AUTHORS’ CONTRIBUTION
I.J. Košir designed the experiments. N. Kočar Mlinarič conducted and monitored all experiments in the brewing house of Pivovarna Laško Union d.o.o., collecting samples and monitoring microbiological conditions. She also organized and led the sensorial assessment of the final beer samples. M. Ocvirk performed chemical analyses and statistical evaluation of the results. I.J. Košir, N. Kočar Mlinarič and M. Ocvirk contributes in the drafting the article. All authors have read and approved the final manuscript. Final approval of the version to be published was made by I.J. Košir.
The production of lager beer includes successive repitchings of a single
The fermentation was monitored during the production of twelve batches of beer, where the starter yeast culture was reused twelve times without any further treatment. The following beer production steps were monitored: wort production, yeast starter culture propagation, primary fermentation, secondary fermentation and the final product. The work was conducted on an industrial scale employing standard process conditions.
Monitoring of the starter culture viability during successive fermentations indicated no reduction in the viability and vitality of the yeast culture. Monitoring of the fermentable wort saccharide concentrations (glucose, fructose, disaccharides and trisaccharides) revealed a correlation between an improvement in saccharide utilisation and starter culture age. Saccharide uptake efficacy proportionally matched the repitching frequency. Successive exploitation of
Results showed the impact of twelve successive wort fermentations on the dynamics of saccharides uptake that gives brewers important information. The added value of the experiment is all the work done on the industrial scale, with control of all processes and usage of exactly the same raw materials. This study contains usable technological data on the behaviour of saccharides during brewing on the industrial scale, which is not yet found in the literature.
Beer production involves the following procedures: wort production, primary and secondary fermentation, filtration and packaging of the final product. The starter culture for lager production is
The number of times a yeast culture can be serially repitched is largely determined by a combination of product quality constraints and company procedures (
Factors other than the pitching yeast starter culture can also influence the fermentation performance and beer quality; these include the wort composition and the wort aeration (oxygenation), the fermentation temperature and the size and geometry of the fermentation vessel (
During primary fermentation, wort saccharides are converted into ethanol, carbon dioxide and glycerol. Yeast biomass is also produced. The manner by which wort saccharides are utilised plays a crucial role in the final quality of the beer and determines the rate and extent of a brewing fermentation (
Glucose is the preferred fermentation substrate compared to all other wort carbohydrates. It moves across the yeast plasma membrane in a non-specific facilitated diffusion path, which modulates its affinity in response to the available glucose. Maltose is transported into the cell by the maltose permease and then hydrolysed by α-
Maltotriose, like maltose, must be internalised by the brewing yeast strain and hydrolysed into glucose molecules before it is metabolised through the glycolytic pathway. Once inside the cell, saccharides are metabolised by the Embden-Meyerhof-Parnas or glycolytic pathway to yield ethanol, carbon dioxide and glycerol as end products. In addition, a complex mixture of flavour active secondary metabolites is produced, of which the higher alcohols (or fusel) and esters are the most important. Secondary fermentation or maturation follows, when the beer is kept for an appropriate period (1–3 weeks) and at reduced temperatures (from -2 to 5 °C). At this stage, flavour maturation occurs, as well as precipitation of haze-forming materials. The residual yeast in suspension may also utilise any remaining slowly fermentable carbohydrates at this stage to generate carbon dioxide and flavour compounds, as well as to remove undesirable flavour by-products of the primary fermentation (for example, diacetyl is an important maturation marker) (
The extract in finished beer consists of approx. 85% carbohydrates (particularly in the form of the dextrins maltotetraose and maltopentaose) (
The aim of this study is to establish the effect of yeast repitching on their saccharide metabolism and the final beer quality. To our knowledge, this work represents the first example of a study at an industrial scale that used 12 successive fermentations with overall use of
Important process parameters, including the fermentation rate, extract consumption, alcohol formation rate, pH value and number and viability of the yeast cells, were assessed. Industrial scale production was conducted in conical fermentation tanks (total volume 4400 hL, working volume 3250 hL). Strict traceability of the ingredients for wort production (malt, corn and hops) was assured. A conventional brewing protocol and fermentation diagram for primary and secondary fermentation was applied. After primary fermentation, the yeast culture was harvested and stored under similar conditions (
The yeast used in the brewing process for all 12 cycles was a lager yeast strain,
Wort samples were taken before the starter yeast culture was inoculated (pitched) into the wort, during primary (every 24 h) and secondary fermentation (at the beginning and the end of this procedure). After beer packaging, the final products were sampled. Samples (
The samples were analysed employing the methods of the European Brewing Convention (EBC). The samples were degassed by shaking for 10 min at 150 rpm on a shaker (HS 50; IKA-Werke Staufen, Germany). The samples were then filtered through a Whatman grade 597½ filter, diameter 240 mm, pore size 4–7 μm (particle retention) (Whatman, Buckinghamshire, UK). Determination of the real, original and apparent extract and original gravity were made on an SP-1m Beer Analyser (Anton Paar, Graz, Austria) instrument according to MEBAK method 2.9.6.3 (
where
The wort and beer pH values were determined at 20 °C according to the Analytica-EBC method 9.35 (
The viability of the yeast cells was determined by methylene blue straining according to the Analytica-Microbiologica–EBC method 3.2.1.11 (
All standard compounds (glucose with 99.5% purity and fructose, maltose and maltotriose with 99.0% purity) were purchased from Fluka (St. Quentin Fallavier Cédex, France). The HPLC method cannot distinguish among different disaccharides or different trisaccharides. The saccharides in beer were detected according to the Analytica EBC method 9.27 (
Sensory evaluation of all beer samples included taste, odour and bitterness rating by seven experts, who gave seven independent marks. Expert panel consisted of 3 females and 4 males, aged between 32 and 56. The evaluation took place in a separate tasting room, with room temperature from 18 to 25 °C and humidity between 50 and 70%. All beer samples tasted fresh one week after the bottling. At the time of tasting, temperature of the samples was between 10 and 11 °C. Evaluation marks ranged from 1 to 5 for each parameter (data not shown).
After sensory evaluation, OriginPro statistical software v. 2020b (
During 12 successive fermentations, 12 samples of wort, 53 samples of the primary fermentation and 24 samples of the secondary fermentation were collected. The sample amounts were sufficient to elucidate the fermentation kinetics and the yeast starter culture behaviour during prolonged exploitation under industrial conditions, which are considerably more stressful for the production yeast culture.
The original extract of wort (from A to L) was between 11.47 and 11.68% (by mass), which is within the normal mass fraction for a regular lager beer. The pH was not significantly different (
Wort sample | pH | ||||||
---|---|---|---|---|---|---|---|
A | 11.6±0.2 | 5.3±0.1 | 0.20±0.01 | 0.67±0.04 | 5.1±0.3 | 1.93±0.03 | 7.9±0.4 |
B | 11.5±0.2 | 5.3±0.1 | 0.20±0.01 | 0.73±0.04 | 5.0±0.3 | 1.96±0.03 | 7.9±0.4 |
C | 11.7±0.2 | 5.3±0.1 | 0.25±0.01 | 0.76±0.04 | 4.7±0.3 | 1.83±0.03 | 7.5±0.3 |
D | 11.7±0.2 | 5.3±0.1 | 0.21±0.01 | 0.60±0.03 | 4.1±0.2 | 1.17±0.02 | 6.1±0.3 |
E | 11.7±0.2 | 5.3±0.1 | 0.12±0.01 | 0.62±0.04 | 4.2±0.2 | 1.20±0.02 | 6.2±0.3 |
F | 11.6±0.2 | 5.2±0.1 | 0.29±0.01 | 1.21±0.07 | 5.7±0.3 | 1.68±0.02 | 8.9±0.4 |
G | 11.7±0.2 | 5.3±0.1 | 0.29±0.01 | 1.22±0.07 | 5.9±0.3 | 1.71±0.02 | 9.2±0.4 |
H | 11.6±0.2 | 5.3±0.1 | 0.30±0.01 | 0.85±0.05 | 5.2±0.3 | 1.45±0.02 | 7.8±0.4 |
I | 11.5±0.2 | 5.3±0.1 | 0.33±0.01 | 1.06±0.06 | 5.8±0.3 | 1.64±0.02 | 8.8±0.4 |
J | 11.5±0.2 | 5.2±0.1 | 0.35±0.01 | 1.17±0.07 | 5.9±0.3 | 1.65±0.02 | 9.0±0.4 |
K | 11.5±0.2 | 5.3±0.1 | 0.34±0.01 | 1.10±0.06 | 5.6±0.3 | 1.56±0.02 | 8.6±0.4 |
L | 11.5±0.2 | 5.2±0.1 | 0.29±0.01 | 1.04±0.06 | 5.4±0.3 | 1.54±0.02 | 8.3±0.4 |
DP2 and DP3=degrees of polymerisation 2 and 3
In total, 77 samples were collected during primary and secondary fermentations. The fermentation rates of all 12 green (immature) beer samples were comparable (82–84%). The alcohol volume fraction followed this kinetics, as expected, and was between 4.90 and 5.05%. The pH decreased from approx. pH=5.25 to 4.10. More than 50% of the pH decrease was achieved during the first 24 h of primary fermentation. The number of yeast cells during the primary fermentation increased and reached a maximum between the second and third days. The number of yeast cells in suspension subsequently decreased to approx. 13∙106 cell/mL. This development was as expected at the end of the primary fermentation in order to enable proper secondary fermentation for the lager beer.
The concentration of fermentable saccharides in wort F at the end of the primary fermentation ranged from 0.29 to 0.58 g/100 mL (data not shown). The only difference was observed for the green beer samples that used yeast starter culture from wort F (1.55 g/100 mL, data not shown) and culture from wort J (1.19 g/100 mL, data not shown). The reason for this difference could be in the initial saccharide concentration in the wort (
In all cases during primary fermentation, the uptake of glucose (from A to L) was completed within 48 h. Glucose was exhausted when the extract mass fraction dropped to 55% of its initial value, which is approx. 5.0%. Fructose kinetics followed that of glucose, and fructose was exhausted when the residual extract reached 3.5%. The saccharides with DP2 and DP3 were not completely exhausted during primary fermentation. However,
Concentrations of fructose, glucose, wort saccharides with the degrees of polymerisation 2 and 3 (DP2 and DP3) and extract during primary and secondary fermentations with the use of yeast starter culture
The monitoring of standard parameters during secondary fermentation revealed only minor changes. The alcohol volume fraction increased between 0.2 and 0.9%, depending on the number of yeast cells that remained in the green beer after racking and the wort saccharide concentration at the beginning of secondary fermentation. The maximum difference between the extract mass fraction was 0.97% (data not shown), which was detected during secondary fermentation of the green beer in wort K. During secondary fermentation, only DP2 and DP3 saccharides remained. Since most DP2 saccharides were nearly consumed during primary fermentation, only an average addition of 3% was consumed during secondary fermentation and no statistical significance was observed regarding the saccharide consumption profile. This was not the case with DP3, where up to 25% differences for DP3 consumption were observed between successive fermentations (
The efficiency of DP2 and DP3 saccharide uptake during primary and secondary fermentation with a successive use of starter culture
The yeast viability was determined for the successive uses of all yeast starter cultures (from starter culture A to starter culture L). The yeast cell viability was consistent and comparable (>95%) during all successive fermentations. These results supported the conclusion that the viability of the yeast starter culture was not affected or reduced by its successive use and suggested that we could have prolonged the fermentation cycle for even further successive fermentations.
Values were normalised to allow observation of the differences in the saccharide consumption dynamics by the yeast. A correlation was made individually for each saccharide. The transparency of the processed data and the efficiency uptake findings led to the formation of the following four groups: group 1: the first, second and third successive uses of the yeast starter culture (A, B and C), group 2: the fourth, fifth and sixth successive uses of yeast starter culture (D, E and F),group 3: the seventh, eighth and ninth successive uses of the yeast starter culture (G, H and I), and group 4: the tenth, eleventh and twelfth successive uses of the yeast starter culture (J, K and L).
The curves that describe the saccharide uptake dynamics during fermentation and the correlation plots fit into an exponential decay curve of the first order with the following equation:
where y is the saccharide mass fraction, x is the extract mass fraction, and A1, t1 and y0 are constants.
The results of glucose consumption during beer production are shown in
The correlation curves for glucose consumption with successive use of yeast starter culture
The uptake of fructose during beer production with successive use of a starter
The correlation curves for fructose consumption with successive use of yeast starter culture
The consumption of DP2 saccharides during the primary and secondary fermentations is shown in
The correlation curves for DP2 sugar consumption with successive use of yeast starter culture
The efficiency of DP3 saccharide (maltotriose) uptake during the fermentations with successive use of
The correlation curves for DP3 sugar consumption with successive use of yeast starter culture
Beer quality is a complex issue, which includes several groupings of parameters: chemical, microbiological and sensory. All the beer that was produced for the purpose of this study was adequate in terms of the quality standards for lager beer (data not shown).
Final product (beer) | ||
---|---|---|
A | 0.12±0.01 | 0.20±0.01 |
B | 0.14±0.01 | 0.21±0.01 |
C | 0.15±0.01 | 0.20±0.01 |
D | 0.18±0.01 | 0.26±0.01 |
E | 0.12±0.01 | 0.21±0.01 |
F | 0.19±0.01 | 0.29±0.01 |
G | 0.14±0.01 | 0.24±0.01 |
H | 0.14±0.01 | 0.23±0.01 |
I | 0.10±0.01 | 0.16±0.01 |
J | 0.10±0.01 | 0.14±0.01 |
K | 0.10±0.01 | 0.16±0.01 |
L | 0.11±0.01 | 0.16±0.01 |
This study confirmed that the propagation of the starter culture in the same medium used in successive fermentations does not develop the maximum potential for yeast fermentation capacity and saccharide uptake efficiency (with the exception of fructose). As a consequence, yeast exploitation can be extended further. This was confirmed by the high viability of the yeast cells in all 12 successive uses of the
This study focused on the impact of successive exploitation of a single
We conclude that the successive use of a single
The authors would like to acknowledge colleagues from the laboratory of quality control and beer production in Pivovarna Laško Union d.o.o. for their help and advice in the course of this study.
FUNDING
Financial support of Pivovarna Laško Union d.o.o. is kindly acknowledged by PhD student N.K.M. The project was financed by the Slovenian Research Agency through project no. L4-8222.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
All supplementary materials are available at:
The relation between glucose and extract mass fractions during beer production with a successively used yeast starter culture (from the first (A) to the last (L) successive use)
The fructose mass fraction in relation to the extract mass fraction during beer production with a successively used yeast starter culture (from the first (A) to the last (L) successive use)
The DP2 saccharide mass fraction in relation to the extract mass fraction during beer production with a successively used yeast starter culture (from the first (A) to the last (L) successive use)
The DP3 saccharide mass fraction in relation to the extract content mass fraction during the beer production with successively used yeast starter culture (from the first (A) to the last (L) successive use)