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1One galactose residue occurs as N-acetygalactosamine

Box 4—3. Exopolysaccharide production by lactic acid bacteria (Continued) Production of Exopolysaccharides

Growth-associated EPS biosynthesis has been observed for several strains of LAB. In Streptococcus thermophilus LY03 and related strains, for example, there is a direct relationship between EPS yield and optimal growth conditions (de Vuyst et al., 1998). In contrast, EPS production in mesophilic LAB appears to be enhanced by sub-optimal temperatures, with maximum EPS production occurring at 20°C (Degeest and de Vuyst, 1999).The increase in EPS production when cells are growing at lower temperatures may be due to an increase in the number of isoprenoid lipid carrier molecules available for EPS formation, as occurs for Gram negative bacteria (Sutherland, 1972).Another possibility is that EPS synthesis may occur as a response to stress when cells are exposed to harmful or sub-optimal environments (Ophir and Gutnick, 1994). In either case, EPS production is likely correlated to EPS gene expression.

Gene organization for Exopolysaccharides

Genes encoding for EPS production exist as clusters, with specific structural, regulatory, and transport functions (Figure 1). In general, these gene clusters are often located on plasmids in mesophilic LAB and on the chromosome in thermophilic LAB (de Vuyst et al., 2001). Genes or gene clusters involved in LAB exopolysaccharide biosynthesis have been sequenced for several yogurt strains, as well as other mesophilic LAB (Broadbent et al., 2003; van Kranenburg et al., 1999b, 1999c).These clusters reveal a common operon structure, with the genes positioned in a particular order (Figure 1).

At the beginning of the gene cluster is epsA, which is suggested to be the region potentially involved in EPS regulation.The central region of the gene cluster, consisting of epsE, epsF, epsG, epsH, and epsI, encodes for enzymes predicted to be involved in the biosynthesis of the EPS repeating unit.The products of epsC, epsD, epsJ, and epsK are responsible for polymerization and export of the EPS. Interestingly, epsA, epsB, and epsC are also present in non-EPS producing

I I Regulation

I I Polymerization and Chain-length Determination

I I Biosynthesis of Repeating Unit

I I Polymerization and Export

I I Unknown, Not Specified or Outside of Cluster

Figure 1. Functional comparison of EPS genes from lactic acid bacteria. 1, Streptococcus thermophilus NCFB 2393; 2, Lactobacillus lactis NIZO B40; 3, Streptococcus thermophilus Sfi6; 4, Lactobacillus delbrueckii subsp. bulgaricus Lif5. Gene maps were adapted from Almirön-Roig et al. 2000; van Kranenburg et al. 1999a; Stingele et al. 1996; and Lamothe et al. 2002.

Box 4—3. Exopolysaccharide production by lactic acid bacteria (Continued)

strains of S. thermophilus, indicating the possibility that a spontaneous genomic deletion of an ancestral EPS gene cluster may have occurred (Bourgoin et al., 1999).This theory is supported by the observation of genetic instability, including deletions, in the S. thermophilus genome.

Regulation of Exopolysaccharide Genes

Production of EPS by LAB is often unstable and yields can be highly variable (Ricciardi and Clementi, 2000). Compared to other Gram positive bacteria, LAB generally produce lower amounts of EPS. Even when grown under optimized conditions, most LAB produce < 3 g per L of EPS, whereas other organisms make as much as 10 g to 15 g per L.Therefore, several approaches have been considered to improve EPS yield and stability (Boels et al., 2003a, 2003b; Levander et al., 2002; Stingele et al., 1999; Kranenburg et al., 1999a).

One strategy involves pathway engineering, such that carbon flow is diverted away from ca-tabolism and toward EPS biosynthesis. For example, synthesis of EPS ordinarily starts when glucose-6-phosphate is isomerized to glucose-1-phosphate by phosphoglucomutase (the product of the pgm gene).The glucose-1-phosphate is then converted, via UDP-glucose pyrophos-phorylase to UDP-glucose, which serves as a precursor for EPS biosynthesis. Over-expression of pgm or galU (encoding for UDP-glucose pyrophosphorylase) led to an increase in the EPS yield by S. thermophilus LY03 (Hugenholtz and Kleerebezem, 1999).

A second approach involves over-expression of EPS genes. For example, over-expression of the epsD gene (encoding the priming glycosyl transferase) in L. lactis resulted in a small increase in EPS production (van Kranbenburg et al., 1999c). Cloning the entire EPS gene cluster on a single plasmid with a high copy number could also have an effect of increasing EPS production, as shown for L. lactis NIZO B40, which produced four times more EPS than the parent strain (Boels et al., 2003).

References

Almiron-Roig, E., Mulholland, F., Gasson, M.J., and A.M. Griffin. 2000.The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide. Microbiol. 146:2793-2802.

Boels, I.C., M. Kleerebezem, and W.M. de Vos. 2003a. Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis.Appl. Environ. Microbiol. 69:1129-1135. Boels, I.C., R. van Kranenburg, M.W. Kanning, B.F. Chong, W.M. de Vos, and M. Kleerebezem. 2003b. Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps Gene Cluster.Appl. Environ. Microbiol. 69:5029-5031. Bourgoin, F,A. Pluvinet, B. Gintz, B. Decaris, and G. Guedon. 1999 Are horizontal transfers involved in the evolution of the Streptococcus thermophilus exopolysaccharide synthesis loci? Gene 233:151-161. Broadbent,J.R., D.J. McMahon, D.L.Welker, C.J. Oberg, and S. Moineau. 2003. Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review. J. Dairy Sci. 86:407-423.

Degeest, B. and L. De Vuyst. 1999. Indication that the nitrogen source influences both amount and size of exopolysaccharides produced by Streptococcus thermophilus LY03 and modelling of the bacterial growth and exopolysaccharide production in a complex medium.Appl. Environ. Microbiol. 65:28632870.

De Vuyst, L., and B. Degeest. 1999. Heteropolysaccharides from lactic acid bacteria. FEMS Microbiol. Rev. 23:157-177.

De Vuyst, L., and F. Vaningelgem. 2003. Developing new polysaccharides, p. 257—320. In B.M. McKenna (ed.), Texture in food, vol. 2. Semi-solid foods. Woodhead Publishing Ltd., Cambridge, United Kingdom. De Vuyst, L., F. De Vin, F. Vaningelgem, and B. Degeest. 2001. Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. Int. Dairy J. 11:687-707.

Box 4—3. Exopolysaccharide production by lactic acid bacteria (Continued)

De Vuyst, L., F. Vanderveken, S. Van de Ven, and B. Degeest. 1998. Production by and isolation of ex-opolysaccharides from Streptococcus thermophilus grown in milk medium and evidence for their growth-associated biosynthesis. J.Appl. Microbiol. 84:1059-1068.

Duboc, P., and B. Mollet. 2001. Applications of exopolysaccharides in the dairy industry. Int. Dairy J. 11: 759-768.

Forde,A., and G.F. Fitzgerald. 1999.Analysis of exopolysaccharide (EPS) production mediated by the bacteriophage adsorption blocking plasmid, pCl658, isolated from Lactococcus lactis subsp. cremoris HO2. Int. Dairy J.9:465-472.

Hugenholtz,J. and Kleerebezem, M. 1999. Metabolic engineering of lactic acid bacteria: overview of the approaches and results of pathway rerouting involved in food fermentations. Curr. Opin. Biotechnol. 10:492-497. Jolly, L., S.J.F. Vincent, I? Duboc, and J.-R. Nesser. 2002. Exploiting exopolysaccharides from lactic acid bacteria.Antonie van Leeuwenhoek 82:367-374.

Lamothe, G.T., L. Jolly, B. Mollet, and F. Stingele. 2002. Genetic and biochemical characterization of ex-opolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus. Arch. Microbiol. 178:218-228.

Levander, F., M. Svensson, and P. Radstrom. 2002. Enhanced exopolysaccharide production of metabolic engineering of Streptococcus thermophilus. Appl. Environ. Microbiol. 68:784-790.

Ophir,T. and Gutnick, D.L. 1994.A role for exopolysaccharides in the protection of microorganisms from desiccation.Appl. Environ. Microbiol. 60:740-745.

Ricciardi,A. and F. Clementi. 2000. Exopolysaccharides from lactic acid bacteria: structure, production and technological applications. Ital.J. Food Sci. 12:23-45.

Ruas-Madiedo, P. and C.G. de los Reyes-Gavilán. 2005. Methods for the screening, isolation, and characterization of exopolysaccharides produced by lactic acid bacteria. J. Dairy Sci. 88:843-856.

Ruas-Madiedo, P, J. Hugenholtz, and P Zoon. 2002.An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. Int. Dairy J. 12:163-171.

Stingele, F., J.-R. Neeser, and B. Mollet. 1996. Identification and characterization of the eps (exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.J. Bacteriol. 178:1680-1690.

Stingele, F., S.Vincent, E.J. Faber,J.W. Newell,J.P. Kamerling, and J.R. Nesser. 1999. Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide. Mol. Microbiol. 32:1287-1295.

Sutherland, I.W. 1972. Bacterial exopolysaccharides.Adv. Microbial Physiol. 8:143-213.

van Kranenburg, R., I.C. Boels, M. Kleerebezem, and W.M. de Vos. 1999a. Genetics and engineering of mi-crobial exopolysaccharides for food: approaches for the production of existing and novel polysaccha-rides. Curr. Opin. Biotechnol. 10:498-504.

van Kranenburg, R., I.I. van Swam, J.D. Marugg, M. Kleerebezem, and W.M. de Vos. 1999c. Exopolysaccharide biosynthesis in Lactococcus lactis NIZO B40: functional analysis of the glycosyltransferase genes involved in synthesis of the polysaccharide backbone. J. Bacteriol. 181:338-340.

van Kranenburg, R., H.R.Vos, I.I. van Swam, M. Kleerebezem, and W.M. de Vos. 1999b. Functional analysis of glycosyltransferase genes from Lactococcus lactis and other gram-positive cocci: complementation, expression, and diversity. J. Bacteriol. 181:6347-6353.

Welman,A.D., and I.S. Maddox. 2003. Exopolysaccharides from lactic acid bacteria: perspectives and chal-lenges.Trends Biotechnol. 21:269-274.

yogurt contain at least 108 viable organisms per gram at the time of manufacture.

Texture, body, and appearance defects are common in yogurt.Two of the more serious defects, weak bodied yogurt and free whey formation (i.e., wheying off), have several causes. They may occur when the solids content is too low or when the mix was pasteurized at too low of a temperature, both of which ultimately influence how much denatured whey proteins are present. The gel structure will shrink and syneresis will occur if the culture produces too much acid too fast during the fermentation, causing the pH to become too low. The same result will also occur if the temperature was lowered too rapidly during cooling. When yo gurt is mixed with fruit or other flavorings, in the case of Swiss or stirred style yogurts, or when stirred by consumers prior to consumption, the gel is further disrupted and thinning and syneresis occurs.

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