Stress markers as indicators of fermentative ability of a Saccharomyces cerevisiae brewery strain

• Main Author: Boudler, Sabrina
• Other Authors: Prior, B.A.
• Format: Others
• Language: en_ZA
• Published: Stellenbosch : University of Stellenbosch 2011
• Subjects: Saccharomyces cerevisiae > Effect of stress on; Fermentation; Brewing > Microbiology; Theses > Microbiology; Dissertations > Microbiology
• Online Access: http://hdl.handle.net/10019.1/16598

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.