Spelling suggestions: "subject:"dissertations -- line biotechnology"" "subject:"dissertations -- eine biotechnology""
71 |
Analysis of endo-polygalacturonase activity in a recombinant yeast containing a reconstituted PGU1 geneVan Wyk, Herine 03 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / The PGU1 gene encodes an endo-polygalacturonase, an enzyme that degrades pectin. Although the presence and function of this gene is well characterized in Saccharomyces cerevisiae, its regulation is very complex and not yet fully understood. Yeast producing a highly active polygalacturonase (PG) during alcoholic fermentation could potentially improve filtration and turbidity and also enhance extraction of certain aroma compounds. This could replace the addition of expensive commercial enzyme preparations that often contain unwanted enzymes.
The first objective of this study was to evaluate PGU1 expression in recombinant strains of S. cerevisiae that originally lacked the PGU1 gene. A functional PGU1 gene and its promoter were successfully re-introduced into their native position in the genomes of five wine strains. Three of these strains recovered PG activity while two did not transcribe the gene and subsequently lacked activity. The three strains that recovered activity were used in microvinification experiments to determine the effect of PG-producing yeast on the aroma profile of the wine. No significant differences were observed in the volatile compounds production between the recombinants and their respective wild types, but some tendencies arose, especially for the monoterpene geraniol.
The second objective of this study was to analyze the PGU1 gene and promoter from Saccharomyces paradoxus RO88 (a strain that exhibits high PG activity) and to compare it to those of S. cerevisiae S288C in order to identify differences that could potentially be responsible for the difference in their PG activities. Comparison of the gene sequences revealed several amino acid differences, one of which was in the peptide secretion signal. Analyses of the promoters also indicated some potentially important differences. Furthermore, S. cerevisiae strain VIN13, RO88 as well as two interspecies hybrids (all displaying varying PG activities) were compared under winemaking conditions. Clear differences were observed for the production of certain compounds. RO88 and the hybrids produced higher concentrations of certain volatile compounds, although they were not strong fermenters. Two recombinants, each containing a PGU1-overexpressing plasmid (one with the PGU1 gene from S. paradoxus and the other from S. cerevisiae), were also used in vinification to determine the effects of the different PGU1 gene on the aroma profile of the wine. Unfortunately, the plasmids were unstable and lost during the fermentation. Nevertheless, some tendencies were observed that indicated possible higher production of certain compounds by the recombinants compared to their wild types.
This study identified that regulation of the PGU1 gene differs between strains with different genetic backgrounds. Certain differences were observed in the PGU1 gene and promoter
sequences between S. cerevisiae and S. paradoxus that could potentially be the reason for the difference in their PG activities. From an oenological point of view, the presence of PGU1 in the genome of a fermenting strain tends to increase the aromatic potential of wine. These results provide a good platform for further studies on the PGU1 gene.
|
72 |
Factors influencing the fermentation performance of commercial wine yeastsFerreira, Jacques 12 1900 (has links)
Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The production of quality wine is influenced by numerous factors of which grape
quality is one of the most important factors. The production of quality wine, however,
is not possible without good winemaking techniques and effective quality control.
Critical control points (CCP) during the winemaking process must be identified to
ensure optimum wine quality. Grape must is a complex medium that contains
different micro-organisms which can be either beneficial or negative to wine quality,
depending on the physical and chemical conditions that prevail in the must. Yeasts
are responsible for alcoholic fermentation, lactic acid bacteria (LAB) for malolactic
fermentation (MLF) and acetic acid bacteria (AAB) for the production acetic acid from
ethanol. Yeasts and certain LAB can also produce acetic acid and thereby increasing
the volatile acidity (VA) of wine. These micro-organisms can influence each other in
complex fashions by competing for growth nutrients and by producing inhibitory
substances.
Most winemakers nowadays use commercial yeast strains to inoculate wine
fermentations. This, however, does not assure a problem-free fermentation and
cases of stuck and sluggish fermentations are annually reported worldwide. In these
or most cases fermentation takes longer than 21 days to complete and the wine
contains a residual sugar concentration of more than 4 g/L, which can be utilised by
wine spoilage micro-organisms such as certain bacteria and other wild yeasts. Stuck
and sluggish fermentations also increase the chances of oxidation due to the
absence of the protective CO2 layer on the surface of the wine, which is formed
during alcoholic fermentation. Another effect of stuck and sluggish fermentations is
that valuable tank space is wasted due to the unexpected time consumption of these
fermentation problems. Many factors during the winemaking process can be
responsible for stuck and sluggish fermentations. In this thesis the different factors is
discussed with the emphasis on the effect of the yeast strain. The way that certain
yeast strains influence AAB and LAB numbers during fermentation and MLF through
the production of inhibiting by-products such as medium chain fatty acids has not
been investigated in detail in the past.
Certain fungicides and pesticides that are used in vineyards to control pests (e.g.
mildew) contain copper which can be inhibiting to yeast growth and alcoholic
fermentation. Legal limits and withholding periods on these sprays are not always
strictly obeyed and can lead to stuck and sluggish fermentations. This motivated us
to evaluate the growth and fermentation activities of a selection of commercial wine
yeasts in the presence of copper levels in the range of maximum legal limits. The
effect of these commercial strains on the LAB and AAB numbers during alcoholic
fermentation and MLF were also investigated.
Our results showed that there was no significant difference on numbers of the
AAB obtained from fermentations inoculated with different commercial wine yeast strains. However, with regards to the LAB numbers, one of the strains produced
significantly more sulphur dioxide (SO2), which led to the inhibition of MLF in that
wine. Our results further indicated which commercial yeast strains were capable of
effectively fermenting high sugar musts and which strains were less effective. From
the strains tested VIN13, N96 & L2056 were able to utilize fructose more effectively
than NT50, RJ11 & D80. We could further distinguish between yeast strains that
produced the lowest (VIN13 & RJ11) and the highest (WE372, NT50 & L2056) VA
concentrations in must containing high sugar levels. Strains that were more tolerant
against high copper levels were also identified. We tested six yeast strains in must
with added copper (0.25 mM cu2+) in the form of CuSO4
.H2O. Three Cu2+-tolerant
(D80, Collection Cepage Cabernet & NT50) yeast strains were distinguished from
three less Cu2+-tolerant yeast strains (VIN13, NT112 & RJ11).
This study made a valuable contribution in knowledge gained about commercially
available wine yeast strains that can ferment effectively under certain stress
conditions. Research such as this, where wine yeasts are evaluated to ferment more
effectively during strenuous winemaking conditions, will be very beneficial to
winemakers. / AFRIKAANSE OPSOMMING: Die produksie van gehalte wyn word deur verskillende faktore beïnvloed waarvan
druifkwaliteit seker die belangrikste is. Die produksie van gehalte wyn is egter nie
moontlik sonder goeie wynmaaktegnieke en effektiewe kwaliteitsbeheer nie. Kritieke
kontrole punte (KKP) tydens die wynmaakproses moet dus geïdentifiseer word om
sodoende ‘n verlaging in wynkwaliteit te vermy. Druiwemos het ‘n komplekse
mikrobiologiese samestelling en bestaan uit verskillende mikroörganismes wat vooren
nadelig vir wynkwaliteit kan wees, afhangende van die fisiese en chemiese
toestande wat in die mos bestaan. Giste is verantwoordelik vir alkoholiese
fermentasie, melksuurbakterieë (MSB) vir appelmelksuurgisting (AMG) en
asynsuurbakterieë (ASB) vir die produksie van asynsuur vanaf etanol. Asynsuur word
egter ook deur giste en MSB geproduseer en dra so by tot die vlugtige suurheid (VS)
van ‘n wyn. Hierdie mikroörganismes kan mekaar op komplekse wyses beïnvloed
deur o.a. te kompeteer vir voedingstowwe asook deur die produksie van inhiberende
verbindings.
Die meeste wynmakers maak gebruik van kommersiële gisrasse om alkoholiese
fermentasies mee uit te voer. Gevalle van sogenaamde slepende en gestaakte
alkoholiese fermentasies, waar suiker nie volledig na etanol en CO2 omgeskakel
word nie, kom egter nog gereeld in die wynbedryf voor. In sulke gevalle neem die
fermentasie gewoonlik langer as 21 dae om te voltooi met ‘n suiker konsentrasie van
meer as 4 g/L wat in die wyn oorbly. Dit is nadelig vir wynkwaliteit aangesien dit nie
net die kanse vir bederf deur bakterieë en giste verhoog nie, maar ook die kanse vir
oksidasie verhoog a.g.v. die verlies van die beskermende CO2 lagie bo-oor die wyn.
Hoe sekere gisrasse, ASB en MSB getalle gedurende fermentasie en AMG
beïnvloed deur die produksie van inhiberende verbindings soos medium ketting
vetsure en SO2, is ook nie baie in die verlede ondersoek nie.
Sommige spuitstowwe wat gebruik word in die bekamping van swamsiektes
bevat koper wat inhiberend kan wees vir gisgroei en alkoholiese fermentasie. Wetlike
maksimum limiete en onthoudingsperiodes op spuitstofresidue word egter nie altyd
gehoorsaam nie en kan lei tot slepende en gestaakte fermentasies. Dit het ons
gemotiveer om ‘n seleksie van kommersiële gisrasse te evalueer in terme van
gisgroei en fermentasie in die teenwoordigheid van kopervlakke naby die maksimum
limiet.
Ons resultate het gewys dat daar nie noemenswaardige verskille in AAB getalle
tydens alkoholiese fermentasie tussen behandelings met verskillende kommersiële
gisrasse was nie. Een van die gisrasse het wel noemenswaardig meer SO2
geproduseer wat gelei het tot inhibering van AMG in hierdie wyn. Ons het verder
uitgewys watter kommersiële gisrasse instaat is om meer effektief in hoër suiker mos
te fermenteer en watter van die rasse minder suksesvol was. Ons het ook rasse
geïdentifiseer wat meer weerstandbiedend is teen hoë kopervlakke in mos en
sodoende groter kans op ‘n suksesvolle fermentasie sal hê in mos wat koperresidue bevat wat afkomstig is van sekere spuitstowwe. Die effek van die ASB en MSB
getalle gedurende fermentasie en AMG is ook ondersoek. Ons resultate het verder
gewys watter kommersiële gisrasse instaat was om mos met hoë suikervlakke meer
effektief te fermenteer. Vam die gisrasse wat getoets was het VIN13, N96 & L2056
fruktose meer effektief benut as NT50, RJ11 & D80. Ons kon verder onderskei
tussen gisrasse wat die laagste (VIN13 & RJ11) en die hoogste (WE372, NT50 &
L2056) vlakke van VS produseer in mos met hoë inisiële suikervlakke. Gisrasse wat
meer tolerant was teen koperresidue in mos is ook geidentifiseer. Ons het ses
gisrasse getoets in mos met bygevoegde koper (0.25 mM Cu2+) in die vorm van
CuSO4
.5H2O. Daar is onderskei tussen drie Cu2+-tolerante (D80, Collection Cepage
Cabernet & NT50) en drie minder Cu2+-tolerante gisrasse (VIN13, NT112 & RJ11).
Hierdie studie lewer ‘n waardevolle bydrae in die invordering van kennis oor
kommersieel beskikbare wyngisrasse wat meer effektief sal fermenteer onder sekere
streskondisies wat in mos voorkom. Inligting soos hierdie is belangrik om die
wynmaker se keuse uit die reeks bestaande kommersiële gisrasse te vergemaklik.
|
73 |
Co-expression of aroma liberating enzymes in a wine yeast strainDe Klerk, Daniel 03 1900 (has links)
Thesis (Msc (Viticulture and Oenology. Institute for Wine Biotechnology))--University of Stellenbosch, 2009. / Monoterpenes are important aroma compounds in certain grape varieties such as Muscat, Gewürztraminer and Riesling and are present as either odourless, glycosidically bound complexes or as free aromatic monoterpenes. These complexes occur as monoglucosides or, when present as diglycosides, most commonly as 6-O-α-L-arabinofuranosyl-β-D-glucopyranosides of mainly linalool, geraniol, nerol and citronellol.
The release of monoterpenes from non-volatile glycosidically bound precursors occurs either by acid hydrolysis or enzymatic hydrolysis. High temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones and a decrease in the aroma and changes in the aromatic characteristics of monoterpenes and is therefore not suitable. Enzymatic hydrolysis does not modify the monoterpene aglycones and can be an efficent method to release potentially volatile monoterpenes.
α-L-arabinofuranosidase and β-glucosidase are important enzymes responsible for the liberation of monoterpene alcohols from their glycosides. Glycosidases from Vitis vinifera and Saccharomyces cerevisiae are severely inhibited by winemaking conditions and this leads to unutilized aroma potential, while commercial preparations of aroma liberating enzymes are crude extracts that often have unwanted and unpredictable side effects. It is therefore of interest to investigate alternative measures to release glycosidically bound monoterpenes to increase the floral aroma of wine without side activities that impact negatively on wine.
Heterologous α-L-arabinofuranosidases and β-glucosidases have previously been expressed in S. cerevisiae and these studies have evaluated and found increased glycosidic activities against both natural and synthetic substrates.
In this study, we expressed the Aspergillus awamori α-L-arabinofuranosidase (AwAbfB) in combination with either the β-glucosidases Bgl2 from Saccharomycopsis fibuligera or the BglA from Aspergillus kawachii in the industrial yeast strain S. cerevisiae VIN13 to facilitate the sequential enzymatic hydrolysis of monoterpene diglycosides. Enzyme assays and GC-FID (Gas Chromatography with a Flame Ionization Detector) results show a significant increase in the amount of free monoterpene concentrations under winemaking conditions in the strain co-expressing the AwAbfB and the Bgl2. The increases in free monoterpene levels obtained were similar to those obtained with a commercial enzyme preparation, LAFAZYM AROM. Sensorial evaluation confirmed the improvement in the wine aroma profile, particularly the floral character. This yeast strain permits a single culture fermentation which improves the sensorial quality and complexity of wine. Further investigations on the factors influencing the stability and reactivity of monoterpenes during alcoholic fermentation are needed.
|
74 |
Over-expression and analysis of two Vitis vinifera carotenoid biosynthetic genes in transgenic ArabidopsisBrackenridge, Anika Elma 03 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / Plants have evolved photosynthetic systems to efficiently harvest sunlight energy for
the production of carbohydrates, but these systems also are extremely susceptible to
an excess of light. To combat the potential damaging effects of light, plants have
developed various mechanisms to control and cope with light stress. These
mechanisms include the movement of either leaves, cells (negative phototaxis) or
chloroplasts to adjust the light-capturing potential, the adjustment of the
light-harvesting antenna size through gene expression or protein degradation, the
removal of excess excitation energy either through an alternative electron transport
pathway or as heat. However, the latter mechanism based on thermal dissipation,
remains the most effective to rid the plant of damaging excess light energy. This
process involves several carotenoid pathway pigments, specifically the de-epoxidised
xanthophyll cycle pigments. The process and extent of thermal dissipation in plants
can be measured and quantified as non-photochemical quenching (NPQ) of
chlorophyll fluorescence by using well-established methodologies. Several
Arabidopsis and Chlamydomonas mutants affected in the xanthophyll cycle have
been isolated. These mutants have provided evidence for the correlation between
the de-epoxidised xanthophyll cycle pigments and NPQ as well as better
understanding of the operation of the xanthophyll cycle and the related carotenoid
biosynthetic enzymes. This key photoprotective role of the xanthophyll cycle is
therefore a promising target for genetic engineering to enhance environmental stress
tolerance in plants. Several genes from the carotenoid biosynthetic pathway of
grapevine (Vitis vinifera L.) were isolated previously in our laboratory. The main aim
of this study was to over-express two xanthophyll cycle genes from grapevine in
Arabidopsis and to analyse the transgenic population with regards to pigment content
and levels as well as certain photosynthetic parameters. The transgenic lines were
compared with wild type Arabidopsis (untransformed) plants and two xanthophyll
cycle mutants under non-limiting conditions as well as a stress condition, specifically
a high light treatment to induce possible photodamage and photoinhibition.
Transgenic Arabidopsis lines over-expressing the two V. vinifera xanthophyll
cycle genes, β-carotene hydroxylase (VvBCH) and zeaxanthin epoxidase (VvZEP),
were established following Agrobacterium transformation. In addition to the
untransformed wild type, two NPQ mutants, npq1 (lacking violaxanthin de-epoxidase)
and npq2 (lacking zeaxanthin epoxidase), were used as controls throughout this
study. The transgenic lines were propagated to a homozygous T3-generation, where
stable integration and expression of the transgenes were confirmed in only 16% and
12% for VvBCH and VvZEP lines, respectively. No phenotypical differences could be
observed for the transgenic lines compared to the wild type, but the npq2 mutant
showed a stunted and ‘wilty’ phenotype, as was previously described. To evaluate the pigment composition of the transgenic lines a reliable and
reproducible method was needed to analyse carotenoids from leafy material. To this
end a new high-performance liquid chromatography (HPLC) method was developed
for the quantitative profiling of eight major carotenoids and chlorophyll a and b.
Emphasis was placed on baseline separation of the xanthophyll pigments, lutein and
zeaxanthin as well as the cis- and trans-forms of violaxanthin and neoxanthin. The
method effectively distinguished Arabidopsis wild type plantlets from the two NPQ
mutant lines (npq1 and 2) and could possibly find application for green leafy tissue
samples in general.
The carotenoid content of the NPQ mutants were in accordance with previous
reports. The lack of zeaxanthin epoxidase activity in the npq2 mutant resulted in the
accumulation of zeaxanthin under both low and high light conditions. This high level
zeaxanthin was found to cause an initial rapid induction of NPQ at low to moderate
light intensities, but this difference disappeared at high light, where zeaxanthin
formation induced considerable NPQ in the wild type. Similarly, the npq1 mutant was
unable to de-epoxidise violaxanthin to zeaxanthin under high light conditions, which
resulted in severe inhibition of NPQ induction. Furthermore, these mutant plantlets
were shown to be more susceptible to photoinhibition compared to that of the wild
type.
The over-expression of VvBCH resulted in a marked increase in the
xanthophyll cycle pool pigments (violaxanthin, antheraxanthin and zeaxanthin) and
reduced β-carotene levels under both low and high light conditions compared to that
the wild type, indicating elevated β-carotene hydroxylase activity possibly due to
over-expression of the VvBCH gene. Similar to the induction of NPQ in the npq2
mutant, the increased levels of zeaxanthin in the VvBCH lines did not offer any
additional photoprotection. This would suggest that the heightened zeaxanthin levels
observed for the VvBCH lines do not necessarily enhance photoprotection, however
may protect the thylakoid membrane against lipid peroxidation as has been shown
previously. The VvZEP lines however, showed reduce levels of zeaxanthin in high
light conditions to that of the wild type, probably due to the competing epoxidation
and de-epoxidation reactions of the xanthophyll cycle. This reduction in zeaxanthin
synthesis in the VvZEP lines resulted in significant reduced NPQ induction compared
that of the wild type, a phenomenon also observed for the npq1 mutant. Similar to
the npq1 mutant, these lines displayed significantly increased photoinhibition, which
may be due to photodamage of the reaction centers if one considers the lowered
photosystem II photochemistry efficiency and reaction center openness of these lines
compared to the wild type. This may suggest that even small reductions in
zeaxanthin amounts can result in an increase in photoinhibition, under high light
conditions.
This study and its results provide fundamental information regarding two
grapevine-derived carotenoid pathway genes and their possible physiological roles.
Moreover, studies like these provide information that is essential when possible biotechnological approaches are planned with this central plant metabolic pathway in
mind. The results highlighted the complex regulation of this pathway, necessitating
attention to flux control, simultaneous manipulation of several pathway genes, and
the measurement of other compounds derived from this pathway when evaluating the
possible applications of the carotenoid pathway of plants.
|
75 |
Screening and characterisation of wine related enzymes produced by wine associated lactic acid bacteriaMtshali, Phillip Senzo 03 1900 (has links)
Thesis (Msc (Viticulture and Oenology))--University of Stellenbosch, 2007. / Among the factors contributing to wine complexity and quality, wine aroma is one of the
most important factors. Wine aroma is the outcome of interaction among different
compounds produced from the grapes, during fermentation as well as during the ageing
process. Apart from its origin from grapes, fungi and yeasts, wine aroma can also be
derived from the metabolic activity of wine lactic acid bacteria (LAB). These
microorganisms are usually associated with malolactic fermentation (MLF) which normally
occurs after alcoholic fermentation. MLF is beneficial to wine due to its contribution to
deacidification, microbiological stabilisation and wine aroma formation, with the latter being
the most important area of interest in our study. The production of volatile aromatic
components in wine can, in part, be achieved through the hydrolytic action of enzymes
produced by LAB associated with wine. These enzymes include β-glucosidase, protease,
esterase, lipase and glucanase. Most of the work done on bacterial enzymes has been on
LAB from food sources other than wine, in which these enzymes contribute to the flavour
development of some cheeses, yoghurt and other fermented foods. The activity of these
enzymes during wine fermentation has mostly been concerned with β-glucosidase from
Oenococcus oeni. Only in recent years has there been a renewed interest in evaluating
the activity of β-glucosidase in other genera of wine LAB.
The overriding goal of this study was to screen and characterise wine-related enzymes
produced by LAB associated with wine. All the LAB isolates tested in this study were
obtained from IWBT culture collection and were previously isolated from five different
wineries situated in the Western Cape region, South Africa. We first screened isolates
using classical methods. The isolates were grown on agar medium supplemented with
appropriate substrate analogues in order to evaluate the activity of enzymes (i.e. β-
glucosidase, glucanase, lipase and esterase). The colonies exhibiting enzymatic activity ...
|
76 |
The breeding of yeast strains for novel oenological outcomesMocke, Bernard A 12 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2005. / The quality of wine is influenced by a variety of factors, most noticeably the quality of
the grapes, winemaking practices and the yeast strains used for alcoholic fermentation.
Although several yeast strains are present in the must at the beginning of fermentation,
strains of S. cerevisiae quickly dominate and survive alcoholic fermentations. This
dominance of S. cerevisiae prompted research that led to the development of a
multitude of industrial yeast starter cultures. Starter cultures are usually capable of quick
and complete fermentations, with minimal production of deleterious substances such as
volatile acidity, H2S, SO2 and ethyl carbamate. Yeast strains should be able to survive
the stressful environment created during alcoholic fermentation, whilst possibly offering
novel oenological benefits such as pectinolytic activity, killer activity and malic acid
degradation. The increased production of volatile esters and higher alcohols may also
be desirable, as this will allow the production of wines that are more aromatic.
In this study, VIN13 was crossed with S. paradoxus strain RO88 and WE14 by using a
micomanipulator. VIN13 was chosen for its fast and complete fermentation ability and
moderate aroma production potential. Other factors such as the presence of killer
activity and low production of volatile sulphur compounds also favoured the selection of
VIN13. S. paradoxus strain RO88 was selected for its ability to degrade malic acid and
the favourable impact on aroma production during fermentation. Hybrids between these
yeasts may have the potential to produce more aromatic wines, with the added bonus of
pectinolytic activity and a strong fermentation capacity. The first crossing yielded 5
hybrids between VIN13 and S. paradoxus strain RO88. Two of these hybrids stood out
in the sense that they were able to degrade more malic acid than VIN13 and they also
possessed killer and pectinolytic activity. Cinsaut wine was made and the 2 hybrids
were shown to have higher aroma compound capacity than the parental yeasts. This
was also confirmed during sensory evaluation. The second crossing between VIN13
and WE14 yielded 10 hybrids with low H2S production potential and killer activity. WE14
was selected for its ability to produce very aromatic wines and also the slower
fermentation capacity. Hybrids between these yeast may have the potential to produce
wines with an increased aromatic content and the fermentation rate might be slower,
thereby improving the aroma profile of the wine. After microvinification, 5 hybrids were
selected on the basis of fermentation rate differing from that of the parental yeasts and
favourable oenological traits, such as fast and complete fermentation, high production of
glycerol and low production of volatile acidity. Pinotage wine was made and it was
shown that some of the hybrids produced more esters and higher alcohols than the
parental yeasts. Sensory evaluation also showed the aroma production potential of the
hybrids, as some of the hybrids were shown to score higher for banana, cherry and
tobacco characteristics.
|
77 |
Discriminating wine yeast strains and their fermented wines : an integrated approachOsborne, Charles D. 12 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / The discrimination between wine yeast strains as well as between their fermented wines
has been investigated in this pilot study. The study was divided in two parts, the first to
investigate the discrimination between wines fermented with five different
Saccharomyces cerevisiae yeast strains, analysed by gas chromatography (GC) and
Fourier transform infrared spectroscopy (FTIR) and the second part to investigate
discrimination between wine yeast strains in different liquid media and in dried form
using FTIR in transmission and attenuated total reflectance (ATR) modes.
Wines from three cultivars (Clairette Blanche, Pinotage and Cabernet Sauvignon)
that were fermented by five Saccharomyces cerevisiae yeast strains (VIN13, WE372,
VIN13-EXS, VIN13-PPK and ML01) were analysed by GC and FTIR. This analysis was
done on individual sample sets that consisted of the wines of each of the mentioned
cultivars and also on samples drawn throughout the ageing process of these wines. The
data obtained were analysed by PLS-Discrimination (PLS-discrim), a chemometric
method. Using the data from both the analytical methods, discrimination was observed
between wines fermented with different yeast strains in each of the two vintages (2005
and 2006) for all the cultivars. When combining the data from the two vintages no
discrimination could be observed between the fermented wines. The discrimination of
the fermented wines was found to be similar when using data from GC and FTIR,
respectively. Since analysis with FTIR is considerably faster than analysis by GC, it
would be recommended that FTIR is used for future studies of similar nature.
Combining the samples into one set consisting of wines fermented with commercial wine
yeast strains and wines fermented from closely related wine yeast strains (the parental
strain and two genetically modified versions thereof (VIN13, VIN13-EXS and VIN13-
PPK), those fermented with closely related stains did not show good discrimination from
each other. Discrimination was found between wines fermented with genetically
modified (GM) wine yeast strains and those fermented with non-GM wine yeast strains.
This was done on a limited number of yeast strains and a larger study is needed to
confirm these results. As this is the first study of this nature and differences seen could
be as result of the different phenotypes.
It was shown that it is possible to use both FTIR-transmission and FTIR-ATR
(attenuated total reflectance) to discriminate between different wine yeast strain
phenotypes. It was shown that when using FTIR-transmission there is discrimination
between yeast samples suspended in yeast-peptone-dextrose (YPD) and in water.
Dried yeast samples could be discriminated when the yeast samples were in a granular, powder form or in a pellet form, using FTIR-ATR. It was possible to discriminate
between the closely related yeast strain phenotypes using FTIR-ATR.
In this pilot study it was shown that there can be discriminated between different wine
yeast strains and also between the wines fermented with different wine yeast strains. It
is recommended that further studies be conducted to refine and expand the study.
|
78 |
The evaluation of Fourier transform infrared (FT-IR) spectroscopy for quantitative and qualitative monitoring of alcoholic wine fermentationMagerman, Cynthia M 12 1900 (has links)
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Fermentation is a complex process in which raw materials are transformed into high-value
products, in this case, grape juice into wine. In this modern and economically competitive
society, it is increasingly important to consistently produce wine to definable specifications and
styles. Process management throughout the production stage is therefore crucial to achieve
effective control over the process and consistent wine quality. Problematic wine fermentations
directly impact on cellar productivity and the quality of wine. Anticipating stuck or sluggish
fermentations, or simply being able to foresee the progress of a given fermentation, would be
extremely useful for an enologist or winemaker, who could then take suitable corrective steps
where necessary, and ensure that vinifications conclude successfully. Conventional methods of
fermentation monitoring are time consuming, sometimes unreliable, and the information limited
to a few parameters only. The current effectiveness of fermentation monitoring in industrial wine
production can be much improved. Winemakers currently lack the tools to identify early signs of
undesirable fermentation behaviour and to take preventive actions.
This study investigated the application of Fourier transform mid infrared (FT-IR)
spectroscopy in transmission mode, for the quantitative and qualitative monitoring of alcoholic
fermentation during industrial wine production. The major research objectives were firstly to
establish a portfolio of quantitative calibration models suitable for quantification of the major
quality determining parameters in fermenting must. The second major research objective
focused on a pilot study aimed at exploring the use of off-line batch multivariate statistical
process control (MSPC) charts for actively fermenting must. This approach used FT-IR spectra
only, for the purpose of qualitative monitoring of alcoholic fermentation in industrial wine
production. Towards these objectives, a total of 284 industrial-scale, individual, actively
fermenting tanks of the seven major white cultivars and blends, and nine major red cultivars, of
Namaqua Wines, Vredendal, South Africa, were sampled and analysed with FT-IR
spectroscopy and appropriate reference methods during vintages 2007 to 2009.
For the quantitative strategy, partial least squares regression (PLS1) calibration models for
determination of the classic wine parameters ethanol, pH, volatile acidity (VA), titratable acidity
(TA) and the total content of glucose plus fructose, were redeveloped to provide a better fit to
local South African samples. New PLS1 models were developed for the must components
glucose, fructose and yeast assimilable nitrogen (YAN), all of which are frequently implicated in
problem fermentations. The regression statistics, that included the standard error of prediction
(SEP), coefficient of determination (R2) and bias, were used to evaluate the performance of the
redeveloped calibration models on local South African samples. Ethanol (SEP = 0.15 %v/v, R2 =
0.999, bias = 0.04 %v/v) showed very good prediction and with a residual predictive deviation
(RPD) of 30, rendered an excellent model for quantitative purposes in fermenting must. The
models for pH (SEP = 0.04, R2 = 0.923, bias = -0.01) and VA (SEP = 0.07 g/L, R2 = 0.894, bias
= -0.01 g/L) with RPD values of 4 and 3 respectively, showed that the models were suitable for
screening purposes. The calibration model for TA (SEP = 0.35 g/L, R2 = 0.797, bias = -0.004
g/L) with a RPD of 2, proved unsatisfactory for quantification purposes, but reasonable for
screening purposes. The calibration model for the total content of glucose plus fructose (SEP =
0.6.19 g/L, R2 = 0.993, bias = 0.02 g/L) with a RPD of 13, showed very good prediction and can
be used to quantify total glucose plus fructose content in fermenting must. The newly developed
calibration models for glucose (SEP = 4.88 g/L, R2 = 0.985, bias = -0.31 g/L) and fructose (SEP
= 4.14 g/L, R2 = 0.989, bias = 0.64 g/L) with RPD values of 8 and 10 respectively, also proved fit
for quantification of these important parameters. The new calibration models of ethanol, total
glucose plus fructose; and glucose and fructose individually, showed an excellent relation to
local South African samples and can be easily implemented by the wider wine industry.
Two calibration models were developed to determine YAN in fermenting must by using
different reference methods, namely the enzyme-linked spectrophotometric assay and Formol
titration method, respectively. The results showed that enzyme-linked assays provided a good
quantitative model for white fermenting must (SEP = 14.10 mg/L, R2 = 0.909, bias = -2.55 mg/L,
RPD = 6), but the regression statistics for predicting YAN in red fermenting must, were less
satisfactory (data not shown). The Formol titration method could be used successfully in both
red- and white fermenting must (SEP = 16.37 mg/L, R2 = 0.912, bias = -1.01 mg/L, RPD = 4). A
minor, but very important finding was made with respect to the storage of must samples that
were taken from tanks, but that could not immediately be analysed with FT-IR spectroscopy or
reference values. Principal component analysis (PCA) of frozen samples showed that must
samples could be stored frozen for up to 3 months and still be used to expand the calibration
sample sets when needed. Therefore, samples can be kept frozen to a later stage if immediate
analyses are not possible.
For the purpose of the pilot study that focused on the use of FT-IR spectroscopy for
qualitative off-line monitoring of alcoholic fermentation, a total of 21 industrial-scale fermentation
tanks were monitored at 8- or 12-hourly intervals, from the onset of fermentation to complete
consumption of the grape sugars. This part of the work excluded quantitative data, and only
used FT-IR spectra. MSPC charts were constructed on the PLS scores of all the FT-IR spectra
taken at the various time intervals of the different batches, using time as the y-variable. The
primary aim of this research objective was to evaluate if the PLS batch models could be used to
discriminate between normal and problem alcoholic fermentations. The models that were
constructed clearly showed the variations in patterns over time, between red- and white wine
alcoholic fermentations. One Colombar tank that was fermented at very low temperature in
order to achieve a specific wine style, was characterised by a fermentation pattern that clearly
differed form the rest of the Colombar fermentations. This atypical fermentation was identified
by the batch models constructed in this study. PLS batch models over all the Colombar
fermentations clearly identified the normal and problem fermentations.
The results obtained in this study showed that FT-IR spectroscopy showed great potential
for effective quantitative and qualitative monitoring of alcoholic fermentation during industrial
wine production. The work done in this project resulted in the development of a portfolio of
calibration models for the most important quality determining parameters in fermenting must.
The quantitative models were subjected to extensive independent test set validation, and have
subsequently been implemented for industrial use at Namaqua Wines. Multivariate batch
monitoring models were established that show good discriminatory power to detect problem
fermentations. This is a very useful diagnostic tool that can be further developed by monitoring
more normal and problem fermentations. Future work in this regard, will focus on further
optimisation and expansion of the quantitative and qualitative calibration models and
implementation of these in the respective wineries of Namaqua Wines. / AFRIKAANSE OPSOMMING: Fermentasie is ‘n komplekse proses waartydens rou material getransformeer word na produkte
van hoë waarde, in hierdie geval, druiwesap na wyn. In die huidige ekonomies-kompeterende
samelewing, is dit al hoe meer belangrik om volhoubaar wyn te produseer wat voldoen aan
definieerbare spesifikasies en style. Goeie prosesbestuur tydens die wynproduksie stadium is
baie belangrik om herhaalbaarheid en gehaltebeheer te verseker. Problematiese
wynfermentasies het ’n direkte impak op beide kelderproduktiwiteit en wynkwaliteit. Die
voorkoming van slepende- of steekfermentasies, of selfs net om probleme te voorsien, sou
uiters bruikbaar wees vir ‘n wynkundige of wynmaker, wat dan die toepaslike regstellende
stappe kan neem waar nodig, om te verseker dat die wynbereiding suksesvol voltooi word.
Konvensionele metodes van monitering van alkoholiese fermentasie is tydrowend, soms
onbetroubaar en die inligting beperk tot ‘n paar parameters. Die huidige effektiwiteit van
fermentasie monitering in industriële wynproduksie kan heelwat verbeter word. Wynmakers
ervaar tans ’n behoete aan tegnologië wat die vroeë tekens van ongunstige fermentasiepatrone
kan identifiseer, en hul doeltreffendheid om moontlike regstellende aksies te neem, is dus
beperk.
Hierdie studie het die toepassing van Fourier transformasie mid-infrarooi (FT-IR)
spektroskopie in transmissie, ondersoek met die oog op kwantitatiewe en kwalitatiewe
monitering van alkoholiese gisting tydens industriële wynproduksie. Die vernaamste
navorsingsdoelwitte was eerstens om ’n portefeulje van kwantitatiewe kalibrasiemodelle te
vestig, wat geskik is om die belangrikste kwaliteitsbepalende parameters in gistende mos te
kwantifiseer. Die tweede hoofnavorsingsdoelwit was ’n loodsstudie wat ondersoek ingestel het
na die opstel van multiveranderlike statistiese proseskontrole grafieke van aktief-gistende mos,
met die oog op aflyn-kwalitatiewe monitering van alkoholiese gisting in industriële
wynproduksie. Hiervoor is slegs FT-IR spektra gebruik. Vir die doel van hierdie studie is
monsters van ’n totaal van 284 individuele, aktief-gistende tenke van die sewe hoof wit kultivars
en hul versnydings en nege hoof rooi kultivars van Namaqua Wyne, Vredendal, Suid Afrika,
geneem. Al die monsters is met toepaslike chemiese metodes en FT-IR spektroskopie analiseer
tydens die parsseisoene van 2007 tot 2009.
Vir die kwantitatiewe strategie is parsiële kleinste kwadraat (PKK1) kalibrasiemodelle vir die
bepaling van die klassieke wynparameters etanol, pH, vlugtige suur (VS), titreerbare suur (TS)
en die totale konsentrasie van glukose plus fruktose herontwikkel, om beter te pas op plaaslike
Suid-Afrikaanse monsters. Nuwe PKK1 kalibrasiemodelle is ontwikkel vir die komponente
glukose, fruktose en gis-assimileerbare stikstof, aangesien hierdie komponente gereelde
aanduidings van probleemgisting is. Die regressiestatistieke het die standaardvoorspellingsfout
(SVF), bepalingskoëffisiënt (R2) en sydigheid ingesluit en was gebruik om die prestasie van die
herontwikkelde kalibrasiemodelle vir plaaslike Suid-Afrikaanse monsters te evalueer. Etanol
(SVF = 0.15 %v/v, R2 = 0.999, sydigheid = 0.04 %v/v) het baie goeie regressiestatistiek getoon
en met ‘n relatiewe voorspellingsafwyking (RVA) van 30, was dit ‘n uitstekende model vir
kwantifisering in gistende mos. Die modelle vir pH en VS met RVA waardes van 4 en 3
onderskeidelik, is geskik vir semi-kwantitatiewe toepassings. Die kalibrasiemodel vir TS met ‘n
RVA waarde van 2, was nie geskik vir akkurate kwantifisering nie, maar wel vir semikwantitatiewe
analises. Die kalibrasiemodel vir die totale glukose plus fruktose inhoud in
gistende mos, met ‘n RVA waarde van 13, het uitstekende regressiestatistiek gegee en is
geskik vir akkurate kwantifiseringsdoeleindes. Die nuut-ontwikkelde kalibrasiemodelle vir
glukose en fruktose, met RVA waardes van onderskeidelik 8 en 10, is geskik vir akkurate
kwantifisering van hierdie belangrike parameters. Die kalibrasiemodelle vir etanol, totale
glukose plus fruktose, en glukose en fruktose afsonderlik, het uitstekende korrelasies getoon
met plaaslike Suid-Afrikaanse monsters en is gereed om toepassing te vind in die wyer
wynindustrie.
Twee kalibrasiemodelle is ontwikkel om gis-assimileerbare stikstof in gistende mos te
bepaal, deur gebruik te maak van verskillende verwysingsmetodes van analise; hierdie metodes
was ‘n ensiem-gekoppelde spektrofotometriese toets en die Formoltitrasie metode. Resultate
het getoon dat goeie regressiestatistiek vir FT-IR spektroskopie-gebaseerde kalibrasiemodelle
waar data wat met die ensiem-gekoppelde toetse verkry is, as verwysingwaardes gebruik is, in
wit gistende mos (SVP = 14.10 mg/L, R2 = 0.909, sydigheid = -2.55 mg/L, RVA = 6), maar nie in
rooi gistende mos nie. Die Formoltitrasie metode as verwysingsmetode, was geskik vir die
ontwikkeling van goeie kalibrasiemodelle in beide rooi- en wit gistende mos (SVP = 16.37 mg/L,
R2 = 0.912, sydigheid = -1.01 mg/L, RVA = 4). ’n Sekondêre, maar baie belangrike bevinding is
gemaak met betrekking tot die stoor van mosmonsters wat geneem is van tenke, maar wat nie
dadelik met die verwysingsmetodes en FT-IR spektroskopie analiseer kon word nie.
Multiveranderlike hoofkomponentanalise op vars en gevriesde sapmonsters het getoon dat
gevriesde monsters gebruik kan word om die kalibrasie datastel uit te brei, wanneer benodig.
Dus, sapmonsters kan gevries word tot ’n later stadium as onmiddelike analises nie moontlik is
nie.
Vir die doel van die tweede navorsingsdoelwit van die studie, naamlik kwalitatiewe af-lyn
monitering van alkoholiese fermentasie met FT-IR spektroskopie, is ‘n totaal van 21 industriëlegrootte
fermentasietenks ge-monitor deur sapmonsters met 8- tot 12-uurlikse intervalle te trek,
vanaf die begin van fermentasie, totdat al die druifsuiker gemetaboliseer is. Vir hierdie deel van
die werk is die kwantitatiewe data nie gebruik nie; slegs die FT-IR spektra. Multiveranderlike
statistiese proseskontrole grafieke is opgestel op grond van die PKK tellings wat bereken is op
al die FT-IR spektra wat gemeet is by die verskillende tydsintervalle. Vir hierdie analise is tyd as
y-veranderlike gebruik. Die vernaamste doel van hierdie ondersoek was om te evalueer of die
PKK-gebaseerde modelle kon onderskei tussen normale en slepende gistings. Die modelle wat
verkry is, het die variasie oor tyd in die fermentasiepatrone tussen wit- en rooiwyn fermentasies
tydens alkoholiese gisting, duidelik uitgewys. Een Colombar tenk wat teen baie lae temperatuur
gefermenteer is om ‘n spesifieke wynstyl te verkry, se fermentasiepatroon het aansienlik verskil
van die ander Colombar tenks wat gemonitor is, en hierdie atipiese patroon is ook deur die
kwalitatiewe modelle identifiseer. ‘n PKK model oor al die Colombar fermentasies kon duidelik
tussen normale en slepende gistings onderskei.
Die resultate wat in hierdie studie verkry is, het getoon dat FT-IR spektroskopie baie goeie
potensiaal toon vir die aanwending van kwantitatiewe en kwalitatiewe monitering van
alkoholiese fermentasie tydens industriële wynproduksie. Die werk wat in hierdie projek gedoen
is, het gelei tot die vestiging van ‘n portefeulje van kalibrasiemodelle vir die belangrikste
kwaliteitsbepalende parameters in fermenterende mos. Die kwantitatiewe modelle is baie
deeglik getoets met onafhanlike toets datastelle, en daarna is die kalibrasiemodelle geimplementeer
vir industriële gebruik by Namaqua Wyne. Multiveranderlike statistiese
proseskontrole grafieke wat baseer is op data wat vanaf 21 verskillende fermentasietenks
verkry is, het baie goeie potensiaal getoon om probleemfermentasies vroeg te identifiseer. Dié
grafieke is ‘n baie nuttige diagnostiese hulpmiddel wat verder ontwikkel kan word om
verskillende tipes probleemfermentasies te monitor. Toekomstige navorsing in hierdie konteks,
sal toegespits word op die optimisering en uitbreiding van die kwantitatiewe en kwalitatiewe
modelle, sowel as toepassing van die tegnieke in die onderskeie kelders van Namaqua Wyne.
|
79 |
Overexpression and evaluation of an antimicrobial peptide from Heuchera sanguinea (Hs-AFP1) for inhibition of fungal pathogens in transgenic tabaccoDe Beer, Abre 04 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Seed germination is the most vulnerable time in a plant's life cycle, since the thick protective
seed coat ruptures and the moist and humid soil environment not only favours seed
germination, but also the growth and development of plant pathogens. Infection of plant seeds
during germination, however, is the exception rather than the rule. Plant seeds have
- - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuse
into the surrounding environment to form a protective layer around the seed. This
protective layer prevents fungal and bacterial pathogens from infecting the young seedling.
Over the last decade, scientists have studied the defense mechanisms of different
seeds in an effort to understand and ultimately to introduce and/or manipulate these
mechanisms in plants as part of the plant's endogenous disease resistance to pathogens.
Various chemical compounds, peptides and proteins that showed strong in vitro activities
against various fungi were isolated in these efforts. The mere demonstration of in vitro activity
alone, however, is not sufficient to assign a defense role to these antifungal agents. Typically,
mutant plants that have lost the ability to produce the antifungal agent, or mutants that are
overproducing the agent, have been used to correlate the mutant phenotype to either a
decline or increase in disease resistance respectively. Genetic transformation and the
subsequent development of transgenic plants have made an unprecedented impact in this
regard, specifically in understanding the role of specific defense-related proteins and their
interaction with plant pathogens.
In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plant
defensin, was evaluated in a heterologous in planta environment as a defense protein with
potential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1
against Botrytis cinerea showed antifungal activities of 88% growth inhibition at a
concentration of 8 J,lg/ml of the purified peptide, while inducing a characteristic
hyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with a
construct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. The
peptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, an
antimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenic
plant systems, two additional constructs were prepared and transformed into tobacco to
anticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putative
peptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and the
antifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A control
construct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2
signal peptide, was also prepared and transformed into tobacco to normalize the background
antifungal activity as a result of the exoglucanase in the fusion construct lines. Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1,
resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgene
expression was confirmed for the THs and THE transgenic lines. The translation of these
transcripts into proteins was also confirmed with Western blot analysis. Moreover, the
heterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance to
B. cinerea in the THs lines in comparison with the untransformed tobacco controls. An
increase of up to 42% in disease resistance was observed in an in planta detached leaf
assay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitative
fungal growth assay. This assay confirmed the results obtained with the disease resistance
assay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. The
incubation of B. cinerea in the presence of crude protein extracts from THs lines resulted in
hyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity.
From these analyses it was clear that the heterologously expressed Hs-AFP1 was
quite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did not
increase the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All the
analyses performed showed the THE lines to be reduced in their ability to inhibit fungal
infection in comparison to the THs line. Also, microscopic analysis of the effects of the crude
THE extracts on B. cinerea growth showed no hyperbranching activity, again confirming the
loss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, in
which sarcotoxin 1A was fused to a reporter gene and also lost activity.
Although integration of the Mj-EXG1 expression cassette was confirmed, no mRNA
levels could be detected with Northern blot or RT-PCR analysis of the TME lines. These lines
also did not show any in vitro antifungal activities, probably indicating post-transcriptional
gene silencing. This silencing was overcome in the fusion constructs that were expressed in
the THE plant lines. These lines also showed EXG1 protein activity, as measured by
~-glucosidase assays. Although the THE lines did not serve the functions originally
envisaged, they fortuitously showed that a fusion strategy might stabilize glucanase
expression in a transgenic environment. A variety of glucanases have been shown to be
prone to gene silencing when overexpressed in a plant environment and the yeast glucanase
can now be added to that list if it is not present as a fusion protein.
Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems and
provided valuable information on the stability of small peptides in a heterologous environment.
The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this study
merits further investigations into the use of this peptide in the engineering of disease-resistant
crops. / AFRIKAANSE OPSOMMING: Saadontkieming is die mees vatbare tyd vir siekteontwikkeling gedurende 'n plant se
lewenssiklus. Die saadhuid bars en die vogtige grondkondisies bevoordeel nie net
saadontkieming nie, maar ook die groei en ontwikkeling van plantpatogene. Infeksie van
plantsade tydens ontkieming is egter die uitsondering eerder as die reël. Plantsade besit
komplekse -veraeaigingsfueganfsmes-reen moontlike - patoqeeninteksies. Die meqanismes
sluit die produksie van antifungiese agense, wat tydens saadontkieming na die omliggende
omgewing diffundeer om 'n beskermende sone om die ontkiemende saad te vorm, in. Die
gevolglike antifungiese sone beskerm die saad teen infeksie deur bakterieë en swamme.
Gedurende die laaste dekade het navorsers baie aandag aan die bestudering van
plantsaadverdedigingsmeganismes gegee. Dié kennis word gebruik om die verdedigingsmeganismes
beter te verstaan, asook om dié meganismes te manipuleer en/of oor te dra aan
plantspesies met inherente swak weerstandsmeganismes wat gereeld aan
plantpatogeeninfeksies onderhewig is. Navorsing op plantsade het tot die isolasie van
verskeie chemiese agense, peptiede en proteïene, wat sterk in vitro aktiwiteite teen 'n wye
reeks swampatogene vertoon, gelei. Die vermoë van dié agense om swamme in 'n in vitro
omgewing te inhibeer, is alleen egter nie 'n bewys dat hulle 'n rol in plantverdeging speel nie.
Studies waar mutante gebruik word, is gewens om addisionele bewys te lewer dat die
substanse 'n rol in plantverdediging vervul. Sodanige mutante sluit plantlyne, waarin die geen
van belang gemuteer is of ooruitgedruk word om so die rol van die geen in 'n in planta
omgewing te bepaal in. In hierdie toepassings het genetiese transformasie en die daarstelling
van transgeniese plante 'n ongeëwenaarde bydrae gelewer.
In dié studie is die antifungiese peptied, Hs-AFP1, wat aan die peptiedgroep van plant-
"defensins" behoort en van Heuchera sanguine a afkomstig is, in 'n heteroloë in planta
omgewing geëvalueer as 'n verdedigingspeptied met die potensiaal om in die generering van
transgeniese siektebestande gewasse gebruik te word. Die antifungiese aktiwiteit van
Hs-AFP1 is teen Botrytis cinerea in 'n in vitro reaksie geëvalueer, waar die toediening van
8 ,",g/mlgesuiwerde Hs-AFP1 peptied aanleiding gegee het tot 'n 88% afname in hifegroei van
B. cinerea. Hipervertakkings van swamhifes, 'n kenmerkende eienskap van Hs-AFP1
aktiwiteit, kon duidelik waargeneem word. Tabakplante is voorts getransformeer met 'n
konstruk, pFAJ3068, wat die koderende geen van Hs-AFP1 onder die sterk konstitutiewe
CaMV 35S promotor bevat het. Die peptied is met behulp van die seinpeptied wat afkomstig
is van die Mirabilis jalapa antimikrobiese peptied, Mj-AMP2, na die apoplastiese omgewing
geteiken. Voorheen is gerapporteer dat transgeniese peptiede in die heteroloë omgewing
soms onstabiel is. Dit het gelei tot die generering van twee addisionele konstrukte om die
moontlikheid van peptiedonstabiliteit te ondervang. 'n Stabiliseringskonstruk, pHs-EXG1, bestaande uit In versmelting tussen Hs-AFP1 en In antifungiese eksoglukanase van
Saccharomyces cerevisiae, gekodeer deur EXG1, is in tabakplante getransformeer. In
Kontrolekonstruk, pMj-EXG1, met die EXG1-geen saam met die Mj-AMP2-seinpeptied, is ook
voorberei en in tabakplante getransformeer. Dit is gebruik om die antifungiese aktiwiteit van
die eksoglukanase in die antifungiese aktiwiteitstoetse van die stabiliseringskonstruk te
kwantifiseer en te normaliseer.
Tabak is suksesvol met pFAJ3068, pHs-EXG1 en pMj-EXG1 getransformeer, wat
onderskeidelik gelei het tot die sogenaamde THs, THE en TME transgeniese tabaklyne.
Transgeentranskripsie en -translasie in die THs en THE tabaklyne is onderskeidelik deur
Noordelike- en Westelike-kladanalises bevestig. Die aktiewe uitdrukking van Hs-AFP1 het die
vermoë van tabakplante om B. cinerea infeksies te weerstaan, met tot 42% verhoog in
vergelyking met ongetransformeerde kontrole tabakplante tydens 'n in planta
siekteweerstandstoets. Totale proteïenekstrakte van THs tabaklyne is voorts ook in In in vitro
inhibisietoets geëvalueer, wat gelei het tot resultate wat goed met dié van die in planta toetse
ooreenstem. Die totale proteïenekstrakte het swamgroei met 40% geïnhibeer en die
kenmerkende hipervertakking van Hs-AFP1-aktiwiteit is ook mikroskopies waargeneem.
Resultate wat verkry is vanaf al die analises wat op die transgeniese THs tabaklyne
uitgevoer is, het aangedui dat Hs-AFP1 baie stabiel in die heteroloë tabakomgewing is en
peptiedstabiliteit was dus nie In probleem, soos verwag is nie. Die fusie tussen Hs-AFP1 en
EXG1 het dus nie die stabiliteit van die reeds stabiele Hs-AFP1 peptied verder verbeter nie,
maar het wel tot die verlies van Hs-AFP1 aktiwiteit gelei. Die antifungiese analises van die
THE tabaklyne het verder bevestig dat dié lyne selfs swakker inhibisie van B. cinereainfeksies
tot gevolg gehad het, as ongetransformeerde tabakplante. Mikroskopiese analises
van totale THE proteïenekstrakte het voorts ook geen kenmerkende hipervertakkings in die
swamhifes vertoon nie, wat alles daarop dui dat die Hs-AFP1-deel van die fusieproteïen as
gevolg van die fusie met EXG1 geïnaktiveer is. Dié resultaat is in lyn met vorige navorsing,
wat getoon het dat In ander peptied, sarcotoxin 1A, sy antifungiese aktiwiteit verloor indien dit
met In verklikkergeen versmelt word.
Alhoewel integrasie van die pMj-EXG1-konstruk in die TME-tabaklyne bevestig is, kon
geen mRNA met Noordelike-klad- of trutranskriptase-PKR (RT-PKR)-analises waargeneem
word nie. Die TME plant het ook geen antifungiese aktiwiteit in in vitro toetse getoon nie en dit
het geblyk dat die pMj-EXG1-konstruk aan geenafskakeling in die heteroloë tabakomgewing
onderworpe was. Dié afskakelingseffek is egter in die THE plante oorkom, aangesien
laasgenoemde sterk EXG1 proteïenaktiwiteit met J3-glukosidase aktiwiteitstoetse vertoon het.
Alhoewel die THE plante nie die stabiliteit van Hs-AFP1 verbeter het nie, het dit onwerwags
tot die stabilisering van EXG1 in In heteroloë omgewing gelei. Versmeltingstegnologie kan
dus moontlik gebruik word as 'n strategie om ander glukanases, wat bekend is vir
geenafskakeling in transgeniese omgewings, heteroloog uit te druk. In die geheel gesien, het dié studie getoon dat Hs-AFP1 'n onbetwiste rol in
plantverdedigingsmeganismes speel en daar is voorts ook meer kennis oor die stabiliteit van
peptiede in 'n heteraloë plantomgewing ingewin. Die positiewe resultate t.o.v. die verhoogde
siekteweerstand in die transgeniese THs plantlyne regverdig ook die verdere bestudering van
dié peptied om transgeniese siekteweerstand in gewasse te bewerkstellig.
|
80 |
n Morfologiese en fisiologiese studie van agt Suid-Afrikaanse gisrasseJoubert, D. J January 1948 (has links)
Thesis (MScAgric)--Stellenbosch University, 1948. / NO ABSTRACT AVAILABLE
|
Page generated in 0.3876 seconds