20.500.12556/RUL-127617 Pretočna citometrija za spremljanje kinetike alkoholne fermentacije grozdnega mošta Monitoring of alcoholic fermentation kinetics in grape must by flow cytometry Namen magistrske naloge je bil ovrednotiti ali lahko pretočna citometrija nadomesti uveljavljene metode spremljanja kinetike alkoholne fermentacije pri pridelavi vina. Spremljali smo potek alkoholne fermentacije preko sproščanja CO2 in sicer v čistih in mešanih kulturah avtohtonih sevov ter komercialnih sevov kvasovk v sterilnem gojišču YM, belem moštu in rdeči drozgi. Ob izbranih časovnih terminih med procesom smo vzorčili in preverjali populacijsko sestavo in živost kvasovk z nacepljanjem na gojišče WL, s pretočno citometrijo in neposrednim avtomatiziranim štetjem pod mikroskopom. Preverili smo skladnost rezultatov merjenja koncentracije kvasovk s pretočno citometrijo in z določanjem kolonijskih enot na agarnih ploščah. Po končani alkoholni fermentaciji smo ovrednotili kakovost vina z analizo fizikalno-kemijskih parametrov z uporabo encimskih testov. Alkoholna fermentacija s komercialnim sevom je bila najhitrejša in vino je imelo največjo vsebnost želenih produktov alkoholne fermentacije. Koncentracije živih celic z vsemi tremi metodami so bile na začetku alkoholne fermentacije primerljive, proti koncu procesa pa so vedno bolj odstopale, kar je bila posledica različnega mehanizma zaznavanja mrtvih celic. Potrdili smo, da je pretočna citometrija hitrejša in tudi natančnejša metoda od tradicionalnih, saj zazna tudi žive, vendar nekultivabilne celice (VBNC). Kvasovka Brettanomyces bruxellensis je najbolj znan kvarljivec vina, ki ga s tradicionalnimi metodami težko zaznamo zaradi domnevnega stanja VBNC ali pa rastejo počasi. Kadar doseže večje koncentracije kvasovka tvori nezaželen 4-etilfenol, ki daje vinu značilno Brett aromo. Z eksperimentalnim delom smo dokazali, da je pretočna citometrija hitrejša, natančnejša, robustna in specifična metoda za dokazovanje kvasnih celic Brettanomyces. The aim of this master thesis is to test whether flow cytometry can replace established methods for monitoring the kinetics of alcoholic fermentation in winemaking. We monitored alcoholic fermentation by the release of CO2 in pure and mixed cultures of indogenous strains and commercial strains in sterile YM medium, white must and red broth. At selected time points during the process, we took samples and checked the population composition of yeasts and viability by plating on WL medium, by flow cytometry and by automated counting under the microscope. Using flow cytometry and by indirect determination of colony units on agar plates, we checked the correlation between the results of yeast concentration measurements. After alcoholic fermentation, we performed an analysis of physicochemical parameters using enzyme assays. Alcoholic fermentation with a commercial strain was the fastest and the wine had the highest content of desirable yeast cell products. Live cell concentrations were comparable for all three methods at the beginning of alcoholic fermentation and differed more towards the end of the process, which can be attributed to the different proportions of dead cells in the different methods. We have confirmed that flow cytometry is a faster and more accurate method than conventional methods as it also detects VBNC cells. Yeast Brettanomyces bruxellensis is a known wine spoilage that is difficult or costly to detect by conventional method. At elevated concentrations, the yeast forms an undesirable 4-ethylphenol that gives it its characteristic Brett aroma. Through experimental work, we have demonstrated that flow cytometry is a faster, more accurate, more robust and specific method for detecting Brettanomyces yeast cells compared to a conventional method. kvasovke Saccharomyces cerevisiae Hanseniaspora uvarum Starmerella bacillaris vino živost dinamika populacije kvasovk inokulirana fermentacija spontana fermentacija pretočna citometrija Brett Count Brettanomyces bruxellensis yeasts Saccharomyces cerevisiae Hanseniaspora uvarum Starmerella bacillaris wine viability yeast population dynamics inoculated fermentation spontaneous fermentation flow cytometry Brett Count Brettanomyces bruxellensis true false false [M. Vodušek] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-06-17 07:15:10 2021-06-17 07:15:28 2022-09-06 03:52:16 0000-00-00 00:00:00 2021 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 663.252.4:579.24+579.26 191248 66988035 14184.pdf 14184.pdf 1 7A50ACA482C52A0E645199B3E0378620 87fdf058154d4665000a2caa48dc443fd357b8d5a049882c24336627a1416f3e f7d8a870-cf2a-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=144082 Biotehniška fakulteta 0 0 0