Harvesting yeast (Saccharomyces cerevisiae) at different physiological phases significantly affects its functionality in bread dough fermentation

Food Microbiology 39 (2014) 108e115

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Harvesting yeast (Saccharomyces cerevisiae) at different physiological phases significantly affects its functionality in bread dough fermentation Mohammad N. Rezaei a, Emmie Dornez a, Pieter Jacobs a, Anali Parsi a, Kevin J. Verstrepen b, Christophe M. Courtin a, * a

Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Molecular and Microbial Systems, KU Leuven, Kasteelpark Arenberg 20, Box 2463, B-3001 Leuven, Belgium VIB Laboratory for Systems Biology & CMPG Laboratory for Genetics and Genomics, KU Leuven, Bio-Incubator, Gaston Geenslaan 1, B-3001 Leuven, Belgium b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 September 2013 Received in revised form 14 November 2013 Accepted 22 November 2013 Available online 1 December 2013

Fermentation of sugars into CO2, ethanol and secondary metabolites by baker’s yeast (Saccharomyces cerevisiae) during bread making leads to leavening of dough and changes in dough rheology. The aim of this study was to increase our understanding of the impact of yeast on dough related aspects by investigating the effect of harvesting yeast at seven different points of the growth profile on its fermentation performance, metabolite production, and the effect on critical dough fermentation parameters, such as gas retention potential. The yeast cells harvested during the diauxic shift and postdiauxic growth phase showed a higher fermentation rate and, consequently, higher maximum dough height than yeast cells harvested in the exponential or stationary growth phase. The results further demonstrate that the onset of CO2 loss from fermenting dough is correlated with the fermentation rate of yeast, but not with the amount of CO2 that accumulated up to the onset point. Analysis of the yeast metabolites produced in dough yielded a possible explanation for this observation, as they are produced in different levels depending on physiological phase and in concentrations that can influence dough matrix properties. Together, our results demonstrate a strong effect of yeast physiology at the time of harvest on subsequent dough fermentation performance, and hint at an important role of yeast metabolites on the subsequent gas holding capacity. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Saccharomyces cerevisiae Yeast Physiological phase Diauxic shift Dough Fermentation rate Rheofermentometer Organic acids pH

1. Introduction Saccharomyces cerevisiae or baker’s yeast is the primary leavening agent in bread products (Liao et al., 1998; Newberry et al., 2002), generating the carbon dioxide (CO2) that is responsible for the distinctive aerated structure. However, the role of yeast in bread making is not limited to gas production (Randez-Gil et al., 1999). Yeast cells are also partly responsible for bread flavor and may affect dough rheology (Liao et al., 1998; Randez-Gil et al., 1999).

List of abbreviations: HPLC, High performance liquid chromatography; OD600, Optical density at 600 nm; YPD, Yeast peptone dextrose; YPS, Yeast peptone sucrose; TCA cycle, Tricarboxylic acid cycle; Yx, yeast cells harvested at harvest point x. * Corresponding author. Tel.: þ32 16 32 19 17; fax: þ32 16 32 19 97. E-mail address: [email protected] (C.M. Courtin). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.11.013

Recent reports suggest that the effect of yeast on dough rheology depends on the production of specific yeast metabolites produced during fermentation, such as ethanol and succinic acid (Jayaram et al., unpublished results, in press) and that this effect is independent from hydrogen peroxide (Rezaei et al., unpublished results), which was previously suggested as a key factor determining dough rheology (Liao et al., 1998). It is well known that the physiological state of yeast cells may affect metabolite production (Gabriela and Daniela, 2010; RandezGil et al., 1999). S. cerevisiae exhibits different growth phases when it is grown on a favorable carbon source (Herman, 2002). After a short adaptation period in the lag phase, where yeast cells adapt to new growth conditions, cells start growing exponentially (Smets et al., 2010). During this exponential growth phase, the cells ferment available sugars such as glucose mainly via glycolysis, resulting in the release of ethanol and acetic acid into the medium (Galdieri et al., 2010; Smets et al., 2010). High glucose concentrations promote carbon catabolite repression, a phenomenon known

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as “Crabtree effect” (Fiaux et al., 2003; Gancedo, 1998). This results in repression of the tricarboxylic acid (TCA) cycle and respiration, even in the presence of oxygen (Blank and Sauer, 2004; Fiaux et al., 2003). Several studies show that such actively growing cells show reduced stress resistance, including reduced tolerance to heatshock, starvation and osmotic stress conditions (Verstrepen et al., 2004; Werner-Washburne et al., 1993). When glucose in the growth medium is depleted, the yeast cells transiently switch metabolism from fermentation of glucose to aerobic utilization of ethanol. This transition from anaerobic growth to aerobic respiration is known as diauxic shift and is accompanied by extensive changes in essential cellular processes (DeRisi et al., 1997; Galdieri et al., 2010; Gasch and WernerWashburne, 2002). Entrance into the diauxic shift is accompanied by an increase in storage molecules such as glycogen (maximum accumulation before diauxic shift) and trehalose (accumulation starts after diauxic shift) (Gasch et al., 2000; Lillie and Pringle, 1980; Taylor and Parks, 1979). After a decrease in cell division during the diauxic shift, the cells resume growth at a slower rate during the subsequent post-diauxic growth period, where they use the ethanol produced during exponential growth as carbon source (Gasch and WernerWashburne, 2002; Herman, 2002; Stahl et al., 2004). During the diauxic shift, components of the electron transport chain and enzymes of the TCA cycle are depressed, resulting in an increase in intermediates of these cycles (e.g. succinic acid) (Liu and Butow, 1999). When the ethanol in the medium is exhausted, the cells stop dividing and enter the stationary phase (Galdieri et al., 2010; Herman, 2002). Cells in stationary phase have thicker and less porous cell walls than cells from exponential growth phase (Werner-Washburne et al., 1993). As the cells approach the stationary phase, different enzymes (e.g. peptidase and invertase) and storage carbohydrates (e.g. glycogen and trehalose) accumulate. Stationary phase cells maintain the ability to continue growth when nutrients become available (Werner-Washburne et al., 1993; Zlotnik et al., 1984). From the above, it is clear that yeast cells in different growth phases show very different physiology, including differences in factors like ribosomes, storage compounds and stress-related proteins (Verstrepen et al., 2004). These differences may in turn affect the metabolite profile and performance of the cells when they are inoculated into fresh medium. To gain insight in the importance of these aspects for bread making, the aim of this study was to investigate the impact of harvest time of yeast during growth on subsequent dough fermentation capacity, the type and level of metabolites produced in dough and their effect on dough gas retention capacity. To this end, yeast was harvested at seven distinct points during growth, characterized and subsequently used in the production of dough. Gas production, dough gas retention capacity and yeast metabolite production were measured throughout the different fermentation tests. 2. Materials and methods 2.1. Materials Commercial wheat flour without additives was obtained from Ceres-Soufflet (Brussels, Belgium). A commercial S. cerevisiae strain (V1116) was obtained from the collection of the VIB Laboratory of Systems Biology (KU Leuven, Belgium). Yeast extract and balanced peptone were procured from Lab M (Brussels, Belgium). All other chemicals, solvents and reagents were purchased from Sigmae Aldrich (Bornem, Belgium) and were of analytical grade unless specified otherwise.

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2.2. Flour characterization Moisture content of the flour was measured according to AACC method 44-19.01 (AACC, 2000). Protein content was determined using an automated Dumas protein analysis system (EAS, VarioMax N/CN, Elt, Gouda, The Netherlands) following the AOAC method 990.03 (AOAC, 1995). The flour had moisture and protein (N  5.7) content of 14.0% and 10.5% (on dry matter base), respectively. Optimal baking absorption and mixing time were determined using Farinograph (Brabender, Duisburg, Germany) and Mixograph (National Manufacturing, Lincoln, NE, USA) analyses according to the AACC Methods 54-40.02 and 54-21.02, respectively (AACC, 2000). All measurements were carried out in triplicate. 2.3. Preparation and growth of the cells The growth profile of the S. cerevisiae yeast strain (V1116) was determined with the Bioscreen C (Thermo Fisher Scientific, Aalst, Belgium) during 70 h of incubation at 30  C with continuous shaking and an optical density measurement at 600 nm (OD600) every 17 min. Yeast cells from a glycerol stock were grown on yeast peptone dextrose (YPD) agar plates with the following composition: 1.0% w/ v yeast extract, 2.0% w/v balanced peptone, 2.0% w/v dextrose and 2.0% w/v agar. Subsequently, they were transferred to yeast peptone sucrose (YPS) medium, a solution of 1.0% w/v yeast extract, 2.0% w/v balanced peptone and 2.0% w/v sucrose, for further propagation of the cells. The above mentioned agar and YP medium were both sterilized by autoclaving (Timo benchtop autoclave, Pbi International, Milan, Italy). Dextrose solution (20.0% w/v) was autoclaved separately prior to addition to YP agar. Sucrose was added to YP medium from a 20% w/v stock solution after filter sterilization (Bottle Top Vacuum Filter, 0.22 mm, Corning B.V. Life Sciences, Amsterdam, The Netherlands). Yeast cells were grown in baffled Erlenmeyer flasks and harvested at seven specific points in the growth profile, by centrifugation using a benchtop centrifuge (model EBA 21, Hettich Lab Technology, Massachusetts, USA) at 870 g for 3 min at room temperature and the yeast cells were immediately used in dough preparation. The number of cells per mL of medium was counted using a hemocytometer (Laboroptik Ltd, Lancing, UK) (Celeromics, Technical Note). Prior to harvesting the yeast cells at each harvest point, the optical density (OD600) corresponding to the cell numbers at that specific harvest point was checked with a Bio-Rad 680 plate reader (Bio-Rad Laboratories Ltd, Hertfordshire, UK). All measurements were performed in duplicate on two different samples. 2.4. Quantification of metabolites and proteins in fermentation medium For quantification of metabolites and protein in fermented growth medium as a function of fermentation time, yeast cells were first removed from the growth medium using an Eppendorf centrifuge 5415D (Eppendorf AG, Hamburg, Germany) at 11,000 g for 3 min. The supernatant was filtered using a Millex-HP 0.22 mm polyethersulfone membrane (Millipore, Carrigtwohill, Ireland). The concentration of glucose, fructose and ethanol in the supernatant were measured with ion-exclusion high performance liquid chromatography (HPLC) using a LC-20AT modular HPLC system (Shimadzu, Kyoto, Japan) connected to a RID-10A refractive index detector (Shimadzu). Separation was carried out using an ion-exclusion ROA-organic acids guard (50  7.8 mm) and analytical (300  7.8 mm) column (Phenomenex, Torrance, CA, USA) and a mobile phase consisting of 2.50 mM H2SO4 at a flow rate of 0.60 mL/

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min. The guard and analytical column were kept in an oven at 60  C. Analyses were performed on two different fermented growth medium samples. Total nitrogen content was measured in duplicate using an automated Dumas protein analysis system, as described above. To this end, the supernatants of fermented growth media (1.0 mL) were dried in an oven at 100  C for 24 h. The dried samples were used for measuring the total nitrogen content. 2.5. Dough preparation Dough was prepared according to the AACC method 10-10.03 using the following formula: 100.0 g flour (on 14% moisture base), 6.0% (w/w) sugar, 1.5% (w/w) sodium chloride, 5.3% (w/w) fresh yeast pellet (16.0  0.5% dry matter) and 52.0% (v/w) water (AACC, 2000). The ingredients were mixed in a 100 g pin bowl mixer (National Manufacturing) for 3 min 50 s. For analysis of metabolites in fermented dough at different time points, the dough was divided into small pieces of 15  1 g and each piece was fermented for different times (0, 30, 60, 90, 120, 150 and 180 min). All doughs were prepared in duplicate. The above-mentioned dough preparation method was downscaled to 10.0 g flour to prepare dough for analysis of metabolites at Tx. The mixing was performed using a 10 g pin bowl mixer (National Manufacturing). All doughs were prepared in duplicate. 2.6. Dough pH measurement Dough pH as a function of fermentation time was measured using a pH probe (HI 9126, Hanna Instruments, Temse, Belgium) that can be placed directly in the dough. 2.7. Dough extraction For quantification of different metabolites in fermented dough, dough extraction was performed by blending the dough with deionized water (two times the volume of the final weight of the dough after fermentation) for 30 s using a Waring 8011E blender (Waring Products, Torrington, CT, USA). The resulting batter was centrifuged using an Eppendorf centrifuge 5415D (11,000 g, 3 min). The supernatant was collected and immediately stored at 18  C until further analysis.

The pressure sensor then measures the gas pressure inside the tank excluding CO2. This gives the total gas retention of dough during the fermentation process. The difference between the direct and indirect cycles shows the amount of CO2 lost by the dough during fermentation. The maximum dough height (Hm, mm), time required to reach the maximum dough height (T1, min), total CO2 production, retention and the volume of CO2 loss at Hm (CO2, mL), maximum fermentation rate (mL of CO2/min), the onset of CO2 release from the dough (Tx, min) and the volume of gas produced by yeast until the onset of CO2 release (CO2 at Tx, mL) were measured. All tests were conducted as single measurements for 6 h at 30  C. The reproducibility of the results was checked by analyzing three doughs made with yeast harvested in diauxic shift on three different days. The percent of error on different parameters is as follows: Hm, 4.5%; T1, 1.0%; maximum fermentation rate, 1.8%; Tx, 4.6%; total CO2 production, 1.7%; total CO2 retention, 1.3%; volume of CO2 loss, 6.5%. 2.10. Statistical analysis Levels of ethanol, glycerol, succinic acid, acetic acid and the dough pH of different harvest points at Tx were analyzed by oneway analysis of variance (ANOVA) using SAS software 9.2 (SAS Institute, Cary, NC) with mean values compared using the Tukey test (P < 0.05). Pearson’s correlation coefficients between the different parameters were also calculated using SAS software 9.2. 3. Results and discussion 3.1. Growing of yeast and harvesting at different physiological phases S. cerevisiae was grown and subsequently harvested at seven different harvest points (labeled AeG) (Fig. 1). The harvested yeast cells are further referred to as YAeYG. The first three harvest points, i.e. A, B and C, were chosen during the exponential growth phase as this is a critical phase in which sugar in the medium is gradually exhausted. Table 1 shows that during the exponential growth phase, yeast cells are converting the available sugar into CO2 and

2.8. Characterization of metabolites in fermented doughs using ion exclusion HPLC Ethanol, glycerol, succinic acid and acetic acid in fermented dough extracts were quantified using ion-exclusion chromatography as described above. 2.9. Rheofermentometer analysis of fermenting dough The Rheofermentometer F3 (Chopin Technologies, Villeneuvela-Garenne, France) was used to follow up fermentation characteristics of fermenting dough following the procedure by Czuchajowska and Pomeranz (1993). Dough (100 g flour basis) was made as described above and placed in the basket of the Rheofermentometer. The Rheofermentometer measures dough height using a displacement sensor. Total gas production and gas retention are measured with a pressure sensor through direct and indirect cycles, respectively. During the direct cycle, the changes in the gas pressure inside the fermentation tank are being recorded and this leads to the total gas production curve. During the indirect cycle, the gas in the fermentation tank is being passed through an absorption tank containing soda lime, in which CO2 is being absorbed.

Fig. 1. The growth of Saccharomyces cerevisiae strain V1116 in yeast peptone sucrose medium (YPS) in the automated Bioscreen C plate reader/incubator, expressed as optical density (OD600) increase as a function of time. The selected harvest points were named from A to G (A, B and C ¼ various harvest points during exponential growth phase; D ¼ at diauxic shift; E ¼ after diauxic shift; F ¼ at early stationary phase; G ¼ at late stationary phase).

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Table 1 Concentrations of glucose, fructose and ethanol (mM) and total nitrogen (w/v %) in yeast peptone sucrose medium (YPS) after removal of the cells from the medium for the cells from harvest points A (YA: exponential growth phase), D (YD: diauxic shift), F (YF: post-diauxic growth) and G (YG: stationary phase). All measurements were performed in duplicate and error margins indicate standard deviations, estimated from the range as described by Nelson (1975). Values with the same letter in rows are not significantly different (p < 0.05). Blank

Point A a

55.7  0.9 54.9  0.6a 0c 0.36  0.01a

Glucose (mM) Fructose (mM) Ethanol (mM) Total nitrogen content (w/v %)

30.8 27.5 99.5 0.33

   

3.6 2.3b 5.2b 0.01ab

ethanol. The yeast cells from harvest point A (YA), for example, only consumed approximately half of the available sugar in their growth medium. Harvest point D was chosen in the diauxic shift and harvest point E in the early post-diauxic growth phase (Fig. 1). Table 1 demonstrates that all available sugar in the growth medium is converted into ethanol at the onset of diauxic shift, and that subsequently, the cell growth resumes with ethanol as carbon source. Harvest points F and G, finally, were selected in early and late stationary phase, respectively (Fig. 1). All carbohydrates and ethanol are depleted at these harvest points and the cells do not have further carbon sources for growing (Table 1). The total remaining nitrogen in the growth media indicated that none of the samples were experiencing nitrogen depletion (Table 1). Table 2 shows the number of cells, the wet weight of the cells per mL of YPS medium, and the number of cells per gram of wet yeast pellet. As expected, the results show that the amount of yeast biomass and the number of cells per mL of YPS medium increase from harvest point AeG. The number of cells per gram of yeast, in contrast, remains relatively constant. The amount of yeast was added to the dough recipe [5.3% (w/w)], corresponds to about 6.0  1010 cells per 100 g of flour for the doughs prepared with the yeast from different harvest points. 3.2. Dough fermentation characteristics Rheofermentometer analysis yields insight into CO2 production, retention and dough height throughout the dough fermentation process and therefore gives a good indication of yeast fermentation performance. Table 3 shows the data obtained from Rheofermentometer experiments performed with YAeYG. From YA to YD, a gradual increase in both total CO2 produced at Hm and maximum fermentation rate can be observed. Later harvest points showed a small decrease in fermentation rate compared to YD and, hence, a slightly lower level of total CO2 at Hm. The cells from YG, harvested 60 h after Table 2 Optical density (OD600) from the Bioscreen as a function of harvest point (A, B and C ¼ during exponential growth; D ¼ at diauxic shift; E ¼ after diauxic shift; F ¼ at early stationary phase; G ¼ at late stationary phase), number of cells per mL of yeast peptone sucrose medium (YPS), weight of yeast cells per mL of YPS and number of cells per one gram of yeast. All measurements were performed in duplicate and error margins indicate standard deviations, estimated from the range as described by Nelson (1975). Values with the same letter in columns are not significantly different (p < 0.05).

Point Point Point Point Point Point Point

A B C D E F G

OD600

Cell#/mL YPS medium ( 108)

0.70 0.85 1.00 1.20 1.30 1.60 1.70

1.2 1.5 2.1 2.4 2.6 3.5 4.6

      

0.1e 0.1de 0.2cd 0.2c 0.1c 0.1b 0.4a

mg Yeast/mL YPS medium 11.0 13.0 16.4 18.2 25.0 35.3 43.1

      

0.1e 0.4e 0.3d 0.4d 1.2c 0.7b 0.5a

Cell#/g yeast ( 1010) 1.1 1.2 1.3 1.3 1.0 1.0 1.1

      

0.1ab 0.1ab 0.1a 0.1a 0.1ab 0.1b 0.1ab

Point D b

0.3 0.3 158.3 0.32

   

Point F c

0.1 0.1c 2.6a 0.01ab

0.3 0.4 1.7 0.29

   

Point G c

0.1 0.1c 0.5c 0.01b

0.5 0.3 1.9 0.28

   

0.1c 0.1c 0.1c 0.01b

inoculation, were less active and probably contained an increased proportion of dead and/or inactive cells (Allen et al., 2006). Together, these results indicate major physiological changes during diauxic shift, which seemingly affect subsequent dough fermentation performance. There is a gradual increase in the maximum dough height from YA to YD. This increase correlates nicely with the total accumulated and retained CO2 at Hm and the maximum fermentation rate of yeast, while the slight decrease in maximum fermentation rate of yeast from YD to YG led to a lower dough height. Although the dough samples prepared with YBeYG reach their maximum height approximately at the same time (similar T1), their maximum dough height (Hm) differed. This difference could be explained by their different CO2 production rate. YA showed the lowest CO2 production rate, which led to a higher T1 and a lower Hm compared to yeasts of the other harvest regions. The total CO2 produced, retained and lost at Hm showed that the doughs prepared with the yeast harvested after diauxic shift lost about 10% of the CO2 produced at Hm while the doughs prepared with the yeast cells harvested before diauxic shift at harvest points A, B and C lost about 2, 2 and 7% of the CO2 produced at Hm, respectively. The time at which the dough cannot retain the CO2 produced by the yeast anymore and starts to lose the gas to the environment (Tx) is another interesting parameter that can be derived from Rheofermentometer analysis. While one would expect that postponing the moment of CO2 loss by the dough (high Tx) would result in a higher maximum dough height (Hm), results in Table 3 surprisingly show the opposite. A negative correlation between Tx and Hm (r ¼ 0.93, p < 0.01) was observed. The occurrence of Tx in this study was mainly correlated with the fermentation rate of yeast. Indeed, the dough made with the yeast displaying the highest fermentation rate (YD) had the earliest appearance of Tx. A negative correlation was also found between Tx and the maximum fermentation rate (r ¼ 0.97, p < 0.01). Interestingly, harvesting yeast at different harvest points does not only affect the subsequent fermentation capacity and dough height, but also influences the gas holding capacity of the dough. The amount of CO2 produced by the yeast at Tx (CO2 at Tx) indicates that the appearance of Tx for different harvest points is not accompanied with the similar levels of CO2 production. There is a difference of about 120 mL of gas between the volume of CO2 produced at Tx for harvest points A and E (Table 3). This indicates that the absolute amount of CO2 produced, and, hence, the fermentation rate itself, are probably not the sole causal factors determining the gas holding capacity of dough and the onset of CO2 release. There are probably underlying factors, perhaps the metabolites that the yeast produces during dough fermentation, that affect dough gas holding capacity and, more generally, dough rheology during fermentation. So in first instance, we looked into the production of dough metabolites during the whole fermentation time by the yeast harvested at different harvest points (3.3) after which we focused on metabolites produced in dough at Tx by these yeasts (3.4).

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Table 3 The Rheofermentometer measurements of doughs prepared with the yeast harvested at seven different harvest points (YA, YB and YC ¼ during exponential growth; YD ¼ at diauxic shift; YE ¼ after diauxic shift; YF ¼ at early stationary phase; YG ¼ at late stationary phase). Hm: maximum dough height; T1: time required to reach the maximum dough height; total CO2 production, retention and loss at Hm: the total CO2 production by the yeast and the volume of CO2 that was retained in dough and the volume of CO2 lost during dough fermentation; Max. fermentation rate: the maximum volume of CO2 produced by yeast in one minute of fermentation; Tx: the onset of CO2 release from the dough; CO2 at Tx: total volume of gas produced by yeast at the onset of CO2 release.

Point Point Point Point Point Point Point

A B C D E F G

Hm (mm)

T1 (min)

Total CO2 produced at Hm (mL)

Total CO2 retained at Hm (mL)

Total CO2 loss at Hm (mL)

Max. fermentation rate (mL CO2/min)

Tx (min)

CO2 at Tx (mL)

30.4 31.5 32.8 35.6 34.0 34.9 33.8

237 194 194 198 182 195 180

600 710 1020 1270 1195 1228 1053

588 695 945 1125 1074 1096 961

12 15 75 145 121 132 92

6.6 8.3 11.2 12.7 13.6 13.2 12.2

185 127 98 65 65 74 78

397 375 342 301 278 285 287

3.3. Analysis of yeast metabolites in dough during fermentation Analysis of yeast metabolites in fermented dough was performed to investigate if certain metabolites are related to the gas retention capacity of dough. Fig. 2 shows the levels of major

metabolites (ethanol, glycerol, succinic acid and acetic acid) produced by YAeYG at 0, 30, 60, 90, 120, 150 and 180 min of fermentation in dough. In keeping with previous observations, YA showed the lowest level of ethanol (Fig. 2a) and glycerol (Fig. 2b) at all fermentation

Fig. 2. Levels of yeast metabolites and pH as a function of fermentation time (0, 30, 60, 90, 120, 150 and 180 min) for fermented dough samples with yeast cells harvested at harvest points A-G: a) ethanol, b) glycerol, c) acetic acid, d) succinic acid and e) pH. All samples were measured in duplicate, and error bars represent the standard deviation between two replicates, estimated from the range as described by Nelson (1975).

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times, pointing to a slower fermentation rate. Harvesting yeast later in the exponential growth phase (YB and YC) resulted in more ethanol and glycerol production (faster fermentation) from 0 to 180 min of fermentation compared to YA. The cells that entered diauxic shift (YD) showed the highest level of ethanol and glycerol produced during fermentation, while the fermentation ability slightly decreased when the cells were harvested after diauxic shift (YE) and in the early and late stationary phase (YF and YG, respectively). This drop in ethanol and glycerol production from YD to YG corresponds to the decline in maximum fermentation rate, total CO2 production at Hm and dough height in the Rheofermentometer analysis (Table 3). While the production of ethanol was more or less linear as a function of time, there was a slight decrease in the level of glycerol production from 120 min onwards for YBeYG. This might be attributed to the fact that the cells harvested at these points are more adapted to the osmotic stress conditions in dough and hence produce glycerol at a lower rate. Fig. 2c and d show the levels of acetic acid and succinic acid in dough during fermentation, respectively. After 180 min of fermentation, dough with YA and YG contained the lowest concentration of acetic acid and dough with YC containing the highest concentration of acetic acid, with a two-fold difference in the acetic acid levels (Fig. 2c). By contrast, the levels of succinic acid showed a different trend (Fig. 2d). Two distinct groups can clearly be distinguished. The cells harvested during exponential growth, i.e. YAeYC, showed little succinic acid production while the cells harvested during or after the diauxic shift, i.e. YDeYG, showed up to four fold higher succinic acid production. The variations in the levels of acetic and succinic acid led to differences in the dough pH (Fig. 2e). Yeast cells harvested during the exponential growth phase produced almost no succinic acid, but there was a gradual increase in the levels of acetic acid, which resulted in a lower dough pH. The yeast cells harvested in later phases, in contrast, produced relatively high levels of both acetic acid and succinic acid which led to a faster drop in dough pH. The variations in acetic and succinic acid levels between yeasts harvested at different points can be explained by the physiological state of yeast at the time of harvest. Indeed, the cells harvested before diauxic shift (YAeYC) are well adapted to fermentative growth. As a consequence, the genes responsible for the TCA cycle in these cells are likely down-regulated (Carreto et al., 2011; Liu and Butow, 1999), leading to less production of TCA cycle intermediates, such as succinic acid. These quickly growing cells also show reduced stress resistance, but when added to dough, the imposed osmotic stress decreases their activity (Nass and Rao, 1999; Reed et al., 1987; Verstrepen et al., 2004). When the cells enter the diauxic shift, the cells respond to the lack of fermentable sugar in the environment and shift their metabolism from fermentation to respiration of ethanol. This switch is also accompanied by accumulation of glycerol and trehalose, resulting in increased stress resistance (Mager and Ferreira, 1993; Verstrepen et al., 2004; Werner-Washburne et al., 1993). The cells harvested between points DeG have shifted to respiration. The high succinic acid production during dough fermentation may therefore be explained by the activation of TCA cycle in the cells harvested at these points (Carreto et al., 2011; Liu and Butow, 1999). It is interesting that the metabolic states at the time of harvest seemingly influence subsequent metabolite production. In this respect, it is important to note that dough fermentation is a relatively fast process (3 h), which explains why cells presumably do not have the time to fully adapt their metabolism to the availability of sugars in fresh dough, and still show properties related to the harvest phase. This was confirmed by analysis of the cell’s transcriptome, which shows that in the first phases of dough fermentation, cells are not yet adapted to dough conditions (Aslankoohi et al., 2013).

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3.4. Effect of yeast metabolites on dough fermentation characteristics The onset of CO2 release (Tx) does not occur at the same total CO2 volume for different harvest points (Table 3) which suggests that the release of CO2 by the dough might not only be affected by the amount of gas produced by yeast. In order to investigate a possible link between the production of yeast metabolites and the onset of CO2 release, we analyzed the levels of metabolites and dough pH at Tx for doughs fermented with YAeYG (Fig. 3). Fig. 3a shows the level of ethanol in dough at Tx for seven different harvest points of yeast. Some variations in the levels of ethanol at Tx can be observed between different harvest points (about 3e5 mmol/g yeast). Jayaram et al. (unpublished results) described that ethanol already at these low levels (3.7 mmol/g yeast) has an impact on dough properties, decreasing dough extensibility and making dough more stiff and tenacious. They hypothesized that ethanol changes gluten property by acting as a good solvent. Fig. 3b shows the level of glycerol in dough produced by yeast harvested at seven different time points at Tx. Although glycerol has been reported to influence moisture distribution and staling of bread during storage (Baik and Chinachoti, 2002; Barrett et al., 2000), it is unlikely that the small difference in glycerol level between YC and YE will have any functional significance in the fermentation phase. Fig. 3c, d and e show the levels of acetic acid, succinic acid and pH in dough at Tx for the seven harvest points of yeast, respectively. The doughs prepared with yeast harvested in the exponential growth phase (YAeYC) showed the same levels of organic acids at Tx, both for acetic acid (Fig. 3c) and succinic acid (Fig. 3d). This led to a pH of about 5.25 (Fig. 3e). From YD to YG, a decrease in the level of acetic acid (Fig. 3c) and an increase in the level of succinic acid (Fig. 3d) were observed, and the pH for these doughs was about 5.10 (Fig. 3e). Previous research by Jayaram et al. (2013) indicated that dough pH is mainly determined by the level of organic acids produced by yeast during dough fermentation. These changes in the level of organic acids produced by the yeast cells harvested at different points and in the dough pH might have an influence on rheological properties of dough such as dough extensibility and therefore, affect the gas holding capacity of dough. Several previous studies investigated the effect of acids and decreases in pH on dough rheology and bread properties. Bennett and Ewart (1962) proposed that pH could change the structure of gluten proteins by modifications in the degree of ionization of specific groups in flour proteins through electrostatic attraction/ repulsion. They found a significant decrease in dough extensibility and increased resistance to extension at low pH. Tanaka et al. (1967) reported that acids have a weakening effect on dough. They showed that a decrease in dough pH through the addition of acetic acid led to a remarkable drop in dough extensibility and increase in dough resistance in the Extensigraph at pH values below 5.1 and explained this effect by cleavage of the inter- and intra-salt linkages of proteins by hydrogen ions. These studies underpin the importance of organic acids produced by the yeast, here shown to vary at various harvest points, and thereby, how these acids influence the dough pH and the rheological properties of dough (Jayaram et al., in press). In summary, it is easy to imagine that there is a threshold CO2 capacity for dough made with various types of flour which depends on different factors such as gluten protein quantity and quality of flour. When the dough reaches this threshold, the dough will rupture and lose part of its retained gas. This threshold might be variable for different dough samples based on the fermentation speed of yeast. However, it is important to

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Fig. 3. Levels of yeast metabolites and pH at the onset of CO2 release (Tx) for fermenting dough samples with yeast cells harvested at harvest regions AeG: a) ethanol, b) glycerol, c) acetic acid (AA), d) succinic acid (SA) and e) pH. All samples were measured in duplicate, and error bars represent the standard deviation between two replicates. All measurements were performed in duplicate and error margins indicate standard deviations, estimated from the range as described by Nelson (1975). Values with the same letter are not significantly different (p < 0.05).

consider the effect of other parameters, such as level of ethanol and organic acids produced by the yeast during dough fermentation, on rheological properties of dough together with the fermentation rate and total volume of accumulated CO2. Jayaram et al. (unpublished results, in press) showed that the relevant levels of ethanol and succinic acid produced by the yeast during dough fermentation are significantly decreasing dough extensibility. The combination of produced metabolites by the yeast during dough fermentation, e.g. ethanol and organic acids, and the fermentation rate of yeast at different harvest points might be the key determining factors on gas holding capacity and the onset of CO2 loss from dough. 4. Conclusion Our results demonstrate the importance of yeast harvest time on the subsequent dough fermentation capacity of yeast, including fermentation rate and metabolite production.

Fermentation rate of yeast and, consequently, dough height were highest when the yeast cells were harvested during or after diauxic shift. The results further showed that the onset of CO2 loss from the fermenting dough (“dough rupture”) was strongly correlated with the fermentation rate of yeast at different harvest points but not determined by the amount of CO2 accumulated at the time of dough rupture. Hence, we postulate that differences in production of metabolites such as ethanol, acetic acid and succinic acid by the yeast during dough fermentation might influence dough extensibility, and therefore affect the gas holding capacity of fermenting dough. Further investigation is needed to confirm the effect of yeast metabolites on gas holding capacity and the onset of CO2 loss in dough. Together, our results illustrate the central role of yeast metabolism in dough fermentation, and open several routes for further research aimed at linking yeast physiology and metabolism to bread quality. Because the fed-batch production of yeast in industry is different from the laboratory scale production, the results obtained in this manuscript should

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be tested in industrial applications to confirm their applicability in industry. Acknowledgments The authors thank Elham Aslankoohi for her advice. The authors acknowledge financial support from the “Fonds voor Wetenschappelijk Onderzoek e Vlaanderen” (FWO, Brussels, Belgium) for project funding (G.0544.10N) and for the postdoctoral fellowship of ED and the financial support from KU Leuven IDO project IDO/12/ 011. Research in the lab of K.J.V. is supported by ERC Starting Grant 241426, VIB, EMBO YIP program, KU Leuven, FWO, IWT and the AB InBev Baillet-Latour foundation. References AACC, 2000. Approved Methods of the AACC, eleventh ed. AACC, St. Paul, MN, USA. Allen, C., Buttner, S., Aragon, A.D., Thomas, J.A., Meirelles, O., Jaetao, J.E., Benn, D., Ruby, S.W., Veenhuis, M., Madeo, F., Werner-Washburne, M., 2006. Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures. J. Cell Biol. 174, 89e100. AOAC, 1995. Official Methods of Analysis. AOAC, Washington, DC, USA. Aslankoohi, E., Zhu, B., Rezaei, M.N., Voordeckers, K., De Maeyer, D., Marchal, K., Dornez, E., Courtin, C.M., Verstrepen, K.J., 2013. Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation. Appl. Environ. Microbiol. 79, 7325e7333. Baik, M.Y., Chinachoti, P., 2002. Effects of glycerol and moisture redistribution on mechanical properties of white bread. Cereal Chem. 79, 376e382. Barrett, A.H., Cardello, A.V., Mair, L., Maguire, P., Lesher, L.L., Richardson, M., Briggs, J., Taub, I.A., 2000. Textural optimization of shelf-stable bread: effects of glycerol content and dough-forming technique. Cereal Chem. 77, 169e176. Bennett, R.J., Ewart, A.D., 1962. The reaction of acids with dough proteins. J. Sci. Food Agric. 13, 15e23. Blank, L.M., Sauer, U., 2004. TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates. Microbiology SGM 150, 1085e1093. Carreto, L., Eiriz, M.F., Domingues, I., Schuller, D., Moura, G.R., Santos, M.A., 2011. Expression variability of co-regulated genes differentiates Saccharomyces cerevisiae strains. BMC Genomics 12, 201. Celeromics, Technical Note. Cell Counting with Neubauer Chamber: Basic Hemocytometer Usage. URL http://celeromics.com/en/resources/docs/Articles/Cellcounting-Neubauer-chamber.pdf (accessed 13.09.13.). Czuchajowska, Z., Pomeranz, Y., 1993. Gas formation and gas retention. I. The system and methodology. Cereal Foods World 38, 499e503. DeRisi, J.L., Iyer, V.R., Brown, P.O., 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680e686. Fiaux, J., Cakar, Z.P., Sonderegger, M., Wuthrich, K., Szyperski, T., Sauer, U., 2003. Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis. Eukaryot. Cell 2, 170e180. Gabriela, C.G., Daniela, V., 2010. The influence of different forms of backery yeast Saccharomyces cerevisie type strain on the concentration of individual sugars and their utilization during fermentation. Romanian Biotechnol. Lett. 15, 5417e 5422. Galdieri, L., Mehrotra, S., Yu, S.A., Vancura, A., 2010. Transcriptional regulation in yeast during diauxic shift and stationary phase. OMICS J. Integr. Biol. 14, 629e638.

115

Gancedo, J.M., 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62, 334e361. Gasch, A.P., Spellman, P.T., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G., Botstein, D., Brown, P.O., 2000. Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell 11, 4241e4257. Gasch, A.P., Werner-Washburne, M., 2002. The genomics of yeast responses to environmental stress and starvation. Funct. Integr. Genomics 2, 181e192. Herman, P.K., 2002. Stationary phase in yeast. Curr. Opin. Microbiol. 5, 602e607. Jayaram, V.B., Cuyvers, S., Lagrain, B., Verstrepen, K.J., Delcour, J.A., Courtin, C.M., 2013a. Mapping of Saccharomyces cerevisiae metabolites in fermenting wheat straight-dough reveals succinic acid as pH-determining factor. Food Chem. 136, 301e308. Jayaram, V.B., Cuyvers, S., Verstrepen, K.J., Delcour, J.A., Courtin, C.M., 2013b. Assessment of the potential impact of yeast’s primary metabolite e ethanol on dough rheology (unpublished results). Jayaram, V.B., Cuyvers, S., Verstrepen, K.J., Delcour, J.A., Courtin, C.M., 2013c. Succinic acid in levels produced by yeast (Saccharomyces cerevisiae) during fermentation strongly impacts wheat bread dough properties. Food Chem.. http://dx.doi.org/10.1016/j.foodchem.2013.11.025 (in press). Liao, Y.E., Miller, R.A., Hoseney, R.C., 1998. Role of hydrogen peroxide produced by Baker’s yeast on dough rheology. Cereal Chem. 75, 612e616. Lillie, S.H., Pringle, J.R., 1980. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J. Bacteriol. 143, 1384e1394. Liu, Z., Butow, R.A., 1999. A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response to a reduction or loss of respiratory function. Mol. Cell Biol. 19, 6720e6728. Mager, W.H., Ferreira, P.M., 1993. Stress response of yeast. Biochem. J. 290, 1e13. Nass, R., Rao, R., 1999. The yeast endosomal Naþ/Hþ exchanger, Nhx1, confers osmotolerance following acute hypertonic shock. Microbiology 145, 3221e3228. Nelson, L.S., 1975. Use of the range to estimate variability. J. Qual. Technol. 7, 46e48. Newberry, M.P., Phan-Thien, N., Larroque, O.R., Tanner, R.I., Larsen, N.G., 2002. Dynamic and elongation rheology of yeasted bread doughs. Cereal Chem. 79, 874e879. Randez-Gil, F., Sanz, P., Prieto, J.A., 1999. Engineering baker’s yeast: room for improvement. Trends Biotechnol. 17, 237e244. Reed, R.H., Chudek, J.A., Foster, R., Gadd, G.M., 1987. Osmotic significance of glycerol accumulation in exponentially growing yeasts. Appl. Environ. Microbiol. 53, 2119e2123. Rezaei, M.N., Dornez, E., Verstrepen, K.J., Courtin, C.M., 2013. Evaluation of hydrogen peroxide production by Saccharomyces cerevisiae during dough fermentation and its effect on rheological properties of dough (unpublished results). Smets, B., Ghillebert, R., De Snijder, P., Binda, M., Swinnen, E., De Virgilio, C., Winderickx, J., 2010. Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae. Curr. Genet. 56, 1e32. Stahl, G., Salem, S.N., Chen, L., Zhao, B., Farabaugh, P.J., 2004. Translational accuracy during exponential, postdiauxic, and stationary growth phases in Saccharomyces cerevisiae. Eukaryot. Cell 3, 331e338. Tanaka, K., Furukawa, K., Matsumoto, H., 1967. The effect of acid and salt on the farinogram and extensigram of dough. Cereal Chem. 44, 675e679. Taylor, F.R., Parks, L.W., 1979. Triacylglycerol metabolism in Saccharomyces cerevisiae relation to phospholipid synthesis. Biochim. Biophys. Acta 575, 204e214. Verstrepen, K.J., Iserentant, D., Malcorps, P., Derdelinckx, G., Van Dijck, P., Winderickx, J., Pretorius, I.S., Thevelein, J.M., Delvaux, F.R., 2004. Glucose and sucrose: hazardous fast-food for industrial yeast? Trends Biotechnol. 22, 531e537. Werner-Washburne, M., Braun, E., Johnston, G.C., Singer, R.A., 1993. Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 57, 383e401. Zlotnik, H., Fernandez, M.P., Bowers, B., Cabib, E., 1984. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J. Bacteriol. 159, 1018e1026.