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For this purpose the AP test has been developed (Opekarová and Sigler 1982; Kara et al. 1988), but in spite of its extensive optimization it has not become ...
Folia Microbiol. 54 (1), 25–29 (2009)

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Yeast Vitality Determination Based on Intracellular NAD(P)H Fluorescence Measurement during Aerobic–Anaerobic Transition M. KUŘECa, G. KUNCOVÁb, T. BRÁNYIKa * aDepartment of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology, 166 28 Prague, Czech Republic bInstitute of Chemical Process Fundamentals, Academy of Sciences, 165 02 Prague, Czech Republic

Received 13 March 2008 Revised version 8 September 2008

ABSTRACT. This work presents a yeast-cell vitality-assessment method based on on-line intracellular fluorescence measurement. The intracellular NAD(P)H fluorescence of a cell suspension is recorded during transition from aerobic to anaerobic conditions and the output signal is evaluated as a measure of yeast vitality (quality). This fluorescence method showed a highly satisfactory correlation with even low dead cell numbers where the acidification power test could not be applied.

Abbreviations NAD(P) NAD(P)H AP

nicotinamide adenine dinucleotide (phosphate) reduced form of nicotinamide adenine dinucleotide (phosphate) acidification power

FI FIrel

μmax

fluorescence intensity relative increase of NAD(P)H fluorescence maximum specific growth rate

The high quality of microbial catalyst, expressed as “vitality“, strongly influences the course of several processes in food industry, such as beer and wine fermentation, dough leavening or cheese making. The term vitality describes a group of characteristics and capabilities of microbial cells related to their metabolic activity and stress resistance (Sigler et al. 2006). Several quantitative methods have been developed to evaluate the vitality of cells including measurements based on intracellular content of different components (Majara et al. 1996; Hutter 2002) or manifestation of metabolic activity (Peddie et al. 1991; Imai et al. 1994) or gravimetric analysis of the course of fermentation (Košin et al. 2008). In fact, none of these methods has found widespread application in industrial biotechnologies. One of the areas where an expeditious and reliable method of yeast vitality estimation would be appreciated is brewing industry. The brewing yeast vitality is an important factor determining the fermentation performance and the formation of sensorially active metabolic by-products (Guido et al. 2004; Brányik et al. 2005). Nevertheless, the methods of yeast vitality estimation developed so far have not met the requirements for routine applicability (Mochaba et al. 1998; Košin et al. 2007). Sensors using intracellular fluorophores (NAD(P)H) have been developed for different application in biotechnology and particularly for bioprocess monitoring (Marose et al. 1998; Podrazký and Kuncová 2005). However, the number of practical applications of fluorometry in biotechnologies has been very limited due to the many factors that affect culture fluorescence (Li et al. 1990). This paper presents a novel application of NAD(P)H-dependent fluorescence monitoring for determination of yeast vitality. The method is based on the monitoring of intensity of NAD(P)H fluorescence during a forced transition from aerobic to anaerobic conditions (AA transition). The relative fluorescence increase (FIrel) change and its rate (dFI/dt) during AA transition was evaluated as a measure of yeast vitality and it was compared with acidification power (AP) test, specific growth rate (μ) measurement and dead cell count. MATERIALS AND METHODS Yeast strain. The industrial bottom-fermenting brewing yeast strain Saccharomyces cerevisiae (carlsbergensis) was supplied by UNICER, S.A. (S. Mamede de Infesta, Portugal) and it was kept on agar

*Corresponding author; fax +420 220 445 051; email [email protected] .

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slant consisting of complex medium (5 g/L KH2PO4, 2 g/L (NH4)2SO4, 0.4 g/L MgSO4, 10 g/L glucose, 2 g/L yeast extract) with 15 g/L bacteriological agar. Storage experiments. During storage experiments 2 L of yeast slurry (total dry mass concentration ca. 100 g/L) was kept in physiological solution (PS; 9 g/L NaCl) either at 20 or 4 °C. Samples for on-line NAD(P)H measurement, µmax determination, acidification power (AP) tests, viable cell count (methylene blue staining) were taken through the experiments. Growth rate determination. The µmax was determined in 100 mL of complex medium on a rotary shaker (200 rpm) at 9 °C. The initial cell concentration was 20 mg/L as determined by absorbance (A600) measurement. Methylene blue staining. Cell viability was measured by counting dead cells stained with methylene blue (Analytica-Microbiologica-EBC 2001). Acidification power test. The acidification power test was done according to Opekarová and Sigler (1982) and Sigler et al. (2006). The overall drop of pH after 20 min is defined as total acidification power (APtot), while the pH drop during 10 min before and after glucose addition as AP10 and AP20, respectively. Measurement of NAD(P)H. Yeast cells (10 mL) were harvested by centrifuging (1050 g, 10 min) and washed twice with physiological solution (9 g/L NaCl). Prior to the measurement the cells were resuspended in PS to the desired concentration (0.5–2.0 g/L dry mass). The change of NAD(P)H fluorescence intensity (FI340/440), measured at 340/440 nm (excitation/emission wavelength) as the response to AA transition can be considered as an indicator of yeast vitality (Podrazký et al. 2003; Brányik et al. 2004). The intracellular NAD(P)H fluorescence of cell suspension (2 mL) was measured in a 10 mm quartz cuvette. The AA transition in yeast was provoked by creating alternately aerobic and anaerobic conditions in cell suspension. This was ensured by sparging either air or nitrogen (0.1 L/min) into the cuvette with suspended yeast. The FI340/440 signal was first recorded on-line every 0.5 s using a fluorescence spectrophotometer (Hitachi F-4500, Japan) during aerobic conditions (air sparging) until a constant output signal was reached (FIAE). Then the air flow was switched to nitrogen which led to step-wise increase of the FI340/440 signal due to oxygen depletion in cells (Fig. 1A). The signal was recorded until a steady-state value was obtained (FIAN). The measurements were performed in rectangular geometric arrangement.

Fig. 1. The course (time in s) of NAD(P)H fluorescence intensity (FI) (AU, A340/440) during aerobic–anaerobic (AA) transition. A: FI signal as recorded by fluorescence spectrophotometer; AS – air sparging, NS – nitrogen sparging. B: The smoothed FI signal (full line) and its first derivative dFI/dt (dashed line); FIAE – fluorescence intensity during aerobic phase, FIAN – fluorescence intensity during anaerobic phase, dFI/dtmax – the maximum value of the FI signal derivation.

Subsequently, the output signal was smoothed by a moving average data analysis tool (Fig. 1B). The yeast vitality was expressed as a relative increase of NAD(P)H fluorescence (FIrel, %) between anaerobic and aerobic value: FIrel = 100 (FIAN – FIAE) / FIAE

(1)

Another method of data treatment was the first derivative (dFI/dt) of the smoothed FINAD(P)H signal during AA transition (Fig. 1B). In this case, the maximum value of dFI/dtmax , i.e. the maximum rate of NAD(P)H fluorescence increase, was considered to be the measure of yeast vitality.

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RESULTS The reduced phosphorylated (NADPH) and nonphosphorylated (NADH) pyridine nucleotides have essentially equivalent fluorescence properties while their oxidized forms are non-fluorescent. Under aerobic conditions the reduced nucleotides in cells are oxidized, e.g., in the respiratory chain, which leads to an equilibrium between the formation and depletion of NAD(P)H. After the sudden alteration from aerobic to anaerobic conditions (AA transition) the yeast are not able to re-oxidize all the reduced NAD(P)H, which can be tracked as an intracellular step-like increase of NAD(P)H FI (Fig. 1). The interpretation of the recorded signal from intact cells is not straightforward and therefore it was necessary to elucidate the influence of biomass concentration on FI signal. It was found that FIrel and dFI/dtmax first increased linearly with the biomass concentration but, after reaching a plateau, both these parameters gradually decreased (Fig. 2). The maximum responses of FIrel and dFI/dtmax were observed in the range of cell concentration 1.1–1.5 and 1.4–1.8 g/L dry mass, respectively (Fig. 2). Based on these results, a cell concentration of 1.5 g/L dry mass was used for AA transition measurements throughout the whole experimental work.

Fig. 2. Relation between cell concentration (X, g/L dry mass) and FIrel (%, closed symbols) and dFI/dtmax (open symbols) revealing the optimum range of cell concentration used for measurement (1.4–1.5 g/L dry mass).

The applicability of the intracellular NAD(P)H measurement during AA transition for yeast vitality monitoring was verified in two storage experiments with cells exposed to starvation and gradual loss of viability (Figs 3 and 4). During the storage of cell suspension for 18 d at 20 °C the initial dead cell number increased from 7 to 23 % (as determined by methylene blue staining). The correlation between dead cell count and different cell vitality estimation methods (FIrel, dFI/dtmax and AP test) was compared. It was found that FIrel showed the best correlation (R2 = 0.89) with dead cell count (Fig. 3).

Fig. 3. Correlation between dead cell count (%) evaluated by methylene blue staining and measured cell vitality estimation methods (FIrel – closed circles, R2 = 0.8907; dFI/dtmax – open circles, R2 = 0.6283; APtot – triangles, R2 = 0.6763). The yeast suspension was stored in physiological solution at 20 °C.

In the following experiment (Fig. 4) the cells were kept under conditions mimicking real storage of pitching yeast in breweries (4 °C, oxygen limitation) for 18 d. During this cold storage the yeast viability decreased from 5 to 9 % dead cells. The alterations in cell vitality were again tracked by FIrel determination and acidification power test (APtot, AP10, AP20). In addition, µmax of the stored yeast population in complex medium at 9 °C was determined. The decrease of yeast viability was registered most reliably by FIrel deter-

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mination while the AP test remained almost unchanged during the whole experiment. The values of µmax slightly decreased when the number of dead cells reached 9 % (Fig. 4).

DISCUSSION In beer fermentation technology the bottom fermenting lager strain begins to degenerate after 10 serial re-pitchings (Jenkins et al. 2001). Before re-pitching (re-inoculation into wort) the yeast are collected, washed and stored in cold water under starvation and oxygen limitation. At the moment of their repeated use they are pitched into an aerated broth rich in nutrients (wort). This represents a sudden alteration (stress situation) from anaerobic to aerobic and starvation to nutrient rich conditions to which the yeast have to adapt swiftly in order to carry out a desirable fermentation performance. Consequently the vitality of brewing yeast can be perceived as their ability to respond to or handle stress situations occurring during pitching.

Fig. 4. The time course (d) of storage experiment (yeast kept at 4 °C in physiological solution) where the cell vitality is expressed as A: FIrel (in %; closed circles, average of 3 repeated measurements) and maximum specific growth rate (μmax; 1/h, diamonds), and B: total (APtot, open circles) and partial (AP10, open squares; AP20, open triangles) acidification power, and cell viability (% dead cells, closed triangles).

Due to the lack of more convenient alternatives, the method most commonly used to eliminate pitching yeast of inferior quality remains the vital staining with methylene blue (Košin et al. 2007). However, the dead-cell number alone cannot assess reliably the yeast quality and predict their fermentation performance. In other words, yeast with low dead cell number need not necessarily perform well during beer fermentation and vice-versa. For this purpose the AP test has been developed (Opekarová and Sigler 1982; Kara et al. 1988), but in spite of its extensive optimization it has not become widely used for yeast quality assessment in breweries (Sigler et al. 2006). One of the reasons is that the AP test is relatively insensitive to “high vitality” yeast (Košin et al. 2007). Similar results were obtained with the application of the intracellular pH measurement (ICP). The “high vitality” pitching yeast (0–10 % dead cell count) showed no correlation between vital staining with methylene blue and ICP method at all (Thiele et al. 2007). The fluorescence intensity of NAD(P)H during AA transition has already been used for the measurement of free and immobilized biomass concentration and activity (Brányik et al. 2005; Podrazký and Kuncová 2005). However, in this work the protocol of the fluorescence measurement was adjusted to the peculiarities of beer fermentation. The measurements were carried out during a forced switch from aerobic to anaerobic conditions (AA transition), which is mimicking one aspect of the wort pitching. The output signal of NAD(P)H fluorescence was evaluated by two methods (FIrel and dFI/dtmax) among which FIrel showed better correlation with dead cell count (Fig. 3). This can presumably be ascribed to signal derivation (dFI/dt) enhancing the errors in the reproducibility of measurements.

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Although NAD(P)H-dependent fluorescence provides a means for non-invasive monitoring of the cell intracellular state, the interpretation of its results has to be very careful due to many factors that affect culture fluorescence (Li and Humprey 1990). For instance by increasing the density of the cell culture, the emitted fluorescence can be re-absorbed due to a phenomenon called inner filter effect (Fig. 2). In order to demonstrate the sensitivity of the fluorescence method towards yeast quality alterations during storage, it was compared with the AP test and μmax determination. The comparison of FIrel with AP tests was clearly in favor of FI measurement as it is shown by the correlation coefficients (Figs 3 and 4). The same applies for the comparison of FI measurement with μmax determination (Fig. 4). Simultaneously, the time and labor requirement of FIrel determination is comparable with the AP tests (ca. 40 min for sample preparation and measurement) while both are faster and less labor demanding than μmax determination. Measurements of NAD(P)H fluorescence intensity and its changes during AA transition well express the decreasing viability of starving yeast suspension. From this perspective, the proposed method outperformed both the acidification power test and the culture-based specific growth rate determination. Although this method can be considered as highly promising, its applicability for yeast vitality estimation in conditions of real brewing practice will have to be further tested. Needless to say, the correlation of this method with the fermentation performance of yeast will be of crucial importance for its future industrial application. The main disadvantage of the proposed method is in the price of the sensitive fluorescence spectrophotometer used in this work. 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