i
  
COMMISSIONING AND OPTIMISATION OF WITS MICRO?BREWERY 
PLANT 
 
 
 
 
 
Ezekiel Makwadinkga Madigoe 
 
 
 
A dissertation submitted to the Faculty of Engineering and the Built Environment, 
University of the Witwatersrand, Johannesburg, in fulfilment of the requirements 
for the degree of Masters of Science in Engineering. 
 
 
Johannesburg, 2009 
 ii
 DECLARATION 
 
 
I declare that this dissertation is my own unaided work. It is being submitted to 
the degree of Masters of Science to the University of the Witwatersrand, 
Johannesburg. It has not been submitted before for any degree or examination to 
any other University. 
 
 
Ezekiel M. Madigoe 
14 June 2009 
 iii
 ABSTRACT 
 
 
The Brewing Industry is facing increasingly challenging times due to global 
competitive business environment and rising costs of raw materials. These factors 
consequently affect the economy of the industry. An effective solution to stay 
competitive is to enhance brewing process efficiency by optimisation of process 
units. The aim of the study was therefore to investigate optimal operating 
conditions that can be implemented in the brewing industry using the Micro-
 Brewery Plant at the University of the Witwatersrand. It was discovered that the 
longer the mashing route, the higher the yield of fermentable sugars. The mash 
regime and liquor to malt ratios were optimised and the carbohydrates obtained 
were analysed at 3.5 L of water per kg of malt and a total mash time of 105 min. 
Carbohydrate and phenolic acid analyses were performed by High Performance 
Liquid Chromatography (HPLC) coupled with a UV-vis detector. The three 
different malts used had a phenolic acid content of 33.25?g/mL, 25.44?g/mL and 
19.98?g/mL for malt A, B and C, respectively. The plot of 1/T versus Ln(t) gave a 
negative slope with activation energy (Ea) = 209 KJ/mol, rate constant (k) = 4.6 x 
10-4 mg/L min, which are comparable to similar data reported in the literature. The 
kinetics studies showed that the optimised mashing temperature of 900C was 
adequate to form 4-Vinylguaiacol by thermal decarboxylation from the 
hydroxycinnamic acids. This study has shown that there is no direct correlation 
between phenolic acids and oxidative flavour stability of beer while the 
corresponding volatile phenols may affect beer flavour. The study showed that 
fermentation rates increase with temperature. Investigated pitching rates showed 
 iv
 insignificant effect on fermentation rates. The study showed that added finings 
during boiling improve beer clarity and have an insignificant effect on 
fermentation rates. Analysis of commissioning strategies at Wits micro-brewery 
plant showed that verbal instruction is always open to error, while the written 
instruction or report is generally not open to error. Hence the commissioning 
documents have been developed in the study, which has helped in reduction of 
errors during brewing. It is therefore concluded that good record keeping and 
documentation is of essential need for successful commissioning of a brewery. 
 v
 DEDICATION 
 
 
In the memory of my late grandfathers Ezekiel Nkosi and Johannes Madigoe, my 
late aunt Anna Madigoe, my late friends Dickson Mapholo and Rangwedi 
Kekana. 
 vi
 ACKNOWLEDGEMENTS 
 
 
I am grateful to Professor Sunny E. Iyuke for dedicating most of his time and 
affording me an opportunity into turning this work into a successful Masters 
research. I am thankful for his guidance and encouragement to develop me into an 
independent researcher. I would also like to thank everyone at Biochemical 
Engineering and Nano-Technology Research Group.   
I would also like to thank John Cluett, former Chairperson of IBD Africa Section 
for his moral, technical support and for giving us opportunity to work with 
industry experts. Support of IBD Africa Section is highly appreciated. I 
acknowledge supply of raw material and sample analysis from SABMiller Alrode, 
South Africa. 
 
Special thank to my mother, father, sisters and brother for their consistent support 
throughout this work and for believing in me. 
 
I would like to acknowledge the financial support of Sasol, National Research 
Fund and Wits University. 
 vii
 TABLE OF CONTENTS 
 
 
 
LIST OF TABLES XIV 
LIST OF TABLES XIV 
CHAPTER ONE 1 
1. INTRODUCTION 1 
1.1 Background and motivation 1 
1.2 Research problem and questions 2 
1.3 Hypothesis 2 
1.4 Justification of the study 2 
1.5 Scope of the project 3 
1.6 Purpose and aims 3 
1.7 Expected contribution to knowledge 4 
1.8 Dissertation outline 4 
CHAPTER TWO 6 
2. LITERATURE REVIEW 6 
 viii
 2.1 Background knowledge on the brewing industry. 6 
2.1.1 History of beer 6 
2.2 Brewing Process 8 
2.3 Beer quality 12 
2.4 Properties of beer 14 
2.4.1 Phenolic acids in beer 14 
2.4.2 Physical properties of organic acids 19 
2.4.3 Chemical properties of organic acids 19 
2.4.4 Physical properties of phenolic acids 20 
2.4.5 Chemical properties of phenolic acids 20 
2.5 Characteristics of Beer Quality 20 
2.5.1 Flavour 21 
2.5.2 Appearance 22 
2.5.3 Wholesomeness 24 
2.6 Review on previous work and available literature 24 
2.6.1 Mashing process 24 
2.6.2 Lautering system 25 
2.6.3 Fermentation process 28 
2.6.4 Fermentation control parameters. 28 
2.6.5 Control of inputs 29 
2.6.6 Filtration in beer processing 30 
2.6.7 Beer Haze/ phenols 31 
 ix
 2.6.8 Primary beer raw material- barley 32 
2.6.9 Clarification by finings 32 
2.6.10 Wort boiling 33 
2.6.11 Sedimentation 39 
2.6.12 Beer spoilage organisms 41 
2.6.13 Process commissioning 44 
CHAPTER THREE 45 
3. EXPERIMETAL 45 
3.1 Materials and Method 45 
3.2 Procedure 45 
CHAPTER FOUR 52 
4. RESULTS AND DISCUSSION 52 
4.1. Optimisation of wort production 52 
4.1.1 Water to malted barley ratios 52 
4.1.2. Different mashing rules 55 
4.2. Beer quality Analysis 57 
4.2.1 Determination of phenolic acids in wort from different malts 57 
4.2.2 Hydroxyl-free radical in different malts 59 
4.2.3 Effect of phenolic acids on beer appearance (colour, clarity, beading and 
foam) 65 
 x
 4.2.4 Decarboxylation of FA during mashing 66 
4.2.5 Effect of fermentation temperature on fermentation rate 67 
4.2.6 Effect of kettle finings on fermentation rate 69 
4.2.7 Optimisation of fermentation by varying pitching rates 70 
4.2.8 Clarification of beer by finings (Kalaginess) 72 
CHAPTER FIVE 73 
5. WITS MICRO-BREWERY COMMISSIONING 73 
5.1 Commissioning Checklist 73 
5.2 Preparation for commissioning 75 
5.4 Brew Sheet 81 
5.5 Fermentation Progress 83 
CHAPTER SIX 84 
6. CONCLUSION AND RECOMMENDATIONS 84 
REFERENCES 87 
CHAPTER SEVEN 94 
7. PUBLICATIONS AND PRESENTATIONS DURING THE 
INVESTIGATION. 94 
7.1 Publications 94 
 xi
 7.2 Presentations 94 
APPENDIX A: SPECIMEN LIST OF SYMBOLS 95 
 xii
 LIST OF FIGURES 
 
 
Figure 2. 1: Flow process diagram for beer production. 11 
Figure 2. 2: Chemical structures of phenolic acids [12]. 14 
Figure 2. 3: Chemical structures of organic (carboxylic acid) and phenolic acids 15 
Figure 2. 4: Decarboxylation of selected phenolic acids and their reduction and 
oxidation products [21, 22]. 17 
Figure 2. 5: Isomerisation of ?-acids 37 
Figure 2. 6: Binding together of small particles by flocculants. 41 
Figure 3. 1:  Experimental set up- (a) schematic of the mashing process flow. (b) 
commissioning of the wits mini brewery plant [39]. 47 
Figure 3. 2:  HPLC chromatograms of the standard mixture of phenolic acids.1, 
gallic acid; 2, p-hydrobenzoic acid; 3, vanillic acid; 4, caffeic,; 5, syringic; 6, 
p-coumaric acid; 7, ferulic acid; 8, m-coumaric acid; 9, isoferulic acid; 10, 
sinapic acid; 11,o-coumaric acid. 50 
 
Figure 4. 1: HPLC chromatogram obtained for carbohydrate analysis; 1- glucose; 
2-fructose; 3-maltose; 4-maltotriose. 53 
Figure 4. 2: Carbohydrate concentration obtained by varying water (litre) to barley 
(kg) ratio. 54 
Figure 4. 3: Various mashing routes investigated. 55 
Figure4.4:The effect of mashing routes on fermentable carbohydrates extraction 56 
Figure 4. 5: Comparison of individual phenolic acid from different malts. 58 
Figure 4. 6:  EPR response of the sample as a function of magnetic field. 60 
 xiii
 Figure 4. 7: The peak-to-peak amplitude of the derivative curve (?m) as a function 
of microwave power. 62 
Figure 4. 8: The linewidth (?b) as a function of microwave power. 63 
Figure 4. 9: Generated OH-radical from three different wort samples as detected 
by the ESR. 64 
Figure 4. 10: Beer samples produced from the researched malts by SAB [39]. 65 
Figure 4. 11: Effect of thermal load during mashing and wort heating on fa 
degradation and 4vg formation. 67 
Figure 4. 12: Change in fermentation rate with fermentation temperature. 68 
Figure 4. 13: Effect of kettle finings on fermentation rate. 69 
Figure 4. 14: Fermentation rates at different pitching rates (a) at temperature 25oC 
and (b) at temperature 16oC. pitching rates used are (a, d) 1x106 cells/ml, (b, 
e) 2x106 cells/ml, (c, f) 3x106 cells/ml. 71 
 Figure 4. 15: Effect of added finings during wort boiling on final beer colour. 72 
 xiv
 LIST OF TABLES 
 
 
Table 2. 1:Optimum conditions for starch hydrolysis enzymes 25 
Table 2. 2: Typical concentrations and size of particulate species. 41 
Table3. 1: mashing routes used in the study. 48 
 
Table 4. 1: Concentrations of various phenolic acids from malt a, b and c, 
respectively. 58 
 
 
 
 
 
 
 
 
 
 
 1
 CHAPTER ONE 
1. INTRODUCTION 
1.1 Background and motivation 
The Brewing Industry is facing increasingly challenging times due to global 
competitive business environment and the costs of feed materials are continuously 
rising, [1] and these factors consequently affect the economy of the industry. An 
effective solution to stay competitive is to enhance brewing process efficiency by 
optimisation of process units. Optimisation can be used to achieve increased 
production yield and reduction in production cost, but these must be done without 
affecting the quality of the beer. Particularly in South Africa with recent power 
crisis, rising energy prices and increasing stringent environmental regulations, 
these call for opportunities to process modifications and efficient operating 
conditions. The project was motivated by problems that were being encountered at 
Wits Micro-brewery plant which are of similar interest and their solution could be 
of much interest to the commercial brewing process. Namely the problems were: 
 
? Poor extraction during mashing resulting in poor usage/ waste of raw 
material. 
? Poor fermentation performance. 
? Unsatisfactory beer quality and shelf-life. 
? Non-availability of commissioning documentation and/or standard 
procedures to carry out a successful brewing process for inexperienced 
brewers. 
 2
 The research aims to provide optimal operating conditions and commissioning 
documentation for the Wits microbrewery plant and investigate the effect of 
phenols on the shelf-life of beer. 
1.2 Research problem and questions 
The research aims to enhance brewing process performance through optimization 
of the brewing units and to study commissioning skills. Thus the research intends 
to answer the following questions: 
? What effect does the mashing temperature profile have on the extraction 
yield of fermentable carbohydrates? 
? What is the optimal malt to liquor for possible high yield of fermentable 
carbohydrates and which factors affect the ratio? 
? Can reduction of phenolic acids in beer increase its shelf life? 
? Which aspects of commissioning are important to minimize material and 
energy waste? 
 
1.3 Hypothesis 
Optimisation of process units can lead to increased process yields, and 
consequently lower waste products.  
 
1.4 Justification of the study 
Several research studies have been reported on brewing process, but not on 
commissioning of micro-breweries. Apart from reports on increasing cost of raw 
materials and running costs of brewing, little work has been reported on the 
optimisation of brew process. The problems that were encountered at Wits micro-
 brewery, which are of similar interest to large production scale were due to non-
 3
 availability of literature on commissioning documentation and documents and 
unsatisfactory performance of the brew process units. This research is expected to 
bring about significant economical and environmental benefit to brewing industry 
because of its cost effectiveness methods and optimal performance of process 
units. Furthermore, commissioning documents to be developed are of great value, 
as they will be essential tools to break the complexity of commissioning into a 
simpler manageable process. 
 
1.5 Scope of the project 
In order to establish an optimised brew process, process units with unsatisfactory 
performance were identified, namely, mashing, boiling and fermentation. Optimal 
operating conditions of these process units will be empirically identified. The 
study will also investigate the effect of these optimal conditions on beer quality. 
After a number of operational conditions have been optimised, proper 
documentation for commissioning of the Wits micro-brewery will be developed.  
1.6 Purpose and aims 
The main purpose of this research is to develop optimum operating conditions and 
develop commissioning documentation for the Micro-Brewery Plant at the 
University of the Witwatersrand through the following objectives: 
 
? Investigate the effect of different mashing temperature profiles on 
extraction of fermentable carbohydrates during mashing. 
? Locate optimum liquor to malt ratio for mashing. 
 4
 ? Investigate the role of phenolic acids on beer quality. 
? Develop commissioning documentation to reduce losses and waste during 
brewing. 
 
1.7 Expected contribution to knowledge 
This work which is aimed at optimizing operating conditions and developing 
commissioning documents for Wits micro-brewery is expected to provide: 
? Information on the effect of mashing, boiling and fermentation parameters 
on beer quality. 
? Proper commissioning documents with structured checklist to reduce the 
risk of omitting important points during commissioning. 
? Information on the role phenolics play in beer quality. 
1.8 Dissertation outline 
Chapter 1 
This chapter discusses the background knowledge and motivation of this study, 
justification of the study, scope of the project, research problem and questions, 
purpose and aims and the expected contribution to knowledge. 
 
Chapter 2 
Literature is reviewed to provide background knowledge on the brewing industry. 
The brewing process is highlighted with detailed information on process units that 
are relevant to the focus of this dissertation. Emphasis is placed on describing key 
 5
 points when describing beer quality and its characteristics. The chapter also 
describes the properties of beer. 
 
Chapter 3 
This chapter explains the experimental procedure of malt crushing, mashing 
routes, boiling as well as analytical procedures. 
 
Chapter 4 
This chapter discusses all experimental results obtained in the optimization of 
Wits micro-brewery. 
 
Chapter 5 
This chapter presents the developed commissioning documents. 
 
Chapter 6 
 This chapter presents the conclusions and recommendations of the dissertation. 
 
Chapter 7 
This chapter list publications that were relevant in the investigation. 
 
 6
 CHAPTER TWO 
2. LITERATURE REVIEW 
2.1 Background knowledge on the brewing industry. 
2.1.1 History of beer 
There is a lot of literature on the history of beer and all the cited literatures agree 
with one another. This section summarises beer history from one of the authors 
[2]
 .Beer is the world?s oldest alcoholic beverage produced from fermentable sugars 
obtained from barley. The oldest proven records that are 6000 years old refer to 
the Sumerians as the first people to start brewing. Sumeria was between the Tigris 
and Euphrates rivers including Mesopotamia and the ancient cities of Babylon and 
Ur. Sumerians discovered brewing by chance and it is thought that a piece of 
bread became wet and the bread began to ferment after a short time. 
 
 The Sumerian empire collapsed during the 2nd millennium and the Babylonians 
became rulers of Mesopotamia. This resulted in the Babylonians mastering the art 
of brewing beer because they derived their culture from the Sumerians. The 
Babylonians started distributing and exporting their beer to Egypt where the 
Egyptians continued with the beer brewing tradition. Egyptians are the first people 
to start using unbaked bread dough for brewing beer. The Grecian and Roman 
empires continued brewing beer where some Romans considered beer as a 
barbarian drink.  
 
 7
 Beer in ancient times was cloudy, produced almost no foam and early civilizations 
found its mood-altering properties supernatural and divine. People sacrificed beer 
to gods and believed beer contained a spirit or a god since drinking it possessed 
the spirit of the drinker, but ancient Germans brewed beer for their own 
enjoyment. Beer played a role in people?s daily lives and was considered a 
valuable foodstuff where workers were often paid with jugs of beer. Beer that was 
brewed before the industrial revolution was sold on a domestic scale although the 
European monasteries produced and sold it by the 7th century.  
 
The baking of bread and brewing was done by women in the first centuries until 
the middle ages. Beer production moved from artisan manufacture to industrial 
scale by the end of the 19th century. From 1000 AD hops were introduced in the 
brewing process and beer making was firmly established as a commercial 
enterprise in Germany, Austria and England. The discovery and development of 
equipment like thermometers and hydrometers changed the way brewing was 
done because it gave the brewers more control over the process. Industrialization 
and the introduction of James Watt?s steam engine invaded brewing where 
breweries using steam power called themselves Steam Beer Breweries. The two 
important inventions that revolutionized beer brewing were James Watt?s steam 
engine and Carl Linde?s refrigeration discovery. It was scientifically proven that 
the brewing of beer required certain temperatures which resulted in brewing 
mostly done in winter because of required low temperatures. The refrigeration 
equipment made beer brewing to be seasonally independent. Most scientific 
research on beer production occurred in the 19th century by people like Louis 
 8
 Pasteur who researched on micro organisms encountered in brewing.  Christian 
Hansen isolated a single yeast cell and induced it to reproduce on an artificial 
culture medium. This resulted in the development of yeast methods that improved 
fermenting processes and beer taste. The brewing industry is presently a huge 
global business with several multinational companies and many smaller producers 
ranging from brewpubs to regional breweries.  
 
2.2 Brewing Process 
Beer production takes place according to the following stages. 
? Malting: After harvest, the barley kernels are allowed to start germinating 
and halted by kiln drying them (now called malted barley). This is the 
process where starch is converted into fermentable sugars and unlocks the 
starches hidden in the barley. The grain and water are initially added to 
storage bin and allowed to soak for plus/minus 40 hours. The grain is 
spread on the germination room to allow rootlets to begin to form. The 
starches within the grain break down into shorter lengths and now the 
grain is called the green malt. Kilning is then used to halt germination 
process. This is achieved by drying the green malt through high 
temperature in the kiln. Temperature increase should take place gradually 
to avoid damage of the grain enzymes. The finished product is now called 
the malt. Different types of malts can be achieved by drying malts at high 
temperatures (pale malt), kilning to a slightly higher temperature (mild 
malt), and using highest temperatures to very flavourful and aromatic 
malts. 
 9
 ? Milling: The malted barley is milled to expose the inner starch endosperm 
to form grits. Milling the malted barley allows it to absorb water that it 
will eventually be mixed with. The milled malt is now called the ?grist?. 
Consideration is given to milling and transporting grist so as not to 
fragment the husks which cause astringent flavours and stability problems. 
? Mashing: Hot brewing water (90oC) is mixed with the grist in the hot 
liquor tank (HLT) as shown in Figure 2.1.  For optimization ratio of mash 
water to milled malt should be varied from 2:1 volume/weight percentage 
(v/w %) to 4:1 v/w% during standard mashing [3].  The grist and water 
mixture is now called the mash. Naturally occurring enzymes of the grain 
are activated to convert the grain?s starch in to fermentable and non-
 fermentable sugars. Depending on the type of beer desired, the mash is 
thoroughly mixed and allowed to rest one to three hours. 
? Lautering: Wort (sweet liquid) is now extracted from the mash in the 
lauter tun. To rinse out the sweet wort from within the wort; hot water is 
sprayed on top of the mash. Operational efficiency of the lauter tun is 
judged on its high yield of the sweet extract and less of the undesired malts 
constituents. At the end of this process the spent grains are discharged 
from the lauter tun. 
? Boiling: The wort is now boiled while the hops are being added to correct 
the beer flavour. Boiling takes place in the brew-kettle during which 
complex chemical changes take place. An evaporation of 7 to 10% of the 
total kettle volume is desirable. 
 10
 ? Whirlpooling: The wort is now allowed to settle until it is clear. The 
substances which coagulated and precipitated during boiling are left at this 
stage. The clear wort can now be separated from the unwanted ?trub or hot 
break?. Whirlpool should be designed to achieve 100% removal of the trub 
from the wort. 
? Cooling: The clear wort is now quickly cooled through heat exchanger to 
fermentation temperature. 
? Fermentation: The yeast in now added to ferment wort into beer by using 
the available oxygen and wort nutrients. Yeast uses up available oxygen 
and increases in numbers by cell division [3].Fermentation takes place at 
low temperatures such as 5oC to 25oC depending on the desired beer style. 
Fermentation may take several weeks depending on the style of beer 
required. 
? Conditioning/maturation: The beer is now chilled to near freezing point 
and held for several days to several weeks (depending on desired beer). 
During this time, chemical processes which make the beer clean and good 
tasting takes place. Extended conditioning naturally clarifies the beer. 
? Clarification: Centrifuge and/or filter are used to clarify the beer. This 
process removes bacteria which may have inadvertently been introduced 
during the brewing process. Sterile filtration can be used to ensure 
microbiological stable beer [4]. Filtration of below one micron should be 
avoided as this may strip away some flavour and mouth feel components 
of the beer. 
 
 11
 Figure 2. 1: Flow Process diagram for beer production. 
Refer to link: Figure2.1_Brew_FlowProcess_diagram.xlsx
 12
 2.3 Beer quality 
 
Three main steps involved in the production of beer are: (i) mashing of grist, (ii) 
boiling of sweet wort with hops, and (iii) addition of yeast fermentation. 
Although, at first glance, the set of flavour compounds in beer should be identical 
with those simply transferred from malt, hops and yeast, numerous biochemical 
reactions during mashing and fermentation as a heat-induced degradation of 
flavour precursors during wort-boiling significantly modify the spectrum of 
flavour compounds in the final product [5]. Beer quality may continue to be 
affected upon storage. One of the major causes of the biochemical reactions is 
phenolic acids content in the beer [6]. 
 
The government is proposing stricter legislation on food safety and hence it is 
necessary to know the effects of organic and phenolic acids content on the beer 
quality. The consumers require awareness and quality of beer they drink. 
Mytocotoxins is one of the most serious contaminants in the food industry. 
Mycotoxins are toxins produced by fungi. Some mycotoxins have been found 
carcinogenic in animal experiments, and a few are believed to have similar effects 
in humans. Examples of mycotoxins that may be important in connection with 
foods are aflatoxins, ochratoxin A, patulin, and trichothecenes. Ochratoxin A may 
occur in malted barley that has been harvested having a high content of water and 
dried inefficiently or too slowly, or in grain that has been stored under humid 
conditions.  
 
 13
  
Phenolic acids in beer are available as bound forms and as free acids [7]. Ferulic, 
caffeic and sinapic acids were present in beer mainly as bound forms, while 4-
 hydroxyphenylacetic acid and p-coumaric acid were present mainly as free forms 
and vanillic acid was present equally in the free and bound forms [8]. Many of the 
reactions involved in flavour instability are due to the activation of oxygen and 
the recent work done in Japan by Uchida & Ono, 1996[9] and Uchida et al., 1996 
[10]
 used electron spin resonance (ESR) to measure hydroxyl free-radicals 
generated by the activation of oxygen. Uchida & Ono, 1996[9] and Uchida et al., 
1996[10] have also shown that the flavour stability in beer is related to its 
generation of hydroxyl free-radicals. This method of measuring flavour stability is 
better than the traditional tasting method which is time consuming as beer needs 
to be stored over a period of weeks to be tasted at various stages. 
 
 Some of the polyphenols found in beer are known to act as antioxidants and free-
 radical quenchers playing an important role in beer flavour stability. Other 
polyphenols, however, are detrimental to flavour and colloidal stability, acting as 
pro-oxidants and being involved in haze formation respectively [2]. Phenolic acids 
may cause haze formation and factors causing haze formation have the potential 
of staling the beer flavour [11]. 
 
Antioxidants are widely used in the food industry to increase the shelf life of the 
products. Previous studies have shown that due to complexity of the oxidative 
reactions it is difficult to control them by a single tool, i.e. using antioxidants. 
 14
 Furthermore, from previous experiments there have not been conclusive 
correlations between antioxidativity, amount of carbonyl compounds and flavour 
stability [11]. 
2.4 Properties of beer 
2.4.1 Phenolic acids in beer 
Phenolic compounds participate in beer stability and sensory properties [3]. 
Organic acids are organic compounds having functional group ?COOH with 
acidic behaviour. The functional group ?COOH is called carboxyl group and thus 
the organic acids are called carboxylic acids. The general formula for carboxylic 
acid is R-COOH where R is and alkyl group as shown in Figure 2.2.  
 
Figure 2. 2: Chemical Structures of phenolic acids [12]. 
 15
 Organic acids are formed during incomplete oxidation of alcohols. Biological 
systems create many complex organic acids such as L-lactic, citric, D-glucuronic 
that contains hydroxyl and carboxyl groups [12]. Phenolic acids are a class of weak 
organic acids with the hydroxyl (-OH) group bonded directly to six-member 
aromatic group [12] and with the functional group ?COOH and alkyl group R as 
shown in Figure 2.3 below.  
 
                                          
 
Figure 2. 3: Chemical structures of organic (carboxylic acid) and phenolic acids 
 
Polyphenols are leached into the wort during mashing process and are derived 
from malt and husks and hops. In wort, 80% of the polyphenols originate from the 
malt and the rest from hops [13]. The most common phenolic acids found in beer 
are m-coumaric followed by ferulic, o-coumaric, p-coumaric and 3-OH-benzoic 
acid. Vanillic, chlorogenic, homovanillic, p-OH-benzoic, 2, 6-dihydroxybenzoic, 
syringic, gallic, protocatechuic, caffeic and finally, 3, 5-dihydroxybenzoic acids 
were present in small quantities [14]. Phenolic acids are known to play significant 
role in beer flavour stability because they act as antioxidants. Activation of 
oxygen initiates many of the reactions involved in flavour instability in beer [3], [8]. 
Uchida et al.1996 [9] and Uchida and Ono, 1996[10] have correlated beer flavour 
Phenolic acid Carboxylic acid 
 16
 stability to the generation of hydroxyl free-radicals. However, other phenolic 
acids are detrimental to beer flavour and colloidal stability, acting as pro-oxidants 
and being involved in haze formation respectively [15]. Phenolic compounds in 
beer production are known to affect the rate and quality of fermentation [16]. 
 
 
Phenolic compounds in beer are believed to be involved in flavour characteristics, 
foam maintenance, physical and chemical stability and shelf life of the beer [17]. 
These phenols are either present in monomeric or in polymeric forms in beer. 
Phenolic monomers in beer include flavonols, phenolic acids and volatile phenols. 
Phenolic acids are simple monocyclic hydroxyl derivatives of benzoic (C6-C1) 
and cinnamic (C6-C3) acids [18]. As much as 20 different derivatives of benzoic 
(e.g. vanillic acid, gallic acid, syringic acid) and cinnamic acid (e.g. p-coumaric 
acid, ferulic acid, sinapic acid, caffeic acid) can be detected in beer [19]. A number 
of them have high threshold values and do not affect the aroma of beer. However, 
they are appreciated for their antioxidant activity. Some of the simple phenolic 
compounds can be formed by yeast activity, namely 4-vinylguaiacol (4VG) and 4-
 vinylphenol (4VP), while most of them originate from the raw materials used in 
the brewing process or from contaminated brewing liquor (e.g., chlorophenols).  
 
Plant cell walls, such as from barley normally contain polysaccharides associated 
with hydroxycinnamic acids. These acids are released from the polysaccharides 
and extracted from malt during brewing. During the process of either thermal[20]  
or enzymic decarboxylation of specific yeast strains [21], these hydroxycinnamic 
 17
 acids, more specifically p-coumaric (pCA) acid and ferulic acid (FA) can be 
transformed into the highly flavour-active volatile phenols 4-vinylphenol (4VP), 
4-vinylguaiacol (4VG) as shown in Figure 2.4.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. 4: Decarboxylation of selected phenolic acids and their reduction and 
oxidation products [21, 22]. 
CO2
 R1
 OH
 R2
 R1 = R2 = H: p-coumaric acid (pC A)
 R1= H; R2 = OMe: ferulic acid (FA)
 R1 = R2 = OMe: sinapic acid (SA)
 R1 = R2 = H: 4-vinylphenol (4VP)
 R1= H; R2 = OMe: 4-vinylguaiacol (4VG)
 R1 = R2 = OMe: 4-vinylsyringol (4VS)
 R
 OH
 R1
 OH
 R2
 COOH
 [2H]
 R
 OH
 R = H: 4-vinylphenol (4VP)
 R = OMe: 4-vinylguaiacol (4VG)
 R = H: 4-ethylphenol (4EP)
 R = OMe: 4-ethylguaiacol (4EG)
 Reduction and oxydation products
 OMe
 OH
 COOH
 R
 OH
 Hydroferulic acid
 4-ethylphenol (R = H)
 4 - ethyl guaiacol (R = OMe)
 R
 OH
 O
 4-OH-benzaldehyde (R = H)
 Vanillin (R = OMe)
 OMe
 OH
 OH3C
 Acetovanillon
 OH
 OMe
 HO
 Vanillin alcohol
 OH
 R
 COOH
 4-OH-benzoic acid (R = H)
 Vanillic acid (R = OMe)
 18
 Vanbeneden et al.22 reported that at mashing temperatures of 90oC and 1000C, 
4VG concentrations increased with the heating time, while Fiddler et al.20 
reported that, in dry air, FA only starts to degrade at 2000C, indicating that the 
thermal decarboxylation is greatly enhanced under aqueous reaction conditions. 
Vanbeneden et al.21, 20 assumed the reaction mechanism of thermal degradation of 
FA is pseudo-zero order and the 4VG concentration in wort may be given as;  
 
0
 44 VG
 t
 VG CktC +=        (2.1) 
where tVGC4  is the final 4VG concentration in the wort after heating, 
0
 4VGC  is the 
initial 4VG concentration in the unheated wort, which is zero in this case,  k is the 
rate constant and t is the time of reaction. The rate constant can be expressed as 
the logarithmic form of the Arrhenius equation as;  
 ??
 ???
 ?
 ?=
 TR
 E
 Ak a 1lnln        (2.2) 
 
where Ea is the activation energy, A Arrhenius frequency factor, R gas constant 
and T is temperature. Rearranging Equation 2.1 and substituting it into (2.2), 
gives, 
 t
 T
 ln1 ?? ?=         (2.3) 
Where
 0
 0
 44
 ln
 E
 R
 CC
 A
 VG
 t
 VG
 ???
 ?
 ???
 ?
 ?
 =? , and
 0E
 R
 =? , the activation energy can then be 
calculated from the plot of 1/T versus lnt. 
 
 
 
 19
 2.4.2 Physical properties of organic acids. 
 
Physical nature: The first three organic acids (vanillic, coumaric and ferulic) are 
colourless at room temperature and those having 4 to 9 carbon atoms are 
colourless oily liquids at room temperature. Carbonic acids with higher carbon 
atoms at room temperatures become colourless wax like substances [23]. 
Odour: The first three organic acids have pungent smell. Organic acids with 4 to 9 
carbon atoms smell pungent like goat?s smell whereas higher molecular organics 
acids are odourless. 
Solubility: The solubility of organic acids in water decreases with increase in their 
molecular weight. The first four organic acids are soluble in water and those with 
10 or more carbon atoms are insoluble in water. All organic acids are soluble in 
organic solvents like benzene. 
Acidic nature: Organic acids are very weak acids with very low H+
  
ions and they 
are only partially ionized in water. 
 
2.4.3 Chemical properties of organic acids. 
 
Action on litmus paper: The first two organic acids turn blue litmus paper to red 
quite easily indicating the acidic nature of the compounds. As molecular weight 
increases their acidity decreases and they do not show this test readily [23]. 
Reaction with alcohols:  Esters can be formed when alcohols are mixed with 
organic acids during fermentation  
 
 20
 2.4.4 Physical properties of phenolic acids 
 
Physical nature: Most phenolic acids are coloured, with their colours varying 
from light yellow to red, brown or purple. 
Solubility: Simple phenolic acids are soluble in water but their solubility 
decreases with an increase in molecular complexity. Increase in number of 
phenolic groups increase molecular complexity. 
Acidic nature: Phenols and hence phenolic acids are weakly acidic with phenol 
itself having ionisation potential pKa = 9.98[24]. 
 
2.4.5 Chemical properties of phenolic acids 
 
Action on litmus paper: Phenolic acids turn blue litmus paper to red and their 
acidity increases with carbonyl groups [24].  
Reactivity: Unless sterically hindered, all phenols (including phenolic acids) take 
part in hydrogen bonding [24]. Hydrogen bonds in phenolic acids stabilize 
particular isomers and usually direct the course of a particular reaction. 
 
2.5 Characteristics of Beer Quality 
This section describes three characteristic which are looked into in this 
investigation. 
 
 21
 2.5.1 Flavour 
Taste 
 
Bitterness in beer is mainly due to hop content and alpha strength. Length of hop 
boil, presence of dark malt and alkaline water are the other causes of beer 
bitterness. Increasing the time that hops are boiled, high temperature and quick 
fermentation and filtration reduces bitterness. Lowering alpha hops, adding hops 
at stages throughout the boil are other processes that can be used to reduce 
bitterness of the beer [23]. 
 
Aroma 
 
Alcoholic content contribute to spicy flavour detected by the nose as vinous 
aroma. The alcohol is caused by the conversion of glucose into carbon dioxide 
and ethyl alcohol. Higher remaining alcohols present contribute to this vinous and 
aromas and flavours. Acetaldehyde formed by oxidation of alcohol to acetic acid 
can also add aroma of green apples to beer. Aroma can be reduced by reducing the 
amount of alcohol in beer. This can be achieved by avoiding large amounts of 
sugars in wort content, reducing fusel fuels by reducing fermentation temperatures 
and pitching adequate amounts of yeast. Hops provide aroma to beer from the hop 
oils [23]. 
 
 
 
 22
  
Mouth-feel 
 
Mouth-feel in beer (sensation of viscosity in the mouth) is caused by the presence 
of non-fermentable sugars or dextrins. Medium length proteins can also contribute 
to palade roundness. Mouth-feel in beer can be increased by use of crystal malt, 
use of lactose, adequate protein rest, flaked wheat, oats or barley in the mash. 
Generally reduction in mouth-feel is not desired but it can be achieved by use of 
large amount of corn sugar in wort, long storage, bacterial breakdown and using 
highly fermentable wort [23]. 
2.5.2 Appearance 
Colour 
 
The colour of the beer is primarily derived from the colour of the malt. Pale malts 
are used to obtain lighter beer colour. Use of sugar or adjuncts and filtration can 
also be used to obtain lighter beer. To obtain darker beer the following are used: 
higher-temperature kilned malts, dark malts, caramelization of the boil or hot side 
aeration and oxidation [24].  
 
Beading and Foam 
 
Beading is a quick way of judging alcoholic strength although it is not precise. 
The beer is closely watched when poured in to the glass. The bigger and longer 
lasting bubbles indicate high alcohol content. The head (foam) and bubbles 
 23
 released when the beer is opened are due to the presences of CO2 dissolved in 
beer during fermentation process.  This characteristic of beer is increased by using 
excessive priming sugars, bacterial contamination, isomerised of hops extract, 
unconverted starch and boiling extract worts. Reduction in beading is achieved by 
not using enough priming sugars, weak or dead yeast when bottling (for long 
lagering) and poor bottle cap seal [23]. 
 
Clarity 
 
Visual clarity in beer contributes to its appeal and hence clarity is one of the most 
important characteristics when looking at beer quality. Beer can be clarified by 
flocculating yeast strains very well; clearing agents such as polyclar, papain, irish 
moss, bentonite, gelatine, etc; filtration; long vigorous boiling and quick chilling, 
lagering and ageing. Decrease in beer clarity can be achieved by weak or mutated 
yeast strains, non-flocculent yeast ,wheat malt, unmalted barley ,poor cold break, 
poor starch conversion in mash, poor malt crush, bacterial contamination, high 
protein content, wild yeast contamination and tannin present in beer due to 
excessive or high temperature sparge. Phenolic and organic acids may cause haze 
formation and factors causing haze formation have the potential of staling the beer 
flavour [25]. 
 
 
 
 
 24
 2.5.3 Wholesomeness 
Absence of hazardous compounds and Presence of useful compounds 
 
Hazardous substances are any material or mixture of substances in beer that 
presents danger to the beer drinker. Undesirable substances that can be found in 
beer are fungal toxins (including mycotoxin A, aflatoxin A and vomitoxin), barley 
storage pesticides, and chloropropanols which are mostly derived from the malt. 
Desirable compounds that can be found in beer include trace metals, vitamins, 
proteins, dietary fibre and antioxidants [26]. 
 
2.6 Review on previous work and available literature 
2.6.1 Mashing process 
Mashing is of prime importance in brewing as it is time consuming and hence 
cost-intensive [27].The objective of the mashing is to extract fermentable 
carbohydrates from the malt grist. This is achieved through mixing and heating of 
malted barley and water. Stirring is essential during mashing to ensure distribution 
of thermal energy as mash is know to be poor conductor of heat. Fine grinding of 
the grist is recommended as it is though to facilitate the penetration of water and 
subsequent gelatinization of starch [28, 29]. Efficient and rapid conversion of starch 
into fermentable carbohydrates depends on various factors including level of 
starch degrading enzymes in the malted barley and temperature program of 
mashing. Number of starch work together to hydrolyse starch and these enzymes 
 25
 include ?-amylase, ?-amylase, limit dextrinase and ?-glucosidase [29]. These 
enzymes are active at particular conditions [30] as depicted in Table 2.1 below. 
 
Table 2. 1: Optimum conditions for starch hydrolysis enzymes 
Enzyme Action Produces Temp pH 
?-Amylase Random starch linkages Sugars 70 oC 5.2 
?-Amylase 
Cleaves maltose from 
 the non-reducing end Maltose 64oC 5.5 
?-Glucanase 
Random ? -glucan 
linkages Sugars 45oC 6 
Proteases Protein linkages 
Peptides and  
amino acids 50oC 5.5 
 
Various breweries use different routes or temperature profiles for mashing, just as 
they use different malt to water ratio. Mashing process requires an estimated 12-
 13Btu/barrel for medium sized breweries [31]. Optimal mashing conditions should 
extract highest possible amount of fermentable carbohydrates. The research will 
investigate different mashing routes and malt to liquor ratio for optimised 
mashing conditions.  
2.6.2 Lautering system 
 
The efficiency of the lautering system is based on three categories: quality of the 
wort in terms of chemical composition and clarity, extract yield and flowrate of 
the filtrate. Optimal performance of the process is based on: 
? High extract efficiency 
? Clear wort with low solids 
 26
 ? Exact/repeatable cycle times 
? Wort with low dissolved oxygen(DO) 
Wort quality from mash filtration is assessed on parameters of clarity, solids, 
dissolved oxygen and minimised pick up of undesirable as haze, starchy 
materials, infection, etc. Extract/filter efficiency is measured as soluble extract 
left in the discharged spent grains. It is influenced by the composition and 
filter bed quality which relates to quality of raw materials, upstream product, 
wort quality required and availability to recycle weak worts.  
 
Filtration of the wort through the bed is explained through Darcy?s Law. 
Equation 2.4 below was established by Henry Darcy in 1856 using a sand 
filter [32]: 
LhhKAQ /)( 21 ?=       (2.4) 
Where 
Q= total volume of liquid percolating in unit time 
K= factor describing filter bed permeability 
A= constant cross sectional area 
?h= Pressure drop across column height L 
 
Huite and Werterman established equation for K: 
  
223 )1(180/ ndnk ?=      (2.5)  
Where 
n= bed porosity (wort volume/bed volume) 
d= effective particle diameter 
 27
 Darcy?s Law has been modified to describe lautering: 
LPKAtVQ ??? // ?==     (2.6) 
Where 
Q= Wort volume flowrate 
K= mash bed permeability 
A= Mash bed cross sectional area 
?P= pressure drop across the mash bed 
?= Viscosity of the wort 
L= mash bed depth 
Using permeability (K) and effective particle size diameter (de)  
Permeability can be defined as follows: 
( )223 1180 ?? ?=
 e
 dK      (2.7) 
Where 
?=bed porosity (wort volume/ bed volume) 
de = effective particle diameter 
Effective particle size diameter is defined as: 
( )? ?= 1/ iie dxd       (2.8) 
Where 
xi = weight fraction (dimensionless) 
di = particle diameter 
 
 
 
 28
 2.6.3 Fermentation process 
 
Fermentable sugars are metabolized by the yeast to produce alcohol and CO2 
during fermentation process. The heat generated during fermentation must be 
dissipated to avoid damage to the yeast by using cooling coils or jackets. 
Fermentation rate is dependent on the yeast strain, targeted taste profile, 
fermentation temperature, oxygen content, yeast content, pitch rate and 
fermentation parameters (like reduction of unwanted diacetyl levels). Objectives 
of fermentation are: 
? Production of the correct beer quality as required by the brewer and 
defined in the specifications. 
? Controlling of yeast metabolism so as to control beer flavour. 
? Yeast growth control as beer loss may be caused by excessive growth, 
whereas inadequate growth may yield poor and incomplete fermentation. 
? Prevention of contamination by micro organisms other than the required 
brewing strain. 
? Meet processing times. 
? Production of alcohol as shown in the following equation. 
2526126 22 COOHHCOHC +?    (2.9) 
2.6.4 Fermentation control parameters. 
 
Fermentation can be controlled through control of inputs or the process itself. To 
achieve the correct beer quality, critical key inputs to be controlled are: 
 29
  
o Composition 
o Wort 
o Aeration/ Oxygenation 
o Sterility 
o Clarity 
o Temperature 
o Volume 
o Yeast addition 
o Fermentation vessel 
 
During the fermentation process three parameters that can be controlled are vessel 
temperature, vessel pressure and fermentation time. Process measurements that 
are used to monitor the process are gravity, alcohol, pH, yeast count and diacetyl.  
 
2.6.5 Control of inputs 
 
After being processed in the brew-house, wort matrix is fixed but has a key 
influence on the outputs of fermentation. Yeast metabolism and hence the final 
beer produced is dependant on the nutritional composition of the wort. Original 
gravity is the amount of extract at the start of fermentation, and that which is 
fermentable is called the limit extract. Limit extract gives an indication of 
potential alcohol that can be produced and how much sweetness or body may be 
left. At the start of fermentation oxygen is added to ensure that there are sufficient 
 30
 live, active yeast cells to complete fermentation. Maximum level of 8-10ppm can 
be achieved if air is used whereas much higher levels can be achieved if sterile 
liquid O2 is used. All living organisms are killed in the brew-house during wort 
boiling except the thermophilic spores of some bacteria, such as Bacillus. Sterile 
status need to be maintained from the kettle via clarification and cooling in to the 
fermentation vessel. The plant need to be cleaned and sanitized and all additions 
made need to be sterilized. Clear wort is required as cloudy wort result in coating 
of yeast by trub particles and can prejudice performance. Excessive yeast growth, 
poor foam and flavour instability to oxidation may be caused by fats from trub. 
Each beer has its own temperature profile and the starting temperature is critical.  
  
 
2.6.6 Filtration in beer processing 
 
Recently membranes have been gaining a wide range of application in brewing, 
chemical and food industries for industrial separations and waste treatment.  
Application of filtration in beer clarification helps to remove bacteria which may 
have inadvertently been introduced during the brewing process. Sterile filtration 
can be used to ensure microbiological stable beer. Filtration can also be used to 
remove yeast, colloidal particles and other haze forming substances. Optimally, 
filtration process should remove best possible amount of unwanted substances 
from the processed stream without effect on the original flavour, aroma, 
nutritional value, functional properties of beer. Overdue application of micro-
 filtration in the commercial brewing industry is owed to its low flux rate, 
 31
 inconsistency in the product, fouling and retention of critical macromolecules for 
essential beer clarification. Filter mediums and aids such as kieselguhr, perlite, 
Silica Hydrogels & Xerogels and Polyvinylpolypyrollidone (PVPP) are used to 
assist with the efficient filtering in filters. Their main objective is to retain an open 
pore structure in the bed to allow easy flow of filtrate while simultaneously 
holding back solids of a size often smaller than the holes in the filter support or 
cloth [33]. Other types of filters are depth filtration filter (sheet filters) and 
centrifuges which are used in breweries for finest (polishing) filtration post 
kieselguhr filter.  
 
2.6.7 Beer Haze/ phenols 
 
Most of the haze-determining instruments are designed to measure turbidity of 
light scattered at 90oC to the incident light at wavelengths of 450-860nm [34]. Beer 
haze is caused by colloidal particles. Colloidal stability at post-packaging can be 
achieved by removal of these hazes and their precursors. Permanent hazes are 
present at both warm and cold temperature whereas chill hazes are present only at 
refrigerated temperatures. The removal of beer haze formation particles poses the 
largest challenge to the industry. Colloidal haze particles originating from protein 
and polyphenols are a major contributor to beer haze formation [35]. Carbonyls are 
responsible for aged character in beer [36]. Polyphenols play positive role by 
reduction of Hydroxyl radicals (highly reactive species) non-enzymically. 
Whereas the oxidation of polyphenols lead to polymerisation and formation of 
coloured materials and complexes with proteins, leading to beer haze. 
 32
 2.6.8 Primary beer raw material- barley 
 
There are various grains that can be used as main raw material or as adjuncts for 
brewing, for this research malted barley was the cereal of choice. Other cereal 
products that can be used include rice, wheat, sorghum and maize. Malted barley 
was chosen as it satisfies the following requirements of the brewer: 
? Yield- Relatively higher extract that the malt can deliver. 
? Fermentability- The amount of fermentable extract delivered by the extract 
is high. One of the brewer?s prime requirements is alcohol production and 
hence reasonable amount of fermentable extract is required. 
? Husk- Its full intact husk is required especially in a case where the mash 
separation in the brew house is achieved by the use of the husk. But it is 
equally important to protect the grain during storage against moisture, 
pests and diseases. 
? Wholesomeness- As a requirement for beer food product quality systems 
such as Hazard analysis and Critical Control Point (HACCP), it is 
particularly important that the brewer ensure the wholesomeness nature of 
this natural product. 
 
2.6.9 Clarification by finings 
 
After fermentation beer undergoes flavour maturation stage and followed by cold 
storage for clarification through sedimentation of particles. Sedimentation of 
particles, mainly protein-polyphenol complexes is enhanced by addition of 
 33
 finings/ isinglass. Protein and unfiltered yeast are other causes of haze formation 
in beer. Isinglass finings has been successfully used for beer clarification and 
stabilization for many years. Isinglass is an amphoteric solubilized molecule that 
can bind both negatively charged yeast cells and positively charged proteins as it 
possesses both negative and positive charged areas. Protein material that 
coagulated during wort boiling can be removed by the use of kettle finings. 
Settling velocity of the particles is increased due to increased size and weight of 
the particles through added finings [37]. Finings may aid brew performance 
through by improving clarification time and hence reduce storage time needed in 
cold tank. And also the achieved clarity has an effect downstream on the beers 
that undergo filtration as it has an effect on the run length achieved by filters. 
Fermentation rate can be affected by the removal of some of the protein nutrients 
required for yeast activity and possibly reduce the level of unsaturated fatty acids 
[38]
 . Hence, it can be noted from literature that there is a trade off to addition of 
kettle finings during brewing as they can positively improve beer clarity or 
negatively affect the fermentation rate.   
 
2.6.10 Wort boiling 
 
In recent years in the brewing industry the focus on this process has been to 
develop methods that strive to reduce boil times and evaporation rates not only to 
reduce cost but also to reduce the precipitation and removal of foam-forming 
proteins. The objectives of wort boiling are to: 
? Halt enzyme activity; 
 34
 ? Sterilise the wort; 
? Remove unwanted volatile substances through evaporation; 
? Concentrate the wort through evaporation; 
? Isomerisation of bitter substances (extract bitterness from hops); 
? Achieve the required colloidal stability; 
? Achieve desired colour and flavour. 
 
Wort boiling is a very energy intensive process that consumes about 39% of total 
energy required for brewery and necessary steps are required to make the process 
as efficient as possible. Although there is no single parameter to judge a ?good 
boil?, its efficiency can be expressed in terms of: 
? The boiling rate; 
? Temperature; 
? Pressure; 
? Evaporation; 
? Trub formation. 
The most common parameters are % evaporation and evaporation rate. Boiling 
requires a lot of energy so, the optimal point/time is required so as not to boil 
more than required, but avoid low evaporation and non-vigorous boils as they 
might cause major product defects. In high gravity brewing the same energy input 
produces more wort and is therefore more cost beneficial.  
 
The polyphenols, from malt and hops, are available in oxidized form and form 
complex products with proteins in the wort called trub. Trub formation can be 
 35
 enhanced by vigorousness of the boil and extended boiling times; it can be 
complete after two hours. The optimum pH for hot break (trub formation) is 5.2. 
The wort pH drops during boiling from 5.8- 5.9 to 5.2-5.4 due to melanoidin 
formation and hop acids but mainly as result of precipitation by calcium 
phosphates and calcium polypeptides complexes as shown is the following 
equations: 
 
 ( ) ?+?+ +? 43421
 ppt
 POCaHPOHCa 243422 23     (2.10) 
 
Similarly with polypeptides: 
 
++ +???+? HCaepolypeptidCaHePolypeptid
 ppt
 22 444 344 21   (2.11) 
It is important to achieve the required decrease in pH as it affects wort and beer 
character. Extra calcium ions in the form of calcium sulphate or calcium chloride 
are added, or alternatively through direct addition of phosphoric or sulphuric acid. 
Lower pH improves protein coagulation, encourages yeast growth, improves beer 
flavour, and inhibits growth contaminating organisms. 
 
At this stage in the process malt enzymes have completed their task of converting 
starch to sugars, converting proteins to amino acids and ?-glucan reduction. The 
heat during wort boiling inactivates the malt enzymes by denaturation. At this 
point, the brewer must have achieved the desired composition as it becomes fixed 
 36
 at this point. Wort boiling is an important step in the brew house as failure to 
carry in out effectively might lead to: 
? Further breakdown and hence change in composition 
? Poor foam due to excessive protein degradation 
? Poor flavour balance 
? Negative mouth feel 
? Potentially higher alcohol levels 
Various unwanted volatiles from hops and malt are removed from the malt during 
this stage. Di-Methyl Sulphide (DMS) is one of the most important compounds. 
Di-Methyl Sulphide arises for s-Methyl Methionine (SMM), a precursor found in 
malt that has an unwanted (but not in all beers) sweet corn/ cooked cabbage 
flavour. SMM is converted to DMS during wort boiling in a reaction dependant 
on time and temperature and the volatile is removed in the evaporating water. 
  
 
Clarity of wort can be improved by the addition of kettle (copper) finings. Finings 
are made from carragenan (derived from seaweed), either in its raw known as 
Irish Moss, pun feed, or processed as powder or pellets. The active ingredient is 
K-carrageenan, which is a negatively charged polymer containing sulphate 
glucose and galactose unit. It is soluble in hot wort but gelatinise on cooling. The 
finings are added at the end of boiling or to the whirlpool. They interact with 
positively charged proteins and aid their precipitation. The timing, dose rate, place 
of addition and product form are key factors in fining addition. By taking freshly 
boiled wort samples and adding finings at different rates and comparing the 
 37
 subsequent wort clarities, comparisons are made to set the dose rate to be used in 
brews for the next period. 
 
During wort boiling, hop ?-acids readily dissolves in boiling wort, but 
isomerisation (hop utilisation) takes considerably longer. Isomerisation is directly 
dependent on temperature, i.e., at 100oC 40 % isomerisation takes 3 hours, at 
135oC it takes two hours. But it is desirable to limit the time that the wort is 
exposed to temperatures>80oC for quality reasons. Usually a boil of 60 to 90 
minutes achieve approximately 40% isomerisation. The ?-acids are easy to access 
in pellets and so isomerisation happens quicker. Boiling converts the compounds 
in to an isomerised, soluble form as shown below: 
 
Figure 2. 5: Isomerisation of ?-acids [24] 
 
The effectiveness of the isomerisation and mixing process during boiling can be 
measured using the concept of Utilisation: 
 38
 % Utilisation = (BU achieved / theoretical BU) x 100  (2.12) 
Where; 
BU= Bitterness 
 
The rate of heat transfer by the wort heater depends upon its surface area (A) in 
contact with the wort, the temperature difference between the heating medium and 
boiling wort (?T), and overall heat transfer coefficient(U) as represented by the 
formula: 
TUAQ ?=         (2.13) 
Heat flux can be increased by an increased area of the heating surface. With our 
brew house, the difficulty of controlling the heat means that there is always wort 
caramelisation which causes characteristic flavour in the final beer. Main 
objective of pasteurisation is to prolong product life by removal of microorganism 
capable of growing and enzymes which might cause undesirable chemical 
changes. 
 
Sterile filtration is an alternative method to pasteurisation for achieving the 
desired microbiological stability in package. The process removes the micro-
 organism which can spoil the product and affect its stated shelf life. Its advantage 
is that unlike heat pasteurisation, it avoids possible product?s flavour deterioration 
from heat treatment. The following process requirements need to be specified for 
the product concerned: 
 
 39
 ? Feedstock maximum microbiological and non-microbiological load 
including concentrations and particle sizes. 
? Filtrate maximum concentrations and details of product spoilage organism 
allowed being present in the filtrate without microbiological failure 
occurring during the stated product life. 
? Product viscosity and flow characteristics  
Microbiological load reduction from feedstock to filtrate is often referred to as 
Log Reduction Value (LRV). Sterile filters should be able to reduce 
microbiological load by 99.999 %. 
 
Key factors affecting flowrate and cartridges are pressure drop and surface area. 
Pressure drop increases with an increase in flowrate and product viscosity, it also 
increases as the pores become blocked. Product solids which are being stopped to 
pass through during cross flow membrane filtration include yeast, proteins, and 
bacteria. High pressure drop across the membranes consumes lots of energy. 
Membrane of 0.5microns is ideal. Membrane should not result in loss of head 
retention. The process should be reliable, repeatable and membrane life should be 
of an important factor during the investigation.  
 
2.6.11 Sedimentation 
 
Yeast in the fermentation tank separates from the beer through sedimentation. 
This is a process whereby particles settle out from the solution due to a force 
either naturally as is gravity or mechanically as in centrifugal acting on each of 
 40
 the particles, forcing them to settle out. The rate of sedimentation depends on 
density of solution and particle, time, temperature, degree of agitation, distance to 
settle out, particle size and size of applied force.  
 
For gravitational settling, terminal velocity of the particle assuming spherical 
particle falling unhindered in solution, can be defined as:  
( ) 21
 3
 4
 ??
 ???
 ? ?
 =
 ?
 ??
 D
 p
 T C
 gd
 V       (2.14) 
Where 
D= diameter of the particle 
?p= density of particle 
?= density of fluid 
CD = drag coefficient 
However, for laminar flow (very slow settling) the above equation can be 
modified to: 
( )
 ??
 ???
 ? ?
 =
 ?
 ??
 18
 2 gd
 V pT
       (2.15) 
This is known as the Stokes? Law.  From equation 16 above it can be seen that the 
following factors need to be considered to influence settling times; particle 
diameter, different in density between liquids and solids, and the settling force. 
Flocculants can be used to aid the settling process by binding smaller particles in 
to larger ones, utilising Van der Waals forces as illustrated in Figure 2.6. 
 41
  
 
 
Figure 2. 6: Binding together of small particles by Flocculants. 
 
Particles that are generally removed from the beer together with their size are 
presented in Table 2.2: 
 
Table 2. 2: Typical concentrations and size of particulate species. 
 
Particulate Species Size(?m) Typical Concentration 
Haze <1 100mg/l 
Bacteria 1-2 10000mg/l 
Yeast 4-6 5million mg/l 
Polyvinylpolypyrrolidone (PVPP) 20 40g/hl 
 
2.6.12 Beer spoilage organisms 
 
Large numbers of micro-organisms, including yeast, bacteria and moulds are 
present in the environment. For beer brewing industry, beer spoilage organisms 
that have been problematic include wild yeasts as Saccharomyces spp, non-
 Particles carry same charge which causes 
separation 
Flocculants consists of long-chain molecules (100 to 1000K 
Daltons) which carries opposite charges and attaches to 
particles 
Particles attracted and form large particles 
 42
 Saccharomyces spp, brettanomyces spp, torulopsis pichia and candida spp, some 
bacteria as Gram positive (lactobacillus spp and pediococcus spp) and Gram 
negative (Acetic acid bacteria, zymomonas spp, pectinatus spp and 
enterobacteriacease spp), and some Lactic acid bacteria as lactobacillus brevis, 
lactobacillus linderi and pedcoccus damnosus. However there are various 
properties in wort/ beer that suppress the growth of these bacteria to make beer 
drinkable, they include: 
? Low pH- The pH of wort drops below tolerable levels for most bacteria as 
fermentation progresses, although acid type bacteria as lactic Acid 
bacteria, acetic acid bacteria and yeast are present. 
?  Hop Antiseptics- many bacteria are suppressed by hop bittering 
substances. Particularly Gram +ve bacteria such as spore formers while 
Gram ?ve bacteria are generally not affected.  
? Alcohol content- The increasingly high alcohol content of fermenting wort 
is inhibitory for many bacteria. 
? Anaerobic conditions- Anaerobic conditions of the wort are unfavourable 
for strictly aerobic bacteria. Anaerobic conditions are achieved within few 
hours although the condition is aerobic at the beginning. Anaerobic 
conditions prevail, throughout storage, filtration and bottling, right up to 
the time of consumption of the beer.  
? Lack of nutrients- the amount of nutrients in wort decrease as fermentation 
continues leaving less than enough to sustain the growth of many bacteria. 
 
 43
 Beer spoilage leads to changes in beer which results in an uncharacteristic flavour, 
odour or appearance of the beer. Physical changes that occur in beer due to 
spoilage are: 
? Haze/ turbidity- It becomes evident when large number of bacteria are 
present.  
? Ropiness- this is the production of slime by certain bacteria, resulting in 
slight viscosity, jelly-like lumps or viscous oily liquids. Strings of slime 
may be seen adhering to the bottom of the bottle when ?ropy? beer is 
inverted.  
Flavour changes that occur in beer due to spoilage are. 
? Acidity- Carbohydrates are broken down by some species of bacteria 
yielding a variety of acids as lactic, formic, acetic, succinic acid, etc. This 
is produced in the mash-tun and carry on to the final product. 
? Diacetyl- Although it is normally present in beer in small percentages, 
some bacteria produce diacetyl in large enough quantities to spoil the beer. 
? DMS- DMS is produced as a by-product by variety of bacteria. 
? Phenolic- Certain bacteria produce phenolic off-flavours. This is mainly 
attributed to infection by wild yeast.  
? Other off-flavours- a variety of other off-flavours will be produced, 
specific to the contaminating micro-organism, e.g. Acetaldehyde, Acetoin 
and 2,3 butanediol. 
 44
 2.6.13 Process commissioning 
 
A successful plant commissioning helps optimise the process and its operation 
and also helps receive full benefit from the plant investment. Plant commissioning 
can only be considered successful if it results in no accidents, no equipment 
damages and on test production within a reasonable time. No commissioning can 
be considered a success if it is not done safely and hence safety has to be stressed 
from the very beginning of pre-commissioning. It has been noted that most brews 
at home brewery and research micro-breweries have not been very successful in 
one way or another, as observed from communications with brewers. These 
observations include: 
? Lack of check list for pre-commissioning; 
? Poor communication pre and during commissioning; 
? Poor preparations of personnel for brew; 
? Poor preparation of materials for brew; 
? Poor documentation of results for full analysis. 
The study will cover different aspects of plant commissioning including planning 
and managerial aspects involved during the start up of process plants and develop 
the documentation to be used for commissioning of the micro-brewery plant at 
Wits. The documentation will include plant specific safety and provide guidance 
on operational requirements for the plant. Developed documentation will help 
supervisors to ensure that brewers have reviewed and understood the procedures 
and hence ready for commissioning.  
 45
 CHAPTER THREE 
3. EXPERIMETAL 
3.1 Materials and Method 
 
The experimental setup used in the study is presented in Figure 3.1. Figure 3.1a is 
the schematic of the brew-house representation of the Wits mini brewery, while 
Figure 3.1b is the commissioned Wits mini brewery plant supplied by Falcon 
Engineering (Pty) Ltd, South Africa.  
Three different kinds of malts supplied by South Africa Brewery (SAB) Alrode 
Maltings were used in the study, which are designated as Roasted malt (Malt A), 
Amber (Malt B) and Lager/Pale (Malt C). Drinking water (tap) was passed 
through water filter (Schleicher & Schuell, Dassel, Germany) and then used 
throughout the experiment. The strain Saccharomyces cerevisae var. uvarum 
(Alfred Jorgensen Laboratories strain AJL 2036, Copenhagen, Denmark) and 
Saaz Hops (SAB, Alrode, South Africa) were used through the experiment. 
 
3.2 Procedure 
 
Malt was crushed by using a 30 cm long wood roller mill (Figure 3.1a). Malted 
barley was milled until husks were left in relatively large pieces and starch in 
granular or fine particles, and this was sent for mashing. Various routes and 
conditions were used to find the optimised mashing procedure.  
 46
 Half a kilogram of malt was milled and mixed with 1.2L of mash water at 50oC in 
the hot liquor tank as shown in Figure 3.1a Mash was taken through the mash 
regimes as shown in Table 3.1 below. Mash liquor was kept at 50oC for 46 min. 
Mash temperature was increased at a rate of 1oC per min until it reached 70oC 
where it was kept for 10 min, and mash liquor temperature was increased to 90oC 
and kept for 10 min, as reported earlier in our previous report [39]. 
 47
  
Figure 3. 1:  Experimental set up- (a) schematic of the mashing process flow. (b) 
Commissioning of the Wits mini brewery plant [39]. 
  
Mash liquor was then sent to the lauter tun for the separation process. Wort was 
separated from the slurry (solid malt particles) using the mash lauter. The 
remaining sugars were recovered by rinsing the solid particles with 550 mL of 
(a) 
(b) 
 48
 mash water at the end of the mash regime, so that in total 1.75 L of water was 
added to 500 g of malt. The solid particles were discarded as shown in Fig 3.1a. 
The volume of the recovered extract was 1.25 L (wort). The wort was left to cool 
at room temperature before the samples were taken for analysis. Table 3.1 shows 
the different mashing routes used in the study. These routes are similar to what is 
practiced in a standard South African brewery  
 
 
Table3. 1: Mashing routes used in the study. 
 
Mash 
Route 
Temperature of 
Mash Water (0C) 
Step 1 Step 2 Step 3 Step 4 Total Mash 
Time(min) 
1 
65 65oC 76oC 80oC   
75  30 min 15 min 15 min   
2 
60 60oC 72oC 80oC 86oC 
105  60 min 10 min 10 min 5 min 
3 
50 50oC 65oC 75oC 90oC 
133  35 min 30 min 20 min 18 min 
4 
 
55 55oC 70oC 80oC   
85 
 
 46 min 10 min 10 min  
 
Anion-exchange chromatography coupled with a UV-vis spectrometer (Diodex, 
Synnyval, California, USA) at 280nm using commercial standards was used to 
identify and quantify the individual carbohydrates in the wort samples. The four 
carbohydrates analysed were glucose, fructose, maltose and maltotriose. One mL 
of wort was added to 500 mL of deionised water and the mixture was filtered 
through a 0.45 micron filter (Schleicher& Schuell, Dassel, Germany). The 
samples were then injected into a high performance liquid chromatography 
(HPLC) with a UV detector operating at the following conditions: column 
temperature 25?C, flow rate 1.0 mL/min, injection volume 20?L and mobile phase 
 49
 A (Acetonitrile) and B (water). Carbohydrate standards were glucose (Sigma G-
 7528), fructose (Fluka-47739), maltose (Sigma M-5885), maltotriose (Sigma M-
 8378), maltotetraose (Sigma M-8253), maltopentaose (Sigma M-8128), 
maltohexaose (Sigma M-9153), and maltoheptaose (Sigma M-7753). Order of 
elution is Glucose, fructose, maltose, maltotriose (maltotri), maltotetraose, 
maltopentaose, maltohexaose and maltoheptaose.  
 
Determination of free and bond phenolic acids in beer and wort was performed by 
using HPLC coupled with a UV-vis spectrometer at 280 nm at the following 
operating conditions: column temperature, 25oC, flow rate: 0.7 mL/min, binary 
solvent phase, 0.1% of (A) formic acid and (B) methanol. Peaks were identified 
by comparison of retention time and UV spectra of commercial standards as 
follows 40: 1. gallic acid, 2. protocatechuic acid, 3. 2,3,4,-trihydroxybenzoic acid, 
4. protocatechuic aldehyde, 5. p-hydroxybenzoic acid, 6. gentisic acid, 7. vanillic 
acid, 8. chlorogenic acid, 9. caffeic acid, 10. vanillin, 11. syringic acid, 12. 
syringealdehyde, 13. p-coumaric acid, 14. ferulic acid, 15. sinapic acid, and 16. m-
 coumaric acid. Preparation of polyphenol standards were made up separately in 
methanol and by sequential dilution of five different concentrations, 20, 40, 60, 
100 pmoles/20 ?l, whereby each sample was injected onto the column. p-
 coumaric acid was used as an internal standard once the system was set up, and its 
final concentration of 100 pmoles/ 20 ?l of sample was added. Their order of 
elution and retention times are shown in figure 3.2 below. 
 50
   
Figure 3. 2:  HPLC Chromatograms of the standard mixture of Phenolic acids.1, 
gallic acid; 2, p-Hydrobenzoic acid; 3, vanillic acid; 4, caffeic,; 5, Syringic; 6, p-
 coumaric acid; 7, ferulic acid; 8, M-coumaric acid; 9, Isoferulic acid; 10, Sinapic 
acid; 11,o-Coumaric acid. 
 
 
The analysis of hydroxyl-free radical in wort samples were conducted using a 
continuous wave electron parametric resonance (EPR) spectrometer (Bruker ESP-
 300E) operating in the X-band, at microwave of 9.81 mW, frequency: 9.43GHz 
and temperature: 25oC. An 807 mg aliquot of alpha-phenyl-N-t-butylnitrone 
(PBN) was weighed and added to 790 ?L of ethanol and then added to 790 ?L of 
deionised water. The PBN solution was stored at 3oC and used throughout the 
experiment. Only wort samples were analysed for OH-radical generation. 
Cuvettes were rinsed with 20% nitric acid and deionised water before use for the 
ESR, to remove any compounds that might interfere with the ESR signal. 
 
 51
 Wort samples were added to vials and mixed with 100 ?L of the PBN solution 
and the amber vials were stoppered. The wort ageing process was conducted by 
placing ESR glass filled with samples in a 60oC water bath. Samples (150 ?L) 
were taken from the vial every 60 min and placed into the ESR cuvette and placed 
into the ESR instrument for the analysis. The ESR was calibrated, the magnetic 
field applied, the sample was scanned, and the signal was printed out. The wort 
samples were analysed over a total period of 180 min. The quantities of the signal 
generated by the OH-radical concentrations were determined and were correlated 
to flavour stability [41]. 
An Ale and lager brewing yeast were used through the experiment and were 
incubated 30oC for 24 hours. Yeast cells were counted using 40 x objective 
microscopes equipped with counting chamber. 
 
Incidents where failing by brewers have been the major contributing cause to 
material waste and brew inefficiency were recorded. Unforeseen eventualities and 
shortfalls by new brewers without commissioning documentation were observed 
and noted. Brewers were then provided with designed commissioning guideline 
document which was iteratively revised and improved until errors are optimally 
reduced. Commissioning of the plant was executed with the assistance of Falcon 
Engineering (Pty) Ltd, South Africa. It was ensured that the document is clear, 
concise and provides accurate commissioning procedure. 
 52
 CHAPTER FOUR 
4. RESULTS AND DISCUSSION 
4.1. Optimisation of wort production 
 
The main objective of the mashing process is to extract the liquid sugars from 
malted barley for fermentation at a later stage. The optimised mashing procedure 
should be the one that yield higher amount of fermentable carbohydrates from the 
same malted barley [42]. The procedures were varied by using different malt to 
liquid ratio, mashing temperature and different mashing rules.  
4.1.1 Water to malted barley ratios 
 
The malted barley to water ratio was varied by adding or reducing mash water and 
malt A was used for this analysis. Breweries usually use the ratio of 2.3:1 to 
4:1(Litres, water: kg, malted barley). To make this analysis representative and to 
keep other variables constant, for this part of investigation, wort was simply 
produced by mixing malt and mash water at 90oC, mashing for 90 minutes and 
analysing the resulting wort. Carbohydrate analysis was done using HPLC 
coupled with UV-s spectrometer. A sample chromatogram obtained from the 
experiment is shown in Figure 4.1 below together with identification of 
carbohydrates. 
 53
   
 
Figure 4. 1: HPLC Chromatogram obtained for carbohydrate analysis; 1- 
Glucose; 2-Fructose; 3-Maltose; 4-Maltotriose. 
 
Figure 4.2 below shows the effect of varying water to malted barley ratio from 
2.3:1 to 4:1. The analyses from four runs gave relative standard deviation of ?1.7 
%, which were also reported [39]. 
1
 2
 3 
4 
Time (min) 
 54
 0
 2
 4
 6
 8
 10
 12
 2.3:1 3.0:1 3.5:1 4.0:1
 Ratio(Litre water:kg malt)
 Ca
 rb
 o
 hy
 dr
 at
 e 
co
 n
 ce
 n
 tr
 at
 io
 n
 (g/
 10
 0m
 l) Maltotriose
 Maltose
 Fructose
 Glucose
  
Figure 4. 2: Carbohydrate concentration obtained by varying water (litre) to 
barley (kg) ratio. 
 
Figure 4.2 shows that the ratio of 3.5:1(litre: kg) resulted in higher total 
concentration of fermentable carbohydrates. Carbohydrate concentration increase 
with an increase in water to malted barley ratio as it is increased from 2.3:1 to 
3.5:1, and there was a decrease in concentration when the ratio is increased to 4:1. 
The trend suggests that more carbohydrates are extracted when mash water is 
increased. The reduction in concentration when mash water is increased to 4 L per 
kg of malt implies that optimal amount of carbohydrates was extracted when the 
ratio reached a value of about 3.5:1. This was then used in all experiments as the 
optimal ratio. 
 55
 4.1.2. Different mashing rules 
 
In terms of mashing rules, various breweries use different routes or temperature 
profiles for mashing, just as they use different malt to water ratio. It was thus 
decided to study different routes in order to use the optimised mashing route for 
sample preparation at a later stage. The established optimum malt to water ratio of 
3.5:1(litre: kg) was used for these analyses. Selection of the optimised mashing 
route was based on higher recovery of fermentable carbohydrates, just as in the 
previous case. Figure 4.3 shows various mashing routes investigated.  
 
0
 20
 40
 60
 80
 100
 0 20 40 60 80 100 120 140 160
 Time(minutes)
 Te
 m
 pe
 ra
 tu
 re
 (oC
 )
 Route1
 Route2
 Route3
 Route4
  
Figure 4. 3: Various mashing routes investigated. 
 
Carbohydrate analysis was performed on wort samples produced from different 
mashing routes illustrated in Figure 4.3 above. The resulting fermentable 
carbohydrates obtained from different mashing route are indicated in Figure 4.4 
below. The analyses gave a standard deviation of ?2.7 %. 
 56
  
0
 2
 4
 6
 8
 10
 12
 14
 16
 1 2 3 4
 Route
 Ca
 rb
 o
 hy
 da
 tr
 e 
co
 n
 ce
 n
 tr
 at
 io
 n
 s(g
 /1
 00
 m
 l)
 Maltotriose
 Maltose
 Fructose
 Glucose
  
 
Figure 4. 4: The effect of mashing routes on fermentable carbohydrates 
extraction. 
 
Figure 4.3 shows that the total mashing time for route one was 75 minutes, and 
this yielded 9.93g/100mL of fermentable carbohydrates, whereas total mashing 
time and consequently fermentable carbohydrates were higher for routes two, 
three and four. Total mashing time for route two and three were 105 minutes and 
133 minutes, respectively, and their fermentable carbohydrate yields were 
13.86g/100ml and 13.82g/ml, respectively. These observations suggest that the 
higher the mashing time the higher yield of fermentable carbohydrates, and also 
suggest that the extended mashing time from 105 minutes to 133 minutes was 
unnecessary, as it did not bring any significant increase in concentration of 
fermentable carbohydrates. Route two was selected as the optimum mashing route 
as it extract higher carbohydrates and has less mashing time than route four.  
 
 57
 Further wort samples for beer quality analysis were prepared from the optimised 
mashing process that was selected from the carbohydrate analysis above. It should 
be kept in mind that the established optimised conditions may vary depending on 
water quality, ambient temperature, and equipment design and probably on 
different malted barley. The optimised experimental conditions were a ratio of 3.5 
L of mash water to 1kg of malt and mashing route as discussed in the preceding 
section. 
 
4.2. Beer quality Analysis 
4.2.1 Determination of phenolic acids in wort from different malts 
Determination of phenolic acids in wort and beer was performed by using high 
performance liquid chromatography (HPLC) coupled with UV-vis detector. 
Phenolics display absorbance at different wavelengths and hence there is no single 
wavelength perfect for monitoring all phenolics [36]. Elution was monitored at 
280nm as it is suggested to be the best alternative for the determination of the 
most phenolic compounds [37]. Qualitative and quantitative analysis of phenolic 
acids were performed on three different malts and Figure 4.1 showed one of the 
selected chromatograms. Three runs per malt were performed and the standard 
deviations were ?7.2 %, ?10.5, ?14.3 % for malt A, B and C, respectively. 
Concentrations of phenolic acids obtained from each malt were calculated and 
Figure 4.5 below shows the amount of phenolic acids detected from different 
types of malts. 
 58
 0
 5
 10
 15
 20
 25
 Malt A Malt B Malt C
 Different malts
 Co
 n
 c
 en
 tr
 a
 tio
 n
  
o
 f a
 c
 id
 s
 (?g
 /m
 L)
 1
 2
 3
 4
 5
 6
 7
 8
 9
 10
 11
  
 
Figure 4. 5: Comparison of individual phenolic acid from different malts. 
 
Table 4. 1: Concentrations of various phenolic acids from malt A, B and C, 
respectively. 
 
  Concentration(?g/mL) 
  Acid Malt A Malt B Malt C 
1 Vanillic acid 0.640 0.447 0.360 
2 Caffeic acid 1.353 0.592 0.612 
3 Syringic acid 0.180 0.814 0.540 
4 p-Coumaric acid 1.920 1.790 1.440 
5 o-Coumaric acid 1.188 0.606 0.360 
6 Ferulic acid 1.664 1.645 0.936 
7 Sinapic acid 0.216 0.244 0.324 
8 Isoferulic acid 0.252 0.492 0.324 
9 M-Coumaric acid 0.416 0.242 0.144 
10 p-Hydrobenzoic acid 23.680 17.899 14.400 
11 Gallic acid 1.740 0.671 0.540 
 Total 33.249 25.443 19.980 
 
The total concentration of phenolic acids in each malt were 33.25 ?g/mL, 25.44 
?g/mL, 19.98 ?g/mL for malt A,B and C respectively as shown in Table 4.1. 
Key: 1- Vanillic acid; 2- Caffeic acid; 3- Syringic acid; 4- p-Coumaric acid; 5- o-Coumaric acid; 6- Ferulic 
acid; 7- Sinapic acid; 8- Isoferulic acid; 9- M-Coumaric acid; 10- p-Hydrobenzoic acid; 11- Gallic acid. 
Key 
 59
 Figure 4.5 shows that p-Hydrobenzoic acid is the most abundant phenolic acid 
across all malts as it contributes more than 65% towards the total phenolic acid 
content. The experiment confirmed that the three malts have different phenolic 
acid content and hence the analysis of the wort produced from the malts can give 
the effect of phenolic acids on parameters under investigation. 
 
4.2.2 Hydroxyl-free radical in different malts 
 
It has been established that more than 80% of phenolic acids in beer are extracted 
from the raw material, malted barley [16]. Recently, research was carried out which 
relates flavour stability of the beer to generation of hydroxyl free-radicals which 
can be measured by Electron Spin Resonance (ESR) [9, 10]. ESR with spin trapping 
method is used to detect the free radicals detected during an oxidative forcing test.  
The advantage of this method is that hydroxyl free radicals can be measured at an 
early stage of beer by the oxidative forcing test, and hence saving time.  
 
Thus in this work, wort samples were analysed. Analysing the wort instead of the 
final beer does not only save time, but eliminates other variables that can be 
introduced at other stages of beer production such as fermentation, clarification, 
addition of yeast and hops, etc. Wort was aged at 70oC and samples we taken and 
analysed after every 60min. The relatively long forcing test of the wort samples 
was due to the fact that with wort, the OH-radical is detected immediately and lag 
phase need relatively long period to be identified [10]. ESR was used to quantify 
 60
 the OH-radical generated for each malt investigated; Figure 4.6 shows the typical 
recorded EPR spectrum 
  
This section reports on the data taken using a Bruker ESP-300E continuous wave 
electron paramagnetic resonance (cw-EPR) spectrometer operating in the X-band.  
A summary of the results and a brief analysis is provided.   
 
Figure 4.6 shows a typical recorded EPR spectrum at room temperature.  It should 
be noted that this is the derivative of the EPR spectrum.  The first measurements 
indicated that we might expect a signal from hydroxyl (OH?) radicals. 
 
Figure 4. 6:  EPR response of the sample as a function of magnetic field. 
 61
 The data have been fitted to a single Lorentzian.  As can be seen, the single 
Lorentzian does not describe the data accurately.  It is suggested that this is 
evidence of at least two inequivalent sites for the paramagnetic species.  The g-
 factor for these two lines are ? 2. 
The following conclusions may be drawn from the EPR spectrum: 
 
? The paramagnetic centre is exhibiting emission rather than absorption of the 
microwave radiation, which indicates that the paramagnetic centre is in an 
excited state.  The reasons for this may be apparent due to the fact that OH-
 radical is in an excited state. 
? A single Lorentzian line does not describe the emission line fully.  This 
suggests that there are at least two sites for the paramagnetic species.  If the 
paramagnetic centre is indeed OH?, then we may be able to ascribe this to a 
hyperfine interaction between the magnetic moment of the ion and the 
hydrogen nucleus. 
? The zero point of the derivative resonance line (resonance field of 
approximately 3479 G) corresponds to a paramagnetic species with g ? 2.  
Accurate determination of the g-factor will require a reference sample to be 
measured simultaneously with the sample.  This particular experiment will 
be performed in the future. 
 
Attention now turned to examining the response of the sample as a function of the 
microwave power.  Using this approach one is able to estimate the spin-lattice 
 62
 relaxation time (T1) of the paramagnetic species.  The results of this measurement 
are summarized in Figure 4.7 and Figure 4.8. 
 
Figure 4. 7: The peak-to-peak amplitude of the derivative curve (?M) as a 
function of microwave power. 
 
The data have been fitted to a power curve of the form ?PMM 0=? , where P is 
the microwave power, and M0 and ? are fit parameters. The results of this fit 
indicate that ? ? 0.5.  This provides conclusive proof that the line shows no sign of 
saturating at the highest microwave power employed.  T1 at room temperature is 
therefore extremely short (of the order of ns). Once again, changing the 
temperature would be informative. 
 
 63
 The peak-to-peak field difference or linewidth (?B) was extracted from the 
results, and these are shown in Figure 4.8.  It should be borne in mind that the 
signal-to-noise ratio deteriorates significantly at low microwave powers. 
 
Figure 4. 8: The linewidth (?B) as a function of microwave power. 
 
The error bars shown are the maximum error estimate for the low power 
measurements (approximately 10%).  The data indicate that the linewidth remains 
constant over the power range observed, and we have extracted the mean and 
standard deviation giving 11.044.2 ?=?B  G. 
 
 
 64
 Figure 4.9 shows the hydroxyl radicals generated from each of three different 
malts with arbitrary units. 
0
 2
 4
 6
 8
 10
 12
 5 55 105 155 205
 Time(minutes)
 O
 H-
 ra
 di
 ca
 l g
 en
 er
 at
 ed
 (ar
 bi
 tr
 ar
 y 
u
 n
 its
 )
 Malt A
 Malt B
 Malt C
  
Figure 4. 9: Generated OH-radical from three different wort samples as detected 
by the ESR.  
 
Figure 4.9 shows that the hydroxyl radicals were detected immediately in all three 
malts under investigation. With beer samples, the definite period after which the 
hydroxyl radical (OH-radical) were generated after forced test is called ?lag time? 
of OH-radical generation [9, 10]. With wort samples, because the OH-radicals are 
identified immediately, there is no observed ?lag time? and hence the quantity of 
OH-radical generated can be used as an index [10]. The lag time or quantity of the 
OH-radical generation is related to an endogenous antioxidant activity of beer 
(EA value) which is related to flavour stability [9]. Three wort samples generated 
similar amounts of OH-radical over a period of 180minutes as seen in Figure 4.9. 
Although the samples have different phenolic acid quantities, Figure 4.9 suggest 
that their endogenous antioxidant activity decrease at the same rate. The results 
imply that there is no relationship between the quantity of phenolic acids and the 
 65
 oxidative flavour stability of beer. The results can be linked to the fact that 
although some of the phenolic acids found in beer are detrimental to beer flavour 
during their act as pro-oxidants, some of them are known to act as antioxidants 
and free radical quenchers [16, 43], and the overall effect is zero in terms of 
oxidative influence on the beer.  
 
4.2.3 Effect of phenolic acids on beer appearance (colour, clarity, beading 
and foam) 
 
Pictures of the three beers that were produced by a brewery from three different 
malts investigated are shown in Figure 4.10. 
 
Figure 4. 10: Beer samples produced from the researched malts by SAB [39]. 
 
Figure 4.10 shows that the three beers have different colours, this was expected 
due to the fact that the three investigated malts have different malt colour and the 
primary colour of the beer is derived from the colour of the malt [26]. It should also 
Malt A 
Malt B Malt C 
 66
 be noted that the above beers went through hop addition, fermentation and 
clarification during their production by the brewery. Hence different content of 
phenolic acids in three malts cannot be accountable for varying beer colour. The 
beading and foam of the final beer is primarily from the yeast and long lagering of 
the beer [26]. Although all the three beers went through clarification, there is an 
increasing trend in haze appearance with increasing phenolic content. Increasing 
haze appearance with phenolic content can be due to the fact that polyphenols 
play a role in haze formation [44]. 
 
4.2.4 Decarboxylation of FA during mashing 
 
The formation of 4VG by thermal decarboxylation of FA during mashing was 
shown in Figure 2.4, while Equation 2.3 presented the dependence of mashing 
temperature on mashing time, activation energy and the concentrations of 4VG. 
However, since 4VG and CO2 concentrations (Figure 2.4) were not measured in 
the study, the temperature profile of route four reported in Figure 4.3 was used to 
evaluate the kinetics of decarboxylation of FA during mashing. The plot of 1/T 
versus lnt is presented in Figure 4.11 and it gives a negative slope as predicted in 
Equation 2.3, with activation energy (Ea) = 209 KJ/mol and rate constant (k) = 4.6 
x10-4mg/L min.  These numbers are comparable to similar data reported by 
Vanbeneden et al. 22 at temperature of 90oC. 
 67
  ? = -0.0057
  ? =  0.0395
 0.000
 0.005
 0.010
 0.015
 0.020
 0.025
 0.030
 0 1 2 3 4 5 6 7
 Ln(t)
 1/
 T 
(o C
 -
 1 )
 0.00
 0.05
 0.10
 0.15
 0.20
 0.25
 0.30
 0 50 100 150 200
 Time (min)
 4V
 G
  
(pp
 m
 )
 Route 3
 Model
 Literature
 Eq. 3
  
Figure 4. 11: Effect of thermal load during mashing and wort heating on FA 
degradation and 4VG Formation. 
 
Figure 4.11 presents the comparison of the predicted kinetics (Equation 2.1) of 
concentration of 4VG with time and the literature 43 at 90oC. A good fit between 
the literature and the model is shown on the secondary axes of Figure 4.11. These 
results support the fact that the optimised mashing temperature of 900C was 
adequate to form 4VG by thermal decarboxylation of the hydroxycinnamic acids 
(i.e., the FA in the malts studied was also found to be a first order reaction 
(Equation 2.1)). It is obvious from Figure 4.11 that the experimental data fits the 
model quite well. 
4.2.5 Effect of fermentation temperature on fermentation rate 
 
A study of the effect of fermentation temperature on the rate of fermentation was 
carried with constant yeast strain (Saccharomyces cerevisae) by varying 
 68
 fermentation temperatures. The effect of this parameter is illustrated in Figure 
4.12. 
 
0
 2
 4
 6
 8
 10
 12
 14
 0 20 40 60 80 100 120
 Ap
 pa
 re
 n
 t E
 x
 tr
 ac
 t(o
 P)
 Fermentation(h)
 15oC
 18oC
 22oC
 29oC
  
Figure 4. 12: Change in fermentation rate with fermentation temperature. 
 
The results (Figure 4.12) suggest that for the yeast strains and materials studied 
here, the rate of conversion of fermentable sugars in to alcohol is dependent on 
temperature. At low temperatures of 15oC to 22oC, there is an insignificant change 
in the rate of fermentation. High fermentation rate is achieved at temperature of 
29oC. Interestingly, the apparent Extract at 29oC remains higher at 4.3 as 
compared to those at lower fermentation temperatures. This means that although 
at low temperatures the fermentation rate is low, yeast cells continue to function 
for a long time. Lower conversion at higher fermentation temperature means that 
high yields of yeast cells loose their viability due to high temperature. This is in 
accordance with what was reported that yeast viability decreases as the 
temperature increases [45].  This decrease in yeast viability is due to production of 
cell toxity due to greater accumulation of intracellular ethanol at high 
 69
 temperatures [45] that alter the structure of membrane and decrease its functionality 
[46]
 . This implies that temperature variance with the studied materials does provide 
viable route for process optimisation. The primary effect of increasing 
temperature is to increase fermentation rate, but it negatively affects viability of 
yeast cells.  
 
4.2.6 Effect of kettle finings on fermentation rate 
 
Figure 4.13 shows fermentation rates associated with and without addition of 
kettle finings. For the solution with kettle finings, 5g of Kalagines was added 
during boiling to enhance flocculation. The experiments were carried out at 
constant temperature of 29oC and pH 5.3 using Saccharomyces cerevisae.  
0
 2
 4
 6
 8
 10
 12
 14
 0 10 20 30 40 50 60 70 80 90 100 110
 Fermentation Time(h)
 Gr
 av
 ity
 (o
 P)
 Added Finings
 No Finings
  
Figure 4. 13: Effect of kettle finings on fermentation rate. 
 
 70
 Kettle finings enhance coagulation of ?trub? and thus remove some amount of 
proteins in the wort to the fermentation tank. The results indicate slight effect of 
kettle finings on the fermentation rate. Improved wort clarity obtained by addition 
of finings could have negative effect on fermentation by removing proteinaceous 
material required for yeast growth during fermentation [47]. Obtained results 
indicate the possibility that these finings might have removed trub that is not 
being used by the yeast, rather than the protein in the form of amino acids and 
small peptides which the yeast utilizes.  
 
4.2.7 Optimisation of fermentation by varying pitching rates 
 
Figure 4.14(a) and Figure 4.14(b) shows variation of fermentation rates with 
pitching rates at temperatures 25oC and 16oC respectively. Investigated pitching 
rates showed insignificant effect on fermentation rates for both temperatures.  
 
 71
 0
 2
 4
 6
 8
 10
 12
 14
 16
 0 20 40 60 80 100 120
 G
 ra
 v
 ity
  
(o
 P)
 Fermentation Time(h)
 D E F
 0
 2
 4
 6
 8
 10
 12
 14
 16
 0 20 40 60 80 100 120
 Fermentation Time(h)
 G
 ar
 v
 ity
  
(oP
 )
 A B C
  
Figure 4. 14: Fermentation rates at different pitching rates (a) at Temperature 
25oC and (b) at Temperature 16oC. Pitching rates used are (A, D) 1x106 cells/mL, 
(B, E) 2x106 cells/mL, (C, F) 3x106 cells/mL. 
 
Contrary to high gravity brewing, fermentation rates did not increase in order of 
increased pitching rates. Various studies in high gravity brewing have reported 
that higher pitching rates led to faster fermentations [48, 49]. However, the results 
obtained are in agreement with reported results by O?Connor-Cox and 
Ingledew[49], that at significantly higher pitching rates (8x107 cells/mL), higher 
fermentation rates are obtainable as compared to insignificant shift in 
fermentation rates at lower pitching rates (1x107 cells/mL).  
(a) 
(b) 
 72
 4.2.8 Clarification of beer by finings (Kalaginess) 
 
Figure 4.15 shows that kettle finings (kalaginess) added during wort boiling have 
an effect on the final colour of beer. Kettle finings are used extensively to remove 
proteinaceous material during wort boiling.  
 
0
 5
 10
 15
 20
 25
 0 2 4 6 8 10 12 14
 kalainess(g/100L)
 Be
 er
  
Co
 lo
 u
 r(E
 BC
 )
  Figure 4. 15: Effect of added finings during wort boiling on final beer colour. 
 
Figure 4.15 shows that for 100Litre batches, an optimal amount of kalaginess 
finings to be added during wort boiling was approximately 5grams as it can be 
seen that beyond this amount there was no improvement in final beer colour. 
Obtained results are in agreement with reported literature that sedimentation of 
particles exhibiting a negative charge is often enhanced by using finings [50]. Since 
a number of operational conditions have been optimised, proper documentation 
for commissioning of the Wits Micro-brewery can be developed as discussed 
next. 
 73
 CHAPTER FIVE 
5. Wits Micro-Brewery Commissioning 
 
Through thorough observations during plant commissioning at Wits Micro-
 brewery plant, the following documents were developed to enhance its 
commissioning. The document addresses important issues which must be given 
careful attention by the brewer, as appropriate, in order to best ensure smooth 
execution of process leading to a successful brew.  
 
Good record keeping during phases of pre-commissioning and commissioning is 
essential in every production industry. Analysis of commissioning strategies at 
Wits Micro-brewery plant showed that verbal instruction is always open to error, 
while the written instruction or report is generally not open error. Through use of 
the developed commissioning documents, errors during brewing have been 
reduced. In the fast tempo of pre-commissioning and commissioning, it was found 
that proper discipline to ensure good record-keeping must be maintained at all 
times. It should be accepted as ?a way of life? rather than ?an unusual effort?. It 
must be remembered that good record-keeping provides a very valuable history of 
the pre-commissioning and commissioning of the brewery, which serves as a 
future reference for the plant, and of similar plants.  
5.1 Commissioning Checklist 
The developed checklist document list issues that must be addressed for any brew, 
should comprise important activities which must be considered at the outset of the 
brew, and receive ongoing attention during brew execution. 
 74
  
 
 
WITS MICRO-BREWERY COMMISSIONING MANUAL 
BREWING No: 001 
DATE OF BREW: 16 April 2008 
 
VALIDITY OF DOCUMENT 
This document will be valid: 
A) If completed by the brew leader and validated by the supervisor. 
B) Validity automatically expires two weeks after verification. 
 
AUTHORIATION 
 
VERIFICATION DATE: 14 April 2008 
 
VERIFIED BY: E.M Madigoe        Date: 14 April 2008   Sign: 
 
APPROVED BY: Prof. S.E Iyuke??Date: 15 April 2008    Sign???. 
 
Wits University Micro-Brewery Plant 
 75
 5.2 Preparation for commissioning 
1. Setting of early brew requirements 
  -Define responsibilities and roles of all involved parties; 
 -Ensure safe working practices are in place for commissioning; 
 -Ensure all safety and environmental considerations are met; 
 -Participants understand knowledge of detailed operating instructions; 
 -Instrumentation verified; 
 -All feedstock and utilities are available. 
 
2. Effluents 
 -Effluents defined and specified; 
 -manner of disposal in place; 
 -disposal destination agreed with all parties concerned; 
 -handling of spillages; 
 -contingent means of disposal for off-spec products. 
 
3. Requirements for chemical cleaning 
 -Early definition and understanding for chemical cleaning; 
 -careful setting and understanding of criteria required of level of 
cleanliness; 
 76
  -use reputable chemical products; 
 -address also requirements for degreasing, e.g., of stainless steel. 
4. Early utilities 
 Establish availability for essential utilities: 
 -Water 
 -Electrical 
5. General matters 
 -Install and connect all system components and verify their conformance; 
 -do dry-run to ensure good condition of all equipments; 
 -check thermo-couples for proper joining of wires, position of elements 
and proper polarity; 
 -do full-plant chemical cleaning and disinfection; 
 -perform plant layout check for freedom of movement; 
 -ensure that all plumbing, electrical and water installations are working; 
 -No overload of electrical circuits; 
 -enough air conditioning; 
 -ensure enough PPE is available for all brewers; 
 -describe beer profile agreed on.  
 
 
 77
 ACTION OK/ 
COMPLETED 
NOT OK/REMARKS SIGN 
A. KETTLES    
1. Heat up at 1oC/min 
 
X   
2. Ensure no water leaks 
 
X Small leak on valve  
3. Ensure electrical cables not exposed. 
 
X   
4. All control devices on kettle working properly X   
5.3 Action Plan 
 78
 5. Set point in accordance with output  Auto heater not working, use manual 
operation 
 
B. MASH LAUTER TUN    
1. Sieve free from old grains X   
2. All control devices on kettle working properly X   
3. Ensure electrical cables not exposed X   
4. Ensure no water leaks 
 
X   
C. REFRIGERATION SYSTEM    
1. Is the refrigeration circuit functioning properly X   
2. Is the refrigerant enough for operation X   
3.Set-point temperature in accordance with the output values X   
 79
 D. PUMPS    
1. Install mechanical seals if required X   
2.Ensure pumps deliver proper capacity X   
3. Ensure pump start and motor works X   
4.Ensure impellers running in the right direction X   
E. PIPING AND HEAT EXCHANGER    
1.Check process lead piping for proper tightness X   
2.Ensure pipes and heat exchanger are visibly clean X   
3.Piping joints/fittings properly installed X   
F.ELECTRICAL    
1.Ensure the plant is not contacting live electrical conductors X   
2. Ensure there is no overload of electrical circuits X   
 80
 3.Ensure there are no damaged or poorly maintained electrical 
leads or cables 
X   
G.RAW MATERIAL    
1. Ensure there is enough grains for the brew X   
2.Yeast ready for brew X   
3.Hydrometer, pH meter, thermometer available X   
4.sample collectors available X   
H. FERMENTER    
1.Check process lead piping for proper tightness X   
2. All control devices on fermenter working properly X   
3. Ensure electrical cables not exposed X   
4. Ensure no water leaks X   
 81
 5.4 Brew Sheet 
Developed brew sheet will ensure that essential data is recorded during brewing to 
provide sufficient data during results analysis and for ease of information accessibility 
for future references.  
 
WITS MICRO-BREWERY BREW SHEET 
 
Date: 08 June 2008? Brew No:  001        Beer Style: American Pale Ale 
 
MASHING 
 
FEED 
Raw Material Amount Volume 
 Pale Malt  13kg   
Crystal Malt   2kg   
 Black malt  200grams   
      
 
MASH ADDITIONS 
Add Amount Time 
 CaSO4  75grams  09:40 
      
      
 
 
 
 
 
 
LAUTER TUN 
 
SPARGING 
Start Finish Volume 
 11:05  11:09  2L 
      
 
 
 
 
 
PARAMETERS 
Mash Start Time  09:00 
Mash end time  10:30 
Total Mashing Time  90min 
Mash liquor Volume  60L 
Mash start ToC  66 
Mash end ToC  57 
WATER QUALITY 
  Amount 
Carbon   
Bicarbonate   
Chlorine   
pH  7.8 
ToC  70 
WORT CIRCULATION 
Flowrate Start Finish 
 0.5L/min  10:35 11:00  
      
 82
 RUN-OFF 
Flowrate Start Finish 
 0.5L/min  11:15  11:45 
      
 
RUN-OFF 
pH ToC Volume collected 
 3.7  55  70Litres 
 
 
WORT BOILING 
 
HOP ADDITION 
Hop Type Amount Time 
 Pre-
 isomerised 
blend  60grams  12:00 
 Amarillo  100grams  12:15 
 Cascade  100grams  12:40 
 
 
 
FERMENTATION 
 
INPUTS 
Initial Volume  60L 
OSG  1.06 
Initial pH  5.2 
Initial ToC  16 
WORT QUALITY 
Gravity Time   
 1.09  11:15 1st Runnings 
 1.08  11:30 2nd Runnings 
 1.06  11:40 3rd Runnings 
 1.02  11:45 Last Runnings 
WORT QUALITY 
Gravity Time pH 
 1.06  12:40  4.8 
      
      
      
ADDITIONS 
Add Amount Time 
      
      
 
 
 
 
  
BOILING 
Boiling Start 11:50  
Boiling end  13:00 
Initial Volume  70L 
Final Volume  60L 
Run-off time  13:10 
Run-off rate  0.8L/min 
 83
  
 
 
5.5 Fermentation Progress 
 
Fermentation progress report document will help to early detect any fermentation 
problems easily. This will ensure that early measures are taken to prevent brew waste.  
 
FERMENTATION PROGRESS 
YEAST 
Type 
 Saccharomyces 
cerevisae 
Volume 1.2L  
Time Added  14:00 
Time            
ToC 16 15 17 16.3 16.6 17 16.4 16.6 16.4 16.3 16.3 
pH 5.3 5 4.87 4.82 4.83 4.75 4.70 4.76 4.8 4.82 4.83 
SG 1.06 1.05 1.044 1.041 1.038 1.036 1.032 1.030 1.029 1.030 1.030 
DO(mg/L) 1.3 0.843 0.84 1.71 1.72 1.98 1.48 0.94 1.29 0.86 1.45 
Alcohol            
Colour            
Other            
 84
 CHAPTER SIX 
6. CONCLUSION AND RECOMMENDATIONS 
 
It can be stated, that the water to malt ratio and the mashing route can influence the 
concentration of malt extracts. Increasing the mashing water of the ratio (water, L: malt, 
kg) increases the concentration of fermentable carbohydrates, but only up to certain 
optimum point where extractable sugars have dissolved and the concentration decreases 
when more water is added. From the mashing routes investigated it can be concluded 
that the longer the mashing time, the more fermentable carbohydrates are extracted. 
From the malts studied which had different phenolic acid amounts, it can be concluded 
that different amounts of phenolic acids have same effect on oxidative flavour stability 
of beer. From the observation of beers produced from malts with different phenolic acid 
content, it can be concluded that phenols are responsible for haze formation in the 
appearance of beer. There are many different mashing systems that can be used; hence 
the investigation has shown that various things need to be taken into consideration when 
designing mashing profile.  
 
Values up to 1.45 ppm p-coumaric acid, 13.10 ppm ferulic acid, 4.65 ppm sinapic acid, 
2.70 ppm 4-vinylphenol and 4.37 ppm vinylguaiacol [51] are common concentrations of 
phenolic compounds in beer. Meilgaard [52] reported that the flavour threshold in beer is 
52 ppm for p-coumaric acid, 66 ppm for ferulic acid (FA) and 0.3 ppm for 
vinylguaiacol. Hence, in this study where the phenolic compound concentrations ranged 
 85
 from 0.18 ppm syringic acid in Malt A, to 23.68 ppm p-hydrobenzoic acid in Malt A, 
they exist within the range reported. Thus this study has shown that there is no direct 
correlation between phenolic acids and oxidative flavour stability of beer while the 
corresponding volatile phenols may affect beer flavour. The concentrations of most of 
the phenolic compounds detected in the study should contribute to the sour, bitter and 
astringent flavours that may be associated with beer prepared from Malt A, B and C. 
The results obtained in this study provide information that is necessary in the control of 
the quality and stability of beer. Since the low molecular weight phenolic compounds 
may affect the sensory properties of the beer, the choice of variety and processing 
conditions are important in determining the quality of the beer.  
 
Fermentation rates increased with an increase in temperature. Hence was concluded that 
optimised fermentation rates can be achieved by increasing fermentation temperature 
within such range when the cells remain viable. Clarity of beers increased with an 
increase in finings up to a certain point, and finings show a slight effect on fermentation 
rates. The results obtained in the study helped to locate the optimum amount of kettle 
finings for beer clarification. Pitching rates investigated showed an insignificant effect 
on fermentation rates, from the study it was concluded that fermentation rates are 
affected by significantly high pitching rates. 
 
Developed commissioning documents helped in reduction of errors during brewing, 
hence it was concluded that good record keeping and documentation is of essential need 
for successful commissioning. 
 86
 The following are recommended for further studies: 
? Increase the difference in phenolic acid content among different malts by 
addition of external phenolic acids because it is suspected that the difference in 
phenolic concentration among our samples was miniature to certain extends. 
? Investigate other methods and or procedures or conditions that can be used to 
detect the phenolic acids in wort and beer with higher precision to prevent major 
losses in phenolic bounds. Addition of ascorbic acid, antioxidant, and 
ethylenediaminetetraacetic acid (EDTA) and a metal chelator can completely 
prevent the loss of phenolics.  
? Further studies are necessary to determine the metabolism of the phenolic 
compounds by lactic acid bacteria and yeasts involved in the preparation of the 
fermented beverage and the effects of any metabolites on overall beer quality 
and stability. 
 87
 REFERENCES 
 
[1] Sten, A. and Nanneman, W. (1999), Cost effective choice of raw materials, 
Master Brewer Association Technical Quarterly.36 (2), 195-197. 
[2] Hornsey, I.S. (2003), A history of beer and brewing, Royal Society of Chemistry, 
Great Britain, 1-160. 
[3] Irwin, A.J., Barker, R.L and Pipasts, P., (1991), The role of copper, oxygen, and 
polyphenols in beer flavour instability, Journal of American Society of Brewing 
Chemists, 49, 140-149. 
[4] Burrell, K.J, Gill, C. Mckenzie, M.T and Murray, J. (1994), Advance in 
separation technology for the brewer: ceramic cross-low filtration micro-
 filtration of rough beer, Master Brewer Association Technical Quarterly. 31 (2), 
42-50. 
[5] Schieberle, P. (2005), Formation of stale flavour compounds in beer: influence 
of raw materials, processing conditions and storage., 30th European Brewery 
Convention, Congress Presentations, June 2005., Prague. 
[6] Nardini, M. and Ghiselli, A, (2003), Determination of free and bound phenolic 
acids in beer, National Institute for Food and Nutrition Research, 546, 137-143. 
[7] Dominic, S., Jacek, P and Zdzilaw, T, (2005), The release of ferulic acid and 
feruloylated oligosaccharides during wort and beer production, Journal Institute 
of Brewing, 111(4), 372-379. 
[8] Bamforth, C. W., Muller, R. E. and Walker, M. D, (1993), Oxygen and oxygen 
radicals in malting and brewing: A review, Journal of American Society of 
Brewing Chemists, 51, 79-88. 
 88
 [9] Uchida, M. and Ono, M, (1996), Improvement of oxidative flavor stability of 
beer-role of OH-radical in beer oxidation. Journal of American Society of 
Brewing Chemists, 54, 198-204. 
[10]  Uchida, M., Suga, S. and Ono, M (1996), Improvement for oxidative flavor 
stability of beer-rapid prediction method for beer flavor stability by electron spin 
resonance spectroscopy ,  Journal of American Society of Brewing Chemists., 
54, 205-211. 
[11]  Kaukovirta-Norja, A., Virtanen, H., Poyri, S., Lehtinen, P., Hartwall, P., 
Reinikainen, P., Siirila, J and Home, S., (2005), The increase of antioxidativity 
during mashing-does it improve beer flavour stability?, 30th European Brewery 
Convention, Congress Presentations, June 2005,  Prague. 
[12] Ough, F., Fermentation rates of grape juice.III. Effects of initial ethyl alcohol, 
pH and fermentation temperature., (1966), American Journal of Ecology and 
Viticulture, 17, 74. 
[13]  Linkens, H.F and Jackson, J.K, (1988), Modern methods of plant analysis, 
Springler-Verlag, Australia, New series ,Vol.7, 109. 
[14]  Siobhan, M., Sangryoul, P., Craig, E.L and Malcolm, R.S, (1998), Detection of 
phenolic acids in beverages by capillary electrophoresis with electrochemical 
detection, The Analyst, 123, 1931-1936. 
[15]  Asano, K., Ohtsu, K., Shinagawa, K. and Hasimoto, N., (1986), Affinity of 
proanthocyanidins and their oxidation products for haze-forming proteins of 
beer and the formation of chill haze, Research Laboratory of Kirin Brewery. 
Co., 29, 31-38. 
 89
 [16]  Bvochora, J.M., Danner, H., Miyafuji, H., Braun, R and Zvauya, R., (2004), 
Variation of phenolic compounds during the preparation of opaque beer, Process 
Biochemistry., 40(2005), 1207-1213. 
[17]  De Stefano, A. and Montanari, L., (1996), Minor components of beer: a review, 
Alcolgia, 8, 43-45. 
[18]  Vanbeneden, N., Gils, F., Delvaux, F. and Delvaux, F. R., (2008), Formation of 
4-vinyl and 4-ethyl derivatives from hydroxycinnamic acids: Occurrence of 
volatile phenolic flavour compounds in beer and distribution of Pad1-activity 
among brewing yeasts, Food Chemistry, 107, 221?230. 
[19]  Floridi, S., Montanari, L., Marconi, O., and Fantozzi, P. Determination of free 
phenolic acids in wort and beer by coulometric array detection, (2003), Journal 
of Agricultural and Food Chemistry, 51(6), 1548?1554. 
[20]  Fiddler, W., Parker, W. E., Wasserman, A. E., and Doerr, R. C. Thermal 
decomposition of ferulic acid., , (1967), Journal of Agricultural and Food 
Chemistry, 15, 757?761. 
[21]  Clausen, M., Lamb, C. L., Megnet, R., and Doerner, P. W. PAD1 encodes 
phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in 
Saccharomyces cerevisiae., (1994), Gene., 142(1), 107?112. 
[22] Vanbeneden, N., Delvaux, F., and Delvaux, F. R. Determination of 
hydroxycinnamic acids and volatile phenols in wort and beer by isocratic high-
 performance liquid chromatography using electrochemical detection. (2006), 
Journal of Chromatography A., 1136(2), 237?242. 
 90
 [23]  Nord, L. I., Vaag, P and O? Duus, J., (2004), Quantification of organic and 
amino acids in beer by NMR spectroscopy. Analytical Chemistry, Carlsberg 
Laboratory, 76, 4790-4798. 
[24]  Harbone, J.B., (1964). Biochemistry of phenolic compounds, Academic Press. 
London and New York, 2-18. 
[25]  Dadic, M, (1976). Current concepts on polyphenols and beer stability. Master 
Brewer Association Technical Quarterly. 13, 182-189. 
[26]  Simpson, W. J., (2001). Good malt-good beer?., 10th Australian Barley 
Technical symposium, June 2001. 
[27]  Meilgarrd, M., (1972), Stale flavour carbonyls in brewing ? a review. Brewers 
Digest, 47(4), 48-52. 
[28]  Hudson J.R., Effect of mashing on the character of beer, (1969), Brewers 
Digest., November, 96-100.  
 
[29]  Manners, D.J., Cereal Food world, (1985), 30, 772. 
[30]  Sjoholm, K., Macri, L.J. and MacGregor, A.W., (1995) Proceedings of the 
European Brewery Convention Progress, Brussels, 1995, 277. 
[31]  Hackensellner, T., (2000), Efficient energy of use in the Brewhouse, The 
Huppman Group, Kitzingen, Germany. 
[32] Bird, R.B., Sterwart, W.E., Lightfoot, E.N., (2002), Transport Phenomena, 
second edition, John Wiley & Sons, Inc, USA, 148. 
[33]  Burrell, K.J. Gill, C. Mckenzie, M.T and Murray, J. (1994). Advance in 
separation technology for the brewer: ceramic cross-low filtration micro-
 91
 filtration of rough beer, Master Brewer Association Technical Quarterly.31 (2), 
42-50. 
[34]  Mundy, A.P and Boley, N. A survey of instrumentation used for the 
determination of haze in beer. (1999) Journal Institute of Brewing, 105(2)., 75-
 78. 
[35] Bamborth, C.W., Beer haze, (1999), Journal of American Society of Brewing 
Chemists, 57(3), 81-90. 
[36]  Schwill-Miedaner, A., Einsiedler, F and Sommer, K, (1998), Barley endosperm 
cells walls: A review of cell wall polysaccharides and cell wall degrading 
enzymes. Master Brewer Association Technical Quarterly., 14(4), 238-243. 
[37]  Paul, S., (2000), Malts Brewing Room Book, 1998-2000, 243. 
[38]  Siebert, K.J., Blum, P.H., Wisk, T.J., Stenroos, L.E and Anklam, W.J., (1986), 
Master Brewer Association Technical Quarterly, 23, 37. 
[39] Iyuke, S.E., Madigoe, E.M., Maponya, R., The Effect of Hydroxycinnamic 
Acids and Volatile Phenols on Beer Quality, (2008), Journal Institute of 
Brewing, 114(40), 300-305. 
[40] Izydorczyk, M. S., Macri, L.J and MacGregor, A.W., (1998), Carbohydrate 
Polymers., 35, 249-258. 
[41]  Bierman, U., (1991), Composition of Wort Fermentation, 4, 229-231. 
 
[42]  Nagodawithana, T.W., Castello, C., Steinkraus, K.H., (1974)., Effects of 
dissolved oxygen, temperature, initial cell count and sugar concentration on the 
viability of Saccharomyces cerevisae in rapid fermentations,  Applied 
Microbiology, 28, 383. 
 92
 [43] Eastmond, R., (1947), The separation and identification of a dimer of catechin 
occurring in beer, Journal Institute of Brewing, 80, 188-200. 
[44] Hirotaka, K, Yukinobu, K, Toshihiko, O, Shunro, K and Minoru, K., (1990), 
Effect of free radicals on haze formation in beer, Journal of Agricultural and 
Food Chemistry, 38, 1909-1912. 
[45] Lucero, P., Penalver, E., Moreno, E., and Lagunas, R., (2000)., Internal trehalose 
protects endocytosis from inhibition by ethanol in Saccharomyces cerevisae,  
Applied and Environmental Microbiology, 66(10), 4456- 4461. 
[46]  Siebert, K.J., Blum, P.H., Wisk, T.J., Stenroos, L.E and Anklam, W.J., (1986), 
Master Brewer Association Technical Quarterly , 23, 37. 
[47]  Edelen, L., Miller, L.J., Patino, H., Effects of yeast pitch rates on the 
fermentation performance and beer quality., (1996),  Master Brewer Association 
Technical Quarterly., 331(1), 30-32. 
[48]  Younis, O.S and Stewart , G.G., Sugar uptake and subsequent ester and higher 
alcohol production by Saccaromyces cerevisiae., (1998),  Journal Institute of 
Brewing., 104, 255-264. 
[49]  O? Connor-Cox, E.S.C and Ingledew, W.M., (1991), Alleviation of the effects of 
nitrogen limitation in high gravity worts through increased inoculation rates, 
Journal of Industrial Microbiology & Biotechnology., 7, 89-96. 
[50]  Burrell, K., Flocculation, Fluid dynamics and fining, (1996), The brewer, 82, 59-
 62. 
[51] Wackerbauer, K., Kramer, P., and Siepert, J, Phenolische aromastoffe in Bier., 
(1982)., Brauwelt, , 15, 618?626. 
 93
 [52] Meilgaard, M. C., (1975).,  Flavor chemistry of beer part II: Flavor and 
threshold of 239 aroma volatiles. Master Brewer Association Technical 
Quarterly, 12, 151?168. 
 94
 CHAPTER SEVEN 
7. Publications and presentations during the investigation. 
7.1 Publications 
[1] Madigoe, E.M., Iyuke., S.E, Maponya, R., (2008), The Effect of 
Hydroxycinnamic Acids and Volatile Phenols on Beer Quality, Journal Institute 
of Brewing, 114(4), 300-305. 
[2] Madigoe, E.M., Iyuke., S.E, (2009), Commissioning and Optimisation of Wits 
Micro-brewery, To be submitted for publication. 
7.2 Presentations 
[1] Madigoe, E.M, Iyuke, S.E, Maponya, R.J., (2007), The effect of Organic and 
Phenolic Acids on Beer Quality, Joint Symposium of Chemical and 
Metallurgical Engineering-SAICHE, University of Pretoria, South Africa, , 02-
 03 August 2007. 
[2] Madigoe, E.M, Iyuke, S.E, Commissioning and optimisation of Wits Micro-
 brewery plant, (2008), SAICHE Postgraduate symposium in Process 
Engineering, Sasol Auditorium, Rosebank, 29 September 2008. 
 
 95
 APPENDIX A: Specimen list of symbols 
 
Abbreviations 
HPLC   High Performance Liquid Chromatography 
HACCP  Hazard Analysis and Critical Control Points 
PVPP   Polyvinylpolypyrrolidone 
ESR   Electron Spin Resonance 
EPR   Electron Paramagnetic Resonance 
BU   Bitterness Units 
 
Symbols 
Q   total volume of liquid percolating in unit time 
K   factor describing filter bed permeability 
A   constant cross sectional area 
?h   Pressure drop across column height L 
n   bed porosity (wort volume/bed volume) 
d   effective particle diameter 
k   mash bed permeability 
?P   pressure drop across the mash bed 
?   Viscosity of the wort 
L   mash bed depth 
?   bed porosity (wort volume/ bed volume) 
de   effective particle diameter 
 96
 xi   weight fraction (dimensionless) 
di   particle diameter 
U   Overall heat transfer coefficient 
D   diameter of the particle 
?p   density of particle 
?   density of fluid 
CD   drag coefficient 
t    time of reaction 
Ea   Activation energy 
A   Arrhenius frequency factor 
R   gas constant