Chapter 7
Conclusions
CHAPTER 7
Conclusions
In the global, increasingly consolidating beer industry, beer often travels large
distances from the brewery to the consumers around the world. Once the beer leaves the
brewery gates the brewer’s influence on transport and storage conditions in the logistics of
the distribution chain, distributor and wholesalers depots and warehouses is often limited.
Thus, beer stability has become increasingly important as the brewing industry shifts from
a local to a global prodution and a stable beer has a better chance to delight the consumer,
be bought again and make money. Beer stability has been the subject of a large number of
previous scientific studies and many areas of beer stability have been explored in great
detail however due to the complex, multifactorial nature of beer stability many questions
remained unanswered. This study attempted to elucidate the influence of raw materials and
yeast quality on beer flavour stability, with a strong focus on beer foam and flavour stability.
Many of the analytical methods routinely employed to evaluate beer quality
parameters have been used for many years and the evolution of these methods has been
slow due to the traditional nature of the brewing industry. The challenges that brewers face
have changed considerably over the years and new research strategies and analytical
methods (possibly looking into other research disciplines for inspiration) should be
harnessed to meet these challenges. Laser scanning confocal microscopy is a cutting edge
imaging technique that is used for cellular studies in biomedical research. This technique
was successfully adapted for imaging physiological parameters and protein dynamics in
brewing yeast. Several yeast compounds and cellular structures, including glycogen,
trehalose, neutral lipids, bud scars, DNA, intracellular proteinase activity and the vacuolar
membrane were visualised. Special focus was given to the imaging of Pr A expression and
dynamics within the yeast cell under a variety of stresses also encountered during brewing
fermentations such as high ethanol concentrations, heat stress and high wort gravities. An
increase of proteinase expression within the vacuole and the cytosol and subsequent
secretion from the cell were successfully visualised and imaged. These results clearly
indicate an exciting future role for confocal fluorescence microscopy to provide new
insights into the molecular mechanisms and vital biological reactions on a single yeast cell
level.
Flow cytometry utilises the same principles of attaching fluorochromes to
molecules, cell organelles or compounds of interest as fluorescence microscopy but allows
the quantification of these parameters in a large number of individual yeast cells. Where a
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traditional analytical technique (for example analysis of glycogen) gives average values
across a yeast population, flow cytometry yields a distribution with the relative fluorescence
(indicative of glycogen content) on the x-axis and the number of cells exhibiting a specific
relative fluorescence on the y-axis. A number of flow cytometric methods were developed
for quantifying parameters such as glycogen, neutral lipids, trehalose, bud scars, DNA, and
intracellular proteinase content. These novel flow cytometric methods are simple assays
that are easily automated with good sensitivity. The practical relevance of these methods
was proven by applying them for evaluating the impact of yeast pitching rates on the
physiological state of yeast such as yeast viability, DNA content, glycogen content and
intracellular proteinase concentration. Yeast handling and pitching procedures are often
based on empirical and basic analytical data. Flow cytometry could be a useful addition to
the existing tools to step-change how we propagate, pitch, ferment and store brewer’s yeast
in order to maximise fermentation performance and beer stability.
Beer head volume and stability are usually the first beer characteristics observed by
the consumer and contribute strongly to his/her appreciation of the beer. The initial right
impression is crucial. It is well established that the foaming ability of beer is largely
influenced by the level of foam-active polypeptides and it is thought that Pr A plays a key
role in the degradation of foam positive hydrophobic polypeptides. If this assumption
holds true sterile filtration (no pasteurisation) should detrimentally impact on beer foam
stability when compared to pasteurised beer since heat sensitive PrA would be inactivated
during pasteurisation. Thus, sterile filtered and pasteurised beers were compared. It was
indeed found that Pr A was denatured during the pasteurisation step and did not survive
into the packaged beer. Consequently, the hydrophobic polypeptides were preserved and
contributed to beer foam stability. In contrast, significant levels of Pr A activity were found
in the sterile filtered beer and, as would be expected, foam stability in this beer declined
over the course of storage.
In a further study, the influence of yeast vitality in bottle conditioned beer on Pr A
activity levels and beer foam stability was investigated. It was shown that employing yeast
in logarithmic growth phase (revitalised) for bottle conditioning does not improve head
retention but has a detrimental effect on head performance, when compared to control
yeast. This may be due mainly to the autolysis of a large amount of dead, immature yeast
buds, which might release foam negative substances (lipids?) into the beer. Based on these
results, it can be suggested that employing a healthy yeast population in stationary growth
phase (G1) for bottle conditioning results in decreased Pr A secretion, increased levels of
hydrophobic polypeptides and consequently improved beer foam stability.
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Beer polypeptides are a complex mixture of proteinaceous molecules of various
molecular sizes and hydrophobicities and several of these polypetides are thought to be
associated with beer foam. However, the exact origin and extent of involvement in head
performance is still an area of heated scientific debate. Thus, a study was conducted to
obtain additional information on the origin and fate of polypeptides throughout production
of sales and higher gravity wort and beer. Special emphasis was placed on the changes in
size and/or hydrophobicity of foam active polypeptides in order to identify stages in the
brewing process where retention of foam active polypeptides and consequently beer foam
stability could be maximised. The present study supports the view that it is not one specific
polypeptide that is solely responsible for beer foam stability. Rather than one polypeptide
being an overriding determinant for beer foam quality, certain groups of polypeptides were
identified that appear to be involved in foam formation. The present findings suggest that
LTP1 is converted into its foam active hydrophobic form more efficiently during a lower
gravity process compared to a high gravity process. Most of this conversion appeared to
take place during wort production. This could be one of the contributing factors for
diminished foam stability of high gravity brewed beer.
The polypeptide distribution in the residues recovered from the emptied fermenters
differed from that of wort or beer. The higher molecular weight fractions of polypeptides
detected in beer were not present in the fermenter residue. However, the low molecular
weight fractions (especially LTP1 and the 17 kDa polypeptide) were present in high
concentrations. These findings support the hypothesis of Cooper et al. (2000), that a large
amount of hydrophobic polypeptides was lost due to lacing of hydrophobic polypeptides
on the fermenter wall when the beer is transferred into conditioning. This factor possibly
accounts for the loss of a proportion of hydrophobic polypeptides during brewing. The
foaming during high gravity fermentations is more pronounced than in sales gravity
fermentations and more foam laces on the fermenter walls thus more hydrophobic
polypeptides are lost. This will also contribute to the diminished beer foam stability of high
gravity brewed beer. Furthermore, as demonstrated in Chapters 3 and 4, yeast Pr A plays a
key role in the degradation of hydrophobic polypeptides responsible for beer foam stability.
Thus, the increased loss of foam positive hydrophobic polypeptides and diminished beer
foam stability in high gravity brewed beers compared to lower gravity brewed beers cannot
be attributed to one factor alone but is likely to be due to both increased yeast Pr A activity
and subsequent degradation of foam positive hydrophobic polypeptides, inefficient
extraction of hydrophobic polypetides in high gravity wort production and the loss of
hydrophobic polypeptides due to lacing in the fermenter. Further research is required to
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quantify losses of hydrophobic polypeptides due to Pr A activity, fermenter foaming and
inefficient extraction during mashing and determine which of these factors is the main
parameter to be manipulated to improve foam stability of high gravity brewed beer.
In addition to foam stability the study and understanding of the mechanisms of
beer oxidation is a longstanding research priority of brewers in order to extend beer shelf
life. The objective of this research was to improve beer shelf life and flavour stability of
beer produced in the 4.5 million hL/annum Foster’s brewery in Yatala, Queensland,
Australia and to shed light on the complex mechanisms involved in beer staling, attempting
to elucidate the mechanisms of ROS (Reactive Oxygen Species) in beer staling and their
interaction with thiol groups in beer proteins.
A biotin-containing, thiol-specific reagent, 3-(N-maleimido-propionyl) (MPB) was
used to detect protein SH groups on Western Blots with sensitivities in the femtomole
range. Beer protein reactivity towards MPB declined as beer was aged since the free SH
groups were oxidised to disulphides which are not detected with MPB. Furthermore, beers
produced from a high gravity wort (18 ºPlato) contained less thiolated proteins than beers
produced from 10 ºPlato and 14 ºPlato worts. The 10 ºPlato beer was brewed with 100%
malt, whereas with higher gravities increasing amounts of sugar syrup were used. Sugar
syrup does not contribute protein, whereas barley malt contains a comparatively large
amount of protein. Furthermore, high gravity brewed beer was diluted with water to sales
gravity. This further decreased the already diminished beer protein content. Thus, it can be
concluded that all malt beers brewed at lower gravities contain more total protein
compared to beers brewed at higher gravity using sugar syrup as an extract source.
Consequently, beers with a lower total protein content will contain reduced protein thiol
levels and might exhibit diminished beer flavour stability. However, higher protein content
also means increased potential for carbonyl formation (see below). Carbonyls are
compounds contributing to the stale character of aged beer. The results so far indicate an
important role for protein thiol groups in beer staling and changes in the extracellular
redox state in packaged beer are reflected in changes in the thiol levels of beer polypeptides,
particularly of some of the low molecular weight beer polypeptides. However, further
research
is
necessary
to
establish
to
what
extent
protein
thiols
have
an
inhibiting/accelerating effect on beer flavour changes during aging.
In addition to the reactions that take place on thiol groups in beer proteins during
aging, free radical-mediated oxidation of proteins in beer results in the formation of
carbonyl groups in quantities that reflect the intensity of oxidative stress. A biomedical
analytical method to assess the formation of carbonyl groups in human diseases was
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adapted to detect carbonyl groups in beer proteins as an oxidation indicator during aging.
The protein carbonyls increased significantly over 3 months storage at 30ºC. The 18 ºPlato
beer C showed less protein carbonylation compared to beer brand A (10 ºPlato) indicating
less oxidative damage to beer constituents in the high gravity beer. However, as already
discussed, high gravity brewed beer contained less protein than beer brewed at lower
gravity. This could explain the lower levels of protein carbonylation found in the 18º Plato
beer. In order to eliminate bias in future research in this study area, the differing total
protein levels in beer have to be taken into account when using the Western Blotting
technique. Sample loading onto the PVDF membrane should not be based on equal
volumes of beer, but rather on equal total protein loading.
Due to the complex reactions taking place in the beer matrix a holistic approach is
paramount in order to reliably measure beer staling. As a consequence, the Peroxide
Challenge Test (PCT) was developed as an alternative to Electron Spin Resonance (ESR).
The principle of the PCT is to mimic oxidation by titrating beer with hydrogen peroxide.
The more hydrogen peroxide a beer can quench, the more resistant it is against oxidation
and flavour deterioration. The PCT allows cost effective, near real-time, high throughput
assessment of beer flavour stability. The PCT is convenient and inexpensive to perform
and the results correlate with ESR measurements and with the perceived aged character of
beer. It was demonstrated that aged beer can be distinguished from fresh beer. Even
differences between production batches can be identified and benchmarking values for
each brand and different breweries defined. Both, PCT and ESR analyses mimic actual
perceived flavour deterioration sufficiently. However, neither of these analytical methods
were able to predict beer flavour deterioration from analysis of a fresh beer sample. Beer C
(which performed worst during aging) was actually rated as the best beer when analysed
fresh with the PCT, and rated on the same level with beer B (which performed best during
aging at 30˚C) when analysed fresh with the ESR method. Based on these findings, it can
be concluded that the ESR and the PCT correlate with perceived flavour deterioration, but
neither allows a reliable prediction of the speed of flavour deterioration in a given beer
based on the analysis of the fresh beer.
A variety of process parameters such as raw material type and amount of hop
addition, boiling system, duration and evaporation rate, wort gravity, yeast strain,
fermentation duration and temperature, cold conditioning duration and temperature,
stabilisation agents and regime, packaging equipment, material and design could be
responsible for the differences in flavour stability observed between beers A, B and C. The
major differencse that set beer C (worst performing) apart from beer A and B is the very
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Conclusions
high gravity process with sugar syrup addition, 18 ˚Plato wort gravity, a high fermentation
temperature of 20˚C and a large dilution ratio to a sales alcohol content of 3.50 % (v/v).
Previous research has found that beer proteins, especially the heavily thiolated low
molecular weight fraction play a fundamental role in preventing oxidation and flavour
deterioration. The thiol groups in these beer proteins have the ability to “cage” reactive
oxygen species and thus prevent oxidation. The use of large amounts of adjunct, the high
gravity process and the significant dilution to sales alcohol content will most likely decrease
the concentration of the lower molecular weight protein fraction that has been shown
beneficial for beer flavour stability. This will negatively impact on beer flavour stability of
beer C. Beers A and B contained a higher proportion of malt as extract source and the wort
gravity was 10 ˚Plato and 14 ˚Plato, respectively. Furthermore, the dilution ratios of beers
A and B were lower compared to beer C. The higher sales alcohol content in beers A and B
might also contribute to their superior flavour stability. Papain, an enzyme with proteolytic
activity, was used as a physical stabilising agent in beers A and C and might have
contributed to increased proteolysis which could have, in turn, diminished the content of
beer proteins protecting flavour stability. Papain was not used during production of beer B.
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