Department of Physics, Chemistry and Biology

Master’s Thesis

Investigation of the Impact on Yeast FermentationPerformance in Production of Pale Lager Beer

through Management Control

Zara SkogsbergLITH-IFM-A-EX--13/2839--SE

Department of Physics, Chemistry and BiologyLinköpings universitet

SE-581 83 Linköping, Sweden

Master’s ThesisLITH-IFM-A-EX--13/2839--SE

Investigation of the Impact on Yeast FermentationPerformance in Production of Pale Lager Beer

through Management Control

Zara Skogsberg

Supervisors: Håkan JohnssonSpendrups Bryggeri AB

Robert Gustavssonifm, Linköping University

Examiner: Carl-Fredrik Mandeniusifm, Linköping University

Linköping, 29 October, 2013

Avdelning, InstitutionDivision, Department

Division of BiotechnologyDepartment of Physics, Chemistry and BiologyLinköpings universitetSE-581 83 Linköping, Sweden

DatumDate

2013-10-29

SpråkLanguage

� Svenska/Swedish� Engelska/English

�

�

RapporttypReport category

� Licentiatavhandling� Examensarbete� C-uppsats� D-uppsats� Övrig rapport�

�

URL för elektronisk versionhttp://www.ifm.liu.se

http://www.ep.liu.se

ISBN—

ISRNLITH-IFM-A-EX--13/2839--SE

Serietitel och serienummerTitle of series, numbering

ISSN—

TitelTitle

Utredning av påverkan på jästfermentering genom hanteringsstyrning vid produk-tion av ljus lagerölInvestigation of the Impact on Yeast Fermentation Performance in Production ofPale Lager Beer through Management Control

FörfattareAuthor

Zara Skogsberg

SammanfattningAbstract

Through a full factorial design experiment, the effects of time between worts,wort aeration and yeast dosage in production of a pale lager beer were exam-ined in the beer process at Spendrups Bryggeri AB. The aim was to learn howdifferent parameters may affect the yeast fermentation performance during beerproduction. Response variables used were the concentrations of ethyl acetate andisoamyl acetate, free amino nitrogen (FAN) degradation and change in extract. Astatistical analysis showed that the concentration of ethyl acetate is dependent onyeast dosage and the interaction between time between worts and aeration whilethe isoamyl acetate concentration is dependent on yeast dosage and time betweenworts. No parameters are statistically significant for FAN degradation while thechange in extract is dependent on the yeast dosage. Due to botched runs, mostlybecause of aeration problems, it was not possible to verify theoretical parametervalues and responses. Since the aeration was not properly performed, the man-agement of the aeration control should be further investigated. Ester analysis andanalysis of FAN were performed as worts entered and exited horizontal fermenta-tion tanks. An additional analysis of ester content was also performed as the earlystage beer was transferred into lagering tanks. Cell viability as well as extract,pH and tank temperature was measured daily to verify the state of fermentation.Statistical calculations showed that when using NucleoCounter YC-100, there is nosignificant difference between analysis made of samples homogenized by a magneticstirrer and samples shaken by hand.

NyckelordKeywords Yeast Fermentation, Full Factorial Design, Wort Aeration, Yeast Dosage, Ester

Production, Yeast Viability

AbstractThrough a full factorial design experiment, the effects of time between worts,wort aeration and yeast dosage in production of a pale lager beer were exam-ined in the beer process at Spendrups Bryggeri AB. The aim was to learn howdifferent parameters may affect the yeast fermentation performance during beerproduction. Response variables used were the concentrations of ethyl acetate andisoamyl acetate, free amino nitrogen (FAN) degradation and change in extract. Astatistical analysis showed that the concentration of ethyl acetate is dependent onyeast dosage and the interaction between time between worts and aeration whilethe isoamyl acetate concentration is dependent on yeast dosage and time betweenworts. No parameters are statistically significant for FAN degradation while thechange in extract is dependent on the yeast dosage. Due to botched runs, mostlybecause of aeration problems, it was not possible to verify theoretical parametervalues and responses. Since the aeration was not properly performed, the man-agement of the aeration control should be further investigated. Ester analysis andanalysis of FAN were performed as worts entered and exited horizontal fermenta-tion tanks. An additional analysis of ester content was also performed as the earlystage beer was transferred into lagering tanks. Cell viability as well as extract,pH and tank temperature was measured daily to verify the state of fermentation.Statistical calculations showed that when using NucleoCounter YC-100, there is nosignificant difference between analysis made of samples homogenized by a magneticstirrer and samples shaken by hand.

v

SammanfattningGenom ett flerfaktorförsök undersöktes effekterna av tid mellan påfyllning av vört-er, beluftning av vörter och jästdosering vid produktion av ljus lageröl i ölprocessenpå Spendrups Bryggeri AB. Syftet var att öka förståelsen för hur olika paramet-rar kan påverka jästens fermenteringsförmåga under ölproduktionen. Responsva-riabler var koncentrationen av estrarna etylacetat och isoamylacetat, nedbrytningav fritt aminokväve (FAN) och extraktsförändring per dag. En statistisk analysvisade att etylacetat beror av jästdosering och interaktionen mellan tid mellanvörter och beluftning medan isoamylacetat beror av jästdosering och tid mellanvörter. Ingen parameter är statistiskt signifikant för nedbrytning av FAN medanextraktsförändringen beror av jästdosering. På grund av illa genomförda experi-ment, till största del orsakade av problem med att erhålla rätt beluftning, var detinte möjligt att kontrollera teoretiska parametervärden och responser. Eftersomkontrollen av beluftning inte var ordentlig, borde hanteringen av den undersökasytterligare. Esteranalyser och analys av FAN gjordes när vörter fördes över tilloch från horisontella fermenteringstankar. Ytterligare en esteranalys gjordes närdet fermenterade ölet fördes över till lagertankar. Cellviabilitet, extrakt, pH ochtanktemperaturer undersöktes dagligen för att kontrollera fermenteringsstadiet.Statistiska beräkningar visade att prov som homogeniserats genom magnetom-rörning inte gav någon större noggrannhet för viabilitetsanalysen med Nucleo-Counter YC-100 än prov som skakats för hand.

vii

Acknowledgments

I would like to express my deepest appreciations to Joachim Fleuchaus, brewmasterat Spendrups Bryggeri, whose ideas and management made this thesis possible.

I also appreciate the guidance given by my supervisors Håkan Johnsson andRobert Gustavsson, whose helpful advices and interesting discussions led me throughthe experimental periods.

Monika Öberg and the workers at Spendrups’ laboratory are given a big thankyou for lending me a workspace and giving me the permission to use the equipmentand material necessary for all the analyses performed.

A special gratitude I give to Therese Rice and Emilie Westberg who proofreadand commented the report more thoroughly than anyone else.

Last but not least, I thank Christoffer Norén with all my heart for the neverending help with the LATEXdocuments.

Zara SkogsbergGrängesberg, September 2013

ix

Contents

Notation xv

1 Introduction 11.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Aim of the Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Beer Production 32.1 Beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.1 The Beer Process . . . . . . . . . . . . . . . . . . . . . . . . 32.2 Tanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.2.1 Horizontal Tanks . . . . . . . . . . . . . . . . . . . . . . . . 42.2.2 Vertical Fermentation Tanks . . . . . . . . . . . . . . . . . 52.2.3 Lagering Tanks . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.3 Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.4 Ester Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.4.1 Ethyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . 62.4.2 Isoamyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . 7

2.5 Free Amino Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3 Yeast Management 93.1 Fermentation Preparations . . . . . . . . . . . . . . . . . . . . . . . 9

3.1.1 Wort Aeration . . . . . . . . . . . . . . . . . . . . . . . . . 93.1.2 Yeast Dosage . . . . . . . . . . . . . . . . . . . . . . . . . . 93.1.3 Yeast Propagation . . . . . . . . . . . . . . . . . . . . . . . 10

3.2 Transfer of Yeast and Beer . . . . . . . . . . . . . . . . . . . . . . 103.2.1 Transfer of Yeast to the Yeast Tank . . . . . . . . . . . . . 103.2.2 Transfer of Worts to Horizontal Tanks . . . . . . . . . . . . 113.2.3 Transfer of Beer to Vertical Fermentation Tanks . . . . . . 123.2.4 Transfer of Beer to Lagering Tanks . . . . . . . . . . . . . . 12

xi

xii Contents

4 Material and Methods 134.1 Yeast Viability Measurement . . . . . . . . . . . . . . . . . . . . . 13

4.1.1 Instrument and Software . . . . . . . . . . . . . . . . . . . . 134.1.2 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.1.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.2 Ester Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154.2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154.2.2 Instrument and Software . . . . . . . . . . . . . . . . . . . . 164.2.3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.3 Free Amino Nitrogen Analysis . . . . . . . . . . . . . . . . . . . . . 164.3.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.3.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174.3.3 Instrument and Calculations . . . . . . . . . . . . . . . . . 18

4.4 Additional Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5 Statistics 195.1 Design of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.1.1 Design Variables . . . . . . . . . . . . . . . . . . . . . . . . 195.1.2 Full Factorial Design . . . . . . . . . . . . . . . . . . . . . . 205.1.3 Response Variables . . . . . . . . . . . . . . . . . . . . . . . 205.1.4 Response Restrictions . . . . . . . . . . . . . . . . . . . . . 215.1.5 Calculation of Responses . . . . . . . . . . . . . . . . . . . 21

5.2 Verification of NucleoCounter R© YC-100TM . . . . . . . . . . . . . 215.3 Examination of Cell Viability . . . . . . . . . . . . . . . . . . . . . 225.4 Examination of Ester Production . . . . . . . . . . . . . . . . . . . 22

6 Results 236.1 Daily Analysis of Worts . . . . . . . . . . . . . . . . . . . . . . . . 236.2 Experimental Design Responses . . . . . . . . . . . . . . . . . . . . 24

6.2.1 Ethyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . 246.2.2 Isoamyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . 256.2.3 FAN Degradation . . . . . . . . . . . . . . . . . . . . . . . . 266.2.4 Change in Extract . . . . . . . . . . . . . . . . . . . . . . . 27

6.3 Verification of NucleoCounter R© YC-100TM . . . . . . . . . . . . . 286.3.1 Total and Dead Cell Count . . . . . . . . . . . . . . . . . . 286.3.2 Percentage of Dead Cells . . . . . . . . . . . . . . . . . . . 29

6.4 Analysis of Cell Viability . . . . . . . . . . . . . . . . . . . . . . . 306.5 Analysis of Ester Production . . . . . . . . . . . . . . . . . . . . . 32

7 Discussion 357.1 Setup of the Experimental Design . . . . . . . . . . . . . . . . . . . 357.2 Experimental Design Responses . . . . . . . . . . . . . . . . . . . . 36

7.2.1 Ethyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . 367.2.2 Isoamyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . 377.2.3 FAN Degradation . . . . . . . . . . . . . . . . . . . . . . . . 377.2.4 Change in Extract . . . . . . . . . . . . . . . . . . . . . . . 38

Contents xiii

7.3 Verification of NucleoCounter R© YC-100TM . . . . . . . . . . . . . 387.4 Analysis of Yeast Viability . . . . . . . . . . . . . . . . . . . . . . . 39

7.4.1 Viability in Beer Fermentation . . . . . . . . . . . . . . . . 397.4.2 Viability after Yeast Transfer . . . . . . . . . . . . . . . . . 39

7.5 Analysis of Ester Production . . . . . . . . . . . . . . . . . . . . . 407.5.1 Ethyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . 407.5.2 Isoamyl Acetate . . . . . . . . . . . . . . . . . . . . . . . . 41

7.6 Improvements and Implementations . . . . . . . . . . . . . . . . . 417.6.1 Improvement of Aeration . . . . . . . . . . . . . . . . . . . 417.6.2 Improvement of Yeast Viability . . . . . . . . . . . . . . . . 41

7.7 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

8 Conclusions 43

Bibliography 45

A Batch Analysis 47

B Tank Exchange 50

C Mathematics 51C.1 Weighted Means . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Notation

Acronyms

Acronym MeaningCCD Charged-coupled deviceCIP Cleaning in placeDNA Deoxyribonucleic acidEBC European Brewing ConventionEDTA Ethylenediaminetetraacetic acidFAN Free amino nitrogenGC FID Gas chromatography with a flame ionization detectorPI Propidium iodide

Chemicals

Molecular Formula MeaningH3PO4 Phosphoric acidNaCl Sodium chlorideNa2HPO4 Disodium hydrogen phosphateNaOH Sodium hydroxideKH2PO4 Monopotassium dihydrogen phosphateKIO3 Potassium iodate

xv

Chapter 1

Introduction

This chapter will present an introduction to this thesis, including background andobjectives. The method used for evaluating production parameters is describedbriefly.

1.1 BackgroundSpendrups Bryggeri AB is a Swedish brewery owned by Spendrups Invest ABand located in Grängesberg, in the south of the Swedish county Dalecarlia. Thebrewery brews its own beer under the names Spendrups Bryggeri, Norrlands Guldand Mariestads, as well as beer from different areas in the world [1].

The management of the type of yeast used in the production is carried out bythe employees, but how the yeast responds to different circumstances is unknown.Still, the knowledge of how the yeast performs is important since it affects beercompounds such as esters and free amino nitrogen (FAN) and these compoundswill in turn affect the finished products. This means that a high quality yeastperformance will result in high quality products.

1.2 Aim of the ThesisSpendrups Bryggeri has noticed a decrease in the amount of the esters ethyl acetateand isoamyl acetate as well as a decrease in viable yeast cells in the productionof pale lager beer. As the yeast is not performing as desired, the brewery isinterested to find out what is affecting the yeast performance in the beer process.Therefore, the aim of this study is to learn and obtain a better understanding ofhow different parameters may affect the yeast fermentation performance duringbeer production and thus determine the important variables. At Spendrups, themajority of production parameters remain constant, but there are certain variablesthat are possible to alter. The following control variables will be analyzed:

• time span between the transfer of the first and second wort to the samehorizontal tank

1

2 Introduction

• aeration of wort batches

• dosage of yeast to the first wort that enters the horizontal tank

1.3 ObjectivesThe objectives of the project are to:

• find parameters that are important for the yeast fermentation performancebased on the responses measured

• find possible improvements in the yeast management that may increase thefermentation performance

• find efficient implementations of the improvements in the beer productionprocess

1.4 LimitationsThis study is limited to the beer fermentation in horizontal and vertical fermenta-tion tanks, with special focus on the horizontal tanks. The statistical experimentswere to proceed until two good runs from each experiment were obtained, withinthe project range of 20 weeks’ time.

1.5 MethodThe main method that will be used to evaluate the different parameters describedin 1.2 is an experimental design. It will use the three parameters of interest ascontinuous design variables described with a lower and an upper level to investigatewhich of these are more important.

Cell viability analysis was performed daily until the yeast was transferred fromthe vertical fermentation tank used. FAN and ester analysis were performed asthe process in the horizontal tank started and ended. As the beer was transferredto lagering tanks a last ester analysis was conducted.

Chapter 2

Beer Production

In this chapter relevant information about beer production at Spendrups Bryggeriwill be found. Among other things, the process of the beer fermentation, theimportance of yeast and the production of esters will be described.

2.1 BeerThe knowledge of beer brewing can be traced back almost 5,000 years. In Europe,it was at the beginning a beloved drink brewed by women in the daily householdsbut it has changed to become an industry driven by large players [2].

2.1.1 The Beer ProcessThe first step of beer production is to soak barley in water and allow it to grow [3].The malt that is formed is transported to the brewery where it is crushed, mixedwith water and heated in a mash tun. This mix is then filtered in a lauter tunwhere spent grains are separated from the liquid. The liquid is then moved to awort kettle where the hops are added after intensive heating. The wort formed istransferred to a whirlpool where proteins are separated, and later it is cooled andtransported to horizontal tanks where the fermentation is started. Afterwards itis transferred to vertical tanks for further fermentation followed by the early stagebeer being moved to lagering tanks. When the beer has matured, it is filteredand stored in pressure tanks, waiting to be filled into bottles and cans. A visualprocess chart can be seen in figure 2.1

For this pale lager, Spendrups Bryggeri has the capacity of producing eight wortbatches per week for two weeks. The production is then limited by the access tofermentation tanks and this gives a production downtime during the third week.During the week where no worts are being produced, new yeast is propagated.Transfer of yeast between the tanks is visualized in Appendix B. In the processthere are four horizontal tanks that can contain two wort batches at a time. Onlythe first wort batch entering each horizontal tank is dosed with yeast, but both ofthem are aerated before being added.

3

4 Beer Production

This study focuses on the process ranging from transfer of wort into horizontaltanks to transfer of the yeast from the vertical fermentation tanks. A verification ofthe ester production is also performed on the contents of the storage tanks. Thesesteps are highlighted in figure 2.1. A study is also made of the yeast transferredto the yeast tank in order to see if there is any difference in yeast viability whereyeast is used for repitching.

Malt crusher

Vertical fermentation

tank

Malt silo

Lagering tank

Horizontal fermentation tank

Lauter tun

Wort kettle

Hops

Whirl-pool

Cooler

Filter

Pressure tank

Packagedbeer

Mash tun

Water

Figure 2.1. The beer process.

2.2 TanksThe tanks of the beer process is located in the basement of the brewery. Over theyears, there have been extensions built where new tanks have been implemented.This has led to different names of the locations, such as basement 20, 90, 100, 200,300, 400, 500 and 600. Basement 20, 90, 100, 200 and some parts of 300 are usedfor vertical fermentation tanks. The rest of basement 300, 400, 500 and 600 areused for lagering tanks.

2.2.1 Horizontal TanksThe horizontal tanks each have a volume of 126 104 L and the temperature ofthe tanks is set to 11 ◦C. Two of the tanks, 8 and 9, are located on the roof ofthe brewery, facilitating sample collection through a sample tap connected to tankpipelines. A prior cleaning in place (CIP) is performed before samples can becollected.

2.3 Yeast 5

Tanks 5 and 6 are located inside the brewery. It is a bit more difficult to collectsamples from these as they are situated above ground and a ladder is used to reachthe sample taps. As the tanks are horizontal it is allowed to collect the sampledirectly from the tap, without discarding any beer.

2.2.2 Vertical Fermentation TanksThe vertical fermentation tanks are of volumes between 120 000 and 130 000 L,depending on the basement used. The pale lager worts are often transferred tothe 20 or 90 basement, in an attempt to not mix the different yeast strains usedin the process. The temperatures are set to 12 ◦C. When collecting samples fromthe tanks, the yeast in the bottom of the tank must be removed. This is done byusing a pitcher, discarding about 6 L of yeast and beer before the sample can becollected.

2.2.3 Lagering TanksLagering tanks hold volumes of between 127 000 and 251 924 L. The temperatureof the tanks is set to approximately 1 ◦C. Prior to collecting samples, a smallamount of beer is discarded.

2.3 YeastMaintaining the quality and health of yeast used in beer productions has in recentyears become a significant issue in the brewing industry [4]. The brewing yeast,often Saccharomyces cerevisiae, is involved in many chemical and biological reac-tions during fermentation and it is known that the condition of the yeast affectsboth the fermentation efficiency and the quality of the final product.

During anaerobic conditions, the yeast mainly produces ethanol and carbondioxide but also influences the levels of other compounds such as esters, higheralcohols and diacetyl [2]. These are compounds which affect the flavor stability ofthe beer. Since flavor stability and high beer quality are important to maintain,many inventions have been made in order to improve and optimize the fermentationtechnology [5].

It is important to sort out the crucial factors that affect yeast fermentationperformance to be able to control the final production results. Several analyseshave been performed with different perspectives and strategies to see how the yeastfermentation performance is affected by certain parameters and how they affectthe final beer quality. For example, impact of top pressure and temperature onhigher alcohols and esters have been investigated [6], as well as the yeast’s abilityto decrease aged beer aroma [7].

Interesting approaches are for example optimization of aroma production usingthe initial yeast concentration as a parameter [8]. Some articles show the influ-ence of aeration on propagated yeast [9] and the flavor control in small-scale beerfermentations [10].

6 Beer Production

Due to, for instance, financial and time aspects, yeasts are often used duringseveral fermentation cycles, known as generations. Although, it is recommendedto propagate fresh yeast every 8-10 generations [11]. Yeast used for brewing thepale lager beer is allowed to be used for a maximum of three cycles of fermentation.Even so, the generation number may sometimes be higher since the yeast is notalways sufficient for the tanks used and due to human error.

As sugar, extract in the malt, is fermented into ethyl alcohol, yeast tends toflocculate and adhere in clumps [12]. These clumps either sediment to the bottomof the fermentation vessel or rise to the surface of the fluid. The yeast will thenbe ready to be transferred to a yeast tank. To be able to repitch yeast, it mustbe determined that growth is sufficient and this can be done through analysis ofviability and vitality. Viability is a term describing whether a cell is dead or alive,while vitality is a cell’s physiological state which describes the metabolic function[13]. The yeast must also be free from contaminants such as bacteria and dirt.

2.4 Ester Synthesis

Esters are compounds much appreciated for their flavoring capacities, which makesthem frequently used in fruit-flavored products, wines and dairy products [14].They are a product of the yeast metabolism and are dependent on the circum-stances affecting yeast. The formation is established by esterification of fattyacids by ethanol and to some extent by esterification of higher alcohols [2]. Prin-cipally, ester concentration increases during the main phase of the fermentationbut has the capacity of doubling during a long secondary fermentation.

Giving important flavors, the esters become the most important aroma com-pounds in the beer. Though, too much esters may give the beer an unpleasanttaste and therefore the desirable amount becomes a consideration. It is known thatesters are affected and increased by for example a restricted wort aeration, a lowerfermentation temperature and worts containing a concentration above 13 ◦P. Onthe other hand, they are decreased by increased wort aeration, higher fermentationtemperatures and lower wort concentration. Acetate esters, such as ethyl acetateand isoamyl acetate which are described below, are acknowledged as importantflavor compounds [14].

2.4.1 Ethyl Acetate

Ethyl acetate is an ester that can be synthesized through esterification of ethylalcohol and acetic acid [15]. The colorless liquid has a fruity odor similar to pearflavor. Apart from being used in the food industry, ethyl acetate is excellent touse in the manufacture of cleaning fluids and nail-polish removers. It is also usedin the pharmacy industry, where it is an important component in extractants forconcentration and purification of antibiotics.

2.5 Free Amino Nitrogen 7

O

OSkeletal formula for ethyl acetate.

2.4.2 Isoamyl AcetateIsoamyl acetate is an ester with a characteristic banana flavor, naturally occurringin bananas, apples and other fruits [16]. It is widely used and of such importancethat a synthesized production of approximately 74 tonnes occurs each year [14].It may be synthesized through esterification of isoamyl alcohol and acetic acid.

O

O

Skeletal formula for isoamyl acetate.

2.5 Free Amino NitrogenAs the yeast consumes between 100 and 140 mg α-amino nitrogen per liter wortduring beer production, there must be a sufficient amount of α-amino acids forthe yeast to utilize. A start concentration of at least 200 mg FAN/L wort hasto be provided or it will affect the production [2]. Lack of FAN may for instanceresult in a reduced cell growth, a retarded fermentation and maturation and thepresence of undesirable immature beer flavor substances. A sufficient amount willalso prevent the yeast from forming a high amount of higher alcohols.

Chapter 3

Yeast Management

This chapter contains information about the yeast management at SpendrupsBryggeri. All the preparations that affect the project as well as the transfersperformed are scrutinized.

3.1 Fermentation PreparationsTo begin beer fermentation, there are several things that need to be considered.Described below are the parameters that affect this thesis.

3.1.1 Wort AerationIt is commonly known that the yeast needs oxygen to multiply and much yeastis needed for the fermentation [2]. A sufficient amount of air must be added tothe wort to stimulate the yeast into multiplying while it also should initiate theanaerobic fermentation stadium. This means that an intense aeration is importantfor the yeast but the amount supplied must be utilized within a few hours for thefermentation to start. For this pale lager beer production, an aeration between25 and 30 mg O2/L is desired. As the wort has been aerated, it is transferred tothe horizontal tank within the decided time limit, which is between 6 and 12 hours.

3.1.2 Yeast DosageThe amount of yeast used during wort fermentation affects the quality of thefinished product. For the investigated beer, the approved amount is between200 and 250 g/hL and to determine the correct dosage it is important to knowthe concentration of the repitching yeast. Whenever a yeast transfer to the yeasttank has been achieved, a sample is taken for analysis of the concentration witha LAB Yeast Analyzer Model 810LC. The yeast counter is capable of measuringsamples with a concentration of up to 30 %, which means that if the sample is toothick it needs to be diluted with wort. When the yeast concentration is known theyeast dosage may be calculated with the formula in (3.1).

9

10 Yeast Management

Y = bc

1000100a

(3.1)

where

Y – kg yeast/2 wortsa – yeast concentration [%]b – amount of yeast per hL [g/hL]c – hL per fermentation tank [hL/tank]

3.1.3 Yeast PropagationEvery third week, when no new worts are being produced, new yeast is propagated.The yeast propagation is initiated at the end of the third week through transferringwort to a rehydrator and heating it to 15 ◦C. 1 kg yeast is added and the mixtureis then homogenized for four hours. Afterwards it is transferred to a small aeratedpropagation tank where it stays for three days.

At the beginning of the first week, the yeast suspension is transferred to thebig propagation tank no 7, at the same time as 40 hL wort at the temperature of11 ◦C arrives from the brewhouse. The mixture is allowed to ferment for a coupleof days before another 140 hL of wort is added. The yeast is then ready to be usedfor wort fermentation in one of the horizontal tanks.

The rehydrator is carefully cleaned with water followed by CIP with a lyesolution, NaOH, both before and after use. Both of the propagation tanks undergoCIP each time they have been emptied after use.

3.2 Transfer of Yeast and BeerAt Spendrups there are pipeline systems that are routed throughout the buildingfor the transfer of beer at different stages. By connecting the pipes between differ-ent locations, it is possible to transfer wort and beer between all the tanks used,from the brewhouse to the fermentation tanks and further towards lagering andpressure tanks.

Where nitrile rubber hoses are handled, these are always cleaned with waterbefore use in any transfer. Once a week the hoses undergo a CIP operation asthey get connected to the pipeline system in the building. Metal pieces used toconnect hoses, known as mountings, are put in a disinfectant solution whenevernot in use.

3.2.1 Transfer of Yeast to the Yeast TankFlocculated yeast is transferred from any vertical fermentation tank into the yeasttank by attaching nitrile rubber hoses between the fermentation tank, a pump andpipes near the yeast tank. To connect the pipeline to the yeast tank, mountingsare used.

3.2 Transfer of Yeast and Beer 11

When opening the fermentation tank and starting the pump, water from thecleaned hoses is pumped into the pipeline and allowed to flush out on the floorthrough an opening. As the yeast starts to pump out, the opening is closed,the tank is opened and the yeast is allowed to enter the tank. When all of theflocculated yeast has entered, the tanks are closed and the pump turned off.

The yeast tank is cleaned both before and after use. When the tank has beenemptied, it is first cleaned with water for ten minutes and then filled with about100 L of water. A container of a disinfectant solution is added to wash the tankfor approximately one hour. After the cleaning, the solution is discarded into thedrain and the tank is rinsed with water for 15 minutes.

Cleaning of the yeast pump is instigated by pumping one disinfectant solutionfor ten minutes and another for five minutes. The pump is always filled withdisinfectant solution when not in use.

3.2.2 Transfer of Worts to Horizontal Tanks

There are two wort pipelines that run from the brewhouse to the beer processand are used to transfer the worts produced in the brewhouse to the horizontaland vertical fermentation tanks. When a horizontal tank is to be used, it is firstcleaned with both water and a CIP solution.

Before transferring the wort, the selected wort pipe is connected to the tanksthrough a set of mountings. Since the horizontal tanks 5 and 6 are located insidethe building, the mountings are connected from the pipelines directly to the tanks.As tanks 8 and 9 are placed on the roof, the mountings create a passage betweenthe brewhouse pipeline and pipes connected to the tanks. This second solution ispreferred because it makes it able to leave the mountings constantly attached tothe tanks. They will be cleaned together with the pipelines during process. Beforeuse, the mountings are cleaned and sterilized using a disinfectant solution to keepthe wort from being contaminated by earlier transferred worts.

When the wort leaves the brewhouse a loud noise sounds in the basement. Thenthe crew allows the wort to enter the tanks through pushing a button that signalsfor the wort to enter the pipeline. It takes approximately one hour to transfer onewort batch to a horizontal tank, the process is often completed within 55 minutes.As the wort arrives, a back pressure is set to 1.8 bar in the pipeline. Though, dueto the pressure being able to push the wort into the yeast tank, ruining the yeastand the tank, this is not implemented until the yeast has been dosed.

After the wort batch has left the brewhouse, the pipelines are filled with water.This is noticed through a color change in a liquid indicator connected to the pipe.When water is detected, the tank will be closed and the pipes will be cleaned fromfor example spent hops as the water continues to the drain.

Whenever a wort batch has passed through one of the pipelines it is cleanedwith cold water followed by hot water. After a number of wort transfers, generallytwo, it also undergoes a CIP with a lye solution.

12 Yeast Management

3.2.3 Transfer of Beer to Vertical Fermentation TanksAs the extract of the beer in the horizontal tanks has decreased to 5.0-5.5 %, thebeer is ready to be transferred to vertical fermentation tanks. The pipeline usedfor pumping the wort will be opened all the way to the selected tank that hasbeen cleaned with water for ten minutes. Necessary valves are opened and thecounter is reset as the water in pipes and hoses are flushed out at the opening ofthe vertical tank. The beer is allowed to enter the tank and the tap at the verticaltank opening is closed. When the pump is switched on, the horizontal tank is leftopened to prevent the production of vacuum from imploding the walls of the tank.

The beer is transferred with a velocity of 500 hL/h. With a volume close to1000 hL this means that the whole tank is emptied in about two hours. To emptythe pipeline and transfer the last of the wort to the tank, water is run through thepipe. Thereafter, the tank is connected to the CIP pipeline and before the nextuse the tank is both cleaned with water and a CIP solution. The transfer pipelineundergoes the CIP operation at the end of the production week.

3.2.4 Transfer of Beer to Lagering TanksThe pale lager beer fermentation progresses during 15 days and the stage of thefermentation is verified by the concentration of diacetyl in the beer. When theconcentration has decreased below the threshold of 100 µl, the beer is ready to betransferred to a lagering tank.

Primarily, yeast that has flocculated and will be discarded is pumped out of thevertical fermentation tank. A hose that has been cleaned with water is connectedfrom the tank to a pipeline that leads to basement 100 where several other hosesare used to connect the pipeline further to a pump, a separator, a wort coolerand finally to mountings on the selected lagering tank. As the lagering tanks arelocated in basement 300, 400, 500 and 600, hoses need to be connected betweenthe different pipeline systems to transfer the beer. A counter, noting the beeramount, is reset before the transfer starts.

The separator is used to centrifuge the beer and separate it from all of theremaining yeast. It is possible to verify the arrival of the beer to the separatoras a turbidity indicator will increase from 0.30 to about 70 units. As the beercontinues to the lagering tank, the cooler will hold a temperature of −1 ◦C. Rinsewater in the pipes is flushed out at the opening of the lager tank and when thebeer arrives, the tank is closed. The temperature of the tank is verified to beabout 1-1.5 ◦C and the tank takes about 3 to 3.5 hours to fill. The pressure of thetank must be decreased to prevent the tank from imploding.

Afterwards, the pipelines, hoses and pump are cleaned with water. The sepa-rator and the cooler undergo a CIP operation every sixth use.

Chapter 4

Material and Methods

This chapter contains information about the different methods used during thestudy. The material and procedures used are also described.

4.1 Yeast Viability MeasurementSince yeast is crucial in the terms of fermentation, it is important to determine theamount of cells that are present in a suspension and the percentage that is viable.This is of importance for both quantities that are transferred to yeast tanks tobe dosed for wort fermentation and wort suspensions that are already undergoingfermentation. Through observing trends of viable cells in time, a good indicationof the yeast quality and performance in fermentation will be given.

In this case the method of yeast counting is based on the cell’s ability to uptakeand be stained by the fluorescent dye propidium iodide (PI). Dead cells have aplasma membrane that is permeable to PI while viable cells will not let the chemicalthrough. Dead cells are counted through dilution of a beer or yeast sample witha 0.9 % solution of NaCl and EDTA, while the total amount is detected throughmixing the diluted sample with a reagent that makes PI staining of the DNA inall cells possible.

A visual verification of the dead cells is performed through microscopy andstaining with methylene blue. The principle of the method is that dead cells willbe stained blue while living cells will degrade the color and remain uncolored.

4.1.1 Instrument and SoftwareYeast cell counting is performed with the use of a ChemoMetec NucleoCounter R©YC-100TM. It is a device that is simple to operate and can be used to count yeastfrom both the pharmaceutical, biotechnology, beer and food industries [17]. Theprinciple of the device is an integrated fluorescence microscope that detects signalsfrom PI bound to DNA. Depending on the sample preparation, the NucleoCountermay display either the total or nonviable cell concentration.

13

14 Material and Methods

The sample is applied to the instrument utilizing the disposable NucleoCassetteTM.This is a sample cartridge containing PI and when it has been loaded with celllyzate, the PI is dissolved and stains the cellular DNA. When placed in the Nu-cleoCounter, 1 µl of the DNA-bound PI solution automatically enters the meas-urement chamber of the cassette and gets excited by green light. A CCD camera forcorrelation then registers emitted red light and converts the result into a cell countof cells/mL. The device has a measurement range of 5 x 103 to 6 x 106 cells/mL,but the optimal range is between 1 x 105 and 2 x 106 cells/mL after dilution.

With the use of the optional software NucleoView, the results may immediatelybe documented, presented and calculated in percentages, with the dilution factortaken into account.

4.1.2 ChemicalsNucleoCounter

For the dead cell count, a 0.9 % NaCl solution is prepared through mixing 9 gNaCl and 9.31 g EDTA in 800 mL of purified water in a 1000 mL volumetric flask.The solution is shaken and the flask is made up to the mark with purified water.This is transferred to a 1000 mL bottle and autoclaved for 20 minutes at 121 ◦C.The EDTA is added to prevent the cells from aggregating in the samples.

The Y100 Reagent is a solution purchased from the company ChemoMetecthat disrupts the plasma membranes and makes the cell nucleus susceptible to PIstaining. Colored solutions like beer or wine may affect the fluorescence intensityin the measurement and therefore a prior dilution of the sample may be necessarybefore the use of Y100 reagent. A 1:10 ratio is recommended by the company touse for volumes of samples and reagent.

Verification by Microscope

A simple solution is mixed, through solving methylene blue in purified water, andlater used for staining. Another solution A, consisting of 0.02 % (m/V) of methy-lene blue in purified water, is prepared through dissolving 100 mg of methyleneblue in 500 mL of the water. Then a solution B and C is prepared from dissolving13.6 g of KH2PO4 and 14.2 g of Na2HPO4 respectively in 500 mL of purified water,both giving a solution of 0.2 M. A solution D is made through mixing 498.75 mLof solution B with 1.25 mL of solution C and finally, an additional solution E iscompleted for staining through a mix of solution A and solution D. Solution Eshould have a pH of about 4.6, which can be adjusted using 0.2 M KH2PO4 orNa2HPO4.

4.1.3 ProcedureNucleoCounter

Samples of about 250 mL are collected from either horizontal or vertical fermen-tation tanks or yeast tanks and measured within an hour. To get a somewhat

4.2 Ester Analysis 15

homogenized sample, the sample bottle is shaken by hand. If the samples arewort undergoing fermentation, an optimal dilution for the dead cell count is often1:5. Therefore 100 µL of the sample is diluted with 400 µL of the NaCl solution.The mixture is homogenized for about 30 seconds using the vibrations of a Hach-Lange TOC-X5. When the sample has been analyzed by the NucleoCounter andNucleoView shows no clumps in the sample, 100 µL from the mixed sample istransferred to 900 µL of the Y100 Reagent. The new sample is mixed using theTOC-X5 and a total cell count is performed using the NucleoCounter.

Start samples taken from the yeast tank often contain a larger amount of yeastthan the wort samples and therefore a stronger dilution is needed. At first adilution of the sample is made through weighing 0.5 g of the yeast and dilutingit with 49.5 g of NaCl solution, which results in a dilution of 1:100. Dependingon the amount of yeast the sample may afterwards be diluted between five andten times more to end up in the range between 1 x 105 and 2 x 106 cells/mL.When an approved analysis has been made, 100 µL of the homogenized sample istransferred to 900 µL of Y100 Reagent just as for the wort samples.

Verification by Microscope

An experiment is conducted through diluting centrifuged yeast to approximately1:100. 2 mL yeast suspension is mixed with 2 mL methylene blue and optionally2 mL of 10 % acetic acid to keep the yeast from aggregating. A counting chamber,Thoma Hellige 0.100 mm 1/400 q mm, is filled with the suspension and coveredwith a cover slip. The number of dead cells stained blue is then counted on4 of 16 squares.

Another experiment is performed through diluting yeast with an equal volumeof the prepared solution E in a test tube. A suspension containing about 100 yeastcells in a microscopic field is desired. After mixing, a small drop is placed ontoa microscopic slide, covered with a cover slip and examined within five to tenminutes. If the contact time is too short or too long, it will result in a too low or atoo high percentage of dead cells. The examination is proceeded through counting500 cells and noting the number of cells stained blue.

4.2 Ester AnalysisSpendrups’ analysis of volatile compounds is able to detect compounds such asesters, acetaldehyde, dimetyl sulphate and higher alcohols. To detect the esters,a reference beer is used to calibrate each analysis. An internal standard is usedin all prepared samples to give a ratio between the amount of compounds in thereference beer and all of the other samples.

4.2.1 ChemicalsThe internal standard is mixed in a 250 mL volumetric flask through addition of12.5 mL ethanol, 3 mL 1-butanol and 0.3 mL 4-heptanone to 200 mL of purifiedwater. The flask is then made up to the mark with purified water. The 1-butanol

16 Material and Methods

is used for detection of the higher alcohols while the 4-heptanone is used to detectall other compounds the method may analyze. The solution has a durability offour weeks if stored in the refrigerator.

4.2.2 Instrument and SoftwareThe method is conducted through the use of a gas chromatograph with a flameionization detector (GC FID). Spendrups Bryggeri uses a Perkin Elmer AutoSys-tem XL Gas Chromatograph with a Headspace Sampler HS40XL. The analysissoftware in use is TotalChrom Navigator.

4.2.3 ProcedureWhere samples are taken from fermentation or storage tanks, some beer is flushedout to clean the sample pipe from yeast that has flocculated respectively to collectcold beer from the tank. The samples are collected in 500 mL bottles, which afterbeing filled to the brim are closed with a lid. If the beer foams excessively, a spiralconnected to a hose can be attached to the sample tap to help lose the foam andfill the bottle with beer.

The sample is then transferred to a 33 cL glass bottle. The volume is decidedby a reference beer that will be used to calibrate the gas chromatograph. Thebottle is filled to approximately the same volume as that for the reference beer.When analyzing packaged beer, 33 cL glass bottles are preferred and therefore cansand 50 cL bottles are also transferred to 33 cL bottles. To each of the samples, aswell as to the reference beer, 2 mL of the mixed internal standard is added. Thebeer bottles are then capped, shaken and put in the freezer for about 30 minutes.

When the internal standard has reacted, 5 mL samples are measured in trip-licates in gas chromatograph vials. The reference beer will be represented in fivevials; two samples at the beginning followed by one replacement calibration sampleand one average calibration sample and at the end of the analysis, one last samplewill be analyzed.

As the oven of the gas chromatograph is heated to 250 ◦C, hydrogen gas isswitched on and the flame of the detector is lit. Each vial is analyzed for about15 minutes.

4.3 Free Amino Nitrogen AnalysisTo determine the content of FAN of wort and packaged beer, a method of colorime-try with ninhydrin is used. It is applicable to all worts, but a color correction needsto be performed if dark worts, EBC > 100, are to be analyzed. The method isknown to measure amino acids, ammonia and to some degree end group α-aminonitrogen in peptides and proteins.

4.3.1 ChemicalsThe chemicals below are all prepared before the analysis is carried out.

4.3 Free Amino Nitrogen Analysis 17

Glycine Stock Solution

A glycine stock solution is prepared in a 100 mL volumetric flask through dissolving0.107 g of glycine in purified water. The flask is then filled to the mark with purifiedwater. If stored cool, 0 to 4 ◦C, the solution has a durability of two months.

Ninhydrin Color Reagent

Ninhydrin color reagent is prepared through mixing 10 g Na2HPO4, 6 g KH2PO4,0.5 g ninhydrin and 0.3 g D-fructose in a 100 mL volumetric flask and dissolving itin purified water. Since the chemicals are poorly soluble, the flask may be heatedcarefully. When the chemicals are fully dissolved, the flask is filled to the markwith purified water. Thereafter the pH of the solution is adjusted to in between6.6 and 6.8 using 25 % NaOH or H3PO4. The color reagent has a durability oftwo weeks if stored cool, 0 to 4 ◦C.

Diluting Solution

Before analysis, the samples are diluted. A diluting solution is prepared throughsolving 0.5 g KIO3 in 155 mL of purified water and adding 95 mL of 99.6 % ethanol.The solution has a durability of one week if stored in room temperature.

4.3.2 ProcedureWort samples are prepared through centrifugation of approximately 15 mL ofeach wort at 1400 rpm for ten minutes. 1 mL is then diluted in purified waterin a 100 mL volumetric flask whereas the flask is filled to the mark with water.Samples of packaged beer are prepared through shaking approximately 100 mLof beer in a 500 mL flask, releasing the carbon dioxide from the fluid. When thefoam has returned to the beer, 1 mL is transferred to a 50 mL volumetric flask anddiluted in purified water. In the meantime, 1 mL of the glycine standard stocksolution is diluted to 100 mL with purified water in a 100 mL volumetric flask toobtain a solution containing 2 mg amino nitrogen per liter. Since it is important toprotect the samples from amino acids from skin and saliva, precautions are takenand gloves are used throughout the whole procedure.

2 mL of the diluted samples are transferred to 16 x 150 mm test tubes and toensure the measuring accuracy, three test tubes of each sample are prepared. Ablank sample triplicate is also made using 2 mL of purified water in each tube.1 mL of color reagent is added to every tube before it is covered with a ceramicmarble.

All tubes are heated in boiling water for exactly 16 minutes and then cooled in20 ◦C water bath for 20 minutes. After pipetting 5 mL of diluting solution to thetubes, the contents are mixed thoroughly through shaking and within 30 minutesthe absorbance of the samples is measured against purified water. SpendrupsBryggeri uses a DR5000TM UV-Vis Spectrophotometer.

The standard and blank samples must be included in every analysis due toany need of compensation for temperature variations in the boiling water. A

18 Material and Methods

verification of the analysis is made through analyzing samples of the same referencebeer as is used in the ester analysis.

4.3.3 Instrument and CalculationsAbsorbance values are investigated through the use of a spectrophotometer at thewavelength of 570 nm, with utilization of 10 mm cuvettes. The average absorbancevalue from the three test tubes is used and FAN is calculated using the formulain (4.1).

FAN (mg/L) = 2d(At−Ab−Ac)As−Ab

(4.1)

where

2 – concentration of glycine standard solution (in mg/L)d – dilution factor (e.g. 100 if dilution was 1 mL to 100 mL)At – average absorbance of diluted wort sampleAb – average absorbance of blanksAc – average absorbance for correction of dark colored samples (for light

colored samples Ac=0)As – average absorbance of glycine standard

The result is expressed in mg/L round to nearest integer.

4.4 Additional AnalysesAdditional analyses are performed in order to verify the state of the fermenta-tion. The tank and pH checks are mostly performed to make sure the process isproceeding as desired.

Wort samples that are collected for the yeast cell count analysis is also used formeasuring extract and pH of the wort. The extract is measured using a DMA 35densitometer from Anton Paar, showing results in degrees Plato ( ◦P). DegreesPlato is a measurement of grams of sugar per 100 grams of wort and is equivalentto % w/w. This means 1 ◦P is equal to 1 % sugar.

To measure the pH of the samples, a Metrohm 691 pH Meter is used. It iscalibrated once a week with the use of buffers of pH 7 and pH 4.

The temperature of the tanks is measured with the help of digital thermometersconnected to the tanks. Only propagation tank no 7 is missing a thermometer andtherefore its temperature is measured using a portable digital thermometer.

Chapter 5

Statistics

This chapter will describe the statistical arrangement of the study. The experi-mental design that will be used for evaluation of the variable process parameterswill be presented together with the response variables obtained.

5.1 Design of ExperimentsAs Spendrups Bryggeri is interested in knowing how yeast fermentation perform-ance is affected by different parameters, it is a good opportunity to verify theallowed ranges of each of these parameters. Since both time between worts, aera-tion and yeast dosage is allowed to be altered more or less, there are quite a fewexperiments that can be setup to obtain the desirable information.

The most efficient way of investigating all possible combinations of factors insuch an experiment is through a factorial design [18]. When having one factorwith s levels and one factor with t levels, the st combinations of treatment willbe investigated in every complete trial and replicate performed. This means thatthe main effects, the primary factors of interest that is, will be examined as wellas interactions between different effects. A factorial design is more efficient thanan experiment where one factor at the time is varied, as the effect of factors ofinterest may be examined at several levels of the other factors. Overall, this maylead to conclusions valid over a range of experimental conditions while misleadingconclusions tend to be avoided.

The experimental design was analyzed through the use of Minitab 16 StatisticalSoftware. For all of the experiments, the assumption was made that there was nodifference between the horizontal tanks.

5.1.1 Design VariablesWhen setting up a full factorial design with three factors, the possibility of varyingthe parameters between at least two levels is desired. Since the three parametersthat may be varied during pale lager beer production have a range of approved

19

20 Statistics

values, the values in the extreme positions are chosen as lower and upper levels.These values can be seen in table 5.1

Design VariablesFactors Definition Lower level (-1) Upper level (1)A Time between worts 6 h 12 hB Aeration 25 mg O2/L 30 mg O2/LC Yeast dosage 200 g/hL 250 g/hL

Table 5.1. The design variables used.

5.1.2 Full Factorial DesignEvery time the yeast is used in a new fermentation cycle it advances one genera-tion. Regardless, yeast used in the same brewery under the same conditions willnot differ much from time to time if the number of generations is limited [19].This makes it possible to compare for example yeast of generation one to laterpropagated yeast in the same situation as well as yeast of other generations. Asthere was not enough time to set up a full factorial design experiment for eachof the different yeast generations, a single one is performed including all of theoccurring generations.

By varying the parameters in table 5.1 between the lower and upper level atdifferent times, the full factorial design and full factorial design with interactioncolumns in table 5.2 are constructed.

Factors Factors and interactionsExp. A B C Exp. A B AB C AC BC ABC

1 -1 -1 -1 1 -1 -1 1 -1 1 1 -12 1 -1 -1 2 1 -1 -1 -1 -1 1 13 -1 1 -1 3 -1 1 -1 -1 1 -1 14 1 1 -1 4 1 1 1 -1 -1 -1 -15 -1 -1 1 5 -1 -1 1 1 -1 -1 16 1 -1 1 6 1 -1 -1 1 1 -1 -17 -1 1 1 7 -1 1 -1 1 -1 1 -18 1 1 1 8 1 1 1 1 1 1 1

Table 5.2. Full factorial design 23 (left) and Full factorial design 23 with interactioncolumns (right).

5.1.3 Response VariablesThe responses that will be measured to describe the outcome of the experimentalruns are:

• concentration of the esters ethyl acetate and isoamyl acetate

• degradation of FAN

5.2 Verification of NucleoCounter R© YC-100TM 21

• change in extract per day

5.1.4 Response RestrictionsWhen producing the pale lager beer, there are some restrictions that need to betaken into account. In the lagering tanks, the ethyl acetate is desired to havereached a concentration of between 24.0 and 33.0 mg/L and the concentration forisoamyl acetate is expected to be between 3.80 and 6.70 mg/L. According to therequirements specification, the optimal value is 31.0 and 4.50 mg/L respectively.For free amino nitrogen, there are no regulations other than that the packagedbeer should contain less than 80 mg FAN/L. The extract is desired to change witha value of 1.6 % ± 0.2 % per day, which gives an indication that the yeast isutilizing the sugar in the wort as desired.

5.1.5 Calculation of ResponsesTo be able to use the different response values in the analysis of the experimentaldesign, some adjustments were performed. The results of the esters were possibleto use unaltered, since assumptions were made that there were no esters presentfrom the beginning. Therefore, the results from the ester analysis of lagering tankswere used as response of ethyl acetate and isoamyl acetate.

For the free amino nitrogen response a calculation was needed. Since therewas a start value of free amino nitrogen per wort, these had to be calculated intoa mean for the whole batch. Because the worts had some differences in volume,this was made through weighted means, see appendix C. Then the value from thesecond FAN analysis was subtracted from the start value and this difference wasused as the response of the FAN degradation.

The start values of extract for each wort was also used to calculate a weightedmean and then a verification of how much the extract changed during the timein the horizontal tanks was made. This was done through using the start valueas minuend and a value between 5.5 % and 5.0 % as subtrahend and dividing thedifference by the number of days it took for the decrease. If the extract had notdecreased below 5.5 % at one daily measurement but was below 5.0 % at the next,an estimation of the time for the value of 5.5 % was made. The quotient valueswere later used as the response of change in extract per day.

5.2 Verification of NucleoCounter R© YC-100TM

With the idea of verifying the deviation of the cell counts achieved with the yeastcounter, a sample was taken from one horizontal tank and one vertical fermentationtank. These samples were homogenized through shaking the sample bottles byhand and thereafter ten samples were taken from each bottle to count both thedead and the total amount of cells.

Since there is a probability that the yeast samples will not be sufficiently ho-mogenized through manually shaking, an experiment was setup where a verticalfermentation sample was homogenized for 30 minutes by a magnetic stirrer. To

22 Statistics

compare the samples solely shaken by hand, ten samples were used to count thetotal and dead number of cells.

The variances from the analysis were analyzed with a significance test at theconfidence level 95 % to see if there was any difference in the accuracy of themeasurements. The hypotheses are formulated below, where H0 is rejected if thep-value is < 0.05.

H0: σ1/σ2 = 1H1: σ1/σ2 6= 1

To verify the percentages of dead cells in the samples, a visual verification wasperformed using a microscope. This procedure is described in section 4.1.

5.3 Examination of Cell ViabilityThe cell viability was measured daily to see if there was any difference betweenthe results from the different experiments. Also, analysis of the yeast viabilitywhen transferring yeast from a vertical fermentation tank to the yeast tank wasperformed to verify the condition of the yeast at the end of the experiments.

When analyzing the yeast viability after a transfer, a sample was taken atthe beginning, in the middle and at the end of the transfer. One hour afterthe transfer, another sample was taken from the yeast tank. The time betweenthese samples could differ depending on how much yeast the fermentation tankcontained. Sometimes, the samples were taken late in the afternoon and storedin the refrigerator overnight before the analysis was performed. Therefore, thereliability of the analysis may be doubtful. A demo version of SIMCA v. 13 fromMKS Umetrics AB, Sweden, was used to verify the different measurements.

5.4 Examination of Ester ProductionAll of the ester analyses performed were examined to see if there were any visi-ble differences between the ester concentrations in time for the batches with thedifferent experimental parameters. Graphs were created using SIMCA v. 13.

Chapter 6

Results

In this chapter results obtained from the statistical experiments will be presented.Included are for instance the verification of the yeast counter, Minitab graphs andgraphs showing the cell viability over time.

6.1 Daily Analysis of WortsIn figure 6.1 examples of the daily measurements performed for the beer batches arevisualized in three graphs. They give an indication of how the different parametershave changed in time. This particular batch is named batch B.

0 2 4 6 8 10 1210

5

106

107

108

Day

Am

ount

Yeast cells

Number of cells [1/ml]Number of dead cells [1/ml]

0 2 4 6 8 10 120

1

2

3

4

5

6

7

Day

Pro

cent

Amount of dead cells in percentage

(a) Cell measurements.

0 2 4 6 8 10 122

4

6

8

10

12

14

Day

Mag

nitu

de

Additional measurements

Tank temperature [°C]pH valueExtract [%]

(b) Additional measurements.

Figure 6.1. Sample measurements of the beer batch named B.

23

24 Results

6.2 Experimental Design Responses

Presented below are graphs from Minitab 16 Statistical Software, created as thefull factorial design was analyzed. Examination of residual plots, pareto charts,half normal and normal plots of the standardized effects, main effects plots andinteraction plots has been performed.

6.2.1 Ethyl Acetate

As can be seen in figure 6.2, the residual plots show normally distributed residualswith random errors. Many results in the same interval are presented in the middleof the histogram. Some of the measurements show a positive correlation for theobservation order, highlighting that there are non-random errors in the order ofthe experiments.

The pareto chart indicates that significant effects are the interaction betweentime between worts and aeration and the effect of yeast dosage.

(a) Residual plots for ethyl acetate. (b) Pareto chart for ethyl acetate.

(c) Half normal plot for ethyl acetate. (d) Normal plot for ethyl acetate.

Figure 6.2. Statistical plots obtained for ethyl acetate.

6.2 Experimental Design Responses 25

(a) Main effects plot for ethyl acetate. (b) Interaction plot for ethyl acetate.

Figure 6.3. Statistical plots obtained for ethyl acetate.

6.2.2 Isoamyl Acetate

Figure 6.4 shows a normal distributed measurement series with random errors.The versus order plot shows some sort of positive correlation in the experimentalorder.

The pareto chart indicates that yeast dosage and time between worts are sig-nificant parameters, which is also shown by the half normal and normal plot infigure 6.5. The main effects plot in figure 6.5 shows how the concentration isaffected by the change of these parameters.

(a) Residual plots for isoamyl acetate. (b) Pareto chart for isoamyl acetate.

Figure 6.4. Statistical plots obtained for isoamyl acetate.

26 Results

(a) Half normal plot for isoamyl acetate. (b) Normal plot for isoamyl acetate.

(c) Main effects plot for isoamyl acetate. (d) Interaction plot for isoamyl acetate.

Figure 6.5. Statistical plots obtained for isoamyl acetate.

6.2.3 FAN DegradationThe plots for FAN degradation, seen in figures 6.6 and 6.7, shows normally dis-tributed residuals with random errors. All of the measurements are evenly spreadin the histogram and the versus order shows no correlation. According to thepareto chart, there are no statistically significant effects.

(a) Residual plots for FAN degradation. (b) Pareto chart for FAN degradation.

Figure 6.6. Statistical plots obtained for FAN degradation.

6.2 Experimental Design Responses 27

(a) Half normal plot for FAN degradation. (b) Normal plot for FAN degradation.

(c) Main effects plot for FAN degradation. (d) Interaction plot for FAN degradation.

Figure 6.7. Statistical plots obtained for FAN degradation.

6.2.4 Change in Extract

Figure 6.8 shows normally distributed residuals with random errors. Though, thehistogram indicates skewness in the measurements. The versus order plot indicatesnon-random errors in the experimental runs.

According to the pareto chart, yeast dosage is statistically significant for thechange in extract. The main effects plot shows the differences in change in extractper day as the yeast dosage increases.

28 Results

(a) Residual plots for change in extract. (b) Pareto chart for change in extract.

(c) Half normal plot for change in extract. (d) Normal plot for change in extract.

(e) Main effects plot for change in extract. (f) Interaction plot for change in extract.

Figure 6.8. Statistical plots obtained for the change in extract.

6.3 Verification of NucleoCounter R© YC-100TM

Below, the results that were obtained with the verification of the yeast counter arepresented.

6.3.1 Total and Dead Cell CountThe number of yeast counts from the same horizontal (HT) and vertical fermenta-tion (VT) tank samples resulted in a verification of the deviations in the measure-

6.3 Verification of NucleoCounter R© YC-100TM 29

ments performed by the yeast counter. The results can be seen in tables 6.1 and 6.2.

Total number of cellsParameters VT stirr. VT HTMean 1.71 x 107 1.68 x107 3.50 x 107

Standard Deviation 1.05 x 106 1.78 x 106 2.11 x 106

Variance 1.10 x 1012 3.16 x 1012 4.46 x 1012

Table 6.1. Statistical parameters of total cell measurements using NucleoCounter.VT = vertical fermentation tank, HT = horizontal fermentation tank.

Dead number of cellsParameters VT stirr. VT HTMean 1.15 x 106 9.88 x 105 7.82 x 105

Standard Deviation 9.48 x 104 8.49 x 104 8.84 x 104

Variance 8.98 x 109 7.21 x 109 7.82 x 109

Table 6.2. Statistical parameters of dead cell measurements using NucleoCounter.VT = vertical fermentation tank, HT = horizontal fermentation tank.

From the statistical analysis of the variances at confidence level 95.0 %, theresults in table 6.3 were obtained. The null hypothesis of σ1/σ2 = 1 is rejected ifp < 0.05.

VT stirr. to VT VT stirr. to HTTotal Dead Total Dead

p-value (F test) 0.131 0.748 0.048 0.840p-value (Levene’s test) 0.153 0.858 0.030 0.930

Table 6.3. P-values for the variances of the NucleoCounter measurements.

6.3.2 Percentage of Dead Cells

The yeast solution was mixed with methylene blue whereas dead cells were stainedblue and counted using a microscope. Below, in table 6.4, the difference betweenthe methylene blue methods and the NucleoCounter analysis is shown.

30 Results

Method MethodSample 1 2 Sample 1 3

1 10.6 % 13.5 % 1 7.9 % 8.0 %2 5.7 % 5.2 % 2 7.3 % 7.4 %3 6.1 % 6.4 % 3 7.4 % 7.8 %4 6.5 % 5.9 % 4 7.3 % 7.4 %

Table 6.4. Verification of the percentage of dead cells using the NucleoCountermethod (1), the simple methylene blue method (2) and the additional methylene bluemethod (3).

6.4 Analysis of Cell Viability

A demo version of SIMCA v. 13 from MKS Umetrics AB, Sweden, was usedto study the different cell viability measurements performed, resulting in fig-ures 6.9, 6.10, 6.11 and 6.12. Though the text to the right in figures 6.9 and 6.10only covers the batches A to N, the graphs really contain the batches A to AM.Batches named unknown in figures 6.11 and 6.12 were not followed during thefermentation stage.

In figure 6.9, it can be seen that batch AF (light pink) and AG (orange) havea higher amount of total cells than the others at day 4, while batch AD (lightblue) is rising to the highest amount on day 5. When it comes to dead cells infigure 6.10(a), batch O (purple) has a higher amount than the others during thewhole measurement period. Batch AF and AG contain a high amount of deadcells on day 4 and 5, but then settles down below average. For the percentage ofdead cells in figure 6.10(b), batch O has the highest values until day 4, when AGpeaks.

Figure 6.9. Plot made in SIMCA of total number of cells/mL for the different experi-ments.

6.4 Analysis of Cell Viability 31

(a) Plot of dead number of cells/mL for the different experiments.

(b) Plot of the percentage of dead cells for the different experiments.

Figure 6.10. The plots are made in SIMCA to verify the difference in yeast viabilityfor the different experiments.

After the transfer of yeast from the AE batch at the third analysis, a higheramount of total cells compared to others was obtained, which can be seen infigure 6.11. Though, the amount of dead cells in figure 6.12(a) was higher too.For the plot of the percentage of dead cells, figure 6.12(b), transfer from batch Ohad a higher percentage than the others, while batch N had a lower percentage.

Figure 6.11. Plot made in SIMCA of total number of cells/mL after transfer to theyeast tank for the different experiments.

32 Results

(a) Plot of dead number of cells/mL after transfer to the yeast tank for the different experi-ments.

(b) Plot of the percentage of dead cells after transfer to the yeast tank for the different exper-iments.

Figure 6.12. The plots are made in SIMCA to verify the difference in yeast viabilityafter transfer to the yeast tank for the different experiments.

6.5 Analysis of Ester Production

As seen in figure 6.13, most of the batches follow the same process, ending upwith a small difference between the end concentrations. In figure 6.13(a), batch N(pink) starts with 12.4 mg ethyl acetate/L but only has 7.0 mg/L in the secondanalysis. Batch A (black) starts with 6.4 mg/L and continues to increase until itstops at 40.2 mg/L in the lagering tanks. Batch AB (dark green) ends up with39.5 mg/L in the lagering tanks, though starting with a smaller concentration.

For isoamyl acetate, 6.13(b), batch AH (orange) and AI (light green) havethe highest start values, but later the concentrations are not increasing as rapidas the remaining batches. Batch AB (dark green) has the highest increase ofconcentration. At the second analysis the concentration was 5.9 mg/L and in thelagering tank 6.2 mg/L.

6.5 Analysis of Ester Production 33

(a) Plot of production of ethyl acetate for the different experiments.

(b) Plot of production of isoamyl acetate for the different experiments.

Figure 6.13. Plots made in SIMCA to verify the production of esters for the differentexperiments.

Chapter 7

Discussion

In the following chapter, discussion of the results from analyses and statisticalcalculations obtained will be found. This section will include both the parameterimpact on the response variables from the statistical analysis and suggestions ofwhich improvements the brewery may implement.

7.1 Setup of the Experimental DesignSince the experimental design was to be performed in tanks which at the timewas producing large amounts of high quality product, there was no possibility tovary the upper and lower levels of the parameters as much as could have beendone in a laboratory scale. This is regretful as a greater variation might havegiven clearer response values. Still, as these are the possible variations that existin the production today, these will also be the variations that can be performed.Therefore, the results obtained are of great importance for the brewery.

The fact that the experiments were performed in sharp production made itdifficult to control the circumstances. Unexpected events including changes inscheduled production, human error and inaccuracies occurred and had to be dealtwith during the project. This contributed to replanning and a prolonged samplingperiod.

A lot of batches that were produced during the period of experiments had thewrong parameters to perform certain experiments, mostly due to a too high wortaeration. This created an imbalance in the experimental design as many batcheswere more fitted to for instance experiment 4 while not that many batches couldbe fitted to experiment 5 or 6. To proceed with a balanced full factorial design thebest batches, which means those with the best matching parameters, were chosento make a 23 full factorial design with 2 replicates.

Because of Spendrups’ industrial production of beer and inability of riskingproduction loss, the order of the experiments was not randomized. The choice ofexperiments was instead picked and controlled by the brewmaster to make surethat the different level experiments were suited for the production. Nonetheless,all of the experiments were performed.

35

36 Discussion

7.2 Experimental Design ResponsesThe experimental design was analyzed and each of the responses were validatedwith regard to the parameters used. Since all of the experimental runs werebotched, due to unsuccessful control of the aeration, there was not a single run atthe extreme points and therefore there was no possibility to proceed with surfaceor contour plots. This is regretful as the surface and contour plots could havegiven information regarding which parameter values should be chosen to obtain acertain result.

7.2.1 Ethyl AcetateWhen analyzing the residual plots from the analysis of the design of experimentsfor ethyl acetate in figure 6.2(a), it can be seen that the residuals are normallydistributed as the normal probability plot is linear. The residual versus fits plotshows a random pattern of residuals which indicates that the errors occurring arerandom. According to the histogram, the results are evenly distributed over themeasurement series.

The residuals versus order plot is describing the possibility of non-randomerrors. Since a positive correlation is indicated by a clustering of residuals with thesame sign, there is a likelihood that some measurements suffer from non-randomerrors. Though, since the order of the experimental runs was not randomized andruns with the best matching parameters were selected after the entire experimentseries, a correlation of 2-3 points are fairly good results.

When viewing the pareto chart for ethyl acetate in figure 6.2(b), it can be seenthat the interaction between time between worts and aeration and the parameterof yeast dosage are the factors that have a statistically significant relationship tothe produced concentration of ethyl acetate. This is understandable as these arethe only parameters that stretch above the red significance threshold and thereforehave a p-value < 0.05.

Verifying the half normal plot in figure 6.2(c), it can be seen that the effectsthat fall far away from the line are the significant effects. Still, many of the othereffects are deviating from the straight line as well, but as they have a p-value > 0.05they are not large enough to be significant. It is noted that the significant effectsare the interaction between time between worts and aeration and the main effectof yeast dosage. Since the interaction is the largest, it is also the most highlysignificant effect. These are the same results that could be viewed in the paretochart.

The difference between the half normal and the normal plot is that the normalplot in figure 6.2(d) may give significant effects on both sides of the line. Inthis case, the yeast dosage effect may be found to the left of the line, while theinteraction of time between worts and aeration is found on the right hand side.

Information from the main effects plot in figure 6.3(a) is mostly interestingfor the parameter that alone affects the concentration of ethyl acetate, namelyyeast dosage. Results show that the mean concentration decreases when the yeastdosage increases. Analysis of the interaction plot in figure 6.3(b) shows that when

7.2 Experimental Design Responses 37

increasing the aeration at 6 h between worts, the mean concentration of ethylacetate will decrease.

Overall, this means that a low aeration combined with 6 h between worts anda low yeast dosage will give a higher mean concentration of ethyl acetate. For theruns used in the experimental design, most of the concentrations of ethyl acetatefollowed the requirements specification and this gives an indication that theseresults are possible to take into account in further production.

It is notable that the aeration does not have a larger impact on ethyl acetatethan through the interaction with time between worts. Since a restricted amountof air should give a higher concentration of esters [2], the range of the aerationmay be too small to give any difference between the upper and lower level.

7.2.2 Isoamyl AcetateTo examine the goodness of model fit, the residual plots in figure 6.4(a) are ana-lyzed for the concentration of isoamyl acetate. A study of the normal probabilityplot gives the indication of a normal measurement distribution. The residualsin the plot of residuals versus fits do not show a recognizable pattern and it istherefore possible to assume that there are no non-random errors. The histogramplot shows high tails in the middle of the chart and this means that many of themeasurement results are located around the same values. According to the plot ofresiduals versus order, there is no sign of correlation in the experimental order.

The pareto chart in figure 6.4(b) gives a hint that the parameters of yeastdosage and time between worts have factor effects large enough to be statisticallysignificant. This is confirmed by the half normal plot while the normal plot in fig-ure 6.5(b) shows that yeast dosage falls on the left hand side of the line and timebetween worts to the right. A verification of the main effects plot in figure 6.5(c)gives the information that the lower level for yeast dosage gives a higher mean con-centration. Also, if the time between worts is set to 12 h, the mean concentrationof isoamyl acetate will slightly increase.

According to the analysis above, to obtain a high mean concentration of isoamylacetate it is important to fill the horizontal tanks with a time gap of 12 h and usea lower yeast dosage. As none of the measurements used contained concentrationsthat were too high, there will not be a problem using these parameter values.

As for ethyl acetate it is notable that the aeration is not statistically significantfor isoamyl acetate. This means that the range between the upper and lower levelmay be too small to show the expected difference in concentration.

7.2.3 FAN DegradationThe measurements of the FAN degradation seem to be normally distributed as theresiduals in figure 6.6(a) form a straight line in the normal probability plot. Theyalso give a random pattern in the versus fits plot, indicating only random errors.The histogram shows that the data is evenly spread with some typical data in themiddle of the measurement series. No indication of non-random errors can be seen

38 Discussion

in the residuals versus order plot. This might be due to the results being close toeach other regardless of which experiment they were included in.

According to the pareto chart for FAN degradation in figure 6.6(b), no factoris statistically significant. This might be true, but can also be an effect of wortssometimes being analyzed to early. As three of four horizontal tanks showed anextract that had decreased below 5.5 % and were to be transferred to verticalfermentation tanks, all of the tanks were analyzed for FAN even if the last tankwas still holding an extract of above 6.0 %. If none of the tanks were to be analyzedawaiting the last to decrease in extract, the other tanks would instead decrease inextract below the interval of interest.

Though there are no significant parameters, it can be seen in the main effectsplot in figure 6.7(c) that a higher aeration will increase the FAN degradation tosome extent while a higher yeast dosage will decrease it. The increase in degrada-tion when increasing the aeration may be due to the yeast being able to use thenitrogen source to produce more yeast cells.

7.2.4 Change in ExtractThe normal probability plot of change in extract in figure 6.8(a) shows residualsfollowing the straight line, which means a normal distribution. Analyzing theversus fits for the residuals, a random pattern indicates random errors. Though,the histogram is a bit skewed, indicating many measurements with the same valueson both sides of the middle. Also, a positive correlation can be seen in the versusorder plot of the measurements from experiment 5 to 10. This means that non-random errors are found. As the experimental order was not randomized andthe runs with the best matching parameters were selected afterwards for the fullfactorial design, this is somewhat expected.

Verification of the pareto chart in figure 6.8(b) shows that the change in extractis dependent on the yeast dosage, which can also be seen in the half normal andnormal plot. This was expected as it is the yeast that utilizes the extract in theworts. Furthermore, the main effects plot in figure 6.8(e) indicates that an increasein the yeast dosage will increase the change in extract.

A high yeast dosage shows a mean of change in extract of about 1.74 %/day.As an change in extract of 1.6 % ± 0.2 % per day is optimal, a lower yeast dosagemay be chosen if the desire is to get closer to 1.6 %.

7.3 Verification of NucleoCounter R© YC-100TM

The statistical analysis of the variance for the yeast count samples seen in ta-bles 6.1 and 6.2 shows that the variance for the magnetically stirred sample differsfrom the horizontal tank sample with regard to the total amount of cells. Thisis understood as the p-value is < 0.05, which can be seen in table 6.3. It meansthat H0 is rejected and there is a significant difference between the sample vari-ances. However, the p-value for the dead cell amount is > 0.05 and therefore H0is accepted for dead cells.

7.4 Analysis of Yeast Viability 39

According to the statistics, to get a representative analysis of the total numberof yeast cells it is helpful to use a magnetic stirrer. Though, due to the lack ofcirculation in the tanks, especially in the horizontal tanks, it will still be hard toobtain a value that is representative for the whole tank. Although, the verticalfermentation tanks obtain some circulation due to temperature differences as thetank cools the wort close to the tank walls while the wort in the middle is kept abit warmer.

Given the results of the visual verification of percentage of dead cells seen intable 6.4, both the manual counting procedures used seem to be close to giving thesame values as is shown by the NucleoCounter. However, as the manual countingis very individual, since the person counting is able to choose which cells to count,the methods are not that reliable.

7.4 Analysis of Yeast ViabilityBelow, batches that were shown deviating in viability in chapter 6 are analyzedbriefly.

7.4.1 Viability in Beer FermentationThe batches AF and AG seem to be the batches having the highest amount oftotal cells in figure 6.9. These are both batches from experiment 7 and the yeastis used for the sixth generation. Experiment 7 included short time between worts,low aeration and high yeast dosage. The largest deviation from the experimentalparameters was the aeration which was 27.5 and 26.9 mg O2/L respectively.

AF and AG were also the batches with the highest amount of dead cells, to-gether with batch O in figure 6.10(a). The O batch had the parameters fromexperiment 4 with 12.25 h between worts, 27.3 mg O2/L and newly propagatedyeast.

The batches with the highest percentages of dead cells in figure 6.10(b) wereagain O, AF and AG indicating that something might have happened to the yeastduring these experiments.

7.4.2 Viability after Yeast TransferAs the yeast is transferred from a vertical fermentation tank, many of the analyzedbatches seem to have approximately the same amount of total and dead cells in thethird measurement made. This is the analysis performed at the end of the transfer.The other measurements are spread out around the mean. Though, in the plotof the total cells, figure 6.11, it can be seen that batch AE has a small deviationfrom the other batches in the third measurement, having a higher total amountof cells/mL. AE is also the one deviating from the other batches in figure 6.12(a),where the dead cells/mL are shown.

For the percentage of dead cells in figure 6.12(b), it is instead batch O thatdeviates with the highest percentage of dead cells. This is a batch that alsodeviated from the other batches in the analysis of the beer fermentation both in

40 Discussion

the amount of dead cells/mL as well as the percentage of dead cells. On the otherhand, after transfer batch N does also seem to deviate from the other batches,having a fewer percentage of dead cells.

It is easy to conclude that something has happened to batch O prior to orduring fermentation, since it was deviating from the other measurements from thebeginning. Regarding batch AE, it seems more reasonable that it was disturbedduring the transfer, as the values during fermentation was as stable as for theother measurements.

7.5 Analysis of Ester Production

In the sections below, the ester concentration during different measurements isbriefly analyzed.

7.5.1 Ethyl Acetate

The samples of produced ethyl acetate that deviate from the others in figure 6.13(a)are those of batch N (pink), A (black) and AB (dark green). Batch N starts witha much higher concentration than the other batches although later it seems todecrease. However, the first value is not correct, since the bottle with the estersample was left in the refrigerator during the weekend to be analyzed at the startof the following week. The fridge temperature must have been insufficient allowingthe newly propagated yeast to utilize the wort to produce a higher concentrationof ethyl acetate than that of the tank in the second measurement.

Batch A seems to start with a slightly higher concentration of ethyl acetatethan the other batches, and the concentration keeps increasing until it stops at40.2 mg/L in the lagering tanks. This is probably due to the experimental param-eters used; the time between worts were 5,58 h, the aeration was 26.3 mg O2/Land the yeast was newly propagated. Since time between worts were short, thenewly propagated yeast must have been more involved in the production of ethylacetate than the reused yeast usually is.

Batch AB starts with a regular concentration but it increases as the processcontinues and in the lagering tanks the concentration is 39.5 mg/L. The batch hada time between worts of 13 h, an aeration of 25.6 mg O2/L and a yeast dosage of250 g yeast/hL. Since the parameter levels are not favoring higher concentrationsof ethyl acetate, the high values were not expected. Unfortunately, the AB batchwas not used for the experimental design and therefore these results can not beexplained.

Both of the concentrations in the lagering tanks for A and AB are too high forthe requirements specification of pale lager lagering tanks. Such high concentra-tions should be avoided and the parameters must be controlled to the point thatthis does not occur.

7.6 Improvements and Implementations 41

7.5.2 Isoamyl AcetateFor isoamyl acetate the batches AH and AI started with rather higher concentra-tions than the other batches, see figure 6.13(b). Since the concentrations continueto increase at the same rate as for the other batches and due to a gas chromato-graph control performed shortly before the analysis, there is reason to believe thatthe first measurements were invalid.

The batch with the concentration that increases the most is AB, just as forethyl acetate, ending with a concentration of 6.2 mg/L. This means that the timeof 13 h between the worts give a high result, just as expected. As 6.2 mg isoamylacetate/L is approved according to the specifications of the pale lager beer, thisspecific ester is not a problem.

7.6 Improvements and ImplementationsAt the beginning of the project, there was a problem with the amount of estersin the lagering tanks. The concentrations were never sufficient, according to thelimits of the concentrations. As the project proceeded and the concentration ofesters became larger, it turned out that the limits of the lagering tanks used waswrong. A much lower threshold value was implemented and suddenly the higherethyl acetate concentration was exceeding the limitations of the finished beer. Onthe other hand, this resulted in isoamyl acetate being almost always sufficient andethyl acetate being either sufficient or too high.

According to the new limitations, Spendrups Bryggeri has got the capacityto produce the right amount of esters desired in the lagering tanks. Though, aconsideration of the parameters used needs to be done. The time between wortsand yeast dosage are stable parameters that are easily controlled, but the aerationcontrol may need alterations in order to stabilize the air supply.

7.6.1 Improvement of AerationAs the worts were aerated, they did not once receive the desirable total amountof oxygen. When the plan was to aerate worts with 30 mg O2/L, the total resultsof two batches were more often closer to 28-29 mg/L or sometimes 31 mg/L andhigher. The dose was never lower than 25.4 mg/L and since the interval of aerationwas rather small from the beginning, the variation in aeration might be negligiblefor the experiments.

Due to the problems with the aeration, a control should be performed to seewhat settings should be used to get a desirable amount of oxygen in the batches.If the aeration is highly dependent on the amount of worts produced, which maybe unknown at the start, a closer theoretical value must be found and used.

7.6.2 Improvement of Yeast ViabilitySince the yeast viability does not differ that much from time to time, thoughdifferent management parameters are used, it is difficult to say what should be

42 Discussion

rearranged for the yeast to increase in viability. Though, it is noticed that themore the yeast get to ferment before it is transferred from the vertical fermentationtanks, the better it gets. It is understood that the tanks and transfer tools mustbe cleaned before and after use and the yeast should not be contaminated by othermicroorganisms or other residues that can affect its viability and vitality.

7.7 Future PerspectivesThrough the factorial design experiment performed during this study, the effectsof different parameters have been investigated. As it is now clear how the differentcontrol variables affect the responses chosen, there is a possibility to setup anexperiment to find the optimal variable values. These should be values that givean ethyl acetate and isoamyl acetate concentration of approximately 31.0 mg/Land 4.50 mg/L respectively in the lagering tanks and a change in extract of 1.6 %per day. It would also be interesting to see how the different parameters affectyeast of different generations, as this was not possible during the study.

The implementation of a better controlled aeration is also something that couldbe performed, especially at the site of Spendrups Bryggeri. If the aeration is keptconstant at the same time as the time between worts and yeast dosage are, thiswill result in equal batches with regard to these parameters. That is a step closerto producing beer of high quality.

Further experiments that may be relevant are those of cell viability and vital-ity. Analyses can be made with the intention of finding parameters that keepsthe viability of the yeast high throughout the whole process. It will also be ofimportance to establish the vitality in the fermentation tanks since the vitality isable to tell how well the living yeast is performing.

Chapter 8

Conclusions

The ultimate purpose of this thesis was to find the important parameters affectingthe responses of ester concentration, FAN degradation and change in extract perday through evaluation of the control variables of time between worts, aerationand yeast dosage. Further objectives were to find possible improvements that mayincrease the fermentation performance and find ways of implementing them in thebeer production.

Based on the mean concentration results from the full factorial design, thevalues of the control variables may be considered to give sufficient results for theresponses. Using a long time span between the worts and a low yeast dosage, asufficient concentration of ethyl acetate and isoamyl acetate will be obtained. Thelow yeast dosage will also keep the change in extract at a level of approximately1.6 % per day. FAN will not be significantly affected.

The aeration effect does not seem to be significantly affecting any of the re-sponses except for being in interaction with time between worts, affecting ethylacetate. Though for the time span of 12 h between worts, the aeration does notseem to give any major differences in the ethyl acetate mean concentration.

Due to botched runs, because of problems with the aeration, it was not possibleto verify the theoretical parameter values and responses of surface and contourplots. Therefore, no theoretical optimal parameter settings could be determined.As the aeration was never correct for any of the experimental runs, either being toohigh or too low, settings and management of the aeration should be further studiedand controlled. This could help to improve the quality of batches produced.

Altogether, this thesis contributed to finding the parameters that are impor-tant during yeast fermentation based on different responses and finding possibleimprovements in the yeast handling. Due to the possible improvements of aerationbeing beyond the experiment analyses, no implementation of the improvementswas investigated.

43

Bibliography

[1] “www.spendrups.se.” Published online. Visited 130813.

[2] W. Kunze, Technology Brewing and Malting. VLB Berlin, Verlagsabteilung,1996.

[3] Bejram&Partner and Livsmedelsbranschernas Utbildningsorgan, LUO, Bryg-geriboken.

[4] L. F. Guido, P. G. Rodrigues, J. A. Rodrigues, C. R. Gonçalves, and A. A.Barros, “The impact of the physiological condition of the pitching yeast onbeer flavour stability: an industrial approach,” Food Chemistry 87, pp. 187-193, 2004.

[5] E. J. Lodolo, J. L. Kock, B. C. Axcell, and M. Brooks, “The yeast Saccha-romyces cerevisiae - the main character in beer brewing,” FEMS Yeast Res8, pp. 1018-1036, 2008.

[6] S. Landaud, E. Latrille, and G. Corrieu, “Top pressure and temperature con-trol the fusel alcohol/ester ratio through yeast growth in beer fermentation,”Journal of the Institute of Brewing, Volume 107, No 2, pp. 107-117, 2001.

[7] D. Saison, D. P. De Schutter, N. Vanbeneden, L. Daenen, F. Delvaux, andF. R. Delvaux, “Decrease of aged beer aroma by the reducing activity ofbrewing yeast,” Journal of Agricultural and Food Chemistry, 58, pp. 3107-3115, 2010.

[8] I. C. Trelea, M. Titica, and G. Corrieu, “Dynamic optimisation of the aromaproduction in brewing fermentation,” Journal of Process Control 14, pp. 1-16,2004.

[9] C. Cheong, K. Wackerbauer, and S. A. Kang, “Influence of aeration duringpropagation of pitching yeast on fermentation and beer flavor,” Journal ofMicrobiology and Biotechnology, 17(2), pp. 297-304, 2007.

[10] A. Brown and J. Hammond, “Flavour control in small-scale beer fermen-tations,” Food and Bioproducts Processing, Volume 81, Issue 1, pp. 40-49,March 2003.

45

46 Bibliography

[11] G. G. Stewart, “Yeast performance and management,” The Guild ReviewPaper - No 3, The Brewer, pp. 211-214, May 1996.

[12] R. A. Speers, Y. Wan, Y. Jin, and R. J. Stewart, “Effects of fermentationparameters and cell wall properties on yeast flocculation,” Journal of theInstitute of Brewing, Volume 112, Issue 3, pp. 246-254, 2006.

[13] D. Kregiel and J. Berlowska, “Evaluation of yeast cell vitality using differentfluorescent dyes,” Food Chemistry and Biotechnology, Vol. 73, No. 1058, pp.5-14, 2009.

[14] T. Yilmaztekin, T. Cabaroglu, and H. Erten, “Effects of fermentation tem-perature and aeration on production of natural isoamyl acetate by Williopsissaturnus var. saturnus,” BioMed Research International, Article ID 870802,pp. 1-6, 2013.

[15] P. Dutia, “Ethyl acetate: A techno-commercial profile,” Chemical Weekly,pp. 179-186, 2004.

[16] L. W. Sutikdja, D. Jelisavac, W. Stahl, and I. Kleinerb, “Structural studieson banana oil, isoamyl acetate, by means of microwave spectroscopy andquantum chemical calculations,” Molecular Physics, Vol. 110, No. 23, pp.2883-2893, 2012.

[17] “Nucleocounter yc-100 data sheet.” Pdf published online. 990-0010 ver 2.

[18] D. C. Montgomery, ’Design and Analysis of Experiments, Fifth Edition. JohnWiley & Sons, Inc., 2001.

[19] B. C. Axcell and E. S. C. O’Connor-Cox, “The concept of yeast vitality - analternative approach,” in Proceedings of the 24th Convention of the Instituteof Brewing (Asia Pacific Section), Singapore, pp. 64-71, 1996.

Appendix A

Batch Analysis

Below, two tables containing information about the different batches can be found.In table A.1 the values of the control variables are visualized and in table A.2the different values of the response variables are presented. The batches in boldtext are those used for analysis of the full factorial design experiment. Esterconcentrations with asterisks are outside of the requirements specification, whichfor ethyl acetate is between 24.0 and 33.0 mg/L and for isoamyl acetate between3.80 and 6.70 mg/L.

Batch ID Time betweenworts (h)

Tot. aeration(mg O2/L)

Yeast dosage(g/hL)

A 5.58 26.3 new yeastB 6.17 25.7 200C 6.08 26.9 199D 6.00 26.2 204E 11.83 25.4 new yeastF 13.23 26.4 200G 12.08 26.4 203H 6.08 29.0 new yeastI 6.33 30.5 197J 6.17 31.0 197K 6.08 31.9 198L 5.83 30.8 197M 6.67 29.8 201N 12.67 29.7 new yeastO 12.25 27.3 new yeastP 12.25 28.5 200Q 11.67 30.9 199R 11.58 31.8 199S 12.00 27.6 204T 11.75 31.2 196

47

48 Batch Analysis

Batch ID Time betweenworts (h)

Tot. aeration(mg O2/L)

Yeast dosage(g/hL)

U 11.42 28.3 201V 11.58 27.4 200W 5.58 27.5 247X 6.08 25.8 247Y 6.00 26.8 251Z 12.00 25.8 248AA 12.17 25.5 249AB 13.00 25.6 250AC 5.83 28.2 247AD 6.67 35.2 249AE 7.08 33.3 248AF 5.92 27.5 249AG 5.58 26.9 248AH 12.25 28.7 246AI 12.67 27.2 249AJ 12.67 28.2 248AK 12.5 28.7 249AL 12.5 28.6 250AM 12.00 37.3 248

Table A.1: Values of the control variables for the different batchesproduced.

BatchID

Ethylacetate(mg/L)

Isoamylacetate(mg/L)

FANdegradation(mg/L)

Extract(-%/day)

A 40.2* 4.43 90 1.5B 33.3* 4.90 104 1.5C 32.2 4.63 107 1.5D 32.1 4.84 109 1.7E 34.3 3.79 81 1.5F 32.6 5.15 98 1.5G 32.1 5.63 117 1.7H 30.0 4.07 109 1.2I 30.4 4.52 112 1.5J 29.1 4.17 87 1.2K 30.6 4.55 103 1.5L 31.5 4.67 104 1.5M 30.3 4.77 119 1.5N 36.3* 4.06 90 1.4

49

BatchID

Ethylacetate(mg/L)

Isoamylacetate(mg/L)

FANdegradation(mg/L)

Extract(-%/day)

O 32.0 3.83 92 1.2P 30.9 4.82 105 1.7Q 32.3 5.02 125 1.7R 29.0 4.29 112 1.7S 33.7* 5.30 116 1.7T 30.3 4.54 104 1.7U 27.6 4.42 111 1.8V 29.8 4.49 117 1.8W 35.8* 4.79 98 1.8X 32.5 4.54 81 1.6Y 28.4 4.24 110 1.8Z 29.5 4.15 97 1.6AA 30.9 4.46 102 1.9AB 39.5* 6.20 102 1.8AC 28.3 4.48 108 1.8AD 25.2 3.75* 118 1.8AE 27.2 4.01 110 1.8AF 29.6 4.43 116 1.8AG 28.1 4.21 115 1.7AH 32.4 4.83 100 1.8AI 31.0 4.43 99 1.7AJ 30.2 5.02 116 1.8AK 31.6 4.45 107 1.9AL 35.3* 5.43 108 1.8AM 26.6 4.25 104 1.8

Table A.2: Values of the response variables for the different batchesproduced. Values with an asterisk are outside of the requirementsspecification.

Appendix B

Tank Exchange

In this appendix, the transfer of yeast between tanks is visualized.

Horizontal 5

Horizontal 6

Horizontal 8

Horizontal 9Yeast Tank

Vertical Fermentation Tanks

Week 2

Week 3

Rehydrator

Propagation tank no 7

1 kg yeast

Week 1

Yeast TankTo be used week 2

Horizontal 5

Horizontal 6

Horizontal 8

Horizontal 9Yeast Tank

Vertical Fermentation Tanks

Figure B.1. The process where yeast is transferred between tanks.

50

Appendix C

Mathematics

C.1 Weighted MeansTotal values of aeration, FAN and extract was calculated through weighted meansof the wort batches as in (C.1).

W =V1

Vtotm+ V2

Vtotn

Vtot(C.1)

where

W – Weighted meanV1 – Volume of the first wort batch [hL]V2 – Volume of the second wort batch [hL]Vtot – Total volume [hL]m – Value of parameter of interest for the first wort batchn – Value of parameter of interest for the second wort batch

51

52 Mathematics

UpphovsrättDetta dokument hålls tillgängligt på Internet — eller dess framtida ersättare —under 25 år från publiceringsdatum under förutsättning att inga extraordinäraomständigheter uppstår.

Tillgång till dokumentet innebär tillstånd för var och en att läsa, ladda ner,skriva ut enstaka kopior för enskilt bruk och att använda det oförändrat för icke-kommersiell forskning och för undervisning. Överföring av upphovsrätten vid ensenare tidpunkt kan inte upphäva detta tillstånd. All annan användning av doku-mentet kräver upphovsmannens medgivande. För att garantera äktheten, säkerhe-ten och tillgängligheten finns det lösningar av teknisk och administrativ art.

Upphovsmannens ideella rätt innefattar rätt att bli nämnd som upphovsmani den omfattning som god sed kräver vid användning av dokumentet på ovan be-skrivna sätt samt skydd mot att dokumentet ändras eller presenteras i sådan formeller i sådant sammanhang som är kränkande för upphovsmannens litterära ellerkonstnärliga anseende eller egenart.

För ytterligare information om Linköping University Electronic Press se förla-gets hemsida http://www.ep.liu.se/

CopyrightThe publishers will keep this document online on the Internet — or its possi-ble replacement — for a period of 25 years from the date of publication barringexceptional circumstances.

The online availability of the document implies a permanent permission foranyone to read, to download, to print out single copies for his/her own use andto use it unchanged for any non-commercial research and educational purpose.Subsequent transfers of copyright cannot revoke this permission. All other uses ofthe document are conditional on the consent of the copyright owner. The publisherhas taken technical and administrative measures to assure authenticity, securityand accessibility.

According to intellectual property law the author has the right to be mentionedwhen his/her work is accessed as described above and to be protected againstinfringement.

For additional information about the Linköping University Electronic Pressand its procedures for publication and for assurance of document integrity, pleaserefer to its www home page: http://www.ep.liu.se/

c© Zara Skogsberg