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Homopolysaccharide metabolism in Lactobacillus reuteri and Pediococcus parvulus

Årsköld, Emma LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Denna avhandling beskriver den centrala metabolismen och produktionen av exopolysackarider (EPS) hos två olika mjölksyrabakterier: Lactobacillus reuteri och Pediococcus parvulus. Mjölksyrabakterier används inom livsmedelsindustrin som startkulturer i olika mjölkbaserade produkter. Generellt utnyttjas en specifik metabol väg för nerbrytningen av sockerkällan till mjölksyra i dessa bakterier. Med hjälp av metabolisk flödesanalys och enzymmätningar visade sig L. reuteri ATCC 55730 innehålla två funktionella metabola vägar där merparten av flödet omvandlades via forsfoketolasvägen och resten via Embden-Meyerhof vägen. Förhållandet mellan NAD(P)H/NAD(P) i kombination med produktionen av extra energi... (More)
Popular Abstract in Swedish

Denna avhandling beskriver den centrala metabolismen och produktionen av exopolysackarider (EPS) hos två olika mjölksyrabakterier: Lactobacillus reuteri och Pediococcus parvulus. Mjölksyrabakterier används inom livsmedelsindustrin som startkulturer i olika mjölkbaserade produkter. Generellt utnyttjas en specifik metabol väg för nerbrytningen av sockerkällan till mjölksyra i dessa bakterier. Med hjälp av metabolisk flödesanalys och enzymmätningar visade sig L. reuteri ATCC 55730 innehålla två funktionella metabola vägar där merparten av flödet omvandlades via forsfoketolasvägen och resten via Embden-Meyerhof vägen. Förhållandet mellan NAD(P)H/NAD(P) i kombination med produktionen av extra energi spekuleras till att vara den bakomliggande kontroll mekanismen. Endast en mindre del av energikällan används till produktionen av EPS, vilket innebär att produktionen är för låg för att EPS ska kunna användas som ett naturligt förtjockningsmedel av livsmedelsindustrin. Optimering av odlingsbetingelserna för både



L. reuteri ATCC 55730 och P. parvulus 2.6 resulterade i en ökad produktionen av EPS. Kombinationen av ökad produktion av biomassa och ett överskott av socker hade en positiv effekt i båda stammarna, vilket gjorde att produktionen fortsatte in i den stationära tillväxt fasen. Produktionen av reuteransucrase, enzymet som är ansvarig för produktionen av EPS i L. reuteri ATCC 55730, visade sig vara kontinuerlig och aktivitets profilen var oberoende av sockerkällan. De viktigaste faktorerna för produktionen av EPS visade sig vara temperatur och pH och bästa produktion av EPS uppnåddes vid 37?C, pH 4.5 och 100 g sukros l-1. Utvecklingen av en matematisk tillväxtmodell resulterade i att under dessa förhållande bibehölls den maximala tillväxten medan sukros transporterades via underlättad diffusion. Högre koncentrationer av sukros resulterade i en lägre produktion av EPS vilket kan bero på den låga produktionen av biomassa och reuteransucrase. Produktionen av EPS i P. Parvulus 2.6 ökades genom att kombinera höga halter av mangan, glukos och etanol. Det visades sig att huvudprodukten mjölksyra inhiberar tillväxten genom att binda in manganet. Genom att öka koncentrationen av mangan i tillväxtmediet hävdes tillväxthämningen. (Less)
Abstract
This thesis deals with the central carbohydrate metabolism and exopolysaccharide (EPS) formation in two different lactic acid bacteria (LAB): Lactobacillus reuteri and Pediococcus parvulus. LAB are traditionally used as starter cultures in the food industry. In general, LAB harbours one glycolytic pathway converting carbohydrates into lactic acid. However, L. reuteri ATCC 55730 was proven to use two functional glycolytic pathways by metabolic flux analysis and enzyme measurements. Surprisingly, the main flux was directed through the phosphoketolase pathway, while the Embden-Meyerhof pathway was used merely as a shunt. A combination of the NAD(P)H/NAD(P) ratio and an extra energy gain is speculated to be the controlling mechanism.... (More)
This thesis deals with the central carbohydrate metabolism and exopolysaccharide (EPS) formation in two different lactic acid bacteria (LAB): Lactobacillus reuteri and Pediococcus parvulus. LAB are traditionally used as starter cultures in the food industry. In general, LAB harbours one glycolytic pathway converting carbohydrates into lactic acid. However, L. reuteri ATCC 55730 was proven to use two functional glycolytic pathways by metabolic flux analysis and enzyme measurements. Surprisingly, the main flux was directed through the phosphoketolase pathway, while the Embden-Meyerhof pathway was used merely as a shunt. A combination of the NAD(P)H/NAD(P) ratio and an extra energy gain is speculated to be the controlling mechanism. Furthermore, only a minor fraction of the utilised carbohydrates is directed into EPS formation. Therefore, the volumetric production of EPS is too low to make it feasible as a natural biothickener in the food industry. In this work enhanced production of EPSs was achieved in both Lactobacillus reuteri ATCC 55730 and Pediococcus parvulus 2.6 by optimisation of the bacterial cultivation conditions. The combination of high biomass yield and surplus of sugar had a positive effect on both bacteria, and resulted in continued production after entering the stationary phase. Reuteransucrase, the enzyme responsible for EPS formation in L. reuteri ATCC 55730, was proven to be constitutively expressed and the enzymatic activity profile was independent of the sugar source in the medium. Temperature and pH were found to be the most important factors for EPS formation. The best EPS production was obtained at 37?C, pH 4.5 and 100 g sucrose l-1. A growth model developed in this work indicates that maximal growth yield is maintained under these conditions while sucrose is taken up by facilitated diffusion. At higher sucrose concentrations lower EPS formation might be due to a relatively lower specific biomass yield and a lower expression of the sucrase gene. In P. parvulus 2.6 EPS production was increased by the combination of high manganese, glucose and ethanol levels. Lactate, the main fermentation product, chelates manganese therby inhibiting growth. This inhibition could be overcome by increasing the manganese concentration in the growth medium. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Fil. Dr Axelsson, Lars, MATFORSK, Norge
organization
publishing date
type
Thesis
publication status
published
subject
keywords
virology, bacteriology, Carbohydrate metabolism, Exopolysaccharides, Lactic acid bacteria, Mikrobiologi, bakteriologi, virologi, mykologi, mycology, Microbiology
pages
100 pages
publisher
Applied Microbiology (LTH)
defense location
Kemicentrum, hörsal A Getingevägen 60 Lunds Tekniska Högskola
defense date
2007-03-30 10:15
ISBN
978-91-628-7112-3
language
English
LU publication?
yes
id
46eadeb3-1479-4457-9498-d8ac14a4b0b5 (old id 548252)
date added to LUP
2007-10-09 09:43:50
date last changed
2016-09-19 08:45:14
@phdthesis{46eadeb3-1479-4457-9498-d8ac14a4b0b5,
  abstract     = {This thesis deals with the central carbohydrate metabolism and exopolysaccharide (EPS) formation in two different lactic acid bacteria (LAB): Lactobacillus reuteri and Pediococcus parvulus. LAB are traditionally used as starter cultures in the food industry. In general, LAB harbours one glycolytic pathway converting carbohydrates into lactic acid. However, L. reuteri ATCC 55730 was proven to use two functional glycolytic pathways by metabolic flux analysis and enzyme measurements. Surprisingly, the main flux was directed through the phosphoketolase pathway, while the Embden-Meyerhof pathway was used merely as a shunt. A combination of the NAD(P)H/NAD(P) ratio and an extra energy gain is speculated to be the controlling mechanism. Furthermore, only a minor fraction of the utilised carbohydrates is directed into EPS formation. Therefore, the volumetric production of EPS is too low to make it feasible as a natural biothickener in the food industry. In this work enhanced production of EPSs was achieved in both Lactobacillus reuteri ATCC 55730 and Pediococcus parvulus 2.6 by optimisation of the bacterial cultivation conditions. The combination of high biomass yield and surplus of sugar had a positive effect on both bacteria, and resulted in continued production after entering the stationary phase. Reuteransucrase, the enzyme responsible for EPS formation in L. reuteri ATCC 55730, was proven to be constitutively expressed and the enzymatic activity profile was independent of the sugar source in the medium. Temperature and pH were found to be the most important factors for EPS formation. The best EPS production was obtained at 37?C, pH 4.5 and 100 g sucrose l-1. A growth model developed in this work indicates that maximal growth yield is maintained under these conditions while sucrose is taken up by facilitated diffusion. At higher sucrose concentrations lower EPS formation might be due to a relatively lower specific biomass yield and a lower expression of the sucrase gene. In P. parvulus 2.6 EPS production was increased by the combination of high manganese, glucose and ethanol levels. Lactate, the main fermentation product, chelates manganese therby inhibiting growth. This inhibition could be overcome by increasing the manganese concentration in the growth medium.},
  author       = {Årsköld, Emma},
  isbn         = {978-91-628-7112-3},
  keyword      = {virology,bacteriology,Carbohydrate metabolism,Exopolysaccharides,Lactic acid bacteria,Mikrobiologi,bakteriologi,virologi,mykologi,mycology,Microbiology},
  language     = {eng},
  pages        = {100},
  publisher    = {Applied Microbiology (LTH)},
  school       = {Lund University},
  title        = {Homopolysaccharide metabolism in Lactobacillus reuteri and Pediococcus parvulus},
  year         = {2007},
}