Temperature Response of New Lager Brewing Strains Isolated From WS34-70

EDITURA ACADEMIEI ROMNE http://www.ear.ro

Romanian Journal of Food Science

Official Journal of the Romanian Association of Food Professionals

http://www.asiar.ro

Romanian Journal of Food Science 2011, 1(1): 2638 26

Temperature response of new lager brewing strains isolated from WS34/70 Irina BOLAT 1, Maria TURTOI 2,* and Michael C. WALSH 1 1 Heineken Supply Chain, Burgemeester Smeetsweg 1, 2382 PH Zoeterwoude, The Netherlands 2 Galati Dunarea de Jos University, 47 Domneasca St., 800008 Galati, Romania Received 5 September 2009; received in revised form 17 March 2010; accepted 21 April 2010.

Abstract

Lager beers cover the largest part of the beer market, hence the increase interest for the lager brewing yeast. The most widely used lager brewing yeast is represented by the WS34/70 population. Previous analysis revealed that the WS34/70 population is actually made of a number of variant strains instead of being a pure culture. Thus, one could find within mixture: variant a 47%, variant b 714%, variant c 4277%, variant d 218%, variant e 17%, variant f 1%, and so on: from samples taken from different locations (breweries as well as agar slant from Weihestephan and Saflager dried yeast). The present study focuses on the fermentation behaviour displayed by two of the selected variants and the initial mixture, represented by the commercial brewers lager yeast WS34/70, applying two different fermentation temperatures: 10C and 15C. The two variants used in this study have disparities as far as the nuber of chromosomes is concerned, the main difference is the lack of one chromosome for one of the variants, while the other possesses a highlighted chromosome band in the corresponding location. The evolution of the attenuation degree, yeast cell multiplication, pH value, fermentation rate, free amino nitrogen (FAN) values, and the diacetyl and 2,3 pentandione production were analysed during the trials, as well as the flavour compounds profile for each strain and each temperature. The results indicate the higher temperature as an accelerator for the extract reduction, pH, number of cells and vicinal diketone reduction for all three strains. Variant a is slightly faster in terms of fermentation rate at 15C and diacetyl reduction both at 10C and 15C. The acetate ester production was higher at 15C, while the acetaldehyde production was favoured by lower fermentation temperatures.

Keywords: brewing yeast, fermentation temperature, fermentation rate, attenuation degree, extract reduction, free amino nitrogen, diacetyl, esters, flavour compounds, yeast cell multiplication, chromosome, strain.

1. Introduction

Lager yeast cultures that are mixtures of very closely related strains are usually employed in the breweries. Each yeast strain may perform differently under a given set of fermentation conditions. The choice of a yeast strain depends on characteristics considered important: attenuation limit, fermentation * Corresponding author: Tel.: +40 744 363190, E-mail: [email protected]

rate, and oxygen requirements etc. (Priest and Stewart, 2006) the extent of transformations during the fermentation process depends on yeast ability to adapt to the new environment (Vesely, 2004).

The desire to increase the capacity of breweries without further investments calls for different measures like high-gravity brewing or accelerated fermentation. Higher temperature represents the most attractive mean to accelerate fermentation by stimulating yeast nitrogen uptake, cell growth and budding but also causes an increase in the formation

Temperature response of new lager brewing strains isolated from WS34/70


of certain flavour active fermentation by-products, producing a beer which is often quite different in flavour from that produced by the same yeast strain from a normal process (Takahashi et al., 1997; Lewis, 1974). Yeast plasma membrane seems to be the probable site of difference between yeasts grown at various temperatures (Lewis, 1974).

Heyse and Piendl (1974) studied the influence of different fermentation temperatures on the activity of some enzymes, grouping them as follows: enzymes whose activities are increased with a rise in fermentation temperature: maltotriase, maltase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase, citrate synthase, isocitrate dehydrogenase, enzymes whose activities decrease with a rise in fermentation temperature: hexokinase, glycerokinase, fumarase and enzymes with intermediate activities at low temperatures and increase activity at high temperatures: phosphoglyceraldehyde dehydrogenase and glucose 6-phosphatedehydrogenase.

An increase in fermentation temperature is assumed to induce higher concentrations of fusel alcohols and esters.

The balance of flavour metabolites is largely a consequence of the combination of raw material quality, yeast strain and wort composition. The essential character of a beer is determined by yeast metabolism and the plethora of yeast metabolic by-products that arise during fermentation (Masschelein, 1981; Boulton and Quain, 2001; Vesely et al., 2004). The flavour-active compounds produced by yeast include: fusel alcohols (810 carbon atoms aliphatic alcohols), glycerol, esters, organic and fatty acids, sulphur compounds, aldehydes and ketones, phenols, amines and a number of miscellaneous compounds (Boulton and Quain, 2001; Vesely et al., 2004; Babb, 2008; Priest and Stewart, 2006).

Saerens et al. (2008 b), monitoring the influence of fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast, noticed that there is good correlation between flavour production and the expression level of specific genes involved in the biosynthesis of aroma compounds.

Yeast contains several enzyme systems with different substrate specificities that positively impact flavour stability by reducing the level of carbonyl compounds present in wort. These attributes vary among the yeast strain according to their genetic background and sensitivity to fermentation

conditions (Boulton and Quain, 2001; Vesely et al., 2004).

Vesely and his team presented the results of their experiments on yeast reductase activity at different temperatures, observing that a 10C fermentation delays the maximum activity of the yeast aldehyde reductase to 4 days, whereas a 15C fermentation determines these enzymes to reach their maximum within 2 days (Vesely et al., 2004).

The most important flavour-active compounds in beer are the esters. They impart characteristic flowery and fruit-like flavours and aromas to beer. They have relatively low taste thresholds that are often attained. Their presence is desirable at appropriate concentrations; however, in higher amounts, they can destroy the flavour balance. (Verstrepen et al., 2003; Hammond, 1986) The flavour-active esters, whose concentrations in beer are considered crucial to product quality, include ethyl acetate (solvent/fruity like), isoamyl acetate (apple, banana like), isobutyl acetate (banana, fruity), ethyl caproate (sour apple) phenyl ethyl acetate (flowery, roses, honey) (Verstrepen et al., 2003; Stewart, 2005; Piendl and Geiger, 1980) The fact that most esters are present in concentrations around the threshold value, implies that minor changes in concentration may have dramatic effects on beer flavour (Verstrepen et al., 2003; Renger et al., 1992).

There are two main groups of flavour-active esters in fermented beverages: the first group contains the acetate esters (such as isoamyl acetate), the second group is the ethyl esters (such as ethyl hexanoate). There are many factors that affect the ester profile, among them the yeast strain and the fermentation temperature has strong effects on the perception of the flavour of the beer produced (Saerens et al., 2008 a; Stewart, 2005).

Fermentation temperatures and ester formation are directly related due to the fact that higher temperatures could make cell membranes more fluid and this could possibly modulate the activity of the membrane-bound alcohol acetyltransferase or simply increase diffusion rates of esters from cells into the beer (Boulton and Quain, 2001).

This paper focuses on the influence of two different temperatures, 10C and 15C respectively, upon the behaviour of new lager yeast strains isolated from WS34/70 population against the initial mixture.

The study follows the fermentation kinetics, yeast behaviour and flavour compounds. This paper represents a part of a larger project concerning the

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Irina BOLAT, Maria TURTOI and Michael C. WALSH


characterization of the lager brewing yeast WS34/70. 2. Materials and methods 2.1. Yeast variants

The two variants used in this trial were isolated and selected from the WS34/70 lager yeast purchased from Weihenstephan (Germany) as yeast agar slant. The WS34/70 population, as well as the variants a and b, selected from WS34/70 as natural variants, were preserved as glycerol stock at 80C.

The identification of the variants within the initial population was done using pulsed field gel electrophoresis, a technique that allows separation of large deoxyribonucleic acid (DNA) molecules, typically ranging in size from 50 to 10 000 kb (Johnston, 1994).

Variant a posses a highlighted chromosome band in the location where variant b lacks the chromosome. Furthermore, variant a is a medium flocculent strain, while variant b is a flocculent strain. 2.2. Fermentation conditions

Fermentations were performed on 16Plato all malt wort, collected from a brewery. Zinc was added into the wort (0.5 ppm final concentration of Zn2+). The fermentations were run in duplicate in 50 mL glass bottles with 30 mL wort, at 10C and 15C respectively, under continuous agitation. The pitching rates are shown in Table 1. Fermentations were stopped after 8 days (at around 80% apparent degree of fermentation).

Table 1. Pitching rate used within the trial

Pitching rate (106 cell/mL) Strain 10C 15C

Control 24.65 0.49 24.20 0.14 Variant a 31.25 3.89 27.05 2.19 Variant b 21.90 0.42 27.35 2.05

2.3. Analysis of different parameters

The extract evolution was analysed using the Anton Paar portable density meter. Yeast cell concentration and yeast viability were determined using the NucleoCounter YC-100 System with its NucleoCassettes containing propidium iodine.

Gas chromatography analysis was used with flame ionization detection (GC/FID) for esters and higher alcohols and an electron capture detector (GC/ECD) for diacetyl and 2,3-pentandione.

Samples were taken at certain intervals; the yeast cells and the fermenting wort were separated by in a refrigerated centrifuge at 2C for 5 min at 3000 rpm.

Free amino nitrogen was determined by the EBC ninhydrin method. 3. Results and discussion 3.1. Degree of fermentation, fermentation rate, cell

multiplication, free amino nitrogen profile during fermentation, pH evolution

The two variants used in this trial were referred to as variant a and variant b. The initial mixture out of which these two yeast strains were selected, was represented by WS34/70 population and referred to as control strain. The fermentation behaviour of these three lager yeast strains was monitored during fermentation in all malt wort at 10C and 15C, at laboratory scale. From the degree of fermentation profiles, as seen in Figure 1 for the trial performed at 10C and the trial performed at 15C, a faster fermentation is seen at 15C, as expected. At 10C, all analysed variants reach a degree of fermentation within 7080% only after 6 days of fermentation, while at 15C after 4 days (Figure 1).

Among the yeasts studied at 10C, variant b stands out as the strain that reached the highest attenuation rate at low temperature. At high temperature, after 8 days, the degree of fermentation overpasses 80%, while at the low temperature it barely gets to 75%. These results are supported by the fermentation speed values in Figure 2. Thus, the fermentation rate was faster at 15C than at 10C, with 0.48Plato/day for the control strain, 0.61Plato/day for variant a and 0.49Plato/day for variant b at high temperature. Moreover, the two variants and the control strain analysed, showed the same value for the fermentation rate at 10C, while small differences among the two variants and the control mixture of strains had been noticed at 15C with a slightly faster rate for variant a (Figure 2).

Yeast cell growth during the two trials was expressed as number of generations (n):

2lnlnln 0NNn

= (1) where: N is the peak cell count/mL; N0 the initial cell count/mL.

The values for this parameter were higher for all the variants during the fermentation at 15C, as shown in Figure 3 than for 10C: with 26% higher

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Figure 1. Attenuation degree during fermentation at 10C and 15C. Mean values of the duplicates.

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Figure 2. Fermentation rate of the two variants and the control strain.

for variant a, 20% for the control sample and 18.5% for variant b.

A strong growth rate entails a greater utilisation of ATP, its inhibiting effect is thus diminished and the enzymes activity is gradually increasing (Heyse and Piendl, 1974).

The values presented in Figure 3 indicate a higher yeast yield of the control mixture both at 10C and 15C. Thus, one can speculate that the

control mixture probably contains other strains with higher yeast yield than the two variants studied herein.

Free amino nitrogen or FAN refers to the free -amino nitrogen and includes all of the amino acids minus proline. Proline, the most plentiful aminoacid in wort, is not an -aminoacid and is not utilizable by Saccharomyces under anaerobic conditions.

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2.02

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Figure 3. Number of generations calculated for the two variants and the control strain.

However, under aerobic laboratory condition,

proline is assimilated after exhaustion of the other aminoacids (Priest and Stewart, 2006). FAN is expressed as milligrams N per liter assimilable nitrogen and affects other fermentation factors, such as cell growth, biomass, viability, pH and attenuation rate.

FAN was reduced with a faster rate at high temperature, the decrease being much more significant at 15C than at 10C (Figure 4). The amino acids rapid uptake at 15C is reflected in the higher rate of yeast growth expressed as number of generations in Figure 3. During the fermentation at higher temperatures there is no evident difference among the two variants and the control yeast regarding the FAN uptake profile, while at 10C variant b stands out with a low uptake rate and a high free amino nitrogen amount after 8 days of fermentation.

There is a good correlation between FAN evolution during the two experiments and the pH profile (Figure 5). The pH characteristically drops during fermentation due to uptake of wort aminoacids and proton extrusion by actively growing and fermenting yeast. A higher amount of amino-acids in wort (the main pH buffering capacity in wort) will lead to higher pH values. High residual quantities of amino nitrogen in beer can cause poor flavour stability and an increase disposition to beer spoiling microorganisms. After 144 hours of fermentation, the pH in the beer fermented at 15C starts to raise, this is due to cell lysis characteristic-

ally within higher temperatures when the stress on the yeast is the most severe. 3.2. Flavour compounds profile during

fermentation: acetaldehyde, isoamyl acetate, ethyl acetate, total higher alcohols

Acetaldehyde is an indicator of the fermentation quality, higher content is less desirable.

Acetaldehyde formation in beer occurs during the period of active yeast growth (Boulton and Quain, 2001) as seen in Figure 6, with an increase in the beginning and a decrease towards the end of fermentation.

The maximum concentration of this compound was registered within 3055% degree of attenuation. The control strain and variant a show a significant decrease in acetaldehyde levels after 48 hours of fermentation, while variant b after 72 hours of fermentation at 10C. During the fermentation at 15C of the control strain and variant a a pronoun-ced decrease in the acetaldehyde amount started earlier, after only 24 hours of fermentation, and after 48 hours for variant b.

After 7 fermentation days, the amount of acetaldehyde within the beers produced at 10C was higher than in the beers produced at 15C; nevertheless, after 8 fermentation days the situation was reversed, with acetaldehyde values lower at 10C than at 15C. Vesely et al. (2004) noticed that longer fermentation time at 10C resulted in slightly lower levels of carbonyl compounds.

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WS34/70 (10C) Variant a (10C) Variant b (10C) WS34/70 (15C) Variant a (15C) Variant b (15C)

Figure 4. Free amino nitrogen utilization during fermentation at 10C (filled markers) and 15C (empty markers).

Values are means of the duplicates at 10C and 15C respectively.

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Figure 5. pH evolution during fermentation at 10C and 15C respectively of the two variants and the control strain.

Values are means of the duplicates at 10C and 15C.

Among the flavour-active compounds in beer, two very important acetate esters, i.e., isoamyl acetate and ethyl acetate were analysed during this trial.

The taste thresholds of these two esters are mentioned in different papers: Isoamyl acetate: 1.6 mg/L (Renger et al., 1992;

Piendl and Geiger, 1980), 0.61.2 mg/L (Verstrespen et al., 2003), 2mg/L (Hammond, 1986), 1.4 mg/L (Babb, 2008);

Ethyl acetate: 33 mg/L (Renger et al., 1992), 2130 mg/L (Verstrespen et al., 2003), 25 mg/L (Hammond, 1986), 30 mg/L (Piendl and Geiger, 1980). A certain ester level is necessary for the normal

flavour of beer, thus beer lacks in fruitiness if the isoamyl acetate level is below 1 mg/L, while it is too high if the level is above 3 mg/L (Piendl and Geiger, 1980).

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Figure 6. Acetaldehyde production during fermentation at 10C and 15C of three lager yeast strains.

Values are means of the duplicates performed at 10C and 15C.

Isoamyl acetate is produced from a reaction between amyl alcohol and acetyl coenzyme A catalyzed by the enzyme isoamyl alcohol acetyl transferase (Quilter et al., 2003).

Within this trial both isoamyl acetate and ethyl acetate were produced in higher amounts during the fermentation performed at 15C than at 10C (Figures 7 and 8).

In the case of isoamyl acetate, the mean value of this ester during fermentation at 15C was higher with 50% for the control strain, with 40% for variant a and 35% for variant b than at 10C.

Smaller differences were noticed for the mean value of the ethyl acetate generated during the fermentation performed at 15C as opposed to 10C: 20% higher for the control strain, 14% for variant a and 15% for variant b.

At the beginning of fermentation, when the active lipid synthesis required for cell growth is taking place, the specific rate of ester synthesis is relatively low (Figures 7 and 8).

Later, as growth becomes restricted by the lack of sterols and unsaturated fatty acids, the specific rate of ester synthesis increases. After 6 days of fermentation, the amount of isoamyl acetate in beer starts to decrease both at 10C and at 15C fermentation temperature.

Engang and Aubert (1977) also found an increase in the concentration of these compounds with temperature when they used 8C, 10C and 12C, both at full scale and laboratory scale. They mentioned through the work of Drews and his team, who found an increase in the isoamyl acetate content when the temperature changed from 9C to 14C, but no difference between 14C and 19C.

The differences in terms of ethyl acetate production at 10C and 15C are rather limited.

The lower the ratio ethyl acetate / isoamyl acetate the better the quality of the product. There is a significant difference of this ratio for the two temperatures (Table 2) with better values obtained when 15C fermentation temperature was applied.

In terms of differences among the two variants and the control strain, variant b seems to produce a beer with the most balanced flavour at 10C, while at 15C the control strain showed the lowest ratio.

The main contribution of higher alcohols to beer flavour is by a general intensification of alcoholic taste and aroma and by imparting a warming character. A second very important role of fusel alcohols is to provide precursors for ester synthesis (Boulton and Quain, 2001). Their formation is linked to amino-acid and carbohydrate metabolism particularly during growth.

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WS34/70 (10C) Variant a (10C) Variant b (10C) WS34/70 (15C) Variant a (15C) Variant b (15C) Taste threshold Poly. (Variant b (10C) ) Poly. (WS34/70 (10C) )Poly. (Variant a (10C) ) Poly. (WS34/70 (15C) ) Poly. (Variant a (15C) )Poly. (Variant b (15C) )

Figure 7. Isoamyl acetate production during fermentation at 10C and 15C of three lager yeast strains.


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Figure 8. Ethyl acetate production during fermentation at 10C and 15C of three lager yeast strains.


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In both cases, the immediate precursors are 2-oxo-acids (-keto acids). In the anabolic route these acids derive from pyruvate or acetyl-CoA as part of amino acid biosynthetic pathways. In the catabolic route or Ehrlich pathway of fusel alcohol formation, the -keto acid is formed by transamination of an amino-acid. The -keto acid is successively decarboxylated and reduced to a fusel alcohol. By starting with leucine as the amino acid, isoamyl alcohol is thus formed. (Boulton and Quain, 2001; Renger et al., 1992) Generally, when low levels of amino acids are available, the anabolic route predominates, and when high concentrations of amino acids are present, the catabolic pathway is favoured (Priest and Stewart, 2006).

Table 2. Ratio R = ethylacetate / isoamyl acetate of the mean values for each strain at 10C and 15C

eamylacetatIsoteEthylacetaR = , mean values,

as a function of temperature t, C Strain

10C 15C Control sample 14.18 9.40 Variant a 14.27 9.60 Variant b 13.60 10.20

High temperatures promote yeast cell growth and

thus higher alcohol production. The more free amino nitrogen and sugars are taken up, the more higher alcohol are produced (Mussche and Mussche, 2009; Priest and Stewart, 2008). Narziss et al. (1981) showed that excessive amounts of amino acids are directly transformed into the relevant alcohols (by the Ehrilch mechanism).

All the yeast strains analysed in the present paper showed increased rates of higher alcohols when fermented at 15C than the same strains fermented at 10C. Variant b produced the lowest amount of total higher alcohols at 10C and variant a at 15C as seen in Figure 9. This is supported by the low capacity of these two variants to take up the free amino nitrogen from the wort in comparison with the other two yeasts analysed (Figure 4).

Engang and Aubert (1977) found during their experiments that an increase in temperature from 8C to 10C is accompanied by an increase in total higher alcohols concentration, but a further increase of the temperature to 12C gave a decrease in concentration. They mention Ayrapaas observation that the effect of temperature largely depends on the amount and type of nitrogenous nutrients in the medium.

2.3. Vicinal diketones profile during fermentation and their correlation with free amino nitrogen content

The two most important members of vicinal diketones (VDK) group with respect to beer are diacetyl and 2,3-pentanedione, which impart a characteristic aroma and taste to beer, described as buttery. These compounds arise as an indirect result of yeast metabolism, being part of the normal fermentation process. Their precursors are -aceto-hydroxy acids, which are intermediates in the biosynthesis of valine (acetolactate) and isoleucine (acetobutyrate) and are being exported into the wort from the yeast during fermentation. Outside the yeast cell they undergo spontaneous oxidative decarboxylation to form diacetyl and 2,3-pen-tanedione. From middle to late fermentation extracellular VDK is converted by yeast cell reductase in acetoin and 2,3-butanediol from diacetyl and 2,3-pentanediol from 2,3-pentanedione.

In Figure 10 the diacetyl profile during fermentation of the two variants and the control strain at 10C and 15C is shown. The peak values were between 241369 ppb at 10C, while at 15C higher values, between 429495 ppb, were registered (mean values of the duplicates). These results are in accordance with Masscheleins (1986) remark that higher starting temperatures increase the initial demand in nitrogenous nutrients leading to beers with high -acetolactate and diacetyl levels. The peak values for diacetyl were reached at 10C after 48h of fermentation for the control yeast and variant a and 96h for variant b. A different pattern was noticed at 15C with the diacetyl peak value after 72h of fermentation for the control strain and variant b and 96h for variant a.

From all the strains studied at 10C variant a produced the lowest amount of diacetyl and reduced it below the taste threshold within 7 days. The same strain synthesised higher amounts of diacetyl at 15C, but was able to reduce it below the taste threshold within the same period of time. This behaviour is supported by its flocculation characteristic as a medium flocculent strain.

During fermentation at 15C the diacetyl reduction is faster than at 10C. At 15C, after the FAN value dropped, the diacetyl level started to increase. It could be possible that an extra need of amino acids was necessary for the cells that started to synthesis them. The 10C temperature slowed the FAN uptake and the cells compensated their need of amino acids by synthesising them.

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Figure 9. Total higher alcohol production during fermentation at 10C and 15C of three lager yeast strains.


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Figure 10. Diacetyl evolution during fermentation at 10C and 15C of three lager yeast strains.

Values are means of the duplicates.

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Moreover, at 10C, the precursor of diacetyl (-acetolactate) is slowly degraded into diacetyl, hence the ratio between the amount of diacetyl reduced by the cells and the newly formed amount is lower than at 15C. To overcome this problem, a high temperature period is used in industry called diacetyl rest.

In parallel, the amount of 2,3-pentandione was considered and measured during both fermentations at 10C and 15C. The evolution of this compound is shown in Figure 11 and resembles the diacetyl profile at the mentioned temperatures. This compound (2,3-pentandione) is produced below its threshold of 900 ppb in all cases.

The peaks registered in the first part of the cultivation period are the result of low amounts of isoleucine available for yeast this being forced to produce it. Similar to the diacetyl reduction, at 10C it can be observed a slow decrease of this compound due to the uptake and release ratio mentioned above. 4. Conclusions

Fermentation temperature represents one of the few factors a brewer can use to influence the process inside an industrial fermenter. Other tools are dissolved oxygen via wort aeration, pitching rate as the intial amount of yeast added to the fermenter and the amount of zinc and overpressure. High temperatures promote the decrease in extract, the absorption of nitrogen substances, the activation of

various enzymes (due to the increase in the intracellular level of NADH + H+ and other substances), stimulate the formation of higher alcohols and esters, while low temperatures result in less intense degradation of the substrate, reduced yeast growth and by-products formation.

Yeast strain may also influence the temperature effect, as well as other factors like wort composition and the scale of fermentation.

The yeast strain itself is a major contributor to the flavour character of beer and many suitable strains are available to the brewer, while some strains of yeast are unacceptable for brewing because of the poor balance of flavour compounds produced. (Priest and Stewart, 2006).

The concentration of the most important esters (ethyl acetate and isoamyl acetate) is influenced by the fermentation method.

High-gravity fermentation and high temperatures produce beers with an estery character. Less estery beers are obtained with fermentations carried out under pressure (Piendl and Geiger, 1980).

As WS34/70 population was proved to be a mixture of very closely related strains, differences among the control and the two variants in terms of fermentation speed when using low temperature could not be seen. At high temperature, a small difference was noticed with the highest fermentation performance assigned to variant a.

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Figure 11. 2,3-pentandione production during fermentation at 10C and 15C of three lager yeast strains.

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The free amino nitrogen is more rapidly assimilated at 15C than at 10C, hence the final amount of free amino nitrogen at the end of the experiment at 10C was twice the amount at 15C. At low fermentation, temperature variant b proved to be the slowest FAN assimilator. Due to this behaviour of all the variants at low temperature, the pH values were also higher at 10C.

An increase in fermentation temperature has been found to lower the level of acetaldehyde in beer. Nevertheless, at the end of 8 days of fermentation, the amount of acetaldehyde in beers produced at 15C was slightly higher than at 10C.

Both control sample and variant a started to reduce acetaldehyde faster than variant b at 10C, as well as at 15C.

Regarding the esters formation, different esters show different temperature dependencies and the balance between various esters is altered when the fermentation temperature is varied (Engang and Aubert, 1977). The esters possess a pleasant fruity flavour by themselves; however, increased amounts of ethyl acetate in beer result in a bitterness associated with hop character, while additional isoamylacetate causes a penetrating fruity ester flavour.

An increase in temperature favours the production of isoamyl acetate and ethyl acetate. The isoamyl acetate produced at 15C was 3550% higher than at 10C and the ethyl acetate amount was 1420% higher than at 10C. After 8 days of fermentation at 10C the beer produced with variant b had the highest level of isoamyl acetate. All the beers at 15C displayed comparable amounts of isoamyl acetate at the end of fermentation

In terms of total higher alcohols their concentration increased with temperature in all three cases. Variant b produced the lowest amount of total higher alcohol at 10C and variant a at 15C, while the control strain produced the highest amount of total higher alcohols both at 10C and 15C. The differences in concentration at 15C were not significant.

As expected, higher concentrations of diacetyl were produced at 15C than at 10C. The most significant difference was noticed for variant b, which produced during the fermentation at 15C more than double the amount synthesised at 10C.

Both at 10C and 15C, variant a was the fastest in reducing the diacetyl, reaching 136 g/L and 140 g/L respectively, after 6 days of fermentation.

Acknowledgments

The authors wish to thank Heineken Supply Chain for their support of this work.

References Babb M. 2008. Brewing Fermentation Flavours.

Presentation to Science Caf, 9.08.2008.

Boulton C. & Quain D. 2001. Brewing Yeast and Fermentation. Blackwell Science Ltd.

Engang S. & Aubert O. 1977. Relations between fermentation temperature and the formation of some flavour components. Proceedings of The European Brewery Convention Amsterdam, 1977, 591607.

Hammond J. 1986. The Contribution of Yeast to Beer Flavour. Brewers Guardian, 115(9): 2728, 3133.

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Abbreviations DNA deoxyribonucleic acid FAN Free amino nitrogen GC/ECD Gas chromatography / electron capture

detector GC/FID Gas chromatography / flame ionization

detection n number of generations ppb part per billion ppm part per million VDK Vicinal diketones

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2011-no1-04-Bolat-Turtoi-p262011-no1-04-Bolat-Turtoi-p27-38

Temperature Response of New Lager Brewing Strains Isolated From WS34-70

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