Removal of Alcohol from Beer Using Membrane Processes

Removal of Alcohol From Beer Using Membrane Processes

Master’s Thesis

Supervisors: Author: Henrik Siegumfeldt Andreas Jakob Wedel Falkenberg 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 months 4/2 - 4/8-2014 30 ECTS

Supervisors: Henrik Siegumfeldt Jens Christian Sørensen

Copies: Printed in 3 copies, as well as being digitally available

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

Written at: Department of Food Science University 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 ﬂavour quality was the original idea of this thesis. An investigation was initiated revealing possible new methods of AFB production. This focusing not only on the process technologies related to alcohol removal from normal alcoholic beer, but in addition looking beyond at the general beer production processes to indicate possible changes resulting in a higher quality AFB with regards to aroma and ﬂavour preservation.

I would like to thank all parties involved from the University of Copenhagen Faculty of 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 Henrik Siegumfeldt and Associate Professor Jens Christian Sørensen for knowledgeable guidance, participation and support. Furthermore, thanks to my fellow student Tobias Emil Jensen and his supervisor Mikael Agerlin Petersen for guidance and permission to run head space gas chromatographic mass spectrometry samples. From Brewhouse Skands A/S a special thanks to Birthe and Morten Skands for guidance, participation and beer donations. From Alfa Laval a special thanks to Anders Bisgaard for guidance, participation, membrane donations and introduction to the newest trends in membrane processing.

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

2 Abstract

The main object of this study is the production of alcohol free beer (AFB) using membrane processes. 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 and rectification or modified brewing and fermentation. These approaches induce pronounced and unwanted changes to the overall flavour profile of beer. Membrane processes for the production of AFB are poorly investigated, however the potential is large because of possible non-thermal selective ethanol removal. A major drawback in membrane processes is the 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%ABV to 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 from nano filtration (NF) to reverse osmosis (RO). The membranes were tested on a Labstak M20-0.72 membrane unit provided by Alfa Laval A/S using Humlefryd 5.5%ABV lager provided by Brewhouse Skands A/S. The M20-0.72 unit was modified to maintain a closed environment with a CO2 pressure of 1-2bar. The alcohol concentration during filtration was determined using high performance liquid chromatography (HPLC), where the flavour profiles before and after filtration were compared using head space gas chromatography mass spectrometry (HS-GC-MS). Additionally, a trained taste panel was used to describe the differences in the membrane filtrated products compared to the original beer.

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

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

Future consideration involving alteration of the brewing and fermentation processes were considered hereby compensating for the aroma losses over RO and NF membranes making this process more proﬁtable.

3 Abbreviations

σ∗ Polar Taft number MCFA Medium chain fatty acid δ Membrane thickness MF Micro filtration AA Amino acids MS Mass spectrometry ABV Alcohol by volume MWCO Molecular weight cut-off ADP Adenosine diphosphate NAD+ Nicotinamide adenine AFB Alcohol free beer dinucleotide ATP Adenosine triphosphate NF Nano filtration Bethanol Solute transport coefficient NFHF Nano filtration membrane type Bwater Solvent transport coefficient NF99HF C∗ Membrane constant OPEX Operational expenditure CA Cellulose acetate ORG Original CAPEX Capital expenditure P Permeate and permeability CCV Cylindroconical vessels PA Polyamide CI Chemical ionization PC Principal component CIP Cleaning in place PCA Principal component analysis CoA-SH Coenzyme A PE Polyester Conc. Concentration PES Polyethersulfone CTA Cellulose triacetate PG Present gravity DAB Danish Brewers’ Association PM Permeability DAM Solutes diffusion coefficient PP Polypropylene DC Direct current PS Polysulfone DF Diafiltration PT Permeate tank E2N (E)-2-Nonenal PVDF Polyvinyllidene fluoride EI Electron ionization Re Retention Es∗ Steric Taft number RF Radio frequency F Flow RI Refractive index FAD+ Flavin adenine dinucleotide RID Refractive index detector FAN Free amino nitrogen RO Reverse osmosis FT Feed tank s∗ Small’s number FTE Feed tank end SD Standard deviation GC Gas chromatography SDME Single drop micro extraction GTP Guanosine triphosphate SPME Solid phase micro extraction HA Higher alcohols TA Trapping agent HF Humlefryd and high flux TCA Tricarboxylic acid cycle HGB High gravity brewing TFC Thin film composition HP High performance/pressure TMC Trimethyl chloride HS Head space TMP Trans membrane pressure J Flux tR Retention time K Distribution ratio between UF Ultra filtration membrane and solution VCF Volume concentration factor KU University of Copenhagen VDK Total vicinal diketones LAB Low alcoholic beer VOC Volatile organic compounds LC Liquid chromatography WCOT Wall coated open tubes

4 Contents

1 Introduction 7

2 Theory 11 2.1 Sedimentation and Filtration ...... 11 2.2 Membrane Processes ...... 13 2.3 Experimental Considerations ...... 23 2.4 Aroma Formation ...... 28 2.5 Methods of Analysis ...... 47

3 Materials and Methods 53 3.1 Equipment ...... 53 3.2 Membranes ...... 55 3.3 Feed Beer ...... 56 3.4 Dia-water ...... 57 3.5 Procedures ...... 57 3.6 Analytical Tools ...... 59

4 Results and Discussion 62 4.1 Preliminary Investigation ...... 62 4.2 Constant Parameters ...... 64 4.3 Membrane Flux ...... 68 4.4 Ethanol Permeability ...... 71 4.5 Aroma Retention ...... 74 4.6 Tasting Results ...... 87 4.7 Overall Results ...... 88 4.8 Aroma Formation ...... 89

5 Future Perspectives 92

6 Conclusion 94

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

5 CONTENTS CONTENTS

A.7 Hops Acids ...... 122 A.8 HS-GC-MS Feed Beer ...... 124 A.9 HPLC Calibration Curve ...... 126 A.10 HPLC Report ...... 127 A.11 HS Sampling Set-up ...... 128 A.12 HS-GC-MS Spectra ...... 129 A.13 Alfa Laval Membrane Classiﬁcation ...... 130 A.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 scientific breakthroughs to prohibition and legislations. With a higher scientific enlightenment alcoholic beverages have been deemed unhealthy by some, while healthy by others. Fur- thermore, the intoxicating effect of alcoholic beverages has caused the need for legislations concerning the intake of alcohol. Alcoholic beverages never seem to go out of fashion, however the public view, legislations and assortments seem to change drastically over the course of time (Gretton, 1929). Beer has gone from being a home made product, enabling personal preferences, to becoming an industrialized production, where supply and demand are in focus. An expand in assortment of beers is caused by a higher competition and a globalization resulting in many different beer styles. In addition, technological development have caused a higher differentiation in beer enabling alcohol removal or reduction. Low alcohol beer (LAB) and alcohol free beer (AFB) are present on the market for the purpose of satisfying customer demands. This demand could be caused by legislation, health issues, religion, prohibition or as an alternative to soft drinks (Ambrosi et al., 2014). For many years health have been the main concern or argument when legislating and banning beer consumption. A high consumption of beer can lead to alcoholism, accidents, brain degeneration, liver failure, cancer, strokes and arteriosclerosis. Many of these illnesses are associated to the alcohol intake when drinking beer. Nevertheless, some positive affects of 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 attributes described by the so-called J-curve as shown in figure 1.1. The figure illustrates a reduction in mortality for people drinking moderately compared to people not drinking. The reduction in mortality associated with moderate alcohol intake is mainly caused by alcohol lowering the risk of coronary diseases. For many years physicians have recommended wine for patients in danger of coronary diseases, when beer is equally sufficient. In fact, beer contributes with constituents with additional positive health effect. This could be an additional reasoning for choosing an AFB or LAB instead of soft drinks where the nutrients and vitamins concentration are low to non-excising (Furbo, 2013), (Groenbaek et al., 1994). Legislation in most European countries concerning alcohol concentration in beer states that 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, especially in the Muslim world, the allowed concentration is most often below 0.05%ABV, only allowing traces of alcohol in beverages (Brányik 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 lines indicate the 95% conﬁdence interval (Groenbaek et al., 1994).

0.5%ABV instead of the previously allowed 0.1%ABV. This change was induced by the Danish Brewers’ Association (DBA) highlighting concerns about the low quality of Danish AFB. According to DBA the low quality and low sales of AFB in Denmark could be altered to the better by allowing a higher concentration of AFB at 0.5%ABV, as observed in other countries such as Germany, Sweden and Spain. In these countries AFB accounts for a higher percentage 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 the demand for LAB and AFB. The current technology of reducing alcohol can be divided into two subsequent methods of respectively physical and biological methods. In figure 1.2 an overview 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 separation using heat and pressure alteration or mechanical separation using membranes. On the other hand, the biological methods entail the use of special yeast, mashing or fermentation methods in the traditional brewing equipment or in new equipment, enabling a short contact time with the wort and yeast. The physical methods often result in a great loss of volatiles which leaves the beer flavourless and watery. On the contrary, the biological methods often leave the beer, worthy and unbalanced. In a review by Brányik et al. (2012) a comparison of physical processes reveal a lower loss of volatiles using membrane processes compared to thermal processes. Furthermore, thermal processes cause irreversible heat damages to the beer resulting in a higher rate of deterioration and unpleasant bitterness formation (Brányik et al., 2012). Membrane processes might cause a high reduction in volatiles because some of the taste and aroma substances are able to pass through the membrane along with alcohol. This could 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 considerably reduced, because this process is performed cold.

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

8 CHAPTER 1. INTRODUCTION sible losses of volatiles during the process. The differences in ethanol and aroma permeation through various membranes ranging from nano filtration (NF) to reverse osmosis (RO) are classified using HPLC and HS-GC-MS. Finally, possible ways of altering the brewing process to compensate for the losses of flavour and aroma compounds during the membrane filtration will be discussed. Hopefully, membrane processes will enable a more flavourful AFB and LAB if the losses over the membrane are standardised and hereby correctly compensated for during the many steps of brewing and fermentation (Brányik 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 readability possible term or word explanations are assembled in appendix A.1.

9 CHAPTER 1. INTRODUCTION Figure 1.2: Diﬀerentexpenditure et (Br´anyik methods al., 2012). of reducing the alcohol concentration in beer. OPEX = operational expenditure, CAPEX = capital

10 Chapter 2

Theory

2.1 Sedimentation and Filtration

Separation of solids and yeast from beer, after fermentation, is normally done using subsequent 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, taste stability, foam stability and uniformity of product based on the customer expectations and equipment availability. The theory of sedimentation and filtration will briefly be mentioned in this thesis as a pretreatment of the beer consequently enabling later membrane filtration without clogging the membranes (Briggs et al., 2004). In the following section the clarification 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 Law given as

(ρ − ρ ) ∗ d2 v = g ∗ P L , (2.1) g 18 ∗ µ

where vg is the velocity of sedimentation, g the gravitational acceleration, ρP the particle density, ρL the liquid density, d the particle diameter and µ is the liquid viscosity. From equation 2.1 it can be deduced that larger particles with high density will sediment fast in liquids with low density and low viscosity. During fermentation the liquid density and viscosity will be lowered as a consequence of the fermentation of sugars to alcohol. Cooling of the beer below temperature of maximum density (2−3oC) will further reduce the density of the beer (Kunze, 2010). Additionally, cooling of the fermenter will promote the formation of 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 size of 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 ”flock particle” enabling sedimentation. Yeast flocculation is highly strain dependent relying on the 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 difference between the strains of lager yeast Saccharomyces pastorianus and ale yeast Saccharomyces cerevisiae 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 a hydrophobic surface. As a result, the hydrophobic surface of ale yeast will interact with hydrophobic CO2 carrying the ale yeast to the top of the liquid, while lager yeast will sediment in the bottom following Stokes law (Walker, 1998). Humlefryd is fermented with lager yeast and will flocculate in the bottom of the fermenter. Nowadays, the design of the cylindroconical vessels (CCV) enables a thermal convection flow, which will force the yeast to 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 after maturation at 7 − 7.5oC. After cooling to 2.5oC secondary cropping is performed. A final cropping is carried out after cooling to filtration temperature at −1.5oC. Cropping in three segments enables sedimentation at three different physical conditions, where different thermal 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 brewhouse 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 three principles; direct interception, inertial interception and electrostatic interactions. Direct interception occurs when the particles are retained because they are larger than the pore size of the filtration medium. Inertial interception is retention of particles smaller than the pore sizes of the filtration medium caused by the momentum of the particle and the fluid flow surrounding the particle and the filtration medium. Particles can also be retained based on the surface charge of the particle and the filter medium hereby creating electrostatic interactions (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 at brewhouse Skands is BECO depth filter sheets. BECO depth filter sheets contains all natural materials 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 Permeability Rate Area Dry Wet ∆p = 1bar g l [µm][µm] [%] [ m2 ][kP a][kP a][ 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 be removed, this indicated with a vertical line in ﬁgure 2.1. Size of brewers yeast is very strain dependent ranging from 2.5-4.5µm in the shortest circumference and up to 10.5-20µm in the largest. All yeast cells are consequently removed by the ﬁltration along with particles with 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 further pre-processing of the beer for the removal of alcohol. In the following section membrane processes 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 0.1µm through dead-end filtration techniques as illustrated in figure 2.1. For the removal of particles and molecules below 0.1µm molecules membrane processes can be applied (Clement et al., 2004). Membrane processes are based on semi-permeable pressure driven membrane filtrations. Micro filtration (MF), ultra filtration (UF), nano filtration (NF) and reverse osmosis (RO) are different membrane processes. The differences are based on pore size and 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 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

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 or cross-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 differentiation over the filter inducing a rapid reduction in the permeate flux. Therefore, Dead-end membrane filtration is mainly used for MF applications, where the retention amount is minimal e.g. removal of yeast and bacteria in membrane sterilisation. Filter cake build-up causing a continuous reduction in permeate flux can be reduced by changing the feed flow from perpendicular to tangential as illustrated in figure 2.3. Cross-flow filtration involves a tangential feed flow resulting in a filter cake washing-off or disruption. Consequently,

14 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY cross-flow filtration will maintain a higher permeate flux throughout the filtration, hereby prolonging the time of filtration. Nevertheless, forcing the entire liquid through the membrane 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 further flow of the retentate, membrane processes can either be continuous or batch. Continuous processes 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-stage processes. Batch processes involve a circulation of the retentate until the desired removal or concentration of compounds have been reached (Smith, 2013b). Considerations concerning batch processes will only be evaluated.

Reverse Osmosis The semi-permeable nature of membranes will involve passing of permeable compounds over the membrane to reach a concentration equilibrium of non-permeable dissolved compounds on both side of the membrane. In the case of membranes, which are only permeable with respect to water, are the movement of water to reach equal chemical potential called osmosis. 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 only concentration dependent, but also dependent on the static pressure on both sides of the membrane. Reversing the flow of permeable compounds from the membrane side with the high concentration of dissolved non-permeable compounds to the side with the low concentration is possible when pressure is applied. The pressure applied on the non-permeable dissolved compounds side of the membrane, to equal out the osmotic flow of water, is called the osmotic pressure (π). Exceeding the osmotic pressure will involve a flow of permeable compounds against the concentration gradient, hereby obtaining reverse osmosis. A simple calculation 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 concentration, R the gas constant and T the absolute temperature (Smith, 2013a), (Clement et 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 permeate pressure (Pp). On the contrary, cross-flow membrane processes are influenced by a pressure loss over the course of the membrane area as illustrated in figure 2.4.

15 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

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 that the retentate pressure (Pr) never falls below the permeate pressure (Pp). As a result of the pressure loss over the membrane, the TMP of cross-flow filtration is calculated as follows P + P TMP = ( f r ) − P (2.3) 2 p (Hausmann et al., 2013).

Membrane Transport Models Movement of water and other solutes through different membranes can be described by two different models dependent on the pore size of the membrane. MF and UF are believed to follow the pore flow model, where the solutes permeates pores in the membrane consequently separated by size. On the other hand, NF and RO follow the solution diffusion model, where separations is based on other factors in addition to size. Factors such as polarity, dipole moment, ionic charge and pH have been proven to affect the rejection of organic molecules in NF (Smith, 2013a). Furthermore, in RO the rejections of organic molecules have been proved 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 on diffusion of other water molecules in a tetrahedral structure within the membrane formed by hydrogen bonding (Smith, 2013a). Additional molecules with a similar structure as water might therefore be able to enter and permeate NF or RO membranes. Furthermore, molecules with a similar molecular size, configuration, polarity and hydrogen bonding abilities as the permeable solutes are able to permeate such membranes. In particular small molecules such as methanol, ethanol, urea and 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 the concentration 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 can be calculated on the basis of the concentrations in the feed stream (cf ) and in the permeate stream (cp), where c − c c Re = f p = 1 − p . (2.4) cf cf

16 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

From equation 2.4 it can be deduced that Re = 1 involves a total rejection (100%) of a speciﬁc 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. Deﬁnitions of retention, rejection and permeability can be found in appendix A.1. (Smith, 2013a), (Cui et al., 2010), (Clement et al., 2004). Flux (J) or permeation rate is the permeation amount (kg) in a given time (min) for a membrane with a given area (m2),

Mass of given compound in permeate kg J = = (2.6) Membrane area ∗ T ime m2 ∗ min (Smith, 2013a), (Cui et al., 2010).

Membrane Selectivity Different polarity between the feed stream and the membrane can influence membrane permeability caused by partitioning. For example a hydrophilic feed stream (beer) filtrated with hydrophobic membranes could result in attraction of the hydrophobic solutes towards the membrane in the feed. Higher alcohols have shown a reduction in rejection possibly caused 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 potential to 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 the polarity effect when considering the solution diffusion model. In addition, the method of MWCO determination is differing greatly, (Cui et al., 2010).

Diafiltration Diafiltration (DF) involves addition of a solvent, usually water, to the feed stream, hereby improving separation of compounds with different rejections in batch systems. The concentration of freely permeating substances in the feed stream, e.g. ethanol, can be reduced by adding a carrier solvent. Regular membrane filtration techniques involves a concentration of the compounds with high rejection factors enhancing possible filter cake build-up. This could result in a higher permeability of unwanted compound into the permeate. Dilution of the feed stream ensures a constant cross-flow filtration disruption, most often resulting in a reduction of possible filter cake build-up. Furthermore, the osmotic pressure will be approximately constant when DF is applied. Maintaining a high permeate flux will enhance washing out of the permeable compounds resulting in a lower final concentration in the retentate stream compared to the concentration involved in regular filtration processes, as illustrated in figure 2.5 (Ferreira et al., 2007) (Hausmann et al., 2013).

17 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

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

The diafiltration factor is the relation between the diafiltration liquid volume (Vd) added to the system and the retentate volume produced after filtration (Vr), where V DF = d . (2.7) Vr DF can be applied continuous or discontinuous respectively involving a constant or a changing volume of retentate. Discontinuous DF often involves a pre-concentration resulting in a desired volume reduction. Continuous DF involves a higher addition of solvent, longer filtration time and a higher flux of permeate, while the same concentration can be reached compared with discontinuous DF. On the contrary, discontinuous DF involves an increase in concentration which can result in a reduction in flux caused by increased viscosity and osmotic pressure. Nevertheless, discontinuous DF will reduce the filtration time and the solvent addition for the removal to identical concentration (Ferreira et al., 2007), (Hausmann et al., 2013), (López et al., 2002).

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

18 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

Membrane Materials The perfect membrane material should have the following physiochemical properties (Pilipovik and 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) membranes with 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 bottom layers serving as support for the upper layer actually separating the molecules. An additional intermediate layer might be introduced, hereby ensuring no penetration of the top layer into the 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 general strong compaction ability plus high resistance to extreme temperatures and pH. PP and PE membranes can be used for MF and UF applications normally in a isotropic membrane structure (Smith, 2013b).

19 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

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) or Cellulose triacetate (CTA)

Polyamide (PA)

Polysulfone (PS) and polyethersulfone (PES) are very resistant membrane polymer materials with a general high strength. In addition, a wide pH range plus high chlorine and temperature resistance gives PS and PES membranes an advantage when it comes to lifetime and cleanability. However, production of PS or PES membranes for RO application is not possible (Smith, 2013b). Polyvinylidene ﬂuoride (PVDF) has a good resistance towards hydrocarbons and oxidising agents with a relatively wide pH range. Nevertheless, PVDF membranes do only exist in the UF and MF range (Smith, 2013b). Cellulose acetate (CA) can be used to produce anisotropic membranes for RO applications. CA membranes are polar. The polarity can be altered by acetylations hereby yielding cellulose triacetate (CTA). As a result of polarity the membrane is hydrophilic enabling a good water permeability, high salt rejection and relatively high strength. The disadvantages of CA membranes include sensibility towards high temperatures (> 40oC) and high chlorine mg concentrations (> 1 l ) plus a narrow pH range (4-7), hereby impeding proper cleaning in place (CIP) coupled with a long lifetime. In addition, drying of CA membranes causes

20 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY 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 Laval A/S. PA is synthesised by mixing trimesoyl chloride (TMC) with diﬀerent amides to form polymers connected by amide bonds, see ﬁgure 2.7 (Tang et al., 2009).

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

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

Ferreira et al. (2007) found CA membranes more efficient for alcohol removal of beer compared to PA membranes during a dialysis membrane process. Comparing different PA and 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). López et al. (2002) also found a higher flux for CA membranes compared to PA membranes, when applying RO for alcohol removal of apple cider. A linear trend was observed between a rise in pressure causing an increase in flux for the PA membranes. However, a loss of linearity was observed for CA membranes at pressures above 35bar, which was associated to a possible membrane compaction. Furthermore, López et al. (2002) did a comparison between aroma retention for CA and PA membranes, with similar NaCl retention, showing a higher ethanol retention for PA membranes as well as aroma retention. Thus, ensuring a higher aroma quality of the end product when applying PA membranes. This phenomenon was explained by the difference in polarity of the membranes, with the CA membranes being more polar than the PA membranes. Therefore, a higher concentration of the polar organic compounds, such as water and ethanol, could be attracted to the more polar CA membrane layer causing higher permeation or lower rejection. PA membranes are more hydrophobic or non-polar than CA membranes and would therefore repel the polar organic compounds while attracting non-polar organic compounds López et al. (2002). In addition, PA membranes do have a higher mechanical strength than CA membranes with a tendency of compaction and collapsing if drying occurs. Finally, PA membranes show a higher durability concerning both pH and temperature resistance enabling a proper CIP and a longer life time (Smith, 2013b).

21 2.2. MEMBRANE PROCESSES CHAPTER 2. THEORY

Membrane Calculations The solution diffusion theory can be used to describe RO and NF membranes, because they can be viewed as a non-porous homogeneous wall in which diffusion of certain compounds are 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, kg the flux of water through the membrane (Jwater,[ m2∗min ]) is dependent on the TMP ([bar]) exceeding the difference in the osmotic pressure over the membrane (∆π,[bar]) and the char- kg acteristics of the membrane itself along with the permeation liquid (Bwater,[ m2∗min∗bar ]), given as Jwater = Bwater ∗ (TMP − ∆π) (2.8) (Hausmann et al., 2013). When operating a membrane filtration with high selectivity for water, in diafiltration mode, the flux of water (Jwater) is approximately equal to the flux of permeate (Jpermeate), which can be found experimentally using the mass of the permeate 2 (mpermeate,[kg]), the area of the effective membranes (Amembrane,[m ]) and the given time (t,[min]), given as mpermeate Jwater ≈ Jpermeate = (2.9) amembrane ∗ t (Ferreira et al., 2007). Solutes, which are partially retained and partially able to permeate the 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)

kg m where Jsolute [ m2∗min ] is the flux of solute through the membrane, Bsolute [ min ] is the solute transport coefficient, cf is the concentration in the feed stream and cp is the concen- kg tration in the permeate [ m3 ]. Jsolute is not influenced by the TMP, but only dependent on the concentration difference in the permeate and the feed stream, as observed from equation 2.10. 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 of m2 the membrane (DAM ,[ min ]), the membrane thickness (δ,[m]) and the distribution ratio between the membrane and the solution (K), resulting in

D B = AM (2.11) solute K ∗ m (López et al., 2002), (Alvarez et al., 1998). According to López et al. (2002) and Alvarez et al. (1998) the solute transport coefficient is dependent on the chemical nature of respectively the 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 and s∗ the Small’s number concerning hydrophobicity. ρ∗, δ∗ and ω∗ are coefficients associated with the importance of the multiplied numbers (López et al., 2002), (Alvarez et al., 1998), (Taft, 1952). The membrane composition constant (C∗) is important when comparing various membranes. This value can only be found experimentally (López 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 general negative or positive polarisation resulting in a difference in acidity. A negative Taft number indicates 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 steric Taft number assumes only negative values. A decrease in value is equivalent to an increase in steric hindrance (López et al., 2002) The polar and steric Taft effects are calculated on 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 only positive values. The higher the Small’s number the more hydrophobic the compound is (López et al., 2002). However, the Small’s number is only considered when the solutes contains 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 effects while ketones, aldehydes and alcohols were separated based on both the polar and steric effects during RO filtration using polyamide (PA) membranes.

A calculation of Bsolute is only possible when the concentration on each side of the membrane, 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 calculation of Bethanol. On the contrary, the aroma compounds were measured in comparison with each other enabling an insight in possible diﬀerence in permeation. In the discussion, the diﬀerence in permeation of aroma compounds will be compared to values of the polar Taft number (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 very limited as a result of this being a fairly new approach. In the light of this, the experimental approaches were often based on intuitive thoughts and on own experience together with the skilled and experienced engineers from Alfa Laval A/S. In figure 2.8 the optimal discontinuous diafiltration process found, in cooperation with these engineers and as a result of the limitations of the experimental set-up, can be observed. The first stages of discontinuous diafiltration is pre-concentration of the beer, where the beer is recirculated within the batch system until the wanted reduction in volume has occurred, see the green line figure 2.8. The volume reduction can be described by the volume concentration factor [VCF] for the pre-concentration

Vstart VCFP re−conc = , (2.13) VDia where Vstart is the initial volume and VDia is the volume maintained during the diafiltration stage. The ethanol concentration will rise during the reduction of the retentate volume, see the blue line in figure 2.8. The rise in ethanol concentration is a result of a higher membrane permeability for water compared to ethanol. The degree of pre-concentration is dependent on the wanted viscosity of the liquid along with the layout of the set-up. In figure 2.8 a pre-concentration degree of 2 is observed resulting in reduction in volume from 5 to 2.5 litres. The second stage is called the diafiltration stage, where the retentate volume is kept constant by addition of dia-water (red line) in the same rate as permeate (purple line) is

23 2.3. EXPERIMENTAL CONSIDERATIONS CHAPTER 2. THEORY

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, initial l alcohol concentration = 5.5%ABV, measured flux (J) through the membrane = 0.519 m2∗min , l 2 flow retentate = 12.8 min , membrane permeability = 85% and membrane area = 0.072m . removed from the system. During the diafiltration stage the concentration of alcohol will steadily decrease in an exponential trend, where the rate of alcohol removal will be reduced as the concentration becomes lower. This phase illustrate the limitation of membrane removal 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 in volume can also be described using VCF

Vstart VCFF inal−conc = , (2.14) VF inal

where Vfinal is the volume after re-dilution. Normally a re-dilution back to the original volume of beer is carried out, hereby reducing the final concentration of alcohol obtaining a VCF of 1, as seen in figure 2.8. The complete re-dilution back to 5 litres results in a final alcohol concentration of 0.5%ABV. The trends observed in figure 2.8 are based on consideration observed in figure 2.9 and experimental data obtained during membrane filtrations. An approximate calculation was performed predicting circulations (loops), time and diafiltration volume needed to obtain the wanted end alcohol concentration. This was used as an indication for the experimental set-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 be equal to one kg (kg = l). The mean permeate flux through the membrane was used, as it never reach a stable level. Finally, the measured water flow, before loading the unit with beer, was used assuming the same flow for the beer. In figure 2.9 the data found experimentally can be observed.

24 2.3. EXPERIMENTAL CONSIDERATIONS CHAPTER 2. THEORY

Figure 2.9: Batch system outlook and corresponding feed, membrane, retentate, permeate and 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]

g • Initial feed alcohol concentration (C(0)) [ l ] l • Water ﬂow of retentate (Fretentate)[ min ]

• Membrane ethanol permeability (Ppermability) [%]

2 • Membrane area (Amembrane)[m ]

l • Mean ﬂux of permeate through the membrane (Jmembrane)[ m2∗min ].

• Pre-concentration degree (VCFP re−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 volume for the three different stages can be calculated. As a result of the batch configuration a change in alcohol concentration, volume and circulation time must be considered for each run-through (loop) over the membrane. For this purpose two different calculation loops were performed in Matlab corresponding to respectively the pre-concentration stage and the diafiltration stage. In appendix A.2 the set-up in Matlab is described for the calculation of the same filtration trend illustrated in figure 2.8. In the following section the background of the calculations will be considered:

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

25 2.3. EXPERIMENTAL CONSIDERATIONS CHAPTER 2. THEORY

• While the volume (V (k)) is above the wanted pre-concentration volume an additional circulated (loop) shall be calculated, hereby stopping the pre-concentration loop when reaching a speciﬁc volume dictated by the VCFP re−conc [l]

V V (k) > start . (2.15) VCFP re−conc • 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]

V (k) t(k) = . (2.16) Fretentate • Alcohol concentration change (C(k)) in the retentate is dependent on the change in g grams of alcohol in relation to the change in volume of the retentate [ l ]

Ppermeability C(k) ∗ Fretentate ∗ t(k) − C(k) ∗ ( ) ∗ Jmean ∗ A ∗ t(k) C(k + 1) = 100 membrane . (Fretentate − Jmean ∗ Amembrane) ∗ t(k) (2.17)

• Volume change (V (k + 1)) in the retentate is dependent on the initial volume going into 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: k Retentate 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 stage V2, C2 and t2. However, during this loop the limitation is not reaching a final volume in 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. The amount of re-circulations or loops in the diafiltration step is described by (n)

Cend C2(n) > . (2.19) VCFP re−conc

• Maintaining a constant volume during the diaﬁltration (V2) will result in a constant time for each loop (t2), because the ﬂow (Fretentate) is kept constant. The only variable per loop is hereby the change in alcohol concentration because of the permeation of g alcohol [ l ]

26 2.3. EXPERIMENTAL CONSIDERATIONS CHAPTER 2. THEORY

Ppermeability C2(n) ∗ Fretentate ∗ t2 − C2(n) ∗ ( 100 ) ∗ Jmean ∗ Amembrane ∗ t2 C2(n + 1) = . V2 (2.20)

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

The amount of loops or circulations: n Total time of the membrane ﬁltration: ttotal = tpre−con + t2 ∗ n Conc. alcohol before re-dilution: C3 = C2(n) Final conc. alcohol after re-dilution: C = C3 final VCFP re−conc Volume of dia-water: Vdia = Jmean ∗ Amembrane ∗ t2 ∗ n Vdia Diaﬁltration factor: DF = V (1)

This approximation will enable a process adjustment without having to run numerous membrane filtrations. It can be used as an evaluation mechanism revealing the optimal trends and furthermore evaluating the actual trends. Evaluation of actual trends was used to describe the membrane potential for alcohol removal by calculating e.g. the ethanol permeability percentage (Ppermeability). The input data throughout the membrane filtration, such as membrane area (Amembrane), could be adjusted simply by adding additional membranes. The flow (Fretentate) could be changed by the speed of the pump. The alcohol permeability 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 produced resulting in a unique flavour and aroma composition of beer. Beer is a complex matrix of various aroma compounds, nonetheless the main constituent is water in concentration of 91-98%. Volatile organic compounds (VOC’s) are most often compounds, which have a tendency to exist on gaseous form because of low solubility in the matrix where they exist. In beer this volatility is induced by the compounds existing in a high 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 different processes in beer production along with possible ways to alter VOC composition by changing physical process parameters. Focus will be given on the fermentation process, where physical parameters are easiest altered, and the raw materials, which can be altered by higher initial addition.

2.4.1 Yeast Metabolism A short introduction to the metabolism of yeast is needed for the purpose of understanding the production of various VOC’s during beer fermentation. Two strains of yeast are normally used for beer production namely Saccharomyces cerevisiae for ale production and Saccharomyces pastorianus for lager production. Brewers yeasts are eukaryote unicellular fungi, reproducing by budding. They belong to the Phylum of Ascomycota, the Family of Saccharomycetaceae, the Genus of Saccharomyces and ﬁnally respectively Species of cerevisiae 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 metabolic reactions 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 to glucose or other monosaccharides, which can enter the glycolysis in diﬀerent stages. Yeast glycolysis is the main source of aroma VOC’s formation as illustrated in ﬁgure 2.10.

28 2.4. AROMA FORMATION CHAPTER 2. THEORY

Figure 2.10: Aerobic and anaerobic glucose metabolism involved in the formation of ﬂavour compounds. 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).

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

29 2.4. AROMA FORMATION CHAPTER 2. THEORY reactions functioning as an oxidising agent (NAD+−→ NADH + H+), or as a reducing agent (NADH + H+−→ NAD+). Many VOC’s come from the glycolysis, which is illustrated in ﬁgure 2.10. During the course of the glycolysis, ending with the formation of two pyruvate, a net formation of two ATP molecules occurs. Furthermore, a net reduction of 2 x NAD+−→ 2 x (NADH + H+) is observed. During alcoholic fermentation the yeast needs to re-oxidise the 2 x (NADH + H+)−→ 2 x NAD+ for the redox balance to be maintained and hereby enable additional glycolytic reactions. The redox balance can also be maintained by producing NAD+ in the formation of glycerol. These catabolic reactions are called substrate level phosphorylation (Briggs et al., 2004). During aerobic respiration the end product of the glycolysis, pyruvate, will be further processed yielding 28 ATP and re-oxidising the NADH + H+ and FADH + H+ formed in the 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 Energy Balance Output Aerobic Glycolysis 2 (NADH + H+) 2 ATP Pyruvate decarboxylation 2 (NADH + H+) TCA cycle 6 (NADH + H+) + 2 (FADH + H+)) 2 ATP (2 GTP) Oxidative phosphorylation 10 NAD+ + 2 FAD+ 24 ATP Total 0 28 ATP Anaerobic Glycolysis 2 (NADH + H+) 2 ATP Fermentation 2 NAD+ Total 0 2 ATP

Aerobic metabolism yields the most ATP and is therefore expected to be the preferred metabolism 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 and fermentation 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 yeast respiration in high glucose concentrations which results in fermentation predominating respiration metabolism of carbohydrates. In addition, the Pasteur effect can, in aerobic conditions with low glucose concentration and limited yeast growth, result in a suppression of the fermentation metabolism hereby yielding more ATP through respiration (Walker, 1998).

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

30 2.4. AROMA FORMATION CHAPTER 2. THEORY

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

• Aggregation or adhesion in clumps and sedimentation in the bottom because of hydrophilic surface of the yeast

• Cellular bridging between cell wall lectins (ﬂocculins) and mannoproteins on adjacent cell 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 the suspended 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 regular lager production.

31 2.4. AROMA FORMATION CHAPTER 2. THEORY

Figure 2.11: Development of diﬀerent substances during batch fermentation of a lager at 15oC. (a) Present gravity (PG) = 1.000+0.004∗[%P lato]. (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 are illustrated.

Table 2.4: Higher alcohols in beer, aroma threshold and corresponding concentration in lager beer (EBC, 2000), (Tan and Siebert, 2004), (Fenaroli, 2005). Compound Compound Aroma Aroma Conc. Name Structure Threshold or Range mg mg [ l ] Taste [ l ] 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 2.4. AROMA FORMATION CHAPTER 2. THEORY

Formation of HAs is a result of amino acid metabolism as illustrated in ﬁgure 2.12. The catabolic pathways for degradation of assimilated amino acids, described by the Erhlich pathway, results in the formation of HAs. In addition, the anabolic pathways, originating from 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 from assimilated amino acids through the Ehrlich pathway (Hazelwood et al., 2008).

After assimilation of amino acids a direct usage of the amino acid in protein synthesis can 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 aminotransferase involving a amino acceptor or enzyme cofactor. In brewing yeast the main amino acceptor is α-oxoglutarate, producing glutamate. In addition, other amino acceptors such as pyridoxal phosphate producing pyridoxamine phosphate and pyruvate producing alanine are observed. The transamination results in a corresponding α-keto acid, see table 2.5. When amino acids deﬁciency occurs, or amino acids not assimilated by the yeast are needed, a biosynthesis pathway originating from pyruvate is possible. From pyruvate the wanted α-keto acid can be synthesised, which hereafter can obtain an amino group through the reverse transamination

33 2.4. AROMA FORMATION CHAPTER 2. THEORY 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. Fusel aldehydes 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 alcohol dehydrogenase to HAs in anaerobic condition with high glucose concentration and phenylalanine as the only nitrogen source. This reaction is valuable in anaerobic conditions because of maintenance on the cellular redox balance, which has a tendency to have surplus NADH + H+ in anaerobic conditions. Glycerol formation is another pathway yielding NAD+, however ATP is needed in this pathway hereby making the Ehrlich pathway more energetically favourable (Hazelwood et al., 2008), (Walker, 1998). The specific HA formed is dependent on the specific amino acids assimilated as illustrated in table 2.5. In aerobic conditions, with limited glucose concentration and various nitrogen sources, an oxidation of the fusel aldehyde to fusel acid has a higher tendency to occur. Once again, for the 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 passive diffusion, 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 Ethanol Threonine α-ketobutyrate n-Propanol Valine α-ketoisovalerate 2-Methylpropanol Isoleucine α-keto-β-methylvalerate 2-Methylbutanol Leucine α-ketoisocaproate 3-Methylbutanol Phenylalanine Phenylpyruvate 2-Phenylethanol Tyrosine p-Hydroxyphenylpyruvate Tyrosol Tryptophan 3-Indole pyruvate Tryptophol

From figure 2.11 (d) it can be seen that the formation of HAs occurs mainly during the yeast initial growth phase, while assimilable free amino nitrogen (FAN) is still present in the suspension, see figure 2.11 (c). For FAN explanation see appendix A.1. The formation of HAs seems to be stagnating when all possible FAN are assimilated and the yeast enters the stationary growth phase. This reveals the formation of HA mainly being a result of the catabolic degradation of amino acids in the Ehrlich pathway. Consequently, factors affecting the levels of HA are closely related to elevated yeast growth and therefore higher assimilation of amino acids. The yeast strain is also an important factor involving different gene expressions resulting in a higher HAs formation in ale yeast than in lager yeast (Briggs et al., 2004). A higher yeast vitality will involve a higher metabolic fitness hereby resulting in a higher formation of HAs. Thus, factors elevating yeast growth will also be factors elevating HA formation (Hammond, 1993). Pitching is important for HAs formation. Yeats pitching must be of a certain volume to obtain a proper growth and fermentation. Insufficient yeast growth related to a low pitching rate is believed to be caused by a lack in inter-cellular signalling to activate cell

34 2.4. AROMA FORMATION CHAPTER 2. THEORY multiplication. The Ehrlich pathway seems to be part of a yeast growth signalling system equivalent to bacteria quorum sensing. Especially 3-methylbutanol (isoamyl alcohol) and 2-phenylethanol have been found as signalling molecules inducing yeast growth and/or environmental adaptation (Hazelwood et al., 2008), (Walker, 1998). The following process parameter can be used to inﬂuence the HA formation:

Process Parameters Inducing 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

35 2.4. AROMA FORMATION CHAPTER 2. THEORY

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

Table 2.6: Esters, aroma threshold and corresponding concentration in lager beer. Ethyl esters (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 mg mg [ l ] Taste [ l ]

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

Ethyl propionate E 25 Rum, Pineapple 5-84

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

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

Ethyl butyrate E 0.4 Fruity, Pineapple -

Isoamyl acetate A 1.2 Fruity, 0.3-3.8 Banana, 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, 0.10-0.73 acetate 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 2.4. AROMA FORMATION CHAPTER 2. THEORY 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 an alcohol and a acyl-group resulting in the formation of ester can be observed. Dependent on the hydrocarbon side chains R2 and R1 various different esters can be formed (Verstrepen et al., 2003). The aroma active esters in beer shown in table 2.6 can be divided into two subgroups based on a difference in substrates (Verstrepen et al., 2003), (Saerens et al., 2008):

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

2. The ethyl esters (E) - The acid group is a medium chain fatty acid (MCFA) or a 1 3 2 oxo-acid (R −CH2R ) while the alcohol group is ethanol (R −CH3).

The most abundant ester is ethyl acetate formed in the reaction between ethanol and acetyl-CoA. Ethanol is obtained directly as a result of substrate level phosphorylation of carbohydrates. Other alcohols involved in ester formation are obtained as a result of HA formation as previously described. In addition, the formation of acetyl is obtained directly as a result of pyruvate decarboxylation with CoA-SH as cofactor, see figure 2.10. The formation of acyl-CoA is a result of fatty acids reaction with CoA-SH during fatty acid catabolism 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 many different 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 generally dependent 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 been proposed. Ester formation could be a mechanism for regulation of the acetyl-CoA to free CoA-SH ratio closely related to lipid synthesis. A decrease in lipid synthesis could induce an increase in concentration of intracellular acetyl-CoA hereby resulting in a rise in ester formation for the purpose of maintaining a constant acetyl-CoA to free CoA-SH ratio. A decrease in ester formation have been reported when supplements of the unsaturated fatty acid, linoleic acid, to wort occur. This suggests a inhibitory effect of fatty acids on ester synthesis. Furthermore, increasing the aeration of the wort, to prolong the lipid synthesis in

37 2.4. AROMA FORMATION CHAPTER 2. THEORY yeast, has shown to decrease ester formation. In figure 2.11 (d) the rate of ester formation is highest right before reaching a peak corresponding to the approximate time where lipid synthesis 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 detoxification mechanism. Fatty acids with a carbon length between C8 to C14 have shown a high toxic effect in yeast, especially if these fatty acids are unsaturated. A possible way to overcome the toxicity of these fatty acids is through fatty acids activation with CoA-SH followed by ester formation, in reaction with alcohols (Hammond, 1993). The difference in ester production observed among different yeast strains is believed to be caused by different AATase activity and cell membrane compositions. The acetate esters are lipid soluble, hereby enabling rapid diffusion through the cell membrane into the fermentation medium. On the contrary, the ethyl esters do not diffuse so easily through the cell membrane with a decreasing ability to pass the cell membrane with increasing chain length (R3). As a result, diffusion of ethyl caproate will happen in a higher rate than ethyl caprylate, while even longer chain esters remain in the cell. The transport of esters through the cell membrane is species dependent. Lager yeast has show a higher tendency of retaining ester within the cell resulting in a lower ester concentration in the final beer (Verstrepen et al., 2003), (Saerens et al., 2008). Specific gravity of the wort has revealed itself as an important factor related to ester formation. High gravity beer (HGB) fermentations have in recent years become an important industrial approach for the purpose of increasing the overall productivity in order to reduce costs. Nevertheless, HGB results in formation of more ester because of the higher sugar concentration (Briggs et al., 2004). The composition of the wort carbohydrates has an effect on ester formation involving glucose and fructose rich worts producing more esters than maltose rich worts. A higher acetyl-CoA and higher alcohols (HAs) formation involved with yeast growing on glucose and fructose compared to maltose could be the reason for a higher ester formation. In addition, glucose could induce stronger expression of ester synthase genes (Saerens et al., 2008). Oxygen depletion results in a decreasing yeast growth mainly observed in the stationary growth phase. Oxygen is essential for yeast production of membrane sterols and unsaturated fatty acids. At the point of oxygen depletion the formation of HA and acetyl-CoA continuous resulting in a higher formation of ester. Free amino nitrogen (FAN) is an important factor for the formation of HA as described in section 2.4.2. For this reason, the presence of FAN’s during growth limitation or oxygen depletion is important for ester formation. Addition of amino acids, assimilated by brewers yeast, during the stationary phase has been proposed as a way of manipulating the final ester production (Saerens et al., 2008). High temperatures induce higher ester production. Esters show different temperature dependency making the fermentation temperature a powerful tool for ester manipulation. The temperature dependency of ester formation is believed to be related to a higher AATase activity as well as a higher HA production. On the contrary, high temperatures during fermentation 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 pressure results in a lower ester production as well as a decrease in yeast growth. Increasing the pressure will result in a higher concentration of dissolved CO2 in the suspension. This will influencing the equilibrium of enzymatic decarboxylation reactions essential for growth and ester formation (Verstrepen et al., 2003), (Saerens et al., 2008). The following process parameter can be used to influence the ester formation:

38 2.4. AROMA FORMATION CHAPTER 2. THEORY

Process Parameters Inducing 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 therefore reduce level of esters

• Pressure increase

• High maltose concentration

• Low FAN

• Lipid or fatty acids addition

39 2.4. AROMA FORMATION CHAPTER 2. THEORY

Aldehydes Aldehydes in beer are mainly considered off flavours connected to either unmature and young beer or to mature stale and oxidised beer. Nonetheless, some aldehydes can contribute with positive flavours and aromas from the raw materials used in the brewing process. In table 2.7 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). Compound Compound Aroma Aroma Conc. Name Structure Threshold or Range mg mg [ l ] Taste [ l ] Acetaldehyde 25 Grassy 2-20 Young beer Apple-like (E)-2-Nonenal 0.00011 Papery 0.00005 Cardboard - 0.00015 Oxidized

Furfural 150 Caramel - Bready Cooked meat Isobutyraldehyde 1 Green malt 1-2 (Isobutanal) Wet cereal (2-Methylpropanal) Husky Straw

Benzaldehyde 2 Marzipan 1-10 Bitter almond

Formation of aldehydes during beer production and ageing can be divided according to the 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 decarboxylation of pyruvate, as illustrated in ﬁgure 2.10. As a part of the anaerobic metabolism of carbohydrates, the formation of acetaldehyde is high during the initial exponential growth phase where carbohydrates are in excess. Boulton (1991) suggested that the excretion of acetaldehyde is a way of controlling the intracellular levels, which is toxic in high concentration. Excretion during the active growth phase is therefore a detoxing mechanism. Subsequently a re-absorption during the stationary phase or the declining growth phase will occur when the carbohydrate sources are limited hereby resulting in the ﬁnal production

40 2.4. AROMA FORMATION CHAPTER 2. THEORY 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 levels detectable 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 to 0.11 ppb. The low aroma threshold involves problems in detection using regular analytical techniques. In this thesis, the analytical techniques used will not be able to detect level in ppb. Nevertheless, a short introduction concerning the formation of E2N is given in appendix A.3 because of the impact in pilsner beer, which is the main beer type used for AFB production.

Maillard reaction, also known as the non-enzymatic browning reaction, results in aromas and flavours of caramel, chocolate, coffee etc., which most often gives positive attribute to the beer. Maillard reactions are heat catalysed reactions between reducing sugars and nitrogen containing compounds such as amines, amino acids, peptides or proteins. Furfural is a Maillard product coming from the reaction with aldopentose, as illustrated in figure 2.14 (Vesely et al., 2003), (Baert et al., 2012).

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

Reaction between one type of aldose sugar with a specific type of nitrogen containing compound can yield a myriad of different compounds. The initial nucleophilic addition of an amino group to the aldehyde of the aldose sugar yields an amino Schiff base. This reaction is favoured in high pH environment. The Schiff base is in equilibrium with other tautomers through rearrangement of the double-bond, as shown in figure 2.14. Through tautomerisation the formation of an enol (1,2-enaminol) and the formation of a ketone is possible. The ketone compounds are called the Amadori compounds, which can be formed

41 2.4. AROMA FORMATION CHAPTER 2. THEORY from many different aldose carbohydrates reacting with a nitrogen containing compound. From the Amadori compound structure the formation of all the different Maillard products is possible. This includes melanoidins, responsible for colour formation, plus different volatiles compounds, where Furfural is the most pronounced in beer. The formation of the different compounds 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),(Vesely et al., 2003), (Baert et al., 2012). Benzealdehyde and isobutyraldehyde are formed by Strecker degradation. In figure 2.15 the transamination of a α-dicabonyl is initiated by a nucleophilic attack by the lone pair in the amino group on the carbonyl group, hereby forming an unstable hemiamine. Electron travel 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 a Strecker aldehyde and a α-ketoamine (Briggs et al., 2004), (Vesely et al., 2003), (Baert et al., 2012).

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

The formation of Strecker aldehydes is dependent on the concentration of corresponding free amino acids. The concentration of valine and phenylalanine is important in the formation of respectively isobutyraldehyde and benzaldehyde. Nevertheless, a large difference in the Strecker aldehydes aroma threshold levels only makes the concentration of specific amino acids important. The formation of phenylacetalehyde from phenylalanine follows the reaction illustrated in figure 2.15. However, oxidation could cause the further reaction into benzaldehyde as illustrated in figure 2.16.

42 2.4. AROMA FORMATION CHAPTER 2. THEORY

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

Most aroma potent Strecker aldehydes formed will either be evaporated during the wort boil 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 reducing the formation of these. Therefore, the following process parameter, which can be used to influence the aldehyde formation, are separated into different formation mechanisms in the following:

Process Parameters Inducing 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

43 2.4. AROMA FORMATION CHAPTER 2. THEORY

• (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 active metabolites will be formed during fermentation or originate form the raw materials. Ketones, sulphur compounds, organic acids and fatty acids are potent flavour and aroma metabolites mainly associated with off flavours of fermentation (EBC, 2000). The most flavourful ketones present in beer are vicinal diketones (VDKs). Diacetyl and pentane-2,3-dione are VDKs found in reasonable concentrations in beer. However, VDKs are not present in detectable concentrations if proper maturation of the beer is performed along with no contamination and oxygen exposure (EBC, 2000). A thorough description of VDK 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 during fermentation or originating from the raw material. Maintaining a proper production will eliminate any possible detection of these compounds in the beer (EBC, 2000). Therefore, sulphur compounds are not expected in Humlefryd. Formation and influential factors can be 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 a decrease in pH as illustrated in figure 2.11. Many organic acids are secondary metabolites excreted 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 are not considered in this report. Nevertheless, formation of various acids plus fatty acids can be 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 originating from the raw materials or as a result of the brewhouse procedures are generally not as difficult 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 the purpose of highlighting possible reduction in aroma, despite the possibility of later addition.

44 2.4. AROMA FORMATION CHAPTER 2. THEORY

Hops Constituents Hops contributes with many important compounds to beer such as aldehydes, resins, oils and polyphenols (Sch¨onberger and Kostelecky, 2011). In table 2.8 some essential hops oils can be viewed.

Table 2.8: Hops constituents, aroma threshold and corresponding concentration in lager beer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989). Compound Compound Aroma Aroma Conc. Name Structure Threshold or Range mg mg [ l ] Taste [ l ]

Myrcene 0.013 Floral 30-1000 Resinous

Geraniol 0.036 Floral 0.004-0.090 Roses Linalool 0.027 Floral 0.0035-0.150 Orange Geranium (pelargonie) Nonanal 0.015 Floral ≈ 3.7 Fruity Perfume

3-Carene - Sweet - Terpentine

α-Humulene 0.120 Hoppy ﬂavours -

trans-Anethole - Anise - Sweet Spicy

Polyphenols can add astringency to beer, however the main concern related to polyphenols is break formation resulting in visible sedimentation by hydrogen bonding to proteins in beer. Furthermore, polyphenols aids in protein precipitation and later foam formation. Therefore, polyphenols are not considered a flavour attributer in this section (Schönberger and Kostelecky, 2011).. Hops contains many different ethereal oils in the lupulin glands, which can be subdivided into different fractions where 50-80% are hydrocarbons, 20-50% are oxygenated hydrocarbons 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 soft resins are given in appendix A.7. Hard resins consist of different polyphenols here amongst flavanoids, proanthocyanidins and tannins in which the bacteriostatic plus health effects of

45 2.4. AROMA FORMATION CHAPTER 2. THEORY hops are located. Nevertheless, the flavour effects of these compounds are minimal. The bitterness donation of hops is not considered in this section because of possible later addition of isomerised products after de-alcoholisation (Schönberger 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 (Schönberger and Kostelecky, 2011). Ethereal hops oils are volatile at low temperatures resulting in a high loss of these oils during 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 fermentation and/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 drastically reduced during fermentation because of an adsorption to the yeast surface. Consequently, the more hydrophobic yeast Saccharomyces cerevisiae must be expected to adsorb more than 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 (Schönberger 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 normally not 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 no sulphur containing compounds are expected within flavour or detection threshold.

46 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 diﬀerent methods of ethanol and aroma detection along with separation techniques applied are shortly presented in the following section.

2.5.1 Sample Separation Separation of different molecules in a complex matrix such as beer can be done using chromatographic methods. Chromatography is based on different interactions of molecules in a mobile phase travelling through a stationary phase. The stationary phase can retain different molecules subsequently releasing them back into the mobile phase resulting in a separation, see figure 2.17. The different chromatographic processes are denominated according to the physical state of the mobile phase. Gas chromatography (GC) entails gas as the mobile phase whereas liquid chromatography (LC) entails liquid as the mobile phase (Moldoveanu and 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 mobile phase through a column typically packed with a stationary phase consistent of small porous particles. HPLC separation is based on a concentration equilibrium of a speciﬁc molecule between the mobile and solid phase. One of the following equilibrium can exist in HPLC columns (Moldoveanu and David, 2013):

Partition equilibrium Separation of two liquid phases involving retention of one liquid phase based on the diﬀerence in polarity. For example a highly polar liquid could form hydrogen bonds with a stationary solid phase hereby immobilising the polar liquid while a less polar mobile phase remains in suspension.

Adsorption equilibrium Separation of molecule species based on the individual polarity of the molecules.

Ion equilibrium Separation involving ionic bonding between ionic species in the mobile phase and ions in the stationary phase. An equilibrium based on the strength of the ionic bond will result in separation.

47 2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY

Size exclusion equilibrium Separation based on molecular size as a result of retention time for the molecule passing a porous structure or channels within the stationary phase. Large molecules not able to enter the channels and pores of the stationary phase will hereby elute earlier than small molecules retain within the stationary phase.

Affinity equilibrium Separation based on molecular recognition or selective non-covalent interaction. This separation technique is most often used for protein purification based on specific tags or antibodies retaining a specific protein in the stationary phase.

Gradient HPLC involves a change in composition of the mobile phase during the separation. Improvement of the separation can be obtained by changing the polarity and/or pH during HPLC. The separation of different molecules within the column will result in different retention times (tR). After separation detection of the different fractions are made possible using a detector, see section 2.5.2. Detection involves a visualization of the different fractions displayed as peaks in a chromatogram. If separation into individual compound peaks is obtained a possible quantitative analysis of the molecular species will be possible based 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 complex food products. Coupling this to a suitable detection source could therefore be a useful quantitative method for measuring ethanol after membrane filtration. Columns packed with a resin based polymeric materials in the stationary phase have been found usable for separation based on ion equilibrium or ion-exchange chromatography. The resin based polymer is protonated with a diluted acid eluent hereby enabling a cation bounding with the anions in the sample solution. Anions will be formed enabling an ionic bonding with the stationary phase based on the different acid dissociation abilities of the compounds in the solution. Further elution with acid will result in an anion-exchange according to the acid-base equilibrium. Consequently, separation occurs according to the pKa value of the different compounds in the sample. Molecules with a low pKa will easier dissociate and therefore 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 vaporised sample in the mobile phase through a column with a stationary phase consistent of either liquid (GLC) or solid (GSC) materials. GLC columns composition can be either packed columns or wall coated open tubes (WCOT) capillary columns, as illustrated in figure 2.18 (Mcnair and Miller, 2009).

48 2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY

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

Nowadays most GC application consists of WCOT with fused silica on which different stationary phases or solvent can be loaded. The stationary liquid or solid phase within the columns separates the different compounds based on intermolecular interactions such as polarity, complexation and hydrogen bonding. The samples are often vaporize by heating hereby enabling separation. The vapour pressure or boiling point of different molecules enables 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 constituents, as described in section 2.4, are ﬂavour and aroma potent compounds with a low vapour pressure enabling vaporisation to occur at atmospheric pressure and ambient temperatures. 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 into the 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 equilibrium HS-GC analysis involve introducing a gas sample directly into the GC from the headspace above a liquid or solid in a closed container. Initially the sealed container is pressurized with the intern carrier gas enabling a later pressure release, after volatile equilibrium, hereby introducing the headspace gas into the GC. The main advantage of static

49 2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY

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 ppb up 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 continuous flow of inert carrier gas through or above a liquid sample will induce a higher volatilisation 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 extraction period. Therefore, the volatiles are adsorbed or trapped within a cartridge containing a suitable adsorbent (TENAX-TA) (Wang et al., 2008). Tenax-TA (trapping agent) consists of a porous 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 VOCs down to ppt levels (Scientific Instrument Services, Inc., 2014). Later thermal desorption is possible releasing the volatiles into a stream of carrier gas onto the GC. Disadvantages with this 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 peak detection methods. Refractive index detectors (RI/RID) can be used for quantitative analysis of HPLC separated solutions. A RI detector is illustrated in ﬁgure 2.21. Detection is based on bending or refraction of light when a beam hits a speciﬁc medium. The refraction index of the sample solution is found by creating an angel of refraction between the sample solution to a reference solution, with a known refraction index. The refraction change between the two solutions causes a beam location change, which is detected by a photoelectric sensor. The rate of beam location change is proportional with the concentration of solute in the solution. As a result, the concentration can be found using standard solutions for calibration (Moldoveanu and David, 2013), (Doyon et al., 1991).

50 2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY

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

Refractive technique does not demand any ﬂuorescence or chromophore groups of the molecule for detection hereby enabling a wider range of detectable substances. Nevertheless, the sensitivity is not as high as the ﬂuorescence detection techniques. Refractive techniques demand a constant elution concentration and temperature, because of the refractive abilities of the eluent, hereby making it impossible to perform gradient elution and temperature change (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 based m on their mass-to-charge ratio ( z ). A mass spectrometer (MS) can be divided into five different components namely a sample inlet, an ioniser, a mass analyser, a detector and finally a data system. In figure 2.22 the ion source, mass analyser and detector of an electron 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 the sample volatility different methods of injection can be applied for the purpose of ionising the sample. Electron ionisation can be applied for gases, liquids and solids where evaporation is possible using heat and vacuum. The vaporised sample enters a vacuum chamber before entering the ioniser. Separation of the sample before introduction using chromatographic methods such as HPLC and GC may demand a specific scanning capability of the MS. The m MS must be able to scan a range of 10-300 ( z ) within seconds before the next molecule

51 2.5. METHODS OF ANALYSIS CHAPTER 2. THEORY 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 energy electron beam is created perpendicular to the sample flow admitted from a super heated filament cathode flowing to an anode on the opposite side of the chamber. The collision between the sample molecule and the electron beam results in an electron strip from the molecule hereby creating a cation. The flow of sample cations is directed to the mass analyser using positively charged repelling plate and negatively charged accelerating plates. Sample molecules which are not ionised are removed by the vacuum, while other molecules which absorb electron creating anions will be adsorbed on the repeller plates. Consequently, the formation or ratio of cations should be based on standard curve calibration of the wanted samples. Fragmentation of the molecules can occur because of the energy applied in the electron beam or electron migration. A short molecular ion lifetime will sometime result in detection of the fragmented ions (Pavia et al., 2009). m The cations are after ionisation separated according to mass-to-charge ratios ( z ) in the mass analyser. Different types of mass analysers exist, with the most common one being 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 beam direction. An oscillating electrostatic field is generated between the rods by application of direct current (DC) and radio frequency (RF) voltage to the rods respectively generating two opposite negatively charged rods and two opposite positive rods. The oscillation in the electric field is dependent on the RF amplitude to the DC voltage. Ions with different m m z will experience different oscillation paths. Ions with a specific z ratio will experience stable oscillation hereby remaining between the quadrupols until reaching the detector. On m the other hand, all other ions with a different z ratio will experience unstable oscillation resulting in a constant increase or decrease in oscillation which will eventually result in an m exit from the electrostatic field. Scanning of a wide range of z is possible by changing the ratio of voltage (Pavia et al., 2009). m Detection happens when the ions, separated into specific z ratio, hit the detector producing a current proportional to the amount of ions striking the detector. An electron multiplier is often installed in the detection system for the purpose of amplifying the signal with 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 the de-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 introduced into the feed tank with a maximum capacity of 9 litre. A modification of the M20 lab unit was made enabling 1-3bar CO2 pressurisation of the beer. A lid of transparent acrylic glass fitted with a gas inlet ensured the possibility of pressurising the feed tank from above the liquid surface. CO2 gas, from a CO2-tank fitted with a pressure release valve, was used for pressure delivery. The beer was pumped from the feed tank into the system by a frequency controlled positive displacement diaphragm Hydra-Cell pump from Wanner Engineering, l Inc. type G10 with a flow of 5 − 24 min and pressure delivery of 0-60bar. The Hydra-Cell pump was connected to a Lenze frequency controlled motor with a hz-inverter enabling rpm control. An Alfa Laval shell and tube heat exchanger ensured a constant temperature before 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 indicator from Alfa Laval revealed the feed pressure (Pf ) in bar.

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

The membrane module consists of respectively spacer and support plates compressed using oil hydraulics. The fluid flow through the system can be observed in figure 3.3. A hydraulic lightweight hand pump from Enerpac was used to pressurise the module to 600bar before filtration was commenced. A permeate outlet from each support plate was collected into a permeate 20l plastic tank. This tank was constantly weighed for the purpose of controlling the flux through the 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 out of the system in the permeate was used to conclude when the point of pre-concentration was reached. On the contrary, the flow out of the system was used to maintain a constant volume during the dia-filtration stage by adding carbonated dia-water. Samples from the permeate were constantly taken, and the volume removed from the system was compensated for in the mass balance. The pressure of the retentate flow leaving the membrane module was constantly measured by an Alfa Laval pressure indicator revealing the retentate pressure (Pr) in bar. A pressure-regulation valve connected directly to the output was used to ensure the right pressure build-up over the membranes. The retentate flow was returned into the feed tank through the inlet pipe going under the surface 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 still being able to take out beer samples and adding carbonated dia-water during the diaﬁltration stage. The sample valve was connected to a hose and a steel pipe ensuring a minimum foaming and release of CO2 while taking samples. The samples were pushed out of the system by the overpressure created above the liquid surface by CO2. Furthermore, the modiﬁcation 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 glass tank 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 arrows are 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 while maintaining 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 9bar 25oC 25oC 25oC 25oC MWCO - - 150-250 - g [ mol ] pH 3-10 3-10 3-9 3-9 Range Operating 15-42 15-42 5-25 15-35 Pressure [Bar] Maximum 55 55 40 41 Pressure [Bar] Temperature 5-50 5-50 2-50 5-45 Range [oC]

3.3 Feed Beer

The feed beer used for all membrane ﬁltrations was Humlefryd pilsner produced on brewhouse Skands A/S. Two diﬀerent productions were used respectively bottled on the 24th of February (HFORG24) and the 11th of April 2014 (HFORG11). The following alcohol concentrations were observed from the HPLC alcohol measurements:

• HFORG24 - 5.71%ABV

• HFORG11 - 5.72%ABV

A sample for alcohol measurement was taken after a couple of runs through the system at ambient CO2 pressure (1-3bar) to illustrate possible dilution in the system. The measured alcohol concentration of this sample was considered the correct start concentration for the filter runs. The dilution amount (l) was calculated based on the change in alcohol concentration from the above illustrated values to the sample taken after mixture. HS-GC-MS spectrum for each original Humlefryd was taken to illustrate possible differences in aroma among the different productions, these spectra can be see in appendix A.8. To compensate for possible production differences each aroma sample was compared to 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 should maintain the same, especially for RO application. Furthermore, the deionised water was carbonated to maintain CO2 pressure within the unit while avoiding oxidation. In table 3.2 pH 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 2.64 Deionised and carbonated dia-water 3.78 52 Milli-q water 5.46 1.69 KU life tap water 6.46 940

The pH of the original beers before membrane ﬁltration were pH 4.36 with a conductivity of 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 water removing all traces of glycol from the membranes. Recirculation was done under standard pressure 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 loading 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 between membrane 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 loaded the 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 pressure and temperature settings of the later membrane filtration. Hereafter the pump settings were maintained enabling an approximation of the beer flow in the unit during membrane filtration. 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 the calculation 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 valve using CO2 pressure on the water surface, see figure 3.2. Water in the system could not be removed entirely due to the pipe into the feed tank did not reach the bottom of the tank plus 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. Instead of compressed air, pressurised CO2 was used to push the beer into the closed feed tank through the inlet pipe, as illustrated in figure 3.2. A couple of circulations at 0bar TMP were done for the purpose of mixing possible water in the system with the beer. Hereafter a sample was taken to determine the new alcohol concentration. The membrane filtration run was then commenced.

3.5.5 Sampling plan Five beers were saved for later analysis of ethanol, aroma, pH and conductivity before initiating 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 Sample Stage Location Time Container Stage time in percent [%] [ml] Pre-concentration > 2.5 L Feed tank (FT) 0 50 Feed tank (FT) 0 2 Permeate (P) 0 2 50 2 100 2 Permeate tank (PT) 0 2 50 2 100 2 Diaﬁltration = 2.5 L Permeate (P) 0 2 20 2 40 2 60 2 80 2 100 2 Permeate tank (PT) 0 2 20 2 40 2 60 2 80 2 100 2 Permeate tank (PT) 100 50 Feed tank (FT) 100 50

All samples were frozen down directly after a membrane ﬁltration run ensuring microbiological stability. The remaining de-alcoholised beer in the feed tank was bottled and equally 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 and aroma substances will be presented.

3.6.1 HPLC - ethanol measurements For the measurement of ethanol a HP1100 HPLC fitted with an Aminex HPX-87H column was used. Each sample was sterile filtered using a nylon sterile syringe filter with a pore size of 0.45µm. Sample dilutions were done ensuring an ethanol concentration within the g range of 0.1-20 l . 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 mixture filtered with a 0.45µm pore size filter using a Sartorius vacuum pump. The following standard setting for the HP1100 HPLC was used for all samples:

Standard setting for the HP1100 HPLC

ml • Eluent ﬂow - 0.5 min • 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 calibration curves can are illustrated in appendix A.9. After 100 samples a new calibration curve was made. A general HPLC report generated for a original beer in the feed tank can be observed in appendix A.10.

3.6.2 HS-GC-MS - aroma measurements Dynamic Headspace (HS) extraction was performed in duplicates on 20ml samples trapping the 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 Tenax specially designed to function as trapping agent (TA). The Tenax traps used contained 250 60 g mg Tenax-TA with a mesh size of 80 and a density of 0.37 ml . Before setting up the sampling each trap was desorbed using a reprogrammed HP 5890A GC set to heat the samples to 250oC while flushing the traps with nitrogen at a flow of ml 50 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 instead placed shortly above the liquid surface. In addition, a magnet was placed within the sample

59 3.6. ANALYTICAL TOOLS CHAPTER 3. MATERIALS AND METHODS container to maintain stirring during sampling. Each sample was diluted to obtain the same concentration of volatiles, based on the original added volume of beer. The sample was placed in a water bath maintaining a constant temperature of 37oC mimicking human body temperature. In the water bath a magnetic stirrer ensured a proper heat transfer between the water and the sample stirring at 200rpm. The carrier gas used was nitrogen with a gas ml flow of 100 h manually controlled and confirmed using a flow-meter on the other side of the trap. Extraction of aroma was done for 15 minutes onto a Tenax-TA aroma trap. Hereafter ml drying was done for 10 minutes using nitrogen at 100 h in the opposite flow direction of the trap. Finally, the traps were sealed with clean caps and maintained in a refrigerator until sampling of the volatiles in the GC-MS, see equipment settings and parameters below in table 3.4:

Table 3.4: Overview of equipment settings and parameters for GC-MS analysis Step/Item Designation Manufacture Agilent Technologies Type 7890A Method Thermal Desorption Column DB-Wax capillary Column dimensions 30 m long x 0.25 mm inner diameter Column ﬁlm thickness 0.50µm Detector 5975C VL Mass Selective Detector (MSD) Column pressure 2.4 psi

Carrier gas H2 mL Initial ﬂow rate 1.2 min Column temperature program 10 min at 30oC o 8oC 30-240 C changing min 5 min at 240 oC Electron ionisation mode 70 eV m Mass-to-charge ratio scanned 15-300 z

The volatiles were identiﬁed using the highest probability or best matching based on their mass spectra compared to a commercial database (Wiley275.L, HP product no. G1035A). MSDChemstation software (Version E.02.00, Agilent Technologies) was used for data analysis. Peaks properly separated from other substances having a high matching with the database fractionation (> 50%) were visually investigated and integrated to obtain a comparable area between the diﬀerent samples. No standards were made for any of the aroma compounds hereby only making internal comparison possible. A standard HS-GC-MS spectra 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 aroma difference in the beer de-alcoholised with different membranes. Seven participants were carefully chosen to represent different backgrounds and knowledge in beer, membranes and general beer tasting. Three participants with a background in brewing, all being educated Diploma 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 3.6. ANALYTICAL TOOLS CHAPTER 3. MATERIALS AND METHODS

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

• HFORG11 - Original Humlefryd bottled on 11th of April Tasting strength: 4.09%ABV

• Sample A - NFHFR1 - NFHF membrane run 1 Tasting strength: 0.71%ABV

• Sample B - RO90R2 - RO90 membrane run 2 Tasting strength: 1.18%ABV

• Sample C - HFORG24 - Original Humlefryd bottled on 24th of March Tasting strength: 4.08%ABV

• Sample D - NFHFR2 - NFHF membrane run 2 Tasting strength: 0.98%ABV

• Sample E - NFR2 - NF membrane run 2 Tasting strength: 0.98%ABV

• Sample F - NF10R1 - NF10 membrane run 1 Tasting strength: 0.57%ABV

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

61 Chapter 4

Results and Discussion

4.1 Preliminary Investigation

Initial considerations concerning the physical parameters during membrane runs were made based on data obtained from Alfa Laval A/S. This data can be see in appendix A.13. Two different nano filtration (NF) membranes and one reverse osmosis (RO) membrane were tested with different applied pressures and temperatures to illustrate possible influences in alcohol 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 area was used as indicator for aroma loss. Principle component analysis (PCA) using Eigenvector in Matlab was done preprocessing the data by mean centering, choosing two components describing 99.3% of the variation in the data. A bi-plot of the two principal components can be observed in figure 4.1.

Figure 4.1: Principal component analysis (PCA) on RO90 data given by Alfa Laval. PC1 describes 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 of ethanol (Alcohol) permeability and flux. Lowering the TMP (Pressure) causes an increase in ethanol permeability as observed in figure 4.1. A rise in ethanol rejection, hereby a reduction in ethanol permeability, caused by a pressure increase have been reported by López et al. (2002) for RO apple cider de-alcoholisation and by Ferreira et al. (2007) for RO dialysis de-alcoholisation of beer. The correlation between the alcohol permeability and TMP dictates 65% of the data variance along the first principal component. The second principal component explains 34% variation in the data mainly being the different temperature during the different runs. Ferreira et al. (2007) illustrated a rise in permeate flux when increasing temperature. Therefore, a rise in temperature also induce a higher alcohol permeability as observed in figure 4.1. The ideal physical parameter settings for RO90 membrane de-alcoholisation therefore seems to be high temperature at 20oC and low pressure 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. PC1 describes 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 = Permeability and PC = Principal component.

The NF membrane filtration bi-plot explains 96% variation in the data. The first component describes the variation in the samples according to both aroma and alcohol permeability, which therefore seems correlated. This correlation indicates that operational settings for high ethanol flux also induces high aroma flux. The second component separates the samples with high temperature and pressure from those with low. From this bi-plot it can be deduced that high pressure and low temperature will cause the best aroma retention in beer. However, the ethanol permeability will equally decrease hereby increasing the process run time and use of dia-water. An increase in alcohol and aroma retention caused by a pressure increase have been illustrated by López et al. (2002) and Ferreira et al. (2007). Especially the retention of methanol and ethanol were changed drastically, while the aroma permeability did not change noteworthy. Therefore, the ideal physical parameters for NF membrane de-alcoholisation seems to be low temperature at 10oC and high pressure at 25bar (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 Alfa Laval. 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 the NF membranes can be observed. However, the correlation between aroma and alcohol permeation is not as clear as for the NF membrane. In figure 4.3 a strong correlation between aroma permeability and temperature is observed. On the contrary, a change in temperature does not seem to change the alcohol permeation as significantly. Changes in pressure seems to have a high influence on the ethanol permeation showing high ethanol permeation at low pressure. Concerning NFHF membranes, focus should be given on reduction in aroma permeation because of a general high flux through NFHF membranes. Therefore, the best settings for de-alcoholisation with NFHF membranes are low temperature 10oC and high pressure 25bar as sample NF99[10][25] in figure 4.3 indicates.

The physical settings chosen for the membrane runs performed in this thesis were based on these considerations along with experimental experience. The physical settings were maintained constant for the purpose of illustrating the potential of the diﬀerent membranes tested. 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 experimental errors and mapping out the reproducibility of the experimental set-up. To enable a comparison of ethanol permeabilities and aroma retentions all physical parameters were kept constant throughout the different membrane filtration runs. Possible deviations can be observed in table 4.1. Furthermore, two additional RO90 membrane runs were performed for the purpose of illustrating the importance of TMP, temperature and pre-concentration before diafiltration.

64 4.2. CONSTANT PARAMETERS CHAPTER 4. RESULTS AND DISCUSSION

An ethanol concentration reduction from 5.71-5.72%ABV to 2.97-3.60%ABV was observed as a consequence of water dilution. This concentration dilution corresponds to a volume between 3.60-4.71 litre water in the system before loading the beer. The water dilution did not influence the possibility of illustrating the membrane potential for alcohol removal 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. Ferreira et al. (2007) proved that an increase in flow caused an increase in permeate flux and aroma retention, while the ethanol retention maintained the same. However, they only reached a l l maximum flow of 7 min limited by the experimental set-up. A flow of 14 min 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 higher pre-concentration. The temperature in the feed tank was maintained using a constant flow of ice water at 0oC in the shell and tube cooler before entering the membrane housing, as illustrated in figure 3.2. A rise in permeate flux as temperature rises has been illustrated by Ferreira et al. (2007). Nevertheless, the effects on the different aroma substances were very different. Higher alcohols (HAs) were more affected by a rise in temperature hereby changing the retention drastically allowing high permeation at temperatures around 20oC. On the contrary, 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 cooling system. Trans membrane pressure (TMP) changed only when adding additional membranes during the RO90 runs. This illustrates a TMP loss of 2bar (20-18bar) when using 8 membranes and a TMP loss of 4bar (20-16bar) when using 20 membranes. A TMP around 18-19bar was chosen 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 this limitation, while still being able to compare the membranes. An increased ethanol retention at 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 retention at higher pressure. On the contrary, López 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, at 0.144m2 while RO90 membrane runs demanded a membrane area of 0.360-0.390m2 to obtain a reasonable membrane filtration run time, enabling a process duration of maximum seven hours equivalent to a days work.

65 4.2. CONSTANT PARAMETERS CHAPTER 4. RESULTS AND DISCUSSION 0 0 SD 0.208 0.565 0.881 0.440 0.036 0.199 5 5 5 5 5 5 15 15 15 14 16 15 1.46 1.35 1.41 1.41 1.37 1.35 0.144 0.144 0.144 0.1441.000 1.482 0.144 0.144 1.683 1.533 1.455 1.471 NFR1 NFR2 NF10R1 NF10R2 HFR1 HFR2 6 18 19 19 19 19 19 19 20 1.65 1.993 10 RO90R3 RO90R4 0.396 0.360 0.360 5 5 5 1719 16 18 16 3.00 3.2914.5 3.25 14 3.38 14.2 3.60 2.94 14 3.32 13.8 3.201.36 13.8 3.10 1.40 14.5 2.97 1.40 14.8 13.1 13.1 0.144 1.428 1.685 1.449 RO90R1 RO90R2 ] ] ] ] ] ] ] 2 ABV l C ] min Start ethanol Water ﬂow Volume Beer Temperature TMP Membrane Pre-conc. Final-conc. Bar VCF VCF l % o m conc. in [ in feed tank [ [ [ [ Area Designation feed tank [ [ [ Table 4.1: Membrane parametersthe and standard constants deviation for (SD) each calculation membrane run. Highlighted number and membrane runs are not considered in

66 4.2. CONSTANT PARAMETERS CHAPTER 4. RESULTS AND DISCUSSION

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 respectively beer and dia-water. Furthermore, a correction for the sample taken was subtracted the 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 4.1 for physical parameters

In figure 4.4 the different masses controlled during two membrane filtrations runs can be observed. 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 in the 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) was initiated and maintained in the same rate as the removal from the system through the permeate, hereby maintaining a constant retentate volume. The dilution was more sever than expected hereby resulting in a low pre-concentration VCF of 1.35-1.46 for the regular membrane runs with 5 litre beer added. Furthermore, a final dilution was observed because the diafiltration volume never came below the initial beer volume reaching volumes of 5.4-7 litre. In addition, the initial water diluted beer was considered in the VCF resulting in starting volumes ranging from 10.16-7.93 litre diluted beer in the system and ending with final volumes ranging from 7-5.4 litre resulting in the VCF observed in table 4.1. An increase in ethanol permeability with increasing VCF factors during pre-concentration was illustrated by López et al. (2002) during de-alcoholisation of apple cider. This illustrates a higher potential for a faster ethanol removal applying a higher VCF ratio.

The constant parameter was maintained as well as possible considering the experimental set-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 shorter process time hence lower cooling load, pump work and storage time. Consequently, a higher flux could possibly lower the OPEX. In addition, a high flux could minimise the membrane area demand thereby contribute positively to a lower CAPEX.

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

A wast difference in flux was observed in figure 4.5 and figure 4.6 depending on the type of membrane applied for the de-alcoholisation. The RO90 membrane showed the lowest flux compared to all types of NF membranes. For RO90R1 and RO90R2 the flux stagnates kg at around 0.150 m2∗min after 150 minutes. RO90R1 was performed under the exact same physical condition as the other NF membrane runs, while RO90R2 was performed using 22 membranes. A reduction in TMP has been shown to lower the retention of ethanol hereby increasing the ethanol permeability percentage (López et al., 2002). Considering this, an additional membrane run 3 (RO90R3) was performed lowering the TMP from 18-19 kg to 10bar. RO90R3 showed the lowest possible flux stagnating at 0.05 m2∗min maintaining a downwards trend. Halving of TMP reduces the flux threefold hereby proving the need to maintain 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 was carried out using a higher pre-concentration at 1.65 VCF and a higher feed tank temperature of 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.

68 4.3. MEMBRANE FLUX CHAPTER 4. RESULTS AND DISCUSSION

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

NFR1 and NFR2 were performed using the same settings. Nevertheless, a lower ﬂux for NFR1 was observed compared to NFR2. This could be a result of a higher dilution of the beer for NFR2 than NFR1, as illustrated in table 4.1. A stagnation was again observed situated kg at 0.375-0.415 m2∗min . The lowest ﬂux was observed for the NF membranes compared to the other NF membrane types. This was also expected since the retention description given by

Alfa Laval in figure 3.1 illustrated a better MgSO4 retention compared to NF10 membranes and a identical retention compared to NFHF membranes. NF10R1 and NF10R2 had a constantly falling trend throughout the membrane filtration never reaching a stable level. However, NF10R2 seamed to become more stable when kg reaching the same flux level as NFR2 around 0.430 m2∗min . The initial flux during the pre- concentration started higher than for the NF runs proving the higher porosity for NF10 as described by the manufacture. Nevertheless, the overall decrease in flux during the membrane runs was more pronounced for the NF10 membranes than respectively for the NF and l RO membranes. The water flow observed in table 4.1 (14.5-14.8 min ) showed that the pump settings for NF10R1 and NF10R2 resulted in a higher flow through the system, which could have resulted in the higher initial flux as well as longer duration for stabilisation compared l to NFR1 and NFR2 (13.8 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) membranes followed by a rapid drop. The first membrane run interestingly had the highest flux throughout the entire run compared to the second membrane run, where the final flux actually came below that of NF10R1. The same trend was observed for the other membrane runs showing a higher flux for run 1 compared to the run 2. In addition, it seems that the greater the pore size of the membrane the greater the difference between run 1 and 2 becomes. The difference between subsequent runs could indicate a possible clogging of the membrane pores, fouling, membrane cake build-up or membrane composition alteration

69 4.3. MEMBRANE FLUX CHAPTER 4. RESULTS AND DISCUSSION caused by CIP or sterilisation. This is most likely caused by internal fouling or clogging of the new membranes. Clogging would explain why the diﬀerence among membranes became larger as the membrane pores size became larger. Finally, the CIP and sterilisation could have resulted in membrane composition alteration, however this would not have been expected as the instructions given by the manufacture were followed. In addition, a CIP was applied before using new or used membranes. This wast alteration between subsequent membrane runs could be used proactive as a kind of priming of the new membranes with a material enabling a higher retention of wanted substances. Membrane selectivity, polarity and composition can hereby be drastically altered.

The solvent permeation coefficient Bwater was not calculated for all membranes because the osmotic pressure of the beer (∆π, [bar]) was unknown. Nonetheless, the RO90 membrane runs 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 the beer can be deduced, as illustrated in figure 4.7.

Figure 4.7: Linear plot of permeate water ﬂux as a function of pressure diﬀerence from equation 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)

kg ⇒ B = 0.0125 [ ] and ∆π = 6 [bar] (4.3) water,RO m2 ∗ min ∗ bar

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

The expected flux patterns were observed yielding the highest flux from the membranes with the highest pore size and the best high flux composition. The consequence of the different 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 ability to permeate ethanol Jethanol. This technical property should be viewed in the light of the ability to retain or reject wanted aroma compounds. In ﬁgure 4.8 the permeate ethanol trend for comparable membrane runs can be viewed.

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

In figure 4.8 an ethanol increase in the permeate is observed during the pre-concentration stage until the diafiltration stage is initiated. During the diafiltration stage there is a steady drop in the slope dependent on the ethanol permeability of the membranes. The decrease in ethanol concentration was illustrated to follow a negative exponential trend during diafiltration (Ferreira et al., 2007). In figure 4.8 the negative exponential trend is not as obvious. Nevertheless, a decrease in the rate of the negative slope during the diafiltration stage is observed for all NF membrane runs. This illustrates the limitation of membrane processes for alcohol removal with the removal rate falling constantly with the ethanol 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 high dilution had not occurred as a consequence of a higher start concentration.

71 4.4. ETHANOL PERMEABILITY CHAPTER 4. RESULTS AND DISCUSSION

Table 4.2: Ethanol retention, permeability and solute transport coeﬃcient for various membrane runs.

Retention Retention Permeability Ethanol Solute Start End Calculated Flux Transport [R][R] [%] Jethanol Coeﬃcient kg [ m2∗min ] Bethanol m [ min ] 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 to an equal amount of membranes were used (8 membranes). In figure 4.8 it can be seen that for RO90R1 both the pre-concentration as well as the diafiltration stage were significantly longer than the other NF membrane runs. All RO90 membrane runs, in table 4.2, had a relatively high retention being lowest at 0.45 while the highest permeability calculated was 85%. A general level of permeability of approximately 50% was observed for the comparable RO90R1 and RO90R2. An ethanol permeability level of 49% for RO90 membranes has also been proved by Alfa Laval doing the membrane filtration at 20oC and 25bar, see appendix A.13. NFR1 and NFR2 had an ethanol permeability of 95% and an ethanol retention ranging from 0-0.14 throughout the filtrations performed, as observed in table 4.2. From figure 4.8 it can be seen that the permeation of ethanol was very similar to the other types of NF membrane filtrations. Nevertheless, NFR2 had the longest duration before entering the diafiltration stage after approximately 50 minutes. This is a result of NF membranes having a 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 rising the pressure to 25bar was found by Alfa Laval, see appendix A.13. NF10 membranes showed an ethanol permeability 91-92% with a retention ranging from 0.10-0.31, as illustrated in table 4.2. The ethanol permeation trend for NF10, see figure 4.8, showed the approximately same rate of ethanol removal as for the other NF membrane types. NFHF membranes also showed a high ethanol permeability at 95% with a ethanol retention ranging from 0-0.29, as illustrated in table 4.2. NFHFR1 showed a different trend compared to the other NF membrane runs. This was most likely caused by the higher water flux through the membrane as earlier described in section 4.3. NFHF membranes have the best ethanol flux and solute transport coefficient as a result of high flux and ethanol permeability. However, there is some concerns in the drastic drop in flux between two adjacent runs as described earlier. A 95% ethanol permeability at 20oC and 10bar changing to 85%

72 4.4. ETHANOL PERMEABILITY CHAPTER 4. RESULTS AND DISCUSSION 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 characteristics a calculation was made predicting possible membrane area and dia-water volume needed to de-alcoholise 5 litre beer from 5.5%ABV to 0.5%ABV within a time span of 4 hours. These calculated values can be observed in table 4.3. This calculation will indicate inﬂuences on CAPEX and OPEX. These values should be considered in connection with the possible aroma 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 and a ﬁnal concentration of 1 VCF resulting in 5 litre of beer with 0.5%ABV within a 4 hour kg operation. A constant ﬂow rate 14 min was used in the calculation.

Designation RO90 NF NF10 NFHF Desired Optimisation Development Mean beer ﬂux 0.173 0.417 0.514 0.717 ⇑ kg [ min∗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 can be deduced from table 4.3. Concerning CAPEX, de-alcholisation using NFHF membranes seems significantly lower compared to RO90 membranes. A more than fivefold increase in 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, moreover demanding 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 for the higher area and possibly piping while maintaining the same TMP. This would influence the OPEX as well as CAPEX. Almost twice the volume of dia-water is needed to reach the final alcohol concentration. All in all, NFHF seem as the most reasonable choice for a membrane process for de-alcoholisation based on permeate flux and ethanol permeability. Nonetheless, the influence on aroma retention among the different membranes needs to be taken 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 based on HS-GC-MS data. As mentioned in section 3.6.2, only properly separated peaks were chosen after a visual confirmation. Consequently, some important aroma components in beer might not be identified because of i) overlapping peaks, ii) to low concentration or iii) to low matching. No absolute quantifications were made because of the high abundance of different aroma compounds in beer. Therefore, only an internal comparison among the different membranes was possible. The integrals of the clearly separated peaks are expected to be equivalent to the relative concentration of the compound. In this assumption a constant dilution of the samples, purge gas volume, column temperature and column pressure is expected during all the HS-GC-MS samples. The different compound integral areas, after membrane 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 compounds [%]. The samples was diluted to ensure that the same volume of original beer was present. 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 observed in appendix A.8. All aroma compounds are illustrated as percentage compared to the mean integrated area of the compound peak in the original beers. Additional aroma compounds might be present, however they were either in too low concentration for MS detection of significance or not fully separated from other compound peaks. A general tendency for a higher aroma compound output was observed for the HFORG11 production compared to HFORG24. Concerning fermentation aroma products, respectively higher alcohols, esters and aldehydes the similarities between the two production do not differ significantly. Nevertheless, the compounds such as decanal and octyl acetate do have a 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 seem to differ significantly which could be caused by different hops addition time, oxidation, productions (crops) or different boiling times. In addition, the deviation observed could illustrate possible deviation in the sampling technique or measurement method. Sampling and measuring errors cannot be ruled out based on duplication of each sample. The standard deviations among duplicates HS-GC-MS samples for all membrane runs and detected aroma compounds can be found in appendix A.14. The different aroma compounds solubility in water will in the following section be used as a tool to evaluate possible partitioning or sorption at or in the more non-polar PA membrane in coherence with polar Taft (σ∗), steric Taft (Es∗) and Small’s number (s∗) found in literature (Alvarez et al., 1998).

74 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

4.5.1 Higher Alcohols

Figure 4.9: Higher alcohol aroma compounds area percentages compared to the original beer 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: Compound name (retention time) - associated aromas.

The higher alcohols (HA) percentage retained in the feed tank beer or permeated into the permeate tank can be observed in respectively figure 4.9 and 4.10. A quick overview reveals a higher retention and lower permeation of aroma compounds by RO90 membranes compared to all the different NF membranes. Especially 1-butanol, n-octanol and benzene ethanol were significantly better retained by RO90 than NF membranes. 1-butanol seems to be completely retained while benzene ethanol actually increases to more than twice the concentration (273%) during the RO90 membrane. The increase of benzene ethanol was investigated in the various HS-GC-MS spectra where no explanation for the increase could be found. In addition, a small permeation was observed when looking at the percentage in the permeate, see illustrated in figure 4.10. Therefore, the increase of benzene alcohol during the RO90 membrane run was considered as almost completely retained. Possible dilution errors during HS sampling were ruled out because the other aroma compounds showed regular trends. In figure 4.10 a decreasing trend for the permeation of linear alcohols was observed as the GC 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 along with an increase in higher molecular mass of linear alcohols (Schutte, 2003).

75 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.10: Higher alcohol aroma compounds area percentages compared to the original beer 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’s Weight In Water Taft Taft number g g ∗ ∗ ∗ [ mol ][ l ] σ Es s Ethanol 46.07 Miscible -0.100 -0.070 ∼ 0 1-Propanol 60.06 Miscible Isobutyl alcohol 74.07 70 -0.125 -0.930 2.24 1-Butanol 74.07 73 -0.130 -0.390 2.17 Isoamyl alcohol 88.09 28 n-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 data will 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 of small alcohols such as 1-propanol, ethanol and methanol (Franks and Ives, 1966). The stability gained by hydrogen bonding with water can compensate for the stability lost by alcohol-alcohol hydrogen bonding and non-polar interaction of the hydrocarbon chain. As the hydrocarbon chain becomes longer the solubility in water is reduced because the stability gained in non-polar interaction becomes higher (Franks and Ives, 1966). 1-Propanol had the highest flux through all different membranes, as observed in figure 4.10. On the contrary, 1-propanol showed a general high retention in the retentate illustrated in figure

76 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

4.9. This might reveal the possibility of 1-propanol to permeate the membranes because of the ability to interact with water. Nonetheless, 1-propanol rejected by the membrane would not partition on or within the membrane as a consequence of a fairly polar nature. Isobutyl alcohol and 1-butanol have a relatively high retention with RO90 membranes. However, NF membranes show significantly different trends in retention, with a high isobutyl alcohol retention and a low 1-butanol retention illustrated in figure 4.9. In table 4.4 it can be observed that 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 isobutyl alcohol and 1-butanol. This indicates that the steric hindrance, caused by a methyl group on 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 compound n-octanol, where the same retention is observed for RO90 membranes while different for NF membranes. The higher permeation of n-octanol by NF membranes could also be a result of partitioning of n-octanol to the membrane caused by non-polar interactions. This tendency for a lower retention of linear compounds only for NF membranes could indicate that NF membranes follow the pore separation model while RO90 membranes follow the solution diffusion model. However, the best retention for RO90 membranes was observed for benzene ethanol which must be considered a result of a high steric hindrance of the molecule. In addition, the lowest retention of NF membranes was observed for benzene ethanol compared to all the other higher alcohols. This could be a result of the cyclic ring in the molecule enabling entrance into the pores despite the cross-flow application. In contrast more linear molecules 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- o l cohol for RO PA membranes (> 97% NaCl retention) at 25bar, 15 C and 3.33 min has been illustrated. Additionally, a lower retention for 1-propanol, isobutyl alcohol and isoamyl alcohol at respectively 25%, 50% and 40% for RO CA membranes (> 95% NaCl retention) at o l 25bar, 15 C and 3.33 min was observed (López et al., 2002).

Considering the HA retention of the diﬀerent membranes a clear superiority was observed for RO90 compared to NF membranes. NF membranes seemed to permeate linear HAs easier than steric hindered compounds while RO90 membranes generally retained the majority of the HAs during de-alcoholisation. The high retention and low permeation of the alcohols through RO90 membranes were connected to the alcohol group with high polarity and ability to form hydrogen bonds.

77 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

4.5.2 Esters

Figure 4.11: Ester aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas.

In figure 4.11 and 4.12 respectively the retention and permeation of different ester by different membranes can be observed. A general higher retention and lower permeation of esters were observed for RO90 compared to NF membranes. Despite a high retention, figure 4.11 indicates some losses esters, only indicating a total retention of ethyl caproate and phenylethyl acetate. On the contrary, the permeate HS-GC-MS data in figure 4.12 illustrate a 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 RO90 permeation 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 were observed for NF and HFNF membranes, while a clear reduction in retention and increase in permeation were observed for NF10. This was considered to be directly related to the higher porosity of NF10 membranes, as seen in table 3.1.

78 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.12: Ester aroma compounds area percentages compared to the original beer in permeate tank (PT) after ended membrane ﬁltration measured by HS-GC-MS. Column legend: 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 (L´opez et al., 2002), (Fenaroli, 2005), (Alvarez et al., 1998).

Compound Molecular Solubility Polar Steric Small’s Weight In Water Taft Taft number g g ∗ ∗ ∗ [ mol ][ l ] σ Es s Ethyl acetate 88.05 Miscible -0.100 -0.070 2.11 Ethyl butyrate 116.08 4.9 -0.215 -0.430 1.54 Isoamyl acetate 130.10 0.02 -0.045 -0.350 1.47 n-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 the difference 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 alcohol group and acid group in coherence with the formation of the ester, see section 2.4.2. Ethyl acetate showed a low retention and high permeation, which were associated to the small hydrocarbon chain causing a polarity enabling a total miscibility in water and a low steric hindrance within the membrane. The same tendency of permeation was observed concerning ethyl acetate for RO90 and NF membranes. This is most likely caused by the ability of ethyl acetate to form hydrogen bonds and thereby interact with water or to permeate the membrane in resemblance to water.

79 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

Esters are generally able to form two hydrogen bonds with respectively the carbonyl oxygen 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. Ester hydrogen bonding with methanol only yields one hydrogen bond to the carbonyl oxygen. Nevertheless, hydrogen bonding with water could yield an additional hydrogen bond to the ether oxygen (Lommerse et al., 1997). According to Shikata and Okuzono (2013) esters are not able to form any hydrogen bond in aqueous solutions. On the contrary, the small esters are miscible with water generally because 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 overall aroma of the beer because of a general association, that this compound contributes with negative solvent aroma. In figure 4.11 the subsequent linear esters (ethyl propionate, propyl acetate and ethyl butyrate) seem to have the same general retention as ethyl acetate while the same esters in figure 4.12 seem to have a lower permeation as the linearity becomes larger. From table 4.5 a lower water solubility for ethyl butyrate can be observed possibly causing partitioning to the non-polar PA membrane. However, a lower polar Taft value shows a higher tendency for ethyl butyrate to gain a negative charge though rearrangement hereby increasing basicity compared to ethyl acetate. A lower Small’s value shows lower hydrophobicity, which could be 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 entails a higher steric hindrance (Es∗). This could explain the permeation reduction observed in figure 4.12 as the chain length increases. Ethyl butyrate has the highest steric Taft in table 4.5, even higher than the branched isoamyl acetate ester. This indicates that the length of the acid part of the ester has a higher influence on steric effects than branching on the alcohol part. Isobutyl acetate and isoamyl acetate do show a higher retention and lower permeation than ethyl butyrate despite a lower steric hindrance for PA membranes according to Alvarez et al. (1998). This could be a result of a lower polar Taft and water solubility resulting in these compounds not being able to permeate the membrane with water or in a similar fashion as water. A remarkable decreasing trend in retention with increasing non-polar hydrocarbon groups and retention time is observed from isoamyl acetate to ethyl caprate for all NF membranes in figure 4.11. For RO90 membranes ethyl caproate and ethyl caprate seem to be excluded from this retention trend. In figure 4.12 contradictory trend seems to be prevalent with a higher permeation of isoamyl acetate decreasing until not detectable. The molecular weight g of these esters are ranging from 130.10-200.18 mol hereby being within the MWCO of the g most porous NF10 membrane at 150-250 mol . Therefore, a NF10 membrane permeation of g g linear compounds such as octyl acetate (172.15 mol ) and ethyl caprate (200.18 mol ) would have been expected. This contradiction in retention and permeability of esters with linear hydrocarbon groups above five carbon in length could be explained by a possible partitioning to or within the PA membrane. A possible non-polar interaction could result in a higher concentration 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 overcome the 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 the same reduced retention of RO90 membranes could be a result of these compounds being to

80 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION bulky to enter the membrane for partitioning hereby being flushed off by the cross flow into the permeate tank. Finally, phenylethyl acetate was almost completely retained by RO90 membranes only detected in the permeate while clear permeation was observed for the various NF membranes. g With a molecular weight of 164.08 mol this compound permeates the NF10 membrane as expected. Phenylethyl acetate contains an aromatic benzene ring in the alcohol group which could be sorped within the PA membrane as a result of non-polar interaction in an equal way as illustrated by Schutte (2003) for phenols. He analysed phenol partitioning or sorption on or within CA, PS and PA membranes. He proved a higher phenol sorption for PA membranes compared to both PS and CA membranes. This could be a result of internal membrane partitioning caused by non-polar interactions. The complete retention by RO90 membranes must be considered to be a result of the size and bulkyness of the molecule being significantly larger than phenol because of the ethyl acetate group.

A 90% retention of ethyl acetate for RO PA membranes (> 97% NaCl retention) at o l 25bar, 15 C and 3.33 min was proven by L´opez et al. (2002). In addition, a lower retention o l at 60% for RO CA membranes (> 95% NaCl retention) at 25bar, 15 C and 3.33 min was observed (L´opez et al., 2002). A 77% retention of ethyl acetate and 68% for isoamyl acetate for RO CA membranes g o l (200 mol MWCO) at 20bar, 5 C and 7 min was illustrated by Ferreira et al. (2007). The retention fell drastically when increasing the pressure.

Conclusively, an increased ester retention was observed comparing RO90 membranes with NF membranes. Among NF membranes, the best retention was observed for NFHF and NF membranes. A general retention above 50% was observed for RO90 membranes highly influenced by the steric hindrance of ester with branching (iso esters). A remarkable reduction in retention among esters, with a linear acid or alcohol group above five carbon in length, was seen which could not be found in the permeate. This could have been caused by a partitioning of these more non-polar substances to the non-polar PA membrane. In the light of this, other membrane materials, pre-coating of the membrane or a higher flow should perhaps be considered for a better ester recovery. Furthermore, an even less porous RO PA membrane could possibly reduce the partitioning and sorption within the membrane resulting in an even lower loss of esters.

81 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

4.5.3 Aldehydes

Figure 4.13: Aldehyde aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas.

In figure 4.13 and 4.14 respectively the retention and permeation of different aldehydes by different membranes can be observed. In figure 4.13 a resembling retention of isobutyr aldehyde and furfural for both RO90 and NF membranes was observed, while a clear difference was 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 for RO90 compared to NF membranes, while benzaldehyde permeation seems to be equal for all membranes. Consequently, the difference among aldehydes detected for RO90 to NF does not seem to be as significant as for other aroma compounds. Isobutyr aldehyde is relatively soluble in water, as illustrated in table 4.6, because of the carbonyl ability to form hydrogen bonds with water. Therefore, a general high flux through the membrane should be expected as a consequence of isobutyr aldehyde being fairly polar while having a short hydrocarbon non-polar end. Nonetheless, the general trend in figure 4.13 and 4.14 illustrates a reasonably high retention and reduced permeation with the RO90 permeation being fairly lower compared to NF membranes. Retention of isobutyr aldehyde is most likely caused by the branching methyl group causing a steric hindrance. In addition, isobutyr aldehyde is not non-polar enough to partition at or in the membrane and will therefore follow the permeate or retentate. Consequently, this aldehyde was separated mainly on structural properties.

82 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.14: Aldehyde aroma compounds area percentages compared to the original beer in permeate tank after ended membrane ﬁltration (PT) measured by HS-GC-MS. Column legend: 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 Solubility Weight In Water g g [ mol ][ l ] Isobutyr aldehyde 72.06 75 Furfural 96.02 83 Benzaldehyde 106.04 6.95

A high permeation of furfural is observed in figure 4.14, which is confirmed with a low retention in figure 4.13. Furfural is likely to form one hydrogen bond with water from the 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 oxygen 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 membrane permeation along with water or in a similar fashion as water. Compared to isobutyr aldehyde a lower retention is observed even though the molecular weight and size is higher. Benzaldehyde also experiences a lower retention and higher permeation than isobutyr aldehyde despite a higher molecular mass. Nevertheless, a higher retention for RO90 membranes is observed in figure 4.13. The reasonable low retention of a bulky molecule with a high molecular weight must once again be a result of a fairly high solubility in water caused by the ability of hydrogen bonding and a dipole moment causing a higher intramolecular polarisation. Indeed Tekin et al. (2004) proved an increasing polarisation of benzaldehyde as the concentration of ethanol was increased. Ethanol favours a strong hydrogen bond with the 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 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION and therefore more water soluble. Further this enables a co-permeation through the membrane. In addition, this would explain the clear difference in retention between RO90 and NF membranes for benzaldehyde possibly caused by the lower ethanol permeation of RO90 membranes. Benzaldehyde is a Strecker aldehyde, which can be formed during the membrane run caused by the presents of oxygen, as illustrated in figure 2.16. Oxygen might be introduced into the feed tank through the dia-water addition where compressed air was used or simply by air diffusion in the permeate tank. A possible explanation for the high concentration of benzaldehyde in the permeate in fiugre 4.14 could be caused by formation in the presence of oxygen 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 aldehydes in accordance to diﬀerent RO90 or NF membrane de-alcoholisations. The more equal retention and permeation could be caused by a tendency to form hydrogen bonds with water and ethanol along with the experience of dipole moments causing polarisation of the aromatic compounds. Furthermore, isobutyr aldehyde experienced the best retention possibly caused by a lesser polarisation of the molecule hereby maintaining a non-polar end with methyl branching.

4.5.4 Hops Constituents

Figure 4.15: Hops aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas.

In figure 4.15 and 4.16 respectively the retention and permeation of different hops constituents by different membranes can be observed. Many of the hops constituents were not detected in the final de-alcoholised beer most likely because of low concentration or loss during membrane filtration. In appendix A.8 the HS-GC-MS of the original beer showed additional detection of geraniol and α-humulene, while figure 4.15 only illustrates detection

84 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION of β-myrcene, nonanal, 3-carene and trans-anethole. However, these compounds represent the membrane potential for hydrocarbon monoterpene retention with β-myrcene and 3- carene plus oxygenated terpene retention with trans-anethole. In addition, nonanal was clearly detected as a positively aroma attributing aldehyde. A general higher retention of hydrocarbon terpenes was observed for all membranes compared to the oxygenated terpene. β-myrcene was almost completely retained only showing permeations for the NF membranes in ﬁgure 4.16.

Figure 4.16: Hops aroma compounds area percentages compared to the original beer in permeate tank after ended membrane ﬁltration (PT) measured by HS-GC-MS. Column legend: 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 in table 4.7, however readily soluble in ethanol. This could entail a β-myrcene co-permeation with ethanol through the membrane. Moreover, the non-polar surface of the PA membrane could entail a partitioning of β-myrcene to the membrane allowing possible permeation if the pore size is adequately large. Indeed, a higher permeation is observed for β-myrcene according to the difference in pore size between RO90 to NF, with NF10 as the membrane with 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 linear compound which could result in a possible permeation through the pores. Nevertheless, this permeation is reduced by the cross-flow application resulting in a tangential direction of the compounds during membrane filtration. 3-Carene is an aromatic monoterpene equally insoluble in water showing an even lower permeation than β-myrcene in figure 4.16. Nevertheless, the retention showed very different values among the different membranes with the lowest retention for RO90 and NF10 membranes, while the highest retention was observed for NFHFR1. A contradiction was observed comparing the permeation to the retention because a higher retention seemed to induce a higher permeation and vice versa. Indeed, this was believed to be a consequence of partitioning within the membrane caused by non-polar interactions resulting in NF10 being the membrane with the highest possibility of 3-carene adsorption within the membrane.

85 4.5. AROMA RETENTION CHAPTER 4. RESULTS AND DISCUSSION

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 is significantly different being more linear than 3-carene with a cyclic structure. This difference could involve an easier membrane entrance of 3-carene compared to β-myrcene. This might be 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 passage through 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 droplets within the cell walls or insoluble constituents (Olle et al., 1997). A possible droplet formation on, or within, the membranes could result in the low retention and low permeation of hydrocarbon oils.

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

Compound Molecular Solubility Weight In Water g g [ mol ][ l ] β-Myrcene 136.24 Insoluble Nonanal 142.24 Insoluble 3-Carene 136.23 Insoluble trans-Anethole 148.21 Insoluble

Nonanal shows an opposite retention and permeation than all previously discussed aroma substances with a higher permeation and lower retention for RO90 membranes compared to NF membranes. Hexanal has the following parameters influencing the solute transport through 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 similar in molecular structure to nonanal only being three carbons shorter in the hydrocarbon chain. The polar and steric Taft are relatively similar to that observed in table 4.4 and 4.5 for respectively linear alcohols and esters. The Small’s number, representing the degree of hydrophobicity, is larger compared to the linear esters and more similar to that of linear 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 to form hydrogen bond with water or alcohol is lower compared to alcohol because aldehydes only carry a hydrogen acceptor in the oxygen of the carbonyl group. This could result in a greater tendency for aldehyde membrane partitioning and thereby lower retention and induced permeation compared to alcohols. In addition, the difference in polarity of the different functional groups (−OH > −CHO > −C−O > −C−O−C) could result in an even higher tendency to partition on, or within, the membrane enabling permeation (Dickson et al., 1975). Trans-anethole was the only oxygenated terpene observed after de-alcoholisation. A poor retention for all NF membranes compared to the RO90 membrane was observed in figure 4.15 while no permeation was observed in figure 4.16. This could likewise be caused by 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

86 4.6. TASTING RESULTS CHAPTER 4. RESULTS AND DISCUSSION cross-ﬂow would not result in diﬃculties in entering the membrane because of a general cyclic and round composition of the molecule.

The hops constituents were generally well retained. The difference between RO90 and NF membranes were not as obvious for these compounds as for the other aroma compounds. Nevertheless, RO90 only showing a better retention of trans-anethole compared to NF membranes. This lack in difference could be a result of a higher PA membrane partitioning because of the general high hydrophobicity of the hops constituents, as illustrated in table 4.7. A tendency of permeation through the different membranes for the more linear structured molecules was observed, while the cyclic molecules showed a lower retention properly 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 original Humlefryd 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 a higher aroma retention for de-alcoholised beer using RO90 compared to NF membranes was observed. Nevertheless, RO90 membrane for de-alcoholisation could induce a higher CAPEX and OPEX which therefore should be compensated for in a higher aroma and flavour retention. The aroma and flavour composition of the beer could be drastically altered as a result of some aromas permeating the membranes more than others hereby changing the beer characteristics. In addition, de-alcoholised beer is often associated with a watery experience mainly caused by the lack of the warming mouth feeling of ethanol. For these reasons, the test participants were asked to rate the beer from 1-10 in accordance with the relative equality and intensity to the original beer. In figure 4.17 the general reproducibility of 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). This was done to validate both the tasting result and the panel, to see if they would give this beer 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 the first original beer sample, could be the course of the low grades of the original beer blind test sample, along with the other samples. In this way the taste panel only expected samples unequal and/or less intense than the original beer. Despite this, higher grades was given the sample produced using RO90 compared to NF membranes hereby confirming the HS-GC- MS results. The additional de-alcoholised beers, produced with NF membranes, were not significantly 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 a larger 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 of CAPEX, OPEX and quality parameters. The choice of membrane application can inﬂuence the 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 tasting results plus the desired optimisation development. All values are mean analytical values of numerous membrane runs. Flux was measured by mass, ethanol by HPLC and aroma compounds by HS-GC-MS. ∗Mean value of percentage esters retained minus benzene ethanol (273%).

Designation RO90 NF NF10 NFHF Desired Optimisation Development Beer Flux 0.173 0.417 0.514 0.717 ⇑ kg [ min∗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 permeation can be observed between RO90 and NFHF membranes. On the contrary, only approximately twice the aroma retention of higher alcohols and ester was observed, while no 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 and esters. 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 produce more ester and higher alcohols during fermentation compensating for the losses observed as retentions in table 4.8. Nonetheless, breweries nowadays seem to be keen on reducing the number of production streams making alteration of the brewing process even harder to influence. If no alteration of the brewing process is made then RO90 membrane would result in the most aromatic and flavourful beer. The brewer could alternatively view the AFB as the final product hereby seeing the membrane loss as a part of the process, which should be accounted for in the beer before membrane filtration.

4.8 Aroma Formation

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

In section 2.4.2 the formation of HAs was described pointing out the close relation to the amino acid metabolism through the Ehrlich pathway. The formation of amino acids was found 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 elevated yeast 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 4.8. AROMA FORMATION CHAPTER 4. RESULTS AND DISCUSSION

• Zn2+ addition ⇒ Yeast growth factor

In section 2.4.2 the formation of esters was described pointing out the formation of two diﬀerent types of ester respectively acetate and ethyl esters. These esters are formed in an enzyme catalysed condensation reaction between alcohols and acids activated in reaction with 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 related to lipid synthesis functioning as an inhibitor on ester synthesis. Furthermore, intracellular detoxiﬁcation of MCFA was proven to be a mechanism resulting in ester formation. To enhance 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 higher fermentation temperature, ii) a higher FAN concentration, iii) Zn2+ addition, iv) applying wort by topping up and v) increasing the pitching rate however vi) reducing the wort aeration seem to be the easiest process parameters to alter. Furthermore, HGB production is a powerful tool for a higher HA and ester production, however this is accompanied with

90 4.8. AROMA FORMATION CHAPTER 4. RESULTS AND DISCUSSION a higher ethanol production. Therefore, this approach would be short-termed as ethanol is the main compound to removal in AFB production. It seems more reasonable to reduce the 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 a process parameter adaptation towards a higher aroma production. Nevertheless, the lower gravity beer production could be coupled with pilsner fermentation using ale yeast at high temperatures with a tendency for higher HA and ester production along with additional process parameter adaptations. This would most likely result in a pilsner rich in HAs and esters hereby being disharmonious before membrane filtration, though more drinkable afterwards. 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 and OPEX influence of the AFB production. As brewers tend to use regular alcoholic beers already in production for AFB production the best approach would be; a higher temperature fermentation, using malt high in FAN, adding Zn2+, applying topping up of wort, a higher pitching rate and a lower aeration of the yeast coupled with RO membrane filtration.

91 Chapter 5

Future Perspectives

The conclusion reached suggest trials where the end product in focus is the alcoholic free beer (AFB) and not the regular alcoholic beer used in membrane filtration. Changing the physical 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 of obtaining a beer capable of compensating for possible membrane aroma losses. Even viewing the AFB after membrane filtration as an intermediate product has potential. Changing the physical parameters of the fermentation, as discussed in the previous section 4.8, while using the same yeast could result in a more aroma rich beer. Trials changing these parameters could be illustrated by measuring HS-GC-MS where only one parameter is changed at the time. Comparing these results would illustrate the potential and powerfulness of simple physical changes in the fermentation. Changing the composition of the initial raw materials could involve a lower gravity production 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 aroma and flavour formation could be coupled with a change in physical parameter of the fermentation. The reduced need for dia-water addition and process duration of the membrane run could result in a lower aroma and flavour loss. Ale yeast has a greater tendency to produce ester and higher alcohols compared to pilsner yeast. This process alteration is equal to ”special yeast” observed as a biological AFB process in figure 1.2. For some brewhouses changing the yeast might be the easiest process alteration. Using an identical wort would mean obtaining some of the same flavours donated from the raw material, while a different yeast might add higher concentrations of the fermentation aromas and flavours. Trials with different ale yeast on the Humlefryd pilsner wort accomplished with HS-GC-MS could illustrate the expected increase in aroma compounds. In this thesis only Humlefryd pilsner was de-alcoholised using membranes. Trials with different start beers such as belgium ales, IPA or bock beer could entail different exciting AFB products. These products would possibly not be as flavourful as the original beer but more flavourful than the AFBs currently on the market. In section 4.5 problems associated with the sorption and partitioning to the non-polar polyamide (PA) membrane were observed. Trials with different membrane layer compositions could reveal polymers with a higher selectivity for specific aroma compounds such as esters and higher alcohols (HAs). Furthermore, pre-coating of the PA membrane with more polar compounds could reduce partitioning of the non-polar aroma compounds to the membrane.

92 CHAPTER 5. FUTURE PERSPECTIVES

If the AFB after membrane filtration was viewed as an intermediate product a possible addition 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 hopping of the dia-water separately before re-dilution back to a VCF of one. Loss of colour and aroma constituents donated by the malt, such as furfural, could be added back to the beer with malt brewing colour products. If a more precise concentration loss of esters and HAs was 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 permeate using well-known techniques of ethanol removal on the permeate, such as evaporation or rectification, as illustrated in figure 1.2. Removing the ethanol gently from the permeate using this as dia-water for re-dilution back to VCF of one could alternatively entail optimal aroma composition of the beer. The optimal process for AFB production could reveal itself to be not only one method of physical or biological processes, but instead the right combination of these processes. The general understanding of AFB as the wanted end product instead of a processed regular beer production is the first milestone to be reached demanding different thinking in the traditional brewing process.

93 Chapter 6

Conclusion

The results obtained in this thesis illustrates the potential of membrane filtration as an application for alcohol free beer (AFB) production. Three different nano filtration membranes, NF, NF10 and NFHF, were tested showing the 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 osmosis membrane (RO90) tested. Applying RO90 for AFB production compared to NFHF induced a more than fivefold increase in membrane area and twice the dia-water volume, if a run time of approximately four hours was wanted. Therefore, the influence of both CAPEX and OPEX for RO90 application could result in a too costly AFB product despite a higher flavour and aroma quality. The polyamide (PA) thin film composition (TFC) of both the NF and RO membranes showed a general tendency to partition hydrophobic compounds on, or within, the membrane in degrees based on structural properties. This resulted in compounds removed from both the retentante and permeate. Less hydrophobic compounds were more clearly separated based on molecular structure with especially branched molecules involving a higher retention. Higher alcohol (HA) and ester retention in beer were clearly different when applying different RO90 and NF membranes for de-alcoholisation. RO90 membranes showed a higher HA and ester retention. On the contrary, aldehyde and hops constituents retention in beer were fairly identical when applying different RO90 and NF membranes for de-alcoholisation. A beer tasting, comparing the membrane filtered products to the original beer, illustrated a higher intensity and equality score for RO90 than NF membranes. Consequently, a high aroma quality product could be produced applying RO90 membranes, however inducing a higher CAPEX and OPEX while still experiencing some aroma losses. Alternatively, the AFB production could be reviewed changing brewing and fermentation processes compensating for the aroma losses over RO90 and NF membranes. Applying a higher fermentation temperature, higher free amino nitrogen (FAN) concentration, higher Zn2+ addition, wort topping up, increasing the pitching rate and reducing the wort aeration were found to be the easiest process alteration to obtain a higher HA and ester formation during fermentation. In addition, trials with i) ale yeast for pilsner production, ii) changing the composition of raw materials, iii) applying different membrane compositions, iv) different membrane pre-coating and v) flavour addition or recovery after membrane filtration would 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. Vertical lines 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 (Brányik 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 (Hausmann et al., 2013)...... 14 2.3 Dead-end and cross-flow filtration processes (Smith, 2013a)...... 14 2.4 Trans-membrane pressure during cross-flow filtration (Hausmann et al., 2013). 16 2.5 Difference in normal filtration processes (left) compared with diafiltration processes (right). The diafiltration liquid added (Vd) results in a lower concentration of the permeating compound in equal retentate volumes (Vr) (Hausmann et al., 2013)...... 18 2.6 The general composition of Alfa Laval RO and NF membranes (Møller, 2014) 19 2.7 Synthesis of polyamide (Tang et al., 2009) ...... 21 2.8 Ideal discontinuous diafiltration process divided into different stages. A math- ematical approximation of the membrane trends. Data used: Feed volume beer = 5 l, initial alcohol concentration = 5.5%ABV, measured flux (J) l l through the membrane = 0.519 m2∗min , flow retentate = 12.8 min , membrane permeability = 85% and membrane area = 0.072m2...... 24 2.9 Batch system outlook and corresponding feed, membrane, retentate, permeate and additional data needed for an approximated calculation of the membrane system...... 25 2.10 Aerobic and anaerobic glucose metabolism involved in the formation of flavour compounds. 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 at 15oC. (a) Present gravity (PG) = 1.000 + 0.004 ∗ [%P lato]. (c) FAN = Free Amino Nitrogen. (d) H. alcohols = Higher alcohols (HA) and VDK = Total vicinal diketones (Briggs et al., 2004)...... 32 2.12 Formation of HA by anabolic routes from pyruvate and catabolic routes from assimilated 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 Maillard product furfural through cascades of reactions (Baert et al., 2012)...... 41 2.15 Strecker aldehyde formation from transamination between a amino acid and a α-dicabonyl (Baert et al., 2012)...... 42 2.16 Formation of benzaldehyde from phenylalanine, (Baert et al., 2012) ...... 43 2.17 Chromatographic separation technique (Moldoveanu and David, 2013). . . . . 47 2.18 Packed and capillary GC columns (Mcnair and Miller, 2009)...... 49 2.19 Static headspace sampling technique (Wang et al., 2008)...... 49 2.20 Dynamic headspace sampling technique (Wang et al., 2008)...... 50 2.21 Refractive index detection technique (Moldoveanu and David, 2013)...... 51 2.22 Electron ionisation quadrupole mass spectrometer (Laboratory, 2012). . . . . 51

3.1 Labstak M20-0.72 Alfa Laval unit...... 53 3.2 Sketch of the experimental set-up with modifications to the Labstak M20-0.72 Alfa Laval labunit...... 54 3.3 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. PC1 describes 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. PC1 describes 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 = Permeability and PC = Principal component...... 63 4.3 Principal component analysis (PCA) on NFHF (NF99HF) data given by Alfa Laval. 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. 64 4.4 Mass trend for membrane run with RO90 and NFHF membranes, see table 4.1 for physical parameters ...... 67 4.5 Flux behaviour for reverse osmosis membrane runs, see table 4.1 for physical parameters...... 68 4.6 Flux behaviour for nano filtration membrane runs, see table 4.1 for physical parameters...... 69 4.7 Linear plot of permeate water flux as a function of pressure difference from equation 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 table 4.1 for physical parameters...... 71 4.9 Higher alcohol aroma compounds area percentages compared to the original beer 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 LIST OF FIGURES LIST OF FIGURES

4.10 Higher alcohol aroma compounds area percentages compared to the original beer in permeate tank (PT) after ended membrane filtration measured by HS- GC-MS. Column legend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compound name (retension time) - associated aromas...... 76 4.11 Ester aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas. . 78 4.12 Ester aroma compounds area percentages compared to the original beer in permeate tank (PT) after ended membrane filtration measured by HS-GC- MS. Column legend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compound name (retention time) - associated aromas. 79 4.13 Aldehyde aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas. 82 4.14 Aldehyde aroma compounds area percentages compared to the original beer in permeate tank after ended membrane filtration (PT) measured by HS-GC- MS. Column legend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compound name (retension time) - associated aromas. 83 4.15 Hops aroma compounds area percentages compared to the original beer in feed 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: Compound name (retension time) - associated aromas. . 84 4.16 Hops aroma compounds area percentages compared to the original beer in permeate tank after ended membrane filtration (PT) measured by HS-GC- MS. Column legend: Membrane name, nr. run, location of sample, stage in process. X-aksis legend: Compound name (retension time) - associated aromas. 85 4.17 Tasting results from a blindtest where the beer was compared to an original Humlefryd beer (HFORG24). Scores from 0-10 given by a skilled taste panel of seven...... 87

A.1 Matlab worksheet for the calculation of membrane processes...... 108 A.2 Formation of hydro peroxide fatty acids from triacylglycerol, (Baert et al., 2012)...... 109 A.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 (Garc´ıaet al., 1994), (Haukeli and Lie, 1978) ...... 111 A.4 Biosynthesis of sulphur containing amino acids. Cysteine and inorganic sulphur assimilation for the anabolism of sulphur containing amino acids. NADPH = Nicotinamide adenine dinucleotide phosphate (Walker, 1998), (Landaud et al., 2008) ...... 115 A.5 Formation routes of dimethyl sulphide, (Briggs et al., 2004) ...... 116 A.6 Formation of Mercaptans in beer, (Vermeulen et al., 2006), (Swiegers and Pretorius, 2007) ...... 117

97 LIST OF FIGURES LIST OF FIGURES

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)...... 123 A.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-aksis ledgend: Compound name (Retension time) - Associated flavours...... 124 A.11 Ester 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-aksis ledgend: Compound name (Retension time) - Associated flavours...... 124 A.12 Aldehyde 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-aksis ledgend: Compound name (Retension time) - Associated flavours...... 125 A.13 Hop aroma compounds area percentages compared to the mean original beer measured 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...... 126 A.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 integral area situated at the ethanol peak...... 127 A.16 Head space sampling set-up. Trapping the aroma in on Tenax-TA traps at 37oC...... 128 A.17 Aroma compounds measured on the GC-MS for original Humlefyrd bottled the 24th of April...... 129 A.18 Dealcoholisation membrane data analysis by Alfa Laval using an HPLC-MS where 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 reduction of ”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.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) ...... 20 2.3 Energy formation for brewers yeast in aerobic and anaerobic conditions (Walker, 1998)...... 30 2.4 Higher alcohols in beer, aroma threshold and corresponding concentration in lager beer (EBC, 2000), (Tan and Siebert, 2004), (Fenaroli, 2005)...... 32 2.5 Amino acids and corresponding alcohols as a result of Ehrlich pathway (Hazel- wood et al., 2008), (EBC, 2000)...... 34 2.6 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 lager beer (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...... 57 3.3 Sampling plan during membrane filtration runs ...... 58 3.4 Overview of equipment settings and parameters for GC-MS analysis . . . . . 60

4.1 Membrane parameters and constants for each membrane run. Highlighted number and membrane runs are not considered in the standard deviation (SD) calculation ...... 66 4.2 Ethanol retention, permeability and solute transport coefficient for various membrane 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-concentration of 2 VCF and a final concentration of 1 VCF resulting in 5 litre of beer with kg 0.5%ABV within a 4 hour operation. A constant flow rate 14 min was used in the calculation...... 73 4.4 Physiochemical data of higher alcohols (Fenaroli, 2005), (Alvarez et al., 1998). 76 4.5 Physiochemical data of esters (López et al., 2002), (Fenaroli, 2005), (Alvarez et al., 1998)...... 79 4.6 Physiochemical data for aldehydes (Fenaroli, 2005)...... 83 4.7 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 tasting results plus the desired optimisation development. All values are mean analytical values of numerous membrane runs. Flux was measured by mass, ethanol by HPLC and aroma compounds by HS-GC-MS. ∗Mean value of percentage esters retained minus benzene ethanol (273%)...... 88

A.1 Ketones in beer, flavour threshold and corresponding concentration in lager beer (EBC, 2000), (Tan and Siebert, 2004)...... 110 A.2 Sulphur compounds in beer, flavour threshold and corresponding concentration 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 concentration in lager beer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989)...... 122

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105 Appendix A

Appendices

A.1 Glossary

Aroma - Sense of smell nasal or retro-nasal. a filter medium while the liquids passes. Absorption - Take-up or assimilation of a FAN - Free amino nitrogen. Individual compound. amino acids and small peptides present in Adsorption - Adhesion or partitioning of a the wort. compound to a surface. Flavour - The total experience of the taste, Centrifugation - Separation of particles aroma and mouth feeling. and liquids caused by the centrifugal force Fermentation - Energy production by making the more dense particles and liquids organisms in the absence of oxygen caused migrate away from the axis during rotary by substrate-level phosphorylation. acceleration. Flocculation - A reversible process of Clarification - The process of making yeast adhering to each other to form something transparent and/or clear. aggregates resulting in easier sedimentation. Cold break - Proteins, polyphenols and Flow - Fluid flow is the mass flow rate of a carbohydrates forming hydrogen bonds and liquid transported within pipelines or over a kg hydrophobic cluster in the beer promoted surfaces such as membranes [ min ]. by cooling. Flux - Fluid flux is the mass flow rate of a Cropping - The process of removing the liquid transported through a given area kg yeast from the fermentation for additional surface such as membranes [ m2∗min ]. fermentation in another fermenter or Lectin - Carbohydrate binding protein. discharge. Sometimes called yeast Mash - Milled grains, mainly barley, mixed harvesting if reused. with water. Diafiltration - Addition of a solvent to the Mashing - The process of degrading the retentate to enable a higher permeation of mash component into usable constituents by permeable solutes through the membrane. optimising gain enzyme processes. Ethereal oils - Volatile oils from plants. Mouth feel - A products physical and Evaporation - Vaporisation of a liquid into chemical interaction in the mouth. a gaseous phase which is not saturated with Partitioning -”The distribution of a the evaporated substance. solute between two immiscible or slightly Filtration - Separation of solid particles miscible solvents in contact with one from liquids by retention of the particles on another, in accordance with its differing 1http://www.oxforddictionaries.com/definition/english/partition

106 A.1. GLOSSARY APPENDIX A. APPENDICES solubility in each”1. retain a specific compound on the retentate Pitching - Addition of yeast to wort hereby side of the membrane [%]. initiating the fermentation. Sedimentation - Sinking or precipitation Propagation - Continuous multiplication of particles with a higher density than the or re-production of an organism. surrounding medium. Rectification - Purification of a liquid by Sorption - The physical or chemical ability distillation. for one substance or compound to become Permeability - The ability of a specific attached to another. compounds to permeate a membrane [%]. Resulting in a flux through the membrane Taste - What you senses inside your into the permeate. mouth. Bitter, sweet, sour, salt and Umami. Permeate - The liquid permeating the Wort - The liquid obtained after removal of membrane. the solids from the mash containing Rejection - The ability of a membrane to fermentable sugars and nutrients for the reject a specific compound hereby causing yeast. the compounds to stay on the retentate side Yeast strain - Brewers yeast belong to the of the membrane [%]. Genus of Saccharomyces and Species of Retentate - The liquid retained by the cerevisae and pastorianus. Different brewers membrane. yeast sub-species can be divided into Retention - The ability of a membrane to different 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 storage lipid in barley accounting for 60-70% of the overall lipid mass in barley. During malting and mashing membrane bound Lipases will have optimum activity hereby hydrolysing triacylglycerol into glycerol and the corresponding fatty acids, see ﬁgure A.2. These fatty acids can subsequently be oxidized into hydro peroxide fatty acids. An additional pathway is an oxidation of the triacylglycerol resulting in Lipid hydro peroxides which then eventually can be hydrolysed by lipase into hydro peroxide fatty acids. The hydro peroxide fatty acids can 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. The presents of oxygen is common for all these reactions to be possible (Vesely et al., 2003), (Baert et al., 2012).

109 A.4. KETONES APPENDIX A. APPENDICES

A.4 Ketones

The most flavourful ketones present in beer are vicinal diketones (VDK’s). Diacetyl and pentane-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 compounds a greater concern for the brewer with flavour flavours associated with butter, toffee-like or honey-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 Concentration Name Structure Threshold or Range mg mg [ l ] Taste [ l ]

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

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

VDK’s are by-products of the amino acids biosynthesis of valine and isoleucine, see Figure A.3. Diacetyl is a by-product of valine anabolism, while pentane-2,3-dione is a by-product of the isoleucine anabolism. All amino acids are essential during the vigorous growth at the beginning of fermentation. Valine and isoleucine are not assimilated by the yeast in the 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 is easily assimilated in the beginning of fermentation (Kunze, 2010), (EBC, 2000).

110 A.4. KETONES APPENDIX A. APPENDICES

Figure A.3: Formation of Diacetyl and 2,3-Pentanedione involved in the Valine and Isoleucine anabolism. TPP = Thiamine pyrophosphate cofactor, NADPH = Nicotinamide adenine dinucleotide phosphate (Garc´ıaet al., 1994), (Haukeli and Lie, 1978)

α-acetolactate (AAL) and α-acetohydroxybutyrate (AAHB) are two intermediate acids in the pathway of respectively valine and isoleucine anabolism. AAL and AAHB are excreted from the yeast cell into the surrounding media. In the beer AAL and AAHB can be oxidatively 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 be converted to diacetyl and pentane-2,3-dione by oxidation. Diacetyl and pentane-2,3-dione will 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 (Haukeli and Lie, 1978), (Garc´ıaet al., 1994). In fermentation the yeasts ability to remove diacetyl is 10 times greater than the rate

111 A.4. KETONES APPENDIX A. APPENDICES of formation. This ability will decrease during secondary fermentation or maturation. The speed of these non-enzymatic oxidation reactions to form VDK’s are temperature, substrate concentration and pH dependent. In addition, the reaction is also dependent on the present of oxygen and oxidative metal ions (Garc´ıaet al., 1994). Factors inducing the rate of the non-enzymatic oxidative decarboxylation to form VDK are considered factors lowering the ﬁnal VDK in beer. The reason for this being the possibility of reacting all present AAL and AAHB in the media while the yeast are still fermentation hereby taking up the VDK rapidly and transforming them into the corresponding ﬂavourless diols. Removing the VDK precursors will hereby ensure not later VDK formation which cannot be removed when the yeast is no longer vital (Garc´ıaet al., 1994).

Factors inﬂuencing VDK concentration in ﬁnal beer

• Fermentation temperature, (Garc´ıaet al., 1994) Higher VDK formation rate during high fermentation temperatures

• Maturation temperature, (Garc´ıaet al., 1994) Higher take-up rate during higher maturation temperatures

• pH, (Garc´ıaet 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, (Garc´ıaet 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), (Godtfredsen and Ottesen, 1982) ALDC catalyses the removal of AAL directly to acetoin Not detected in brewers yeast

112 A.4. KETONES APPENDIX A. APPENDICES

• 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 concentration in lager beer (EBC, 2000), (Tan and Siebert, 2004), (Landaud et al., 2008), (Briggs et al., 2004). Compound Compound Flavor Aroma Concentration Name Structure Threshold or Range µg µg [ l ] Taste [ l ]

Hydrogen sulphide 8 Rotten eggs 1-200 Sulphidic

Sulphur dioxide 10000 Pungent 200-20000 Burnt matches Dimethyl sulphide 30 Sweet corn 10-150 DMS Cooked vegetable, Tomato sauce

Mercaptans Drain

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

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

The sources of sulphur in brewers yeast are respectively inorganic sulphate or organic proteins, amino acids or vitamins containing sulphur. Sulphur is not only essential in amino acids 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 cysteine and methionine, see ﬁgure A.4. Yeast prefers assimilation of organic sulphur compounds mainly sulphur containing amino acids. However, if no free sulphur containing amino acids are available then yeast are able to assimilate inorganic sulphate for the anabolism of the essential sulphur containing amino acids (Walker, 1998), (Landaud et al., 2008).

114 A.5. SULPHUR COMPOUNDS APPENDIX A. APPENDICES

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

Factors inﬂuencing hydrogen sulphide concentration in ﬁnal beer, (Briggs et al., 2004), (EBC, 2000) • Induced formation with induced yeast growth • Induced formation with high cysteine concentration • Induced formation with high sulphate concentration

115 A.5. SULPHUR COMPOUNDS APPENDIX A. APPENDICES

• 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 2– Reacts with oxygen to form SO3 • 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 and brewing 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 and hereafter evaporated because of high volatility. The DMSO formed are on the contrary less volatile hereby mostly retained in the malt. Some yeast strains, wild yeast and bacteria are able 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)

116 A.5. SULPHUR COMPOUNDS APPENDIX A. APPENDICES

• 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 solution and yeast metabolites such as methanol, ethanol, higher alcohols and acetaldehyde. In ﬁgure A.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 and Pretorius, 2007)

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

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

• 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 from hops. Hops are added to the wort boil mainly to enhance the aroma profile. β- and α-acids are hops components adding bitterness to the beer after isomerations into the corresponding iso-β- and iso-α-acids. For a more detailed description of additional hops components see section 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 result in a Norrish cleavage reaction in one of the carbon-carbon bonds situated next to carbonyl in 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 A.5. SULPHUR COMPOUNDS APPENDIX A. APPENDICES compound A and D in ﬁgure A.7. Moreover, the formation of a corresponding acyl radicals will 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 or secondary from decarbonylation of the formed 4-methyl pent-3-enal acyl radical. Radical A can react directly with hydrogen sulphide radicals which originate from free hydrogen sulphide or scavenged from a thiol group from sulphur amino acids or proteins. Finally, this result 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 contamination. However, the majority is produced during fermentation inducing a decrease in pH as observed in figure 2.11. Many organic acids are secondary metabolites excreted during rapid yeast growth and some re-assimilated later in the fermentation. In table A.3 some of the most 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). Compound Compound Flavor Aroma Concentration Name Structure Threshold or Range mg mg [ l ] Taste [ l ]

Acetic acid 130 Vinegar 30-200 Acidic

Lactic acid 400 Milky 10-1362 Sour ﬂavour

Butyric acid 2-3 Rancid 0.5-1.5 Baby sick Sour milk

Citric acid 170 Lemon 90-300 Acidic Sour

Fatty acid Soapy Fatty Octanoic acid R = (CH2)6CH3 13 3-10 Decanoic 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 cycle during anaerobic growth of brewing yeast, see ﬁgure A.8. Moreover, organic acids can be formed from amino acids where the yeast have removed the amino group to form α-keto acids for later amino acid synthesis within the cell, as observed in ﬁgure 2.12, (EBC, 2000).

119 A.6. ACIDS APPENDIX A. APPENDICES

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 reaction of 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 to microbial growth. Furthermore, lactic acid can be formed directly by reduction of Pyruvate yielding NAD+ (Klopper et al., 1986), (Briggs et al., 2004). Butyric acid is mainly caused by bacterial contamination (Klopper et al., 1986), (Briggs et al., 2004)). Citric acid can be formed directly by protonation of citrate an intermediate in the TCA cycle (Klopper et al., 1986), (Briggs et al., 2004). Fatty acids in beer are either liberated from triglyceride in the wort by lipolytic enzymes

120 A.6. ACIDS APPENDIX A. APPENDICES 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), (Briggs et 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 in lager beer (EBC, 2000), (Caballero et al., 2012), (Briggs et al., 2004), (Irwin, 1989). Compound Compound Flavor Aroma Conc. Name Structure Threshold or Range mg mg [ l ] Taste [ l ]

Iso-α-acid 5 Bitter 10-100

Humulone R−CH2CH(CH3)2 Cohumulone R−CH(CH3)2 Adhumulone R−CH(CH3)CH2CH3

Iso-β-acid - Bitter -

Lupulone R−CH2CH(CH3)2 Colupulone R−CH(CH3)2 Adlupulone R−CH(CH3)CH2CH3

Soft resins namely α and β-acids are responsible for the bittering effect of hops. α and β-acids are nearly insoluble in beer leaving only traces of α-acids within the beer after hops addition. Therefore, hops boiling is commenced for the purpose of isomerizing the different α and β-acids into iso-acids as observed in table A.4. The isomerization reaction observed in figure A.9 is induced by heat, low pH, high gravity and a high Mg2+ concentration resulting in two diastereoisomers differing from each other as cis/trans isomers. This isomerisation 68 cis reaction is a acyloin-type ring contraction. Generally, a 32 ratio between trans 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, which is therefore not considered in table A.4 in relation to flavour threshold and concentration range (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 mean original 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 (Retension time) - Associated ﬂavours.

Figure A.11: Ester 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-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 original 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 (Retension time) - Associated ﬂavours.

Figure A.13: Hop 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-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 ﬁltration was initiated. Retention time in minutes, at the top of the peak, and integral area situated at 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 at 37oC.

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.

Figure A.17: Aroma compounds measured on the GC-MS for original Humlefyrd bottled the 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-MS where 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 reduction of ”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 SAMPLESAPPENDIXA. APPENDICES

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

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

131