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Removal of Alcohol From Beer UsingMembrane Processes

Master’s Thesis

Author:Andreas Jakob Wedel Falkenberg

Supervisors:Henrik Siegumfeldt

Jens Christian Sørensen

In collaboration with:

July 31, 2014

Title page

Title:

Removal of Alcohol From Beer Using Membrane Processes

Author:

Andreas Jakob Wedel Falkenberg (zdg243)

Duration:

6 months4/2 - 4/8-201430 ECTS

Supervisors:

Henrik SiegumfeldtJens Christian Sørensen

Copies:

Printed in 3 copies, as well as being digitally available

Thesis:

Master’s Thesis in Brewing Science and TechnologyNumber of pages: 94Written in LATEX

Written at:

Department of Food ScienceUniversity of Copenhagen Faculty of Science

In collaboration with:

Brewhouse Skands A/S and Alfa Laval Nakskov A/S

1

Preface and Acknowledgement

Rethinking the process for alcohol free beer (AFB) production focusing on aroma and flavourquality was the original idea of this thesis. An investigation was initiated revealing possiblenew methods of AFB production. This focusing not only on the process technologies re-lated to alcohol removal from normal alcoholic beer, but in addition looking beyond at thegeneral beer production processes to indicate possible changes resulting in a higher qualityAFB with regards to aroma and flavour preservation.

I would like to thank all parties involved from the University of Copenhagen Facultyof Science, Brewhouse Skands A/S and Alfa Laval A/S. From the University of Copen-hagen Faculty of Science a special thanks to my supervisors Associate Professor HenrikSiegumfeldt and Associate Professor Jens Christian Sørensen for knowledgeable guidance,participation and support. Furthermore, thanks to my fellow student Tobias Emil Jensenand his supervisor Mikael Agerlin Petersen for guidance and permission to run head spacegas chromatographic mass spectrometry samples. From Brewhouse Skands A/S a specialthanks to Birthe and Morten Skands for guidance, participation and beer donations. FromAlfa Laval a special thanks to Anders Bisgaard for guidance, participation, membrane do-nations and introduction to the newest trends in membrane processing.

Furthermore, I would like to thank M.Sc. in Chemical Engineering Jascha Rosenbaumand M.Sc. in Physical Engineering Christoffer Klærke for support and proof reading of thethesis. Thanks to Diploma Master Brewer Anders Nielsen for participation in beer tastingand proof reading. Finally, I would like to thank my family and friends for being supportivein the process of producing this thesis.

2

Abstract

The main object of this study is the production of alcohol free beer (AFB) using membraneprocesses. Although beer is perceived by the public as unhealthy, due to the alcohol content,it actually contains numerous nutrients.

Traditionally AFB is produced using either thermal processes such as evaporation andrectification or modified brewing and fermentation. These approaches induce pronouncedand unwanted changes to the overall flavour profile of beer. Membrane processes for theproduction of AFB are poorly investigated, however the potential is large because of pos-sible non-thermal selective ethanol removal. A major drawback in membrane processes isthe difficulty in removing ethanol below 0.5% alcohol by volume (ABV) without high ex-penses. In Denmark the legal limit for AFB labelling was recently changed from 0.1%ABVto 0.5%ABV making membrane processes a viable alternative for future AFB production.

This study compares the potential of four different membranes ranging in pore size fromnano filtration (NF) to reverse osmosis (RO). The membranes were tested on a LabstakM20-0.72 membrane unit provided by Alfa Laval A/S using Humlefryd 5.5%ABV lager pro-vided by Brewhouse Skands A/S. The M20-0.72 unit was modified to maintain a closedenvironment with a CO2 pressure of 1-2bar. The alcohol concentration during filtration wasdetermined using high performance liquid chromatography (HPLC), where the flavour pro-files before and after filtration were compared using head space gas chromatography massspectrometry (HS-GC-MS). Additionally, a trained taste panel was used to describe thedifferences in the membrane filtrated products compared to the original beer.

Investigations showed that RO membrane filtration provided a good aroma retentionwhile the ethanol permeability and flux through the membrane were low. On the otherhand, the different NF membranes had a higher ethanol permeability while a higher loss ofaroma was observed.

As a result, production of AFB using RO membranes will induce a higher capital expen-diture (CAPEX) for membranes and tanks plus a higher operational expenditure (OPEX)for pump work, cooling and water consumption, however the product will have a higheraroma quality. On the contrary, NF membranes will lower both the CAPEX and the OPEXas well as the quality.

Future consideration involving alteration of the brewing and fermentation processes wereconsidered hereby compensating for the aroma losses over RO and NF membranes makingthis process more profitable.

3

Abbreviations

σ∗ Polar Taft numberδ Membrane thicknessAA Amino acidsABV Alcohol by volumeADP Adenosine diphosphateAFB Alcohol free beerATP Adenosine triphosphateBethanol Solute transport coefficientBwater Solvent transport coefficientC∗ Membrane constantCA Cellulose acetateCAPEX Capital expenditureCCV Cylindroconical vesselsCI Chemical ionizationCIP Cleaning in placeCoA-SH Coenzyme AConc. ConcentrationCTA Cellulose triacetateDAB Danish Brewers’ AssociationDAM Solutes diffusion coefficientDC Direct currentDF DiafiltrationE2N (E)-2-NonenalEI Electron ionizationEs∗ Steric Taft numberF FlowFAD+ Flavin adenine dinucleotideFAN Free amino nitrogenFT Feed tankFTE Feed tank endGC Gas chromatographyGTP Guanosine triphosphateHA Higher alcoholsHF Humlefryd and high fluxHGB High gravity brewingHP High performance/pressureHS Head spaceJ FluxK Distribution ratio between

membrane and solutionKU University of CopenhagenLAB Low alcoholic beerLC Liquid chromatography

MCFA Medium chain fatty acidMF Micro filtrationMS Mass spectrometryMWCO Molecular weight cut-offNAD+ Nicotinamide adenine

dinucleotideNF Nano filtrationNFHF Nano filtration membrane type

NF99HFOPEX Operational expenditureORG OriginalP Permeate and permeabilityPA PolyamidePC Principal componentPCA Principal component analysisPE PolyesterPES PolyethersulfonePG Present gravityPM PermeabilityPP PolypropylenePS PolysulfonePT Permeate tankPVDF Polyvinyllidene fluorideRe RetentionRF Radio frequencyRI Refractive indexRID Refractive index detectorRO Reverse osmosiss∗ Small’s numberSD Standard deviationSDME Single drop micro extractionSPME Solid phase micro extractionTA Trapping agentTCA Tricarboxylic acid cycleTFC Thin film compositionTMC Trimethyl chlorideTMP Trans membrane pressuretR Retention timeUF Ultra filtrationVCF Volume concentration factorVDK Total vicinal diketonesVOC Volatile organic compoundsWCOT Wall coated open tubes

4

Contents

1 Introduction 7

2 Theory 112.1 Sedimentation and Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.2 Membrane Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.3 Experimental Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.4 Aroma Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.5 Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3 Materials and Methods 533.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.2 Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553.3 Feed Beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563.4 Dia-water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573.5 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573.6 Analytical Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4 Results and Discussion 624.1 Preliminary Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624.2 Constant Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644.3 Membrane Flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684.4 Ethanol Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714.5 Aroma Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744.6 Tasting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 874.7 Overall Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884.8 Aroma Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

5 Future Perspectives 92

6 Conclusion 94

A Appendices 106A.1 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106A.2 Diafiltration Approximation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108A.3 E2N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109A.4 Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110A.5 Sulphur Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114A.6 Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

5

CONTENTS CONTENTS

A.7 Hops Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122A.8 HS-GC-MS Feed Beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124A.9 HPLC Calibration Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126A.10 HPLC Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127A.11 HS Sampling Set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128A.12 HS-GC-MS Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129A.13 Alfa Laval Membrane Classification . . . . . . . . . . . . . . . . . . . . . . . . 130A.14 Standard Deviation of HS-GC-MS Samples . . . . . . . . . . . . . . . . . . . 131

6

Chapter 1

Introduction

The evolution of alcoholic beverages have changed the course of history ranging from scien-tific breakthroughs to prohibition and legislations. With a higher scientific enlightenmentalcoholic beverages have been deemed unhealthy by some, while healthy by others. Fur-thermore, the intoxicating effect of alcoholic beverages has caused the need for legislationsconcerning the intake of alcohol. Alcoholic beverages never seem to go out of fashion, how-ever the public view, legislations and assortments seem to change drastically over the courseof time (Gretton, 1929).

Beer has gone from being a home made product, enabling personal preferences, to be-coming an industrialized production, where supply and demand are in focus. An expandin assortment of beers is caused by a higher competition and a globalization resulting inmany different beer styles. In addition, technological development have caused a higherdifferentiation in beer enabling alcohol removal or reduction. Low alcohol beer (LAB) andalcohol free beer (AFB) are present on the market for the purpose of satisfying customerdemands. This demand could be caused by legislation, health issues, religion, prohibitionor as an alternative to soft drinks (Ambrosi et al., 2014).

For many years health have been the main concern or argument when legislating andbanning beer consumption. A high consumption of beer can lead to alcoholism, accidents,brain degeneration, liver failure, cancer, strokes and arteriosclerosis. Many of these illnessesare associated to the alcohol intake when drinking beer. Nevertheless, some positive affectsof beer drinking have been observed when drinking moderately or drinking LAB or AFB.Drinking one to six regular alcoholic beers a week have shown to have positive attributesdescribed by the so-called J-curve as shown in figure 1.1. The figure illustrates a reduc-tion in mortality for people drinking moderately compared to people not drinking. Thereduction in mortality associated with moderate alcohol intake is mainly caused by alcohollowering the risk of coronary diseases. For many years physicians have recommended winefor patients in danger of coronary diseases, when beer is equally sufficient. In fact, beercontributes with constituents with additional positive health effect. This could be an addi-tional reasoning for choosing an AFB or LAB instead of soft drinks where the nutrients andvitamins concentration are low to non-excising (Furbo, 2013), (Groenbaek et al., 1994).

Legislation in most European countries concerning alcohol concentration in beer statesthat beer with an alcohol concentration below 0.5%ABV are allowed to be labelled AFB,while beer below 1.2%ABV are allowed to be labelled LAB. In relation to religion, especiallyin the Muslim world, the allowed concentration is most often below 0.05%ABV, only allowingtraces of alcohol in beverages (Branyik et al., 2012).

In Denmark the legislation concerning AFB labelling was recently changed allowing

7

CHAPTER 1. INTRODUCTION

Figure 1.1: Relative risk of mortality compared to the weekly alcohol intake. Vertical linesindicate the 95% confidence interval (Groenbaek et al., 1994).

0.5%ABV instead of the previously allowed 0.1%ABV. This change was induced by theDanish Brewers’ Association (DBA) highlighting concerns about the low quality of DanishAFB. According to DBA the low quality and low sales of AFB in Denmark could be alteredto the better by allowing a higher concentration of AFB at 0.5%ABV, as observed in othercountries such as Germany, Sweden and Spain. In these countries AFB accounts for a higherpercentage of the market shares hereby increasing the accessibility of these product (Quass,2013), (Gormsen, 2013).

As stated above, legislation, health and religion are some of the factors creating thedemand for LAB and AFB. The current technology of reducing alcohol can be divided intotwo subsequent methods of respectively physical and biological methods. In figure 1.2 anoverview and summary of these two methods of producing AFB and LAB can be observed.The physical methods entail gentle removal of already created alcohol in the beer by separa-tion using heat and pressure alteration or mechanical separation using membranes. On theother hand, the biological methods entail the use of special yeast, mashing or fermentationmethods in the traditional brewing equipment or in new equipment, enabling a short contacttime with the wort and yeast. The physical methods often result in a great loss of volatileswhich leaves the beer flavourless and watery. On the contrary, the biological methods oftenleave the beer, worthy and unbalanced. In a review by Branyik et al. (2012) a comparisonof physical processes reveal a lower loss of volatiles using membrane processes compared tothermal processes. Furthermore, thermal processes cause irreversible heat damages to thebeer resulting in a higher rate of deterioration and unpleasant bitterness formation (Branyiket al., 2012).

Membrane processes might cause a high reduction in volatiles because some of the tasteand aroma substances are able to pass through the membrane along with alcohol. Thiscould result in a loss of mouth feel, taste, body and aroma, see appendix A.1 for glossary.Nevertheless, the irreversible alteration of flavour and aroma compounds is considerablyreduced, because this process is performed cold.

Membrane processes are investigated in this thesis for the purpose of characterising pos-

8


sible losses of volatiles during the process. The differences in ethanol and aroma permeationthrough various membranes ranging from nano filtration (NF) to reverse osmosis (RO) areclassified using HPLC and HS-GC-MS. Finally, possible ways of altering the brewing processto compensate for the losses of flavour and aroma compounds during the membrane filtrationwill be discussed. Hopefully, membrane processes will enable a more flavourful AFB andLAB if the losses over the membrane are standardised and hereby correctly compensatedfor during the many steps of brewing and fermentation (Branyik et al., 2012).

Throughout this thesis different technological brewing and process terms might be used,which are not defined directly in the text. For the purpose of simplification and readabilitypossible term or word explanations are assembled in appendix A.1.

9


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10

Chapter 2

Theory

2.1 Sedimentation and Filtration

Separation of solids and yeast from beer, after fermentation, is normally done using sub-sequent methods of clarifications. Different methods such as sedimentation, centrifugation,filtration and membrane processes are applied on beer to obtain different levels of clarity.The clarification methods are often dependent on the wanted level of clarity, shelf life, tastestability, foam stability and uniformity of product based on the customer expectations andequipment availability. The theory of sedimentation and filtration will briefly be mentionedin this thesis as a pretreatment of the beer consequently enabling later membrane filtra-tion without clogging the membranes (Briggs et al., 2004). In the following section theclarification technique applied on Humlefryd will be presented.

Sedimentation of yeast and cold break are done directly in the fermenter aided by cooling,which promotes yeast flocculation and cold break formation.

The speed of passive sedimentation in the fermenter can be described by Stokes Lawgiven as

vg = g ∗ (ρP − ρL) ∗ d2

18 ∗ µ, (2.1)

where vg is the velocity of sedimentation, g the gravitational acceleration, ρP the particledensity, ρL the liquid density, d the particle diameter and µ is the liquid viscosity. Fromequation 2.1 it can be deduced that larger particles with high density will sediment fastin liquids with low density and low viscosity. During fermentation the liquid density andviscosity will be lowered as a consequence of the fermentation of sugars to alcohol. Cooling ofthe beer below temperature of maximum density (2−3oC) will further reduce the density ofthe beer (Kunze, 2010). Additionally, cooling of the fermenter will promote the formationof cold break. Cooling will lower the solubility of the cold break, especially the protein,making the cold break visible, hence the name cold break. Cold break have a particle sizeof approximately 0.5-1mm (Briggs et al., 2004), (Clement et al., 2004).

Yeast cells have a tendency to form flocks, hereby increasing the diameter of the ”flockparticle” enabling sedimentation. Yeast flocculation is highly strain dependent relying onthe expression of certain flocculation genes. Different factors such as nutrient deficiency,calcium concentration, wort oxygen concentration, temperature, pH, ethanol concentration,cell size, cell age and yeast generation can affect the flocculation ability. A clear differencebetween the strains of lager yeast Saccharomyces pastorianus and ale yeast Saccharomycescerevisiae in their flocculation behaviour is indeed the reason for the separation into bottom

11

2.1. SEDIMENTATION AND FILTRATION CHAPTER 2. THEORY

and top fermenting yeast. Lager yeast has a hydrophilic surface whereas ale yeast has ahydrophobic surface. As a result, the hydrophobic surface of ale yeast will interact withhydrophobic CO2 carrying the ale yeast to the top of the liquid, while lager yeast willsediment in the bottom following Stokes law (Walker, 1998). Humlefryd is fermented withlager yeast and will flocculate in the bottom of the fermenter. Nowadays, the design of thecylindroconical vessels (CCV) enables a thermal convection flow, which will force the yeastto the bottom despite the yeast being a top fermenting ale yeast (Verstrepen et al., 2003),(Briggs et al., 2004). The definitions of flow and flux can be found in appendix A.1.

At brewhouse Skands the procedure is to first crop the tank bottom of Humlefryd aftermaturation at 7 − 7.5oC. After cooling to 2.5oC secondary cropping is performed. A finalcropping is carried out after cooling to filtration temperature at −1.5oC. Cropping inthree segments enables sedimentation at three different physical conditions, where differentthermal convection flow, flocculation and cold break formation are induced.

Sedimentation will remove flocculated and sedimented yeast plus cold break. Neverthe-less, additional yeast and particles will still be in suspension causing a unclear final beer.Therefore, filtration is applied for the purpose of obtaining a clear end-product. At brew-house Skands a plate and frame filter using filter sheets for dead-end depth filtration is used.Depth filtration is a mechanical process removing solid particles from a liquid based on threeprinciples; direct interception, inertial interception and electrostatic interactions. Direct in-terception occurs when the particles are retained because they are larger than the pore sizeof the filtration medium. Inertial interception is retention of particles smaller than the poresizes of the filtration medium caused by the momentum of the particle and the fluid flowsurrounding the particle and the filtration medium. Particles can also be retained basedon the surface charge of the particle and the filter medium hereby creating electrostaticinteractions (Kunze, 2010), (Briggs et al., 2004), (Clement et al., 2004), (Smith, 2013a),(Hlavacek and Bouchet, 1993).

The filtration medium used for the dead-end filtration in the plate and frame filter atbrewhouse Skands is BECO depth filter sheets. BECO depth filter sheets contains all naturalmaterials such as cellulose, hardwood, softwood, kieselguhr and perlite plus cationic resins.The filter sheets used are type KD 7, with the following physical data, see table 2.1 (BEKO,2004).

Table 2.1: Physical data on BECO filter sheet. Type KD7, Article no. 22070 (BEKO, 2004)Nominal Thickness Ash Mass Bursting Bursting Water

Retention Content Per unit Strength Strength PermeabilityRate Area Dry Wet ∆p = 1bar

[µm] [µm] [%] [ gm2 ] [kPa] [kPa] [ l

m2∗min ]

1.5 3.8 50.0 1281 > 250 > 50 240

With a nominal retention rate of 1.5µm all particles visible for the naked eye will beremoved, this indicated with a vertical line in figure 2.1. Size of brewers yeast is very straindependent ranging from 2.5-4.5µm in the shortest circumference and up to 10.5-20µm inthe largest. All yeast cells are consequently removed by the filtration along with particleswith a higher circumference than 1.5µm (Kunze, 2010), (Briggs et al., 2004).

Removal of all yeast and the majority of the haze causing particles enables furtherpre-processing of the beer for the removal of alcohol. In the following section membraneprocesses for the purpose of alcohol removal will be evaluated.

12

2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

Figure 2.1: Different separation processes along with particle size retention (Askew et al.,2008).

2.2 Membrane Processes

Conventional liquid filtrations are able to remove particles down to approximately 0.1µmthrough dead-end filtration techniques as illustrated in figure 2.1. For the removal of par-ticles and molecules below 0.1µm molecules membrane processes can be applied (Clementet al., 2004). Membrane processes are based on semi-permeable pressure driven membranefiltrations. Micro filtration (MF), ultra filtration (UF), nano filtration (NF) and reverseosmosis (RO) are different membrane processes. The differences are based on pore sizeand the pressure demand to permeate the membranes as shown in figure 2.2 (Askew et al.,2008), (Hausmann et al., 2013), (Cui et al., 2010). For definition of permeability, permeate,rejection, retentate and retention see appendix A.1.

13


Figure 2.2: Pore size and pressure range for different membrane processes (Hausmann et al.,2013).

Flow Regimes

Membrane processes can be performed using two techniques of filtration, either dead-end orcross-flow filtration as illustrated in figure 2.3 (Cui et al., 2010).

Figure 2.3: Dead-end and cross-flow filtration processes (Smith, 2013a).

Dead-end filtration causes a filter cake build-up which leads to a large pressure differen-tiation over the filter inducing a rapid reduction in the permeate flux. Therefore, Dead-endmembrane filtration is mainly used for MF applications, where the retention amount isminimal e.g. removal of yeast and bacteria in membrane sterilisation. Filter cake build-upcausing a continuous reduction in permeate flux can be reduced by changing the feed flowfrom perpendicular to tangential as illustrated in figure 2.3. Cross-flow filtration involvesa tangential feed flow resulting in a filter cake washing-off or disruption. Consequently,

14


cross-flow filtration will maintain a higher permeate flux throughout the filtration, herebyprolonging the time of filtration. Nevertheless, forcing the entire liquid through the mem-brane as permeate is not possible in cross-flow membrane filtration, which results in a con-centrated suspension denominated the retentate (Smith, 2013a). Dependent on the furtherflow of the retentate, membrane processes can either be continuous or batch. Continuousprocesses involve the entire retentate or proportions continuously removed from the process.This can be done in a single pass, recirculation loop with feed and bleed or in multi-stageprocesses. Batch processes involve a circulation of the retentate until the desired removal orconcentration of compounds have been reached (Smith, 2013b). Considerations concerningbatch processes will only be evaluated.

Reverse Osmosis

The semi-permeable nature of membranes will involve passing of permeable compounds overthe membrane to reach a concentration equilibrium of non-permeable dissolved compoundson both side of the membrane. In the case of membranes, which are only permeable withrespect to water, are the movement of water to reach equal chemical potential called os-mosis. Different chemical potentials on each side of the membrane could be caused by e.g.different concentration of salts on each side of the membrane. This equilibrium is not onlyconcentration dependent, but also dependent on the static pressure on both sides of themembrane. Reversing the flow of permeable compounds from the membrane side with thehigh concentration of dissolved non-permeable compounds to the side with the low concen-tration is possible when pressure is applied. The pressure applied on the non-permeabledissolved compounds side of the membrane, to equal out the osmotic flow of water, is calledthe osmotic pressure (π). Exceeding the osmotic pressure will involve a flow of permeablecompounds against the concentration gradient, hereby obtaining reverse osmosis. A simplecalculation of the osmotic pressure (π) can be done, based on the ideal gas law (PV = nRT )given as

π = icRT, (2.2)

where π is the pressure, i is the ion dissociation correction factor, c is the molar con-centration, R the gas constant and T the absolute temperature (Smith, 2013a), (Clementet al., 2004).

Transmembrane Pressure

Transmembrane pressure (TMP) is the pressure across the membrane enabling separation.For dead-end processes TMP is the difference in the feed pressure (Pf ) to the permeatepressure (Pp). On the contrary, cross-flow membrane processes are influenced by a pressureloss over the course of the membrane area as illustrated in figure 2.4.

15


Figure 2.4: Trans-membrane pressure during cross-flow filtration (Hausmann et al., 2013).

To obtain filtration throughout the length of the membrane module it is important thatthe retentate pressure (Pr) never falls below the permeate pressure (Pp). As a result of thepressure loss over the membrane, the TMP of cross-flow filtration is calculated as follows

TMP = (Pf + Pr

2)− Pp (2.3)

(Hausmann et al., 2013).

Membrane Transport Models

Movement of water and other solutes through different membranes can be described by twodifferent models dependent on the pore size of the membrane. MF and UF are believed tofollow the pore flow model, where the solutes permeates pores in the membrane consequentlyseparated by size. On the other hand, NF and RO follow the solution diffusion model, whereseparations is based on other factors in addition to size. Factors such as polarity, dipolemoment, ionic charge and pH have been proven to affect the rejection of organic moleculesin NF (Smith, 2013a). Furthermore, in RO the rejections of organic molecules have beenproved to be dependent on solubility, acidity and the ability to form hydrogen bonds (Ben-David et al., 2006). Water is indeed believed to permeate RO and NF membranes based ondiffusion of other water molecules in a tetrahedral structure within the membrane formedby hydrogen bonding (Smith, 2013a).

Additional molecules with a similar structure as water might therefore be able to enterand permeate NF or RO membranes. Furthermore, molecules with a similar molecular size,configuration, polarity and hydrogen bonding abilities as the permeable solutes are able topermeate such membranes. In particular small molecules such as methanol, ethanol, ureaand lactic acid might be able to permeate the membrane with selective water permeability.Based on this theory solute passage over the membrane will only be influenced by theconcentration gradient and hence not the pressure, which only will enhance the water passage(Smith, 2013a).

Retention, Rejection and Flux

For a given membrane the retention or rejection (Re) for a specific compound of interest canbe calculated on the basis of the concentrations in the feed stream (cf ) and in the permeatestream (cp), where

Re =cf − cpcf

= 1− cpcf. (2.4)

16


From equation 2.4 it can be deduced that Re = 1 involves a total rejection (100%) of aspecific compound, while Re = 0 involves total permeability (P) (100%) of the compound,where

P = 1−Re. (2.5)

Rejection and permeability are dependent on the operation conditions. Therefore, compar-isons between experiments needs to be done over similar operation conditions. Definitionsof retention, rejection and permeability can be found in appendix A.1. (Smith, 2013a), (Cuiet al., 2010), (Clement et al., 2004).

Flux (J) or permeation rate is the permeation amount (kg) in a given time (min) for amembrane with a given area (m2),

J =Mass of given compound in permeate

Membrane area ∗ Time=

kg

m2 ∗min(2.6)

(Smith, 2013a), (Cui et al., 2010).

Membrane Selectivity

Different polarity between the feed stream and the membrane can influence membranepermeability caused by partitioning. For example a hydrophilic feed stream (beer) filtratedwith hydrophobic membranes could result in attraction of the hydrophobic solutes towardsthe membrane in the feed. Higher alcohols have shown a reduction in rejection possiblycaused by attraction to thin film polyamid (PA) membranes with a hydrophobic surface(Ben-David et al., 2006).

Molecular weight cut-off (MWCO) is often used to characterize a membranes potentialto reject 90-97% of any compound based on the molecular weight. Nevertheless, a charac-terisation based on MWCO will neither account for the steric structure of the molecules,causing an easier passage for linear molecules compared to branched molecules, nor thepolarity effect when considering the solution diffusion model. In addition, the method ofMWCO determination is differing greatly, (Cui et al., 2010).

Diafiltration

Diafiltration (DF) involves addition of a solvent, usually water, to the feed stream, herebyimproving separation of compounds with different rejections in batch systems. The concen-tration of freely permeating substances in the feed stream, e.g. ethanol, can be reduced byadding a carrier solvent. Regular membrane filtration techniques involves a concentrationof the compounds with high rejection factors enhancing possible filter cake build-up. Thiscould result in a higher permeability of unwanted compound into the permeate. Dilutionof the feed stream ensures a constant cross-flow filtration disruption, most often resultingin a reduction of possible filter cake build-up. Furthermore, the osmotic pressure will beapproximately constant when DF is applied. Maintaining a high permeate flux will enhancewashing out of the permeable compounds resulting in a lower final concentration in theretentate stream compared to the concentration involved in regular filtration processes, asillustrated in figure 2.5 (Ferreira et al., 2007) (Hausmann et al., 2013).

17


Figure 2.5: Difference in normal filtration processes (left) compared with diafiltration pro-cesses (right). The diafiltration liquid added (Vd) results in a lower concentration of thepermeating compound in equal retentate volumes (Vr) (Hausmann et al., 2013).

The diafiltration factor is the relation between the diafiltration liquid volume (Vd) addedto the system and the retentate volume produced after filtration (Vr), where

DF =VdVr. (2.7)

DF can be applied continuous or discontinuous respectively involving a constant or a chang-ing volume of retentate. Discontinuous DF often involves a pre-concentration resulting ina desired volume reduction. Continuous DF involves a higher addition of solvent, longerfiltration time and a higher flux of permeate, while the same concentration can be reachedcompared with discontinuous DF. On the contrary, discontinuous DF involves an increasein concentration which can result in a reduction in flux caused by increased viscosity andosmotic pressure. Nevertheless, discontinuous DF will reduce the filtration time and the sol-vent addition for the removal to identical concentration (Ferreira et al., 2007), (Hausmannet al., 2013), (Lopez et al., 2002).

Membrane Structure

The structure of membranes is often divided into isotropic membranes with a symmetricstructure throughout the membrane and anisotropic membranes with asymmetric structuresor layers (Smith, 2013a). Membrane polarity is an important factor involving the perme-ability or so-called wettability. Membranes with polar or hydrophilic surfaces are generallypreferred when operating with an aqueous feed stream, while non-polar or hydrophobicsurfaces are preferred when operating with e.g. protein, oil or other hydrophobic molecule(Hausmann et al., 2013).

18


Membrane Materials

The perfect membrane material should have the following physiochemical properties (Pilipovikand Riverol, 2005):

• High mechanical strength (durable)

• High porosity

• Sharp cut-off (selectivity)

• High temperature and chemical resistance

• Wide pH range

• Cleanable

• High fouling resistance

• High packing density - high membrane area-to-volume ratio

• Long and reliable lifetime

Alfa Laval Nakskov A/S produces anisotropic thin-film composition (TFC) membraneswith the layer compositions shown in table 2.2, (Møller, 2014), (Smith, 2013b).

TFC membranes are built in different layers, as illustrated in figure 2.6, with bottomlayers serving as support for the upper layer actually separating the molecules. An additionalintermediate layer might be introduced, hereby ensuring no penetration of the top layer intothe more porous bottom support layer during manufacturing (Smith, 2013b), (Tang et al.,2009).

Figure 2.6: The general composition of Alfa Laval RO and NF membranes (Møller, 2014)

Polypropylene (PP) and polyesters (PE) are used as support layers because of a generalstrong compaction ability plus high resistance to extreme temperatures and pH. PP andPE membranes can be used for MF and UF applications normally in a isotropic membranestructure (Smith, 2013b).

19


Table 2.2: Alfa Laval TFC membrane layer composition (Møller, 2014)Support layers Name Structure

Polypropylene (PP)

Polyester (PE)

Membrane layers Name Structure

Polysulfone (PS)

Polyethersulfone (PES)

Polyvinylidene fluoride (PVDF)

Cellulose acetate (CA) orCellulose triacetate (CTA)

Polyamide (PA)

Polysulfone (PS) and polyethersulfone (PES) are very resistant membrane polymer ma-terials with a general high strength. In addition, a wide pH range plus high chlorine andtemperature resistance gives PS and PES membranes an advantage when it comes to life-time and cleanability. However, production of PS or PES membranes for RO application isnot possible (Smith, 2013b).

Polyvinylidene fluoride (PVDF) has a good resistance towards hydrocarbons and oxidis-ing agents with a relatively wide pH range. Nevertheless, PVDF membranes do only existin the UF and MF range (Smith, 2013b).

Cellulose acetate (CA) can be used to produce anisotropic membranes for RO applica-tions. CA membranes are polar. The polarity can be altered by acetylations hereby yieldingcellulose triacetate (CTA). As a result of polarity the membrane is hydrophilic enabling agood water permeability, high salt rejection and relatively high strength. The disadvantagesof CA membranes include sensibility towards high temperatures (> 40oC) and high chlorineconcentrations (> 1mgl ) plus a narrow pH range (4-7), hereby impeding proper cleaningin place (CIP) coupled with a long lifetime. In addition, drying of CA membranes causes

20


an irreversible collapse of the spongy structure resulting in a loss of permeability (Smith,2013b).

Polyamide (PA) membranes are mainly used for RO and NF applications by Alfa LavalA/S. PA is synthesised by mixing trimesoyl chloride (TMC) with different amides to formpolymers connected by amide bonds, see figure 2.7 (Tang et al., 2009).

Figure 2.7: Synthesis of polyamide (Tang et al., 2009)

PA membranes are highly chlorine intolerable, only capable of tolerating up to 0.1mgl atpH below eight. On the contrary, PA membranes have a wider pH range (4-10) and highertemperature (< 50oC) tolerance than CA membranes plus the ability of being reused afterdrying. Furthermore, PA membranes have a higher mechanical strength and resistance tooxidants than CA membranes (Smith, 2013b) (Tang et al., 2009).

Ferreira et al. (2007) found CA membranes more efficient for alcohol removal of beercompared to PA membranes during a dialysis membrane process. Comparing different PAand CA membranes by general permeate flux (Jpermeate) and ethanol rejection (Reethanol)a higher flux and ethanol rejection for CA than PA membranes was found (Ferreira et al.,2007).

Lopez et al. (2002) also found a higher flux for CA membranes compared to PA mem-branes, when applying RO for alcohol removal of apple cider. A linear trend was observedbetween a rise in pressure causing an increase in flux for the PA membranes. However, a lossof linearity was observed for CA membranes at pressures above 35bar, which was associatedto a possible membrane compaction. Furthermore, Lopez et al. (2002) did a comparisonbetween aroma retention for CA and PA membranes, with similar NaCl retention, showinga higher ethanol retention for PA membranes as well as aroma retention. Thus, ensuring ahigher aroma quality of the end product when applying PA membranes. This phenomenonwas explained by the difference in polarity of the membranes, with the CA membranes beingmore polar than the PA membranes. Therefore, a higher concentration of the polar organiccompounds, such as water and ethanol, could be attracted to the more polar CA membranelayer causing higher permeation or lower rejection. PA membranes are more hydrophobic ornon-polar than CA membranes and would therefore repel the polar organic compounds whileattracting non-polar organic compounds Lopez et al. (2002). In addition, PA membranesdo have a higher mechanical strength than CA membranes with a tendency of compactionand collapsing if drying occurs. Finally, PA membranes show a higher durability concerningboth pH and temperature resistance enabling a proper CIP and a longer life time (Smith,2013b).

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Membrane Calculations

The solution diffusion theory can be used to describe RO and NF membranes, because theycan be viewed as a non-porous homogeneous wall in which diffusion of certain compoundsare possible, as mentioned earlier. This theory indicates that the flux of solvent (water)and solute (eg. ethanol and aroma compounds) are independent of each other. Therefore,the flux of water through the membrane (Jwater, [ kg

m2∗min ]) is dependent on the TMP ([bar])exceeding the difference in the osmotic pressure over the membrane (∆π, [bar]) and the char-acteristics of the membrane itself along with the permeation liquid (Bwater, [ kg

m2∗min∗bar ]),given as

Jwater = Bwater ∗ (TMP −∆π) (2.8)

(Hausmann et al., 2013). When operating a membrane filtration with high selectivity forwater, in diafiltration mode, the flux of water (Jwater) is approximately equal to the fluxof permeate (Jpermeate), which can be found experimentally using the mass of the permeate(mpermeate, [kg]), the area of the effective membranes (Amembrane, [m2]) and the given time(t, [min]), given as

Jwater ≈ Jpermeate =mpermeate

amembrane ∗ t(2.9)

(Ferreira et al., 2007). Solutes, which are partially retained and partially able to permeatethe membrane primary dependent on the concentration on each side of the membrane, e.g.ethanol and aroma compounds, can be described by

Jsolute = Bsolute ∗ (cf − cp) = Bsolute ∗∆csolute, (2.10)

where Jsolute [ kgm2∗min ] is the flux of solute through the membrane, Bsolute [ m

min ] is thesolute transport coefficient, cf is the concentration in the feed stream and cp is the concen-

tration in the permeate [ kgm3 ]. Jsolute is not influenced by the TMP, but only dependent on

the concentration difference in the permeate and the feed stream, as observed from equation2.10. However, pressure has been observed to influence the flux of solutes (Ferreira et al.,2007), (Hausmann et al., 2013), (Clement et al., 2004).

The solute transport coefficient Bsolute is dependent the solute diffusion coefficient ofthe membrane (DAM , [ m

2

min ]), the membrane thickness (δ, [m]) and the distribution ratiobetween the membrane and the solution (K), resulting in

Bsolute =DAM

K ∗m(2.11)

(Lopez et al., 2002), (Alvarez et al., 1998). According to Lopez et al. (2002) and Alvarez et al.(1998) the solute transport coefficient is dependent on the chemical nature of respectivelythe membrane and the solute

ln(Bsolute) = ln(C∗) + ρ∗ ∗ σ∗ + δ∗ ∗ Es∗ + ω∗ ∗ s∗, (2.12)

where C∗ is a membrane constant, σ∗ the polar Taft number, Es∗ the steric Taft number ands∗ the Small’s number concerning hydrophobicity. ρ∗, δ∗ and ω∗ are coefficients associatedwith the importance of the multiplied numbers (Lopez et al., 2002), (Alvarez et al., 1998),(Taft, 1952).

The membrane composition constant (C∗) is important when comparing various mem-branes. This value can only be found experimentally (Lopez et al., 2002).

22

2.3. EXPERIMENTAL CONSIDERATIONS CHAPTER 2. THEORY

The polar Taft number illustrates the tendency of a specific molecule to have a generalnegative or positive polarisation resulting in a difference in acidity. A negative Taft numberindicates a more alkaline behaviour of the molecule, while a positive indicates a more acidic.The steric Taft number is a measure for the steric hindrance of the molecule. The stericTaft number assumes only negative values. A decrease in value is equivalent to an increasein steric hindrance (Lopez et al., 2002) The polar and steric Taft effects are calculatedon behalf of the different substituents (R-groups) effect on the hydrolysis of methyl ester(R−COOCH3) (Taft, 1952).

The Small’s number is a measure of the hydrophobicity of the compound assuming onlypositive values. The higher the Small’s number the more hydrophobic the compound is(Lopez et al., 2002). However, the Small’s number is only considered when the solutescontains a hydrocarbon chain above three carbons in length (Alvarez et al., 1998).

Dickson et al. (1975) found that ethers were separated as a function of the steric effectswhile ketones, aldehydes and alcohols were separated based on both the polar and stericeffects during RO filtration using polyamide (PA) membranes.

A calculation of Bsolute is only possible when the concentration on each side of themembrane, at a current moment in stable state, is known, as illustrated in equation 2.10.Nevertheless, only the concentration of ethanol was measured, which only enabled a calcula-tion of Bethanol. On the contrary, the aroma compounds were measured in comparison witheach other enabling an insight in possible difference in permeation. In the discussion, thedifference in permeation of aroma compounds will be compared to values of the polar Taftnumber (σ∗), the steric Taft number (Es∗) and the Small’s number (s∗) found in literature.

2.3 Experimental Considerations

The quantity of literature concerning AFB production using membrane processes is verylimited as a result of this being a fairly new approach. In the light of this, the experimentalapproaches were often based on intuitive thoughts and on own experience together with theskilled and experienced engineers from Alfa Laval A/S. In figure 2.8 the optimal discontin-uous diafiltration process found, in cooperation with these engineers and as a result of thelimitations of the experimental set-up, can be observed.

The first stages of discontinuous diafiltration is pre-concentration of the beer, wherethe beer is recirculated within the batch system until the wanted reduction in volume hasoccurred, see the green line figure 2.8. The volume reduction can be described by the volumeconcentration factor [VCF] for the pre-concentration

V CFPre−conc =VstartVDia

, (2.13)

where Vstart is the initial volume and VDia is the volume maintained during the diafiltrationstage. The ethanol concentration will rise during the reduction of the retentate volume, seethe blue line in figure 2.8. The rise in ethanol concentration is a result of a higher membranepermeability for water compared to ethanol. The degree of pre-concentration is dependenton the wanted viscosity of the liquid along with the layout of the set-up. In figure 2.8 apre-concentration degree of 2 is observed resulting in reduction in volume from 5 to 2.5litres.

The second stage is called the diafiltration stage, where the retentate volume is keptconstant by addition of dia-water (red line) in the same rate as permeate (purple line) is

23


Figure 2.8: Ideal discontinuous diafiltration process divided into different stages. A mathe-matical approximation of the membrane trends. Data used: Feed volume beer = 5 l, initialalcohol concentration = 5.5%ABV, measured flux (J) through the membrane = 0.519 l

m2∗min ,

flow retentate = 12.8 lmin , membrane permeability = 85% and membrane area = 0.072m2.

removed from the system. During the diafiltration stage the concentration of alcohol willsteadily decrease in an exponential trend, where the rate of alcohol removal will be reducedas the concentration becomes lower. This phase illustrate the limitation of membrane re-moval of alcohol from beer. Reaching an alcohol concentration below 0.5%ABV is possible,however costly in respect to both capital expenditure (CAPEX) and operational expenditure(OPEX).

The third an final stage is the re-dilution or final concentration stage. This change involume can also be described using VCF

V CFFinal−conc =VstartVFinal

, (2.14)

where Vfinal is the volume after re-dilution. Normally a re-dilution back to the originalvolume of beer is carried out, hereby reducing the final concentration of alcohol obtaininga VCF of 1, as seen in figure 2.8. The complete re-dilution back to 5 litres results in a finalalcohol concentration of 0.5%ABV.

The trends observed in figure 2.8 are based on consideration observed in figure 2.9 andexperimental data obtained during membrane filtrations. An approximate calculation wasperformed predicting circulations (loops), time and diafiltration volume needed to obtainthe wanted end alcohol concentration. This was used as an indication for the experimentalset-up and as a validation of the membrane potential for alcohol removal. Some simplifica-tions were made for this calculation to be possible. One litre of beer was estimated to beequal to one kg (kg = l). The mean permeate flux through the membrane was used, as itnever reach a stable level. Finally, the measured water flow, before loading the unit withbeer, was used assuming the same flow for the beer. In figure 2.9 the data found experi-mentally can be observed.

24


Figure 2.9: Batch system outlook and corresponding feed, membrane, retentate, permeateand additional data needed for an approximated calculation of the membrane system.

Data input for approximation of membrane filtration, see figure 2.9:

• Initial feed volume (Vstart) [l]

• Initial feed alcohol concentration (C(0)) [gl ]

• Water flow of retentate (Fretentate) [ lmin ]

• Membrane ethanol permeability (Ppermability) [%]

• Membrane area (Amembrane)[m2]

• Mean flux of permeate through the membrane (Jmembrane) [ lm2∗min ] .

• Pre-concentration degree (V CFPre−conc)

Based on the data input described above and consideration related to figure 2.9, a predic-tion of the change in concentration, retentate volume, time per stage and dia-water volumefor the three different stages can be calculated. As a result of the batch configuration achange in alcohol concentration, volume and circulation time must be considered for eachrun-through (loop) over the membrane. For this purpose two different calculation loopswere performed in Matlab corresponding to respectively the pre-concentration stage andthe diafiltration stage. In appendix A.2 the set-up in Matlab is described for the calculationof the same filtration trend illustrated in figure 2.8. In the following section the backgroundof the calculations will be considered:

1. Pre-concentration loopEach run-through or circulation (k) is described setting up the following boundaries.

25


• While the volume (V (k)) is above the wanted pre-concentration volume an additionalcirculated (loop) shall be calculated, hereby stopping the pre-concentration loop whenreaching a specific volume dictated by the V CFPre−conc [l]

V (k) >Vstart

V CFPre−conc. (2.15)

• Time per circulation (t(k)) of the retentate is dependent on the volume change (V (k))while the flow (Fretentate) is kept constant by the pump settings [min]

t(k) =V (k)

Fretentate. (2.16)

• Alcohol concentration change (C(k)) in the retentate is dependent on the change ingrams of alcohol in relation to the change in volume of the retentate [gl ]

C(k + 1) =C(k) ∗ Fretentate ∗ t(k)− C(k) ∗ (

Ppermeability

100 ) ∗ Jmean ∗Amembrane ∗ t(k)

(Fretentate − Jmean ∗Amembrane) ∗ t(k).

(2.17)

• Volume change (V (k + 1)) in the retentate is dependent on the initial volume goinginto the loop minus the permeate volume going out of the loop [l]

V (k + 1) = V (k)− Jmean ∗Amembrane ∗ t(k). (2.18)

The output of the pre-concentration loop will reveal the following process parameters:

Overall time of the pre-concentration stage: tpre−con = sum(t)The amount of loops or circulations: kRetentate volume: V2 = V (k − 1)Concentration of alcohol: C2 = C(k)Time of the final loop: t2 = t(k − 1)

2. Diafiltration loop

• The inputs into the diafiltration stage are outputs from the pre-concentration stageV2, C2 and t2. However, during this loop the limitation is not reaching a final volumein the retentate, but on the contrary reaching a final concentration of alcohol (Cend).In this set-up, the final alcohol concentration will be reached after re-dilution. Theamount of re-circulations or loops in the diafiltration step is described by (n)

C2(n) >Cend

V CFPre−conc. (2.19)

• Maintaining a constant volume during the diafiltration (V2) will result in a constanttime for each loop (t2), because the flow (Fretentate) is kept constant. The only variableper loop is hereby the change in alcohol concentration because of the permeation ofalcohol [gl ]

26


C2(n+ 1) =C2(n) ∗ Fretentate ∗ t2 − C2(n) ∗ (

Ppermeability

100 ) ∗ Jmean ∗Amembrane ∗ t2V2

.

(2.20)

The output of the diafiltration loop will reveal the following process parameters:

The amount of loops or circulations: nTotal time of the membrane filtration: ttotal = tpre−con + t2 ∗ nConc. alcohol before re-dilution: C3 = C2(n)Final conc. alcohol after re-dilution: Cfinal = C3

V CFPre−conc

Volume of dia-water: Vdia = Jmean ∗Amembrane ∗ t2 ∗ nDiafiltration factor: DF = Vdia

V (1)

This approximation will enable a process adjustment without having to run numerousmembrane filtrations. It can be used as an evaluation mechanism revealing the optimaltrends and furthermore evaluating the actual trends. Evaluation of actual trends was usedto describe the membrane potential for alcohol removal by calculating e.g. the ethanol per-meability percentage (Ppermeability). The input data throughout the membrane filtration,such as membrane area (Amembrane), could be adjusted simply by adding additional mem-branes. The flow (Fretentate) could be changed by the speed of the pump. The alcoholpermeability could be changed by changing membrane, TMP, flow or diafiltration degree.

27

2.4. AROMA FORMATION CHAPTER 2. THEORY

2.4 Aroma Formation

During the process of brewing many volatile organic compounds (VOC’s) are producedresulting in a unique flavour and aroma composition of beer.

Beer is a complex matrix of various aroma compounds, nonetheless the main constituentis water in concentration of 91-98%. Volatile organic compounds (VOC’s) are most oftencompounds, which have a tendency to exist on gaseous form because of low solubility in thematrix where they exist. In beer this volatility is induced by the compounds existing in ahigh water concentration where the VOC’s solubility is low (Briggs et al., 2004).

In the following sections the formation of specific VOC’s will be related to the differentprocesses in beer production along with possible ways to alter VOC composition by changingphysical process parameters. Focus will be given on the fermentation process, where physicalparameters are easiest altered, and the raw materials, which can be altered by higher initialaddition.

2.4.1 Yeast Metabolism

A short introduction to the metabolism of yeast is needed for the purpose of understand-ing the production of various VOC’s during beer fermentation. Two strains of yeast arenormally used for beer production namely Saccharomyces cerevisiae for ale production andSaccharomyces pastorianus for lager production. Brewers yeasts are eukaryote unicellularfungi, reproducing by budding. They belong to the Phylum of Ascomycota, the Family ofSaccharomycetaceae, the Genus of Saccharomyces and finally respectively Species of cere-visiae and pastorianus with subset strains (Walker, 1998), (Briggs et al., 2004)).

Lager yeast (Bottom yeast) - Saccharomyces pastorianus, (Walker, 1998)

Melibase positive

No growth at 37oC

Flocculates at the bottom because of hydrophilic surface - dose not integrate CO2

(hydrophobic)

Ale yeast (Top yeast) - Saccharomyces cerevisiae , (Walker, 1998)

Melibase negative

Growth at 37oC

Flocculates at the top because of hydrophobic surface - does integrate CO2

Brewers yeasts are facultative aerobic fermenters involving the ability to perform metabolicreactions both oxidative in aerobic conditions and fermentative in anaerobic conditions (La-gunas, 1986).

Carbohydrates are the main source of energy in brewing yeast. They are degraded toglucose or other monosaccharides, which can enter the glycolysis in different stages. Yeastglycolysis is the main source of aroma VOC’s formation as illustrated in figure 2.10.

28


Figure 2.10: Aerobic and anaerobic glucose metabolism involved in the formation of flavourcompounds. ATP = Adenosine triphosphate, ADP = adenosine diphosphate, NAD+ =Nicotinamide adenine dinucleotide, NADH + H+ = Reduced NAD+, CoA-SH = CoenzymeA, AA = Amino acids, VDK = Vicinal diketones, FAD+ = Flavin adenine dinucleotide,FADH + H+ = Reduced FAD+, GTP = Guanosine triphosphate and TCA = TricarboxylicAcid Cycle (Kunze, 2010).

Enzymatic reduction and oxidation are important for different metabolic reactions tooccur. Nicotine amide adenine dinucleotide (NAD+) is a cofactor often used in enzymatic

29


reactions functioning as an oxidising agent (NAD+−→ NADH + H+), or as a reducingagent (NADH + H+−→ NAD+). Many VOC’s come from the glycolysis, which is illus-trated in figure 2.10. During the course of the glycolysis, ending with the formation of twopyruvate, a net formation of two ATP molecules occurs. Furthermore, a net reduction of2 x NAD+−→ 2 x (NADH + H+) is observed. During alcoholic fermentation the yeastneeds to re-oxidise the 2 x (NADH + H+)−→ 2 x NAD+ for the redox balance to bemaintained and hereby enable additional glycolytic reactions. The redox balance can alsobe maintained by producing NAD+ in the formation of glycerol. These catabolic reactionsare called substrate level phosphorylation (Briggs et al., 2004).

During aerobic respiration the end product of the glycolysis, pyruvate, will be furtherprocessed yielding 28 ATP and re-oxidising the NADH + H+ and FADH + H+ formed inthe electron transport chain by the aid of oxygen, see table 2.3 (Hammond, 1993), (Walker,1998), (Briggs et al., 2004), (Madigan et al., 2012).

Table 2.3: Energy formation for brewers yeast in aerobic and anaerobic conditions (Walker,1998).

Condition Metabolism Redox EnergyBalance Output

Aerobic Glycolysis 2 (NADH + H+) 2 ATPPyruvatedecarboxylation 2 (NADH + H+)TCAcycle 6 (NADH + H+) + 2 (FADH + H+)) 2 ATP (2 GTP)Oxidativephosphorylation 10 NAD+ + 2 FAD+ 24 ATPTotal 0 28 ATP

Anaerobic Glycolysis 2 (NADH + H+) 2 ATPFermentation 2 NAD+

Total 0 2 ATP

Aerobic metabolism yields the most ATP and is therefore expected to be the preferredmetabolism compared to anaerobic fermentation as observed in figure 2.10 and table 2.3.On the contrary, brewing yeast has shown the ability to perform both respiration andfermentation in aerobic conditions also called respirofermentation, where

Glucose + O2 −−→ Cells + Ethanol + CO2·

The respirofermentative degradation of glucose in aerobic conditions is related to the so-called Crabtree effect of brewing yeast. The Crabtree effect is the suppression of yeastrespiration in high glucose concentrations which results in fermentation predominating res-piration metabolism of carbohydrates. In addition, the Pasteur effect can, in aerobic con-ditions with low glucose concentration and limited yeast growth, result in a suppression ofthe fermentation metabolism hereby yielding more ATP through respiration (Walker, 1998).

Different strain dependent technological characters are in addition to the general glu-cose metabolism of brewers yeast important in beer production. The following technologicalcharacters are relevant (Walker, 1998), (Briggs et al., 2004):

30


Flocculation (Walker, 1998), (Briggs et al., 2004)

• Aggregation or adhesion in clumps and sedimentation in the bottom because of hy-drophilic surface of the yeast

• Cellular bridging between cell wall lectins (flocculins) and mannoproteins on adjacentcell involving Ca2+ ions

• Initiated by Ca2+ ions

• Inhibited by mannose and glucose

• Strain dependent expression of specific FLO genes

Reproduction (Walker, 1998), (Briggs et al., 2004)

• Propagation - Biomass production and adaptation to brewing conditions

• Pitching - Rate, aeration, generation and initial stress factors such as osmotic pressure,temperature, pressure and pH

• Submerged liquid batch fermentation growth phases

Lag - Adaptation time in new environment

Acceleration - Rate of specific growth (µ) is accelerating

Exponential - Logarithmic cell doubling with the specific growth rate (µmax)

Declining - Rate of µ is decelerating.

Stationary - Yeast biomass is constant death rate equals growth rate

Death - Death rate exceeds the growth rate

• Viability - Yeast cells alive or dead

• Vitality - Yeast cells physiological condition

The different growth phases of yeast can be observed graphically in figure 2.11 (a) as thesuspended cell count. Figure 2.11 (a-c) illustrates the connection of the glucose metabolism,cell growth, nitrogen assimilation, acid formation and aroma formation during a regularlager production.

31


Figure 2.11: Development of different substances during batch fermentation of a lager at15oC. (a) Present gravity (PG) = 1.000+0.004∗[%Plato]. (c) FAN = Free Amino Nitrogen.(d) H. alcohols = Higher alcohols (HA) and VDK = Total vicinal diketones (Briggs et al.,2004).

2.4.2 Fermentation Products

Higher Alcohols

Higher alcohols (HA) or fusel alcohols are mainly viewed as off flavours in lager production,while considered as a positive attribute in ales. In table 2.4 the most essential HAs areillustrated.

Table 2.4: Higher alcohols in beer, aroma threshold and corresponding concentration inlager beer (EBC, 2000), (Tan and Siebert, 2004), (Fenaroli, 2005).

Compound Compound Aroma Aroma Conc.Name Structure Threshold or Range

[mgl ] Taste [mgl ]

1-Propanol 800 Alcoholic 7-9

1-Butanol 450 Alcoholic 6-11

1-Octanol 0.9 Orange -Rose

2-Methylpropanol 200 Alcoholic 4-20(Isobutanol)

(Isobutyl alcohol)

3-Methylbutanol 70 Pungent 25-75(Isoamyl alcohol)

2-Phenylethanol 125 Roses 11-51(Benzene ethanol) Sweet

32


Formation of HAs is a result of amino acid metabolism as illustrated in figure 2.12. Thecatabolic pathways for degradation of assimilated amino acids, described by the Erhlichpathway, results in the formation of HAs. In addition, the anabolic pathways, originatingfrom pyruvate, can also result in the formation of HAs (Hazelwood et al., 2008).

Figure 2.12: Formation of HA by anabolic routes from pyruvate and catabolic routes fromassimilated amino acids through the Ehrlich pathway (Hazelwood et al., 2008).

After assimilation of amino acids a direct usage of the amino acid in protein synthesiscan occur. Degradation of the assimilated amino acids is also observed in figure 2.12,which involves a transamination where the amino group are removed by aminotransferaseinvolving a amino acceptor or enzyme cofactor. In brewing yeast the main amino acceptor isα-oxoglutarate, producing glutamate. In addition, other amino acceptors such as pyridoxalphosphate producing pyridoxamine phosphate and pyruvate producing alanine are observed.The transamination results in a corresponding α-keto acid, see table 2.5. When amino acidsdeficiency occurs, or amino acids not assimilated by the yeast are needed, a biosynthesispathway originating from pyruvate is possible. From pyruvate the wanted α-keto acid can besynthesised, which hereafter can obtain an amino group through the reverse transamination

33


where glutamate mostly function as an amino donor in yeast (Hazelwood et al., 2008).The different α-keto acids make up the oxo-acid pool of yeast, see table 2.5, from which

the formation of fusel aldehydes occur enzymatically using α-keto acid decarboxylate. Fuselaldehydes are able to diffuse out of the yeast cell (Briggs et al., 2004).

The fusel aldehydes can be oxidised to fusel acids or reduced to fusel alcohols (HA)dependent on the growth conditions. 90% of the fusel aldehydes will be reduced by al-cohol dehydrogenase to HAs in anaerobic condition with high glucose concentration andphenylalanine as the only nitrogen source. This reaction is valuable in anaerobic conditionsbecause of maintenance on the cellular redox balance, which has a tendency to have sur-plus NADH + H+ in anaerobic conditions. Glycerol formation is another pathway yieldingNAD+, however ATP is needed in this pathway hereby making the Ehrlich pathway moreenergetically favourable (Hazelwood et al., 2008), (Walker, 1998). The specific HA formedis dependent on the specific amino acids assimilated as illustrated in table 2.5.

In aerobic conditions, with limited glucose concentration and various nitrogen sources, anoxidation of the fusel aldehyde to fusel acid has a higher tendency to occur. Once again, forthe purpose of maintaining a redox balance which has a tendency towards surplus NAD+,caused by respiration. HAs are believed to be exported by the yeast cell using passivediffusion, while fusel acids involve a plasma membrane transporter (Hazelwood et al., 2008),(EBC, 2000), (Walker, 1998), (Briggs et al., 2004).

Table 2.5: Amino acids and corresponding alcohols as a result of Ehrlich pathway (Hazel-wood et al., 2008), (EBC, 2000).

Amino acid α-keto acid/ Oxo-acid Alcohol

Alanine Pyruvic acid EthanolThreonine α-ketobutyrate n-PropanolValine α-ketoisovalerate 2-MethylpropanolIsoleucine α-keto-β-methylvalerate 2-MethylbutanolLeucine α-ketoisocaproate 3-MethylbutanolPhenylalanine Phenylpyruvate 2-PhenylethanolTyrosine p-Hydroxyphenylpyruvate TyrosolTryptophan 3-Indole pyruvate Tryptophol

From figure 2.11 (d) it can be seen that the formation of HAs occurs mainly during theyeast initial growth phase, while assimilable free amino nitrogen (FAN) is still present inthe suspension, see figure 2.11 (c). For FAN explanation see appendix A.1. The formationof HAs seems to be stagnating when all possible FAN are assimilated and the yeast entersthe stationary growth phase. This reveals the formation of HA mainly being a result ofthe catabolic degradation of amino acids in the Ehrlich pathway. Consequently, factorsaffecting the levels of HA are closely related to elevated yeast growth and therefore higherassimilation of amino acids. The yeast strain is also an important factor involving differentgene expressions resulting in a higher HAs formation in ale yeast than in lager yeast (Briggset al., 2004). A higher yeast vitality will involve a higher metabolic fitness hereby resultingin a higher formation of HAs. Thus, factors elevating yeast growth will also be factorselevating HA formation (Hammond, 1993).

Pitching is important for HAs formation. Yeats pitching must be of a certain volumeto obtain a proper growth and fermentation. Insufficient yeast growth related to a lowpitching rate is believed to be caused by a lack in inter-cellular signalling to activate cell

34


multiplication. The Ehrlich pathway seems to be part of a yeast growth signalling sys-tem equivalent to bacteria quorum sensing. Especially 3-methylbutanol (isoamyl alcohol)and 2-phenylethanol have been found as signalling molecules inducing yeast growth and/orenvironmental adaptation (Hazelwood et al., 2008), (Walker, 1998). The following processparameter can be used to influence the HA formation:

Process ParametersInducing HA formation, (Briggs et al., 2004)

• High FAN

• High fermentation temperatures

• High wort aeration

• Continuous agitation (Iso-mix)

• Topping up

• High gravity beer fermentation (HGB)

• Yeast strain - ale yeast has a higher HA formation

Reducing HA formation, (Briggs et al., 2004)

• Increase pitching rate causing low growth

• Low fermentation temperatures

• Pressure fermentations

• Low aeration

• Low FAN

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Esters

Esters are considered one of the most important flavour contributors in beer responsible formainly fruity, floral and solvent-like flavours, as illustrated in table 2.6.

Table 2.6: Esters, aroma threshold and corresponding concentration in lager beer. Ethylesters (E) and acetate ester (A) (EBC, 2000), (Tan and Siebert, 2004), (Fenaroli, 2005).

Compound Ester Compound Aroma Aroma Conc.Name Type Structure Threshold or Range

[mgl ] Taste [mgl ]

Ethyl acetate E+A 30 Fruity, Solvent 8-32

Ethyl propionate E 25 Rum, Pineapple 5-84

Propyl acetate A 10 Fruity, Banana, 7-22Honey

Isobutyl acetate A 1.6 Fruity, Floral, -Pear, Hyacinth

Ethyl butyrate E 0.4 Fruity, Pineapple -

Isoamyl acetate A 1.2 Fruity, 0.3-3.8Banana, Pear

Ethyl caproate E 0.21 Apple, 0.05-0.3(Ethyl hexanoate) Aniseeds

Hexyl acetate A 3.5 Fruity, -Apple, Pear

Ethyl heptanoate E 0.4 Cognac, Wine, -Brandy

Ethyl caprylate E 0.9 Apple 0.04-0.53(Ethyl octaanoate)

Octyl acetate A 0.5 Neroli, Jasmine, -Peach

Ethyl caprate E 1.5 Grape, Cognac, -(Ethyl decanoate) Brandy

2-Phenylethyl A 3.8 Rose, Honey, 0.10-0.73acetate Apple, Sweetish

In table 2.6 some aroma thresholds appear higher than possible detected in lager beer.However, these aromas are still considered important in aroma contribution because of the

36


synergistic aroma effect with the additional esters (Verstrepen et al., 2003).

Figure 2.13: Formation of ester by enzyme-catalysed coenzyme A (CoA-SH) condensation.R = hydrocarbon side chain (Verstrepen et al., 2003).

In figure 2.13 the overall enzyme catalysed CoA-SH condensation reaction between analcohol and a acyl-group resulting in the formation of ester can be observed. Dependent onthe hydrocarbon side chains R2 and R1 various different esters can be formed (Verstrepenet al., 2003).

The aroma active esters in beer shown in table 2.6 can be divided into two subgroupsbased on a difference in substrates (Verstrepen et al., 2003), (Saerens et al., 2008):

1. The acetate esters (A) - The acid group is acetate (R1−−CH3) while the alcoholgroup is ethanol or a HA.

2. The ethyl esters (E) - The acid group is a medium chain fatty acid (MCFA) or aoxo-acid (R1−−CH2R

3) while the alcohol group is ethanol (R2−−CH3).

The most abundant ester is ethyl acetate formed in the reaction between ethanol andacetyl-CoA. Ethanol is obtained directly as a result of substrate level phosphorylation ofcarbohydrates. Other alcohols involved in ester formation are obtained as a result of HAformation as previously described. In addition, the formation of acetyl is obtained directlyas a result of pyruvate decarboxylation with CoA-SH as cofactor, see figure 2.10. Theformation of acyl-CoA is a result of fatty acids reaction with CoA-SH during fatty acidcatabolism and anabolism or oxo-acid reaction with CoA-SH (Verstrepen et al., 2003).

The enzymatic condensation reactions, shown in figure 2.13, are catalysed by manydifferent enzymes, based on the substrates, where alcohol acetyl transferase I (AATase I)and II (AATase II) are the most characterised. The rate of ester formation is generallydependent on the abundance of the two substrates and the associated enzyme activity(Verstrepen et al., 2003).

Many different theories concerning the metabolic function of ester formation have beenproposed. Ester formation could be a mechanism for regulation of the acetyl-CoA to freeCoA-SH ratio closely related to lipid synthesis. A decrease in lipid synthesis could inducean increase in concentration of intracellular acetyl-CoA hereby resulting in a rise in esterformation for the purpose of maintaining a constant acetyl-CoA to free CoA-SH ratio. Adecrease in ester formation have been reported when supplements of the unsaturated fattyacid, linoleic acid, to wort occur. This suggests a inhibitory effect of fatty acids on estersynthesis. Furthermore, increasing the aeration of the wort, to prolong the lipid synthesis in

37


yeast, has shown to decrease ester formation. In figure 2.11 (d) the rate of ester formationis highest right before reaching a peak corresponding to the approximate time where lipidsynthesis rate decreases and the yeast enters a stationary growth phase (Briggs et al., 2004),(Hammond, 1993). Another possible metabolic function of ester formation is a detoxificationmechanism. Fatty acids with a carbon length between C8 to C14 have shown a high toxiceffect in yeast, especially if these fatty acids are unsaturated. A possible way to overcomethe toxicity of these fatty acids is through fatty acids activation with CoA-SH followed byester formation, in reaction with alcohols (Hammond, 1993).

The difference in ester production observed among different yeast strains is believedto be caused by different AATase activity and cell membrane compositions. The acetateesters are lipid soluble, hereby enabling rapid diffusion through the cell membrane into thefermentation medium. On the contrary, the ethyl esters do not diffuse so easily through thecell membrane with a decreasing ability to pass the cell membrane with increasing chainlength (R3). As a result, diffusion of ethyl caproate will happen in a higher rate than ethylcaprylate, while even longer chain esters remain in the cell. The transport of esters throughthe cell membrane is species dependent. Lager yeast has show a higher tendency of retainingester within the cell resulting in a lower ester concentration in the final beer (Verstrepenet al., 2003), (Saerens et al., 2008).

Specific gravity of the wort has revealed itself as an important factor related to esterformation. High gravity beer (HGB) fermentations have in recent years become an importantindustrial approach for the purpose of increasing the overall productivity in order to reducecosts. Nevertheless, HGB results in formation of more ester because of the higher sugarconcentration (Briggs et al., 2004). The composition of the wort carbohydrates has an effecton ester formation involving glucose and fructose rich worts producing more esters thanmaltose rich worts. A higher acetyl-CoA and higher alcohols (HAs) formation involved withyeast growing on glucose and fructose compared to maltose could be the reason for a higherester formation. In addition, glucose could induce stronger expression of ester synthasegenes (Saerens et al., 2008).

Oxygen depletion results in a decreasing yeast growth mainly observed in the stationarygrowth phase. Oxygen is essential for yeast production of membrane sterols and unsaturatedfatty acids. At the point of oxygen depletion the formation of HA and acetyl-CoA continuousresulting in a higher formation of ester. Free amino nitrogen (FAN) is an important factorfor the formation of HA as described in section 2.4.2. For this reason, the presence of FAN’sduring growth limitation or oxygen depletion is important for ester formation. Addition ofamino acids, assimilated by brewers yeast, during the stationary phase has been proposedas a way of manipulating the final ester production (Saerens et al., 2008).

High temperatures induce higher ester production. Esters show different temperaturedependency making the fermentation temperature a powerful tool for ester manipulation.The temperature dependency of ester formation is believed to be related to a higher AATaseactivity as well as a higher HA production. On the contrary, high temperatures duringfermentation will result in a higher evaporation rate of volatile organic compounds (VOC’s)(Verstrepen et al., 2003), (Saerens et al., 2008).

High top pressure caused by hydrostatic pressure in high fermenters or top pressureresults in a lower ester production as well as a decrease in yeast growth. Increasing thepressure will result in a higher concentration of dissolved CO2 in the suspension. Thiswill influencing the equilibrium of enzymatic decarboxylation reactions essential for growthand ester formation (Verstrepen et al., 2003), (Saerens et al., 2008). The following processparameter can be used to influence the ester formation:

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Process ParametersInducing ester formation (Verstrepen et al., 2003), (Saerens et al., 2008)

• Increasing gravity - High gravity brewing (HGB)

• Increasing attenuation

• Restricting wort aeration

• High fermentation temperature

• Less pressure - Horizontal fermenters

• Zinc addition - Yeast growth factor which stimulates HA production

• High glucose and fructose concentration

• Drauflassen or topping up without aeration - Longer AATase activity

• High FAN

Reducing ester formation (Verstrepen et al., 2003), (Saerens et al., 2008)

• Lower wort gravity

• Lower attenuation

• Increased wort aeration

• Agitation or movements during fermentation will increase yeast growth and thereforereduce level of esters

• Pressure increase

• High maltose concentration

• Low FAN

• Lipid or fatty acids addition

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Aldehydes

Aldehydes in beer are mainly considered off flavours connected to either unmature and youngbeer or to mature stale and oxidised beer. Nonetheless, some aldehydes can contribute withpositive flavours and aromas from the raw materials used in the brewing process. In table2.7 the main aroma aldehyde contributors in beer is presented.

Table 2.7: Aldehydes, aroma threshold and corresponding concentration in lager beer (EBC,2000), (Tan and Siebert, 2004).


[mgl ] Taste [mgl ]

Acetaldehyde 25 Grassy 2-20Young beerApple-like

(E)-2-Nonenal 0.00011 Papery 0.00005Cardboard - 0.00015Oxidized

Furfural 150 Caramel -Bready

Cooked meat

Isobutyraldehyde 1 Green malt 1-2(Isobutanal) Wet cereal

(2-Methylpropanal) HuskyStraw

Benzaldehyde 2 Marzipan 1-10Bitter almond

Formation of aldehydes during beer production and ageing can be divided according tothe mechanism of formation:

1. Glycolysis byproduct - Acetaldehyde

2. Fatty acid oxidation product - (E)-2-Nonenal

3. Maillard reaction product - Furfural

4. Strecker degradation product - Isobutyraldehyde and Benzealdehyde

Acetaldehyde is a glycolysis intermediate formed as a result of the enzymatic decarboxy-lation of pyruvate, as illustrated in figure 2.10. As a part of the anaerobic metabolism ofcarbohydrates, the formation of acetaldehyde is high during the initial exponential growthphase where carbohydrates are in excess. Boulton (1991) suggested that the excretion ofacetaldehyde is a way of controlling the intracellular levels, which is toxic in high con-centration. Excretion during the active growth phase is therefore a detoxing mechanism.Subsequently a re-absorption during the stationary phase or the declining growth phase willoccur when the carbohydrate sources are limited hereby resulting in the final production

40


of NAD+ and ethanol removing the green flavours associated with acetaldehyde (Boulton,1991), (EBC, 2000), (Baert et al., 2012). Therefore, acetaldehyde is not expected in levelsdetectable in the finished beer.

(E)-2-Nonenal (E2N) is responsible for the stale off flavour mainly associated with ex-port pilsners. As seen in table 2.7 the aroma threshold is very low being detectable down to0.11 ppb. The low aroma threshold involves problems in detection using regular analyticaltechniques. In this thesis, the analytical techniques used will not be able to detect levelin ppb. Nevertheless, a short introduction concerning the formation of E2N is given inappendix A.3 because of the impact in pilsner beer, which is the main beer type used forAFB production.

Maillard reaction, also known as the non-enzymatic browning reaction, results in aromasand flavours of caramel, chocolate, coffee etc., which most often gives positive attribute tothe beer. Maillard reactions are heat catalysed reactions between reducing sugars andnitrogen containing compounds such as amines, amino acids, peptides or proteins. Furfuralis a Maillard product coming from the reaction with aldopentose, as illustrated in figure2.14 (Vesely et al., 2003), (Baert et al., 2012).

Figure 2.14: Pentose reaction with a nitrogen containing compounds to form the Maillardproduct furfural through cascades of reactions (Baert et al., 2012).

Reaction between one type of aldose sugar with a specific type of nitrogen containingcompound can yield a myriad of different compounds. The initial nucleophilic additionof an amino group to the aldehyde of the aldose sugar yields an amino Schiff base. Thisreaction is favoured in high pH environment. The Schiff base is in equilibrium with othertautomers through rearrangement of the double-bond, as shown in figure 2.14. Throughtautomerisation the formation of an enol (1,2-enaminol) and the formation of a ketone ispossible. The ketone compounds are called the Amadori compounds, which can be formed

41


from many different aldose carbohydrates reacting with a nitrogen containing compound.From the Amadori compound structure the formation of all the different Maillard products ispossible. This includes melanoidins, responsible for colour formation, plus different volatilescompounds, where Furfural is the most pronounced in beer. The formation of the differentcompounds is very pH dependent, e.g. is the formation of furfural increased by low pH(pH<5). Free protons are needed for the formation of furfural, as illustrated in figure 2.14.pH>7 will induce the formation of other Maillard products (Briggs et al., 2004),(Veselyet al., 2003), (Baert et al., 2012).

Benzealdehyde and isobutyraldehyde are formed by Strecker degradation. In figure 2.15the transamination of a α-dicabonyl is initiated by a nucleophilic attack by the lone pair inthe amino group on the carbonyl group, hereby forming an unstable hemiamine. Electrontravel within the hemiamine will induce the loss of water resulting in imine formation.The zwitterion form of the imine can be formed by irreversible decarboxylation. Finally,a reaction with water will result in an unstable amino alcohol which will degrade into aStrecker aldehyde and a α-ketoamine (Briggs et al., 2004), (Vesely et al., 2003), (Baertet al., 2012).

Figure 2.15: Strecker aldehyde formation from transamination between a amino acid and aα-dicabonyl (Baert et al., 2012).

The formation of Strecker aldehydes is dependent on the concentration of correspondingfree amino acids. The concentration of valine and phenylalanine is important in the for-mation of respectively isobutyraldehyde and benzaldehyde. Nevertheless, a large differencein the Strecker aldehydes aroma threshold levels only makes the concentration of specificamino acids important. The formation of phenylacetalehyde from phenylalanine follows thereaction illustrated in figure 2.15. However, oxidation could cause the further reaction intobenzaldehyde as illustrated in figure 2.16.

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Figure 2.16: Formation of benzaldehyde from phenylalanine, (Baert et al., 2012)

Most aroma potent Strecker aldehydes formed will either be evaporated during the wortboil or reduced to alcohol later during fermentation (Briggs et al., 2004), (Vesely et al.,2003), (Baert et al., 2012)).

The different methods of aldehyde formation entails different factors inducing and re-ducing the formation of these. Therefore, the following process parameter, which can beused to influence the aldehyde formation, are separated into different formation mechanismsin the following:

Process ParametersInducing aldehyde formation (Vesely et al., 2003), (Baert et al., 2012)

• Acetaldehyde

High metabolic fitness during exponential growth phase

No or short maturation period before cooling or filtrating the beer

Premature flocculation or sedimentation

• (E)-2-Nonenal

High lipid concentration in the barley or malt

Oxygen exposure during mashing, lautering and storage

Light exposure during storage

• Furfural

High heat exposure during malting and boiling

Low pH

• Benzaldehyde and isobutyraldehyde

High concentration of amino acids especially valine and phenylalanine

Oxygen exposure or other oxidative compounds present

Reducing aldehyde formation (Vesely et al., 2003), (Baert et al., 2012)

• Acetaldehyde

Low metabolic fitness during exponential growth phase

Long maturation period before cooling or filtrating the beer.

Weak flocculation or sedimentation

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• (E)-2-Nonenal

Low lipid concentration in the barley or malt

No oxygen exposure during mashing, lautering and storage.

Inert gas in mashing and lautering and proper CO2 purge before packaging.

No light exposure during storage

• Furfural

Low heat exposure during malting and boiling

High pH

• Benzaldehyde and isobutyraldehyde

Low concentration of amino acids especially valine and phenylalanine

No oxygen exposure or other oxidative compounds present

Additional Fermentation Products

In addition to higher alcohols (HA), esters and aldehydes other aroma and flavour activemetabolites will be formed during fermentation or originate form the raw materials. Ketones,sulphur compounds, organic acids and fatty acids are potent flavour and aroma metabolitesmainly associated with off flavours of fermentation (EBC, 2000).

The most flavourful ketones present in beer are vicinal diketones (VDKs). Diacetyl andpentane-2,3-dione are VDKs found in reasonable concentrations in beer. However, VDKsare not present in detectable concentrations if proper maturation of the beer is performedalong with no contamination and oxygen exposure (EBC, 2000). A thorough description ofVDK formation and influential factors can be found in appendix A.4.

Sulphur compounds such as hydrogen sulphide, sulphur dioxide, dimethyl sulphide (DMS),mercaptans and 3-methyl-but-2-ene-thiols (sun struck) are all off flavours in beer formed dur-ing fermentation or originating from the raw material. Maintaining a proper production willeliminate any possible detection of these compounds in the beer (EBC, 2000). Therefore,sulphur compounds are not expected in Humlefryd. Formation and influential factors canbe found in appendix A.5.

Acids in beer may come from different raw materials in the wort or bacterial contami-nations. Nevertheless, the majority of acids are produced during fermentation inducing adecrease in pH as illustrated in figure 2.11. Many organic acids are secondary metabolitesexcreted during rapid yeast growth and some re-assimilated later in the fermentation (EBC,2000). Acids in beer are generally not volatile, except for acetic acid. Therefore, they arenot considered in this report. Nevertheless, formation of various acids plus fatty acids canbe found in appendix A.6.

2.4.3 Raw Materials Products

Maillard product, such as furfural, generally originating form malting or wort boiling. There-fore, these compounds should not be considered as fermentation products. Aromas originat-ing from the raw materials or as a result of the brewhouse procedures are generally not asdifficult to influence as aroma compounds formed during the fermentation. Consequently,the majority of these aromas and flavours will not be discussed in this thesis. Nonetheless,hops constituents attributing to beer aroma are considered in the following section for thepurpose of highlighting possible reduction in aroma, despite the possibility of later addition.

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Hops Constituents

Hops contributes with many important compounds to beer such as aldehydes, resins, oilsand polyphenols (Schonberger and Kostelecky, 2011). In table 2.8 some essential hops oilscan be viewed.

Table 2.8: Hops constituents, aroma threshold and corresponding concentration in lagerbeer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989).


[mgl ] Taste [mgl ]

Myrcene 0.013 Floral 30-1000Resinous

Geraniol 0.036 Floral 0.004-0.090Roses

Linalool 0.027 Floral 0.0035-0.150Orange

Geranium (pelargonie)

Nonanal 0.015 Floral ≈ 3.7Fruity

Perfume

3-Carene - Sweet -Terpentine

α-Humulene 0.120 Hoppy flavours -

trans-Anethole - Anise -SweetSpicy

Polyphenols can add astringency to beer, however the main concern related to polyphe-nols is break formation resulting in visible sedimentation by hydrogen bonding to proteinsin beer. Furthermore, polyphenols aids in protein precipitation and later foam formation.Therefore, polyphenols are not considered a flavour attributer in this section (Schonbergerand Kostelecky, 2011)..

Hops contains many different ethereal oils in the lupulin glands, which can be subdividedinto different fractions where 50-80% are hydrocarbons, 20-50% are oxygenated hydrocar-bons and < 1% contain sulphur. In table 2.8 some important aroma oils are illustrated(Briggs et al., 2004), (Praet et al., 2012).

Hops resins can be subdivided into soft and hard. The most important soft resins is α-and β-acids, which adds the distinct bitterness to the beer. A thorough description of softresins are given in appendix A.7. Hard resins consist of different polyphenols here amongstflavanoids, proanthocyanidins and tannins in which the bacteriostatic plus health effects of

45


hops are located. Nevertheless, the flavour effects of these compounds are minimal. Thebitterness donation of hops is not considered in this section because of possible later additionof isomerised products after de-alcoholisation (Schonberger and Kostelecky, 2011).

Aldehydes from hops are mostly associated with the green and grassy aromas of hops,however some aldehydes, such as nonanal, can contribute positively to beer (Schonbergerand Kostelecky, 2011).

Ethereal hops oils are volatile at low temperatures resulting in a high loss of these oilsduring wort boiling. Generally addition of hops, for the purpose of adding oils to the beer,is done late in the wort boiling, in the whirlpool or even as dry hopping during fermentationand/or maturation (Briggs et al., 2004).

Hydrocarbon oils with the highest abundance in hops are monoterpene, e.g. myrcene,and the sesquiterpene , e.g. α-humulene. The concentration of hydrocarbon oil is drasticallyreduced during fermentation because of an adsorption to the yeast surface. Consequently,the more hydrophobic yeast Saccharomyces cerevisiae must be expected to adsorb morethan the less hydrohobic yeast Saccharomyces pastorianus used for the production of Hum-lefryd. No transformation of these hydrocarbon oils by the yeast occurs during fermentation(Schonberger and Kostelecky, 2011).

Oxygenated oil compounds in hops consist of terpene alcohols, aldehydes, epoxides,esters and ethers. Terpene alcohols, such as geraniol and linalool and ethers such as trans-anethole, are among a wide array of aroma substances detected in hops and hence beer(Praet et al., 2012).

Sulphur containing hops derived compounds, such as thioesters and sulfides, are normallynot detectable in beer if hop addition is done during boiling because of rapid volatilisation(Praet et al., 2012). Hops addition for Humlefryd is done during boiling and therefore nosulphur containing compounds are expected within flavour or detection threshold.

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2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY

2.5 Methods of Analysis

Beer is a complex matrix of various metabolic and raw material originating compounds,which involves a high demand for separation before detection is possible. The differentmethods of ethanol and aroma detection along with separation techniques applied are shortlypresented in the following section.

2.5.1 Sample Separation

Separation of different molecules in a complex matrix such as beer can be done using chro-matographic methods. Chromatography is based on different interactions of molecules in amobile phase travelling through a stationary phase. The stationary phase can retain differentmolecules subsequently releasing them back into the mobile phase resulting in a separation,see figure 2.17. The different chromatographic processes are denominated according to thephysical state of the mobile phase. Gas chromatography (GC) entails gas as the mobilephase whereas liquid chromatography (LC) entails liquid as the mobile phase (Moldoveanuand David, 2013), (Mcnair and Miller, 2009).

Figure 2.17: Chromatographic separation technique (Moldoveanu and David, 2013).

High performance/pressure liquid chromatography (HPLC) involves pumping of the mo-bile phase through a column typically packed with a stationary phase consistent of smallporous particles. HPLC separation is based on a concentration equilibrium of a specificmolecule between the mobile and solid phase. One of the following equilibrium can exist inHPLC columns (Moldoveanu and David, 2013):

Partition equilibriumSeparation of two liquid phases involving retention of one liquid phase based on thedifference in polarity. For example a highly polar liquid could form hydrogen bondswith a stationary solid phase hereby immobilising the polar liquid while a less polarmobile phase remains in suspension.

Adsorption equilibriumSeparation of molecule species based on the individual polarity of the molecules.

Ion equilibriumSeparation involving ionic bonding between ionic species in the mobile phase and ionsin the stationary phase. An equilibrium based on the strength of the ionic bond willresult in separation.

47


Size exclusion equilibriumSeparation based on molecular size as a result of retention time for the molecule passinga porous structure or channels within the stationary phase. Large molecules not ableto enter the channels and pores of the stationary phase will hereby elute earlier thansmall molecules retain within the stationary phase.

Affinity equilibriumSeparation based on molecular recognition or selective non-covalent interaction. Thisseparation technique is most often used for protein purification based on specific tagsor antibodies retaining a specific protein in the stationary phase.

Gradient HPLC involves a change in composition of the mobile phase during the sep-aration. Improvement of the separation can be obtained by changing the polarity and/orpH during HPLC. The separation of different molecules within the column will result indifferent retention times (tR). After separation detection of the different fractions are madepossible using a detector, see section 2.5.2. Detection involves a visualization of the differ-ent fractions displayed as peaks in a chromatogram. If separation into individual compoundpeaks is obtained a possible quantitative analysis of the molecular species will be possiblebased on the peak areas and the amount of sample injected (Moldoveanu and David, 2013).

HPLC has proven usable for separation of organic acids, sugars and alcohols in complexfood products. Coupling this to a suitable detection source could therefore be a usefulquantitative method for measuring ethanol after membrane filtration. Columns packedwith a resin based polymeric materials in the stationary phase have been found usablefor separation based on ion equilibrium or ion-exchange chromatography. The resin basedpolymer is protonated with a diluted acid eluent hereby enabling a cation bounding withthe anions in the sample solution. Anions will be formed enabling an ionic bonding withthe stationary phase based on the different acid dissociation abilities of the compounds inthe solution. Further elution with acid will result in an anion-exchange according to theacid-base equilibrium. Consequently, separation occurs according to the pKa value of thedifferent compounds in the sample. Molecules with a low pKa will easier dissociate andtherefore retain longer in the column than the molecules with higher pKa (Bio-Rad, 2012),(Moldoveanu and David, 2013), (Doyon et al., 1991), (Klein and Leubolt, 1993), (Bio-Rad,2014).

In GC application inert gases such as helium, nitrogen, etc. are carrying a vaporisedsample in the mobile phase through a column with a stationary phase consistent of eitherliquid (GLC) or solid (GSC) materials. GLC columns composition can be either packedcolumns or wall coated open tubes (WCOT) capillary columns, as illustrated in figure 2.18(Mcnair and Miller, 2009).

48


Figure 2.18: Packed and capillary GC columns (Mcnair and Miller, 2009).

Nowadays most GC application consists of WCOT with fused silica on which differentstationary phases or solvent can be loaded. The stationary liquid or solid phase withinthe columns separates the different compounds based on intermolecular interactions such aspolarity, complexation and hydrogen bonding. The samples are often vaporize by heatinghereby enabling separation. The vapour pressure or boiling point of different moleculesenables a further separation based on the point of vaporisation at different temperatures(Blumberg, 2012), (Mcnair and Miller, 2009), (Abraham et al., 1999).

Volatile organic compounds (VOCs) including alcohols, aldehydes, esters and hop con-stituents, as described in section 2.4, are flavour and aroma potent compounds with a lowvapour pressure enabling vaporisation to occur at atmospheric pressure and ambient temper-atures. Volatiles with low vapour pressure will accordingly exist in a gas-liquid equilibrium,described by Henry’s law, hereby making it possible to introduce the volatiles directly intothe column without thermal vaporisation, as shown in figure 2.19 (Wang et al., 2008).

Figure 2.19: Static headspace sampling technique (Wang et al., 2008).

In figure 2.19 an outline of static headspace (HS) GC can be observed. Static or equi-librium HS-GC analysis involve introducing a gas sample directly into the GC from theheadspace above a liquid or solid in a closed container. Initially the sealed container ispressurized with the intern carrier gas enabling a later pressure release, after volatile equi-librium, hereby introducing the headspace gas into the GC. The main advantage of static

49


HS-GC is the equivalence to the concentrations of volatiles when sniffing the open container.On the contrary, a clear disadvantage is observed in the low sensibility limited to high ppbup till percentage concentration ranges (Kolb, 1999), (Wang et al., 2008).

Figure 2.20: Dynamic headspace sampling technique (Wang et al., 2008).

In figure 2.20 a dynamic headspace (dynamic HS) process is outlined. Applying a con-tinuous flow of inert carrier gas through or above a liquid sample will induce a highervolatilisation hereby enabling higher sensibility of detection reaching low ppb levels. Di-rect introduction of the volatiles into the GC is not possible due to a prolonged extractionperiod. Therefore, the volatiles are adsorbed or trapped within a cartridge containing a suit-able adsorbent (TENAX-TA) (Wang et al., 2008). Tenax-TA (trapping agent) consists of aporous polymer resin based on 2,6-diphenylphenylene oxide. With a low affinity for water,Tenax-TA is very useful for volatiles from water samples with the ability to detect VOCsdown to ppt levels (Scientific Instrument Services, Inc., 2014). Later thermal desorption ispossible releasing the volatiles into a stream of carrier gas onto the GC. Disadvantages withthis technique include high dilution of the gas sample caused by the desorption procedure(Kolb, 1999), (Wang et al., 2008).

2.5.2 Sample Detection

If clear separation is obtained quantitative analyses are possible based on the single peakdetection methods. Refractive index detectors (RI/RID) can be used for quantitative anal-ysis of HPLC separated solutions. A RI detector is illustrated in figure 2.21. Detection isbased on bending or refraction of light when a beam hits a specific medium. The refractionindex of the sample solution is found by creating an angel of refraction between the samplesolution to a reference solution, with a known refraction index. The refraction change be-tween the two solutions causes a beam location change, which is detected by a photoelectricsensor. The rate of beam location change is proportional with the concentration of solutein the solution. As a result, the concentration can be found using standard solutions forcalibration (Moldoveanu and David, 2013), (Doyon et al., 1991).

50


Figure 2.21: Refractive index detection technique (Moldoveanu and David, 2013).

Refractive technique does not demand any fluorescence or chromophore groups of themolecule for detection hereby enabling a wider range of detectable substances. Nevertheless,the sensitivity is not as high as the fluorescence detection techniques. Refractive techniquesdemand a constant elution concentration and temperature, because of the refractive abili-ties of the eluent, hereby making it impossible to perform gradient elution and temperaturechange (Moldoveanu and David, 2013).

Figure 2.22: Electron ionisation quadrupole mass spectrometer (Laboratory, 2012).

Mass spectrometry is an analytical method separating ionised molecules or atoms basedon their mass-to-charge ratio (mz ). A mass spectrometer (MS) can be divided into fivedifferent components namely a sample inlet, an ioniser, a mass analyser, a detector andfinally a data system. In figure 2.22 the ion source, mass analyser and detector of anelectron ionisation (EI) MS can be observed (Pavia et al., 2009).

Sample injection into the MS can be done as gas, liquid or solid. Dependent on thesample volatility different methods of injection can be applied for the purpose of ionisingthe sample. Electron ionisation can be applied for gases, liquids and solids where evaporationis possible using heat and vacuum. The vaporised sample enters a vacuum chamber beforeentering the ioniser. Separation of the sample before introduction using chromatographicmethods such as HPLC and GC may demand a specific scanning capability of the MS. TheMS must be able to scan a range of 10-300 (mz ) within seconds before the next molecule

51


enters the MS from the separation technique (Pavia et al., 2009).Ionisation of the vaporised sample can be achieved using electron ionisation (EI) or

chemical ionisation (CI). In this thesis only electron ionisation is applied and described.The vaporised sample is drawn into the EI chamber using high vacuum. An high energyelectron beam is created perpendicular to the sample flow admitted from a super heatedfilament cathode flowing to an anode on the opposite side of the chamber. The collisionbetween the sample molecule and the electron beam results in an electron strip from themolecule hereby creating a cation. The flow of sample cations is directed to the mass analyserusing positively charged repelling plate and negatively charged accelerating plates. Samplemolecules which are not ionised are removed by the vacuum, while other molecules whichabsorb electron creating anions will be adsorbed on the repeller plates. Consequently, theformation or ratio of cations should be based on standard curve calibration of the wantedsamples. Fragmentation of the molecules can occur because of the energy applied in theelectron beam or electron migration. A short molecular ion lifetime will sometime result indetection of the fragmented ions (Pavia et al., 2009).

The cations are after ionisation separated according to mass-to-charge ratios (mz ) inthe mass analyser. Different types of mass analysers exist, with the most common onebeing quadrupole mass analysers for laboratory applications, as illustrated in figure 2.22.Quadrupole mass analysers consists of four solid rods running parallel to the sample ion beamdirection. An oscillating electrostatic field is generated between the rods by application ofdirect current (DC) and radio frequency (RF) voltage to the rods respectively generatingtwo opposite negatively charged rods and two opposite positive rods. The oscillation inthe electric field is dependent on the RF amplitude to the DC voltage. Ions with differentmz will experience different oscillation paths. Ions with a specific m

z ratio will experiencestable oscillation hereby remaining between the quadrupols until reaching the detector. Onthe other hand, all other ions with a different m

z ratio will experience unstable oscillationresulting in a constant increase or decrease in oscillation which will eventually result in anexit from the electrostatic field. Scanning of a wide range of m

z is possible by changing theratio of voltage (Pavia et al., 2009).

Detection happens when the ions, separated into specific mz ratio, hit the detector pro-

ducing a current proportional to the amount of ions striking the detector. An electronmultiplier is often installed in the detection system for the purpose of amplifying the signalwith a typical factor of 105 to 106. Electron multiplication occurs when ions hit a glass,coated with lead oxide, resulting in an ejection of two electron (Pavia et al., 2009).

52

Chapter 3

Materials and Methods

3.1 Equipment

A LabStak M20-0.72 Alfa Laval unit, as illustrated in figure 3.1, was used to conduct thede-alcoholisation of beer.

Figure 3.1: Labstak M20-0.72 Alfa Laval unit.

A sketch of the pilot scale set-up can be viewed in figure 3.2. The beer was introducedinto the feed tank with a maximum capacity of 9 litre. A modification of the M20 lab unitwas made enabling 1-3bar CO2 pressurisation of the beer. A lid of transparent acrylic glassfitted with a gas inlet ensured the possibility of pressurising the feed tank from above theliquid surface. CO2 gas, from a CO2-tank fitted with a pressure release valve, was used forpressure delivery. The beer was pumped from the feed tank into the system by a frequencycontrolled positive displacement diaphragm Hydra-Cell pump from Wanner Engineering,Inc. type G10 with a flow of 5 − 24 l

min and pressure delivery of 0-60bar. The Hydra-Cellpump was connected to a Lenze frequency controlled motor with a hz-inverter enablingrpm control. An Alfa Laval shell and tube heat exchanger ensured a constant temperaturebefore entering the membrane module. Cooling water at 0oC was used as cooling medium at

53

3.1. EQUIPMENT CHAPTER 3. MATERIALS AND METHODS

different speeds through the heat exchanger. Right before the beer inlet a pressure indicatorfrom Alfa Laval revealed the feed pressure (Pf ) in bar.

Figure 3.2: Sketch of the experimental set-up with modifications to the Labstak M20-0.72Alfa Laval labunit.

The membrane module consists of respectively spacer and support plates compressedusing oil hydraulics. The fluid flow through the system can be observed in figure 3.3. Ahydraulic lightweight hand pump from Enerpac was used to pressurise the module to 600barbefore filtration was commenced.

A permeate outlet from each support plate was collected into a permeate 20l plastictank. This tank was constantly weighed for the purpose of controlling the flux throughthe membranes while ensuring the right mass balance in the system, both during the pre-concentration and the diafiltration stage. During the pre-concentration stage the flow outof the system in the permeate was used to conclude when the point of pre-concentrationwas reached. On the contrary, the flow out of the system was used to maintain a constantvolume during the dia-filtration stage by adding carbonated dia-water. Samples from thepermeate were constantly taken, and the volume removed from the system was compensatedfor in the mass balance.

The pressure of the retentate flow leaving the membrane module was constantly mea-sured by an Alfa Laval pressure indicator revealing the retentate pressure (Pr) in bar. Apressure-regulation valve connected directly to the output was used to ensure the rightpressure build-up over the membranes.

The retentate flow was returned into the feed tank through the inlet pipe going under thesurface of the beer hereby ensuring a reduction in foaming and volatile release. Modification

54

3.2. MEMBRANES CHAPTER 3. MATERIALS AND METHODS

to this pipe was done for the purpose of enabling pressurisation of the feed tank while stillbeing able to take out beer samples and adding carbonated dia-water during the diafiltrationstage. The sample valve was connected to a hose and a steel pipe ensuring a minimumfoaming and release of CO2 while taking samples. The samples were pushed out of thesystem by the overpressure created above the liquid surface by CO2. Furthermore, themodification involved a possible addition of carbonated water without loosing the pressure.An additional valve for the carbonated water inlet was connected to a pressurised 2l glasstank where the carbonated water was pushed out by compressed air at 2-3bar pressure.

Figure 3.3: Membrane module flow of Labstak M20-0.72 Alfa Laval labunit. Black arrowsare retentate flow and green arrows are permeate flow.

3.2 Membranes

Different membranes were tested for the purpose of finding the best flux of alcohol whilemaintaining a proper retention of flavour and aroma substances. In table 3.1 the specifica-tions of the membranes are shown.

55

3.3. FEED BEER CHAPTER 3. MATERIALS AND METHODS

Table 3.1: Physical data on thin film composition (TFC) membranes from Alfa Laval (Laval,2014a), (Laval, 2014b), (Laval, 2014c).

Designation RO90 NF NF10 NF99HF (NFHF)

Membrane layer Polyamide Polyamide Polyamide Polyamide

Support layer Polyester Polyester Polyester Polyester

Rejection 90% 98% 98% 98%2000 [ppm] NaCl MgSO4 MgSO4 MgSO4

9bar 9bar 7.6bar 9bar25oC 25oC 25oC 25oC

MWCO - - 150-250 -[ gmol ]

pH 3-10 3-10 3-9 3-9Range

Operating 15-42 15-42 5-25 15-35Pressure[Bar]

Maximum 55 55 40 41Pressure[Bar]

Temperature 5-50 5-50 2-50 5-45Range[oC]

3.3 Feed Beer

The feed beer used for all membrane filtrations was Humlefryd pilsner produced on brew-house Skands A/S. Two different productions were used respectively bottled on the 24th ofFebruary (HFORG24) and the 11th of April 2014 (HFORG11).

The following alcohol concentrations were observed from the HPLC alcohol measure-ments:

• HFORG24 - 5.71%ABV

• HFORG11 - 5.72%ABV

A sample for alcohol measurement was taken after a couple of runs through the systemat ambient CO2 pressure (1-3bar) to illustrate possible dilution in the system. The mea-sured alcohol concentration of this sample was considered the correct start concentrationfor the filter runs. The dilution amount (l) was calculated based on the change in alcoholconcentration from the above illustrated values to the sample taken after mixture.

HS-GC-MS spectrum for each original Humlefryd was taken to illustrate possible dif-ferences in aroma among the different productions, these spectra can be see in appendixA.8. To compensate for possible production differences each aroma sample was comparedto the original beer in the feed tank of the unit, before initiation of the membrane filtration,hereby only comparing the same beer before and after de-alcoholisation.

56

3.4. DIA-WATER CHAPTER 3. MATERIALS AND METHODS

3.4 Dia-water

Deionised water and carbonated was used as dia-water during all membrane filtration runs.Deionised water was chosen because the mineral composition of the beer hereby shouldmaintain the same, especially for RO application. Furthermore, the deionised water wascarbonated to maintain CO2 pressure within the unit while avoiding oxidation. In table 3.2pH and conductivity measured can be observed for various different water sources.

Table 3.2: pH and conductivity of different water sources.Water source pH Conductivity

µs

Deionised dia-water 5.00 2.64Deionised and carbonated dia-water 3.78 52Milli-q water 5.46 1.69KU life tap water 6.46 940

The pH of the original beers before membrane filtration were pH 4.36 with a conductivityof 2.38µs.

3.5 Procedures

3.5.1 Loading the membranes

The membranes were loaded into the membrane module and hereafter pressurised to 600bar.The membranes were flushed five times the volume of the system (9l) with deionised waterremoving all traces of glycol from the membranes. Recirculation was done under standardpressure 8-10bar until reaching a temperature of 30-50oC.

3.5.2 CIP of membranes

Cleaning in place (CIP) was done between every membrane filtration run and when load-ing the module with new membranes. 27.05% NaOH was added the water reaching 0.1%NaOH and pH 9-10 within the unit. 30 minutes circulation was done under 10bar pressure.The system was flushed until reaching pH below 8. In case of a longer duration betweenmembrane runs an additional sterilisation was done after CIP.

3.5.3 Membrane sterilisation

Sterilisation was done using a 0.1% H2O2 solution within the unit. The solution was loadedthe closed system to maintain proper microbiological stability.

3.5.4 Start-up procedure

Measurement of water flow (Fretentate) through the system was done with the pressureand temperature settings of the later membrane filtration. Hereafter the pump settingswere maintained enabling an approximation of the beer flow in the unit during membranefiltration. Furthermore, a measurement of the water flux through the membrane (Fpermeate)was done. An estimation of filtration time and dia-water volume were done using thecalculation presented in section 2.3 and an on-line calculation tool by Alfa Laval Møller

57

3.5. PROCEDURES CHAPTER 3. MATERIALS AND METHODS

(2013). The deionised water was removed by displacement throughout the sample valveusing CO2 pressure on the water surface, see figure 3.2. Water in the system could not beremoved entirely due to the pipe into the feed tank did not reach the bottom of the tankplus additional water allocated in the piping, pump and membrane housing of the system.Addition of 5l beer into the tank was done using the carbonated water tank. Insteadof compressed air, pressurised CO2 was used to push the beer into the closed feed tankthrough the inlet pipe, as illustrated in figure 3.2. A couple of circulations at 0bar TMPwere done for the purpose of mixing possible water in the system with the beer. Hereaftera sample was taken to determine the new alcohol concentration. The membrane filtrationrun was then commenced.

3.5.5 Sampling plan

Five beers were saved for later analysis of ethanol, aroma, pH and conductivity beforeinitiating the filtration. The following sampling plan was used through all the experiments,see table 3.3:

Table 3.3: Sampling plan during membrane filtration runs

Process Sample Sample SampleStage Location Time Container

Stage time in percent [%] [ml]

Pre-concentration > 2.5 L Feed tank (FT) 0 50Feed tank (FT) 0 2Permeate (P) 0 2

50 2100 2

Permeate tank (PT) 0 250 2100 2

Diafiltration = 2.5 L Permeate (P) 0 220 240 260 280 2100 2

Permeate tank (PT) 0 220 240 260 280 2100 2

Permeate tank (PT) 100 50Feed tank (FT) 100 50

All samples were frozen down directly after a membrane filtration run ensuring micro-biological stability. The remaining de-alcoholised beer in the feed tank was bottled andequally frozen down. The re-concentration was done before analyses or tasting, based on

58

3.6. ANALYTICAL TOOLS CHAPTER 3. MATERIALS AND METHODS

the reached alcohol concentration in the concentrate.

3.6 Analytical Tools

In the following section the materials and methods used for the measurement of ethanol andaroma substances will be presented.

3.6.1 HPLC - ethanol measurements

For the measurement of ethanol a HP1100 HPLC fitted with an Aminex HPX-87H columnwas used. Each sample was sterile filtered using a nylon sterile syringe filter with a poresize of 0.45µm. Sample dilutions were done ensuring an ethanol concentration within therange of 0.1-20gl . An eluent was prepared with 1mM H2SO4 and 0.3mM ethylenedinitrilote-traacetic acid (EDTA) solution reaching a pH of 2.75 ± 0.05. The eluent was after mixturefiltered with a 0.45µm pore size filter using a Sartorius vacuum pump. The following stan-dard setting for the HP1100 HPLC was used for all samples:

Standard setting for the HP1100 HPLC

• Eluent flow - 0.5 mlmin

• Injection volume - 10µl

• Detection temperature - 30oC

• Running time per sample - 32min

• Post run time - 2min

• Maximum pressure - 400bar

• Minimum pressure - 0bar

Calibration samples were prepared using 96%ABV ethanol dilutions. The calibrationcurves can are illustrated in appendix A.9. After 100 samples a new calibration curve wasmade. A general HPLC report generated for a original beer in the feed tank can be observedin appendix A.10.

3.6.2 HS-GC-MS - aroma measurements

Dynamic Headspace (HS) extraction was performed in duplicates on 20ml samples trappingthe volatiles onto a Tenax-TA trap, in appendix A.11 the sampling set-up is illustrated.Each trap contained a resin based porous polymer of 2,6-diphenylene oxide called Tenaxspecially designed to function as trapping agent (TA). The Tenax traps used contained 250mg Tenax-TA with a mesh size of 60

80 and a density of 0.37 gml .

Before setting up the sampling each trap was desorbed using a reprogrammed HP 5890AGC set to heat the samples to 250oC while flushing the traps with nitrogen at a flow of50 ml

min for 20 minutes.The sample container had a general outlook as illustrated in figure 2.20, section 2.5.1.

Nevertheless, the purge gas outlet was not placed under the sample surface, but insteadplaced shortly above the liquid surface. In addition, a magnet was placed within the sample

59


container to maintain stirring during sampling. Each sample was diluted to obtain the sameconcentration of volatiles, based on the original added volume of beer. The sample wasplaced in a water bath maintaining a constant temperature of 37oC mimicking human bodytemperature. In the water bath a magnetic stirrer ensured a proper heat transfer betweenthe water and the sample stirring at 200rpm. The carrier gas used was nitrogen with a gasflow of 100mlh manually controlled and confirmed using a flow-meter on the other side of thetrap. Extraction of aroma was done for 15 minutes onto a Tenax-TA aroma trap. Hereafterdrying was done for 10 minutes using nitrogen at 100mlh in the opposite flow direction ofthe trap. Finally, the traps were sealed with clean caps and maintained in a refrigeratoruntil sampling of the volatiles in the GC-MS, see equipment settings and parameters belowin table 3.4:

Table 3.4: Overview of equipment settings and parameters for GC-MS analysisStep/Item Designation

Manufacture Agilent TechnologiesType 7890AMethod Thermal DesorptionColumn DB-Wax capillaryColumn dimensions 30 m long x 0.25 mm inner diameterColumn film thickness 0.50µmDetector 5975C VL Mass Selective Detector (MSD)Column pressure 2.4 psiCarrier gas H2

Initial flow rate 1.2 mLmin

Column temperature program 10 min at 30oC

30-240oC changing 8oCmin

5 min at 240 oCElectron ionisation mode 70 eVMass-to-charge ratio scanned 15-300 m

z

The volatiles were identified using the highest probability or best matching based on theirmass spectra compared to a commercial database (Wiley275.L, HP product no. G1035A).MSDChemstation software (Version E.02.00, Agilent Technologies) was used for data anal-ysis. Peaks properly separated from other substances having a high matching with thedatabase fractionation (> 50%) were visually investigated and integrated to obtain a com-parable area between the different samples. No standards were made for any of the aromacompounds hereby only making internal comparison possible. A standard HS-GC-MS spec-tra can be observed in appendix A.12 for Humlefryd original bottled the 24th of April(HFORG24).

3.6.3 Beer Tasting

A beer tasting was performed for the purpose of revealing a possible flavour and aromadifference in the beer de-alcoholised with different membranes. Seven participants werecarefully chosen to represent different backgrounds and knowledge in beer, membranes andgeneral beer tasting. Three participants with a background in brewing, all being educatedDiploma Master Brewers and trained tasters, along with two technical engineers, one Asso-ciate Professor in chemistry and one Associate Professor in Food Microbiology, participated.

60


The tasting was performed as a blind test where the participants were asked to rate the beerin strength and resemblance to an original Humlefryd. The original Humlefryd was treatedin a identical way as the samples after de-alcoholisation. Caused by dilution in the systema decision was made to dilute all the beers to equal strength with deionised and carbonatedwater, causing the same concentration of aroma compounds in all samples correspondingto a dilution of the original beer from 5 to 7 litres. Despite the dilution equal alcohol con-centrations were not achieved because of difference in experiments. The following sampleswere tasted:

• HFORG11 - Original Humlefryd bottled on 11th of April

Tasting strength: 4.09%ABV

• Sample A - NFHFR1 - NFHF membrane run 1


• Sample B - RO90R2 - RO90 membrane run 2


• Sample C - HFORG24 - Original Humlefryd bottled on 24th of March


• Sample D - NFHFR2 - NFHF membrane run 2


• Sample E - NFR2 - NF membrane run 2


• Sample F - NF10R1 - NF10 membrane run 1


The results of this blind test were used as an indicator of possible detection of differenceamong membranes. The results will later be discussed in correlation to measured aromaconcentrations of the samples.

61

Chapter 4

Results and Discussion

4.1 Preliminary Investigation

Initial considerations concerning the physical parameters during membrane runs were madebased on data obtained from Alfa Laval A/S. This data can be see in appendix A.13. Twodifferent nano filtration (NF) membranes and one reverse osmosis (RO) membrane weretested with different applied pressures and temperatures to illustrate possible influences inalcohol and aroma permeability. Ethanol reduction was analysed by use of HPLC-MS. Ad-ditional HPLC-MS peaks were regarded as aroma peaks where reduction of total peak areawas used as indicator for aroma loss. Principle component analysis (PCA) using Eigenvectorin Matlab was done preprocessing the data by mean centering, choosing two componentsdescribing 99.3% of the variation in the data. A bi-plot of the two principal componentscan be observed in figure 4.1.

Figure 4.1: Principal component analysis (PCA) on RO90 data given by Alfa Laval. PC1describes 65% of the variation in the data while PC2 describes 34%. Sample (red triangle)name RO90[Temperature in oC][Pressure in bar]. Loadings (blue square), PM = Perme-ability. PC = Principal component.

No reduction in aroma designated peaks was observed for all the different temperature

62

4.1. PRELIMINARY INVESTIGATION CHAPTER 4. RESULTS AND DISCUSSION

and pressure settings. Therefore, a higher attention can be given to the optimisation ofethanol (Alcohol) permeability and flux. Lowering the TMP (Pressure) causes an increasein ethanol permeability as observed in figure 4.1. A rise in ethanol rejection, hereby areduction in ethanol permeability, caused by a pressure increase have been reported byLopez et al. (2002) for RO apple cider de-alcoholisation and by Ferreira et al. (2007) forRO dialysis de-alcoholisation of beer. The correlation between the alcohol permeabilityand TMP dictates 65% of the data variance along the first principal component. Thesecond principal component explains 34% variation in the data mainly being the differenttemperature during the different runs. Ferreira et al. (2007) illustrated a rise in permeateflux when increasing temperature. Therefore, a rise in temperature also induce a higheralcohol permeability as observed in figure 4.1. The ideal physical parameter settings forRO90 membrane de-alcoholisation therefore seems to be high temperature at 20oC and lowpressure at 10bar, such as sample RO90[20][10].

In figure 4.2 an identical PCA performed on NF data is illustrated.

Figure 4.2: Principal component analysis (PCA) on NF data given by Alfa Laval. PC1describes 67% of the variation in the data while PC2 describes 29%. Sample (red triangle)name NF[Temperature in oC][Pressure in bar]. Loadings (blue square), PM = Permeabilityand PC = Principal component.

The NF membrane filtration bi-plot explains 96% variation in the data. The first compo-nent describes the variation in the samples according to both aroma and alcohol permeabil-ity, which therefore seems correlated. This correlation indicates that operational settingsfor high ethanol flux also induces high aroma flux. The second component separates thesamples with high temperature and pressure from those with low. From this bi-plot it canbe deduced that high pressure and low temperature will cause the best aroma retention inbeer. However, the ethanol permeability will equally decrease hereby increasing the processrun time and use of dia-water. An increase in alcohol and aroma retention caused by apressure increase have been illustrated by Lopez et al. (2002) and Ferreira et al. (2007).Especially the retention of methanol and ethanol were changed drastically, while the aromapermeability did not change noteworthy. Therefore, the ideal physical parameters for NFmembrane de-alcoholisation seems to be low temperature at 10oC and high pressure at25bar (NF[10][25]), if focus is on high aroma quality, or high temperature at 20oC and low

63

4.2. CONSTANT PARAMETERS CHAPTER 4. RESULTS AND DISCUSSION

pressure at 10bar (NF[20][10]) if high alcohol permeation and flux are the priorities.

Figure 4.3: Principal component analysis (PCA) on NFHF (NF99HF) data given by AlfaLaval. PC1 describes 59% of the variation in the data while PC2 describes 36%. Sample(red triangle) name NF[Temperature in oC][Pressure in bar]. Loadings (blue square), PM= Permeability and PC = Principal component.

From the bi-plot in figure 4.3 the same general tendency for NFHF (NF99HF) as for theNF membranes can be observed. However, the correlation between aroma and alcohol per-meation is not as clear as for the NF membrane. In figure 4.3 a strong correlation betweenaroma permeability and temperature is observed. On the contrary, a change in temperaturedoes not seem to change the alcohol permeation as significantly. Changes in pressure seemsto have a high influence on the ethanol permeation showing high ethanol permeation atlow pressure. Concerning NFHF membranes, focus should be given on reduction in aromapermeation because of a general high flux through NFHF membranes. Therefore, the bestsettings for de-alcoholisation with NFHF membranes are low temperature 10oC and highpressure 25bar as sample NF99[10][25] in figure 4.3 indicates.

The physical settings chosen for the membrane runs performed in this thesis were basedon these considerations along with experimental experience. The physical settings weremaintained constant for the purpose of illustrating the potential of the different membranestested. In the following section the physical settings will be presented.

4.2 Constant Parameters

Each membrane was tested in duplicates for the purpose of reducing possible experimentalerrors and mapping out the reproducibility of the experimental set-up. To enable a com-parison of ethanol permeabilities and aroma retentions all physical parameters were keptconstant throughout the different membrane filtration runs. Possible deviations can be ob-served in table 4.1. Furthermore, two additional RO90 membrane runs were performed forthe purpose of illustrating the importance of TMP, temperature and pre-concentration be-fore diafiltration.

64


An ethanol concentration reduction from 5.71-5.72%ABV to 2.97-3.60%ABV was ob-served as a consequence of water dilution. This concentration dilution corresponds to avolume between 3.60-4.71 litre water in the system before loading the beer. The waterdilution did not influence the possibility of illustrating the membrane potential for alcoholremoval as well as the aroma retention.

The flow through the system was measured before each membrane run using water.The pump settings were maintained throughout all the different membrane runs. Ferreiraet al. (2007) proved that an increase in flow caused an increase in permeate flux and aromaretention, while the ethanol retention maintained the same. However, they only reached amaximum flow of 7 l

min limited by the experimental set-up. A flow of 14 lmin was chosen for

this set-up to increase the water and ethanol permeation plus aroma retention.The volume of beer added to the system was maintained constant by adding 10x0.5 litre

bottles per run. With the exception of run ROR4 where 6 litres were used to obtain a higherpre-concentration.

The temperature in the feed tank was maintained using a constant flow of ice waterat 0oC in the shell and tube cooler before entering the membrane housing, as illustratedin figure 3.2. A rise in permeate flux as temperature rises has been illustrated by Ferreiraet al. (2007). Nevertheless, the effects on the different aroma substances were very differ-ent. Higher alcohols (HAs) were more affected by a rise in temperature hereby changingthe retention drastically allowing high permeation at temperatures around 20oC. On thecontrary, ethanol and esters retention did not change as drastically (Ferreira et al., 2007).A temperature around 15oC was chosen mainly because of the limitation of the coolingsystem.

Trans membrane pressure (TMP) changed only when adding additional membranes dur-ing the RO90 runs. This illustrates a TMP loss of 2bar (20-18bar) when using 8 membranesand a TMP loss of 4bar (20-16bar) when using 20 membranes. A TMP around 18-19bar waschosen because of the limitation of NF10 and NFHF at 5-25bar, as illustrated in table 3.1.In addition, the choice of TMP was done for the purpose of being in the higher end of thislimitation, while still being able to compare the membranes. An increased ethanol retentionat increased TMP has been illustrated. Ferreira et al. (2007), using cellulose acetate (CA)membranes, showed a decrease in ester retention and an increase in higher alcohol retentionat higher pressure. On the contrary, Lopez et al. (2002), using Polyamide (PA) membranes,illustrated a minimal influence on aroma substances at the highest possible pressure.

The membrane area was maintained, throughout the different NF membrane runs, at0.144m2 while RO90 membrane runs demanded a membrane area of 0.360-0.390m2 to obtaina reasonable membrane filtration run time, enabling a process duration of maximum sevenhours equivalent to a days work.

65


Tab

le4.

1:M

emb

ran

ep

aram

eter

san

dco

nst

ants

for

each

mem

bra

ne

run

.H

igh

ligh

ted

nu

mb

eran

dm

emb

ran

eru

ns

are

not

con

sid

ered

inth

est

and

ard

dev

iati

on(S

D)

calc

ula

tion

Desi

gn

ati

on

RO

90R

1R

O90

R2

RO

90R

3R

O90R

4N

FR

1N

FR

2N

F10

R1

NF

10R

2H

FR

1H

FR

2S

D

Sta

rteth

an

ol

3.00

3.29

3.25

3.38

3.60

2.94

3.32

3.20

3.1

02.9

70.2

08

con

c.

infe

ed

tan

k[%ABV

]

Wate

rfl

ow

14.5

1414

.214

13.8

13.8

14.5

14.8

13.1

13.1

0.5

65

[l

min

]

Volu

me

Beer

55

56

55

55

55

0[l] Tem

pera

ture

1716

1620

1515

1514

16

15

0.8

81

infe

ed

tan

k[oC

]

TM

P19

1810

1819

1919

1919

19

0.4

40

[Bar]

Mem

bra

ne

0.14

40.3

96

0.3

60

0.3

60

0.14

40.

144

0.14

40.

144

0.1

44

0.1

44

0A

rea

[m2]

Pre

-con

c.

1.36

1.40

1.40

1.6

51.

461.

351.

411.

411.3

71.3

50.0

36

[VCF

]

Fin

al-

con

c.

1.42

81.

685

1.44

91.9

93

1.00

01.

482

1.68

31.

533

1.4

55

1.4

71

0.1

99

[VCF

]

66


During all membrane filtrations the mass balance in the system was controlled by weigh-ing the mass leaving the system in the permeate, while knowing the amount added in respec-tively beer and dia-water. Furthermore, a correction for the sample taken was subtractedthe permeate volume to ensure a correct mass balance, as illustrated in figure 4.4.

Figure 4.4: Mass trend for membrane run with RO90 and NFHF membranes, see table 4.1for physical parameters

In figure 4.4 the different masses controlled during two membrane filtrations runs can beobserved. Initially 2.5kg permeate was removed from the beer during the pre-concentration.The end of the pre-concentration stages can be observed, in figure 4.4, as a stagnation inthe retentate volumes (orange and green) when the permeate volumes (turquoise and red)reaches approximately 2.5kg. At this point, addition of dia-water (purple and blue) wasinitiated and maintained in the same rate as the removal from the system through thepermeate, hereby maintaining a constant retentate volume. The dilution was more severthan expected hereby resulting in a low pre-concentration VCF of 1.35-1.46 for the regularmembrane runs with 5 litre beer added. Furthermore, a final dilution was observed becausethe diafiltration volume never came below the initial beer volume reaching volumes of 5.4-7litre. In addition, the initial water diluted beer was considered in the VCF resulting instarting volumes ranging from 10.16-7.93 litre diluted beer in the system and ending withfinal volumes ranging from 7-5.4 litre resulting in the VCF observed in table 4.1. An in-crease in ethanol permeability with increasing VCF factors during pre-concentration wasillustrated by Lopez et al. (2002) during de-alcoholisation of apple cider. This illustrates ahigher potential for a faster ethanol removal applying a higher VCF ratio.

The constant parameter was maintained as well as possible considering the experimentalset-up.

67

4.3. MEMBRANE FLUX CHAPTER 4. RESULTS AND DISCUSSION

4.3 Membrane Flux

Membrane flux is important for the process profitability. Having a high flux entails shorterprocess time hence lower cooling load, pump work and storage time. Consequently, a higherflux could possibly lower the OPEX. In addition, a high flux could minimise the membranearea demand thereby contribute positively to a lower CAPEX.

Figure 4.5: Flux behaviour for reverse osmosis membrane runs, see table 4.1 for physicalparameters.

A wast difference in flux was observed in figure 4.5 and figure 4.6 depending on the typeof membrane applied for the de-alcoholisation. The RO90 membrane showed the lowestflux compared to all types of NF membranes. For RO90R1 and RO90R2 the flux stagnatesat around 0.150 kg

m2∗min after 150 minutes. RO90R1 was performed under the exact samephysical condition as the other NF membrane runs, while RO90R2 was performed using22 membranes. A reduction in TMP has been shown to lower the retention of ethanolhereby increasing the ethanol permeability percentage (Lopez et al., 2002). Consideringthis, an additional membrane run 3 (RO90R3) was performed lowering the TMP from 18-19to 10bar. RO90R3 showed the lowest possible flux stagnating at 0.05 kg

m2∗min maintaining adownwards trend. Halving of TMP reduces the flux threefold hereby proving the need tomaintain a high pressure to make a membrane filtration using RO90 membranes possible.Ethanol flux increases when the temperature is raised (Ferreira et al., 2007). RO90R4 wascarried out using a higher pre-concentration at 1.65 VCF and a higher feed tank temperatureof 20oC. This membrane run showed no drop in flux caused by a higher pre-concentration.It rather showed a rise most likely caused by the higher temperature in the feed tank.

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Figure 4.6: Flux behaviour for nano filtration membrane runs, see table 4.1 for physicalparameters.

NFR1 and NFR2 were performed using the same settings. Nevertheless, a lower flux forNFR1 was observed compared to NFR2. This could be a result of a higher dilution of the beerfor NFR2 than NFR1, as illustrated in table 4.1. A stagnation was again observed situatedat 0.375-0.415 kg

m2∗min . The lowest flux was observed for the NF membranes compared to theother NF membrane types. This was also expected since the retention description given byAlfa Laval in figure 3.1 illustrated a better MgSO4 retention compared to NF10 membranesand a identical retention compared to NFHF membranes.

NF10R1 and NF10R2 had a constantly falling trend throughout the membrane filtra-tion never reaching a stable level. However, NF10R2 seamed to become more stable whenreaching the same flux level as NFR2 around 0.430 kg

m2∗min . The initial flux during the pre-concentration started higher than for the NF runs proving the higher porosity for NF10 asdescribed by the manufacture. Nevertheless, the overall decrease in flux during the mem-brane runs was more pronounced for the NF10 membranes than respectively for the NF andRO membranes. The water flow observed in table 4.1 (14.5-14.8 l

min) showed that the pumpsettings for NF10R1 and NF10R2 resulted in a higher flow through the system, which couldhave resulted in the higher initial flux as well as longer duration for stabilisation comparedto NFR1 and NFR2 (13.8 l

min).NFHFR1 and NFHFR2 showed strikingly different trends despite equal parameters, as

shown in table 4.1. The highest initial flux was observed for these high flux (HF) mem-branes followed by a rapid drop. The first membrane run interestingly had the highest fluxthroughout the entire run compared to the second membrane run, where the final flux ac-tually came below that of NF10R1. The same trend was observed for the other membraneruns showing a higher flux for run 1 compared to the run 2. In addition, it seems thatthe greater the pore size of the membrane the greater the difference between run 1 and2 becomes. The difference between subsequent runs could indicate a possible clogging ofthe membrane pores, fouling, membrane cake build-up or membrane composition alteration

69


caused by CIP or sterilisation. This is most likely caused by internal fouling or cloggingof the new membranes. Clogging would explain why the difference among membranes be-came larger as the membrane pores size became larger. Finally, the CIP and sterilisationcould have resulted in membrane composition alteration, however this would not have beenexpected as the instructions given by the manufacture were followed. In addition, a CIPwas applied before using new or used membranes. This wast alteration between subsequentmembrane runs could be used proactive as a kind of priming of the new membranes with amaterial enabling a higher retention of wanted substances. Membrane selectivity, polarityand composition can hereby be drastically altered.

The solvent permeation coefficient Bwater was not calculated for all membranes becausethe osmotic pressure of the beer (∆π, [bar]) was unknown. Nonetheless, the RO90 membraneruns were carried out at two different pressures (RO90R2 and RO90R3 at 10 and 18bar,respectively) enabling a linear plot from where the Bwater,RO and osmotic pressure of thebeer can be deduced, as illustrated in figure 4.7.

Figure 4.7: Linear plot of permeate water flux as a function of pressure difference fromequation 4.2. Other physical parameters can be viewed in table 4.1.

Jwater,RO = Bwater,RO ∗ (TMP −∆π) (4.1)

⇒ Jwater,RO = Bwater,RO ∗ TMP −Bwater,RO ∗∆π (4.2)

⇒ Bwater,RO = 0.0125 [kg

m2 ∗min ∗ bar] and ∆π = 6 [bar] (4.3)

The Bwater,RO found based on two pressure differences is not yielding as a precise num-ber as if various different TMP runs were performed. Therefore, the flux of water is notdiscussed based on Bwater of the different membranes. The general Jwater was as applicablea membrane assessment factor as Bwater because the same ∆π and TMP was applied amongthe different membrane runs.

The expected flux patterns were observed yielding the highest flux from the membraneswith the highest pore size and the best high flux composition. The consequence of thedifferent flux patterns along with the ethanol permeability can be viewed in table 4.8.

70

4.4. ETHANOL PERMEABILITY CHAPTER 4. RESULTS AND DISCUSSION

4.4 Ethanol Permeability

One important technical property of membranes used for de-alcoholisation is the abilityto permeate ethanol Jethanol. This technical property should be viewed in the light of theability to retain or reject wanted aroma compounds. In figure 4.8 the permeate ethanoltrend for comparable membrane runs can be viewed.

Figure 4.8: Permeate ethanol trend. (Start-End %ABV of beer in feed tank). See table 4.1for physical parameters.

In figure 4.8 an ethanol increase in the permeate is observed during the pre-concentrationstage until the diafiltration stage is initiated. During the diafiltration stage there is asteady drop in the slope dependent on the ethanol permeability of the membranes. Thedecrease in ethanol concentration was illustrated to follow a negative exponential trendduring diafiltration (Ferreira et al., 2007). In figure 4.8 the negative exponential trendis not as obvious. Nevertheless, a decrease in the rate of the negative slope during thediafiltration stage is observed for all NF membrane runs. This illustrates the limitation ofmembrane processes for alcohol removal with the removal rate falling constantly with theethanol concentration limiting the alcohol removal of beer to 0.45%ABV (Ferreira et al.,2007). A more drastic change in the removal rate would have been illustrated if the highdilution had not occurred as a consequence of a higher start concentration.

71


Table 4.2: Ethanol retention, permeability and solute transport coefficient for various mem-brane runs.

Retention Retention Permeability Ethanol SoluteStart End Calculated Flux Transport

[R] [R] [%] Jethanol Coefficient

[ kgm2∗min ] Bethanol

[ mmin ]

RO90R1 0.45 0.51 54 1.83∗10−3 17.1∗10−5

RO90R2 - 0.60 51 1.57∗10−3 6.46∗10−5

RO90R3 - 0.29 75 0.761∗10−3 2.97∗10−5

RO90R4 0.62 0.49 85 2.89∗10−3 17.3∗10−5

NFR1 0 - 95 - -

NFR2 - 0.14 95 6.26∗10−3 36.7∗10−5

NF10R1 0.11 0.31 91 7.09∗10−3 251.6∗10−5

NF10R2 0.10 0.23 92 6.13∗10−3 255.5∗10−5

NFHFR1 0 0.068 95 12.90∗10−3 3014∗10−5

NFHFR2 0.067 0.29 95 7.40∗10−3 474.3∗10−5

RO90R1 is the only RO90 run comparable to the others NF runs in figure 4.8 due toan equal amount of membranes were used (8 membranes). In figure 4.8 it can be seen thatfor RO90R1 both the pre-concentration as well as the diafiltration stage were significantlylonger than the other NF membrane runs. All RO90 membrane runs, in table 4.2, had arelatively high retention being lowest at 0.45 while the highest permeability calculated was85%. A general level of permeability of approximately 50% was observed for the comparableRO90R1 and RO90R2. An ethanol permeability level of 49% for RO90 membranes has alsobeen proved by Alfa Laval doing the membrane filtration at 20oC and 25bar, see appendixA.13.

NFR1 and NFR2 had an ethanol permeability of 95% and an ethanol retention rangingfrom 0-0.14 throughout the filtrations performed, as observed in table 4.2. From figure 4.8it can be seen that the permeation of ethanol was very similar to the other types of NFmembrane filtrations. Nevertheless, NFR2 had the longest duration before entering thediafiltration stage after approximately 50 minutes. This is a result of NF membranes havinga lower flux compared to NFHF and NF10 membranes, see illustrated in figure 4.6. A 97%ethanol permeability for NF membranes at 20oC and 10bar changing to 88% when risingthe pressure to 25bar was found by Alfa Laval, see appendix A.13.

NF10 membranes showed an ethanol permeability 91-92% with a retention ranging from0.10-0.31, as illustrated in table 4.2. The ethanol permeation trend for NF10, see figure4.8, showed the approximately same rate of ethanol removal as for the other NF membranetypes.

NFHF membranes also showed a high ethanol permeability at 95% with a ethanol re-tention ranging from 0-0.29, as illustrated in table 4.2. NFHFR1 showed a different trendcompared to the other NF membrane runs. This was most likely caused by the higher waterflux through the membrane as earlier described in section 4.3. NFHF membranes have thebest ethanol flux and solute transport coefficient as a result of high flux and ethanol per-meability. However, there is some concerns in the drastic drop in flux between two adjacentruns as described earlier. A 95% ethanol permeability at 20oC and 10bar changing to 85%

72


when increasing the pressure to 15bar were observed by Alfa Laval, see appendix A.13.

For the purpose of illustrating the consequence of the different membrane characteristicsa calculation was made predicting possible membrane area and dia-water volume needed tode-alcoholise 5 litre beer from 5.5%ABV to 0.5%ABV within a time span of 4 hours. Thesecalculated values can be observed in table 4.3. This calculation will indicate influences onCAPEX and OPEX. These values should be considered in connection with the possiblearoma losses over the membrane discussed in the following section.

Table 4.3: A calculated approximation of membrane area and dia-water needed to de-alcoholise 5 litre of beer from 5.5%ABV to 1%ABV with a pre-concentration of 2 VCF anda final concentration of 1 VCF resulting in 5 litre of beer with 0.5%ABV within a 4 houroperation. A constant flow rate 14 kg

min was used in the calculation.

Designation RO90 NF NF10 NFHF DesiredOptimisationDevelopment

Mean beer flux 0.173 0.417 0.514 0.717 ⇑[ kgmin∗m2 ]

Mean ethanol permeability 54 95 92 95 ⇑[%]

Membrane amount 16 4 4 3 ⇓0.018m2

membrane[n]

Membrane area 0.288 0.072 0.072 0.054 ⇓[m2]

Dia-water volume 9.4 4.6 4.8 4.6 ⇓[l]

Exact run time 4.1 3.9 3.6 3.4 ⇓[h]

A clear difference among the membranes influence on both OPEX and CAPEX canbe deduced from table 4.3. Concerning CAPEX, de-alcholisation using NFHF membranesseems significantly lower compared to RO90 membranes. A more than fivefold increasein membrane investment should be considered if choosing RO90 over NFHF membranes.In addition, a possible buffer tank for the dia-water should have twice the size, moreoverdemanding treatment, e.g. sterile filtration or ion exchange, of twice the volume of water.Increased area causes an increase in pump work for RO90 application to compensate forthe higher area and possibly piping while maintaining the same TMP. This would influencethe OPEX as well as CAPEX. Almost twice the volume of dia-water is needed to reachthe final alcohol concentration. All in all, NFHF seem as the most reasonable choice for amembrane process for de-alcoholisation based on permeate flux and ethanol permeability.Nonetheless, the influence on aroma retention among the different membranes needs to betaken into account.

73

4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

4.5 Aroma Retention

In this section the aroma retention of the different membranes will be considered basedon HS-GC-MS data. As mentioned in section 3.6.2, only properly separated peaks werechosen after a visual confirmation. Consequently, some important aroma components inbeer might not be identified because of i) overlapping peaks, ii) to low concentration oriii) to low matching. No absolute quantifications were made because of the high abundanceof different aroma compounds in beer. Therefore, only an internal comparison among thedifferent membranes was possible. The integrals of the clearly separated peaks are expectedto be equivalent to the relative concentration of the compound. In this assumption a con-stant dilution of the samples, purge gas volume, column temperature and column pressureis expected during all the HS-GC-MS samples. The different compound integral areas, aftermembrane filtration, were in any case compared to the original beer compound integrals,from the start beer in the feed tank. This will yielded an integral area ratio. Consequently,all results are presented as compounds area percentage of the original beers aroma com-pounds [%]. The samples was diluted to ensure that the same volume of original beer waspresent.

The HS-GC-MS aroma profiles of respectively Humlefryd bottled on the 24th of Feburary(HFORG24) and Humlefryd bottled on the 11th of April 2014 (HFORG11) can be observedin appendix A.8. All aroma compounds are illustrated as percentage compared to the meanintegrated area of the compound peak in the original beers. Additional aroma compoundsmight be present, however they were either in too low concentration for MS detection ofsignificance or not fully separated from other compound peaks.

A general tendency for a higher aroma compound output was observed for the HFORG11production compared to HFORG24. Concerning fermentation aroma products, respectivelyhigher alcohols, esters and aldehydes the similarities between the two production do notdiffer significantly. Nevertheless, the compounds such as decanal and octyl acetate do havea standard deviation of 49% and 44%. This might be caused by difference in yeast generation,temperature, aeration or other fermentation factors. Aroma products from hops all seemto differ significantly which could be caused by different hops addition time, oxidation,productions (crops) or different boiling times. In addition, the deviation observed couldillustrate possible deviation in the sampling technique or measurement method. Samplingand measuring errors cannot be ruled out based on duplication of each sample. The standarddeviations among duplicates HS-GC-MS samples for all membrane runs and detected aromacompounds can be found in appendix A.14.

The different aroma compounds solubility in water will in the following section be usedas a tool to evaluate possible partitioning or sorption at or in the more non-polar PAmembrane in coherence with polar Taft (σ∗), steric Taft (Es∗) and Small’s number (s∗)found in literature (Alvarez et al., 1998).

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4.5.1 Higher Alcohols

Figure 4.9: Higher alcohol aroma compounds area percentages compared to the original beerafter ended membrane filtration (FTE) measured by HS-GC-MS. Column legend: Membranename, nr. run, location of sample, stage in process. X-aksis legend: Compound name(retention time) - associated aromas.

The higher alcohols (HA) percentage retained in the feed tank beer or permeated intothe permeate tank can be observed in respectively figure 4.9 and 4.10. A quick overviewreveals a higher retention and lower permeation of aroma compounds by RO90 membranescompared to all the different NF membranes. Especially 1-butanol, n-octanol and benzeneethanol were significantly better retained by RO90 than NF membranes. 1-butanol seemsto be completely retained while benzene ethanol actually increases to more than twice theconcentration (273%) during the RO90 membrane. The increase of benzene ethanol wasinvestigated in the various HS-GC-MS spectra where no explanation for the increase couldbe found. In addition, a small permeation was observed when looking at the percentagein the permeate, see illustrated in figure 4.10. Therefore, the increase of benzene alcoholduring the RO90 membrane run was considered as almost completely retained. Possibledilution errors during HS sampling were ruled out because the other aroma compoundsshowed regular trends.

In figure 4.10 a decreasing trend for the permeation of linear alcohols was observed as theGC retention time (tR) and molecular mass increased (1-propanol→1-butanol→n-octanol).PA membrane filtration of pure alcohol solutions have shown an increase in retention alongwith an increase in higher molecular mass of linear alcohols (Schutte, 2003).

75


Figure 4.10: Higher alcohol aroma compounds area percentages compared to the originalbeer in permeate tank (PT) after ended membrane filtration measured by HS-GC-MS. Col-umn legend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend:Compound name (retension time) - associated aromas.

Table 4.4: Physiochemical data of higher alcohols (Fenaroli, 2005), (Alvarez et al., 1998).

Compound Molecular Solubility Polar Steric Small’sWeight In Water Taft Taft number

[ gmol ] [gl ] σ∗ Es∗ s∗

Ethanol 46.07 Miscible -0.100 -0.070 ∼ 01-Propanol 60.06 Miscible

Isobutyl alcohol 74.07 70 -0.125 -0.930 2.241-Butanol 74.07 73 -0.130 -0.390 2.17

Isoamyl alcohol 88.09 28n-Octanol 130.14 0.46

Benzene ethanol 108.06 4.3

In table 4.4 some physiochemical data of selected alcohols can be observed. These datawill be used to evaluate the observed retention in figure 4.9 and 4.10. The hydroxy (−OH)group of alcohols can form two hydrogen bonds with water enabling a total miscibility ofsmall alcohols such as 1-propanol, ethanol and methanol (Franks and Ives, 1966). Thestability gained by hydrogen bonding with water can compensate for the stability lost byalcohol-alcohol hydrogen bonding and non-polar interaction of the hydrocarbon chain. Asthe hydrocarbon chain becomes longer the solubility in water is reduced because the sta-bility gained in non-polar interaction becomes higher (Franks and Ives, 1966). 1-Propanolhad the highest flux through all different membranes, as observed in figure 4.10. On thecontrary, 1-propanol showed a general high retention in the retentate illustrated in figure

76


4.9. This might reveal the possibility of 1-propanol to permeate the membranes because ofthe ability to interact with water. Nonetheless, 1-propanol rejected by the membrane wouldnot partition on or within the membrane as a consequence of a fairly polar nature. Isobutylalcohol and 1-butanol have a relatively high retention with RO90 membranes. However, NFmembranes show significantly different trends in retention, with a high isobutyl alcohol re-tention and a low 1-butanol retention illustrated in figure 4.9. In table 4.4 it can be observedthat the solubility in water, the polar Taft (σ∗) and hydrophobicity Small’s number (s∗)is approximately the same while the steric Taft (Es∗) differs significantly between isobutylalcohol and 1-butanol. This indicates that the steric hindrance, caused by a methyl groupon carbon-2 (C2) in isobutyl alcohol, causes a lower permeation and a higher retention.The same tendency is observed comparing isoamyl alcohol to the more linear compoundn-octanol, where the same retention is observed for RO90 membranes while different for NFmembranes. The higher permeation of n-octanol by NF membranes could also be a result ofpartitioning of n-octanol to the membrane caused by non-polar interactions. This tendencyfor a lower retention of linear compounds only for NF membranes could indicate that NFmembranes follow the pore separation model while RO90 membranes follow the solution dif-fusion model. However, the best retention for RO90 membranes was observed for benzeneethanol which must be considered a result of a high steric hindrance of the molecule. Inaddition, the lowest retention of NF membranes was observed for benzene ethanol comparedto all the other higher alcohols. This could be a result of the cyclic ring in the moleculeenabling entrance into the pores despite the cross-flow application. In contrast more linearmolecules would pass the pores in a tangential fashion hereby not entering them.

A 100% retention of 1-propanol and isobutyl alcohol plus a 90% retention of isoamyl al-cohol for RO PA membranes (> 97% NaCl retention) at 25bar, 15oC and 3.33 l

min has beenillustrated. Additionally, a lower retention for 1-propanol, isobutyl alcohol and isoamyl al-cohol at respectively 25%, 50% and 40% for RO CA membranes (> 95% NaCl retention) at25bar, 15oC and 3.33 l

min was observed (Lopez et al., 2002).

Considering the HA retention of the different membranes a clear superiority was observedfor RO90 compared to NF membranes. NF membranes seemed to permeate linear HAs easierthan steric hindered compounds while RO90 membranes generally retained the majority ofthe HAs during de-alcoholisation. The high retention and low permeation of the alcoholsthrough RO90 membranes were connected to the alcohol group with high polarity and abilityto form hydrogen bonds.

77


4.5.2 Esters

Figure 4.11: Ester aroma compounds area percentages compared to the original beer infeed tank after ended membrane filtration (FTE) measured by HS-GC-MS. Column legend:Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compoundname (retension time) - associated aromas.

In figure 4.11 and 4.12 respectively the retention and permeation of different ester by differentmembranes can be observed. A general higher retention and lower permeation of esterswere observed for RO90 compared to NF membranes. Despite a high retention, figure4.11 indicates some losses esters, only indicating a total retention of ethyl caproate andphenylethyl acetate. On the contrary, the permeate HS-GC-MS data in figure 4.12 illustratea different trend for the RO90 membranes indicating no permeation of ethyl caproate, n-hexyl acetate, ethyl heptanoate, octyl acetate and ethyl caprate. In addition, the RO90permeation was below 10% for all esters with a longer retention time than 5.1 minute.Among the different NF membranes the best aroma retention and lowest permeation wereobserved for NF and HFNF membranes, while a clear reduction in retention and increasein permeation were observed for NF10. This was considered to be directly related to thehigher porosity of NF10 membranes, as seen in table 3.1.

78


Figure 4.12: Ester aroma compounds area percentages compared to the original beer inpermeate tank (PT) after ended membrane filtration measured by HS-GC-MS. Columnlegend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend:Compound name (retention time) - associated aromas.

Table 4.5: Physiochemical data of esters (Lopez et al., 2002), (Fenaroli, 2005), (Alvarezet al., 1998).

Compound Molecular Solubility Polar Steric Small’sWeight In Water Taft Taft number

[ gmol ] [gl ] σ∗ Es∗ s∗

Ethyl acetate 88.05 Miscible -0.100 -0.070 2.11Ethyl butyrate 116.08 4.9 -0.215 -0.430 1.54Isoamyl acetate 130.10 0.02 -0.045 -0.350 1.47n-Hexyl acetate 144.12 0.4 -0.133 -0.40 1.85

In table 4.5 the physiochemical data of selected esters were chosen to illustrate thedifference among acetate esters and ethyl esters along with the different lengths of the non-polar hydrocarbon parts. The different hydrocarbon groups will be referred to as the alcoholgroup and acid group in coherence with the formation of the ester, see section 2.4.2. Ethylacetate showed a low retention and high permeation, which were associated to the smallhydrocarbon chain causing a polarity enabling a total miscibility in water and a low sterichindrance within the membrane. The same tendency of permeation was observed concerningethyl acetate for RO90 and NF membranes. This is most likely caused by the ability ofethyl acetate to form hydrogen bonds and thereby interact with water or to permeate themembrane in resemblance to water.

79


Esters are generally able to form two hydrogen bonds with respectively the carbonyloxygen and ether oxygen as hydrogen bond acceptors (Lommerse et al., 1997). However,the strength of the carbonyl hydrogen bond is much stronger than that of the ether. Esterhydrogen bonding with methanol only yields one hydrogen bond to the carbonyl oxygen.Nevertheless, hydrogen bonding with water could yield an additional hydrogen bond to theether oxygen (Lommerse et al., 1997).

According to Shikata and Okuzono (2013) esters are not able to form any hydrogen bondin aqueous solutions. On the contrary, the small esters are miscible with water generallybecause of an intrinsic dipole moment behaving as proton acceptors in aqueous solutions(Shikata and Okuzono, 2013).

High permeation of ethyl acetate should not be viewed upon as critical for the overallaroma of the beer because of a general association, that this compound contributes withnegative solvent aroma.

In figure 4.11 the subsequent linear esters (ethyl propionate, propyl acetate and ethylbutyrate) seem to have the same general retention as ethyl acetate while the same esters infigure 4.12 seem to have a lower permeation as the linearity becomes larger. From table 4.5a lower water solubility for ethyl butyrate can be observed possibly causing partitioning tothe non-polar PA membrane. However, a lower polar Taft value shows a higher tendency forethyl butyrate to gain a negative charge though rearrangement hereby increasing basicitycompared to ethyl acetate. A lower Small’s value shows lower hydrophobicity, which couldbe connected to the ability of molecular rearrangement resulting in an intermolecular charge.However, the additional methyl groups on both the acid and ester side of the esters entailsa higher steric hindrance (Es∗). This could explain the permeation reduction observedin figure 4.12 as the chain length increases. Ethyl butyrate has the highest steric Taft intable 4.5, even higher than the branched isoamyl acetate ester. This indicates that thelength of the acid part of the ester has a higher influence on steric effects than branchingon the alcohol part. Isobutyl acetate and isoamyl acetate do show a higher retention andlower permeation than ethyl butyrate despite a lower steric hindrance for PA membranesaccording to Alvarez et al. (1998). This could be a result of a lower polar Taft and watersolubility resulting in these compounds not being able to permeate the membrane with wateror in a similar fashion as water.

A remarkable decreasing trend in retention with increasing non-polar hydrocarbon groupsand retention time is observed from isoamyl acetate to ethyl caprate for all NF membranesin figure 4.11. For RO90 membranes ethyl caproate and ethyl caprate seem to be excludedfrom this retention trend. In figure 4.12 contradictory trend seems to be prevalent with ahigher permeation of isoamyl acetate decreasing until not detectable. The molecular weightof these esters are ranging from 130.10-200.18 g

mol hereby being within the MWCO of themost porous NF10 membrane at 150-250 g

mol . Therefore, a NF10 membrane permeation oflinear compounds such as octyl acetate (172.15 g

mol ) and ethyl caprate (200.18 gmol ) would

have been expected. This contradiction in retention and permeability of esters with linearhydrocarbon groups above five carbon in length could be explained by a possible partition-ing to or within the PA membrane. A possible non-polar interaction could result in a higherconcentration polarisation close to the membrane surface or within the membrane structure.Both the flow and the flux through the membranes could have been too low to overcomethe non-polar interaction leaving the esters as fouling on or in the membrane structure.This would result in a reduction of esters in both the feed tank beer and in the permeate.A possible explanation for the ethyl caprate and phenylethyl acetate not experiencing thesame reduced retention of RO90 membranes could be a result of these compounds being to

80


bulky to enter the membrane for partitioning hereby being flushed off by the cross flow intothe permeate tank.

Finally, phenylethyl acetate was almost completely retained by RO90 membranes onlydetected in the permeate while clear permeation was observed for the various NF membranes.With a molecular weight of 164.08 g

mol this compound permeates the NF10 membrane as ex-pected. Phenylethyl acetate contains an aromatic benzene ring in the alcohol group whichcould be sorped within the PA membrane as a result of non-polar interaction in an equalway as illustrated by Schutte (2003) for phenols. He analysed phenol partitioning or sorp-tion on or within CA, PS and PA membranes. He proved a higher phenol sorption for PAmembranes compared to both PS and CA membranes. This could be a result of internalmembrane partitioning caused by non-polar interactions. The complete retention by RO90membranes must be considered to be a result of the size and bulkyness of the molecule beingsignificantly larger than phenol because of the ethyl acetate group.

A 90% retention of ethyl acetate for RO PA membranes (> 97% NaCl retention) at25bar, 15oC and 3.33 l

min was proven by Lopez et al. (2002). In addition, a lower retention

at 60% for RO CA membranes (> 95% NaCl retention) at 25bar, 15oC and 3.33 lmin was

observed (Lopez et al., 2002).A 77% retention of ethyl acetate and 68% for isoamyl acetate for RO CA membranes

(200 gmol MWCO) at 20bar, 5oC and 7 l

min was illustrated by Ferreira et al. (2007). Theretention fell drastically when increasing the pressure.

Conclusively, an increased ester retention was observed comparing RO90 membraneswith NF membranes. Among NF membranes, the best retention was observed for NFHFand NF membranes. A general retention above 50% was observed for RO90 membraneshighly influenced by the steric hindrance of ester with branching (iso esters). A remarkablereduction in retention among esters, with a linear acid or alcohol group above five carbonin length, was seen which could not be found in the permeate. This could have been causedby a partitioning of these more non-polar substances to the non-polar PA membrane. Inthe light of this, other membrane materials, pre-coating of the membrane or a higher flowshould perhaps be considered for a better ester recovery. Furthermore, an even less porousRO PA membrane could possibly reduce the partitioning and sorption within the membraneresulting in an even lower loss of esters.

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4.5.3 Aldehydes

Figure 4.13: Aldehyde aroma compounds area percentages compared to the original beer infeed tank after ended membrane filtration (FTE) measured by HS-GC-MS. Column legend:Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compoundname (retension time) - associated aromas.

In figure 4.13 and 4.14 respectively the retention and permeation of different aldehydes bydifferent membranes can be observed. In figure 4.13 a resembling retention of isobutyr alde-hyde and furfural for both RO90 and NF membranes was observed, while a clear differencewas observed for benzaldehyde. On the contrary, the permeation of the same compounds,illustrated in figure 4.14, shows a lower permeation of isobutyr aldehyde and furfural forRO90 compared to NF membranes, while benzaldehyde permeation seems to be equal for allmembranes. Consequently, the difference among aldehydes detected for RO90 to NF doesnot seem to be as significant as for other aroma compounds.

Isobutyr aldehyde is relatively soluble in water, as illustrated in table 4.6, because ofthe carbonyl ability to form hydrogen bonds with water. Therefore, a general high fluxthrough the membrane should be expected as a consequence of isobutyr aldehyde beingfairly polar while having a short hydrocarbon non-polar end. Nonetheless, the general trendin figure 4.13 and 4.14 illustrates a reasonably high retention and reduced permeation withthe RO90 permeation being fairly lower compared to NF membranes. Retention of isobutyraldehyde is most likely caused by the branching methyl group causing a steric hindrance. Inaddition, isobutyr aldehyde is not non-polar enough to partition at or in the membrane andwill therefore follow the permeate or retentate. Consequently, this aldehyde was separatedmainly on structural properties.

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Figure 4.14: Aldehyde aroma compounds area percentages compared to the original beerin permeate tank after ended membrane filtration (PT) measured by HS-GC-MS. Columnlegend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend:Compound name (retension time) - associated aromas.

Table 4.6: Physiochemical data for aldehydes (Fenaroli, 2005).

Compound Molecular SolubilityWeight In Water

[ gmol ] [gl ]

Isobutyr aldehyde 72.06 75Furfural 96.02 83

Benzaldehyde 106.04 6.95

A high permeation of furfural is observed in figure 4.14, which is confirmed with a lowretention in figure 4.13. Furfural is likely to form one hydrogen bond with water fromthe carbonyl group enabling a relatively high solubility, as seen in table 4.6. Furthermore,furfural has a relatively high dipole moment caused by a high electron negativity of the oxy-gen atoms attracting the π-electrons from the aromatic furan ring (Rivelino et al., 2004).Furfural hydrogen binding abilities and overall dipole moment might cause a higher mem-brane permeation along with water or in a similar fashion as water. Compared to isobutyraldehyde a lower retention is observed even though the molecular weight and size is higher.

Benzaldehyde also experiences a lower retention and higher permeation than isobutyraldehyde despite a higher molecular mass. Nevertheless, a higher retention for RO90 mem-branes is observed in figure 4.13. The reasonable low retention of a bulky molecule with ahigh molecular weight must once again be a result of a fairly high solubility in water causedby the ability of hydrogen bonding and a dipole moment causing a higher intramolecularpolarisation. Indeed Tekin et al. (2004) proved an increasing polarisation of benzaldehydeas the concentration of ethanol was increased. Ethanol favours a strong hydrogen bond withthe carbonyl oxygen in benzaldehyde being a Lewis base acceptor for the alcohol hydrogen.This clearly indicates a tendency to interact with ethanol hereby becoming more polarised

83


and therefore more water soluble. Further this enables a co-permeation through the mem-brane. In addition, this would explain the clear difference in retention between RO90 andNF membranes for benzaldehyde possibly caused by the lower ethanol permeation of RO90membranes.

Benzaldehyde is a Strecker aldehyde, which can be formed during the membrane runcaused by the presents of oxygen, as illustrated in figure 2.16. Oxygen might be introducedinto the feed tank through the dia-water addition where compressed air was used or simplyby air diffusion in the permeate tank. A possible explanation for the high concentration ofbenzaldehyde in the permeate in fiugre 4.14 could be caused by formation in the presence ofoxygen after permeation of phenyl alanine or phenyl acetaldehyde along with α-decarbonyl.

In summery, the difference in retention and permeation was not as prevalent for aldehy-des in accordance to different RO90 or NF membrane de-alcoholisations. The more equalretention and permeation could be caused by a tendency to form hydrogen bonds with waterand ethanol along with the experience of dipole moments causing polarisation of the aro-matic compounds. Furthermore, isobutyr aldehyde experienced the best retention possiblycaused by a lesser polarisation of the molecule hereby maintaining a non-polar end withmethyl branching.

4.5.4 Hops Constituents

Figure 4.15: Hops aroma compounds area percentages compared to the original beer infeed tank after ended membrane filtration (FTE) measured by HS-GC-MS. Column legend:Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compoundname (retension time) - associated aromas.

In figure 4.15 and 4.16 respectively the retention and permeation of different hops con-stituents by different membranes can be observed. Many of the hops constituents were notdetected in the final de-alcoholised beer most likely because of low concentration or lossduring membrane filtration. In appendix A.8 the HS-GC-MS of the original beer showedadditional detection of geraniol and α-humulene, while figure 4.15 only illustrates detection

84


of β-myrcene, nonanal, 3-carene and trans-anethole. However, these compounds representthe membrane potential for hydrocarbon monoterpene retention with β-myrcene and 3-carene plus oxygenated terpene retention with trans-anethole. In addition, nonanal wasclearly detected as a positively aroma attributing aldehyde. A general higher retention ofhydrocarbon terpenes was observed for all membranes compared to the oxygenated terpene.β-myrcene was almost completely retained only showing permeations for the NF membranesin figure 4.16.

Figure 4.16: Hops aroma compounds area percentages compared to the original beer inpermeate tank after ended membrane filtration (PT) measured by HS-GC-MS. Columnlegend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend:Compound name (retension time) - associated aromas.

β-Myrcene is a non-polar molecule almost completely insoluble in water, as illustrated intable 4.7, however readily soluble in ethanol. This could entail a β-myrcene co-permeationwith ethanol through the membrane. Moreover, the non-polar surface of the PA membranecould entail a partitioning of β-myrcene to the membrane allowing possible permeation ifthe pore size is adequately large. Indeed, a higher permeation is observed for β-myrceneaccording to the difference in pore size between RO90 to NF, with NF10 as the membranewith the largest pore size. Therefore, a structural separation seems most likely. β-Myrcene(7-methyl-3-methyleneocta-1,6-diene) is despite a methylene and methyl group a fairly linearcompound which could result in a possible permeation through the pores. Nevertheless, thispermeation is reduced by the cross-flow application resulting in a tangential direction of thecompounds during membrane filtration.

3-Carene is an aromatic monoterpene equally insoluble in water showing an even lowerpermeation than β-myrcene in figure 4.16. Nevertheless, the retention showed very differ-ent values among the different membranes with the lowest retention for RO90 and NF10membranes, while the highest retention was observed for NFHFR1. A contradiction wasobserved comparing the permeation to the retention because a higher retention seemed toinduce a higher permeation and vice versa. Indeed, this was believed to be a consequence ofpartitioning within the membrane caused by non-polar interactions resulting in NF10 beingthe membrane with the highest possibility of 3-carene adsorption within the membrane.

85


As a consequence 3-Carene will be removed from both the retentate and the permeate. 3-Carene has the same molecular weight as β-myrcene. However, the molecular structure issignificantly different being more linear than 3-carene with a cyclic structure. This differencecould involve an easier membrane entrance of 3-carene compared to β-myrcene. This mightbe a result of the cross-flow application resulting in a lower retention of 3-carene. Thus,the more linear β-myrcene, which has entered the membrane, could have a easier passagethrough the membrane resulting in the higher permeability observed.

In literature an almost complete retention of β-myrcene in the retentate after microfil-tation of ripe mango pure has been illustrated, possibly caused by entrapment of dropletswithin the cell walls or insoluble constituents (Olle et al., 1997). A possible droplet forma-tion on, or within, the membranes could result in the low retention and low permeation ofhydrocarbon oils.

Table 4.7: Physiochemical data for hops constituents (Fenaroli, 2005).

Compound Molecular SolubilityWeight In Water

[ gmol ] [gl ]

β-Myrcene 136.24 InsolubleNonanal 142.24 Insoluble3-Carene 136.23 Insoluble

trans-Anethole 148.21 Insoluble

Nonanal shows an opposite retention and permeation than all previously discussed aromasubstances with a higher permeation and lower retention for RO90 membranes comparedto NF membranes. Hexanal has the following parameters influencing the solute transportthrough the membrane with respectively a i) polar Taft (σ∗) of -0.133, ii) steric Taft (Es∗)of -0.40 and iii) a Small’s number (s∗) of 2.19 (Alvarez et al., 1998). Hexanal is very simi-lar in molecular structure to nonanal only being three carbons shorter in the hydrocarbonchain. The polar and steric Taft are relatively similar to that observed in table 4.4 and4.5 for respectively linear alcohols and esters. The Small’s number, representing the de-gree of hydrophobicity, is larger compared to the linear esters and more similar to that oflinear alcohols. Therefore, aldehydes such as nonanal seem to have a high hydrophobicity,which could result in a partitioning to the PA membrane. The tendency for aldehydes toform hydrogen bond with water or alcohol is lower compared to alcohol because aldehydesonly carry a hydrogen acceptor in the oxygen of the carbonyl group. This could result ina greater tendency for aldehyde membrane partitioning and thereby lower retention andinduced permeation compared to alcohols. In addition, the difference in polarity of thedifferent functional groups (−OH > −CHO > −C−−O > −C−O−C) could result in an evenhigher tendency to partition on, or within, the membrane enabling permeation (Dicksonet al., 1975).

Trans-anethole was the only oxygenated terpene observed after de-alcoholisation. Apoor retention for all NF membranes compared to the RO90 membrane was observed infigure 4.15 while no permeation was observed in figure 4.16. This could likewise be causedby compound partitioning with the membrane as a result of a relative non-polar molecule.This cyclic compound could possibly be able to partition within the pores of NF membranes,while only partitioning on the surface of the RO90 membrane. Compared to β-myrcene the

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4.6. TASTING RESULTS CHAPTER 4. RESULTS AND DISCUSSION

cross-flow would not result in difficulties in entering the membrane because of a generalcyclic and round composition of the molecule.

The hops constituents were generally well retained. The difference between RO90 andNF membranes were not as obvious for these compounds as for the other aroma com-pounds. Nevertheless, RO90 only showing a better retention of trans-anethole compared toNF membranes. This lack in difference could be a result of a higher PA membrane parti-tioning because of the general high hydrophobicity of the hops constituents, as illustratedin table 4.7. A tendency of permeation through the different membranes for the more lin-ear structured molecules was observed, while the cyclic molecules showed a lower retentionproperly caused by an easier entrance into the membrane in the cross-flow application.

4.6 Tasting Results

Figure 4.17: Tasting results from a blindtest where the beer was compared to an originalHumlefryd beer (HFORG24). Scores from 0-10 given by a skilled taste panel of seven.

The results of the tasting can be observed in figure 4.17. From the previous section ahigher aroma retention for de-alcoholised beer using RO90 compared to NF membraneswas observed. Nevertheless, RO90 membrane for de-alcoholisation could induce a higherCAPEX and OPEX which therefore should be compensated for in a higher aroma andflavour retention. The aroma and flavour composition of the beer could be drastically alteredas a result of some aromas permeating the membranes more than others hereby changingthe beer characteristics. In addition, de-alcoholised beer is often associated with a wateryexperience mainly caused by the lack of the warming mouth feeling of ethanol. For thesereasons, the test participants were asked to rate the beer from 1-10 in accordance with therelative equality and intensity to the original beer. In figure 4.17 the general reproducibilityof the panel was tested by placing an original beer (HFORG24) sample within the de-alcoholised samples. The original sample scored significantly lower than the identical beer,which the panel tasted as the standard sample, representing the highest grade (10). Thiswas done to validate both the tasting result and the panel, to see if they would give thisbeer the highest score of 10 as would be expected. Nonetheless, this sample was given the

87

4.7. OVERALL RESULTS CHAPTER 4. RESULTS AND DISCUSSION

highest overall rating. The design of experiment, asking the panel to rate only below thefirst original beer sample, could be the course of the low grades of the original beer blind testsample, along with the other samples. In this way the taste panel only expected samplesunequal and/or less intense than the original beer. Despite this, higher grades was given thesample produced using RO90 compared to NF membranes hereby confirming the HS-GC-MS results. The additional de-alcoholised beers, produced with NF membranes, were notsignificantly different, however NFR2 did seem to be the best among the NF membranes.A significant variation in grades given was observed in figure 4.17 revealing the need for alarger taste panel, with more than seven participants, to obtain statistic significance.

4.7 Overall Results

Evaluating membrane potential for de-alcoholisation of beer should be done on the basis ofCAPEX, OPEX and quality parameters. The choice of membrane application can influencethe end product drastically or involve a final production cost entailing a too costly product.In table 4.8 the overall results are illustrated.

Table 4.8: Overall results for beer flux, ethanol permeability, aroma retention and tastingresults plus the desired optimisation development. All values are mean analytical valuesof numerous membrane runs. Flux was measured by mass, ethanol by HPLC and aromacompounds by HS-GC-MS. ∗Mean value of percentage esters retained minus benzene ethanol(273%).

Designation RO90 NF NF10 NFHF DesiredOptimisationDevelopment

Beer Flux 0.173 0.417 0.514 0.717 ⇑[ kgmin∗m2 ]

Ethanol 54 95 92 95 ⇑Permeability [%]Higher alcohol 96∗ 58 52 56 ⇑Retention [%]Ester 63 40 23 37 ⇑Retention [%]Aldehyde 70 68 44 73 ⇑Retention [%]Hops constituents 77 64 55 73 ⇑Retention [%]Tasting 6.8 6.3 5.8 5.5 ⇑Grade [1− 10]

From table 4.8 an approximate seven-fold increase in flux plus twice the ethanol per-meation can be observed between RO90 and NFHF membranes. On the contrary, onlyapproximately twice the aroma retention of higher alcohols and ester was observed, whileno significant retention difference was observed for aldehydes and hops constituents. There-fore, RO90 application seems as a high cost for the purpose of retaining higher alcohols andesters. Nevertheless, these aroma substances, especially esters, are considered extremely im-

88

4.8. AROMA FORMATION CHAPTER 4. RESULTS AND DISCUSSION

portant in beers. From this point of view, it might seem more reasonable simply to producemore ester and higher alcohols during fermentation compensating for the losses observedas retentions in table 4.8. Nonetheless, breweries nowadays seem to be keen on reducingthe number of production streams making alteration of the brewing process even harder toinfluence. If no alteration of the brewing process is made then RO90 membrane would resultin the most aromatic and flavourful beer. The brewer could alternatively view the AFB asthe final product hereby seeing the membrane loss as a part of the process, which should beaccounted for in the beer before membrane filtration.

4.8 Aroma Formation

Dependent on membrane application the degree of aroma loss should be considered beforethe beer is membrane filtered to alcohol free beer (AFB). Fermentation is an influentialprocess where different parameters could be altered to compensate for later aroma losses.The main loss of aroma compounds during membrane filtration was experienced in regardsto esters and higher alcohols (HAs). Especially short linear esters and HAs had a tendencyto permeate the membranes along with water and ethanol. In addition, longer linear estersand HAs was removed both from the retentate and the permeate as a result of a possiblepartitioning within the membrane. A partitioning on or within the membrane was alsoobserved for cyclic compounds of esters and HAs.

In section 2.4.2 the formation of HAs was described pointing out the close relation tothe amino acid metabolism through the Ehrlich pathway. The formation of amino acids wasfound to occur mainly in the initial yeast growth phases during free amino nitrogen (FAN)assimilation. Factors affecting HA concentration were found to be closely related to elevatedyeast growth. To enhance HA formation the following process parameters could be altered:

• Higher fermentation temperature

⇒ More active metabolism releases more metabolites

• Higher FAN

⇒ Higher FAN assimilation and degradation to HA

• Topping up

⇒ Maintaining the yeast growth phase for a longer period

• High gravity brewing (HGB)

⇒ Higher FAN concentration and more active metabolism

• Increase pitching rate

⇒ Induced metabolite formation as a consequence of higher yeast concentration

• Higher wort aeration

⇒ A higher yeast growth

• Continuous agitation (Iso-mix)

⇒ More active metabolism releases more metabolites

89


• Zn2+ addition

⇒ Yeast growth factor

In section 2.4.2 the formation of esters was described pointing out the formation of twodifferent types of ester respectively acetate and ethyl esters. These esters are formed in anenzyme catalysed condensation reaction between alcohols and acids activated in reactionwith CoA-SH. Ethanol and HAs can participate in this reaction along with acetate, oxo-acids and medium chain fatty acids (MCFA). Ester formation seems to be closely relatedto lipid synthesis functioning as an inhibitor on ester synthesis. Furthermore, intracellulardetoxification of MCFA was proven to be a mechanism resulting in ester formation. Toenhance ester formation the following process parameters could be altered:

• Higer fermentation temperature

⇒ Higher AATase activity and higher HA production

• Higher FAN

⇒ FAN addition during stationary phase induces ester formation

• Topping up and increasing attenuation

⇒ Longer AATase activity

• Reduced wort aeration

⇒ During oxygen depletion formation of HA and acetyl-CoA continuous

• HGB production

⇒ Higher acetyl-CoA and HA formation

• Reduced medium chain fatty concentration in the wort

⇒ Linoleic acid inhibits ester formation

• Using ale yeast for pilsner production

⇒ Higher AATase activity and easier membrane ester permeation

• Low pressure

⇒ Less dissolved CO2

• High glucose and fructose concentrations

⇒ Higher acetyl-CoA and HA formation

⇒ Stronger exression of ester synthase genes

• Zn2+ addition

⇒ Yeast growth factor stimulation HA formation

Conclusively, to enhance both HA and ester formation in the fermentation i) a higherfermentation temperature, ii) a higher FAN concentration, iii) Zn2+ addition, iv) applyingwort by topping up and v) increasing the pitching rate however vi) reducing the wortaeration seem to be the easiest process parameters to alter. Furthermore, HGB productionis a powerful tool for a higher HA and ester production, however this is accompanied with

90


a higher ethanol production. Therefore, this approach would be short-termed as ethanol isthe main compound to removal in AFB production. It seems more reasonable to reducethe gravity of the fermenting wort, thus producing less alcohol during the fermentation.A wort gravity reduction could possibly result in an additional aroma reduction despite aprocess parameter adaptation towards a higher aroma production. Nevertheless, the lowergravity beer production could be coupled with pilsner fermentation using ale yeast at hightemperatures with a tendency for higher HA and ester production along with additionalprocess parameter adaptations. This would most likely result in a pilsner rich in HAsand esters hereby being disharmonious before membrane filtration, though more drinkableafterwards. Applying this approach could entail a possible application of NF membranes,despite the higher aroma flux, instead of RO membranes resulting in a lesser CAPEX andOPEX influence of the AFB production. As brewers tend to use regular alcoholic beersalready in production for AFB production the best approach would be; a higher temperaturefermentation, using malt high in FAN, adding Zn2+, applying topping up of wort, a higherpitching rate and a lower aeration of the yeast coupled with RO membrane filtration.

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Chapter 5

Future Perspectives

The conclusion reached suggest trials where the end product in focus is the alcoholic freebeer (AFB) and not the regular alcoholic beer used in membrane filtration. Changing thephysical parameters of the fermentation, the composition of the raw material, the yeast,the start beer, the membrane composition or even the entire brewing process are ways ofobtaining a beer capable of compensating for possible membrane aroma losses. Even viewingthe AFB after membrane filtration as an intermediate product has potential.

Changing the physical parameters of the fermentation, as discussed in the previoussection 4.8, while using the same yeast could result in a more aroma rich beer. Trialschanging these parameters could be illustrated by measuring HS-GC-MS where only oneparameter is changed at the time. Comparing these results would illustrate the potentialand powerfulness of simple physical changes in the fermentation.

Changing the composition of the initial raw materials could involve a lower gravity pro-duction causing a lower %ABV beer. This process alteration is equal to ”changed mashing”observed as a biological AFB process in figure 1.2 page 10. A possible reduction in aromaand flavour formation could be coupled with a change in physical parameter of the fermen-tation. The reduced need for dia-water addition and process duration of the membrane runcould result in a lower aroma and flavour loss.

Ale yeast has a greater tendency to produce ester and higher alcohols compared topilsner yeast. This process alteration is equal to ”special yeast” observed as a biologicalAFB process in figure 1.2. For some brewhouses changing the yeast might be the easiestprocess alteration. Using an identical wort would mean obtaining some of the same flavoursdonated from the raw material, while a different yeast might add higher concentrations ofthe fermentation aromas and flavours. Trials with different ale yeast on the Humlefrydpilsner wort accomplished with HS-GC-MS could illustrate the expected increase in aromacompounds.

In this thesis only Humlefryd pilsner was de-alcoholised using membranes. Trials withdifferent start beers such as belgium ales, IPA or bock beer could entail different excitingAFB products. These products would possibly not be as flavourful as the original beer butmore flavourful than the AFBs currently on the market.

In section 4.5 problems associated with the sorption and partitioning to the non-polarpolyamide (PA) membrane were observed. Trials with different membrane layer compo-sitions could reveal polymers with a higher selectivity for specific aroma compounds suchas esters and higher alcohols (HAs). Furthermore, pre-coating of the PA membrane withmore polar compounds could reduce partitioning of the non-polar aroma compounds to themembrane.

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CHAPTER 5. FUTURE PERSPECTIVES

If the AFB after membrane filtration was viewed as an intermediate product a possibleaddition of aroma and flavours to the AFB could result in a higher quality end product.Loss of hops constituents could for example be compensated by hop boiling or dry hoppingof the dia-water separately before re-dilution back to a VCF of one. Loss of colour andaroma constituents donated by the malt, such as furfural, could be added back to the beerwith malt brewing colour products. If a more precise concentration loss of esters and HAswas obtained, then food grade esters and HAs could be added to the AFB in the dia-water.

The HA and ester loss to the permeate could alternatively be regained from the permeateusing well-known techniques of ethanol removal on the permeate, such as evaporation orrectification, as illustrated in figure 1.2. Removing the ethanol gently from the permeateusing this as dia-water for re-dilution back to VCF of one could alternatively entail optimalaroma composition of the beer.

The optimal process for AFB production could reveal itself to be not only one method ofphysical or biological processes, but instead the right combination of these processes. Thegeneral understanding of AFB as the wanted end product instead of a processed regularbeer production is the first milestone to be reached demanding different thinking in thetraditional brewing process.

93

Chapter 6

Conclusion

The results obtained in this thesis illustrates the potential of membrane filtration as anapplication for alcohol free beer (AFB) production.

Three different nano filtration membranes, NF, NF10 and NFHF, were tested showingthe best flux and ethanol permeability through NFHF membranes. NFHF showed a sev-enfold higher flux and twice the ethanol permeability compared to the one reverse osmosismembrane (RO90) tested. Applying RO90 for AFB production compared to NFHF induceda more than fivefold increase in membrane area and twice the dia-water volume, if a runtime of approximately four hours was wanted. Therefore, the influence of both CAPEXand OPEX for RO90 application could result in a too costly AFB product despite a higherflavour and aroma quality.

The polyamide (PA) thin film composition (TFC) of both the NF and RO membranesshowed a general tendency to partition hydrophobic compounds on, or within, the mem-brane in degrees based on structural properties. This resulted in compounds removed fromboth the retentante and permeate. Less hydrophobic compounds were more clearly sepa-rated based on molecular structure with especially branched molecules involving a higherretention.

Higher alcohol (HA) and ester retention in beer were clearly different when applyingdifferent RO90 and NF membranes for de-alcoholisation. RO90 membranes showed a higherHA and ester retention. On the contrary, aldehyde and hops constituents retention in beerwere fairly identical when applying different RO90 and NF membranes for de-alcoholisation.

A beer tasting, comparing the membrane filtered products to the original beer, illustrateda higher intensity and equality score for RO90 than NF membranes.

Consequently, a high aroma quality product could be produced applying RO90 mem-branes, however inducing a higher CAPEX and OPEX while still experiencing some aromalosses. Alternatively, the AFB production could be reviewed changing brewing and fermen-tation processes compensating for the aroma losses over RO90 and NF membranes. Applyinga higher fermentation temperature, higher free amino nitrogen (FAN) concentration, higherZn2+ addition, wort topping up, increasing the pitching rate and reducing the wort aerationwere found to be the easiest process alteration to obtain a higher HA and ester formationduring fermentation. In addition, trials with i) ale yeast for pilsner production, ii) changingthe composition of raw materials, iii) applying different membrane compositions, iv) dif-ferent membrane pre-coating and v) flavour addition or recovery after membrane filtrationwould be alternative ways of producing a high quality AFB.

.

94

List of Figures

1.1 Relative risk of mortality compared to the weekly alcohol intake. Verticallines indicate the 95% confidence interval (Groenbaek et al., 1994). . . . . . . 8

1.2 Different methods of reducing the alcohol concentration in beer. OPEX =operational expenditure, CAPEX = capital expenditure (Branyik et al., 2012). 10

2.1 Different separation processes along with particle size retention (Askew et al.,2008). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2 Pore size and pressure range for different membrane processes (Hausmannet al., 2013). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.3 Dead-end and cross-flow filtration processes (Smith, 2013a). . . . . . . . . . . 142.4 Trans-membrane pressure during cross-flow filtration (Hausmann et al., 2013). 162.5 Difference in normal filtration processes (left) compared with diafiltration pro-

cesses (right). The diafiltration liquid added (Vd) results in a lower concentra-tion of the permeating compound in equal retentate volumes (Vr) (Hausmannet al., 2013). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.6 The general composition of Alfa Laval RO and NF membranes (Møller, 2014) 192.7 Synthesis of polyamide (Tang et al., 2009) . . . . . . . . . . . . . . . . . . . . 212.8 Ideal discontinuous diafiltration process divided into different stages. A math-

ematical approximation of the membrane trends. Data used: Feed volumebeer = 5 l, initial alcohol concentration = 5.5%ABV, measured flux (J)through the membrane = 0.519 l

m2∗min , flow retentate = 12.8 lmin , membrane

permeability = 85% and membrane area = 0.072m2. . . . . . . . . . . . . . . 242.9 Batch system outlook and corresponding feed, membrane, retentate, per-

meate and additional data needed for an approximated calculation of themembrane system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.10 Aerobic and anaerobic glucose metabolism involved in the formation of flavourcompounds. ATP = Adenosine triphosphate, ADP = adenosine diphosphate,NAD+ = Nicotinamide adenine dinucleotide, NADH+H+ = Reduced NAD+,CoA-SH = Coenzyme A, AA = Amino acids, VDK = Vicinal diketones,FAD+ = Flavin adenine dinucleotide, FADH + H+ = Reduced FAD+, GTP= Guanosine triphosphate and TCA = Tricarboxylic Acid Cycle (Kunze, 2010). 29

2.11 Development of different substances during batch fermentation of a lager at15oC. (a) Present gravity (PG) = 1.000 + 0.004 ∗ [%Plato]. (c) FAN = FreeAmino Nitrogen. (d) H. alcohols = Higher alcohols (HA) and VDK = Totalvicinal diketones (Briggs et al., 2004). . . . . . . . . . . . . . . . . . . . . . . 32

2.12 Formation of HA by anabolic routes from pyruvate and catabolic routes fromassimilated amino acids through the Ehrlich pathway (Hazelwood et al., 2008). 33

95

LIST OF FIGURES LIST OF FIGURES

2.13 Formation of ester by enzyme-catalysed coenzyme A (CoA-SH) condensation.R = hydrocarbon side chain (Verstrepen et al., 2003). . . . . . . . . . . . . . 37

2.14 Pentose reaction with a nitrogen containing compounds to form the Maillardproduct furfural through cascades of reactions (Baert et al., 2012). . . . . . . 41

2.15 Strecker aldehyde formation from transamination between a amino acid anda α-dicabonyl (Baert et al., 2012). . . . . . . . . . . . . . . . . . . . . . . . . 42

2.16 Formation of benzaldehyde from phenylalanine, (Baert et al., 2012) . . . . . . 432.17 Chromatographic separation technique (Moldoveanu and David, 2013). . . . . 472.18 Packed and capillary GC columns (Mcnair and Miller, 2009). . . . . . . . . . 492.19 Static headspace sampling technique (Wang et al., 2008). . . . . . . . . . . . 492.20 Dynamic headspace sampling technique (Wang et al., 2008). . . . . . . . . . . 502.21 Refractive index detection technique (Moldoveanu and David, 2013). . . . . . 512.22 Electron ionisation quadrupole mass spectrometer (Laboratory, 2012). . . . . 51

3.1 Labstak M20-0.72 Alfa Laval unit. . . . . . . . . . . . . . . . . . . . . . . . . 533.2 Sketch of the experimental set-up with modifications to the Labstak M20-0.72

Alfa Laval labunit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543.3 Membrane module flow of Labstak M20-0.72 Alfa Laval labunit. Black arrows

are retentate flow and green arrows are permeate flow. . . . . . . . . . . . . 55

4.1 Principal component analysis (PCA) on RO90 data given by Alfa Laval. PC1describes 65% of the variation in the data while PC2 describes 34%. Sample(red triangle) name RO90[Temperature in oC][Pressure in bar]. Loadings(blue square), PM = Permeability. PC = Principal component. . . . . . . . . 62

4.2 Principal component analysis (PCA) on NF data given by Alfa Laval. PC1describes 67% of the variation in the data while PC2 describes 29%. Sample(red triangle) name NF[Temperature in oC][Pressure in bar]. Loadings (bluesquare), PM = Permeability and PC = Principal component. . . . . . . . . . 63

4.3 Principal component analysis (PCA) on NFHF (NF99HF) data given by AlfaLaval. PC1 describes 59% of the variation in the data while PC2 describes36%. Sample (red triangle) name NF[Temperature in oC][Pressure in bar].Loadings (blue square), PM = Permeability and PC = Principal component. 64

4.4 Mass trend for membrane run with RO90 and NFHF membranes, see table4.1 for physical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4.5 Flux behaviour for reverse osmosis membrane runs, see table 4.1 for physicalparameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.6 Flux behaviour for nano filtration membrane runs, see table 4.1 for physicalparameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

4.7 Linear plot of permeate water flux as a function of pressure difference fromequation 4.2. Other physical parameters can be viewed in table 4.1. . . . . . 70

4.8 Permeate ethanol trend. (Start-End %ABV of beer in feed tank). See table4.1 for physical parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4.9 Higher alcohol aroma compounds area percentages compared to the originalbeer after ended membrane filtration (FTE) measured by HS-GC-MS. Col-umn legend: Membrane name, nr. run, location of sample, stage in process.X-aksis legend: Compound name (retention time) - associated aromas. . . . . 75

96


4.10 Higher alcohol aroma compounds area percentages compared to the originalbeer in permeate tank (PT) after ended membrane filtration measured by HS-GC-MS. Column legend: Membrane name, nr. run, location of sample, stagein process. X-aksis legend: Compound name (retension time) - associatedaromas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

4.11 Ester aroma compounds area percentages compared to the original beer infeed tank after ended membrane filtration (FTE) measured by HS-GC-MS.Column legend: Membrane name, nr. run, location of sample, stage in pro-cess. X-aksis legend: Compound name (retension time) - associated aromas. . 78

4.12 Ester aroma compounds area percentages compared to the original beer inpermeate tank (PT) after ended membrane filtration measured by HS-GC-MS. Column legend: Membrane name, nr. run, location of sample, stage inprocess. X-aksis legend: Compound name (retention time) - associated aromas. 79

4.13 Aldehyde aroma compounds area percentages compared to the original beerin feed tank after ended membrane filtration (FTE) measured by HS-GC-MS. Column legend: Membrane name, nr. run, location of sample, stage inprocess. X-aksis legend: Compound name (retension time) - associated aromas. 82

4.14 Aldehyde aroma compounds area percentages compared to the original beerin permeate tank after ended membrane filtration (PT) measured by HS-GC-MS. Column legend: Membrane name, nr. run, location of sample, stage inprocess. X-aksis legend: Compound name (retension time) - associated aromas. 83

4.15 Hops aroma compounds area percentages compared to the original beer infeed tank after ended membrane filtration (FTE) measured by HS-GC-MS.Column legend: Membrane name, nr. run, location of sample, stage in pro-cess. X-aksis legend: Compound name (retension time) - associated aromas. . 84

4.16 Hops aroma compounds area percentages compared to the original beer inpermeate tank after ended membrane filtration (PT) measured by HS-GC-MS. Column legend: Membrane name, nr. run, location of sample, stage inprocess. X-aksis legend: Compound name (retension time) - associated aromas. 85

4.17 Tasting results from a blindtest where the beer was compared to an originalHumlefryd beer (HFORG24). Scores from 0-10 given by a skilled taste panelof seven. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

A.1 Matlab worksheet for the calculation of membrane processes. . . . . . . . . . 108A.2 Formation of hydro peroxide fatty acids from triacylglycerol, (Baert et al.,

2012). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109A.3 Formation of Diacetyl and 2,3-Pentanedione involved in the Valine and Isoleucine

anabolism. TPP = Thiamine pyrophosphate cofactor, NADPH = Nicoti-namide adenine dinucleotide phosphate (Garcıa et al., 1994), (Haukeli andLie, 1978) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

A.4 Biosynthesis of sulphur containing amino acids. Cysteine and inorganic sul-phur assimilation for the anabolism of sulphur containing amino acids. NADPH= Nicotinamide adenine dinucleotide phosphate (Walker, 1998), (Landaudet al., 2008) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

A.5 Formation routes of dimethyl sulphide, (Briggs et al., 2004) . . . . . . . . . . 116A.6 Formation of Mercaptans in beer, (Vermeulen et al., 2006), (Swiegers and

Pretorius, 2007) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

97


A.7 Formation of 3-methylbut-2-ene-1-thiol, the sunstruck flavour, (Burns et al.,2001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

A.8 TCA cycle and corresponding acids excreted by brewing yeast (Briggs et al.,2004). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

A.9 Isomerization reaction of α-acid, (Briggs et al., 2004). . . . . . . . . . . . . . 123A.10 Higher alcohol aroma compounds area percentages compared to the mean

original beer measured by HS-GC-MS. Column ledgend: HF = Humlefryd ,ORG = Orignal , 24 = 24th of February and 11 = 11th of April. X-aksisledgend: Compound name (Retension time) - Associated flavours. . . . . . . 124

A.11 Ester aroma compounds area percentages compared to the mean originalbeer measured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG =Orignal , 24 = 24th of February and 11 = 11th of April. X-aksis ledgend:Compound name (Retension time) - Associated flavours. . . . . . . . . . . . . 124

A.12 Aldehyde aroma compounds area percentages compared to the mean originalbeer measured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG =Orignal , 24 = 24th of February and 11 = 11th of April. X-aksis ledgend:Compound name (Retension time) - Associated flavours. . . . . . . . . . . . . 125

A.13 Hop aroma compounds area percentages compared to the mean original beermeasured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG = Orig-nal , 24 = 24th of February and 11 = 11th of April. X-aksis ledgend: Com-pound name (Retension time) - Associated flavours. . . . . . . . . . . . . . . 125

A.14 Calibration curve for alcohol measurement on HPLC. . . . . . . . . . . . . . . 126A.15 HPLC report of the beer in the feed tank before NFHF membrane filtration

was initiated. Retention time in minutes, at the top of the peak, and integralarea situated at the ethanol peak. . . . . . . . . . . . . . . . . . . . . . . . . . 127

A.16 Head space sampling set-up. Trapping the aroma in on Tenax-TA traps at37oC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

A.17 Aroma compounds measured on the GC-MS for original Humlefyrd bottledthe 24th of April. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

A.18 Dealcoholisation membrane data analysis by Alfa Laval using an HPLC-MSwhere the ethanol peak was used to dictate the ethanol reduction. The addi-tional HPLC-MS peaks were viewed upon as aroma peaks resulting in a possi-ble percentage reduction of ”aroma” peaks. Analyse = analysis, bestemmelse= determination, af = of, alkohol = alcohol, aromastoffer = aroma com-pounds, i = in, øl = beer, prøve=sample, tryk = pressure, konc. = conc.,areal = area and PM = permeability [%]. . . . . . . . . . . . . . . . . . . . . 130

A.19 Standard deviation percentage [%] between duplicates samples on HS-GC-MS.131

98

List of Tables

2.1 Physical data on BECO filter sheet. Type KD7, Article no. 22070 (BEKO,2004) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.2 Alfa Laval TFC membrane layer composition (Møller, 2014) . . . . . . . . . . 202.3 Energy formation for brewers yeast in aerobic and anaerobic conditions (Walker,

1998). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.4 Higher alcohols in beer, aroma threshold and corresponding concentration in

lager beer (EBC, 2000), (Tan and Siebert, 2004), (Fenaroli, 2005). . . . . . . 322.5 Amino acids and corresponding alcohols as a result of Ehrlich pathway (Hazel-

wood et al., 2008), (EBC, 2000). . . . . . . . . . . . . . . . . . . . . . . . . . 342.6 Esters, aroma threshold and corresponding concentration in lager beer. Ethyl

esters (E) and acetate ester (A) (EBC, 2000), (Tan and Siebert, 2004), (Fe-naroli, 2005). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

2.7 Aldehydes, aroma threshold and corresponding concentration in lager beer(EBC, 2000), (Tan and Siebert, 2004). . . . . . . . . . . . . . . . . . . . . . . 40

2.8 Hops constituents, aroma threshold and corresponding concentration in lagerbeer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989). 45

3.1 Physical data on thin film composition (TFC) membranes from Alfa Laval(Laval, 2014a), (Laval, 2014b), (Laval, 2014c). . . . . . . . . . . . . . . . . . . 56

3.2 pH and conductivity of different water sources. . . . . . . . . . . . . . . . . . 573.3 Sampling plan during membrane filtration runs . . . . . . . . . . . . . . . . . 583.4 Overview of equipment settings and parameters for GC-MS analysis . . . . . 60

4.1 Membrane parameters and constants for each membrane run. Highlightednumber and membrane runs are not considered in the standard deviation(SD) calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

4.2 Ethanol retention, permeability and solute transport coefficient for variousmembrane runs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4.3 A calculated approximation of membrane area and dia-water needed to de-alcoholise 5 litre of beer from 5.5%ABV to 1%ABV with a pre-concentrationof 2 VCF and a final concentration of 1 VCF resulting in 5 litre of beer with0.5%ABV within a 4 hour operation. A constant flow rate 14 kg

min was usedin the calculation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4.4 Physiochemical data of higher alcohols (Fenaroli, 2005), (Alvarez et al., 1998). 764.5 Physiochemical data of esters (Lopez et al., 2002), (Fenaroli, 2005), (Alvarez

et al., 1998). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794.6 Physiochemical data for aldehydes (Fenaroli, 2005). . . . . . . . . . . . . . . . 834.7 Physiochemical data for hops constituents (Fenaroli, 2005). . . . . . . . . . . 86

99

LIST OF TABLES LIST OF TABLES

4.8 Overall results for beer flux, ethanol permeability, aroma retention and tast-ing results plus the desired optimisation development. All values are meananalytical values of numerous membrane runs. Flux was measured by mass,ethanol by HPLC and aroma compounds by HS-GC-MS. ∗Mean value of per-centage esters retained minus benzene ethanol (273%). . . . . . . . . . . . . . 88

A.1 Ketones in beer, flavour threshold and corresponding concentration in lagerbeer (EBC, 2000), (Tan and Siebert, 2004). . . . . . . . . . . . . . . . . . . . 110

A.2 Sulphur compounds in beer, flavour threshold and corresponding concentra-tion in lager beer (EBC, 2000), (Tan and Siebert, 2004), (Landaud et al.,2008), (Briggs et al., 2004). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

A.3 Acids in beer, flavour threshold and corresponding concentration in lager beer(EBC, 2000), (Tan and Siebert, 2004), (Klopper et al., 1986), (Siebert, 1999),(Kunze, 2010). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

A.4 Hops constituents in beer, flavour threshold and corresponding concentrationin lager beer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004),(Irwin, 1989). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

100

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Appendix A

Appendices

A.1 Glossary

Aroma - Sense of smell nasal or retro-nasal.

Absorption - Take-up or assimilation of acompound.

Adsorption - Adhesion or partitioning of acompound to a surface.

Centrifugation - Separation of particlesand liquids caused by the centrifugal forcemaking the more dense particles and liquidsmigrate away from the axis during rotaryacceleration.

Clarification - The process of makingsomething transparent and/or clear.

Cold break - Proteins, polyphenols andcarbohydrates forming hydrogen bonds andhydrophobic cluster in the beer promotedby cooling.

Cropping - The process of removing theyeast from the fermentation for additionalfermentation in another fermenter ordischarge. Sometimes called yeastharvesting if reused.

Diafiltration - Addition of a solvent to theretentate to enable a higher permeation ofpermeable solutes through the membrane.

Ethereal oils - Volatile oils from plants.

Evaporation - Vaporisation of a liquid intoa gaseous phase which is not saturated withthe evaporated substance.

Filtration - Separation of solid particlesfrom liquids by retention of the particles on

a filter medium while the liquids passes.

FAN - Free amino nitrogen. Individualamino acids and small peptides present inthe wort.

Flavour - The total experience of the taste,aroma and mouth feeling.

Fermentation - Energy production byorganisms in the absence of oxygen causedby substrate-level phosphorylation.

Flocculation - A reversible process ofyeast adhering to each other to formaggregates resulting in easier sedimentation.

Flow - Fluid flow is the mass flow rate of aliquid transported within pipelines or over asurfaces such as membranes [ kgmin ].

Flux - Fluid flux is the mass flow rate of aliquid transported through a given areasurface such as membranes [ kg

m2∗min ].

Lectin - Carbohydrate binding protein.

Mash - Milled grains, mainly barley, mixedwith water.

Mashing - The process of degrading themash component into usable constituents byoptimising gain enzyme processes.

Mouth feel - A products physical andchemical interaction in the mouth.

Partitioning - ”The distribution of asolute between two immiscible or slightlymiscible solvents in contact with oneanother, in accordance with its differing

1http://www.oxforddictionaries.com/definition/english/partition

106

A.1. GLOSSARY APPENDIX A. APPENDICES

solubility in each”1.Pitching - Addition of yeast to wort herebyinitiating the fermentation.Propagation - Continuous multiplicationor re-production of an organism.Rectification - Purification of a liquid bydistillation.Permeability - The ability of a specificcompounds to permeate a membrane [%].Resulting in a flux through the membraneinto the permeate.Permeate - The liquid permeating themembrane.Rejection - The ability of a membrane toreject a specific compound hereby causingthe compounds to stay on the retentate sideof the membrane [%].Retentate - The liquid retained by themembrane.Retention - The ability of a membrane to

retain a specific compound on the retentateside of the membrane [%].

Sedimentation - Sinking or precipitationof particles with a higher density than thesurrounding medium.

Sorption - The physical or chemical abilityfor one substance or compound to becomeattached to another.

Taste - What you senses inside yourmouth. Bitter, sweet, sour, salt and Umami.

Wort - The liquid obtained after removal ofthe solids from the mash containingfermentable sugars and nutrients for theyeast.

Yeast strain - Brewers yeast belong to theGenus of Saccharomyces and Species ofcerevisae and pastorianus. Different brewersyeast sub-species can be divided intodifferent yeast strains.

107

A.2. DIAFILTRATION APPROXIMATION APPENDIX A. APPENDICES

A.2 Diafiltration Approximation

Figure A.1: Matlab worksheet for the calculation of membrane processes.

108

A.3. E2N APPENDIX A. APPENDICES

A.3 E2N

E2N is formed by oxidation of fatty acids. Triacylglycerol is the most abundant storagelipid in barley accounting for 60-70% of the overall lipid mass in barley. During maltingand mashing membrane bound Lipases will have optimum activity hereby hydrolysing tria-cylglycerol into glycerol and the corresponding fatty acids, see figure A.2. These fatty acidscan subsequently be oxidized into hydro peroxide fatty acids. An additional pathway is anoxidation of the triacylglycerol resulting in Lipid hydro peroxides which then eventually canbe hydrolysed by lipase into hydro peroxide fatty acids. The hydro peroxide fatty acidscan be transformed into E2N through a pathways of enzymatic and non-enzymatic reaction(Baert et al., 2012)).

Figure A.2: Formation of hydro peroxide fatty acids from triacylglycerol, (Baert et al.,2012).

Different types of oxidation can occur respectively enzymatic oxidation involving lipoxy-genase, autoxidation involving radicals and photo-oxidation involving light irradiation. Thepresents of oxygen is common for all these reactions to be possible (Vesely et al., 2003),(Baert et al., 2012).

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A.4. KETONES APPENDIX A. APPENDICES

A.4 Ketones

The most flavourful ketones present in beer are vicinal diketones (VDK’s). Diacetyl andpentane-2,3-dione are VDK’s found in reasonable concentrations in beer, see table A.1 (EBC,2000).

Pentane-2,3-dione has a very high flavour threshold above normal levels found in beer.On the contrary, the threshold for diacetyl is often detected in beer making this compoundsa greater concern for the brewer with flavour flavours associated with butter, toffee-like orhoney-like flavours (Kunze, 2010), (EBC, 2000).

Table A.1: Ketones in beer, flavour threshold and corresponding concentration in lager beer(EBC, 2000), (Tan and Siebert, 2004).

Compound Compound Flavor Aroma ConcentrationName Structure Threshold or Range

[mgl ] Taste [mgl ]

Diacetyl 0.05-0.15 Buttery 0.008-0.6(Butane-2,3-dione) Butterscotch

Toffee

Pentane-2,3-dione 0.9 Buttery 0.008-0.6Butterscotch

Toffee

VDK’s are by-products of the amino acids biosynthesis of valine and isoleucine, see FigureA.3. Diacetyl is a by-product of valine anabolism, while pentane-2,3-dione is a by-productof the isoleucine anabolism. All amino acids are essential during the vigorous growth atthe beginning of fermentation. Valine and isoleucine are not assimilated by the yeast inthe beginning of fermentation inducing the yeast to synthesise these essential amino acids.These amino acids can be formed from pyruvate and the amino acid threonine, which iseasily assimilated in the beginning of fermentation (Kunze, 2010), (EBC, 2000).

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Figure A.3: Formation of Diacetyl and 2,3-Pentanedione involved in the Valine andIsoleucine anabolism. TPP = Thiamine pyrophosphate cofactor, NADPH = Nicotinamideadenine dinucleotide phosphate (Garcıa et al., 1994), (Haukeli and Lie, 1978)

α-acetolactate (AAL) and α-acetohydroxybutyrate (AAHB) are two intermediate acidsin the pathway of respectively valine and isoleucine anabolism. AAL and AAHB are ex-creted from the yeast cell into the surrounding media. In the beer AAL and AAHB can beoxidatively decarboxylated to diacetyl and pentane-2,3-dione in a non-enzymatic reaction.These reactions are very slow and therefore dictating the levels of diacetyl and pentane-2,3-dione in the beer. During the entire length of fermentation AAL and AAHB will beconverted to diacetyl and pentane-2,3-dione by oxidation. Diacetyl and pentane-2,3-dionewill be taken up by the yeast and converted into acetoin in a faster enzymatic reaction oxi-dizing NADH + H+−→ NAD+ which can be reused for reduction in the Glycolysis (Haukeliand Lie, 1978), (Garcıa et al., 1994).

In fermentation the yeasts ability to remove diacetyl is 10 times greater than the rate

111


of formation. This ability will decrease during secondary fermentation or maturation. Thespeed of these non-enzymatic oxidation reactions to form VDK’s are temperature, substrateconcentration and pH dependent. In addition, the reaction is also dependent on the presentof oxygen and oxidative metal ions (Garcıa et al., 1994). Factors inducing the rate of thenon-enzymatic oxidative decarboxylation to form VDK are considered factors lowering thefinal VDK in beer. The reason for this being the possibility of reacting all present AALand AAHB in the media while the yeast are still fermentation hereby taking up the VDKrapidly and transforming them into the corresponding flavourless diols. Removing the VDKprecursors will hereby ensure not later VDK formation which cannot be removed when theyeast is no longer vital (Garcıa et al., 1994).

Factors influencing VDK concentration in final beer

• Fermentation temperature, (Garcıa et al., 1994)

Higher VDK formation rate during high fermentation temperatures

• Maturation temperature, (Garcıa et al., 1994)

Higher take-up rate during higher maturation temperatures

• pH, (Garcıa et al., 1994)

Higher formation of AAL and AAHB in high pH

Higher conversion rate of AAL and AAHB to VDK in high pH

• Oxygen and metal ions (Cu2+, Al3+, Fe2+), (Haukeli and Lie, 1978)

Lowering final VDK when introduced in the early stage of fermentation

Raising final VDK when introduced after maturation and yeast removal

Introduced during tank change, packaging and/or transportation

• Flocculation, (Kunze, 2010)

Premature flocculation causes VDK take-up to be lowered

• Pitching rate, (Kunze, 2010)

High pitching rate will induce a higher formation of VDK during fermentation

High pitching rate will also induce a higher take-up rate during maturation

• Pasteurization, (Garcıa et al., 1994), (Haukeli and Lie, 1978), (Chuang and Collins,1972), (Godtfredsen and Ottesen, 1982)

Enhance the formation of VDK’s if precursors (AAL or AAHB) are present

• Bacteria (Kunze, 2010)

Pediococcus or wild yeast contamination can cause a increase in VDK

• Adding α-acetolactate decarboxylase (ALDC) (Donalies et al., 2008), (Godtfredsenand Ottesen, 1982)

ALDC catalyses the removal of AAL directly to acetoin

Not detected in brewers yeast

112


• Free Amino Nitrogen (FAN), (Chuang and Collins, 1972)

High FAN equals high Valine and Isoleucine concentration

High FAN lowers VDK formation during fermentation

High FAN however increases final VDK formation during maturation

113

A.5. SULPHUR COMPOUNDS APPENDIX A. APPENDICES

A.5 Sulphur Compounds

Table A.2: Sulphur compounds in beer, flavour threshold and corresponding concentrationin lager beer (EBC, 2000), (Tan and Siebert, 2004), (Landaud et al., 2008), (Briggs et al.,2004).


[µgl ] Taste [µgl ]

Hydrogen sulphide 8 Rotten eggs 1-200Sulphidic

Sulphur dioxide 10000 Pungent 200-20000Burnt matches

Dimethyl sulphide 30 Sweet corn 10-150DMS Cooked vegetable,

Tomato sauce

Mercaptans DrainMethanethiol R−−CH3 2 Rotten vegetable 0.2-15Ethanethiol R−−CH2CH3 1.7 0-20Propanethiol R−−(CH2)2CH3 0.15 0.1-0.2

3-methyl- 0.004 Sun struck 0.001-1.500but-2-ene-1-thiol Skunky

The sources of sulphur in brewers yeast are respectively inorganic sulphate or organic pro-teins, amino acids or vitamins containing sulphur. Sulphur is not only essential in aminoacids and proteins but also in vitamins a coenzymes such as Coenzyme A (CoA) (Walker,1998).

Hydrogen sulphide and sulphur dioxide are by-products of yeast metabolism of cysteineand methionine, see figure A.4. Yeast prefers assimilation of organic sulphur compoundsmainly sulphur containing amino acids. However, if no free sulphur containing amino acidsare available then yeast are able to assimilate inorganic sulphate for the anabolism of theessential sulphur containing amino acids (Walker, 1998), (Landaud et al., 2008).

114


Figure A.4: Biosynthesis of sulphur containing amino acids. Cysteine and inorganic sulphurassimilation for the anabolism of sulphur containing amino acids. NADPH = Nicotinamideadenine dinucleotide phosphate (Walker, 1998), (Landaud et al., 2008)

Factors influencing hydrogen sulphide concentration in final beer, (Briggset al., 2004), (EBC, 2000)

• Induced formation with induced yeast growth

• Induced formation with high cysteine concentration

• Induced formation with high sulphate concentration

115


• Induced formation at higher temperature caused by yeast autolysis

• Induced formation if high FAN/protein concentration

• Reduced formation with high methionine concentration

• Reduced formation with higher Zn+ concentration

• Reduced by oxidation with metals (copper) to form metal sulphides

• Taken up by the yeast during maturation

Factors influencing sulphur dioxide concentration in final beer, (Kunze, 2010),(EBC, 2000)

• Induced when the need for sulphur containing amino acids is reduced

• Induced when low aeration

Reacts with oxygen to form SO2–3

• Induced when low lipid level

A reduction in growth while the sulphur containing metabolism continuous

Dimethyl sulphide (DMS) can be formed by two different routes during malting andbrewing respectively from s-sethyl methionine (SMM) and dimethyl sulphoxide (DMSO),see figure A.5. SMM is formed during germination by methylation of methionine in barley.SMM can hereafter be thermally converted into DMS or DMSO during kilning of the malt.Dependent on the temperature and duration of the kilning SMM is converted into DMS andhereafter evaporated because of high volatility. The DMSO formed are on the contrary lessvolatile hereby mostly retained in the malt. Some yeast strains, wild yeast and bacteria areable to reduce the DMSO in wort to DMS hereby releasing the flavourful DMS into the beer(EBC, 2000), (Briggs et al., 2004), (White and Wainwright, 1977).

Figure A.5: Formation routes of dimethyl sulphide, (Briggs et al., 2004)

Factors influencing DMS concentration in final beer, (Briggs et al., 2004),(White and Wainwright, 1977)

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• SMM content in malts

The lower SMM concentration in malt the lower DMS and DMSO formation

Light malts have a higher SMM concentration than dark malts

• Kilning temperature (> 60oC) and duration

Longer kilning at higher temperature reduced SMM, DMS and DMSO in malt

• Boiling duration

Long boiling duration lowers the amount of SMM, DMS and DMSO in wort

• Whirlpool stand time before cooling

Holding time for conversion of SMM without any evaporation

• Yeast metabolism of SMM and DMSO

SMM assimilated by some yeast and converted to methionine

DMSO assimilation by reduction to from DMS

Linear mercaptans are formed as a reaction between hydrogen sulphide in the solutionand yeast metabolites such as methanol, ethanol, higher alcohols and acetaldehyde. In figureA.6 the general formation of different mercaptans can be viewed. (Vermeulen et al., 2006),(Swiegers and Pretorius, 2007).

Figure A.6: Formation of Mercaptans in beer, (Vermeulen et al., 2006), (Swiegers andPretorius, 2007)

Factors influencing linear mercaptans concentration in final beer, (Vermeulenet al., 2006)

• Factors inducing the hydrogen sulphide formation and alcohol will induce mercaptanformation

• Nutrient deficiency may lead to higher formation especially nitrogen deficiency

• Copper reduces the formation of hydrogen sulphide

The sun struck flavour, mostly associated with a skunky aroma and taste, originates fromhops. Hops are added to the wort boil mainly to enhance the aroma profile. β- and α-acidsare hops components adding bitterness to the beer after isomerations into the correspondingiso-β- and iso-α-acids. For a more detailed description of additional hops components seesection A.7 (Burns et al., 2001).

Mainly iso-α-acid is isomerized hereby being the main source of 3-methyl-3-but-1-ene-thiol formation, see figure A.7. Sun exposure or photolysis of iso-α-acid in beer may resultin a Norrish cleavage reaction in one of the carbon-carbon bonds situated next to carbonylin the 4-methylpent-3-enoyl group. In both cleavage reaction the formation of radicals stabi-lized by π-electron de-location form a neighbouring double bond will occur, see respectively

117


compound A and D in figure A.7. Moreover, the formation of a corresponding acyl radicalswill occur, see respectively compounds B and C in figure A.7 (Burns et al., 2001).

Figure A.7: Formation of 3-methylbut-2-ene-1-thiol, the sunstruck flavour, (Burns et al.,2001)

3-methyl-2-butenyl allyl radical A are formed directly from the cleavage of iso-α-acid orsecondary from decarbonylation of the formed 4-methyl pent-3-enal acyl radical. RadicalA can react directly with hydrogen sulphide radicals which originate from free hydrogensulphide or scavenged from a thiol group from sulphur amino acids or proteins. Finally, thisresult in the formation of 3-methylbut-2-ene-1-thiol (Burns et al., 2001), (Briggs et al., 2004).

Factor influencing 3-methylbut-2-ene-1-thiol formation in beer, (Burns et al.,2001), (Briggs et al., 2004))

• Sun exposure of the beer enhances formation

Colour of the bottle glass is important

• Higher IBU enhances formation

• Higher sulphur compounds concentration enhances formation

• Reduction of the carbonyl group in iso-α-acids 3-methylpent-3-enoyl group

Donated ρ-iso-α-acid

The secondary alcohol formed in the reduction causes cleavage not to occur

Sodium borohydride (NABH4) can be used for the reduction

118

A.6. ACIDS APPENDIX A. APPENDICES

A.6 Acids

Acids in beer may origin from different raw materials in the wort or bacterial contamina-tion. However, the majority is produced during fermentation inducing a decrease in pH asobserved in figure 2.11. Many organic acids are secondary metabolites excreted during rapidyeast growth and some re-assimilated later in the fermentation. In table A.3 some of themost flavourful acids can be observed (EBC, 2000).

Table A.3: Acids in beer, flavour threshold and corresponding concentration in lager beer(EBC, 2000), (Tan and Siebert, 2004), (Klopper et al., 1986), (Siebert, 1999), (Kunze, 2010).


[mgl ] Taste [mgl ]

Acetic acid 130 Vinegar 30-200Acidic

Lactic acid 400 Milky 10-1362Sour flavour

Butyric acid 2-3 Rancid 0.5-1.5Baby sickSour milk

Citric acid 170 Lemon 90-300AcidicSour

Fatty acid SoapyFatty

Octanoic acid R = (CH2)6CH3 13 3-10Decanoic acid R = (CH2)8CH3 10 0.8

Dodecanoic acid R = (CH2)10CH3 6 0.1-0.5

Most acids observed in table A.3 are formed as a result of an incomplete TCA cycleduring anaerobic growth of brewing yeast, see figure A.8. Moreover, organic acids can beformed from amino acids where the yeast have removed the amino group to form α-ketoacids for later amino acid synthesis within the cell, as observed in figure 2.12, (EBC, 2000).

119


Figure A.8: TCA cycle and corresponding acids excreted by brewing yeast (Briggs et al.,2004).

Acetic acid can be formed in beer by acetic acid bacteria contamination or by the reactionof Acetyl-CoA with water releasing CoASH as observed in figure A.8 (Klopper et al., 1986),(Briggs et al., 2004).

Lactic acid may be formed during germination of the barley and fermentation due tomicrobial growth. Furthermore, lactic acid can be formed directly by reduction of Pyruvateyielding NAD+ (Klopper et al., 1986), (Briggs et al., 2004).

Butyric acid is mainly caused by bacterial contamination (Klopper et al., 1986), (Briggset al., 2004)).

Citric acid can be formed directly by protonation of citrate an intermediate in the TCAcycle (Klopper et al., 1986), (Briggs et al., 2004).

Fatty acids in beer are either liberated from triglyceride in the wort by lipolytic enzymes

120


or synthesized by the yeast from from Acetyl-CoA by β-oxidation in the peroxisomes (Klop-per et al., 1986), (Briggs et al., 2004).

Factors influencing organic acid formation in beer (Klopper et al., 1986),(Briggs et al., 2004).

• Fermentation rate

Sluggish fermentation result in low levels of organic acids excreted

Secondary metabolites in excess during rapid growth

• Wort composition

Buffering ability of the wort

• Yeast strain

Absorption abilities

Excretion rate of protons

Factors influencing fatty acid formation in beer (Klopper et al., 1986), (Briggset al., 2004)).

• Beer conditioning temperature

High temperatures can cause excretion of fatty acids

Could be caused by loss of cell wall integrity

121

A.7. HOPS ACIDS APPENDIX A. APPENDICES

A.7 Hops Acids

Table A.4: Hops constituents in beer, flavour threshold and corresponding concentration inlager beer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989).

Compound Compound Flavor Aroma Conc.Name Structure Threshold or Range

[mgl ] Taste [mgl ]

Iso-α-acid 5 Bitter 10-100Humulone R−−CH2CH(CH3)2

Cohumulone R−−CH(CH3)2Adhumulone R−−CH(CH3)CH2CH3

Iso-β-acid - Bitter -Lupulone R−−CH2CH(CH3)2

Colupulone R−−CH(CH3)2Adlupulone R−−CH(CH3)CH2CH3

Soft resins namely α and β-acids are responsible for the bittering effect of hops. α andβ-acids are nearly insoluble in beer leaving only traces of α-acids within the beer after hopsaddition. Therefore, hops boiling is commenced for the purpose of isomerizing the differentα and β-acids into iso-acids as observed in table A.4. The isomerization reaction observed infigure A.9 is induced by heat, low pH, high gravity and a high Mg2+ concentration resultingin two diastereoisomers differing from each other as cis/trans isomers. This isomerisationreaction is a acyloin-type ring contraction. Generally, a 68

32 ratio between cistrans isomers are

observed cis-isomers generally being more bitter and more stable than the trans-isomers.The α-acids fraction is nine times more bittering compared to the β-acids fraction, whichis therefore not considered in table A.4 in relation to flavour threshold and concentrationrange (Briggs et al., 2004), (Praet et al., 2012).

122

A.7. HOPS ACIDS APPENDIX A. APPENDICES

Figure A.9: Isomerization reaction of α-acid, (Briggs et al., 2004).

123

A.8. HS-GC-MS FEED BEER APPENDIX A. APPENDICES

A.8 HS-GC-MS Feed Beer

Figure A.10: Higher alcohol aroma compounds area percentages compared to the meanoriginal beer measured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG = Orignal ,24 = 24th of February and 11 = 11th of April. X-aksis ledgend: Compound name (Retensiontime) - Associated flavours.

Figure A.11: Ester aroma compounds area percentages compared to the mean original beermeasured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG = Orignal , 24 = 24thof February and 11 = 11th of April. X-aksis ledgend: Compound name (Retension time) -Associated flavours.

124

A.8. HS-GC-MS FEED BEER APPENDIX A. APPENDICES

Figure A.12: Aldehyde aroma compounds area percentages compared to the mean originalbeer measured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG = Orignal , 24= 24th of February and 11 = 11th of April. X-aksis ledgend: Compound name (Retensiontime) - Associated flavours.

Figure A.13: Hop aroma compounds area percentages compared to the mean original beermeasured by HS-GC-MS. Column ledgend: HF = Humlefryd , ORG = Orignal , 24 = 24thof February and 11 = 11th of April. X-aksis ledgend: Compound name (Retension time) -Associated flavours.

125

A.9. HPLC CALIBRATION CURVE APPENDIX A. APPENDICES

A.9 HPLC Calibration Curve

Figure A.14: Calibration curve for alcohol measurement on HPLC.

126

A.10. HPLC REPORT APPENDIX A. APPENDICES

A.10 HPLC Report

Figure A.15: HPLC report of the beer in the feed tank before NFHF membrane filtrationwas initiated. Retention time in minutes, at the top of the peak, and integral area situatedat the ethanol peak.

127

A.11. HS SAMPLING SET-UP APPENDIX A. APPENDICES

A.11 HS Sampling Set-up

Figure A.16: Head space sampling set-up. Trapping the aroma in on Tenax-TA traps at37oC.

128

A.12. HS-GC-MS SPECTRA APPENDIX A. APPENDICES

A.12 HS-GC-MS Spectra

(a) Retention time (tR) 0-13 minutes.

(b) Retention time (tR) 13-23 minutes.

(c) Retention time (tR) 23-29 minutes.

Figure A.17: Aroma compounds measured on the GC-MS for original Humlefyrd bottledthe 24th of April.

129

A.13. ALFA LAVAL MEMBRANE CLASSIFICATION APPENDIX A. APPENDICES

A.13 Alfa Laval Membrane Classification

Figure A.18: Dealcoholisation membrane data analysis by Alfa Laval using an HPLC-MSwhere the ethanol peak was used to dictate the ethanol reduction. The additional HPLC-MS peaks were viewed upon as aroma peaks resulting in a possible percentage reductionof ”aroma” peaks. Analyse = analysis, bestemmelse = determination, af = of, alkohol =alcohol, aromastoffer = aroma compounds, i = in, øl = beer, prøve=sample, tryk = pressure,konc. = conc., areal = area and PM = permeability [%].

130

A.14. STANDARD DEVIATION OF HS-GC-MS SAMPLESAPPENDIX A. APPENDICES

A.14 Standard Deviation of HS-GC-MS Samples

Figure A.19: Standard deviation percentage [%] between duplicates samples on HS-GC-MS.

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