| Summary: | Thesis (MSc)--University of Stellenbosch, 2005. === ENGLISH ABSTRACT: In the brewing industry yeast cells are re-used in successive fermentations. Consequently,
the state of the cells at the end of each successive fermentation could impact on the
quality of the subsequent fermentations. The use of markers to evaluate the fermentative
ability of yeast to resist stress enables brewers to select populations of yeast for brewing.
Yeasts are typically exposed to osmotic-, ethanol- and cold-stress during the high-gravity
brewing process. In this study the vitality of the yeast cells was monitored during and
after each successive high-gravity brewing fermentation. This was done by measuring the
cell metabolites, which included glycerol, trehalose and glycogen. Others markers that
were evaluated for yeast viability were the number of budding scars, the levels of activity
of the enzymes neutral trehalase and esterase and the expression level of the heat shock
protein Hsp12p. Coupled to these evaluations, the growth of the yeast and the utilisation
of the sugars glucose, fructose, maltose and maltotriose were monitored during the
fermentations. The experiments were conducted in 2-litre E.B.C. tubes at either 14 oC or
at 18oC using standard techniques.
Comparable growth patterns were obtained for different re-pitching fermentations, with
fermentation 1 at 18ºC and 5 and 6 at 14°C being the most active fermentations. The
higher temperature encouraged more rapid growth and a greater numbers of cells. The
wort attenuation was more rapid at 18°C than at 14°C. Glucose and fructose in wort were
utilised prior to maltose and maltotriose. At 18°C the yeast consumed the sugars faster,
with mean utilisation values of 97.3% glucose, 100% fructose, 59.9% maltose and 65.6%
maltotriose. At the lower temperature of 14°C high concentrations of residual sugars
remained at the end of the fermentation. All re-pitching fermentations revealed lower
viabilities at 18°C in comparison to the 14°C fermentations.
Simultaneously, a number of other markers were evaluated. The intracellular trehalose
concentration per cell varied considerably with each fermentation. Trehalose levels at
18°C gradually increased in concentration from 48h until the end of the stationary phase. Much lower trehalose concentrations were observed in fermentations conducted at 14°C.
Higher and more consistent glycerol concentrations were found in fermentations at 14°C
with mean concentrations of 12 mg/g dry weight at pitching. The expression of the heat
shock protein Hsp12p level increased during the fermentation but no sharp increase was
detected in any particular fermentation. No increase in yeast budding scar number was
observed during re-pitching fermentations. Neutral trehalase and esterase activities in
fermentations at 18°C were especially high at pitching. Neutral trehalase activities at
14°C were all generally lower than in the case of fermentations at 18°C.
The fermentation ability of flocculated yeast in slurry and yeast suspended in beer was
investigated after exposure to various stresses. The aged yeast present in the slurry was
generally found to be more resistant to stress, in particularly to osmotic stress, throughout
the serial re-pitching process. The fermentation rates of both yeast types were especially
sensitive to prior exposure to ethanol stress. === AFRIKAANSE OPSOMMING: In die broubedryf word gisselle herhaaldelik gebruik vir agtereenvolgende fermentasies.
Derhalwe kan die toestand van die gisselle teen die einde van elke agtereenvolgende
fermentasie ‘n invloed hê op die kwaliteit van die daaropvolgende fermentasies. Deur
gebruik te maak van merkers om die fermentasievermoë van gis om stres te weerstaan te
evalueer, stel dit bierbrouers in staat om gispopulasies te selekteer. Gedurende die hoëdigtheid
brouproses word giste tipies aan osmotiese-, etanol- en koue-stres blootgestel. In
hierdie studie, gedurende hoë-digtheid fermentasies, is die lewensvatbaarheid van die
gisselle gedurende en na elke agtereenvolgende fermentasie gemonitor deur die volgende
selmetaboliete te bepaal: gliserol, trehalose en glikogeen. Bykomende merkers vir gis
lewensvatbaarheidsbepalings was: die aantal botselletsels, die vlakke van aktiwiteit van
die neutrale trehalose en esterase ensieme, en die uitdrukkingsvlak van die
hitteskokprotein Hsp12p. As aanvullende evaluasies is die groei van die gis en die
gebruik van die suikers glukose, fruktose, maltose en maltotriose gedurende fermentasies
gemonitor:. Die proewe is in 2-liter E.B.C. buise uitgevoer, by ‘n temperatuur van 14oC
of 18oC, deur van standaard tegnieke gebruik te maak.
Die groeipatrone van die verskillende herhaaldelike-inokulasie gistings was ongeveer
dieselfde. Fermentasie 1 by 18ºC en fermentasies 5 en 6 by 14°C was die mees aktiewe
fermentasies. Die hoër temperatuur het vinniger groei en ‘n groter aantal selle begunstig.
Die wortattenuasie was vinniger by 18°C as by 14°C. Glukose en fruktose in mout is
voor die maltose and maltotriose opgebruik. By 18°C het die gis die suikers vinniger
opgebruik. Gemiddelde gebruikswaardes vir die sewe reeksgewyse fermentasies was die
volgende: 97.3% glukose, 100% fruktose, 59.9% maltose en 65.6% maltotriose. Teen die
einde van fermentasie by 14°C was daar hoë konsentrasies van die oorblywende suikers,
hoofsaaklik na fermentasie 1. Alle herhaaldelike inokulasie fermentasies het lae
lewensvatbaarheid by 18°C in vergelyking met 14°C fermentasies getoon.
Ander merkers is ook gelyktydig gebruik. In die verskillende fermentasies was daar ‘n
groot verskil in die intrasellulêre trehalose konsentrasie per sel. Trehalose konsentrasies
by 18°C het geleidelik toegeneem, vanaf 48 uur tot aan die einde van die stationêre fase. Baie laer trehalose konsentrasies is gemeet vir fermentasies by 14°C. In fermentasies by
14°C was die gliserolkonsentrasies hoër en meer konstant. Gemiddelde konsentrasies was
12mg/g 14°droë gewig by inokulasie. Die uitdrukking van die hitteskokproteien Hsp12p
vlak het gedurende fermentasie toegeneem, maar daar was geen skerp toename vir die
afsonderlike fermentasies nie. Die bepaling van die aantal botselletsels per sel het daarop
gewys dat die gemiddelde aantal nie toegeneem het met die veroudering van die gis
gedurende reeksgewyse herhaaldelike inokulasie nie. Neutrale trehalase aktiwiteite in
fermentasies by 18°C was besonders hoog, veral by inokulasie. Die neutrale trehalase
aktiwiteite in die fermentasies by 14°C was in die algemeen laer as die by 18°C.
Die fermentasievermoë van die geflokkuleerde gis in die sediment en gesuspendeerde gis
in die bier is ondersoek na blootstelling aan verskeie tipes stres. Die verouderde gis
teenwoordig in die sediment was in die algemeen meer bestand teen stres, veral aan
osmotiese stres, dwarsdeur die reeksgewyse herhaaldelike inokulasie proses. Etanolstres
het die gistingstempo van beide giste dieselfde geaffekteer.
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