Exopolysaccharides from lactic acid bacteria are an alternative class of biothickeners, having potential for development and exploitation as functional food ingredients with both health and economic benefits, explain Harpreet K. Khurana and S.K. Kanawjia

The role of microbes in producing fermented dairy products has evolved from a chance of discovery to a highly elaborated process involving the production of specialized “starter” of bacteria that function consistently in large cultures. The primary function of almost all starter cultures is to develop acid in the product. The secondary effects of acid production include coagulation, the expulsion of moisture, texture formation and initiation of flavor production. Starters also help in imparting pleasant acid taste, conferring protection against potential pathogens and providing a longer shelf life to the product.

Exopolysaccharide Producing Lactic Acid Bacteria

The food industry uses polysaccharides as thickeners, emulsifiers, gelling agents and stabilizers. The demand for these ingredients is mostly met by alginates, carrageenan, cellulose, pectins, starches etc. There is a growing interest for all-natural, healthy food products. Moreover, in various countries, the amount of stabilizers being used is regulated. In this respect, the lactic acid bacteria (LAB) have great potential, as many of its representatives are known to produce exopolysaccharides (EPS). EPS from LAB are an alternative class of biothickeners, having potential for development and exploitation as functional food ingredients with both health and economic benefits. Consumer demand for products with low fat or sugar content and low levels of additives, as well as cost factors, make EPS a promising and viable alternative as these contribute to texture, mouth-feel, taste perception and stability of the final product.

Definition of exopolysaccharides

The name exopolysaccharides (EPS) as proposed by Sutherland (1972) provides a general term for various forms of bacterial polysaccharides found outside the cell wall. These extracellular, long-chain, high-molecular-mass polymers dissolve or disperse in water to give thickening or gelling properties and are indispensable tools in food product formulation. They consist of branched, repeating units of sugars or sugar derivatives. These sugar units are mainly glucose, galactose and rhamnose, in different ratios. They are secreted into their surroundings during growth and are not attached permanently to the surface of the microbial cell. This distinguishes them from the structurally similar capsular polysaccharides (CPS), which do remain permanently attached to the surface of the cell.

Classification of exopolysaccharides from lab

EPS occur widely among bacteria and microalgae and less among yeasts and fungi. EPS from LAB can be subdivided into two groups:

  1. Homopolysaccharides: these constitute four subgroups, namely;
    (a) a-D-glucans, i.e. dextrans (Leuconostoc mesenteroides subsp. mesenteroides and Leuc. mesenteroides subsp. dextranicum), mainly composed of a-1,6-linked glucose residues with variable (strain specific) degrees of branching at position 3, and less frequently at positions 2 and 4, and alternan (Leuc. mesenteroides) and mutans (Streptococcus mutans and Streptococcus sobrinus), both composed of a-1,3- and a-1,6-linkages; (b) b-D-glucans composed of b-1,3-linked glucose molecules with b-1,2 branches, produced by Pediococcus spp. and Streptococcus spp.; (c) Fructans, mainly composed of b-2, 6-linked D-fructose molecules, such as levan produced by S. salivarius; and (d) Others, like Polygalactans, composed of structurally identical repeating units with different glycosidic linkages; and
  2. Heteropolysaccharides produced by mesophilic (Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, Lactobacillus casei, Lb. sake, Lb. rhamnosus, etc.) and thermophilic (Lb. acidophilus, Lb. delbrueckii subsp. bulgaricus, Lb.helveticus and S. thermophilus) LAB strains. This group of EPS is extremely important, since they play an important role in the rheology, body/texture and mouthfeel of fermented milks.

Applications of EPS producing cultures in fermented dairy products

A large variety of EPS can be produced by LAB employed for production of fermented dairy products. In particular for the production of yoghurt, drinking yoghurt, cheese, fermented cream, milk-based desserts, EPS producing LAB play a significant role. They play a major role in the production of fermented dairy products in Northern Europe, Eastern Europe and Asia.

EPS producing lactic cultures have also been successfully used for the manufacture of Nordic ropy milks. Scandinavian fermented milk drinks display a firm thick, slimy consistency and these rely on the souring capacity of mesophillic ropy strains of Lactococcus lactis subsp. lactis and ssp. cremoris and concomitant production of heterotype EPS for texture. Early studies on the isolates from commercially produced Swedish ‘‘Langlfil” and Finnish ‘‘Viili’’ suggested involvement of a slime or capsular material in developing the typical viscoelastic properties of these products (Macura and Townsley, 1984).

The success of the application of an EPS is determined by its ability to bind water, interact with proteins, and to increase the viscosity of the milk serum phase. EPS may act as texturizers and stabilizers and consequently avoid the use of food additives. Creamy, smooth texture is one of the aspects of yoghurt quality, which seems to be improved by the ability of the yoghurt bacteria to produce EPS. In addition, EPS from LAB have one of the largest technical potentials for development of novel and improved products such as low-milk-solid yoghurts, low-fat yoghurts, creamier yoghurts and other fermented milk products. EPS producing lactic cultures are also useful for stirred fermented milk products as stirred products possess a smooth, creamy texture and are made by mild homogenization of the coagulum after fermentation. The presence of EPS in stirred-type fermented milks makes them less susceptible to mechanical damage from pumping, blending and filling machines. Mechanical processing steps also increase syneresis of the final product, but use of EPS producing LAB can help to control this defect.

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Effect of EPS producing lactic cultures for fermented dairy products

EPS produced by LAB have themselves no taste. These, however, increase the time that milk product spends in the mouth and hence impart an enhanced perception of taste through an improved volatilization of the intrinsic flavours.

Various investigations have been made to study the impact of EPS on fermented milks. It has been seen that the EPS produced by LAB modify the microstructure of fermented milks and these changes are then manifested as changes in rheological and other physical properties. The microstructure of set type fermented milks consists of a matrix of aggregated casein particles in which fat globules are embedded. The cavities of the gels are filled with serum and bacterial cells. In case of fermented milks containing EPS producing cultures, an envelope of EPS is observed surrounding the bacterial starter strains, by which ropy cells attach to the protein matrix via a web of filaments. It has been observed that the attachment of cells to the protein matrix is more pronounced in set-type fermented milks than in stirred products. Various reports have also suggested that the protein gels obtained with the ropy strains showed a homogenous structure with randomly distributed small cavities whereas gels obtained with non-ropy strains showed larger cavities filled with bacteria and serum (Zoon and van Marle, 1998; Khurana, 2006).

The use of ropy cultures, results in yoghurts with a more stable texture for shear and on subjecting to a shear force, changes occur in the microstructure of ropy yoghurt. An increase of shear rate disrupts the attachment of polymer to the bacterial surface. However since the polysaccharide material remains incorporated within the casein, it continues to influence viscosity of yoghurt. It has been observed that stirring of fermented milks induces the segregation of proteins and EPS, causing the EPS to self-interact and to collect in large agglomerates hence, accounting for the increased ropiness found in stirred products. The use of EPS producing starter cultures during fermentation results in a product with increased viscosity.

However, no clear correlation between this apparent viscosity and the concentration of EPS is found for stirred fermented milks produced with ropy, moderately ropy and non-ropy cultures. The physical property most immediately apparent in EPS producing yoghurts is the ropiness or extensibility of the product. Many studies have also indicated that use of EPS producing starter cultures resulted in yoghurts with decreased susceptibility to syneresis. This could be attributed to the high water-binding capacity of EPS as well as due to the modification of yoghurt microstructure by the use of EPS+ starter cultures.

Two types of microstructures have been generally found using Confocal Laser Scanning Microscopic studies in yoghurt. In one type, the EPS were associated with proteins surrounding the pores in the protein network, and in the other type, incompatibility between the EPS and protein seemed to exist causing the EPS to be placed inside the pores. These two types of microstructures correspond to separate sensory profiles. The yoghurts with EPS inside the pores were found to be less ropy, with higher serum separation and showed significant increase in mouth thickness as a result of stirring. In contrast, the yoghurts with EPS associated with protein had higher ropiness, lower serum separation and appeared to be more resistant to stirring.

Health benefits of EPS produced by lab

In addition to technological benefits certain EPS produced by LAB are also claimed to have beneficial physiological effects on the consumer. It is speculated that the increased viscosity of EPS containing foods may increase the residence time of ingested fermented milk in the gastrointestinal tract and therefore be beneficial to transient colonization by probiotic bacteria.

Another example of a suggested health benefit of some EPS is, the generation of short-chain fatty acids (SCFAs) upon degradation in the gut by the colonic microflora. SCFAs provide energy to epithelial cells and some have been claimed to play a role in the prevention of colon cancer. In vivo studies showed that EPS produced by Lactococcus lactis subsp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi 20 and L. helveticus 59 were not degraded by fecal microorganisms (Ruijssenaars et al., 2000).

Further beneficial health effects of EPS were postulated in the literature for e.g. an anti-tumor effect of EPS produced by Lactobacillus, a cholesterol-lowering effect by fermented milk ‘viili’ and immune-modulatory effects from Bifidobacterium adolescentis M101-4. It has also been found that the structure of a polysaccharide produced by Streptococcus macedonicus Sc136 that contains the trisaccharide sequence b-D-GlcpNAc-(1-3)-b-D-Galp(1-4)-b-D-Glcp which corresponds to an internal EPS backbone of lacto-N-tetraose and lacto-N-neotetraose. The same two trioses have also been identified in the structure of several human milk oligosaccharides that are important for healthy infant nutrition. It is thus quite convincing that the future will show a shift from pure technical and texturing applications of selected EPS to more and more targeted applications of specially developed EPS for improved consumer health benefits (Vincent et al., 2001).

Future challenges

Commercial application of exopolysaccharide producing strains for fermented milk manufacture must address specific problems. If the exopolysaccharide is to enable manufactures to reduce the amount of milk solids needed for a well-textured yoghurt, then the exopolysaccharide should positively influence the gel strength and the ‘‘yield value’’. If, however, there is a requirement to prevent syneresis, an exopolysaccharide that produces a softer gel and results in less whey drainage on application of external pressure will be required.

However, various investigators have reported via rheology and penetrometry studies that inclusion of a ropy strain will not always lead to improved texture attributes (Rawson and Marshall, 1997). Some reports also reveal that while ropy strains may increase viscosity and impart adhesiveness they might not influence firmness and elasticity that are more influenced by protein-protein interactions. Moreover, in yoghurt, which is a complex system we still need to determine how much of a particular exopolysaccharide is required for good viscosity and improved stability, as well as assessing acceptability for the consumer. Additionally, the cultures should also perform in a consistent manner in commercial fermentation vessels.

The genetic instability of EPS production is a serious problem to industrial applications. Several studies reported loss or reduction in production, or a change in the EPS composition (Bouzar et al., 1996). The instability was observed both for strains harboring plasmids encoding genes for EPS production and for thermophilic S. thermophilus and L. delbrueckii subsp. bulgaricus strains which do not contain such plasmids.

The benefits of EPS are detectable at extremely low concentrations. The aim is to obtain an appealing visual appearance (gloss) of a product, to prevent syneresis, to have a creamy and firm texture, and to give a pleasant mouth-feel. However, since production of one kind of EPS may not satisfy all texture specifications, the production of several EPS, by one or several starter cultures, may be required. As such it is possible to precisely tailor the texture of an end product and to match the consumer preferences that can vary from one country to another. The fermentation production process is a dynamic one, where exopolysaccharide synthesis will influence and be influenced by the dispersed milk polymers as fermentation progresses and finally consumer acceptance will be the ultimate test of the application.

References

  1. Bouzar F., Cerning J., Desmazeaud M. 1996 Exopolysaccaride production in milk by Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187 and by two colonial variants. J. Dairy Sci., 79: 205-211.
  2. Khurana, H.K. 2006. development of technology for extended shelf life fruit lassi. Ph.D. Thesis, NDRI Deemed University, Karnal (Haryana) – India.
  3. Macura, D. and Townsley, P.M. 1984. Scandinavian ropy milk: Identification and characterization of endogenous ropy lactic streptococci and their extracellular excretion. J. Dairy Sci., 67: 735–744.
  4. Rawson, H.L. and Marshall, V.M. 1997. Effect of ‘ropy’ strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt. Intl. J. Food Sci. Technol., 32: 213-220.
  5. Ruijssenaars, H. J., Stingele, F. and Hartmans, S. 2000. Biodegradability of food-associated extracellular polysaccharides. Current Microbiol., 40: 194-199.
  6. Sutherland, I.W. 1972. Bacterial exopolysaccharides. Adv. Microb. Physiol., 8:143-212.
  7. Vincent, S.J., Faber, E.J., Neeser, J.R., Stingele, F. and Kamerling, J.P. 2001. Structure and properties of the exopolysaccharide produced by Streptococcus macedonicus Sc136. Glycobiology, 11: 131–139.
  8. Zoon, P. and Van Marle, M.E. 1998. Relation between the consistency of stirred yoghurt and the structure of the yoghurt gel. Texture of fermented milk products and dairy desserts, May 5-6, 1997. Vicenza, Italy.
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