microbiology applied to a microbrewery: from yeast management … · a cerveja é uma bebida...
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Microbiology applied to a microbrewery: from yeast management to quality control
Melissa Badrudin
Thesis to obtain the Master of Science Degree in
Microbiology
Supervisors:
Professor Rogério Paulo de Andrade Tenreiro
Professor Isabel Maria de Sá Correia Leite de Almeida
Examination committee:
Chairperson: Professor Ana Cristina Anjinho Madeira Viegas
Supervisor: Professor Rogério Paulo de Andrade Tenreiro
Member of the committee: Doctor Margarida Isabel Rosa Bento Palma
November, 2019
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The work presented in this thesis was performed at the Bugworkers Lab in Tec Labs (Lisbon, Portugal),
during the period September 2017 – July 2019, under the supervision of Professor Rogério Paulo de Andrade Tenreiro. The thesis was co-supervised at Instituto Superior Técnico by Professor Isabel Maria de
Sá Correia Leite de Almeida.
Declaration
I declare that this document is an original work of my own authorship and that it fulfills all the requirements
of the Code of Conduct and Good Practices of the Universidade de Lisboa.
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ACKNOWLEDGMENTS
I dedicate this thesis to my parents, two fierce and incredible beings, whom I love dearly.
I would like to thank Professor Rogério Tenreiro, by providing the opportunity to develop this thesis
but mostly for the patience, guidance and learning. Being able to work and learn under your supervisioning as been a goal of mine since the first microbiology lecture in the second year of university.
To Professor Ana Tenreiro, thank you so much for the guidance and learning. Your cheerful mood
is contagious, from 8AM to 8PM.
To the Dois Corvos co-owners, Scott Steffens and Susana Cascais, thank you so much for the trust
and the opportunity. It is a true pleasure to work in such an interesting and enthusiastic environment.
I would like to thank Professor Isabel Sá-Correia, for the availability and help during this thesis.
To Paulo, my informal supervisor, and dear friend, thank you for the guidance, the shared
knowledge and thank you in advance for the book you will be offering me this Christmas.
To my Dois Corvos family: Pawel, David, Tiago, Marta, Mateus, Jorge, Joana, César, (and Paulo
again) for welcoming and integrating me in the team.
To my friends and family, thank you for the support and the good times. Thank you Adriana and
Miguel for the feedback and for helping me to keep my cool during the writing; Filipe Hanson, for being my
bae and just for existing and being my partner in crime; Ana, Brotini, Inês, Sara and Ana Rafael, for the sisterhood; Ana Casian, Arnaldo Tema and Cristiana Reis for being my core.
A special thank you my little brother Omar, and my favorite victim of society, Eric.
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ABSTRACT
Beer is a fermented alcoholic beverage made from 4 primary ingredients: water, yeast, malted
cereals and hops. The brewing process, and philosophy, separates the industrial from the craft beer
producers, as the latter are mostly interested in developing a more personal and sensory experience for
the consumer.
In order to optimize the production process and guarantee the quality of the final product, quality
procedures should be followed, and the yeast should be correctly handled to maintain its viability and be used in subsequent fermentations. Several quality check-points were implemented: ATP testing, pH
monitoring during wort production, wort stability test, pH and specific density monitoring during fermentation
and microbiological testing in selective media (HLP, WLD and LCSM), for the detection of the most common
contaminants. At the same time, an in-house yeast propagation system was assembled and serial re-
pitching started to be practiced. In order to avoid slow and incomplete fermentations the viability
(percentage of live cells) started to be measured by staining with methylene blue. Further, the results were
compared with flow cytometry viability assays, performed using a combination of SYTO9+PI and the individual use of DIBAC4(3).
The viability results with methylene blue staining appear to be reliable, as they strongly correlate
with the results of the flow cytometry assays and the theoretical viability value, provided by the
manufacturer. A subsequent study also demonstrated that, if handled and stored properly, the yeast can
maintain a high viability through generations (at least until generation 4). With the propagation system, 12
brewing yeast strains were propagated and several parameters were monitored and further analyzed, in
order to investigate how the dynamics of the specific propagation system work.
Key-words: craft beer, microbrewery, quality control, yeast management, yeast propagation.
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RESUMO A cerveja é uma bebida alcoólica fermentada, produzida a partir de 4 ingredientes: água, levedura, cereais
maltados e lúpulo. Alguns passos no processo e a filosofia de produção distinguem a cerveja industrial da
cerveja artesanal, sendo que a artesanal é desenvolvida com o objetivo de oferecer uma experiência mais
personalizada e sensorial ao consumidor.
De modo a otimizar o processo de produção e garantir a qualidade do produto final, devem ser
implementados procedimentos para avaliar qualidade ao longo do processo e a levedura deve ser manuseada de forma a manter a viabilidade para que possa ser utilizada em fermentações em série. Foram
implementados pontos de avaliação de qualidade: o teste de ATP, a monitorização do pH durante a
produção do mosto, o teste de estabilidade de mosto, a monitorização do pH e da gravidade específica
durante a fermentação e testes microbiológicos recorrendo ao uso de meios seletivos (HLP, WLD e LCSM)
para a deteção de contaminações mais comuns. Em paralelo, foi construído um sistema para propagação
de levedura e iniciou-se a prática de reutilização de levedura. Para evitar fermentações incompletas ou
com características menos favoráveis, a viabilidade (percentagem de células vivas) começou a ser
calculada recorrendo à coloração com o azul de metileno. De modo a inferir sobre a veracidade dos resultados obtidos com esta metodologia, a viabilidade foi também calculada recorrendo à marcação com
fluoróforos e posterior análise por citometria de fluxo.
Os resultados obtidos demonstraram uma boa correlação entre metodologias e entre a viabilidade
experimental e a viabilidade teórica. Posteriormente foi também concluído que a levedura consegue manter
a viabilidade entre fermentações, pelo menos até à quarta geração. Com sistema de propagação, 12
estirpes de levedura foram alvo de propagação e monitorização. Os dados obtidos foram submetidos a
uma análise de modo a inferir sobre a dinâmica das propagações no sistema em concreto. Palavras-chave: cerveja artesanal, microcervejeira, controlo de qualidade, gestão de levedura,
propagação de levedura.
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INDEX
ACKNOWLEDGMENTS ............................................................................................................................ I ABSTRACT ............................................................................................................................................... II RESUMO ................................................................................................................................................. III INDEX ...................................................................................................................................................... IV LIST OF FIGURES .................................................................................................................................. VII LIST OF TABLES ................................................................................................................................... VIII LIST OF ABBREVIATIONS ...................................................................................................................... IX
INTRODUCTION ......................................................................................................................................... 1
1. BEER IN SOCIETY AND CULTURE .......................................................................................................... 1 2. CRAFT BEER INDUSTRY ....................................................................................................................... 1
2.1. Microbreweries ......................................................................................................................... 1 3. OVERVIEW OF THE BREWING PROCESS ................................................................................................ 2
3.1. Malting ...................................................................................................................................... 2 3.2. Milling ....................................................................................................................................... 2 3.3. Mashing .................................................................................................................................... 2 3.4. Lautering .................................................................................................................................. 2 3.5. Boiling ...................................................................................................................................... 3 3.6. Cooling down ........................................................................................................................... 3 3.7. Fermentation ............................................................................................................................ 3 3.8. Maturation and conditioning ..................................................................................................... 4 3.9. Brightening and packaging ...................................................................................................... 4 3.10. Additional steps ........................................................................................................................ 4
4. QUALITY CONTROL IN A MICROBREWERY .............................................................................................. 4 4.1. Defining quality ........................................................................................................................ 5 4.2. How to achieve quality ............................................................................................................. 5
5. YEASTS ............................................................................................................................................. 7 5.1. Domestication of the brewing yeast ......................................................................................... 8 5.2. Brewing yeast strains ............................................................................................................... 8 5.3. Yeast management .................................................................................................................. 9
6. THESIS SCOPE AND GOALS ................................................................................................................ 15
MATERIALS AND METHODS .................................................................................................................. 16
PART I: YEAST MANAGEMENT .............................................................................................................. 16
1. YEAST LONG TERM STORAGE ........................................................................................................... 16 1.1. Sampling ................................................................................................................................ 16
1.2. ISOLATION .................................................................................................................................... 16 1.3. STORAGE ..................................................................................................................................... 16
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2. PROPAGATION ................................................................................................................................. 16 2.1. Laboratory propagation .......................................................................................................... 16 2.2. Brewery propagation stage .................................................................................................... 17
3. FLOW CYTOMETRY ASSAY ................................................................................................................. 19 3.1. Yeast samples ........................................................................................................................ 19 3.2. Sample washing ..................................................................................................................... 19 3.3. Staining .................................................................................................................................. 19 3.4. Flow cytometry analysis ......................................................................................................... 20
4. YEAST CELLS HARVEST AND RE-PITCHING .......................................................................................... 20 4.1. Sanitization............................................................................................................................. 20 4.2. Harvest ................................................................................................................................... 21 4.3. Re-pitching ............................................................................................................................. 21
5. PRINCIPAL COMPONENT ANALYSIS ..................................................................................................... 21
PART II: QUALITY CONTROL IMPLEMENTATION ................................................................................ 22
1. ADENOSINE TRIPHOSPHATE TEST ...................................................................................................... 22 1.1. Sampling ................................................................................................................................ 22 1.2. Luminometer .......................................................................................................................... 23
2. PH MEASUREMENT ........................................................................................................................... 23 2.1. Calibration .............................................................................................................................. 23 2.2. Readings ................................................................................................................................ 23
3. WORT STABILITY TEST ...................................................................................................................... 23 3.1. Sample port sanitizing ............................................................................................................ 23 3.2. Sampling ................................................................................................................................ 24
4. MICROBIOLOGICAL ANALYSIS ............................................................................................................. 24 4.1. Sampling ................................................................................................................................ 24 4.2. Culture media ......................................................................................................................... 24 4.3. Colonies characterization ....................................................................................................... 25
5. FERMENTATION MONITORING ............................................................................................................ 25 5.1. Specific gravity ....................................................................................................................... 25 5.2. Forced fermentations ............................................................................................................. 25
6. PRODUCT STABILITY ANALYSIS .......................................................................................................... 26 7. COMPLAINTS TRACKING .................................................................................................................... 26
RESULTS AND DISCUSSION .................................................................................................................. 27
PART I: YEAST MANAGEMENT .............................................................................................................. 27
1. VALIDATION OF METHYLENE BLUE STAINING METHOD .......................................................................... 27 1.1. Determination of viability by flow cytometry with SYTO9+PI ................................................. 28 1.2. Determination of viability by flow cytometry with DIBAC4(3) .................................................. 29 1.3. Experimental vs. theoretical viability ...................................................................................... 32 1.4. Correlation between the staining with methylene blue and flow cytometry staining with a conjugation of SYTO9+PI and DIBAC4(3) .......................................................................................... 33 1.5. Assessing yeast viability through generations ....................................................................... 34
2. PROPAGATIONS ................................................................................................................................ 35 2.1. Principal component analysis ................................................................................................ 35
FINAL REMARKS ..................................................................................................................................... 44
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PART II: QUALITY CONTROL IMPLEMENTATION ................................................................................ 45
1. ATP TESTING ................................................................................................................................... 45 2. FERMENTATION MONITORING ............................................................................................................ 46
2.1. Determine the beer final specific gravity ................................................................................ 47 2.2. Contamination awareness ..................................................................................................... 47
3. PH MONITORING DURING WORT PRODUCTION ..................................................................................... 47 4. MICROBIOLOGICAL TESTING OPTIMIZATION ......................................................................................... 48 5. WORT STABILITY TEST ...................................................................................................................... 49 6. PRODUCT STABILITY MONITORING ...................................................................................................... 49 7. COMPLAINT RECORDS ....................................................................................................................... 49
FINAL REMARKS ..................................................................................................................................... 50
REFERENCES .......................................................................................................................................... 52
APPENDIX ................................................................................................................................................. 57
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LIST OF FIGURES
Figure 1: Viability percentage assessed by the methylene blue staining method. .................................... 27 Figure 2: Graphic example of the viability analysis using flow cytometry. Yeast cells were co-stained with SYTO9 (green fluorescence detected on FL1) and PI (red fluorescence detected on FL3). (A) Pseudocolor dotplots of side scatter (SSC) vs. FL3; (B) Pseudocolor dotplots of FL3 vs. FL1. ................ 28 Figure 3: Viability percentage assessed by the flow cytometry analysis using SYTO9 as staining and PI as counterstain. ......................................................................................................................................... 29 Figure 4: Viability using flow cytometry. Yeast cells were co-stained with DIBAC4(3) (fluorescence measured on FL3 channel). On the left aligned scatter plots of FL3 vs. frontal scatter (FSC) and on the right aligned scatter plots of FL1 vs. FL3. ................................................................................................. 30 Figure 5: Viability percentage assessed by the flow cytometry analysis using DIBAC4(3) as a staining. . 31 Figure 6: Graphic representation of the enhanced green fluorescence of cells treated with FCCP. ......... 32 Figure 7: Correlation between the viability measured by the three methodologies presented and the theoretical viability. .................................................................................................................................... 32 Figure 8: Correlation analysis between viability by methylene blue and viability by FCM. Left: Correlation between viability by methylene blue and FCM using SYTO9 + PI. Right: Correlation between viability by methylene blue and FCM using DIBAC4(3). .............................................................................................. 33 Figure 9: Comparison of the viability percentage measured by methylene blue, FCM with SYTO9+PI as staining, and FCM with DIBAC4(3) as staining from generation 0 to generation 4. .................................. 34 Figure 10: Distribution of the variables in the space of principal component 1 (PC1) and principal component 2 (PC2). ................................................................................................................................... 39 Figure 11: Distribution of the variables in the space of principal component 1 (PC1) and principal component 3 (PC3). ................................................................................................................................... 39 Figure 12: Distribution of the propagations in the PC1 vs. PC2 space. Each propagation is represented by a dot and polygons A, B, C, and D represent independent propagations of the same yeast strain. Clusters (1,2, and 3) represent propagations with the same yeast genus. .............................................................. 41 Figure 13: Distribution of the propagations in the PC1 vs. PC3 space. Each propagation is represented by a dot and polygons A, B, C, and D represent independent propagations of the same yeast strain. Clusters (1,2, and 3) represent propagations with the same yeast genus. .............................................................. 41 Figure 14: Overview of the Dois Corvos production flow and each quality testing point ........................... 45
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LIST OF TABLES
Table 1: Enumeration of quality at source type tests and the processes step where they can be
implemented ................................................................................................................................................ 6
Table 2: Correlation between variables. *(p≤0.05) **(p≤0.01) .................................................................. 36
Table 3: Identification of the propagated yeast strains .............................................................................. 40
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LIST OF ABBREVIATIONS
CIP: Clean In Place
SOP: Standard Operating Procedure
LCSM: Lin’s Cupric Sulfate media
WLD: Wallerstein Differential
HLP: Hsu’s Lactobacillus/Pediococcus
MB: Methylene Blue
FCM: Flow Cytometry
FAN: Free Amino Nitrogen SSC: Side Scatter light
FSC: Front Scatter light
YEPGA: Yeast Extract Peptone Glucose Agar
DME: Dry Malt Extract
PBS: Phosphate Buffered Saline
RLU: Relative Light Units
DMSO: Dimethyl sulfoxide
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INTRODUCTION
1. Beer in society and culture
Beer is one of the oldest cultural achievements of mankind and one of the most popular alcoholic
beverages in the world (Arnold & John, 2005). According to the German Beer Purity Law, beer is considered
a fermented alcoholic beverage made of malted cereals and water flavored with hops (Nelson, 2005).
Despite its unknown origin, the oldest mentions to it lead back to the Mesopotamian, in the year
2800 BC, and ancient Egypt, where it was noticed that, consumption of beer was safer than water and,
therefore, was the natural thirst-quencher for the common man (Kunze, 2014). In Europe, beer is associated
with German tribes, Scythians, and the Celts, as it was brewed as a daily household by women. Later in history, Christian monasteries started to produce beer to sell beyond than their own consumption, a moment
that can be defined as the origin of the brewing industry (Nelson, 2005).
During the industrial revolution, beer production moved from the artisan domestic scale to an
industrial dimension, which continued to be explored until the modern times. Nowadays, the beer industry
is a global business, dominated by several industrial multinational companies but also with the existence
of thousands of smaller craft producers, preferred by those who seek a more personal experience.
2. Craft beer industry
According to the American Brewers Association (2019), a craft brewery has three core characteristics that
differentiate it from an industrial brewery: it is small, with “an annual production of six million barrels of beer (or less)”; independent, with “less than 25 percent of the brewery being owned or controlled by a member
of the alcoholic beverages industry that is not a craft brewer”; and traditional, having “beers with flavors that
derive from traditional or innovative ingredients, and their fermentation, as the majority of its total production
volume”.
2.1. Microbreweries
In the craft beer industry, there are four business segments: brewpubs, microbreweries, regional
craft breweries, and contract brewing companies.
A microbrewery is a brewery that produces less than 17,600 hectoliters of beer per year, with 75
percent of more of it being sold off-site. Microbreweries can sell craft beer to the consumer directly through
tap rooms, and can also sell it to local markets by providing it to distributors, retail stores and restaurants
(Brewers Association, 2019).
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3. Overview of the brewing process
3.1. Malting
Malt production from barley is the first step in beer production (Bokulich and Bamforth, 2013). In
stored barley, the relevant enzymes for the malting process have either a greatly reduced activity or are
still inactive. The purpose of malting is to produce these enzymes in the germinating barley kernel and to
cause certain changes in its chemical constituents (Kunze, 2014).
This process comprises three primary steps: steeping, germination and kilning (Bokulich and
Bamforth, 2013). During steeping, the barley absorbs the water necessary for germination and it is then germinated in large tanks or chambers known as boxes. Finally, the germination is terminated by a drying
procedure at a high temperature, known as kilning, and any seeds sticking to the malt are removed (Kunze,
2014).
3.2. Milling
Milling designates the step where the malted grains are crushed into a coarse flour known as the
grist. This process is important as it allows for the breakage of the endosperm and the release of the
enzymes found in the aleurone layer (Barth, 2013). This leads to an optimization of the mashing step
whereas the endosperm can be in contact with the water and the enzymes that break down the starch into
fermentable sugars.
3.3. Mashing
In the mashing step, the grist is suspended in hot water (45-72 ºC) for 1-2 hours in the mash vessel,
allowing enzymatic hydrolysis to take place, converting starch into fermentable carbohydrates (Boulton &
Quain, 2006). Depending on the type of beer being produced, the mash temperature and duration can be
adjusted.
3.4. Lautering
In the lautering step, the former mixture is transferred to the lautering vessel and is subjected to a
raise of temperature to stop the enzyme-catalyzed breakdown of starch into fermentable carbohydrates. A
perforated bottom plate usually acts as a strainer, allowing the solids to be retained and form a grist bed
filter. There is a brief recirculation of the liquid phase to obtain clarification and the liquor is then moved to another vessel for boiling, while hot water is sparged from above to allow for optimal extraction of
carbohydrates from the grain (Bleier, Callahan & Farmer, 2014).
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3.5. Boiling
In the boiling kettle, hops are added to the wort and the mixture is subjected to heat treatment.
During this step, the wort is reduced and the high temperature isomerizes the alpha acids from the
hops, which are responsible for bitterness, eliminating possible microbiological contaminants and
contributing to the loss of undesirable vegetable-like odors (Boulton & Quain, 2006).
When boiling is completed, the wort is clarified by a whirlpool where solids in the form of trub (a
mixture of coagulated proteins and heavy fats) and hop material are separated from the hot wort.
3.6. Cooling down
This stage is performed utilizing a heat-exchanger, where hot wort flows between one side of the
plates and cold water flows on the other side of them, allowing the transference of heat from the hot wort
to the cold water, which results in a rapid decrease to the desired wort casting temperature.
3.7. Fermentation
At the desired temperature (4-12 ºC for lager beers, 14-25 ºC for ale beers), the wort is aerated
while it is passing to the fermentation vessel, since oxygen is essential to the initial stage of fermentation.
At this point, yeast is inoculated directly into the fermentation vessel, having all the favorable conditions for fermentation to begin.
Lag phase: In this aerobic phase, the dissolved oxygen in the wort is absorbed by the yeast, enabling the
synthesis of essential cell membrane components (sterols and unsaturated fatty acids) (Aries & Kirsop,
1977). There is a change in the expression of the genetic pattern and protein synthesis, which enables the
end of the yeast inactive metabolic state.
Growing phase: In this phase, there is a drop in pH and the oxygen content in wort decreases. The produced and stored fatty acids, together with the amino acids and other essential nutrients in the wort, are consumed
during intensive yeast multiplication (Higgins, Beckhouse, Oliver, Roger & Dawes, 2003).
Fermentation phase: This anaerobic process is characterized by the conversion of fermentable
carbohydrates in ethanol, carbon dioxide and other by-products. As fermentation proceeds and the content
of fermentable carbohydrates decreases, yeast enters a less active stage and begins to flocculate.
Flocculation is a reversible form of aggregation that consists in the ability of the yeast to form clumps of cells (‘flocs’) through the synthesis of lectin-like proteins (‘flocculins’) associated with the cell wall, that bind
to the cell wall mannans of nearby yeast cells (Vidgren & Londesborough, 2011; Bokulich & Bamforth,
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2017). By the end of this phase, yeast activates the production of glycogen reserves, necessary to survive
the following dormancy state (Boulton & Quain, 2006).
Stationary phase: At this point, the yeast drastically reduces the metabolic activity and enters a dormancy state, accumulating, flocculated, in the cone of the fermentation vessel.
3.8. Maturation and conditioning
Maturation and conditioning is achieved by gradually dropping the beer temperature to 2 ºC. During fermentation, yeast produces by-products that give the beer an unclean and unbalanced taste and aroma,
if found in high concentrations (Kunzee, 2014). The purpose of the maturation stage is to allow for these
by-products to be biochemically removed from the beer and to clarify it, as the yeast begins to accumulate
in the bottom of the fermentation vessel.
3.9. Brightening and packaging
The beer is aseptically transferred from the fermenting to the brightening vessels, where it is
carbonated and packaged into bottles or cans, and stored in kegs. In these steps, it is extremely important
the absence of oxygen, as its presence has a deleterious effect in beer flavour and shelf life (Cahil, 1999).
3.10. Additional steps
After the conditioning, some breweries filter or centrifuge the beer prior to packaging, in order to
remove all the yeast in suspension and obtain a clarified product. Pasteurization is commonly used in
industrial brewing but rarely employed in the craft beer industry. It increases the product stability by
diminishing the chances of contamination.
4. Quality control in a microbrewery
Quality control in a microbrewery works towards the consistent production of high quality beer. It is
not just a set of criteria that defines good beer, but it is also a system of policies, procedures, specifications,
and empowering employees at all levels to maintain a correct process.
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4.1. Defining quality
Defining quality according to the traditional view: According to the Brewers Association (2019), quality in
brewing corresponds to a beer that is responsibly produced using wholesome ingredients, consistent brewing techniques, and good manufacturing practices, exhibiting flavor characteristics that are
consistently aligned with both the brewer and beer drinker expectations. Therefore, quality beer is safe to
consume and has a system to monitor the process from raw material selection through brewing and
fermentation, from packaging to serving the end consumer.
Defining quality as “free from defect” :Another traditional way to define quality is “free from defect”, meaning
that the quality goals are to prevent off-flavors and meet government requirements, such as bottle volume,
alcoholic percentage, sulfite concentrations, and that it is made in a food-safe manner (Pellettieri, 2015).
Defining quality as “fitness for use”: Defining quality as “fitness for use” assumes that the customer helps
to define what a good quality beer is. This relies on the parameters such as color, bitterness, alcohol, and
foam, which reflect the parameters that are most valued by consumers in beer (Neely, 2005).
Defining quality as esoteric: This definition takes into consideration the art of brewing and the brand values,
allowing to articulate values to a quality manual in terms of criteria as part of how each brewery defines brand quality (Pellettieri, 2015).
4.2. How to achieve quality
4.2.1. Risk assessment
The risk assessment analysis can be done by mapping the brewing process, identifying the major
risks and vulnerabilities of each step and reviewing the quality procedures.
4.2.2. The quality system and quality checks
The quality system is a term used to define the overarching system of process, policy, and record
keeping that maintains control of the desired quality output. In a brewery, quality should be found at every
step of the process (Pellettieri, 2015). Each step, from wort boiling to fermentation to finishing to packaging, works together in a chain. At the connections of the chain links are the quality checks that assure that the
process was in control.
Quality at source checks: These types of quality checks are summarized in Table 1 and can be performed
at the site during the process and the results are instantaneous.
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Table 1: Enumeration of quality at source type tests and the processes step where they can be implemented
Test Process step
Specific gravity measurement Wort production Fermentation
pH measurement Wort production Fermentation
Dissolved oxygen measurement Fermentation Packaging
Dissolved carbon dioxide measurement Brightening Packaging
Sensory analysis Fermentation Final product
Yeast count and viability Yeast management
ATP measurement Clean in place (CIP) Packaging
4.2.2.1. Microbial quality checks
Microbiological contamination is one of the most common problems found in the craft beer industry.
Since craft beer is rarely pasteurized at the end of the production process, it is crucial to have good
sanitation practices to avoid possible contaminations.
Unlike the checks at the source, microbial quality checks have to be conducted in a controlled
environment (as a laboratory) and the results are not provided in the same day, but provide an indication about contamination by potential beer spoilers.
4.2.2.1.1. Beer contaminating microorganisms
Beer has a high microbiological stability given its properties: relatively low nutritional status, ethanol
concentration (0.5-10%) (w/w), low pH (3.8-4.7), and high concentration of hop iso-alpha-acids with
antimicrobial properties (17-55 ppm) (Roadhouse & Carbonero, 2017). However, some microorganisms are able to survive and proliferate in that environment, possibly causing undesirable changes in the final
product. These microorganisms can be divided in two main categories: bacteria and yeast.
Bacterial contaminations: The most common beer contaminating bacteria are lactic acid bacteria (LAB)
from the genera Pediococcus and Lactobacillus (Roadhouse & Carbonero, 2017). Contamination by these
Gram-positive bacteria is problematic and capable of giving the beer an acidic or buttery taste, due to the
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production of lactic acid and acetyl. Another relevant bacterial contamination is caused by the Gram-
negative acetic acid bacteria (AAB), which give a “vinegary” taste to the beer, due to the production of
acetic acid (Hill, 2017).
Yeast contaminations: Besides being responsible for fermentation, yeast can also be considered a
contaminating microorganism, if it is found in a beer where it was not deliberately used. In brewing, the
most commonly used yeasts are Saccharomyces cerevisiae for ale beers and Saccharomyces pastorianus
for lager beers, although the use of yeast from genera Brettanomyces is increasing and becoming
popularized to ferment different styles of beer. Saccharomyces cerevisiae diastatic strains can also be a
problematic contamination, as the presence of the active STA genes encode for a glucoamylase that
degrades otherwise non-fermentable carbohydrates, which may lead to super attenuation (Bokulich &
Bamforth, 2017).
4.2.2.2. Setting the frequency of the check
After defining the quality checks, it is important to define the frequency needed for each quality
control check. This frequency depends on the initial risk assessment, the process variation, and the ease
of access to the measurement, and should also consider the costs and difficulty of the check (Pellettieri,
2015).
4.2.3. Corrective action
When a quality parameter fails to meet a specification, corrective action can be applied by stopping
the process, adjusting or correcting it, and then documenting the action. The less time that passes between
error detection and correction, the better the quality of the beer will be.
4.2.4. Ease access to data
The history of results of all the quality checks should be available and organized, facilitating the
trace of the problem source and contributing for quick solution. From time to time, the analysis of this bulk
of data will also infer on the state of the quality in the brewery.
5. Yeasts
Yeasts are eukaryotic, unicellular organisms that belong to the Fungi kingdom. The
Saccharomyces genus is the most commercially exploited, being used in the bread, wine, and beer industry,
due to its attractive fermentative skills (Stewart, 2017).
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5.1. Domestication of the brewing yeast
In order to thrive in “man-made environments”, such as wort, wild yeast species suffered a process
of domestication through human selection and breeding, resulting in the industrial yeasts that are in use nowadays. These industrial strains are genetically and phenotypically distinct from the wild yeast strains,
having evolved from a small set of ancestrals (Stewart, Hill & Russel, 2013).
The abrupt change from a natural environment to wort rich in nutrients allowed for specialized
adaptations: the duplication of the MAL (maltase; α-D-glucosidase) genes, which endorsed an efficient
fermentation, and the nonsense mutation in PAD1 (phenylacrylic acid decarboxylase) and FDC1 (ferulic
acid decarboxylase) genes, that led to a reduction of the beer off-flavor 4-vinylguaicol (Kopecká, Nemec &
Matoulková, 2016). Ultimately, domestication led to genome decay, aneuploidy (a chromosome number
that deviates from a multiple of the haploid set) and loss of a functional sexual cycle (Kopecká, Nemec &
Matoulková, 2016).
It is important to realize that this was not a quick process, but the outcome of centuries of human
domestication, shaped by the industrial application and geographical origin.
5.2. Brewing yeast strains
Brewing yeast commonly belongs to the genus Saccharomyces, saving the exception of the use of
Brettanomyces genus in lambics and barrel aged styles. The species employed depends on the type of beer being produced, whereas Saccharomyces cerevisiae is used in ale and Saccharomyces pastorianus
is used in lager beers.
Lager strains exhibit poorer temperature tolerance than ale strains, as ale strains will grow at 37 ºC
and lager strains do not grow above 34 ºC, and under normal brewing conditions, lager strains produce
considerably more sulfur dioxide than ale strains (Kunze, 2014).
The uptake of carbohydrates differentiates with the lager strains capacity of metabolize the sugar
melibiose into glucose and galactose by the enzyme melibiase (𝛼- galactosidase). This enzyme is produced
in lager strains probably due the presence of one or more MEL genes in the genome, absent in the ale
strains (Stewart, Hill & Russel, 2013).
5.2.1. Ale versus lager fermentation
Ale fermentations are commonly referred to as top fermentations, as the yeast rises to the surface
during fermentation due to strong hydrophobic characteristics in its cell wall, and as a result of the presence
of CO2 in the fermentation. The temperature of ale fermentations is typically in the range of 15-23 ºC
(Boulton and Quain, 2006).
9
Lager fermentations are commonly referred to as bottom fermentations, as the yeast has the
tendency to settle in the bottom of the fermentation vessel, given the hydrophobic characteristics of the
membrane. The temperature of lager fermentations is typically in the range of 8-18 ºC (Boulton and Quain,
2006).
5.3. Yeast management
Yeast has a central role in brewing, being responsible for the fermentation process. To maximize
the use of the yeast and diminish costs, it is a common brewing practice to do yeast propagations and to reutilize the yeast from one fermentation to another several times (serial repitching). For a successful yeast
yeast management, the yeast needs to maintain good viability, and to be correctly propagated, harvested,
stored, and re-pitched. Understanding and controlling these steps in order to meet the brewery yeast needs
is what can be considered as the definition of yeast management.
5.3.1. Yeast propagation
Propagation is a stepwise aerobic process whose purpose is to multiply the yeast until it is sufficient
to pitch in a fermentation vessel (Kunze, 2014). The requirements for fresh propagated yeast cultures is
that it is non-stressed, highly viable and free of contaminant organisms. Therefore this process must be handled in an aseptic manner and, before the use of the yeast in a fermentation step, its viability should be
determined, to assure that the physiological state of the yeast is appropriate to perform a healthy
fermentation.
This process can be divided in two stages: laboratory and brewery propagation. Laboratory
propagation refers to the step where the chosen yeast strain is removed from storage and inoculated in a
small volume of oxygenated wort, in order to grow, and is successively transferred to another vessel
containing up to 10 times as much wort, until it reaches a total volume of 5 -10 L (Kunze, 2014). At this
stage, the scale up is usually done using a Carlsberg Flask. The Carlsberg Flask is a vessel made of chrome nickel steel with capacity between 8-10 L (small Carlsberg Flask) or 20-25 L (large Carlsberg Flask),
which allows the continuity of the propagation in an aseptic manner, as it enables the sterilization of wort
prior to yeast inoculation (Kunze, 2014). The scale up to higher volumes is done in the brewery, in yeast
propagation plants, composed of closed vessels of chrome-nickel steel. This system can be constituted by
one tank (propagator only) or two tanks (propagator and sterilizer), where the wort is sterilized firstly and
cooled before it’s pumped into the propagator (Lodolo, Kock, Axcell & Brooks, 2008).
10
5.3.1.1. Propagation essential requirements
The propagation success relies on having a sanitary propagation plant, with an aeration system
and wort with the right nutrients composition. However, even with optimized conditions, it is only possible to achieve relatively low cell numbers. If the propagation media has a too high concentration of glucose,
the yeast can switch to ethanol producing anaerobic metabolism, even under highly aerated conditions.
The suppression of respiration by the presence of high glucose concentrations is known as the “Crabtree”
effect (Narendranath et al., 2015). To increase the propagation yield, some breweries adapted a fed batch
system, where the sugar concentration is maintained at a consistently low level but not too low to avoid the
yeast growing purely aerobically that can lead to the loss of fermentation characteristics (Stewart, Hill &
Russel, 2013).
Nutrient availability will significantly impact the yeast growth and stress tolerance. In brewing, wort functions as both growth media for propagations and fermentation medium for the production of ethanol,
carbon dioxide and other metabolic products. It is mainly composed by:
Carbohydrates (90 - 92 %): in which maltose is the most abundant, followed by glucose, fructose, sucrose,
maltotriose, and dextrin, which is non-fermentable (only utilized by Saccharomyces diastaticus). Yeast cells
utilize these nutrients in an orderly manner but with some overlaps, starting with sucrose, glucose, fructose
and latter maltose and maltotriose (Kunze, 2014);
Nitrogenous components (4 - 5%): nitrogen is an essential element for yeast growth and function but not all nitrogen in the wort can be utilized by yeast. Free amino nitrogen (FAN) is the fraction of nitrogen
components available for yeast consumption and it is consists of individual amino acids, ammonium ions,
small peptides (di-, tripeptides) (Kunze, 2014);
A wide range of compounds (the remaining percentage): vitamins, minerals, inorganic ions, fatty acids,
trace elements, and other minor constituents (Stewart, Hill & Russel, 2013).
The amino acids and trace elements contained in the wort are sufficient for the fermentation process but
not to propagations. The multiplying of yeast cells demands a higher concentration of these elements
(Kunze, 2014). The supply of oxygen (or aeration) is essential for propagations, as it allows the yeast to maintain
the use of aerobic metabolic pathways, essential for a higher biomass yield. Moreover, yeast is auxotrophic
for oxygen even in strictly anaerobic conditions, for the synthesis of sterols and unsaturated fatty acids,
constituents of cell membranes (Englezvos et al., 2018).
The temperature factor can also influence the propagation yield as it has been seen that at 30 ºC,
the same final cell number can be obtained in half of the time compared to with at room temperature (around
20 ºC) (Stewart, Hill & Russel, 2013).
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5.3.2. Yeast serial repitching
Yeast serial repitching is commonly done in breweries in order to reduce the costs associated with
yeast. This process includes pitching (inoculating the yeast in the fermentation vessel), harvesting or cropping (collecting yeast from the fermentation vessel), evaluating the yeast viability, and re-pitching
(pitching the collected yeast in a subsequent fermentation vessel). The process of inoculating wort with yeast is called pitching and has a direct impact on the
fermentation outcome (Kunze, 2014). To perform a correct pitching, the pitching rate has first to be
calculated. This rate is influenced by the wort original gravity, where high gravity worts ask for higher
pitching rates, and by the yeast strain employed (Stewart, 2017). The use of a correct pitching rate
contributes to a healthy fermentation and a clean fermentation profile.
The term cropping is utilized to describe the yeast harvesting from fermentation vessel to a yeast collection vessel (Boulton & Quain, 2004). As the yeast ceases its activity, the fermentation vessel
temperature should be reduced to minimize yeast physiological changes. With the end of the fermentation,
yeast flocculates and sediments in the bottom of the fermentation cone, ready to be harvested. This process
should be done shortly after the conclusion of fermentation, as the yeast is exposed to a stressful
environment with high ethanol concentrations and nutrient limitation. If the yeast is not harvested in the first
days after fermentation conclusion, it will begin to autolyze and excrete proteinase A and other substances
into the beer, which will have a negative effect on flavor, aroma, and foam stability (Stewart, 2017) . The harvested yeast should be stored in an aseptic manner at a temperature of 2-4 ºC as the
lowering temperature (but not too low in order to prevent freezing) leads to a more ordered membrane
structure, consequently reducing membrane fluidity. The yeast should be suspended in fermented wort with
a 4 - 5 % alcohol (v/v) concentration and slow agitation should be employed in order to maintain the yeast
cells in suspension.Vigorous agitation has been associated with loss of viability, glycogen breakdown, and
poor fermentation performance (Stewart, Hill & Russel, 2013).
5.3.2.1. Yeast viability
The yeast performance during propagation and the alcoholic fermentation depends on its
physiological state. The brewery environment exposes the yeast to physical (temperature and osmotic
pressure) and chemical (ethanol and oxygen) stresses that can destabilize the cell physiology and lead to
the deterioration of the cells, influencing the levels of organic acids, esters, higher alcohols, aldehydes and
diacetyl in fermentation and maturation (Heggart et al., 2000). The oscillation of these levels has a negative
effect on the flavour stability, an important quality criteria for beer. Therefore the critical parameter for all
stages of yeast management is to maintain the viability (percentage of live cells) and vitality (state of the living cells) of cultures, ensuring that when the yeast is pitched into wort, the lag phase is kept to a minimum.
For pitching yeast, both viability and vitality should be high as possible.
12
To determine viability, one can use standard procedures such as plate counts, slide culture, and
vital staining. Plate counts are inexpensive and accessible but the method takes 24-48h to yield results,
which is not ideal for practical reasons in the brewing industry. Moreover, this methodology could
underestimate the viability, as some cells can be viable but not culturable, due to exposure to nutrient starvation and general stress (Smart, Chambers, Lambert, Jenkins & Smart, 1999).
The bright field observation of cells stained with methylene blue (MB) is the most commonly used
method by brewers to rapidly assess viability, whereas the metabolically active yeast cells convert
methylene blue to a colorless solution through cellular dehydrogenase activities, while dead yeast cells
remain blue (Smart, Chambers, Lambert, Jenkins & Smart, 1999). Although relatively inexpensive and easy
to perform, this method has a few drawbacks: analysis is subjective, with often variable interpretations of
weakly stained cells; and relatively low numbers of cells are counted, usually less than 400, which is not
very representative of the entire population (Boyd et al., 2003). As an alternative to MB, flow cytometry (FC), with selected fluorescent probes, can be employed
to assess the yeast viability and vitality. In this technique, cells carried by a fluid stream pass through a light
beam and three parameters are measured: forward angle light scatter (FSC), for the determination of
relative size; side angle light scatter (SSC), for the determination of granularity/internal complexity;
fluorescence at selected wavelengths, for the detection of fluorescent dye labels (Herlem & Hua, 2010).
The combined data the three parameters allows the user to determine the heterogeneity of the population.
FC also stands out by yielding a result that is representative of the entire population as it analyses a range
of 106 to 107 cells in a matter of seconds (Herlem & Hua, 2010). Several criteria may be used to estimate the physiological state of the cell by FC, such as cell
membrane integrity, membrane potential and cell enzymatic activities, requiring the use of different
fluorescent probes (Díaz, Herrero, García & Quirós, 2010).
5.3.3. Yeast exposure to stress in the brewing environment
In the course of brewing, yeast is exposed to multiple stress factors, that may influence its
physiological state, and consequently, the yeast management.
The initial high concentration of carbohydrates in wort (90 – 92 % of the total wort solids) sets an
imbalance of intracellular and extracellular osmolarities (osmotic stress), sufficient to cause a deleterious change in yeast physiology (Csonka & Hanson, 1991). Further, the depletion of fermentable carbohydrates
and/or assimilable nitrogen (nutritional stress), enables the yeast entering in stationary phase, in which
proliferation ceases, and individual cells exit cell cycle and enter the G0 or quiescent cycle, remaining in
this state until the depleted nutrients become available (Briggs, Boulton, Brookes & Stevens, 2014). The
quiescent state is characterized by the cessation of cell proliferation, thickening of cell walls, reduced cell
porosity and increased resistance to lytic enzymes, intracellular accumulation of proteases, accumulation
of polyphosphates within vacuoles and a general increase in storage carbohydrates (trehalose and glycerol)
13
(Gray et al., 2004). The switch to this state is a yeast common response to nutrient starvation, and it is
necessary for the maintenance of viability over extended periods in the absence of all the necessary
elements for growth, such as during yeast storage.
Carbon and nitrogen starvation are also known to trigger flocculation (the aggregation of dispersed cells into flocs) which is essential for yeast sedimentation and further cropping. This aggregation occurs by
the binding of cell surface lectins (flocculins), to carbohydrates residues on the walls of surrounding cells.
Flocculation is also determined by the presence of calcium ions, the absence of mannose (and glucose,
sucrose and maltose in some strains), oxygen content or the presence of compounds produced during
aerobic metabolism (such as ergosterol), changes in pH and temperature, presence of premature yeast
flocculation-induction factor and ethanol concentration. In brewing, the flocculation is cyclical as yeast
unflocculates by entering a nutrient rich media (wort), and starts to flocculate by the end of exponential
growth phase (Soares & Mota, 1996). Besides nutritional stress, yeast is also submitted to oxidative stress in the course of propagation
and early stages of fermentation. As it has been mentioned, the supply of oxygen (or aeration) is essential
for propagations and the initial state of fermentation. However, the reactive oxygen species (ROS) produced
endogenously by the cell can damage cell components (lipid peroxidation, protein inactivation and nucleic
acid damage), possibly leading to cell loss of viability (Gibson, Lawrence, Leclaire, Powel & Smart, 2007).
The production of ethanol is one the purposes in the brewing fermentation, but a viable yeast population
that can be used in subsequent fermentations should also be seen as a product of fermentation. As ethanol
increases throughout fermentation (it can reach up to 16 % in brewing), the yeast is exposed to increasing levels of ethanol toxicity that will negatively affect the yeast viability.
This toxicity has several targets but the cellular membrane is one of the most affected. The major effects
of ethanol toxicity are growth inhibition, reduced cell size, reduced viability, reduced respiration and glucose
uptake, reduced fermentation, enzyme inactivation, lipid modification, loss of the proton motive force across
the cellular membrane and increased membrane permeability (Stewart, Hill & Russel, 2013).
The complex array of stresses that the brewing yeast cell is exposed to during brewery handling is
matched by the array of defense mechanisms that are possessed by the cell. Yeast has two major pathways to respond to stress: heat shock response (HSR), which is activated by sublethal yeast stress, and general
stress response (GSR), activated by oxidative, pH, heat and osmotic stresses and nitrogen starvation
(Gibson, Lawrence, Leclair, Powell & Smart, 2007). The activation of these responses increase the yeast
ability to survive in adverse conditions.
5.3.4. Yeast long-term preservation
Yeast long-term preservation by cryogenic storage is used as an alternative to continuous
subculture. The yeast is frozen with a cryoprotector (glycerol or inositol) and stored at -80 ºC (Lodolo, Kock,
Axcell & Brooks, 2008). Cryogenic storage has several advantages over other preservation techniques: it
14
is simple, reproducible, and the storage can last up to three years, although it is advisable to provide
maintenance within one year (Bokulich & Bamforth, 2017).
Having the yeast strains in bank has many advantages, such as always having them available, and
the lowering of the yeast-associated costs.
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6. Thesis scope and goals
This project was developed as a collaboration between BioISI Microbiology & Biotechnology Research
Group (Lab Bugworkers|M&B – BioISI) and the Dois Corvos craft brewing company.
The Dois Corvos brewery has been in constant growth since its beginning in 2012. As the brand created a
reputation, in the Portuguese and foreign markets, it developed growing necessity to control the quality of
the beers being produced and optimize the overall production process.
For this reason, the goals of the present dissertation were:
❏ Start a yeast management program by implementing in-house propagations, serial yeast
repitching;
❏ Cryopreserve valuable yeast strains for further use;
❏ Validate the serial yeast repitching protocol implemented;
❏ Study the propagations variables in order to optimize the biomass yield;
❏ Introduce monitoring points throughout the production work-flow;
❏ Use the monitored data to optimize the production process and improve the product quality.
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MATERIALS AND METHODS
Part I: Yeast Management
1. Yeast Long Term Storage
1.1. Sampling
Samples were taken aseptically from a fermentation vessel following the American Society of
Brewing Chemists (2018b) method.
The sampling port was open completely in order to let the beer flow out for ten seconds. The
surfaces form the sampling port that will come into contact with the sampled beer were scrubbed with a cotton swab soaked in 70% alcohol. The same surfaces were then flamed with the use of a butane torch
until it became incandescent. In order to cool the previously heated surfaces the sample port is opened and
beer is let to flow for five seconds. A 50mL sample is collected into a sterile container.
1.2. Isolation
One hundred microliters of beer from the sample taken from the vessel were inoculated in yeast
extract, peptone, glucose and agar media (YEPGA) plates and incubated at 30 ºC for 48-72 hours. After
the growth of colonies, one was subcultured into a new YEPGA plate and incubated once more for 48-
72 hours at 30 ºC. This last step was repeated three times.
1.3. Storage
The cells from the plate were scraped into a cryotube with 1mL sterile glycerol 20% and stored at
-80 ºC.
2. Propagation
2.1. Laboratory propagation
2.1.1. Plating and cell growth
Working in aseptic conditions, 100 microliters of a cell suspension of the desired strain were plated
in YEPGA plates and incubated at 28ºC for 48h-72h.
17
2.1.2. First step up
In the laminar flow chamber, colonies were scraped from the YEPGA plate and suspended in a
sterile 500mL Erlenmeyer with a sterile solution of dextrose malt extract (DME) and water with the density of 1.040 in order to indulge biomass creation instead of favoring fermentation. The sample was left in an
orbital shaker at 30 ºC and 300 rpm for 48-72h.
2.1.3. Second step up
In the laminar flow chamber, the sample prepared in 2.1.2 is inoculated to a sterile 1L schott flask
and another 500mL of sterile DME and water solution were added. The sample was left in the orbital shaker
with the same features as in the step before for a period of 48-72h.
2.2. Brewery propagation stage
2.2.1. Propagation system assembly
To a cylindrical-conical vessel with 200 L capacity it was adapted a sample port and two open/close valves, one at the top and one at the bottom of the vessel. The adapted propagator was cleaned in place
(CIP) and properly sanitized according to the Dois Corvos standard operating procedures (SOP). A sanitary
hose was sanitized by recirculation with 80ºC water for 40 minutes and was added to the system,
connecting with the top and bottom valves.
2.2.2. Propagation conditions
Wort: For propagations, wort was always transferred from the batch being brewed at the brewery that
day, therefore its composition depended on the recipe/style of beer being brewed.
Temperature: Contrary to the fermenting tanks, the propagation tank did not allow temperature control.
Wort was introduced at a temperature of 20 ºC and propagations were carried at room temperature, which
oscillated between approximately 15 to 28 ºC.
Aeration: Oxygen was provided by mechanical agitation of the propagation medium using a peristaltic
pump with a hose connected to the top and bottom valves of the propagation vessel.
Addition of trace elements: Yeast Vit (YVIT-5K from Murphy & Sons, UK) was added at a rate of 4.50
g/HL. Zinc sulphate heptahydrate (ZINSUL-5K from Murphy & Sons, UK) was added at a rate of 2.15 g/HL.
18
2.2.3. Yeast
Yeast for propagation was sourced in the form of pure pitch purchased from White Labs or obtained
from the laboratory propagation of a banked yeast strain.
2.2.4. Propagation step up
At the Dois Corvos facilities, the propagation system assembled in method 2.2.3 was filled with
approximately 50 L of sterile wort previously cooled down to 20ºC. The 1 L inoculum was added carefully
in the most aseptic manner to the 50 L of wort.
Zinc was added at the rate of 0.5 ppm and yeast nutrient was added at the rate of 4.5 g/hL. A
peristaltic pump was connected to the system by the hose and with the valves open the recirculation was
activated at 50 rpm.
2.2.5. Yeast Viability and counting
A representative sample was collected from the yeast source (e.a fermenter or propagator). A first
dilution of 1 ml of yeast sample to 9 ml of sterile wort was performed, mechanically agitated, followed by a
second dilution of 1 ml of the previous dilution to 8 ml of distilled water.
The use of sterile wort in the first dilution, instead of the commonly used distilled water, promotes
yeast desegregation, and allows an homogenous spread of cells in the hemocytometer chamber. After
mixing by agitation, 1 ml of 0.1% methylene blue staining solution was added, stirred and led to sit
undisturbed for 3 min, to allow metabolization of the dye prior to counting.
With a Pasteur pipette, a single drop of the mixture was placed in the tip of the hemocytometer chamber, allowing capillary action to pull the sample under the glass cover slip and avoiding flooding the
moats. With bright field imaging, cells evenly distributed inside the 25 squares of the hemocytometer
chamber were counted and defined as total cells. Single cells stained in blue were defined as non-viable
cells, single non stained cells and all the budding cells were defined as viable cells. Hence, the total of cells
is obtained by the sum of non-viable and viable cells. The process was repeated with heat-killed cells
(submitted to 80ºC for 20 min) to act as a negative control and cells diluted with no staining to act as positive
control.
Percentage of viability was determined by the formula:
%𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝑇𝑜𝑡𝑎𝑙𝑐𝑒𝑙𝑙𝑠 − 𝑁𝑜𝑛𝑉𝑖𝑎𝑏𝑙𝑒𝐶𝑒𝑙𝑙𝑠
𝑇𝑜𝑡𝑎𝑙𝑐𝑒𝑙𝑙𝑠 × 100
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Number of cells per milliliter was determined by the formula:
𝐶𝑒𝑙𝑙𝐶𝑜𝑢𝑛𝑡(𝑐𝑒𝑙𝑙𝑠/𝑚𝑙) = 𝑇𝑜𝑡𝑎𝑙𝑐𝑒𝑙𝑙𝑠 × 𝐷𝑓 × 10?
Whereas Df represents the dilution factor and 104 represents the inverse of the volume of the chamber
(0.1 mm3 or 10-4 ml). To estimate the total number of cells in the propagation the number of cells per milliliter
was then multiplied by the volume of the propagation.
3. Flow cytometry assay
3.1. Yeast samples
Studies were performed with WLP007 “Dry English Ale” (Whitelabs, Copenhagen) in the form of yeast slurry.
3.2. Sample washing
In a 15 ml falcon tube, the yeast slurry was diluted in a proportion of 1 to 10 with phosphate buffered saline (PBS) solution. To achieve the ideal cell count for the cytometer, a second dilution of 1 to 10 was
performed with the same buffer. In order to remove all the trub, which mainly consisted of fats, hops debris
and coagulated proteins, the yeast slurry was first submitted to a washing process, prior to the cytometer
analysis. The washing consisted of adding PBS to the already diluted sample, resuspending first with a
pipette, and posteriorly with a vortex, centrifuging (Centrifuge 5810 R, Eppendorf) at 4000 rpm for 10 min
after which, a pellet was obtained and the supernatant was discarded. This washing process was repeated
3 times per sample.
3.3. Staining
Staining for flow cytometry was performed in 1 ml final reaction volumes, in which cell density was
approximately 107 yeast cells ml -1 . After staining, cells were vortexed for 10 s immediately prior to analysis.
For calibration, three replicates of five fresh and heat-killed volumetric series of proportions (100, 75, 50,
25 and 0%) were analyzed.
3.3.1. Fungalight staining
The FungalightTM yeast viability kit (L34952 from Thermo Fisher Scientific) was used to stain yeast
cells. 1 µL of SYTO9 stock solution (3.34 mM in DMSO) and 1 µL of PI stock solution ( 2.20 mM in DMSO)
20
were added to 1 mL yeast cell suspensions. The samples were incubated at room temperature, protected
from light, for 15min. The effect of dyes staining was examined by flow cytometry.
3.3.2. Oxonol staining
Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC4(3)), (B-438 from Thermo Fisher
Scientific) was used to stain yeast cells. 1 µL of DiBAC4(3) stock solution (5 mg/mL in DMSO) was added to 1 mL yeast cell suspensions and the samples were incubated at room temperature, protected from light,
for 15min. For positive control, cells were stained with 2 µL of carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (FCCP) (5 mM in ethanol), (C2920 from Sigma Aldrich). The effect of
dye staining was examined by flow cytometry.
3.4. Flow cytometry analysis
FC analysis was performed in a CyFlow Space flow cytometer (Sysmex Partec GmbH, Germany),
equipped with a blue solid-state laser (20 mW at 488 nm) and 5 optical detectors (FSC, SSC, FL1, FL2 and
FL3). Samples were excited using a 488 nm solid-state laser and fluorescence was detected using green
fluorescence detector FL1 (536/40 nm) and red fluorescence detector FL3 (610/30 nm). Logarithmic
amplification was always used. The data was analyzed using FlowJo™ v10.6.1, yeast populations were
identified on an FSC vs SSC pseudocolor dotplot and gated for presentation on an FL3 vs FL1, FSC vs FL3 and SSC vs FL3 dotplots , where live and dead cells were identified.
4. Yeast cells harvest and re-pitching
Yeast harvest from cylindro-condrical vessels is made into yeast brinks, 10 L plastic jugs (Kartell,
Italy) in which the yeast were stored at 2-4 ºC until further use.
4.1. Sanitization
All the pieces involved in the harvest have to be well sanitized to prevent any type of contamination
that can potentially leave the yeast inept to being used in a future brew.
The yeast brinks were sanitized with 85 ºC water for 30 min. A tube in the shape of a ‘S’ was assembled
with parts of stainless steel joined by sanitary gaskets and tri clamps. The ‘S’ tube was soaked in a solution
of MIDA CHRIOX 5 (1%) for 15 min and then connected to the chosen fermenter.
21
4.2. Harvest
The yeast was pulled down to the yeast brink by the tube. The first 10 L were always discarded
and the collected yeast was stored at a temperature ranging from 2 to 4 ºC. Also at the 10 L mark a 100 ml sample was collected for a cell count and viability assay.
4.3. Re-pitching
The re-pitching process was composed by a theoretical part of defining the pitching rate to calculate
the necessary number of cells, and a challenging practical part of inoculating this immense quantity of yeast in a sanitary manner into the fermenting vessel.
The pitching rate is read in millions of cells per milliliter per degree of Plato and it translates into
the quantity of yeast necessary to indulge a healthy fermentation. After optimization, the pitching rate for
ales was set for 0.5 million cells per milliliter per degree of Plato, and for lagers, 1.0 million cells per milliliter
per degree of Plato. With this set rate, the original density (in Plato) and the quantity (in milliliters) of beer
being produced, the number of cells needed for inoculation was calculated. The correlation between the
number of cells and liters of yeast slurry was given by the initial counting with the hemocytometer as
explained in method 2.2.5. To inoculate, the yeast was removed from the storage, 2 to 5 h prior to inoculation, so it could reach
room temperature (around 20 ºC) and start coming out of the dormant stage. Inoculation was made through
the fermenter man hole, cleaned with alcohol wipes beforehand, 10 to 15 minutes before filling the tank
with wort.
This process of serial repitching was be repeated as many times as desired as the yeast maintained
its viability.
5. Principal component analysis
Principal component analysis (PCA) was performed with the software NTSYS 2.1 (Applied
Biostatistics Inc.). The following variables were used for characterization of the analyzed set of fermentations:
● Time (Time_h): represents the time in hours from the beginning to the end of the propagation; ● Growth rate (GR) : calculated by the formula: (Final yeast cells / Initial yeast cells) / time (h)
representing the number of cell duplications per hour; ● Yeast final biomass (logFB): yeast biomass in the end of propagation was first represented in
trillions of cells. This wide range of values was converted to a logarithmic scale to facilitate the
comparison of values. Therefore final biomass represents the yeast biomass in the end of
propagation in logarithmic scale;
22
● Percentage of viability (%V): viability by the methylene blue staining method measures were taken in the same day the propagated yeast was used to inoculate wort;
● Upscale step (UpS): quantifies the number of times more wort was added to the propagation system; ● Laboratory propagation stage (LPS): it is a Boolean variable that describes if the propagation started
with a yeast bank pre-inoculum in the laboratory, or started one step ahead, at the Dois Corvos
facilities, with a pre-inoculum purchased from White Labs (Copenhagen); ● Expiration date (VAL): pre-inoculums purchased from White Labs, or other yeast selling sources,
have expiration dates, through which the viability of the yeast decreases. According to the source,
a pre-inoculum before the expiration date has a 95% viability. After the expiration date, this value is
no longer assured and viability is expected to diminish 3.2% per month. This variable indicates the
number of months by which the yeast had expired, when it was used for a propagation;
● Consumed carbohydrates (PLATO): calculated by the difference between the initial and the final specific gravity, in the plato scale. This measurement estimates the amount of carbohydrates
consumed by the yeast during propagation;
● Initial pH (pH_I): indicates the wort initial pH;
● Final pH (ph_F): indicates the propagation final initial pH;
● Fermentation outcome (End_F): yeast biomass, resulting from the propagations, was always used for beer production. This Boolean variable indicates if the yeast was able to perform a successful
fermentation;
● S_smell: indicates presence or absence of the sulfur like smell, a recurrent off-flavor found in propagations and fermentations;
Part II: Quality Control Implementation
1. Adenosine triphosphate test
1.1. Sampling
Samples were swabbed directly from the testing points with the use of the Hygiena Ultrasnap
surface ATP Test. The swab is removed from the tube, a ten by ten centimeters area is swabbed in a
crisscross pattern, swabbing the area with even coverage and rotating the swab tip to ensure maximum sample collection. The swab is replaced in the tube and the ultrasnap is activated by breaking the snap
valve. To expel liquid into the tube, the bulb is squeezed twice and the sample is shaken for five seconds
and then inserted into the luminometer.
23
1.2. Luminometer
The Luminometer should be turned on before starting the actual test to make sure the device has time
run the self-calibration. The swab is inserted into the reading chamber and the lid is closed. With the luminometer held up right, the OK button is pressed to initiate the test. The result is displayed in fifteen
seconds in relative light units (RLU). The maximum allowable RLU for a result to pass the ATP test for
cleanliness is 60.
2. pH Measurement
2.1. Calibration
The pH meter is calibrated before each use to ensure accurate readings. Calibration is done with
a two calibration standard points: an acidic 4.0 and a neutral 7.0 buffer solutions. The probe is removed
from the storage solution and rinsed with deionized water. The calibration mode is selected in the device
and the probe is placed in a beaker with 4.0 standard buffer solution. Once the stable icon appears, the
probe is rinsed once again with deionized water and then placed in a beaker with the standard 7.0 buffer
solution. Once the measurement is stable the device returns automatically to measurement mode. The
probe is rinsed with deionized water and placed in the storage solution ready to use.
2.2. Readings
To take a pH measurements the samples were taken to a beaker from the interest points, degassed
if needed, and cooled down to a temperature below 40 ºC. The pH probe was inserted into the beaker with
the sample and the reading was registered once it was stable.
3. Wort stability test
3.1. Sample port sanitizing
Samples were taken aseptically from a port placed immediately after the heat exchanger following
the American Society of Brewing Chemists (2018b) method.
The sampling port was open completely in order to let the wort flow out for ten seconds. The surfaces from the sampling port that will come into contact with the sampled wort were scrubbed with a
cotton swab soaked in 70% alcohol. The same surfaces were then flamed with the use of a butane torch
until it became incandescent. In order to cool the previously heated surfaces the sample port is opened and
wort is let to flow for five seconds.
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3.2. Sampling
A sample of 120 ml of wort was taken from the sterile port into the sterile Whirl Pak Stand Up Bag. The
bag is carefully handled to remove the maximum air and then closed and left at room temperature for 7 days. Positive results were characterized by a swelling of the bag, whereas the absence of swelling points
to a negative result.
4. Microbiological analysis
4.1. Sampling
Samples were taken aseptically from the fermentation vessels following the American Society of Brewing Chemists (2018b) method.
The sampling port was open completely in order to let the beer flow out for ten seconds. The
surfaces from the sampling port that will come into contact with the sampled beer were scrubbed with a
cotton swab soaked in 70% alcohol. The same surfaces were then flamed with the use of a butane torch
until it became incandescent. In order to cool the previously heated surfaces the sample port is opened
and beer is let to flow for five seconds. A 50 ml sample is collected into a sterile container.
4.2. Culture media
Samples taken accordingly to the methodology described in 4.1 were inoculated in three media
recommended by the American Society of Brewing Chemists:
1. Hsu’s Lactobacillus and Pediococcus (HLP) medium: 1ml of each sample was inoculated on sterile
falcon tubes containing 10 ml of HLP medium (Lallemand, USA) and incubated at 28 ºC for 9 days.
This medium is commonly utilized in the brewing industry for the selective detection of lactic acid
bacteria. Yeast growth is suppressed by cycloheximide contained in the media and the
mercaptoacetic acid creates an anaerobic environment in the tube.
2. Wallerstein Differential (WLD) medium: 100 μl of each sample were inoculated on plates containing
20 ml of WLD medium (Hardy Diagnostics, USA). The plates were incubated at 37ºC, both aerobically and anaerobically, for 72 hours. This medium inhibits yeast growth using cycloheximide
in its composition and was used exclusively for the detection of bacteria.
3. Lin’s Cupric Sulfate (LCS) medium: 100 μl of each sample were inoculated on plates containing 20
ml of LCSM (prepared in house) and incubated at 25 ºC aerobically for 5 days. This medium was
25
used for the detection of non-Saccharomyces yeasts. This is possible due to the cupric sulfate
concentration in its composition that inhibits the growth of bacteria and the majority of
Saccharomyces spp. yeasts.
4.3. Colonies characterization
In case of a positive result in either of the media described in 4.2, the colonies were observed under
the light microscope Leica CM E (Leica Microsystems, Germany) so bacteria were distinguished from yeast
and morphology was described. Subsequently, to bacteria colonies, KOH and catalase test were performed.
5. Fermentation monitoring
5.1. Specific gravity
5.1.1. Sampling
Samples were taken from the fermenters daily through the fermentation and maturation stages of
the beer. The sampling port was open to maximum flow and a first sample of 100 ml was taken and
discarded to enhance representativity. A second sample of 100 ml was then collected.
5.1.2. Sample degassing filtering
The sample collected in method 5.1.1 was first degassed mechanically by pouring back and forth
for 2 minutes (American Society of Brewing Chemists, 2018a). Secondly the sample was filtered with the
use of 0.45 μm filter paper in order to avoid yeast and particles in suspension influencing the specific gravity readings.
5.1.3. Specific gravity readings
Specific gravity readings were performed with DMATM 35 (Anton Paar, Austria) portable density
meter. The device was turned on and calibrated daily with distilled water. After the water check, three
readings of each sample were taken and registered on the cellar logs next to each fermenter.
5.2. Forced fermentations
One liter of wort from each brew was aseptically transferred to a sterile 2 L Erlenmeyer flask.
Approximately 5 ml of yeast slurry were added to the wort. The flask was incubated at room temperature,
with agitation of 1500 rpm, for 48h. Gravity readings were performed according to method 5.1.
26
6. Product stability analysis
A box of 24 bottles from each batch was taken and labelled from 1 to 6 according to the filling tube
number in the bottling machine (Meheen, USA). Microbiological analysis, according to method 4.2 and 4.3,
was done to 6 bottles (one from each filling tube), two weeks, two months and six months after bottling.
7. Complaints Tracking
In order to keep track of all the complaints related to the beer quality problems, a file was created
so every situation is registered and a proper follow up is given. The file mostly contains information as batch number, bottling date, type of container and a summary of the occurrence.
27
RESULTS AND DISCUSSION
Part I: Yeast Management
1. Validation of methylene blue staining method
Viability assays are crucial for deciding whether to keep or discard harvested yeast. In a brewery, this
type of decision as to be taken on a daily basis, using a simple and affordable approach.
Direct observation using bright-field microscopy of yeast cells stained with methylene blue has been the
elected method for years by microbreweries (Lee, Robinson & Wang, 1981). Despite the simplicity of the
method, existing protocols often present a lack of consensus on the methylene blue concentration to be
employed, the yeast dilution factor, and the solvent for the dilutions.
Going further, some researchers theorize that the method is only reliable with apparent viability above 90%, suggesting that damaged cell membranes may result in the occurrence of variable cell shading
and provoke inconsistent viability counts (Smart, Chambers, Lambert, Jenkins & Smart, 1999). To validate the adjustments made to the method and inquire about the veracity of viability counts
with apparent low viability, the methylene blue method was tested and the results shown on Figure 1 were
compared against viability assessments by flow cytometry (FCM) using three different dyes.
Figure 1: Viability percentage assessed by the methylene blue staining method.
28
1.1. Determination of viability by flow cytometry with SYTO9+PI
Staining procedures were optimized using different proportions of fresh and heat-killed cells. Cells
were stained with permeant green fluorescent nucleic acid stain (SYTO9) and impermeant red-fluorescent
nucleic acid counterstain propidium iodide (PI), in order to differentiate between live and dead
Saccharomyces cerevisiae cells. The display of a distinctive and reproducible fluorescence pattern
observed with flow cytometry is shown in Figure 2 and the of the viability assay results are shown in Figure 3.
Figure 2: Graphic example of the viability analysis using flow cytometry. Yeast cells were co-stained with SYTO9 (green
fluorescence detected on FL1) and PI (red fluorescence detected on FL3). (A) Pseudocolor dotplots of side scatter (SSC) vs. FL3; (B) Pseudocolor dotplots of FL3 vs. FL1.
Different cellular states were illustrated, as cells with intact membranes (subset1 – Fig 2A and Q3
– Fig 2B) emitted a strong green fluorescence (SYTO9) and weak red fluorescence (PI), while a cell
A B
29
population with damaged or permeable membranes (subset3 – Fig 2A and Q2 – Fig 2B) emitted weak
green fluorescence (FL1) and strong red fluorescence (FL3). This shift in fluorescence was gradual and
followed the change in the fresh/heat-killed cells ratio, as expected.
It was also possible to observe a subset2, with different intracellular concentrations of SYTO9 and PI, suggesting the existence of a intermediate state (Berney, Hammes, Bosshard, Weilenmann & Egli,
2007). Translating into a viability context, one can associate cells in subset1 with viable cells and cells in
subset3 with non-viable cells. The subset2 can be associated with “injured” cells with an apparent partial
(perhaps temporary) loss of membrane integrity.
Figure 3: Viability percentage assessed by the flow cytometry analysis using SYTO9 as staining and PI as counterstain.
1.2. Determination of viability by flow cytometry with DIBAC4(3)
Figure 4 shows an example of the validation of the use of DiBAC4 as a marker of cell viability in
yeasts and Figure 5 shows the results of the viability assay. This marker can also be associated with the
measure of cell vitality as the increase in fluorescence intensity occurs upon the depolarization of the
membrane.
30
Figure 4: Graphic example of the viability analysis using flow cytometry. Yeast cells were co-stained with DIBAC4(3) ( green fluorescence detected on FL1 and a red spectral shift detected on FL3). (A) Pseudocolor dotplots frontal scatter (FSC) vs. FL3; (B)
Pseudocolor dotplots of FL3 vs. FL1.
A B
31
Figure 5: Viability percentage assessed by the flow cytometry analysis using DIBAC4(3) as a staining.
With suspensions of different proportions of fresh and heat-killed cells, distinct DiBAC4(3) fluorescence distributions were observed. This green lipophilic fluorescent probe can only enter cells if their
membranes are depolarized and binds to intracellular proteins or membrane exhibitng enhanced
fluorescence and a red spectral shift. (Hernlem & Hua, 2010). Viable cells (subset1- Fig4A) possessed
intact polarized cytoplasmic membranes; in contrast, injured or stressed (subset2 – Fig4B), or dead cells
(subset3 – Fig4B), with depolarized cytoplasmic membranes, emitted strong green fluorescence and a red
spectral shift.
As a positive control, fresh cells were also treated with FCCP (carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone), which is an ionophore, uncoupler of oxidative phosphorylation which dissipates the
membrane potential (Perry, Norman, Barbieri, Brown & Gelbard, 2011). Figure 6 shows the increase of the
green fluorescence when compared to the one on the untreated cells, demonstrated that, indeed,
DiBAC4(3) fluorescence is due to a membrane potential-dependent
32
Figure 6: Graphic representation of the enhanced green fluorescence of cells treated with FCCP. (A) Pseudocolor dotplots frontal
scatter (FSC) vs. FL3; (B) Pseudocolor dotplots of FL3 vs. FL1.
1.3. Experimental vs. theoretical viability
The viability assessments by 3 different methodologies of five fresh and heat-killed volumetric
series of proportions (100, 75, 50, 25, and 0%) were compared to the theoretical viability values provided
by the manufacturer (Whitelabs, Copenhagen) and the results are shown in Figure 7.
Figure 7: Correlation between the viability measured by the three methodologies presented and the theoretical viability.
Since the theoretical viability of the fresh cells was approximately 84.2% (Whitelabs, Copenhagen),
the cells represented as subset2 in the flow cytometry assays were accounted as non-viable for the assay
with SYTO9+PI and accounted as viable for the assay with DIBAC4(3); other way, the experimental viability
value with SYTO9+PI would be superior (94.2%) to the value provided by the manufacturer, and the
A B
33
experimental viability value with DIBAC4(3) would be too inferior and less related (75.5%) to the value also
provided by it.
Results (Figure 7) indicated a strong (p≤0.95) correlation of 99.6% between theoretical and
experimental viability using MB, a strong (p≤0.95) correlation of 97.9% between theoretical and
experimental viability using FCM with SYTO9+PI as staining, and a correlation of 89.1% between
theoretical and experimental viability using FCM with DIBAC4(3) as staining, which suggested that viability
measured by the two first methodologies may be more reliable.
1.4. Correlation between the staining with methylene blue and flow cytometry staining with a conjugation of SYTO9+PI and DIBAC4(3)
Results of both flow cytometry assays were correlated with the results obtained with methylene
blue staining to validate the use of this method in the brewery quotidian. It was demonstrated (Figure 8) a
strong correlation (p≤95) between the viability measured with the methylene blue and FCM using
SYTO9+PI as staining (97%), and a mild correlation (84%) between methylene blue and FCM using
DIBAC4(3) as staining.
Figure 8: Correlation analysis between viability by methylene blue and viability by FCM. Left: Correlation between viability by
methylene blue and FCM using SYTO9 + PI. Right: Correlation between viability by methylene blue and FCM using DIBAC4(3).
Staining with methylene blue is the simplest and most cost effective methodology to apply in a
brewery environment, as it only requires for an optical microscope and basic laboratory materials. Along with the fact that the yielded results are almost instantaneous and demonstrated a strong correlation with
results obtained in the FCM assay, the implementation of the method in the Dois Corvos brewery was more
than justified.
However, it should be registered that the low viability populations were composed by a combination
of healthy yeast and yeast submitted to conditions that are designed to reduce the viability of a control
population to zero. These conditions are unlikely to be representative of the damage caused to the
proportion of cells in a population that lose viability when exposed to less severe environmental conditions (Amor et al., 2002) as in the brewing environment.
34
Independently on how viability decreases, the loss of membrane permeabilization in non-viable
cells is always a constant, meaning that the viability assay by FCM of cells stained with a conjugation of
SYTO9+PI or DIBAC4(3) should always be reliable.
As for the methylene blue assay, the custom loss of viability might have an influence on the results, as the metabolically active live yeast cells convert methylene blue to a colorless compound through cellular
dehydrogenase activities, while the dead yeast cells remain blue. Throughout this experiment, the viable
cells were not exposed to stress conditions and the reduction of methylene blue was not affected.
Therefore, viable (colorless) and non-viable (dark blue) yeast cells were easily distinguishable. When stress
is applied to the total population, if it may affect the reduction of methylene blue in cells that are still viable,
it can also produce a pattern of different blue intensities that can lead to wrong estimation of viability.
In this case, it may be safe to say that the reliability of the method depends on the experience and
critical sense of the user employing it, as it can be prone to human error and lead to user-dependent variations.
1.5. Assessing yeast viability through generations
Viability percentage was assessed by the three formerly explained procedures throughout four
yeast generations. The results are shown on Figure 9 and the graphic representation of the viability by flow cytometry is represented on Appendix a and Appendix b.
Figure 9: Comparison of the viability percentage measured by methylene blue, FCM with SYTO9+PI as staining, and FCM with
DIBAC4(3) as staining from generation 0 to generation 4.
0
20
40
60
80
100
Generation0 Generation1 Generation2 Generation3 Generation4
ExperimentalViability(%)
METHYLENEBLUE FCMSYTO9+PI FCMDIBAC4(3)
35
The viability of the generation 0 is the viability of the liquid yeast pure pitched from the manufacturer
Whitelabs. If transported and stored in proper conditions, the pure pitch viability can be estimated by
subtracting 3.2% (Whitelabs, USA) by each month after the product packaging in the Whitelabs facilities. This pure pitch, correctly transported and stored, was opened 4 months after packaging. Therefore, its
estimated viability was 84.28%.
For the later generations, the yeast was always harvested from fermenting tanks five days after
inoculation. Results show an expected constant high viability level from generations 1 to 4 (Luarasi, Troja
& Pinguli, 2016), which validates the methodology of harvest and re-pitching employed as a part of the
yeast management.
During the FCM assay of the fourth generation, a second population of yeasts was detected, predicting
a possible yeast contamination. To avoid the contamination of future batches, this yeast was discarded, the assay was terminated and a new pure pitch was used in the next fermentation.
2. Propagations
Throughout the experimental work of this thesis, several yeast strains were propagated. The aim was
to start with a small cell biomass and finish with enough yeast cells to pitch a batch of beer with volumes
comprehended between 900 - 2000 L.
During propagations, 12 variables were monitored and the data were posteriorly a subject of principal
component analysis. The aim was to discover the correlation between the variables, study the significant
ones, and define strategies leading towards a higher propagation performance.
2.1. Principal component analysis
PCA is a multivariate data analysis method which provides a visually interpretable overview of the main
information of multidimensional and often complex data sets. By plotting principal components, sample
groupings may be detected and analyzed, and relationships between variables may also be assessed
(Rossouw, Olivares-Hernandes, Nielsen & Bauer, 2009). The following variables were considered: growth rate (GR), logarithm of final biomass (logFB), time in hours (Time_h), viability percentage (%V), upscale
(UpS), lab propagation phase (LPS), months after expiration date (VAL), final density (PLATO), initial pH
(pH_I), final pH (pH_F), fermentation conclusion (End_F), and sulfur smell (S_smell).
2.1.1. Variables Correlation Matrix
Analysis of correlation between each pair of variables (Table 2) shows that some variables are
significantly correlated.
36
Table 2: Correlation between variables. *(p≤0.05) **(p≤0.01)
GR logFB Time_h %V UpS LPS VAL Plato pH_I pH_F End_F S_smell
GR 1
logFB -0.31 1
Time_h 0.51* 0.23 1
%V -0.1 -0.41 -0.68** 1
UpS 0.71** -0.25 0.48* -0.3 1
LPS 0.07 0.23 0.09 -0.44* 0.33 1
VAL 0.04 0.34 -0.05 -0.14 -0.23 -0.05 1
Plato 0.06 0.11 0.02 -0.04 -0.2 -0.26 0.42 1
pH_I -0.02 0.15 0.15 -0.01 -0.22 -0.19 0.17 0.42 1
pH_F 0.18 -0.46 -0.56* 0.6** -0.02 -0.1 0.28 -0.03 -0.29 1
End_F 0.09 0.04 0.15 -0.24 0.15 0.18 0.2 -0.24 -0.07 -0.19 1
S_smell 0.32 -0.41 0.13 0.2 -0.01 -0.38 -0.17 0.19 -0.03 0.17 -0.46* 1
Time and growth rate have an expected strong correlation. When all the conditions are reunited,
wort with the correct supply of oxygen, amino acids, and trace elements, at a suitable temperature (20-25
ºC), viable yeast just needs time to multiply.
Time and the percentage of viability have an expected strong negative correlation (Table 2).
Freshly propagated yeast has a very good viability, as it just finished several replications. When propagation
ceases and the yeast is not promptly used or stored in appropriate conditions, it stays exposed to multiple
stresses, such as lack of nutrients, ethanol stress and thermal stress. As a consequence, cell autolysis can occur, leading to excretion of cell contents and intracellular enzymes, particularly proteinase A (PrA), a
protein known to have a negative effect on beer foam stability (Stewart, 2015). As the number of death cells
increases, the viability percentage decreases.
The upscale step and the growth rate are the strongest correlation according to the data (Table 2).
In yeast propagation, one of the constraints is the anaerobic metabolization of sugars due to catabolite
repression and Crabtree effect that leads to a low biomass yield on substrate (Dynesen, 1998). To control
this metabolic tendency, the golden standard is the use of a fed-batch system, allowing control over the total sugar concentration (Vieira, Andrietta & Andrietta, 2012). Adapting a fed-batch system at the Dois
37
Corvos facilities was not a possibility, although there is a desire to do so in the future. A different option to
try to diminish the constraints and obtain a high yield propagation is to do volume upscales (Kunze, 2014).
This consists of starting with a small volume and consistently adding up to 10 times as much aerated wort
to the propagator during the yeast exponential growth phase, increasing the total volume of the propagation until the desired amount of yeast is obtained. Therefore, a strong correlation between these two variables
is an indicator that the upscaling process is suitable for improving the efficiency of our propagation system.
The variable upscale step also has a strong correlation with the variable time (Table 2), which is
expected. Starting with smaller volumes and consistently upscaling the process is more time consuming
than a one-step propagation.
The laboratory propagation step and percentage of viability have a strong negative correlation (Table 2), which was not expected. The occurrence of a laboratory stage propagation is associated with
the propagation of yeast strains stored in the yeast bank at the TecLabs facilities. When there is a laboratory
propagation stage, the process starts with a smaller initial inoculum and, therefore, it has a tendency to be
upscaled a superior number of times in comparison with propagations where this laboratory stage does not
occur. As it was shown, upscaling and growth rate have a strong positive correlation (Table 2). Thus, it was
expected that with a better growth rate as a consequence of the upscaling process, yeast would have better
viability. However, the data points in the opposite direction.
In a microbrewery reality, where these propagations where conducted, yeast is often not inoculated in the ideal timing (as soon as it finishes to propagate). These situations are common due to fermenter
unavailability or simply because more urgent tasks take all the available time. It is also important to refer
that during this study the variable viability was always measured in the same day as the yeast was
inoculated, which means that in cases when the propagated yeast was not inoculated during its prime, the
viability probably diminished as a consequence of the waiting step, and it has been shown that viability has
a negative correlation with time. If the viability had been measured as soon as the propagation was
concluded, it would be possible that this correlation would be a strong positive one, as expected.
Yeast final biomass and final pH have an expected strong negative correlation (Table 2). During
propagation, yeast works to produce biomass and other metabolites and, consequently, pH decreases.
This decrease is particularly accentuated in the initial and exponential growth phases due to formation of
organic acids by deamination, the use of primary phosphate ions by yeast, as well as the uptake of
ammonium and potassium ions with release of hydrogen ions into the beer (Kunze, 2014).
Final pH seems to have a strong correlation with the percentage of viability (Table 2), which proposes that propagations that end with a higher final pH have a better percentage of viable cells.
Observing the initial data, throughout the propagations, the final pH oscillates between the values 4.2 and
38
3.8. These values are within the range of values (3.5-6.5) considered optimal for yeast propagation
(Narendranath, 2015). Considering the discussion of the previous correlation, where it is hypothesized that
the pH decreases as the biomass increases, one can interpret that a superior percentage of viable cells
would be associated with an inferior biomass, which by its turn is associated with shorter propagation time. As it was clarified before, the percentage of viable cells was measured before inoculation, whereas some
propagations where let on hold between the end of propagation stage and the inoculation step. Thus, it is
possible that this correlation only exists because propagations with shorter propagation time (and,
consequently, less biomass, leading to higher pH) and inoculated in their prime, have better viability.
Supposing that viability had been measured in the end of propagations, these two variables would
presumptively show no correlation.
Finally, there is a strong negative correlation between the fermentation outcome and the sulfur smell (Table 2), proposing that if the yeast ferments all the possible fermentable sugars, its product is less
expected to have a detectable quantity of the off-flavor, while yeast that does not complete the fermentation
is more prone to have a product with detectable sulfur compounds.
Sulfur-like smell can be present due to a range of sulfur volatile compounds, from which the most
common are sulfite, hydrogen sulfite, and dimethyl sulfide (Kunze, 2014). A variety of these compounds
are produced during fermentation, but others derive directly from raw materials, malt and hops (Ferreira &
Guido, 2018). To take any further conclusions, one must be able to first identity (e.g., using gas
chromatography) which of these compounds was responsible for the off-flavor, and then analyze the correspondent metabolic pathway, in order to postulate assumptions that would justify this correlation.
Without this identification, and excluding the compounds that derive from any source prior to the
fermentation stage to elaborate a train of thought, the most reasonable explanation for this sulfur-like smell
is the predominance of residual hydrogen sulfite as a result of a less-vigorous fermentation derived from
low quality yeast (Ferreira & Guido, 2018). As for unfinished fermentations, low quality yeast is also the
logical explanation, with this correlation being expected.
2.1.2. Distribution of the variables in the principal component space
The variables were distributed in each of the 12 principal components space. Since graphic representation is only possible up to 3 dimensions (Figure 10 and Figure 11), analysis will focus on the
principal component number one (PC1) which retained 24.67% of the original system variance, principal
component number two (PC2) which retained 20.03% of the original system variance, and principal
component number three (PC3) which retained 16.39% of the original system variance. Hence, the three
principal components retained a cumulative variance of 61.09%.
39
Figure 10: Distribution of the variables in the space of principal component 1 (PC1) and principal component 2 (PC2).
Figure 11: Distribution of the variables in the space of principal component 1 (PC1) and principal component 3 (PC3).
40
Observing PC1 (Figure 10 and Figure 11), it is noticeable that the viability percentage (%V) and
the final pH (pH_F) are in opposition with the propagation final time (Time_h) and the laboratory propagation
stage (LPS). Regarding PC2 (Figure 10), it is clear that the final biomass (logFB) is in opposition with the
growth rate (GR) and the upscale (UpS). The distribution of variables in PC3 (Figure 11) establishes a gradient of relative density (PLATO), initial pH (pH_I) and detectable sulphur smell (smell_S).
2.1.3. Projection of the propagations in the principal component space
2.1.3.1. Propagation Identification
Throughout the experimental phase of this thesis, different yeast strains belonging to the genus Saccharomyces and genus Brettanomyces were propagated, as summarized in Table 3.
Table 3: Identification of the propagated yeast strains
Yeast Code Identification
WLP001 “California Ale Yeast” - Saccharomyces cerevisase
WLP007 “Dry English Yeast” - Saccharomyces cerevisase
WLP029 “German Ale/ Kolsch Yeast” - Saccharomyces cerevisase
WLP066 “London Fog Ale” - Saccharomyces cerevisae
WLP644 Brettanomyces bruxellensis troix, recently renamed to
Saccharomyces cerevisae
WLP648 Brettanomyces bruxellensis trois vrai
WLP650 Brettanomyces bruxellensis
WLP670 “Farmhouse Blend” - a mixture of Saccharomyces cerevisae
and Brettanomyces spp.
WLP830 “German Lager” - Saccharomyces pastorianus
WLP835 “German X Lager/ Bavarian Lager” - Saccharomyces
pastorianus
WLP4638 Brettanomyces spp. TYB184
WY5526 Brettanomyces lambicus
41
The distribution of these several propagations in the space of the three first principal components
is represented in Figure 12 (PC1 x PC2) and Figure 13 (PC1 x PC3).
Figure 12: Distribution of the propagations in the PC1 vs. PC2 space. Each propagation is represented by a dot and polygons A, B, C, and D represent independent propagations of the same yeast strain. Clusters (1,2, and 3) represent propagations with the same
yeast genus.
Figure 13: Distribution of the propagations in the PC1 vs. PC3 space. Each propagation is represented by a dot and polygons A, B, C, and D represent independent propagations of the same yeast strain. Clusters (1,2, and 3) represent propagations with the same
yeast genus.
42
Polygons A, B, C, and D (Figure 12 and Figure 13) point to the conclusion that there is
heterogeneity between propagations with the same yeast strain, which highlights the importance of
understanding and controlling the variables in a propagation system.
Subsequently, 3 clusters were outlined in the new orthogonal space: Cluster 1: includes WL001, WL007, WL029, and WL066;
Cluster 2: includes WL830, and WL835;
Cluster 3: includes WLP644, WLP648, WLP650, WLP4638, and WY5526.
This clustering was simply based on yeast taxonomy, as Cluster 1 represents Saccharomyces
cerevisiae, Cluster 2 represents Saccharomyces pastorianus, and Cluster 3 represents Brettanomyces spp
(Figure 12 and Figure 13). Since WLP670 is a mixed propagation of Saccharomyces cerevisiae and
Brettanomyces spp., it can be included in both clusters 1 and 3 (Figure 12 and Figure 13).
The formation of clusters enhances a clearer view of the distribution of propagations of yeasts strains from the same genus or even the same species, as is the case of clusters 1 and 2.
In each cluster, the bulk of propagations was primarily scanned in order to select the propagations
who had a better fitness (e.a. higher final biomass, growth rate, and viability). In these, a detailed analysis
of the variables was driven to understand the reasons, create assumptions, and, lastly, reach conclusions.
Cluster 1 This cluster groups together the propagations of four Saccharomyces cerevisiae strains that are
used to ferment a wide variety of beer styles ranging from Indian Pale Ales to Imperial Stouts. Scanning
through the cluster, it is noticeable that one propagation in particular, WLP066, has the best final biomass
outcome. In a comparative analysis with other propagations in the cluster, it is quickly revealed that this
propagation consumed a higher amount of fermentable sugars and started with a higher initial pH. A higher amount of sugars consumed can have two explanations: the propagation was submitted to
one or more upscale steps, or the wort added to the propagation was the wort being brewed for a high
gravity beer and presented a high sugar content.
In this particular case, the propagation was not upscaled, leading to the assumption that a propagation
with high gravity wort has given a better yield of biomass. As it is known, high gravity wort is not advised
for propagations - in fact, the wort employed for propagations should not be over 12ºP (Stewart, 2017).
Even with low gravity wort, the high concentration of fermentable sugars severely limits the yeast aerobic growth (as a consequence of the Crabtree effect) to a point where the yield factor is only between 5 to 10%
(Stewart, 2017). Therefore, biomass yield of high gravity wort should usually not surpass the biomass yield
of a low gravity wort propagation.
Unable to find a theoretical explanation, it is time to question the practical side. The sampling for the
counting of the final biomass was consistently done from the sampling port adapted to the propagation
system. However, if vigorous agitation is not being supplied when the sample is taken, the majority of yeast
cells accumulates in the bottom of the propagation vessel, making the counting biased for more yeast cells.
43
In the Dois Corvos brewery, only one peristaltic pump is used for more tasks other than providing agitation
for the yeast propagation system, and although this type of situation is consistently avoided, it might have
happened as a lapse.
A higher initial pH is also pointed as a difference between this propagation and the remaining from the same cluster. As it was mentioned before, yeast grows best in an acidic pH of 4 (Salari, 2017) but the wort
should have an optimal pH between 5.2-5.6 for several reasons, such as good precipitation of protein-
polyphenol complexes, less increase in color, and a clean taste hop bitterness (Kunze, 2014). Since wort
was used for propagations, the initial pH never deviated much from these optimal values. Also, it is known
that medium pH does not have any significant effect on the specific growth rate of yeast at any particular
concentration of dissolved solids in the wort (Narendranath, 2004).
Cluster 2 The second cluster is formed by propagations of Saccharomyces pastorianus which are commonly
employed to fermentate lager beers.
An overview of the spatial distribution shows that WLP835-2 stands out from the bulk with a
superior growth rate. Apart from the other three propagations in the cluster, this propagation had three upscale steps and consumed more fermentable sugars.
In this case, it is knowledgeable that the higher consumption of fermentable sugars can be a
consequence of a superior propagation volume, as more wort was used and more fermentable sugars were
available. As it was discussed in the variable correlation section, the upscale steps consisted of adding wort
to the propagation system by phases, increasing the propagation volume up to ten times, instead of
combining a small volume inoculum with the total wort in one step. The results suggest that performing a
higher number of upscale steps, therefore submitting the yeast to lower concentrations of nutrients, can be
beneficial and can increase the biomass yield.
Cluster 3 The third and final cluster is the one with the largest set of cases and includes strains belonging to
Brettanomyces spp.. This genus is highly related to lambic and barrel aged beer, commonly being
addressed as wild yeast in the beer industry.
Among these propagations, WLP644-1 stands out with the highest growth rate. The existence of a
laboratory propagation stage and four upscale steps differentiate this propagation from the others in the
cluster. The results suggest once again that the repeated addition of nutrients and, therefore, the
maintenance of a lower nutrient concentration in the propagation system, is favorable to a higher growth
rate.
44
Final Remarks
Yeast management and in house propagations have a significant impact in the brewery as they
decrease the beer producing cost and increase the product quality. However, these processes require a
high level of sanitary practices and knowledge about the yeast metabolism. Assembling a propagation system and successfully propagating yeast in the Dois Corvos
production reality was one of the major goals of this work, and although the existing system satisfies the
brewery basic yeast needs, there is still work to be done in terms of optimization.
The conducted analysis of the monitored data proposes that the upscaling step might have a
positive influence on the propagations yield. Therefore, it will be considered in future propagations. The
study results also revealed that some variables (%viability, time_h, log_FB) were not always correctly
monitored, which influenced the results with the formation of false correlations. Thus, in the future, variable
monitoring will be extremely rigorous.
More than addressing possible optimizations, this analysis demonstrated that the variability of the
yeast strains employed and the oscillation of the several variables worked as an obstacle in the moment of
building conclusions. Each propagation was shaped towards the brewery yeast necessities and was built
with the daily available resources. Therefore, these oscillations were difficult to avoid.
In spite of the industrial environment at a microbrewery may not be the most suitable for studying
strategies to enhance the output of future propagations in comparison to a laboratory controlled setup, the application of principal component analysis, as an exploratory data analysis strategy, enabled us to get a
comparative overview of the several propagations, to unveil the major underlying issues and to identify
some factors that could be responsible for the higher or lower performance of propagations.
As for yeast management, it was implemented with great success in the Dois Corvos reality. The
methodology for assessing the viability in site was validated in the laboratory against the flow cytometry
assays. The methodologies of harvest, counting, and re-pitching are now a part of the production daily tasks
and the yeast has been used up to ten generations without a decrease in viability.
In the future, the methodology for storing harvested yeast should be addressed, seeking an
approach that will maintain the yeast viability through a higher number of storing days.
45
Part II: Quality Control Implementation
Several quality points were implemented in the Dois Corvos production daily routine, whereas other
existing points were further optimized. Figure 14 summarizes the quality control testing points of production
flow at Dois Corvos brewery.
Figure 14: Overview of the Dois Corvos production flow and each quality testing point
1. ATP testing
Tank cleaning or clean-in-place (CIP) procedures are conducted with a high frequency. CIP
standard operating procedures were written by the production manager and include all the important
information such as cleaning chemical types, temperatures, flow rate, water chemistry, and even spray ball
maintenance. However, there was an absence of a validation step (besides visual assessment), a critical
aspect of sanitation that should not be ignored.
46
After a study of the situation and discussion with the management, the decision was to purchase
the device SystemSURE Plus Luminometer from Hygiena (USA). This test utilizes the reaction of adenosine
triphosphate (ATP) with luciferin/luciferase to generate quantifiable bioluminescence, with results being
expressed in relative light units (RLU). The employment of adenosine triphosphate (ATP) swabs allowed for a rapid real-time assessment of microbial load in cleaning water and tank surfaces.
After the purchase, each fermentation vessel was visually inspected and hot spots (areas that do
not get well cleaned) identified. As a result, between three to five (depending on tank architecture) ATP
surface tests are performed after a CIP, and a sample of the rinse water after the cleaning was also tested.
The ATP testing parameter was added to the CIP log (Appendix c).
During the first week, the testing revealed constant positive results, leading to the conclusion that
CIP was not being effective. It was discovered that the cold water used in the final rinse had a high microbial
load. As a solution, the final water rinse is now performed with 80 ºC water. An introduction of an additional step of acid washing to dissolve mineral salts and deposits left by hard water (Lees, 2009), as well as an
increase in the percentage of the caustic cycle chemical (from 1 to 2%), was tested and managed to
decrease positive results.
With a CIP optimized and the step of validation implemented, the production gained a new level of
trust and the spectre of contamination sources was narrowed.
2. Fermentation monitoring
Specific gravity and pH started to be measured daily for each beer. Together with tank temperature, the data is recorded on a log (Appendix d) that is attached to the tank, making the information accessible
to the whole production team. Prior to beginning of the quality program, these two parameters were only
measured occasionally.
With these data, one was able to optimize towards a rapid, healthy fermentation, which can
contribute to the reduction of processing time. Through the analysis of the specific gravity profile, one can
estimate how much time fermentation took to start based on the accentuated decrease in the specific
gravity.
Following its inoculation in aerated wort, yeast begins to activate its metabolism in a period designed by lag phase, followed by a period of intense growth. The length of this phase and the growth rate
are positively influenced by the yeast nutritional state and by the temperature of the system (ideally 25 to
30 ºC to genus Saccharomyces) (Kunze, 2014). Considering that the temperature is always set to the value
advised by the yeast manufacturer for each strain, it was attempted to improve the fermentation
performance by raising the zinc concentration in the wort. The importance of this trace element goes further
than the structural and functional role in proteins and nucleic acids (Nicola, 2009), as it is used as a cofactor
in numerous enzymes, namely alcohol dehydrogenase for the terminal enzymatic step in ethanologenesis
(Zhao, 2011). Preceding this attempt, zinc was being supplied only as a constituent of yeast nutrient (a
47
blend of organic salts, vitamins, amino acids, and trace elements, along with an approved organic source
of nitrogen). To adjust the concentration and reach the estimated target of 0.25 mg per liter (Nicola, 2011),
the adding of zinc sulphate directly into the tank was implemented. It should be emphasized that although
the supplemented zinc quantity is well known, the total zinc concentration in the wort is not, as one is currently unable to estimate with precision the wort initial zinc concentration. That specific measurement
can only be given by atomic absorption spectrometry, a method that was not feasible in the course of this
thesis. Therefore, the value 0.25 mg/L can only be considered an estimate, and one can only affirm that
the zinc concentration is equal or superior to 0.25 mg/L. It was noticeable that fermentation was optimized as there was a decrease in the period of time between
the pitching and the fermentation start from 2-4 days (P. Custódio, personal communication, November
2017) to 0.5-1 day (Guerreiro, 2018). In the same period of time, yeast viability started to be monitored,
assuring the use of yeast with a viability superior to 90%. Therefore this optimization can be attributed to either of these variables or even a synergistic effect of both.
2.1. Determine the beer final specific gravity
Considering the wort original gravity and the attenuation percentage characteristic of the yeast being employed, one can estimate the beer final specific gravity. This information enables a faster transition
from the fermenting to the cold maturation stage, as one can program the temperature drop as soon as the
daily specific gravity value equals the known final specific gravity value, avoiding extra processing time.
2.2. Contamination awareness
Data from consistent measures of specific gravity and pH of beers that follow the same recipe and
are fermented with the same yeast strain are eligible to the tracing of fermentation profiles. Significant
differences between subsequent fermentations and the fermentation profile can be an indicator of
contamination. This approach is not further described, as it was explored by a fellow master student and
implemented in the Dois Corvos brewery.
3. pH monitoring during wort production
Additionally to measuring the pH during fermentation and maturation stages in the fermentation vessel, this measure was also revised in the wort production as several processes are dependent on the
pH value. As a result, pH is now measured in four stages during wort production: during the mash step, in
the vorlauf, and both before and after the boiling step.
48
This implementation of pH readings lead to pH adjustments (using calcium carbonate to raise the pH and
calcium sulfate to lower the pH) in order to achieve the production of wort with final optimum pH (between
5.2 and 5.6), which has the following benefits (Kunze, 2014):
1) Improves the enzyme activity during the mash, leading to a better conversion of starches to simple sugars;
2) Lowers the pH in the finished wort, which improves yeast health during fermentation and inhibits
bacterial growth;
3) Improves the extraction of hops bitterness in the boil;
4) Leads to a reduced chill haze and a superior clarity in the finished beer;
5) Enhances the stability of flavor and clarity during the beer aging process.
4. Microbiological testing optimization
Microbiological testing was already implemented prior to the beginning of this work. Samples from each tank were collected weekly and analyzed in the laboratory with the use of HLP, WLN, and LCSM
media. In this step, optimization focused in three points:
1) Increase the reliability of the results by formulating the medium LCSM in house.
Although this selective media is used for the detection of non-Saccharomyces yeast, results often
exhibited false positives characterized by the presence of Saccharomyces cerevisiae colonies in
the plates. The selectiveness resides in the concentration of cupric sulfate that inhibits yeast from
the genus Saccharomyces to grow (Lin, 1981). By formulating the media in house, it was possible
to adjust the concentration of cupric sulfate from 0.06% (Lin, 1981) to 0.09% to inhibit the growth of Saccharomyces yeast strains used in the brewery, and allow the growth of non-Saccharomyces
yeast strains in order to get reliable results.
2) Implementing microbial testing of the final bottled product.
It is known that contamination can occur downstream from the fermenting and brightening tanks,
namely during the packaging process. Hence, sample bottles from every batch of packaged beer
are now subject to microbiological analysis. This measure contributes by facilitating the tracing of
contamination source when it occurs and works as a validation to the bottling machine sanitation
process. 3) Implementing microbial testing on barrel aged beers.
As the Dois Corvos barrel aged beer program expanded and the beer started to be packaged and
exported, there was a necessity of monitoring the quality. A sample from each barrel is tested at
the end of the aging time, before packaging.
49
5. Wort stability test
This simple test of collecting a wort sample aseptically into a sterile bag following by a week
incubation at room temperature produces results with great importance.
In the case of a positive result, it indicates the existence of a contamination source upstream from
the sampling point, namely in the heat exchanger or in the brewhouse. As the wort is subject to a boiling
step, brewhouse contaminations are very rare. On the contrary, heat exchanger contaminations are quite common due to the complexity in its maintenance and sanitary process efficiency.
In the case of a negative result, it validates the heat exchanger sanitation step and facilitates the
tracing of contaminations by excluding the sources upstream from the testing point.
6. Product stability monitoring
The beer produced in Dois Corvos is considered a live product, as it is not pasteurized. Therefore,
it continues to change during the aging process. Monitoring these changes helps to understand what
upstream processes should be optimized to minimize the beer aging and maximize the shelf life. Therefore,
it was decided to store 12 samples from every batch of beer packaged in bottles. One, three and six months after bottling, these samples are the subject of sensory analysis by a member of the production team, a
member of the management, and the quality manager. The results of the tasting are discussed and a plan
is traced to address the problems found.
7. Complaint records
The last component providing support to a quality program is to record and monitor complaints. It
was actively instructed to the whole Dois Corvos team to forward every complaint to the quality department
in order to record the data. Weekly reviews of the summarized data help to get to the root of the problem
and address concerns. One can also postulate that this information validates the rest of the quality program, as the lack of complaints might be related to an increase in beer quality.
50
Final remarks
The expected outcome of a quality program is to produce high quality products in a consistent
manner. In a brewery, this translates into producing high quality beer every day. Beer quality can be defined
by several parameters, as presence or absence of microbiological contamination, shelf life determination, sensory quality, and customer expectations. The goal of a quality program is to set criteria that define high
quality beer in each parameter and create a system of policies, procedures, and specifications to meet the
criteria. Starting a program with so many ambitions can be overwhelming, time consuming and has
associated costs. Therefore, it is best to tackle one parameter at the time and work progressively towards
the whole.
In the Dois Corvos quality program, the first approach was towards the reduction of microbiological
contaminants. Microbiological testing provided information about the contamination of each batch, and was useful to determine whether the product should be commercialized or not. After implementing the testing
that defined the presence or absence of microbiological contamination, the next step was to gradually
implement tests that facilitate the tracing of the contamination source when one is present. For this purpose,
several quality checks were distributed along the production line, starting with the wort stability test and
validation of the tanks cleaning process, and finishing with the microbiological analysis of bottled beer and
complaints recording. Nowadays, when a contamination is detected, one can easily look at the bulk of
recorded data, make an assumption about the contamination source and focus on the resolution, instead of questioning the entire production line. This saves time and resources leading to an increase in the
efficiency of the production.
The fitness of the fermentation was the second parameter to be tackled as it downgrading the
sensory quality of the final product. The correction of the zinc concentration and the control of the yeast
quality by viability monitoring contributed to the reduction of detectable off-flavors during the fermentation
step.
Thus it is safe to affirm that both the consistent absence of contamination and fermentation-caused
off-flavors led to an increase in the overall product quality, but it should also be noted that there is still space for improvement, as the work of a quality manager is never finished. Currently, it is being studied an
approach to reduce the level of oxygen in packaged product as the current levels are excessively oxidizing
the beer and speeding the beer aging process. This is causing a decrease in the product sensory quality
during the shelf life, especially in hoppy beers.
In the near future, it is also being considered to acquire a centrifuge to reduce the solid residues in
the packaged beer and reduce the overall processing time.
51
To finish this chapter, it is important to emphasize that quality control is essential to the brewing
industry as it is for the food industry, and its importance should not be underestimated.
52
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Appendix a Graphic example of the viability analysis using flow cytometry from generation 0 to 4. Yeast cells were co-stained with SYTO9 (green fluorescence detected on FL1) and PI (red fluorescence detected on FL3). (A) Pseudocolor dotplots of side scatter (SSC) vs.
FL3; (B) Pseudocolor dotplots of FL3 vs. FL1
APPENDIX
A Graph
B Graph
Generation 0
Generation 1
Generation 2
Generation 3
Generation 4
58
Appendix b Graphic example of the viability analysis using flow cytometry form generation 0 to 4.. Yeast cells were co-stained with DIBAC4(3) ( green
fluorescence detected on FL1 and a red spectral shift detected on FL3). (A) Pseudocolor dotplots frontal scatter (FSC) vs. FL3; (B)Pseudocolor dotplots of FL3 vs. FL1.
A Graphic
B Graphic
Generation 0
Generation 1
Generation 2
Generation 3
Generation 4
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Appendix c: CIP log used daily at the Dois Corvos production site.
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Appendix d: Cellar log for fermentation monitoring used daily at the Dois Corvos production site