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Studies on regulation of the plantaricin 423 geneCohen, Francisca 12 1900 (has links)
Thesis (MSc) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing
organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds
are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow
spectrum of activity and are usually only active against bacteria from the same ecological niche.
The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as
natural food preservatives. The ideal would be to replace or reduce chemical preservatives such
as sulfur dioxide, nitrates and nitrites.
Bacteriocins are classified into four groups according to their structural and functional
characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable,
plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The
peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus
spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp.,
Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria
monocytogenes.
Production of bacteriocins may occur constitutively or may be regulated by a cell-density
dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic
growth, with no apparent change in production levels when the producer strain is cultured in the
presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe
that plantaricin 423 may be produced constitutively.
A reporter system was constructed which consisted of the plantaricin 423 promoter, P423,
fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed
plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B}
Despite several repeats, no luciferase activity was recorded and no RNA homologous to the
luxAB genes was detected.
The region necessary for expression of plantaricin 423 may be located stream-up of the -80
region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins.
Inclusion of the latter region in the reporter construct may result in the successful expression of
luxAB. / AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur
die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile
antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste
bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is
meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur
proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik
van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese
preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met
bakteriosiene.
Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele
karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum
423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder
die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer
Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni,
Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus
cereus, Clostridium spp. en Listeria monocytogenes.
Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids-
afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur
logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die
produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en
Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423
moontlik konstitutief geproduseer word.
'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die
luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde
plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum
(stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit
opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor
word nie.
Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van
die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II
bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot
die suksesvolle uitdrukking van luxAB.
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Characterisation of biogenic amine genes in lactic acid bacteria isolated from wineDowning, Lynn,1978- 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of
yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the
quality and wholesomeness of the final product. Yeasts are primarily responsible for
alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is
conducted by lactic acid bacteria. These bacteria are important in winemaking and
can have a positive or negative effect on the wine quality. Biogenic amines are one
of the compounds produced by lactic acid bacteria, which affect the hygienic quality
and wholesomeness of the wine negatively and directly pose a health risk to the
consumer. The demand of consumers for higher quality and healthier foods has led
to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide
variety of food products, such as cheese, dried sausage, sauerkraut, fishery
products, chocolates, wine and beer. This thesis focussed on the presence of
biogenic amines in wine.
The first objective of the study was to determine the ability of lactic acid bacteria
isolated from South African wine to produce biogenic amines, using a decarboxylase
screening plate method. The potential to produce the biogenic amines histamine,
tyramine, putrescine and cadaverine was investigated. The results obtained showed
that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be
the lactic acid bacteria responsible for tyramine and putrescine production and that it
can contribute significantly to the overall biogenic amine content in wines. The
results also suggest that amine production is strain dependent and not species
specific. None of the lactic acid bacteria tested had the ability to produce histamine
or cadaverine. It is important to remember that the ability of the lactic acid bacteria to
produce biogenic amines has only been investigated in synthetic media and that it
does not necessarily imply similar behaviour in wine. Wine represents a complex
environment with a wide number of factors influencing microbial growth and
decarboxylase activity and, thus, further investigation is necessary to determine if
these amine-producing bacteria behave similarly in wine conditions.
In addition, the polymerase chain reaction (PCR) amplification method was
used for the identification of the tyrosine decarboxylase (TOe) gene in some of the
tyramine-producing lactic acid bacteria. This was followed by the sequencing of the
amplified products, which are partial TOe gene sequences, of two L. brevis strains
and of a L. hilgardii strain. Only one tdc gene sequence has been described for
bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis
lOEB 9809 was described. An amino acid sequence alignment of the three TOe
gene fragments, obtained in this study, with the known TOe gene fragment of
L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of
relatedness and conserved regions.
To meet consumer demands, procedures are necessary to prevent the
formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species
involved in the winemaking process. Another possible way would be to develop a
rapid detection method for bacteria carrying amino acid decarboxylase genes. The
results of this study provide knowledge about which lactic acid bacteria in the
winemaking process could contribute to the production of biogenic amines and the
sequencing of additional partial TOe genes could possibly assist in the development
of a rapid detection method for tyramine-producing lactic acid bacteria in food
products. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van
giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit
en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir
alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en
word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak
van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê.
Biogeniese amiene is een van die komponente wat deur melksuurbakterieë
geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn
benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van
verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in
studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye
verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade,
wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in
wyn.
Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid-
Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op
dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene
histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die
resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en
Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat
hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in
wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die
ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die
vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in
ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs
in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn
sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat
die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere
studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde
gedrag in wyn sal toon.
Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie
van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende
melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling
van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee
L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir
bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde
vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie
TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en
gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809
en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van
amiene in voedselprodukte voorkom word. Een manier van voorkoming is om
amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke
is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die
opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van
die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die
produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele
gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige
opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte
bydra.
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Characterization of the adhesion genes of probiotic lactic acid bacteriaRamiah, Kamini 03 1900 (has links)
Thesis (PhD (Microbiology))--Stellenbosch University, 2008. / One of the key selection criteria for potential probiotics is the ability to adhere and
colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at
the host intestinal surface, preventing the colonisation of pathogens, thereby
protecting the host from infection. In addition, several important intestinal functions
are mediated by the binding of probiotics to host tissue. However, the molecular
mechanisms and genotypic characterization of adhesive elements have not received as
much attention as other aspects of probiotic research. The present study aims to
contribute to this area of research.
The first part of the study focused on monitoring the expression of mucus
adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of
Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of
Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile,
pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain
423 were up-regulated in the presence of mucus and expression increased under
increasing concentrations of mucus. Expression of mapA was up-regulated under
normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher
levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated
in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu
and plaA remained unchanged. At pH 4.0, the expression of mub and mapA
remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA
was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene
is regulated by a mechanism of transcription attenuation that involves cysteine. In the
case of L. acidophilus ATCC 4356, none of the genes were up-regulated under
increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated
under normal and stressful gut conditions in vitro.
In the second part of the study, male Wistar rats were used to evaluate which
section of the gastrointestinal tract are colonised by L. plantarum 423 and
Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ
hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated
strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum
and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time
PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon.
Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any
toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing
gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species
whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA
agglutinated yeast cells in vitro, indicating the possible presence of mannose
receptors. It is well known that these receptors play a crucial role in the elimination of
type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors
may have played a role in diminishing the enterobacterial content in the gut.
The third part of the study encompassed characterization of cell surface proteins of
L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the
typical surface layer protein whilst a multifunctional “intracellular” protein,
elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected.
Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by
40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to
competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and
Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of
C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and
90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells
by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by
60% and displaced from Caco-2 cells by 90%. Despite removal of the surface
proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells
and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG
13566 by 70%.
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Investigation of bacteriocins from lactic acid bacteria and their impact in winemakingKnoll, Caroline 12 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine.
The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation.
The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni.
The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected.
The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours.
The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed.
This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.
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