Thermal resistance of Saccharomyces yeast ascospores in beers

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Chapter 2. Thermal resistance of Saccharomyces yeast ascospores in beers

Chapter Abstract

The industrial production of beer ends with a process of thermal pasteurization. Saccharomyces cerevisiae and Saccharomyces pastorianus are yeasts used to produce top and bottom fermenting beers, respectively. In this research, first the percentage of sporulation of 12 Saccharomyces strains was studied. Then, the thermal resistance of ascospores of four S. cerevisiae strains (DSMZ 1848, DSMZ 70487, ATCC 9080, Ethanol Red®) was determined in 4% (v/v) ethanol lager beer. D60˚C-values of 11.2, 7.5, 4.6, and 6.0 min and z- values of 11.7, 14.3, 12.4, and 12.7°C were determined for DSMZ 1848, DSMZ 70487, ATCC 9080, and Ethanol Red®, respectively. Lastly, experiments with 0 and 7% (v/v) beers were carried out to investigate the effect of ethanol content on the thermal resistance of S. cerevisiae (DSMZ 1848). D55°C-values of 34.2 and 15.3 min were obtained for 0 and 7% beers, respectively, indicating lower thermal resistance in the more alcoholic beer.
These results demonstrate similar spore thermal resistance for different Saccharomyces strains and will assist in the design of appropriate thermal pasteurization conditions for preserving beers with different alcohol contents.
Keywords: pasteurization; ethanol; heat resistance; strain; sporulation method; percentage of sporulation


A Sumerian tablet found in Mesopotamia dated 6000 years ago is the oldest evidence of beer production (Mirsky, 2007; Nelson, 2014). Beer is an alcoholic beverage obtained by yeast fermentation of the sugar from malted cereal grains (e.g. barley, wheat). The production of beer consists of several stages: the transformation of barley water extract to malt (malting), the conversion of malt to wort (mashing), yeast pitching, fermentation of sugars to ethanol and post-fermentation operations. The main post-fermentation operations are beer clarification/filtration, packaging, and pasteurization. The hops added during production are responsible for the bitter flavour and contribute to its natural preservation. The beer ingredients (e.g. water, cereal, hops, and yeast) can be combined in different ways to create different styles of beers such as ale, lager, stout, pilsner, etc. A few regions such as Senne Valley in Belgium still use wild yeasts for spontaneous fermentation. Ale and lager are the two major classes of beers, obtained with top and bottom fermentation yeasts, respectively. An ale beer ferments with top-cropping Saccharomyces cerevisiae at temperatures around 15 to 20°C. A lager beer is fermented by bottom-cropping yeasts such as Saccharomyces carlsbergensis (pastorianus) or Saccharomyces uvarum at temperatures ranging between 8 and 13°C (Hardwick et al., 1995; Hornsey, 2003). Brewer’s yeast has been the focus of several studies (Hammond, 1993; Linko et al., 1998; Priest and Yeasts, 2006; Stewart and Russell, 1986). Dengis and Rouxhet (1997) studied the surface properties of top- and bottom-fermenting yeast and Fleet (1998) reviewed the microbiology of alcoholic beverages.
The production of industrial bottled beer ends with a process of thermal pasteurization. This thermal process aims to inactivate the fermenting yeast used as starter along with potential spoilage microorganisms such as wild yeasts, Lactobacillus, Pediococcus, Leuconostoc and other bacteria that can contaminate the beer during the fermentation (Priest, 2003; Priest and Yeasts, 2006). The pasteurization enables the stabilization of the beverage for a longer period, increasing the beer shelf-life. The thermal pasteurization measure for the beer is the pasteurization unit (PU): 1 PU is equivalent to 1 min at 60ºC. The minimum thermal pasteurization applied by breweries is 15 PU = 15 min at 60°C, which was established based on the thermal resistance of the brewing yeast in the vegetative form. The processing time for 15 PU at other temperatures can be estimated based on the yeast z-value of 7.0°C (Del Vecchio et al., 1951; Portno, 1968). Beer contains carbon dioxide, alcohol, and hops, all of which are natural antimicrobials, so a mild pasteurization is effective for its stabilization at room temperature (Silva & Gibbs, 2009; Silva et al., 2014). Higher S. cerevisiae percentage of sporulation was registered when beer, barley, and malt extracts were added to the sporulation agar (Lin 1978; 1979). This suggests it is possible to find yeast ascospores during brewing, especially due to the adverse conditions created by the ethanol, hops and carbon dioxide, all antimicrobial beer components. Ascospores are more resistant to thermal processing than vegetative cells, so inactivation of the ascospores will also inactivate the vegetative cells (Milani et al. 2015a).
King et al. (1978) found that flash pasteurization at 71°C for 30 s did not fully inactivate the beer spoilage organisms such as Lactobacillus brevis, Pediococcus cerevisiae, and a wild yeast Saccharomyces diastaticus. Normally, bottled beer is processed at 65-68°C for 20 min or 72-75°C for 1-4 min, equivalent to 10-20 PU (Fricker, 1984), since beer is carbonated, contains ethanol, has a low pH from 3 to 4.2 (Horn et al., 1997) and is bittered with hops, which are all natural antimicrobials. Therefore, thermal pasteurization is effective for its stabilization at room temperature (Silva & Gibbs, 2009). However, concerns have been expressed, especially in ethanol-free and in less bitter beers, the last being a trend in consumer preference. L’Anthoen and Ingledew (1996) reported that the D-value of lactic acid bacteria was four- to seven-fold higher in ethanol-free beer compared to 5% (v/v) ethanol beer. In addition, pathogens such Escherichia coli O:157:H7 and Salmonella typhimurium were also more heat resistant by three to seventeen times in alcohol-free beer. Presently, the beer industry applies a more severe pasteurization process (e.g. 120 to 300 PU), to cope with on-going modifications in the traditional beer composition (Silva et al., 2014).
The thermal inactivation of microorganisms is often described by first order kinetics, with D-and z-values being the parameters estimated. Buzrul (2007) used first order kinetics for modelling S. carlsbergensis vegetative cell survivors in beer. D-value is the time required at a given temperature to inactivate 90% of the studied microorganisms and z-value is the temperature required for a one-log reduction in the D-value (Bigelow and Esty, 1920; Silva and Gibbs, 2009). The D- and z- values are used to define beer pasteurization times at different temperatures.
Although some researchers have determined S. cerevisiae thermal resistance parameters, only one performed tests in beer and these experiments were carried out with vegetative cells (Tsang and Ingledew, 1982). Past work with S. cerevisiae in fruit juices (Put et al., 1976; Put and Jong, 1982) demonstrated that the ascospores are 25 to 350 times more heat resistant than vegetative cells, and the highest D60°C-value for ascospores (among the 21 strains tested) was
19.2 min. Considering the huge difference between the thermal resistance of ascospore and vegetative cells, one can assume that if spores are destroyed, all the vegetative cells will also be. Lin (1979) obtained higher percentage of sporulation of S. cerevisiae when beer, barley, and malt extracts were added to the sporulation agar. This suggests it is possible to find yeast ascospores during brewing, especially due to the adverse conditions created by the ethanol, hops and carbon dioxide, all natural antimicrobial beer components. In another study using Pulsed Electric Fields, we have observed that the inactivation of S. cerevisiae ascospores was easier in high-alcohol beers (Milani et al., 2015b; Chapter 6). Hence, the study of the effect of beer alcohol content on the thermal inactivation of yeast ascospores is also important to investigate.
Therefore, the objectives of this work were to determine: (i) the percentage of sporulation of different brewing and non-brewing Saccharomyces yeast strains; (ii) the thermal resistance (D- and z-values) of ascospores of four S. cerevisiae strains in beer; (iii) the effect of beer alcohol content on the thermal resistance of S. cerevisiae DSMZ 1848 ascospores.

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Material and methods

Yeast strains

The eight strains of S. cerevisiae and four strains of S. pastorianus used in this investigation were obtained from different culture collection described in Table 2.1. ATCC 9080, CBS 1171 (top fermenting yeast, neo type strain isolated from beer), CBS 1503 (type strain bottom fermenting), CBS 1538 (neo type strain isolated from beer), DSMZ 1848 (hybrid isolated from bottom fermenting beer), DSMZ 70487 (isolated from super attenuated beer), Wyeast 1469 (commercial bottom fermenting brewing yeast) and Wyeast 2278 (commercial top fermenting brewing yeast). In addition the following strains from the School of Biology Sciences of the University of Auckland were used because of their good sporulation: BC186 (natural isolate from oak trees), SK1 (=NCYC 3265, lab strain isolated from soil; Liti et al., 2009), Zymaflore F15 (commercial wine yeast; Harsch & Gardner, 2013), and Lesaffre
Ethanol Red® (industrial fermentation).
All the strains were tested for sporulation while for the thermal inactivation experiments the strains DSMZ 1848, DSMZ 70487, ATCC 9080, and Ethanol Red® were used.

Yeast enumeration

Colony formation was used for yeast enumeration. Yeast Extract Peptone Glucose (YEPG) medium was prepared by mixing 0.5% (w/v) yeast extract, 1.0% (w/v) peptone, 2.0% (w/v) glucose, 2.0% (w/v) agar. The agar medium was autoclaved at 121°C for 10 min. A volume of 100 µL of appropriately diluted beer samples containing the yeast was spread into duplicate agar plates and colonies were counted after 2 days of incubation at 28°C.

Ascospores production

The culture stored at -80°C was streaked on YEPG agar and after growth a fresh single colony was inoculated into 50 mL of presporulation sterilised liquid (121°C, 10 min) composed of 0.8% yeast extract, 0.3% peptone, 10% glucose, and zinc sulphate 25 mg/L. After inoculation, the presporulation flasks (500 mL) were incubated overnight in incubators (with rotary shaking at 168 rpm) at 28°C. When optical density (PG Instrument T60 set at 600 nm) reached around 0.2 to 0.8, an appropriate portion of the presporulation broth (ca. 1.5 mL) was inoculated into sterile sporulation broth (10 mL) to yield 107 cfu/mL. Sporulation broth consisted of potassium acetate 1% (w/v), bacto yeast extract 0.1% (w/v), glucose 0.05% (w/v), zinc sulphate 25 mg/L. The mixture was incubated at 18°C for 14 days (with rotary shaking at 230 rpm) in 1-L Erlenmeyer flasks. The solution was split in 1-mL Eppendorf tubes and the spores were extracted from the vegetative (parental cells) by adding 100 µL Zymolyase solution (5 mg/mL solid Zymolase in pH 7.2 buffer containing 1.2 M sorbitol and 0.1 M KH2PO4), 900 µL spheroblasting buffer (2.2 M sorbitol), and 800 µL softening buffer (100 mM Tris-SO4, pH 9.4, 10 mM dithiothreitol (DTT) solution). Then, the mixture was incubated at 30°C in a water bath for 2 h and the Eppendorfs were gently inverted every 20 min to accelerate the break-up of tetrads into single ascospores. The spores were harvested by centrifuging three times at 9700 g (rotor F-45-12-11) for 1 min and resuspending in 200 µL of 0.5% Triton X-100 to ensure total removal of the enzyme. After the last resuspension, 4 µL DTT was added to the Eppendorfs containing the spore solution. Then, the Eppendorfs were sonicated three times at 6 Hz for 2 min, both to break up tetrads into single ascospores and to kill any vegetative cells remaining in the medium. Finally, 1 mL of salt triton dithiothreitol (STD) solution (0.1 g NaCl in 10 mL of 0.05% Triton X-100) was added to the spore solution to avoid spore aggregation (Xiao, 2006).


Determination of percentage of sporulation

The percentage of sporulation was determined after 7 days of incubation and reassessed after 10 and 14 days. Strains showed different behaviours during sporulation. Some strains changed into tetrads, some triads, some dyads, and others stayed as vegetative cells. In order to measure the percentage of sporulation, a portion of 50 µL of the spores was diluted into 950 µL of a 1:1 mixture of sterile water and methylene blue (ca 107 cfu/ml) and the spores were counted under a microscope using a haemocytometer. Adding the methylene blue to the spore suspension allowed differentiating the live from dead cells, due to permeation of the methylene blue through the cell walls of dead cells. Blue-staining (dead) cells were not counted. Percentage of sporulation was calculated as the percentage of tetrads and/or triads divided by the total cell counts (tetrads, triads, dyads, and vegetative cells). Four replicate counts were carried out for each strain and the percentage of sporulation average ± standard deviation was determined. ANOVA was used to investigate significant differences between yeast strains (Statistica version 8, USA), and when differences were detected (p<0.05), Tukey’s Honest Significant Difference (HSD) test was carried out to separate the average values.

Saccharomyces thermal inactivation experiments

Ethanol is the major alcohol of beer fermentation by yeast. Alcohol by volume abbreviated as ABV, abv, or alc/vol is a standard measure of how much alcohol (ethanol) is contained in a given volume of an alcoholic beverage. It is expressed as a volume percent and defined as the number of millilitres of pure ethanol present in 100 mL of beer at 20°C, (% v/v ethanol). Commercial beers with 0, 4 and 7% ethanol were selected for the thermal inactivation studies, since they represent the minimum, standard and maximum alcohol concentrations found in commercial beers (Turner, 1990; Priest & Stewart, 2006). The alcohol content was read from the beer bottle label. For the comparison of the thermal resistance of the four strains’ ascospores, 4% alc/vol beer was used. With respect to the effect of alcohol content on the thermal resistance, the strain DSMZ 1848 was used in 0 and 7% alc/vol beers.
A preliminary experiment was initially carried out to investigate the degree of difference in thermal resistance between ascospores and vegetative cells of DSMZ 1848 S. cerevisiae in 4% alc/vol beer, and the D55°C-value was determined for vegetative and ascospore cells.
Then, the main experiments were carried out at 50, 55, 60 and 65°C with ascospores of S. cerevisiae DSMZ 1848, DSMZ 70487, and Ethanol Red® and S. pastorianus ATCC 9080 (also named S. cerevisiae) using 4% alc/vol beer. In the last set of experiments, spores of the most thermal resistant yeast, S. cerevisiae DSMZ 1848, were used in 0 and 7% alc/vol beers to investigate the effect of beer ethanol content on the ascospores D-value at 50 and 55°C.
Each yeast ascospore solution was centrifuged to remove the STD solution. Filter-sterilized beer was mixed with the spore pellet to yield a final ascospore concentration of ca. 106-107 cfu/mL. The clustering and the large size of ascospores did not allow the use of higher initial spore concentration. Five millilitres of beer samples containing the yeast spore were vacuum packed in 5×5 cm heat-resistant pouches that had been previously sterilized (Caspak, New Zealand). The removal of the air inside the bag increased the heat transfer and produced more reliable results, with less variation. The 154-µm thick film can withstand temperatures up to 110°C and was composed of linear low density poly ethylene (LLDPE) and poly ethylene therephthalate (PET). A thermostatic water bath (W28 Grant Instruments, Cambridge, Ltd, England) equipped with stirring ensured uniform temperature throughout the bath during thermal experiments. After setting the water bath temperature to the desired treatment temperature, the packed beer samples were fully submerged in the water bath for pre-specified times between 3 and 90 min. For each time point, two replicates of beer samples were removed and placed immediately into an ice container to avoid more spore killing. The yeast spore survivors were enumerated

Chapter 1. Literature review
1.1. Beer
1.2. High pressure processing (HPP)
1.3. Power ultrasound
1.4. Pulsed Electric Fields (PEF)
Chapter 2. Thermal resistance of Saccharomyces yeast ascospores in beers
2.1. Introduction
2.2. Material and methods
2.3. Results and discussion
2.4. Conclusions
Chapter 3. High pressure processing and thermosonication of beer: comparing the energy requirements and Saccharomyces cerevisiae ascospores inactivation with thermal processing and modelling
3.1. Introduction
3.2. Material and methods
3.3. Results and discussion
3.4. Conclusions
Chapter 4. Nonthermal pasteurization of beer by high pressure processing: Modelling the inactivation of Saccharomyces cerevisiae ascospores in different alcohol beers
4.1. Introduction
4.2. Material and methods
4.3. Results and discussion
4.4. Conclusion
Chapter 5. Ultrasound pasteurization of beers with different alcohol levels: Modelling the inactivation kinetics of Saccharomyces cerevisiae ascospores
5.1. Introduction
5.2. Material and methods
5.3. Results and discussion
5.4. Conclusion
Chapter 6. Pulsed Electric Field continuous pasteurization of different types of beers
6.1. Introduction
6.2. Material and methods
6.3. Results and discussion
6.4. Conclusion
Chapter 7. Studies on the mechanisms of Saccharomyces cerevisiae spores inactivation by scanning electron microscope observations
7.1. Introduction
7.2. Material and methods
7.3. Results and Discussion
7.4. Conclusions

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