Assessment of the Physiological Status of Yeast During High- and Low-Gravity Wort Fermentations Determined by Flow Cytometry
MBAA TQ vol. 44, no. 4, 2007, pp.
286-295 |
VIEW ARTICLE Paul H. Chlup, Tao Wang, Eung Gwan Lee, and Graham G. Stewart. The
International Centre for Brewing and Distilling (ICBD), Heriot-Watt University,
Riccarton, Edinburgh, EH14 4AS, Scotland.
Abstract
The use of flow cytometry as an analytical control tool during high-gravity
(20�P) and low-gravity (12�P) wort fermentations was investigated. A flow
cytometer is an instrument capable of determining the physiological status of
Saccharomyces cerevisiae cultures in near real time. Flow cytometry uses
technology that simultaneously performs multiparametric analyses of the physical
and chemical characteristics of the yeast based on cell size, relative
granularity, and fluorescence. The differentiation of subpopulations permitted
the classification of viable and depleted cell populations. Flow cytometry and
fluorescent dyes provided a rapid and accurate means to monitor the viability
and vitality of yeast throughout fermentation. The viability method
distinguished between dead, live, and damaged cells. Propidium iodide and
fluorescein diacetate probes were used as markers to determine functioning
cells. The vitality of the cells was determined based on their intracellular pH
using the pH-dependent fluorescent probe carboxy SNARF-4F (SNARF). The SNARF
possesses two inversely related emission signals at two different wavelengths,
which made it possible to calculate the pH from the ratio between the
fluorescence intensities measured at the two wavelengths. Predictors of yeast
performance such as intracellular glycogen and trehalose concentrations, assayed
by flow cytometry, were established. The results indicate that flow cytometry
provides an innovative, rapid, and viable option for brewers to evaluate yeast
cells during fermentation.
Keywords: flow cytometer, glycogen, high-gravity
wort, intracellular pH, trehalose, yeast physiology
S�ntesis
Se investig� el uso de la citometr�a de flujo como una herramienta anal�tica
de control en las fermentaciones de mosto de alta (20�P) y baja (12�P) gravedad
(extracto original). Un cit�metro de flujo es un instrumento capaz de determinar
el estatus fisiol�gico de cultivos de Saccharomyces cerevisiae en casi
tiempo real. La citometr�a de flujo utiliza tecnolog�a que simult�neamente
realiza an�lisis multiparam�tricos de las caracter�sticas qu�micas y f�sicas de
una levadura basado sobre el tama�o de la c�lula, la granularidad relativa y su
fluorescencia. La diferenciaci�n de subpoblaciones permite clasificarlos seg�n
sean poblaciones viables o agotadas. La citometr�a de flujo y colorantes
fluorescentes provee un m�todo r�pido y preciso de monitorear la viabilidad y
vitalidad de una levadura a lo largo de una fermentaci�n. La viabilidad hace una
distinci�n entre c�lulas vivas, muertas o da�adas. Sensores de yoduro propidio y
de fluoresce�na diacetato fueron usados como marcadores para determinar la
funcionabilidad de las c�lulas. La vitalidad de las c�lulas se determin� sobre
la base de su pH intracelular utilizando una sonda fluorescente dependiente del
pH, un carboxilo SNARF-4F. El SNARF posee dos se�ales de emisi�n con una
relaci�n inversa a dos diferentes longitudes de onda, haciendo posible calcular
el pH seg�n la raz�n entre las intensidades de fluorescencia medidas a dos
longitudes de onda. Se determin� la concentraci�n de glic�geno intracelular y de
trehalosa mediante la citometr�a de flujo para pronosticar el desempe�o de una
levadura. Los resultados indican que la citometr�a de flujo es una opci�n
viable, r�pida e innovativa para que los cerveceros puedan evaluar la levadura
durante la fermentaci�n.
Palabras claves: citometr�a de flujo, fisiolog�a de
levadura, glic�geno, mosto �high gravity,� pH intracelular, trehalosa
