The production of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus

G. Grobben

Research output: Thesisinternal PhD, WU

Abstract

<p>Extracellular polysaccharides (EPS) produced by lactic acid bacteria have gained great popularity in the food industry because of their thickening and stabilizing properties. Since lactic acid bacteria have the GRAS ( <u>G</u> enerally <u>R</u> ecognized <u>A</u> s <u>S</u> afe) status, these organisms and their products can be applied as food additives. In particular in the dairy industry, EPS producing lactic acid bacteria are used to improve the texture and viscosity of fermented milk products, like yogurt, viili and långfil and the prevention of syneresis (wheying-off).</p><p>Many lactic acid bacteria producing EPS have already been studied, but since they have numerous medium requirements, including carbohydrates, amino acids, vitamins, purines, pyrimidines and mineral salts, most investigations on the production of EPS were performed using very complex growth media, like milk, whey ultrafiltrate or complex synthetic media. However, these media are often not suitable when the influence of individual medium components on growth and EPS production is studied. Therefore, a chemically defined growth medium is required, containing all components necessary for growth and EPS production.</p><p>In this thesis, the influence of physiological factors and the medium composition on the growth, EPS production and the sugar composition of the EPS produced by <em>Lactobacillus delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772 is studied in detail. This lactic acid bacterium is, together with <em>Streptococcus thermophilus</em> , used in the manufacture of yogurt. A chemically defined medium is composed, in which the strain was able to grow and produce EPS. The exopolysaccharides were composed of glucose, galactose and rhamnose in the ratio 1 : 6.8 : 0.7. The EPS production was growth-related, and an excess of carbohydrate did not result in an increased polymer production. It was found that production of EPS was higher with glucose or lactose as the carbohydrate source, than when fructose was used. The amount and composition of the EPS produced on glucose and lactose were identical, the galactose monomer from lactose was not used for growth and polymer production. Limiting amounts of manganese and phosphate in the growth medium resulted in a lower EPS production by <em>Lb. delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772. The amount of rhamnose monomer in the EPS was slightly lower than when manganese and phosphate were present in non-limiting concentrations.</p><p>The medium requirements of <em>Lb. delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772 were studied using the technique of single and multiple omissions of medium components. Surprisingly, a multiple omission of vitamins from the growth medium caused a significant enhancement of the EPS production. A simplified defined medium was composed, from which many components were omitted which were individually not required for growth. Although the strain grew less well in the simplified medium, the amount of EPS produced was twofold higher than in the complete medium. Furthermore, the EPS production beyond the stationary growth phase was stronger when grown in the simplified medium.</p><p>The influence of the carbohydrate source on the production of EPS and the activities of enzymes involved in the production of sugar nucleotides as precursors for EPS biosynthesis is described. <em>Lb. delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772 produced four times more EPS when grown on glucose than when fructose was used and it was found that the EPS produced on fructose contained no rhamnose monomer. Activities of enzymes involved in the synthesis of UDP-glucose, UDP-galactose and dTDP-rhamnose were observed when the strain was grown on glucose. But when grown on fructose, no activities were detected of enzymes leading to the production of dTDP-rhamnose and this may lead to the absence of rhamnose in the EPS produced on fructose. On the other hand, the amount of UDP-glucose and UDP-galactose in extracts of cells grown on fructose and glucose were comparable. Apparently the differences in the amount of EPS produced on glucose and fructose are not caused by the activities of the enzymes leading to the production of these sugar nucleotides.</p><em><p>Lb. delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772 grown on glucose produces two types of EPS concurrently and in almost equal amounts, with molecular weights of 1.7 × 10 <sup>6</SUP>and 4 × 10 <sup>4</SUP>. The exopolysaccharides produced with fructose as the carbohydrate source were composed of mainly a fraction with a low molecular weight of 4 x 10 <sup>4</SUP>. The high- molecular-weight fractions were composed of branched repeating units with glucose, galactose and rhamnose in the ratio 1 : 5 : 1, whereas the low-molecular-weight fractions were more linear and contained glucose, galactose and rhamnose in the ratio 1 : 11 : 0.4. The production of the high-molecular-weight fraction was dependent on the carbohydrate source, whereas the low-molecular-weight fraction was produced more continuously. Since a low amount of rhamnose monomer was found in the EPS produced on fructose, the enzymes involved in the production of dTDP-rhamnose are active, in contrast to the results obtained in Chapter 5, but these activities may be below the detection level of the enzyme assays performed.</p><p><em>Lb. delbrueckii</em> subsp. <em>bulgaricus</em> NCFB 2772 produces EPS when grown in a defined medium and the amount of EPS is dependent on the carbohydrate source and the presence or absence of several vitamins. For future research, it would be interesting to study the genetics and enzymology of the assembly of the different EPS repeating units in fructose-grown and glucose-grown cells and in cells grown at low optical densities. Furthermore, the mechanisms of the enhanced EPS production as a result of multiple vitamin omissions may be an interesting topic for future research.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • de Bont, J.A.M., Promotor
  • Sikkema, J., Promotor, External person
  • Smith, M.R., Promotor, External person
Award date22 May 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054858270
Publication statusPublished - 1998

Keywords

  • nutrition
  • disaccharides

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