated for Lb. rhamnosus RW-9595M strain (2.8 g/l), Lb. kefiranofaciens WT-2B strain (2.5 g/l) and Lb. plantarum BR2 strain (2.8 g/l) [58, 60, 79].

EPS production is determined by the growth stage of bacteria, composition of culture medium (type of carbon and nitrogen sources, and presence of other nutrients), temperature, and pH, and/or by the presence of adjuvant microflora [2, 3, 62, 72, 82, 86, 92, 93] (Tab. I). The concentration of produced EPS is largely affected by conditions of growth of bacteria which synthesize them, whereas the monosaccharide composition of most of the EPS does not depend on the available source of carbon. Interesting – especially from the perspective of practical application – seems to be the fact that the same LAB strain may produce different EPS under various growth conditions [39].

Ample studies have addressed the effect of culture medium composition on the concentration of EPS produced by LAB [69, 72, 82, 86, 92]. The Lb. casei and Lb. bulgaricus strains were shown to produce EPS in the concentration below 0.6 g/l when cultured in fermented milk, and at 1.5 g/l when grown in M17 broth enriched with various sources of carbon and nitrogen [69]. In turn, Rabha et al. [72] demonstrated milk to be a better medium for EPS production by S. thermophilus than MRS or M17 broths. Investigations on the effect of carbon source on EPS concentration have demonstrated that increased synthesis of kefiran by LAB engaged in kefir production was achieved with disaccharides used as sources of carbon. The enrichment of culture medium in saccharose or lactose enabled achieving kefiran content in kefir grains at 3.8% and 4.3%, respectively. When fructose or glucose were used in the culture medium as the source of carbon, the respective values were lower and reached 2.7% and 2.1% [92]. Lb. fermentum F6 produced greater amounts of EPS when glucose was used as a carbon source in the culture medium (ca. 0.035 g/l), compared to fructose >lactose > galactose [93]. S. thermophilus strain 23, isolated from homemade yoghurt in Bulgaria produced more EPS in the presence of sucrose (0.13 g/l) than in the presence of lactose (0.083 g/l) [83]. Results of this research show milk to be a good culture medium for EPS production by LAB, especially when the milk composition is modified by addition of a source of carbon. EPS production by LAB is also affected by the source of nitrogen in the culture medium. The highest concentration of kefiran was produced in the presence of organic nitrogen, e.g., casein (1.78 g kefiran per l), peptone (1.65 g/l), tryptone (1.64 g/l), or yeast extract (1.64 g/l), whereas a significantly lower amounts were noted when the culture medium was supplemented with inorganic nitrogen (urea – 0.89 g kefiran per l, ammonium chloride – 0.73 g/l, ammonium sulfate – 0.69 g/l) [86]. Similar results were reported by Zajšek et al. [92]. In their study, the content of kefiran in kefir grains was significantly lower when ammonium chloride was used as the source of nitrogen in the culture medium (1.3%), whereas its content increased to 1.8% when skim milk was used as the source of organic nitrogen (casein) [92]. Additional enrichment of the skim milk-based culture medium with peptone and yeast extract improved EPS production also by S. thermophilus (2 to several times more, depending on the strain) [82].

Another factor influencing EPS synthesis by LAB is temperature. Many studies have shown the highest production of EPS at the so-called sub-optimal temperature, i.e. at few °C lower than the optimal temperature for growth of a given LAB species, by both mesophilic and thermophilic species [62, 72]. The overproduction of EPS at the sub-optimal temperature is a likely response of a bacterial cell to the physiological stress induced by the decreased temperature, especially in species or strains defective in proteolytic activity (e.g., S. thermophilus) [12, 72, 82]. In their research on thermophilic S. thermophilus BN1 with the optimal growth temperature at 42°C, Rabha et al. [72] demonstrated a significantly higher EPS production by this strain at a slightly lower than optimal growth temperature (37°C), irrespective of the culture medium composition. For example, in skim milk, EPS production by this strain reached 0.097 g PDM (polymer dry mass) per l at 42°C, but was by over 5-fold higher at 37°C. In their study on kefiran synthesis, Zajšek et al. [92] demonstrated the highest content of EPS in kefir grains at the temperature of 25°C (2.75%), and the lowest one – at 37°C (1.3%). In turn, the latter temperature turned out to be optimal for the growth of the kefir grains. In addition, the temperature of fermentation was found to influence the galactose to glucose ratio in the produced kefiran. At the temperature of 25°C, this ratio was lower (ca. 1.3) than at the temperature not facilitating EPS production (ca. 1.4). The temperature lower than the optimal growth temperature was also reported to enhance EPS production by Lb. sake 0–1 [13]. In turn, Zhang et al. [93] demonstrated that Lb. fermentum F6 produced the highest concentration of EPS when cultured at the optimal temperature (37°C). Likewise, strains of Lb. fermentum TDS030603 and Lb. casei CRL 870 were shown to produce the highest concentrations of EPS at the optimal temperature for their growth, i.e. 37°C [49]. Also Mende et al. [62] demonstrated higher EPS production by the Lb. delbrüeckii subsp. bulgaricus DSM 20081 strain at temperatures from 30°C to 40°C (optimal growth temperature for this strain is 40°C), while almost by half lower EPS production at 45°C. Thus, it may be concluded that, unlike the sub-optimal temperature, the temperature exceeding the optimal growth temperature does not enhance EPS synthesis as a form of cell response to physiological stress. Ruas-Madiedo et al. [76] showed no effect of temperature on EPS concentration in milk fermented by Lc. lactis subsp. cremoris strains. In turn, they observed significant differences in EPS concentrations produced in replications of the same experiment. This may result from the fact that the capability to produce EPS is not a permanent trait, especially when strains are subjected to multiple proliferation due to the applied experimental method, which may lead to the loss of genes responsible for EPS synthesis that are located on plasmids. In addition, prolonged incubation may induce hydrolytic degradation of EPS.

Another factor influencing LAB ability to produce EPS is the time of incubation. Zhang et al. [93] showed the growth stage of Lb. fermentum F6 to be associated with EPS synthesis, the concentration of which decreased at the end of fermentation in the stationary phase of growth (after 32 h). Presumably, the Lb. fermentum F6 strain produces glycohydrolase which catalyzes degradation of polysaccharides, thereby decreasing EPS synthesis. EPS degradation after prolonged incubation was also observed in cultures of other LAB strains [12, 13]. Decreased EPS degradation was demonstrated when bacteria were incubated at a lower temperature and pH than the ones optimal for their growth [14, 53]. In the case of some LAB (e.g., S. thermophilus ST111), the concentration of EPS did not change over time, as the maximum yield of their synthesis occurred at the end of fermentation [82].

The optimal pH value for EPS production varies between species and between LAB strains, however usually reaches around 6 [12]. The intensive growth and maximum capability to produce EPS by S. thermophilus ST111 were determined in the culture medium with active acidity of 6.2 [82]. In turn, the optimal pH for EPS synthesis by Lb. delbrüeckii subsp. bulgaricus is similar to the optimal pH for their growth (ca. 6.5) [39]. Finally, Zhang et al. [93] demonstrated the highest EPS production by the Lb. fermentum F6 strain at pH 6.5, and a higher cell count of these bacteria at a slightly higher pH value (ca. 7.0).

One of the factors which influence EPS synthesis by some LAB strains is the simultaneous presence of other LAB in the culture medium. This issue is of high importance as mixed cultures constituted by several strains of the same LAB species or by different species are usually used in the industrial practice. Mechanisms of their interactions may be based on cooperation (e.g., Lb. bulgaricus and S. thermophilus in yoghurt), but also on competition or even growth inhibition (production of bacteriocins). Investigations on the effect of the mixed culture of the EPS(+) strain Lb. kefiranofaciens ZW3 with the EPS(–) strains: Lb. bulgaricus and S. thermophilus, demonstrated that the ZW3 strain produced EPS of a different structure compared to the EPS from a culture without yoghurt bacteria [3]. The mixed culture of EPS(+) and EPS(–) strains may, therefore, offer the possibility of changing the structure and type of the produced EPS and – indirectly – of inducing highly specified, desired changes in the final product.

The capability of LAB to produce EPS in milk during fermentation is an especially important trait for the dairy industry as these compounds increase the apparent viscosity and improve the texture and mouthfeel of the dairy products as well as inhibit syneresis even at their low concentrations (from 0.1 to 0.4 g/l) [17]. The presence of EPS synthesized by LAB strains has a significant effect on changes in various properties of dairy products, including: yoghurt, kefir and many other fermented milk drinks, sour cream and cheeses [15, 28, 32, 46].

The consumption of milk desserts, yoghurts and snacks is observed to successively increase in the United States and also in EU Member States. Products of this type contain additives which affect their rheological properties; but, on the other hand, they have to meet consumers demands for natural and healthy foods [54]. In Great Britain, the addition of stabilizers is regulated by law, e.g., the addition of starch and other stabilizers to yoghurts should not exceed 1% and 0.5%, respectively. It seems that in this respect the EPS(+) strains of LAB may arise interest of the dairy industry. The use of EPS(+) starters strongly inscribes itself into the “clean label” trend which is rather a permanent and irreversible trend that needs to be taken into account by food producers [57].

The EPS(+) LAB strains are applied as adjunct starters in the manufacture of fermented products or are incorporated into mixed starters (Tab. II). The effect of EPS on the rheological properties of fermented milk is more tangible and yields better outcomes when EPS are synthesized in situ in the product than when they are added as one of the components [15, 46]. Such an approach responds to economic concerns but may be applied mainly in the case of fermented products.

Viscosity of milk gels formed during fermentation by the EPS(+) LAB strains depends not only on the quantity of EPS products but also, to a significant extent, on their primary structure (stiffness of the EPS backbone), molecular weight, molecule rotation and charge [21, 22, 74, 75], character of bonds inside the molecule, and potential presence of side chains [21, 22, 46, 47]. At the same molecular weight, EPS molecules with a linear structure occupy a larger volume in solution compared to the branched EPS, owing to which they have a greater impact on increasing the viscosity of a solution. Also the stiffness of the EPS backbone contributes to the increase in the viscosity of the EPS-containing solutions by preventing potential deformations of the molecules. In milk gels, the presence of EPS with high molecular weight, stiff and only slightly branched has a positive impact on their viscosity and stability, and on their reduced syneresis [21]. The high degree of branching and the flexibility of the backbone leads to the “compactness” of the EPS, which results in a decreased viscosity of the solution. The microstructure of a milk gel formed with LAB strains producing EPS of this type is similar to the microstructure of a gel made with EPS(–) cultures [22].

Another factor affecting the rheological properties of milk gels produced with ESP(+) LAB strains is the charge of the EPS molecule. The use of strains synthesizing anionic EPS in starter cultures enabled achieving milk gels with higher values of the elastic modulus (G’) compared to the starters containing LAB strains synthesizing neutral EPS. The improvement of milk gels elasticity is probably due to electrostatic interactions between the anionic EPS and the positively charged molecules of casein, which strengthen the casein network [22]. Apart from dynamic changes in casein micelles, colloidal calcium phosphate, whey proteins and other milk constituents which cause the formation of a protein network, the process of milk fermentation by EPS(+) bacteria involves also EPS synthesis and incorporation into the protein network. Due to the successive release of EPS by LAB, increased acidity and change in environment conditions, interactions between EPS and milk proteins are also likely. The equilibrium between repulsion/attraction of EPS and milk proteins varies throughout fermentation and depends mainly on the environment pH as the released casein micelles bear a negative charge in milk with pH>4.6–6.65, a neutral charge in milk with pH 4.6, and a positive charge in milk with pH below 4.6, whilst the process of fermentation may terminate at different pH values. Understanding the dynamics of this phenomenon allows modifying and controlling final properties of milk gels [23, 46].

The EPS may influence the formation of a casein gel structure by acting as a “bond”, and their effect on the protein matrix and structure depends on their concentration, interactions with proteins and characteristics of a molecule [22, 70]. They may positively co-act with milk proteins, thus increasing values of the viscoelastic moduli and firmness of milk gels [70]. This may be caused by electrostatic interactions between casein and EPS, which besides protein-protein interactions may additionally reinforce the structure of a casein gel [6, 21, 23, 70]. Interactions between milk proteins and EPS in a complicated system, like, e.g., fermented milk, are poorly recognized. Research addressing this problem need to take into account the differences in the mechanisms of formation of these compounds during fermentation compared to the process aided with stabilizers (modified starches and pectins) added to milk prior to its souring. Some studies related to the protein-EPS interactions were conducted in model systems, wherein purified EPS preparations were added to milk before fermentation [23]. However, milks produced in this way had different, less beneficial rheological properties than the fermented milks in which EPS were synthesized by LAB in situ during fermentation [15]. This may result from the loss of certain properties of EPS (for example ropy character) during purification and drying of their preparations [23]. Results of investigations on the interactions between proteins and EPS in skim milk media do not fully reflect the complexity of milk composition which, apart from casein and whey proteins, includes also fat, lactose and mineral salts. Ayala-Hernández et al. [5, 6] in their studies on interactions between whey proteins and EPS with a well-known structure produced in situ by Lc. lactis in a simplified model of milk (milk permeate) demonstrated interactions between anionic EPS and whey proteins occurring in the amount of 2–8%, at pH 4.5 after 12 h of fermentation at a temperature of 30°C. The interactions between EPS and proteins (both casein and whey proteins) during fermentation of a dairy model system were also confirmed by Gentés et al. [22].

A largely significant aspect from the practical perspective is the capability of milk gels to “recover” after stirring and pumping during production of e.g., stirred yoghurts. Studies addressing the effect of stirring milk gels produced using EPS(+) strains and for comparison using EPS(–) strains demonstrated that although the EPS-containing gels were indeed more compact, a greater decrease was noted in the value of their elastic modulus (G’) after stirring than in the gels free of EPS [31, 32, 46]. The non-stirred gel formed by the EPS(+) strain showed a network of thick, continuous aggregates of milk with large void spaces, whereas the gel formed by the EPS(–) strain showed a network of fine protein strands and small void spaces around. Provided that a considerable amount of EPS is produced after the onset of aggregation of casein micelles, it would be entrapped in pockets between casein clusters and constrained around the bacteria cells. Such spatial constrain would reduce the probability of possible interactions of EPS with casein micelles and induce an intensification of the mutual interactions of casein particles. Stirring changes the structure of the EPS-containing gel, causing the formation of a larger number of pores and channels and of less compact protein aggregates. In addition, it causes the formation of channels with EPS-containing serum concentrated in larger strands than those observed in the undisturbed gels. This was attributable to the intense mutual interactions and aggregation of EPS molecules in the continuous serum phase due to repulsion with casein micelles. After stirring, the microstructure of the gel formed by the EPS(+) strain showed a significantly lower connectivity between protein aggregates, most likely as a result of the void spaces filled with EPS which surrounded casein. In the EPS(–) gels, stirring resulted in a more aggregated protein network with more connectivity between protein strands and smaller pores compared to the stirred gels containing EPS. The structure of the stirred EPS(–) gel did not differ significantly from its structure before stirring, but was more dense and aggregated [31, 32, 46]. Kristo et al. [46] concluded that the time needed to reach the gelation point (T_gel) in the case of milk fermented by the EPS(+) Lc. lactis subsp. cremoris JFR1 strain and time needed to reach pH 4.6 (T_pH4.6) were longer than for the milk fermented by the EPS(–) strain. However, the presence of EPS caused no significant difference in gelation pH (pH_gel).

Apart from the sensory benefits stemming from the EPS presence in dairy products, many EPS(+) LAB strains exhibit traits of probiotics [12, 16]. The probiotic activity of LAB strains is believed to be partly associated with the activity of biopolymers they produce [84]. The probiotic effect is then due not only to the activity of viable microorganisms, but also to the activity of their metabolites, including EPS (the so-called postbiotics). Beside the prebiotic effect of EPS [78], they are also claimed to have antibacterial activities [35, 41] and many health-promoting properties like: anti-carcinogenic, antioxidative, immunomodulatory, and reducing blood cholesterol [24, 26, 42, 43, 50, 51, 56, 59, 64, 65, 79, 84].

The mechanism of the anti-carcinogenic activity of EPS has not been fully elucidated, yet. According to one of the theories, EPS induce apoptosis of cancer cells by removing reactive oxygen species from their mitochondria [24, 51, 94]. The oxidative stress plays a key role in cancer pathogenesis. Levels of antioxidants and reactive oxygen species are correlated with the development and malignant transformation of cancer cells [37, 51]. Given that the EPS are capable of promoting antioxidative transformations and removing reactive oxygen species in cancer cells, they may as well inhibit their proliferation [51]. An EPS isolated from Lb. helveticus MB2-1 and built of three fractions: LHEPS-1, LHEPS-2 and LHEPS-3, inhibited the proliferation of human stomach cancer cells (cell line BGC-823). This activity was revealed for crude EPS (non-purified) and for all its three fractions. All of the three fractions were also capable of scavenging free radicals and chelating iron ions [50–52]. Liu et al. [56] demonstrated that EPS produced by Lb. casei 01, in the concentration from 0.005 to 0.050 g/l, exhibited a high antiproliferative against HT-29 human colon cancer cells, but simultaneously had no adverse effect on enteric cells. It is speculated that this activity involves both regulating enteric cells work and reducing cytotoxicity of procarcinogens. EPS of the probiotic Lb. plantarum NRRL B-4496 was active in vitro against tumor cell lines: Caco (intestinal carcinoma cell line), HeLa (cervical carcinoma cell line), HCT116 (colon carcinoma cell line), Hep-G2 (liver carcinoma cell line), MCF-7 (breast carcinoma cell line), and HEp2 (larynx carcinoma cell line) [29]. Results of the above-cited works allow hypothesizing that the anti-carcinogenic properties of EPS are ascribed to their activity as effectors inducing the immune response of the host body.

The slime EPS produced by Lb. paraplantarum BGCG11 was shown to exhibit anti-inflammatory and anti-suppressive properties [64]. This in vitro study demonstrated that during stimulation of peripheral blood mononuclear cells with purified EPS, the cytokine profile was similar to that induced by stimulation with viable cells of Lb. paraplantarum BGCG11.

Furthermore, EPS isolated from Lc. lactis subsp. lactis, and its derivative with selenium (Se-EPS) were capable of scavenging free superoxide anions (O₂^–) and hydroxyl radicals. In addition, they enhanced the activity of selected enzymes, e.g., catalase, superoxide dismutase and glutathione peroxidase, while reducing the level of malondialdehyde (MDA; an indicator of adverse lipid peroxidation) in blood serum and liver, and displaying immunomodulatory properties [26]. The hydrogen peroxide radical scavenging activity was also reported for EPS produced by S. thermophilus CC30 [40]. Investigations on the immunomodulatory properties of EPS produced by LAB (Lc. lactis subsp. cremoris, Lb. delbrüeckii subsp. bulgaricus, and Ln. mesenteroides) demonstrated some EPS to be capable of inducing cytokine synthesis [84] and modifying selected functions of macrophages and splenocytes [42]. EPS derived from yogurt obtained by fermentation using Lb. delbrüeckii subsp. bulgaricus OLL1073R-1 exerted immunostimulatory effects in mice [61]. Yogurt and semi-hard cheeses represent suitable food matrices for the delivery of the hypocholesterolemic EPS-producer strain Lb. mucosae DPC 6426 [77]. Works addressing kefiran indicate its antibacterial properties, ability to accelerate wound healing [73], and its potential to reduce blood pressure and cholesterol level in blood serum [59].

Apart from the longstanding health benefits resulting from the use of EPS(+) strains in the manufacture of dairy products, the EPS may also beneficially affect consumer physiology. Presumably, increased viscosity of EPS-containing fermented milk may prolong the time of its retention in the gastrointestinal tract, which is beneficial for, e.g., temporary gut colonization by probiotic bacteria. Another example of the putative health benefits of some EPS is their degradation in the colon to short-chain fatty acids (SCFAs) by enteric microflora. The SCFAs, and butyric acid in particular, provide energy to intestinal epithelium cells, and some of them prevent colon cancer [95].

In the food industry, the role of LAB capable of producing EPS may increase considering their effects on the rheological and textural properties of fermented food products. The EPS synthesized by LAB differ in their chemical composition and structure. Their production is relatively low (up to 0.1%), however they improve the consistency, stability and the widely understood quality of the final product. LAB capable of EPS production may find application in the manufacture of fermented milk products in countries in which the use of stabilizers is either limited or banned by law. The application of adjunct EPS(+) cultures in the production of fermented milks allows reducing the addition of milk powder and other thickening agents and offers vast possibilities for diversifying production, inscribing into the “clean label” trend, and meeting consumer demands for health-promoting and/or dedicated foods. EPS(+) cultures could also be applied in cheesemaking, especially in the case of low-fat fresh cheeses. Such a solution would prevent whey syneresis, but simultaneously give the sensation of some “fatness”. Besides technological benefits, the use of EPS(+) LAB for the manufacture of fermented dairy products has a positive effect on human health, involving mainly elongation of fermented milk retention in the gastrointestinal tract and thereby promoting gut colonization by probiotic bacteria. The EPS are usually prebiotics, however they also exhibit anti-ulcer, immunomodulatory, anti-carcinogenic activities and reduce blood cholesterol.