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Yeast chitin enrichment for haze protection
Thesis (MScAgric)--Stellenbosch University, 2022.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613989255512064 |
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| access_status_str | Open Access |
| author | Chuene, Lesiba Tyrone |
| author2 | Bauer, Florian |
| author_browse | Bauer, Florian Chuene, Lesiba Tyrone |
| author_facet | Bauer, Florian Chuene, Lesiba Tyrone |
| author_sort | Chuene, Lesiba Tyrone |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125120 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:53.996Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125120 Yeast chitin enrichment for haze protection Chuene, Lesiba Tyrone Bauer, Florian Rossouw, Debra Naidoo, Rene Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Yeast Mutagenesis Yeast -- Effect of stress on Saccharomyces cerevisiae -- Effect of stress on Chitin Phenotype Haze Wine -- Protein content Flow cytometry Strains and stresses -- Data processing UCTD Thesis (MScAgric)--Stellenbosch University, 2022. ENGLISH ABSTRACT: The formation of protein haze in white wine is a major global problem for the wine industry. While the risk of haze can be reduced or eliminated by various treatments, collectively referred to as “fining” or “filtering”, such treatments are costly and may impact wine quality. Treatment with bentonite clay is currently the most commonly used method. Alternative approaches have been explored to replace bentonite by using yeast cell wall chitin. Previously it was shown that yeast cell wall chitin can lessen protein related haze development. The data suggested that the group of proteins that are primarily accountable for haze formation, grape chitinases, can bind to yeast cell walls, and that yeast strains with increased cell wall chitin levels significantly reduce haze formation more efficiently than strains with lower cell wall chitin levels. In this work, we describe an original selection strategy to isolate mutant strains of commercial wine yeast strains with increased cell wall chitin. For this purpose, the commercial wine yeast strain Saccharomyces cerevisiae EC 1118® was mutagenized with ethyl-methanosulphonate (EMS). The mutagenized population was stained with calcofluor white, and cells with the highest chitin levels were separated from the rest of the population using Fluorescence Activated Cell Sorting (FACS). These selected cells were reinoculated into new growth media, and after growing for several generations in batch cultures, were again stained and subjected to sorting to select for cells with the highest chitin level. This cycle of sequential batch cultures and sorting was repeated six times to enrich the mutagenized population with mutant strains exhibiting high chitin levels. The data show that this method indeed selected for strains with higher chitin levels than the wild type, and that such strains reduced commercial chitinase activity in solutions more efficiently than the wild type. Phenotypic characterisation of these strains however indicated a reduction in growth rate, suggesting that high chitin levels exert a significant metabolic or physical burden on these cells. These cells were also more resistant to hypersaline conditions. A high phenotypic reversion rate of the high chitin mutant phenotype was also observed. In conclusion, strains with increased cell wall chitin levels can be selected with the strategy proposed here, but the application of such strains may be limited by a reduced growth rate and possible lack of phenotype stability. More studies should be conducted to further understand the phenotypic impact of chitin levels and to improve the biotechnological application of strains with high chitin levels. AFRIKAANSE OPSOMMING: Die vorming van proteïen waas in witwyn is 'n groot wêreldwye probleem vir die wynbedryf. Alhoewel die risiko van waas deur verskillende behandelings verminder of uitgeskakel kan word, gesamentlik na verwys as "vining" of "filter", is sulke behandelings duur en kan dit wynkwaliteit beïnvloed. Behandeling met bentoniet klei is tans die mees gebruikte metode. Alternatiewe benaderings is ondersoek om bentoniet te vervang deur gisselwand chitien te gebruik. Voorheen is getoon dat gisselwand chitien proteïenverwante waasontwikkeling kan verminder. Die data het voorgestel dat die groep proteïene wat hoofsaaklik aanspreeklik is vir waasvorming, druiwe chitinases, kan bind aan gisselmure, en dat gisstamme met verhoogde selwand chitienvlakke die waasvorming meer doeltreffend verminder as stamme met laer selwand chitienvlakke. In hierdie werk beskryf ons 'n oorspronklike keuringstrategie om mutante stamme van kommersiële wyngisstamme met verhoogde selwand chitien te isoleer. Vir hierdie doel, die kommersiële wyn gis stam Saccharomyces cerevisiae EC 1118® is gemuteer met etiel- metanosulphonate (EMS). Die gemuteerde bevolking is bevlek met kalcofluor wit, en selle met die hoogste chitienvlakke is geskei van die res van die bevolking met behulp van Fluorescence Activated Cell Sorting (FACS). Hierdie geselekteerde selle is herinokuleer in nuwe groeimedia, en nadat dit vir verskeie generasies in bondelkulture gegroei het, is dit weer gekleur en onderworpe aan sortering om vir selle met die hoogste chitienvlak te kies. Hierdie siklus van opeenvolgende bondelkulture en sortering is ses keer herhaal om die gemuteerde bevolking te verryk met mutant stamme wat hoë chitienvlakke vertoon. Die data toon dat hierdie metode inderdaad gekies is vir stamme met hoër chitienvlakke as die wilde tipe, en dat sulke stamme kommersiële chitinase-aktiwiteit in oplossings doeltreffender verminder het as die wilde tipe. Fenotipiese karakterisering van hierdie stamme het egter 'n afname in groeikoers aangedui, wat daarop dui dat hoë chitienvlakke 'n beduidende metaboliese of fisiese las op hierdie selle uitoefen. Hierdie selle was ook meer bestand teen hipersaline toestande. 'N Hoë fenotipiese omkeringskoers van die hoë chitienmutant fenotipe is ook waargeneem. Ten slotte kan stamme met verhoogde selwand chitienvlakke gekies word met die strategie wat hier voorgestel word, maar die toepassing van sulke stamme kan beperk word deur 'n verminderde groeikoers en moontlike gebrek aan fenotipe stabiliteit. Meer studies moet gedoen word om die fenotipiese impak van chitienvlakke verder te verstaan en om die biotegnologiese toepassing van stamme met hoë chitienvlakke te verbeter. Masters 2022-03-10T18:48:15Z 2022-04-29T12:54:50Z 2023-03-31T03:00:11Z 2022-03 Thesis http://hdl.handle.net/10019.1/125120 en_ZA Stellenbosch University vii, 88 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Yeast Mutagenesis Yeast -- Effect of stress on Saccharomyces cerevisiae -- Effect of stress on Chitin Phenotype Haze Wine -- Protein content Flow cytometry Strains and stresses -- Data processing UCTD Chuene, Lesiba Tyrone Yeast chitin enrichment for haze protection |
| title | Yeast chitin enrichment for haze protection |
| title_full | Yeast chitin enrichment for haze protection |
| title_fullStr | Yeast chitin enrichment for haze protection |
| title_full_unstemmed | Yeast chitin enrichment for haze protection |
| title_short | Yeast chitin enrichment for haze protection |
| title_sort | yeast chitin enrichment for haze protection |
| topic | Yeast Mutagenesis Yeast -- Effect of stress on Saccharomyces cerevisiae -- Effect of stress on Chitin Phenotype Haze Wine -- Protein content Flow cytometry Strains and stresses -- Data processing UCTD |
| url | http://hdl.handle.net/10019.1/125120 |
| work_keys_str_mv | AT chuenelesibatyrone yeastchitinenrichmentforhazeprotection |