Addis Ababa University Addis Ababa Institute of Technology School of Chemical and Bioengineering

ADDIS ABABA UNIVERSITY ADDIS ABABA INSTITUTE OF TECHNOLOGY SCHOOL OF CHEMICAL AND BIOENGINEERING

QUALITY IMPROVEMENT AND SHELF STABILTY OF BEER BY CONTROLLED MALT BLENDING RATIOS WITH RESPECT TO MALT PROTEIN CONTENT

A Thesis Submitted to the School of Graduate Studies, Addis Ababa Institute of Technology, for Partial Fulfillment of the Requirement for the Degree of Master of Science in Chemical Engineering (Food Engineering)

By: - Yidnekachew Hailu

Advisor: - Dr. Eng Shimelis Admassu

(Associate Professor)

Addis Ababa, Ethiopia January, 2020

Declaration

I ,the undersigned, declare that this thesis presented for the attainment of the degree of Master of science in Chemical Engineering(Food Engineering) has been composed solely by myself and has not been presented or submitted ,in part or as whole, for a degree in any other institution or university. The Thesis presented is my original work and all sources of information or materials used for the thesis have been duly acknowledged.

Mr.Yidnekachew Hailu Tadesse Signature: ______Date: ______

Msc Candidate

This Thesis has been submitted for examination with my approval and done under my supervision as University advisor.

Dr.Eng Shimelis Admasu Emire Signature: ______Date: ______

Advisor

The Undersigned member of the thesi9s examining board appointed to examine the thesis of Mr.Yidnekache Hailu is submitted for the degree of Master of Science in Chemical Engineering (Food Engineering) confirmed that the thesis fulfills the requirements of the graduate program and approved it to be accepted.

Approved by the examining Board Signature Date ______

Chairman, School’s Head

Mr.AdamuZegeye ______

Internal Examiner

Dr.Helen W/Gabriel ______

External Examiner

Acknowledgements I am so indebt to my Advisor, Dr. Engineer Shemelis Admasu , for his closest follow-up and leadership next to the Almighty GOD. I would like to offer my sincere thanks to the School of Chemical Bioengineering, Addis Ababa Institute of Technology and BGI Ethiopia Plc, Special thanks goes to the manager, Mr. Workneh Birhanu. I would like to extend my gratitude towards the following individuals too: Ato Kebena Bula, Ato Elias Hailu, Ato Yizengaw Eniyew, Ato Derejen Tilahun, Ato Belachew Assefa, Ato Wasyhun Yihunie, W/o Azeb G / Giorgis and Ato Mesfin Shumye. I would like to thank also my family members for their consistent support and inspiration namely: my mother (W/o Nunu Altaseb), my brothers Ato Abenezer Amdie and Ato Esayas Hailu. I am also greatful to my classmates for the care and teamwork. I want to give credit to staff members of Center for Food Science and Nutrition of Addis Ababa University (Dr.Paulos Getachew, Dr. Kaleab Baye, Ato Kelbessa Urga ,Ato Debebe Hailu), for their support. I also acknowledge staff members of Ethiopian Public Health Institute: Ato Biniyam Tesfaye, Ato Daniel Abera and Ato Mesay Getachew, among others.

Table of Contents

Title Page Numbers Acknowledgements ii Table of contents iii Abbreviations vi List of Tables vii List of Figures viii Abstract- ix Chapter-1 1 1. Introduction 1 1.1. Background 1 1.2. Statement of the problem 2 1.3. Significance of the study 3 1.4. General Objective 3 1.5. Specific Objectives 3 Chapter-2 4 2. Literature review 4 2.1. Introduction 4 2.2. Impact of malt protein on brewing optimization 4 2.3. Overview of beer manufacturing in Ethiopia 5 2.4. Beer Production process technology 7 2.4.1. Malted Grains 7 2.4.2. Beer Quality and Stability 12 2.4.3. The Nature of beer Particles 12 2.4.4. Origin and control of Particles in brewing process 13 2.4.4.1. Mashing 13 2.4.4.2. Wort Boiling 14 2.4.4.3. Wort Cooling 14 2.4.4.4. Fermentation 14 2.4.5. Wort and beer Clarification 15 2.5. Settlements/Sedimentation of Solids 15

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2.5.1. Colloidal Stability 16 2.5.2. Turbidity and Haze Formation 17 2.5.2.1. Components of Haze particles 18 2.5.2.2. Polyphenol 19 2.5.2.3. Prolamin storage protein: General properties 20 2.5.2.4. Regulation of prolamin gene expression 21 2.5.3. Proteomic Analysis of different extracts from barley grains 22 2.5.4. Particles Contributing to Turbidity in Beer 22 2.6. Accelerated Shelf- Life Testing 24 2.6.1. Predicting the Shelf-Life of Beer 24 2.6.2. Predicting Test 25 2.6.2.1. Accelerated beer aging test 25 2.6.2.2. Predicting Test 26 2.6.2.3. Sensitive protein content 26 2.6.2.4. Chilling Test 26 2.6.2.5. Polyphenol (Tannoid) content titrating with pvpp 26 Chapter-3 29 3. Material and Methods 29 3.1. Materials 29 3.1.1. Chemicals 29 3.2. Experimental Design Summary 30 3.2.1. Factors, the corresponding ranges and levels 30 3.2.2. Response Summary 30 3.2.3. Several blending ratios responses 30 3.2.4. Optimization of process for mixture D-optimal 31 3.2.4.1. Constraints applied for the Optimization 31 3.2.4.2. Selected Optimum Solution 31 3.2.4.3. Validation of Optimal Combination (Comparison of Products) 31 3.3. Malt Analysis 32 3.3.1. Total Protein Content 33 3.3.2. Moisture Content 34

3.4. Wort Analysis 34 3.4.1. Extract Yield by Mashing 34 3.4.2. Total Polyphenol 36 3.4.3. Free Amino Nitrogen (FAN) 36 3.5. Design of the Experiment 37 3.5.1. Pysico-chemical and instrument Analysis of beer 37 3.5.2. Bitterness 37 3.5.3. VDK 38 3.5.4. Total Polyphenol 38 3.5.5. Determination of Calcium ion ( Ca 2+) 38 3.5.6. Determination of Foam Stability Value 39 3.5.7. Pasteurization 39 3.6. Microbial Analysis of the beer 39 3.6.1. Saccharomyces Wild yeast 39 3.6.2. Total Count 40 3.6.3. Lactic Acid Bacteria 40 3.7. Sensory Analysis 41 Chapter-4 43 4. Results and Discussion 43 4.1. Moisture Content 43 4.2. Protein Fractionation of Malt 44 4.3. Particle size distribution 45 4.4. Pysico-chemical, Instrumental and microbial Analysis results of the beer 45 4.5. Accelerated Shelf-Life /Forcing Test Results 46 4.6. Sensory Analysis 47 Chapter-5 49 5. Conclusion and Recommendations 49 References 51 Appendix A 54 Appendix B 57 Appendix C 58

Abbreviations

EBC European Brewery Convention OG Original Gravity Al(w/w) Alcohol Weight by Weight

Al (v/v) Alcohol Volume by Volume

Er Real Extract

Ea Apparent Extract

ADOF Apparent Degree of Fermentation SG Specific Gravity BU Bitterness Unit VDK Vicinal Diketones FAO Food & Agricultural Organization LAB Lactic Acid Bacteria DMS Dimethyl Sulphide AAB Acetic Acid Bacteria GMP Good Manufacturing Practice HA Haze Active PVPP Polyvinyl Poly Pryollidon FAN Free Amino Nitrogen

List of Tables

Table 1 Main Malt Producing Countries...... 8 Table 2 Import of Malt in Ton...... 9 Table3 Blending Ratio Response...... 31 Table 4 Validation of Optimal Combination...... 33 Table 5 Protein Fractionation...... 46 Table 6 Particle Size Distribution...... 48 Table 7 Chemical Analysis results of the optimized Beer...... 49 Table 8 Chill Haze Measurement after Forcing Test...... 50

Table 9 sensory Analysis result...... 51

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List of Figures

Figure 1 Malt handling and cleaning Flowchart...... 11

Figure 2 Beer Production Flow Sheet...... 14

Figure 3 Concept of Protein Polyphenol interactions leading to Haze...... 42

Figure 4 Flavonoid Phenolic and Proanthocyanidin (Condensed Tannin) Structure...... 42

Figure 5 Mode of Combination of Proteins with Polyphenols...... 43

Figure 6 Similarities of Polyproline and Polyvinylpyrrolidine (PVPP)...... 43

Figure 7 Flavan-3-ol Monomers found in Malt, Hops and Beer...... 44

Figure 8 Flavanol 3’-4’ Ortho-hydroxy Chelation of Metals...... 44 Figure 9 The Oxidation of Flavan-3-ols....……………………………………………………...... 45

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Abstract The beer industry has begun its wider demand of raw materials to accomplish the vast production .Ethiopia has many breweries being opened, among the raw materials that are needed ,the Malt has been praised as the soul of the beer. Both imported and local malts are used in the beer production. Besides the maximum quality and quantity imported malt contributes for beer production, it also takes much of our foreign currency. Based on the proximate analysis of both malt variants the different parameters essential for the malt types have been analyzed. Taking the critical parameters in this research which are proteins which occur as polyphenol complexes, Free Amino Nitrogen (FAN) in The extract yield of each variety of malt apart from pure of the Malt types, several blending ratios of local to imported malts have been taken as factors in several runs of Experiments were designed by using Mixture Design, D-optimal after undertaking several runs and analyzing the results, the optimized malt ratio has been estimated to be 64 % imported and 36% local malt with critical values of polyphenol as 109.8 ppm, extract yield as 8.8 European Brewey Convention and FAN as 198.9 ppm with desirability of 57%. After the improved beer produced was analyzed both physico-chemical, microbial and accelerated shelf-life study were conducted beside other quality parameters. Thus the optimized beer has showed the following results :original gravity 11.99 oP Ea 2.24 Op Alc(v/v) 5.18 oP,apparent degree of fermetation 81.34 Foam Stability 170 ,Haziness 0.77/0.06,Biterness 14.9 Bu, Polyphenol 119 ppm ,Color 8.0 EBC,VDK 0.215 EBC, Smell 0(1),+1(3),Taste+1(4)Appearance +1(4) The colloidal Chill haze formed after forcing test and calculating the haze difference from initial and final showed a lower haze value than the standard 2 EBC according to Harp Method, i.e.1.77 EBC, as expected. This research initiates impacts on alleviating the malt scarcity problem for the several breweries including facilitation of the genetic manipulation of reducing the haze active protein, Herdoin, in the organic local malt (which has displayed a greater Herdoin amount (0.35 % ), more than double, than the imported Malt’s( 0.84%). Furthermore, describing good agricultural practice, good manufacturing practice, good storage and delivery system of the produced malt variant also may help. This shows several beer properties can be controlled for ensuring consistent sensory qualities of beer while it becomes compulsory to use different kinds of malt strains in homogeneous or heterogeneous mixes. A further research on the genetic manipulation to reduce herdoin protein of the local malt is expected in the near future in order to fill the gap between the herdoin amounts of that it has with imported malt that will affect shelf stability of beer to be produced. Key Words: Beer, Malt, Protein quality, Shelf-Stability,

Chapter -1

1. Introduction

1.1. Background

From the time of antiquity to tomb excavations and archeological discoveries, all revealed that beer has been well drunk by common man of different civilizations including Egypt, Mesopotamia and Babylon, since 5000BC until now. From homemade to commercial scale, beer production has been recorded throughout history. After the industrial revolution however, a scientific and technological discoveries and inventions, respectively contributed much on the vast beer production as a breakthrough throughout Europe, America and the rest of the world. Today beer is produced and drunk in almost every country in the world, although, widely in different amounts (Kunz, 1999).

In comparison to neighboring countries, Ethiopia produces much less amount of beer per person, annually. Average annual per capita consumption of beer in Ethiopia is less than 6 liters against 12 liters in Kenya and 59 liters in South Africa. But demand for beer is expanding by 15% per annum. Ethiopia is second among the African countries (After Nigeria) where the population size exploded outrageously. A very young demographic (44 percent of the population is under 15 and 73 percent under 30) which is greatly favored from the customers market gap side, underutilized .Due to the current income generated, demographic changes, the trend of internal migrations towards cities, increasing urbanization and the remarkable high-density living arrangements .Ethiopia’s urban population has, for instance, recently crossed the 100 million threshold. Thus, figuring out the demand-supply gap, many different brewery companies are flooding into the market. These days, there are even more than seven breweries operating in the country. But the quality of the available raw materials has a decisive influence on the quality of the beer produced thus on the success of these breweries for this the knowledge of properties of the raw material and their effects on the process and final product provides basis for their production and handlings (Elias, 2016).

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Four raw materials are required for beer production barley malt, hop, water and yeast. Barely (malt) is the most important raw material being prominently responsible for the beers sensory and colloidal qualities (AMF, 2012). While hope gives beer its bitter taste and have an effect on its aroma. The quality of beer is greatly affected by the quality of its hop and other important content is water. The alcoholic behavior of beer is achieved through yeast in the process of fermentation.

1.2 Statement of the Problem Among the four main raw materials that are used for beer production, the malt has been praised as ‘the soul of the beer’; this is due to the fact that it contributes to the Flavor, Foam, Aroma and body fullness of Beer. Ethiopian Breweries use both Local Malt and also Imported Malt type that take most of our foreign Exchange.The Local and Imported Malt Types have difference in both quality and yield of the Beer produced. The imported malt is the first choice of malt type among the breweries. However, the shortage of Foreign Currency to import the desired Malt has led several researchers to focusing on partial substituting of the Imported Malt with Local Adjuncts such as Maize, Sorghum, Millet and Un-malted Barley (Kunz,1999).Due to lack of practical significance of previous researches done ,the unavailability of Scientific Methods, technological advancements ,skilled human power and natural growing conditions for growing Barely and malting for filling the gap between the local an imported malt variants, this research focuses on describing ,technologically ,the optimization of the local to imported Malt ratios which will be computed with respect to the quality ,turbidity and sensory qualities of the beer produced.This research will have an impact on maximizing the ratio of local malt usage, thus saving valued foreign currency and also enhancing the shelf stability of the Beer that has been problem by those attempt of replacing the local adjuncts that leads to colloidal instability .Hopefully, this research could serve as a benchmark for future research concerning the prevalent issue. By completion of this study the results would utterly contributes to the Ethiopian beer industry by indicating major quality improvement possibilities, hopefully together with better Shelf Stability with reduced cost production of our Beer by practical for malt types, blend ratios with desired qualities and yield of beer.

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1.3 Significance of the Study This research investigates the possibilities of achieving higher shelf stability of beer, clarity and better taste among other important issues, through blending of the best Imported Barley Malt type and the Local ones. To maximize the usage of the locally produced malt type without changing the Sensory qualities the consumers have become accustomed to and to improve the colloidal stability of the Current Ethiopian Beer with underlining benefit of reducing the usage of imported malt. If all the above attempts result negative outcomes, this Research will conclude on with scientifically deducing correct proportion of local and imported malt types blending Ratio and producing improved beer quality with possible measures to reduce the formation of protein- polyphenol complexes, that impacts the shelf-life of the beer produced.

1.4 General objective To produce an improved quality and shelf stable beer through optimization of the malt type..

1.5 Specific Objectives  To evaluate the proximate composition of local (Assela) and imported malt types.  To investigate and compare the quality parameters of extract between Local (L), Imported (I) and Blended Malt Ratios.  To produce an improved beer using the optimal blending ratio.  To evaluate the quality of beer produced in terms of physico-chemical and microbiological assays.  To predict the shelf-life of the beer by accelerating shelf-life study.

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Chapter-2

2. Literature Review

2.1 Introduction

Barely (Hordeum volgare L.) is highly adoptable cereal grain that is produced in climate region from sub-arctic to subtropical. At present only 2%of barely is used for human food worldwide (Baik and Olrich, 2008).The greatest use of barely is for malting purpose mostly for brewing industry. The increased competition within the brewing industry needs maximizing the raw materials. Barely is the basic raw material for brewing, its chemical composition is highly affected the beer quality and the economic efficiency of the brewing process. A large number of parameters have been utilized to define malting quality. The texture of endosperm influences the malt modification process by affecting water uptake and enzyme synthesis with in the endosperm (Chandara et al, 1999).The malting huskless barely present a number of challenges due to differences in chemical and physical changes (Shewry , 1993).The structural changes and Bio- Chemical degradation of the endosperm modification (Gunkel et al,2002). Therefore different modification of different Kernel properties have been identified as a factor affecting water uptake during stepping of barely, proteins, starch granule size and distribution of enzymes are affecting the hardness of the endosperm. A superior Malt Quality for the improved varieties with the old varieties was obtained that is reliable for Breeder as well as Maltsers and Brewers to be used as raw material.

2.2. Impact of Malt Protein on Brewing Process Optimization

Malt is a barely seed partially separated which had been previously heated and dried.Barely cannot be used for production of beer because it has not developed enzymatic system, which transforms starch into sugar during moistening process.During malting process occurs hydrolyzing of barely protein, white barely contains Proteins white barely contains proteins which makes the beer turbid. (Tanja Kumbari et al ,4 ).Proteins are long chains or polymers with large molecular weight composed from amino acids connect to each other via peptide bond. Grain proteins have been classified either according to their Physio-Chemical properties as

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Albumins, Globulins, Gluteline and Prolamines or according to their function as catalyst (enzymes), structural, and storage and defense proteins respectively (Janja Kumbari, 13).Proteins are among barely components that are essential for the quality of malt and beer. First high protein contents decrease available carbohydrates, with a negative influence on the brewing process and second, (proteolysis protease hydrolysis producing amino acids and peptides from hordeins) during malting and mashing is necessary for yeast metabolism.Finally soluble proteins are important in beer head retention and stability. They are necessary in enzymatic process of malting, Wort production and affect directly the consistency and foam. Proteins are composed of nitrogen bearing compounds such as amino acids. The nitrogenous constituents of barely are of great interest to Malters and Brewers. It is very important to determine protein content before using malt for beer production. Malt is the main source of protein in beer and proteins are made from compounds with nitrogen bases such as amino acids 1% Nitrogen is equal to 6.25% in Protein.

The amount of soluble protein or nitrogen, expressed in percentage by weight of malt and this indicator will be used to calculate the amount of Nitrogen dissolved. Soluble Nitrogen is determined by method of European Beer Convention . Industrial and experimental yield is calculated based on the values of soluble nitrogen. All malts that exceed the protein content by 12 % (1.9 TN), cause problems in boiling process or in turbidity of beer .European Malt Lager or Ale type have a protein content below 10%.The amount of dissolved Nitrogen is a very important indicator for modification of malt. The higher this value is, the more the malt will be modifiable. Protein content in malt grow to the extent 9-14% compared to barely. Furthermore, the quality of beer flavor tastefulness, tendency to form haze is influenced by the nitrogen content of the Malt from which it is made. Nitrogen compounds present Wort may have different molar mass and this profile influences the brewing process and the quality of the final product. Protein with higher molar mass (>106 Pa) contributes to the Beer texture and the formation of foam, although those Protein might be related to Haze formation in the determination of Viscosity (kunz, 1999).

2.3 Overview of Beer Manufacturing in Ethiopia Beer is defined as a fermented alcoholic beverage produced from malted cereals (usually barley), water, hops and yeast .The cereal used must firstly undergo modification during the malting and

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mashing steps to yield sufficiently simple carbohydrate that the yeast is able to utilize it by converting the yielded carbohydrate into Ethanol and Carbon dioxide .The traditional fermentation process that has been discovered in ancient times has not been changed, entirely. Despite the large scale technological growth that separates the old time homemade brewing from today’s high-tech breweries, primitive human beings didn’t knew the complex biochemical steps involved in the process. Yeast are the major biocatalyst that are used to make beer worldwide .The last but not the least, Water is the major raw material used to make beer. Supply and preparation of the water is due to the expensive commodities of main raw materials such as barley malt, many countries allow additional materials to be incorporated as substitution in place of malted barley with Adjuncts like corn, rice, sorghum or wheat. In additional to reducing costs, the use of un-malted cereals gave palatable flavors to the beer. Hope is the dried flowers from the female hop (Humulus Sluplus) plant and contributes flavor and anti-bacterial compounds to beer.

Particularly important to the brewers because the water quality affects the quality of the beer produced. Beer is the most popular alcoholic beverage in Ethiopia having a larger numbers of consumers .The first brewery in Ethiopia was inaugurated in 1922 E.C by St. George beer (Named after the patron saint of Ethiopia).Brands like Meta and Bedele are also Older brands but have since been acquired by foreign Companies like Heineken and Diageo meta Abo and rebranded. The beer industry has gone through tremendous growth in the last two decades. It transformed into one of the most competitive industry with millions of beer spent on advertisements alone. The competitiveness of the Industry has led to more investments the farming sectors such as malt production (Sisay, 2015).

Currently, Ethiopia’s beer Industry is comprises of seven major brewery plants .These are: BGI Ethiopia (Addis Ababa, Kombolcha and Hawassa Plants), Diageo Meta Abo, Heineken(Addis Ababa, Bedele and Harar Plants), Dashen ( Debre Birhan and Gonder Plants),Habesha ,Raya and Zebidar Breweries. There are more than 15 Beer Brands brewed from these different Breweries operating in the Country .To numerate them: Meta, Bedele, St.George Beer, St. George Amber,Dashen,Jano,Habesha,Harar,Walia,Raya, Zebidar Beer,Heineken,Castel,Hakim Stout, Garden Brau Ebony ,Garden Brau Blondy. The big players in Ethiopia beer industry are the French BGI Ethiopia plc, Heineken, Dashen, Bavaria,Diageo Meta Abo that owns bigger market share ,respectively.BGI Ethiopia is the largest brewing company and unit of the French Castel

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Group; it has bought Zebidar Brewery, recently. It grants ownership of the plant that sits at 150,000 sq.m plot of land and has an annual production capacity of 350,000 hectoliters. The brewery is located in SNNP .close to Welkite Town, 167 Kms from Addis Ababa.BGI also has offered 2.5 billion birr to fully acquire Raya Brewery ,a brewery located in the Northern part of Ethiopia .Its expansion plan was brought by the success of its product, St.George , Ethiopia’s oldest beer brand that Castel Group bought in 1998 and two other Brands added were Amber and Castel.Though BGI is reported to own majority of the Ethiopian market, up to 48 %, However it is facing fierce competition from Heineken popular brand, Walia and two other Brands, Habesha ,unit of the privately-owned Dutch Brewer, Bavaria and also from Dashen beer.

Meta Abo Diageo that acquired Meta Abo Brewery in 2012 for $225 million is also still in the game with 17% share[Ethiopia Observer newspapaer,2018] .Meta Abo is the second largest Brewery with annual production capacity of 0.8 million hectoliters. Dashen owns 14 % of the Market Share.But when comes to the beer Industry in the capital/Addis Ababa/BGI is still the leader with 64 % of the market share with Meta ,Dashen, Harar and Bedele hold 12 %,11 %,7% and 5% Market Share consecutively[Fortunenewspaper,2011].Heineken Officially inaugurated its new Brewery at a Greenfield site in Kilinto on the outskirt of Addis Ababa with a total capacity of 1.5 million Hectoliters,the Kilinto brewery is already producing the recently launched Walia beer together with Bedele and Harar Beer Brands.Heineken has bought Bedele and Harar Breweries in 2011.It key’s brands are Bedele Special, Bedele Regular Harar, Hakim Stout and Safi Malt. Aiming at improving the quality and quantity of barley grown in Ethiopia Heineken also entered public and private partnership to improve access to Market for Local Farmers.

2.4 Beer Production Processing Technology The four raw materials required for beer production include water, malted grains, hops and yeast.

2.4.1 Malted Grains

Malted Grains are providing the sugars that are fermented by Yeast to produce Alcohol and CO2. Also malted grains provide the essential Nutrients for Yeast that is needed to reproduce. They are the primary source of Color in Beer. Malted Grains are a major contributor to the flavor and aroma profiles of Beer. The malting process is simply a controlled sprouting and kilning of the Grain. The sprouting begins to break down Starches contained in the Grain making them accessible to the brewer and the kilning provides Color and Flavor. Grains kilned at higher

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temperatures or longer periods of time are darker. Malted Barley is by far the most widely used Grain in Beer making, but it is not the only one. Depending on the species of the barley, the Plant will expose one or more kernel per node of the Ear. Mainly, Two Species of barley are used in brewing: the two-row barley (with one Grain per Node) and the six-row barley (with three Grains per Node). To put it simple, the fewer are the Kernels per Node, the bigger and richer in Starch they are. Conversely, the six-row barley has less Starch but higher protein Content. Therefore, if the Brewer wants to increase the Extract Content, the Two-row Barley is the best Option, whereas if enzymatic strength is the aim, the Six row will be the best Choice (Wunderlich and Back ,2009). Worldwide, there is few malt processing companies. For instance, Malt Europe (France), the largest Malting Company in the World with a current annual production Capacity of more than 2.2million Tons. Belgium has taken over the biggest US brewing Anheuser-Busch and this newly created Company in Belgium is the Leading global Brewer and one of the world’s top Five Consumer product Companies. The Top Ten Malting Companies produce approximately 9.4 Million Tons or 44% of global Malt Production ( FAO , 2009). Main Malt Producing Countries ( Thousand Tons). Table 1 Main Malt producing Countries

Country 2000 2001 2002 2003 2004 2005 Trend 2005/2004 World Total 17820 18487 18296 18640 19140 19704 +3% 1. China 2870 2954 2870 2380 2730 3222 +18% 2. USA 2404 2960 1952 1923 1990 2086 +5% 3. Germany 1635 2000 2000 2072 1797 1436 -20% 4. UK 1452 1490 1477 1501 1425 1332 -7% 5. France 1155 1162 1183 1211 1211 1225 +1% Source: FAOSTAT internal follow-up, 2008 In Ethiopia, there are two malt plants i.e. Assela Malt Factory -located in Assela Town of Oromiya regional State,which was established in 1984 with the aim of supplying malt to the ocal Breweries and the Plant is located in area where Barley can be grown potentially, whereas Gonder malt factory located in Gonder town of Amhara regional state,which was recently established Factory and it had almost the same potential to Assela Malt Factory by producing

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malt. Since the existing plants in the country do not satisfy the demand for Malt, Breweries are forced to Import from Abroad as shown in Table 4 (Malt profile in Ethiopia). Table 2 Import of Malt in Ton Year Quantity (in Tones ) Value (birr)

2000 3042 13,117,368 2001 9624 36,749,085 2002 5509 21,557,400 2003 5428 21,153,453 2004 6184 30,032,464 2005 10913 52,942,054 2006 26967 126,449,016 Source: Customs Authority Worldwide, most Breweries use alternative Starch Sources (Adjuncts) in addition to malted Barley. Adjuncts are used to reduce the final cost of the recipe and/ or improve beer’s color and flavor/aroma. The Enzyme potential of Malt ( Diastatic Power) is sufficient to catabolize additional Starch. Consequently ,throughout the World ,usually 15 to 20 % part of the Pilsner Malt is replaced by unmated Cereal .This un-malted Cereal is cheaper than the relatively expensive Malt .The lower Extract Yield must be set against the Low Price compared with Malt ,problem may be caused because ,as result of the absence of the malting process ,the β- glucan is not dissolved and is not sufficiently degraded during mashing .In such a case, Filtration problems are to be expected (kunze,1999).The climatic conditions in many African countries are unsuitable for cultivating brewing Barley.It is quite natural that in many African Countries attempts are increasingly being made to use the indigenous Raw Materials ,Sorghum, as source of Extract for Beer Production and also to Malt it, in order to economies on expensive Malt Imports[kunze,1999].Both the growing and harvesting of Sorghum occurs in the rainy Season .Extensive contamination by Microorganisms ,especially Fungi, must therefore be expected ( kunze,1999).

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Table Sorghum Parameters Protein Content 11-12.6%

Germinating Energy at 5days 90% Hl Weight 70g Thousand Corn Weight >25gm Fat Content (Dry Weight) 2.0-6.0 % Starch Content (Dry Weight) 67%

Source:Kunz ,Technology of malting and Brewing To produce 1 hl of Beer having an Original Extract of 11%, approximately 17 kg of Malt is required [Kunze,1999].For economic or various reasons, when brewing Beer, part of the Malt is often replaced by un-malted Cereals or other Sources of Carbohydrates .This reduces the total amount of Malt required[Kunz,199].Adequate supplies of malting Barley and the ability to make large amounts of Malt made Europe and Australia the largest Malt Exporters in the World. Others large Malt Exporters are Canada and Turkey.Also the Large amount of Barley and Malt are exported to South America, Africa and in particular to East Asia where a flourishing Brewing Industry has recently developed (Kunz,1999). The most common adjuncts are un-malted barley, wheat, rice, or corn, but other sugar sources such as Starch, Sucrose, Glucose, and corresponding Syrup are also used. The use of Adjuncts is only Feasible because Light Malts (i.e. Pilsner Malt) have enough Enzymes (Higher Diastatic Power) to break down up to twice their Weight of Starch Granules. Among un-malted raw Materials Maize Components are the most reliable in processing. However Maize Grits and fine grits, used as malt substitute, require Gelatinization. This process facilitates enzymatic alteration mainly of Starch, while the efficiency of obtaining non-Carbohydrates from un-Malted Raw Materials is Low [Hug & Pfenninger, 1976a, b, c].During Germination, the Embryo grows at the expense of reserve Material stored in the Kernel. As Soon as the Grain makes contact with suitable Conditions during Steeping (Moist and adequate Temperature), all enzymatic Apparatus is gradually activated to break the reserves of Starch and Proteins to form a New Plant. Here lie the crucial roles of malting, which are enriching the Malt with enzymes ( amylolytic, proteolytic, etc.), modification of Kernel Endosperm, and Formation of Flavor and Aroma Compounds.

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Starch-degrading Enzymes (such as α-amylase, β-amylase, α-glucosidase, and limit dextrinase) produced during Germination are better characterized than them proteolytic counterparts (Schmitt et al. 2013).

Figure 1 Malt Handling and Cleaning Flowchart

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2.4.2 Beer Quality and Stability

Beer contains number of barely proteins that have been modified both chemically & proteolytically during the malting and brewing & can influence haze stability. Proteins with high levels of Proline and polyphenols with higher degree of polymerization are most likely to form haze. There are number of time points and components of the brewing process, from raw material right through packaging and storage that can influence the colloidal stability of beer. During the brewing process HA proteins and polyphenols are normally removed from beer during wort boiling, fermentation, maturation and filtration.

Haze-active proteins (HA) isolated from beer have been found to be derived primarily from fragments of the barely storage protein group the Hordeins. These protein fragments consist of several different molecular weights and are relatively rich in Proline. Haze formation in beer has shown to occur at a rate that is a function of the product of the concentration of the HA and of the HA polyphenols at the time of packaging therefore to improve the colloidal stability of beer the residual HA protein and HA polyphenols or portion of both needs to be removed. This is typically achieved by using stabilization treatment such as silica hydrogel (HA proteins) or polyvinylpoly-pyroliden (pvpp) HA polyphenols. An alternative to the use of these stabilization techniques is the removal of HA materials and there has been the development proanthocyanidin free (ant-free) barely variety that significantly improve haze stability. Colloidal haze does not affect the taste and should not affect the beer quality: but “Most Consumers” drinks with their eyes”, They are more often willing to accept a glass of beer which does not taste quite right, over glass of beer which is hazy” (T.O’ Rourke, 2002). Indeed the brilliance of beer in probably is the most “objective” parameter. So, most of the consumers will simply reject a beer that is not bright, that is hazy.

2.4.3 The Nature of Beer Particles

Instead of infection is clear sterile process, yeast cells and non-microbiological particles (NMP) largely comprises the beer clarity. In reality the settlement or clarity different yeast strain contribute different flocculation characteristics and as result exhibit slightly different problem in settlement. The term non-microbiologically particles comprise multitudes of compositional

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species, although they are roughly classified as proteins, usually complex with polyphenols and other molecules such as lipids, carbohydrates and/or metal ions. Practical observation of particles by microscope, fitted with a calibrated eyepiece and haemocytometer slide, present in most breweries NMp have been classified in three size fractions, <2mm, 2-10mm and >10mm also size consideration of NMP surface charge has been examined as a tool to characterize beer particles(Denis Bilge,2015).

2.4.4 Origin and Control of Particles in the Brewing Process

Non-microbiological particles are produced and removed at five different stages of the brewing process. Understanding of the how the particles affect particle formation and their will allow the brewer to move easily control the process to succeed at achieving consistent and optimum level of beer particles leading to a more consistent and efficient clarification process whether the end product is cask or brewery conditioned .

2.4.4.1 Mashing

Milling of grist materials results in the generation of numerous fine dusty starch and husk particles. These are usually removed during mash separation. However if the wort is not re- circulated through the mash bed prior to run-off, or excessive pressure are applied to a mash filter, these grist particles will carry through into the sweet wort. This is particularly true in the case of lautering, where frequent, rapid or excessively deep raking will disturb the mash bed releasing the numerous entrapped particles. In addition it is not unknown for lauter plates to become damaged wrapped or incorrectly re-laid allowing the passage of larger particles in to the wort. Coagulation of mash particles is favored by an increase in final mash temperature though this may also increase wort viscosity which tends to offset the beneficial effect of coagulation on the run of rates. Certain materials have been shown to coagulate mash particles enhancing run off rates and reducing the number of particles carried over in to the wort. Over- sparging has also been shown to wash excessive levels of undesirables such as lipids from the mash which have deleterious effect upon the particle levels and hence final clarities or filtration performance.

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2.4.4.2 Wort Boiling During the wort boiling process thermal denaturation causes coagulation of protein to form hot break. Efficient coagulate is favored by a high Wort pH, the presence of sufficient protein and good wort boiling conditions i.e. a minimum of 102 0C at atmospheric pressure to maximize denaturation. Under these conditions hot break is break formed as large flocks that are relatively easily removed in the whirlpool or hop-back. If coagulation is inefficient fine flocs will be formed which may remain in suspension and be carried over in to subsequent downstream stages of brewing process. As well as protein removal the boiling stage also extracts polyphenol materials from the hops although that may not be implicated in the hot break formation playing an important role downstream in the formation cold break and chill haze. The contribution of hops to the total polyphenol level of wort depends upon the variety used. It has been reported that the derivation of high proportion of bitterness from extracts or oils at the expense of plant materials can lead to sufficiently low levels of polyphenols as to cause poor protein removal during cold break formation.

2.4.4.3 Wort Cooling On cooling wort proteins interact with polyphenols to precipitate as cold break. This material consists of very fine particles that are slow to settle and consequently likely to survive in to finished beer. Both combining both wort boiling and cooling 17-35% of the total protein content are removed depends upon the malts variety and hop products variety used. Cold break formation is temperature dependent only forming in significant quantities below 20-300C and increasing dramatically in quality as the temperature is further decreased. The removal of these cold break particles can be facilitated and enhanced by kettle fining.

2.4.4.4 Fermentation Several physical changes occur which both produce particles and facilitate their removal. Yeast reproduction starts resulting in an increase in the number of yeast cells in the beer the pH is reduced by 1.0-1.5 pH units facilitating the interaction of protein and polyphenol mioties to form NMP. This results in the removal of 45-65% of the total soluble protein and 20-30 % of the soluble anthocyanogen content of bitter wort. Streaming current measurement suggest that acidic proteins are selectively removed at this stage.

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In addition as the concentration of alcohol increases the viscosity and density of the wort are reduced increasing the rate of sedimentation of any particles present (stocks law ) this together with the long period of time associated with fermentation permits the removal of a certain amount Cold break with the yeast fermenter bottoms.

2.4.5 Wort and Beer Clarification In case of cask beer the brewer is totally reliant upon the use of findings to achieve optimum clarity. The process ruling clarification of beer are not fully understood and given the complex mixture of constituents that is beer, it is likely to be some time before they are packed. Observation, optimization, empirical determination, and monitoring will be required to ensure efficient fining application. By identifying critical factors that will influence clarification efficiency, monitoring and recording observation surrounding these factors any outrage from the normal for whatever reason will directly imply problem in near future of process. The essential palliative actions may then be taken before the beer is processed or packaged, thus avoiding high levels of reprocessing, embarrassing trade complaints or costly product recalls.

2.5 Settlement/Sedimentation of Solids

Particle sediment or precipitate under the influence of gravity as described by stokes law. Stokes law states that the rate of sedimentation of an idealized spherical particle is directly proportional to the difference in the density of the particle and the liquid medium, the acceleration due to gravity and the square of the radius of the particle is inversely proportional to the viscosity of the liquid. Thus, if the beer is kept for a sufficiently long time, it will clarify itself is the basis of the lagering process.

Equation 1 Determination of viscosity

푟1− 푟2 V=2 푟. 푔 푔ℎ Where, V – Rate of Sedimentation of a Spherical Particle

r1- density of particle

r2 – density of medium of beer

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r – Radius of the sphere g – Acceleration due to gravity h – Viscosity of the medium Stokes law suggest two possible strategies for increasing the rate of clarification. The g term may effectively be increased by means of a centrifuge or the radius of the particle may be increased by the use of finings. Centrifuges are particularly effective at removing yeast but generally less effective on the very small particle that fining are particularly good at removing.  In commercial lager both fining centrifuges are complementary.  Since the speed of sediment is proportional to the square of the radius a modest increase in particle size yield a profound decrease in settlement time.  This therefore, makes increasing particle size by flocculation a very useful technique of decreasing settlement time.  Coagulation is not an easy process and depends upon the nature of the particulates and the liquid.

2.5.1 Colloidal stability Beer colloidal stability is an important parameter to have under control because most of the consumers “drink with their eyes”. Rotation period in these distribution channels is longer, poor transport and storage conditions, more colored beers; haze is more visible than in colored beers, commercial shelf life for lagers: 3 months up to one year or more, a non-stabilized (full) malt beer does not achieve these targets easily.

Beer stability is defined as the degree to which a beer tastes and looks as good at the end of its shelf life it did when it was first package (T.O’ Rourke, 2002).Beer stability relates to “no” changes in sensory characteristics.

 Flavor (taste-odour-aroma-texture-Co2)  Color  Foam  Brilliance-clarity-brightness or more pragmatically, it relates to “acceptable” changes.

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Beer is generally packaged bright, if haze is measured it is generally due to Biological causes: yeast and Bacteria, Non-biological causes: carbohydrates (starch, dextrin, glycogen, β-glucan, pentosan), mono-proteins, polyphenols-proteins, iron, calcium-oxidase. But during storage, beer haze almost always increases. There is visual brightness instability: if no biological, the reasons are mostly due to colloidal haze formed by polyphenols and protein interactions. During storage, beer (even pasteurized) become hazy due to colloidal haze formation. This turbidity is caused predominantly by interacting substances such as polyphenols and protein that can form visible colloids. Haze development is observed at low T o c (hill haze) and high T o c (permanent haze).Follow up of colloidal stability is defined here as the follow up of chill haze development (EBC) during storage (months).The colloidal stability corresponds to the period of time which the beer is steel acceptable. Accelerating beer aging , particular haze formation, by subjecting the packaged beer to storage at elevated temperature for a shorter time: months at 37oc, days at 50, 60oc (Tim O’Rourke,2002).

2.5.2 Turbidity and Haze Formation

Beer is a complex mixture of over 450 Constituents; in addition it contains macromolecules such as proteins, nucleic acids, polysaccharides and lipids. Proteins influence the entire brewing process width regard to enzyme, which degrade starch B-glucans and proteins with protein- protein linkages that stabilize foam and are responsible for mouth feel and flavor stability and in combination with polyphenols thought to form haze. Several substances in beer are responsible for haze formation. Organic components such as proteins, polyphenols and carbohydrates (alpha- glucan, beta-glucans) are known to from haze. Haze formation is an important problem in beer, as it affects the quality of the end product .Beer consists of various ingredients such as proteins, carbohydrates, polyphenols, fatty acids, nucleic acid, amino acids. These ingredients can precipitate and haze is formed. Malted barley contains 70-85% total carbohydrates, 10.5-11% proteins, 2-4% inorganic matter, 1.5-2% fat and 1-2% other substances. Beer haze consists of several components the most common organic parts are proteins (40-75%), polyphenols (in combination with proteins) and to a smaller proportion of carbohydrates (2-15%).There exists two form of haze: cold break (chill haze) and age-related haze. Cold break forms at 00C and dissolves at higher temperatures. If cold break haze does not dissolve, age related haze which is

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irreversible develops. Chill haze is formed when polypeptides and polyphenols are non- covalently bond. Permanent haze forms in the same manner initially, but covalent bonds are soon formed and insoluble complexes are created that will not dissolve when heated. Haze formation can be due to residual starch pentosans from wheat derived adjuncts, oxlate form calcium deficient wort. Beta-glucan foam in adequately modified malt, carbohydrates protein foam autolysed yeast, lubricants from can lids and dead bacteria from Malt. Haze particles can show different appearances glenrister et.al published a classification for haze particles in beer as follows

 Native Particles, which originates from the beer by coagulation/precipitation.  Foreign Particles, which enters the beer as accidental contaminants.  Process Particles, which originates from materials (e.g. Filter Aids) added during the process.

These particles can have a shape of flakes, ribbons and grains. Flakes are thin, film like particles with no regular formation. When flakes are precipitated ribbons are formed. Grains can be mixed up with singular cells and bacteria. Bamforth et. al also divided haze into several types visible haze seen as “bits” that contain protein and perhaps petsans is thought to arise as the skin around foam generated within the package. Visible haze formation can limit the shelf-life of products, since the consumer expects a clear beer. There are also the invisible hazes, which also called “psedo-hazes” these are caused by very small particles (<0.1um) that cause high levels of light scatter when measure at 900 to incident.

2.5.2.1 Components of Haze Particles

Beer contains 500ml/g of protein material including a variety of polyphenols with molecular masses ranging from <5 to >100k da. The content of only 2mg/l protein is enough to cause haze, only about 20% of the total grain proteins are water soluble. Barely water soluble proteins are believed to be resistant to proteolysis and heat coagulation and hence pass through the processing steps, intact or somewhat modified to beer. These polypeptides, which mainly originated from barely proteins, are the product of the proteolytic and chemical modification that occurs during brewing. Only one third of the total protein content passes in to finished beer. Proteins play a major role besides polyphenols part of colloidal haze. Proteins as the main cause of haze

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formation in beer, are divided in to two main groups; first proteins and second their breakdown products are characterized by always being soluble in water and they do not precipitate during boiling. Finished beer contains primarily finished breakdown products. A beer protein may be defined as a more or less heterogeneous mixture of molecules containing some core peptide structure and originating from only one district protein present in the brewing materials. Several aspects of the brewing process are affected by soluble proteins peptide and/or amino acids that are released .Many studied have been conducted on the identification of haze and foam active proteins. (Nummiet et.al) suggested that the acidic proteins derived from albumins and globulins of barely are responsible for chill haze formation. Researches have proven that proline-rich proteins are involved in haze formation. With the help of wort boiling process, fermentation and maturation proteins particles can be removed. Proteins coagulate during the whir pool. The pH decreases during fermentation and proteins can be separated as cold-trub. Protein during maturation adheres to yeast and can’t be discarded with yeast.

2.5.2.2 Polyphenols Phenolic components which also can participate in haze formation, reach the beer through hops and malt.

Figure 3.Flavan-3-ol monomers found in malt, hops and beer

They exert an influences on several beer quality attribute, such as the colloidal stability of beer. Protein and polyphenolic compounds can combine to form soluble complexes.

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Figure 4.Concept of protein polyphenol interactions leading to haze; originally proposed by Siebert and Lynn104.

These can grow to colloidal size at which lead to sediment formation. The protein/polyphenol ratio has a strong influence on the amount of haze formed; the largest amount occurs when the numbers of polyphenols binding sites are nearly equal (O’Rourke T, 2002).

2.5.2.3 Prolamin Storage Proteins: General Properties

With the exception of oats and rice, the major endosperm storage proteins of all Cereal Grains are Prolamins. This name was originally based on the observation that they are generally rich in Proline and Amide Nitrogen derived from Glutamine, but it is now known that the combined proportions of these amino acids actually vary from about 30-70% of the total among different cereals and protein groups. Although Prolamins were originally defined as soluble in alcohol water mixtures (e.g. 60-70% V/v) Ethanol, 50-55% (V/v), Propanol or Propan -2-1, some occur in alcohol –insoluble polymers. Nevertheless, all individual prolamin polypeptides are alcohol- soluble in the reduced state. The Prolamins vary greatly, from about 10,000 to almost 100,000 in their molecular masses. In some Prolamin groups also the amino acids Histidins, Glycine, Methionine, and Phenylalanine are found in higher proportions apart from Proline. Although, the above mentioned classification of barely protein are assigned by the solubility differences in series of Solvents for example Albumin in water, Globulin in Salin, Proamix in Alcohol and

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Glutidine in caustic, modern classification assigns all of the Prolamins of barely to three broad groups; sulphur- rich (s-rich), sulphur –poor (s-poor) and high molecular weight (HMW) prolamins, with several subgroups within the s- rich group. These groups do not correspond directly to the polymeric and monomeric fractions in barely (glutenins and gliandins, respectively) recognized by cereal chemists as both monomeric and polymeric forms of s-rich and s-poor prolamins occur. Difference in protein distribution throughout the endosperm also occur in barely although the developmental basis for this is unclear. The sub-aleurone cells of barely is rich in proteins, but immune cytho chemical and pearling studies has shown that they contain mainly s-rich and s-poor Prolamins (principally B and C Hordeins), with HMW Prolamin (D Hordein ) only occurring in significant amounts below the sub-aleueone (shewry et. al 1996) teci et. al 2000). This may have significance for the utilization of barely as D hordeins is a major component of gel protein fraction that may limit the modification of barely during malting (smith and lister, 1983).

2.5.2.4 Regulation of Prolamin Gene Expression It is obvious fact that Prolamin Gene in barely are subject to tissue –specific and developmental regulation, being expressed exclusively in the starchy endosperm during mid and late development and nutritional regulation responding sensitivity to the availability of nitrogen and sulphur in the grain (duffus and Cochrane, 1992; giese and hopp,1984).

This control of gene expression is exerted primary at the transcriptional level (bartels and Thompson, 1986 sorensen et. al 1989).The sequence and the location of Prolamin gene promoter is identified thus manipulation of storage protein through genetic engineering thus utilized to improve the barely variety. The storage proteins of cereals are of immense importance in determining the quality and end use properties of the grain. Understanding the structures of these proteins, their biophysical and functional properties, and the biological mechanisms which determine their synthesis, traffic king and deposition in the grain is important to underpin future attempts to improve the end use quality of grain by genetic engineering.

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2.5.3 Proteomic Analysis of Different Extracts from Barely Grains

Cereal grains have a long storage life under favorable conditions because they are harvested at relatively low moisture content and compromise stable components. The largest morphological components of all grains in the endosperm and approximately 80% of this is Starch. Other important compounds are the storage proteins which make the next largest contribution to endosperm dry weight. Proteins are important both as nutrient and as active chemicals because they include enzymes that although making a small contribution to grain weight can have a marked effect on the quality. The classification system of seed grain proteins is based largely on the pioneering work of Osbourne who recognized that seed proteins differ in their solubility properties (et. al Osborne 1895). Although the classical Osborne scheme can be criticized as being insufficiently rigorous in modern terms, as it provides a useful way of classifying seed proteins and it is still used by most workers in this field (Cooke ,1984). There are thus considered four categories of proteins occurring in seeds.

 Albumins, which are soluble in water and compromise mostly enzymatic proteins;  Globulins, which are soluble in dilute salt solution and generally occur in protein bodies (i.e. they can be considered as storage proteins in the strict sense);  Prolamins, which are soluble in aqueous ethanol solution and are also found in proteins as true storage proteins;  Glutelins, which are soluble in alkaline or acid solutions, or in detergents and are probably mainly structural proteins, although some of them may have metablic functions.

The extraction procedure not only differentiates various categories of proteins occurring in seeds but also simplifies the proteins mixture for characterization.

2.5.4 Particles Contributing to Turbidity in Beer The appearance of beverage plays a significant role in the enjoyment of the drinking experience. With the advent of transparent, colorless glasses, clarity has become a sign of quality. Cloudy hazy beverages are considered as unappetizing and spoiled. If a products shelf life is just slightly over their expiration date, that is a reason enough for them to be rejected by consumers. Wine is considered the sparkling beer referred as bright and water described as crystal clear. Though sterilization and pasteurization of beer is microbiologically stable for the most part to the actual

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preservative effect due to the actual preservation and due to the substances contained in it is limited. The influences of the raw materials, the brewing process and the filling equipment has been extensively investigated. However to this day, brewing scientists have yet to exhaust the possibilities for further research in to the interactions occurring among the proteins, polyphenols, oxygen’s, their pH, molecular weight and the electrical charges they exchange.

The ever wider distribution of food and the changed corresponding necessity for longer shelf life time has demands placed on the quality. This began with the change from and agrarian to industrial society, culminating in the service and trade based society of today. In the year 1900, 450 farmers provided food for 1000 citizens today it is three farmers per 1000 citizens. Parallels can be concluded in the beer market as well; beer often has an expiration date of year and more.

Turbidity is measured in EBC formazine turbidity unit (EBC). According to Ludwig et. al a beer is classified as clear at <0.2 EBC, clear to <1 EBC, opalscent to <4 EBC and cloudy at all higher values; however the sensory taste of individuals do not always correspond to the measured values for turbidity(Denize Bilge,2015).

According to several turbid beer analyses with the exception of beer exhibiting early stability problems, these beers can be divided in to three groups on the basis of the following characteristics

 Extremely rapid turbidity development during aging  Increased turbidity values at the filter outlet  Visible particles in the packaged beer

Turbidity measurement is carried out at angels of 900 and 250 at a temperature of 200c. During testing it is remarked that turbidity is known to occur most frequently by beta-glucans this is measured primarily using the 900 measurement.

Turbidity that occur as a result of beta-glucans is followed by carbohydrates and proteins, which is recognized by using both 900 and 250. It has been repeatedly stated that the influence of prolin is over-estimated, estimated since only four percent of the improvement could be linked to the addition of prolinase .Apart from changing the kieslghur composition to finer addition of alpha

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and/or amyloglucanese to reduce the turbidity value. It is suspected that up to 95% of turbidity particles consist of glycogen from yeast.

A rapid increase in turbidity in packaged beer and the associated decline in shelf life can be monitored and controlled using quality assurance plan in conjunction with the appropriate correctional measures. The currently accepted strategy is to modify brew house particles to adapt to the raw materials and to carryout stabilization with silica jel pvpp. Oxalate precipitation is avoided by adding calcium ions during mashing. Though oxalate precipitation has been observed in bright beer tanks particularly after long period of storage. The major concern is turbidity that is caused by particular matter, visible in form of individual flakes, fibers or scales after shaking. If the cause is not microbiological in nature, it is very difficult to identify and more challenging to prevent. This problem is common and much speculation exists to is source mainly blame six- row barely.It is remarked that the residues of foam drinking to the wall of the filler or from the neck of bottle down into the filled beer is probable and should be ignored, this phenomenon strongly affects non-alcoholic and reduced alcohol beers.

2.6 Accelerated Shelf Life Testing The purpose of this scheme is to investigate accelerated beer shelf-life of light filtrated and unfiltered pasteurized beer with alcohol content 5.2% and the degree of turbidity of beer after 3 hours storage at 0 °C using a turbidity meter.

 Accelerated shelf-life testing (ASLT) involves storing the products under a controlled set

of storage conditions designed to accelerate the rate of deterioration of the product.  The results can be used to devise models to predict shelf life in different storage conditions.  The deteriorative changes must be the same as those occurring under normal conditions.

2.6.1 Predicting the Shelf -Life of Beer Beer instability can be easily observed by customers since most customer drinks with their eyes. They are often more willing to accept a glass of beer which does not taste quite right, over a glass of beer which is hazy. Hence, colloidal stabilization is often considered as a more important attribute than flavor stability.

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Figure 5.The oxidation of flavan-3-ols predominantly produces Semiquinone radicals.

In this research, though the flavor instability created by oxidation reactions principally during after packaging is prevalent, I focused on the measurement and prediction of colloidal stability of beer produced. When estimating colloidal stability of beer from the duration of its shelf life I used the accelerated shelf life and predicting method of shelf life rather than the time-consuming absolute accuracy, which requires maintaining the beer at ambient and measuring haze at the end of it stated shelf life. (Best before date, e.g. one year). By utilizing reliable indicators to form some of accelerating aging (forcing test) on the packed beer e.g. four weeks at 370 C is equivalent to 1 month of storage of ambient and relatively to date of absolute result.

2.6.2 Predicting Test Using a measurement usually related to the proteins or polyphenols content of the beer to predict the probable rate of production of the haze and hence the shelf life. Typically bright beer is packaged with an EBC haze of less than 0.8 units. The critical haze for stored beer is usually less than two or three EBC units for beer at 00C. Keeping beer to the end of shelf- life to evaluate as an assurance exercise but it is essential to calibrate rapid prediction methods. (Tim O’Rourke, 2002).

2.6.2.1 Accelerated Beer Aging Test These are aimed at stressing the beer usually by subjecting the beer to either hot or cold conditions to produce accelerated aging. The turbidity of the beer is measured at 0 0 C, samples are placed in a water bath at 60 0C for Seven days. The samples are cooled and kept at 00C overnight. The turbidity is measured at 0 0C. The bottles are placed in upright position and without agitation at 60 0C for 48 hr. the bottles are then placed in a refrigerator bath at 00C overnight. The final are read at 00C the result will be reported as the means of the initial and final in EBC units (EBC, 930)

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2.6.2.2 Predicting Test There are number of factors that are considered in order to predict the colloidal stability and therefore, the expected shelf life of beer however with other parameters controlled like given similar brands and brewery equipment, the principal variables will come from the protein and polyphenols content of the beer. These are usually measured when predicting the colloidal stability (Tim O’ Rouke, 2002).

2.6.2.3 Sensitive Protein Content

2.6.2.4 Chilling Test

A sample beer is chilled below 00C to as low as -80C without freezing often alcohol, 3% added) and kept for 8 hours and chilled is measured. The chill haze is principally the protein fraction.

A predictive test can be used to produce rapid results for beer prior to packaging but the results have to be correlated with actual storage data. For best results the data should be setup per brand to reduce the amount of outside influences distorting the stabilization results. As well as predicting the potential shelf life of a beer these methods are useful in determining the optimum dosage rate of beer stabilizer (Tim O’Rourke, 2002).

2.6.2.5 Polyphenol (Tannoid) Content Titrating with pvpp

This is a Nephelometric titrations of soluble pvpp (poly vinyl pyollidone) solution. Pvpp has a similar structure to a protein molecule and readily forms an insoluble precipitate with polyphenols particularly medium size molecular weight polyphenols often called Tannoids which are known to be haze active. When the pvpp is titrated in a beer haze is formed. This increases to a maximum and decreased by dilution effects as pvpp additions continues. The peak value gives a measure of the “tanoids” which can be correlated with chill stability. Using modern brewing technology, brewers can now produce beer with a colloidal stability exceeding a year. Much has been learned about the different hazes that can be formed in a beer of these the most intensively studied has been chill haze (Keneth A.Leiper.etal,2005).

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Figure 6.Similarity of polyproline and polyvinylpyrrolidine (PVPP); both possess a five-membered nitrogen containing ring capable of hydrogen bonding.

The greater part of this non-biological haze is proteineceous but existing analytical methods do not yet permit the specific measurement of this componenet. (Murrough et. al, 1992).Much more information has been obtained in polyphenolic constituents of chill haze which exists in beer not only as simple monomers and dimers but also as more complex associations with other substances, especially proteins. The flavonoid polyphenols have been referred loosely as anthocyanogens, but the term proanthocyanidins has gained favor for identified structures such as prodelphinidin B3 and procyanidin B3. On hydrolysis proanthocyanidins gives anthocyanidinis total polyphenol. In brewing often encountered anthocyanidins are delphinidin, cyaniding, and pelagoridin. Although the chemical mechanical remains obscure, haze stability is influenced by the effects of major haze precursors on one or another. The stabilization agents silica hydrogel (SHG) and polyvinyl poly pyrolidone (pvpp) remove by the sorbing proteins and polyphenols respectively (Kenneth A.Leiper,etal,2005).

Figure 7.Flavonoid Phenolic and Proanthocyanidin (Condensed Tannin) Structures

L. chapon stressed the importance of the equilibrium between the quantities of protein (p) and tannin (T) in formation of chill haze. (M .moll, 1984).

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Moll has further elaborated and devised automated instrument for measuring “sensitive proteins” and tannoid reactants in beer. The rate of haze development should be proportional to the product of the reactants in accordance with second order reaction kinetics such that for reaction example as.

A+B K [AB]

Rate=K [A] [B]

Values for haze development rate can be plotted therefore against the products of sensitive protein contents and dimeric proanthocyanidin contents.These correlations suggest that the rate of chill haze development is closely dependent on the content of sensitive protein and dimeric proanthocyanidin present in the beer at bottling and that analytical method less specific than HPLC reveals that connection less clearly.

The key polyphenolic constituents in the determination of colloidal stability are the dimeric proanthocyanidins among the total polyphenols, total flavonoids anthocyanogen, Oxidizable polyphenols catechins e.t.c (Kenneth A.Leiper. etal, 2005) .

Figure 8.Flavanol 3’-4’ Ortho- hydroxy Chelation of Metals

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Chapter-3

3. Materials and methods 3.1. Materials The major raw materials used in the study including barely, barely malt, brewery yeast, hops, distilled and brewing water are from BGI Ethiopia Plc.

3.1.1. Chemicals All the reagents used in this study were analytical grade.

Chemicals/ Reagents: H2SO4, CaCl2, CaSO4, Iso-octane, O-toludine, EBT indicator, phenolphthalein (1%), NaOH, Phenol EDTA (0.01%), Iodine solution, Zinc sulfate, etc.

Equipments: the following equipment were used in this study

 Mash bath (Congress, temperature programmed, RS232, Czech),  Buhler-Miag disc miller (0.2-1.0 mm, DLFU-1980, Germany),  Spectrophotometer, HACH Large DR 3900  Kjeldahl apparatus (Gerhardt, Germany)  Magnetic Stirrer  Anton Paar DMA 450 digital density meter and alcoholyzer (Austria-Europe),  Beaker, oven, volumetric flasks, Pipette , burettes , desiccator  Analytical balance  Stop watch  pH meter  Density meter  Digital thermometer  Goggles for eyes protection  Protective gloves for hand  Filter paper watt man  Food grade acids for pH adjustment  Haze meter

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For this paper there were two main works. The first one was optimization work. In this stage, the response variables were the extract contents, Polyphenol and Free amino nitrogen of mashed wort from Congress Mash. Here the Extracts were Optimized to get higher values of Extract content, Lower Polyphenol and Higher Free Amino Nitrogen (FAN), using EBC Method. The second work was the production of an improved beer from optimized malt ratio made by fermenting the boiled with hope and Cooled wort fermentation by adding of Yeast and Analyzing the produced Beer Quality at BGI Ethiopia Plc , Laboratory. 3.2 Experimental Design Summary 3.2.1 Factors, the Corresponding ranges and Levels

Table 8 Factors, the Corresponding Ranges and Levels Factors Units Ranges Levels

Low(-) High(+) Local Malt g/kg 30%-70% 30 70 Imported Malt g/kg 30%-70% 30 70

3.2.2 Response Summary

Table 9 Response Summary

Response Unit Minimum Maximum Extract Yield % 8.4 9.4 FAN mg/l 156.8 242 Polyphenol Mg/l 83.5 310.2 3.2.3 Several Blending Ratio Responses Table 10 Blending Ratio Response Std Run Code % Local to PPH Extract FAN order order Imported % Yield (mg/l) (%,EBC) 8 1 +1 100% imported 83.48 8.81 242 11 2 -1 100% local 310.13 8.72 157.5 10 3 30L:70I 85.40 8.67 226.34 4 4 40L:60I 89.30 8.43 216.8 7 5 50L:50I 78.55 8.46 176.2 5 6 60L:40I 88.66 8.96 163 6 7 70L:30I 84.34 9.21 171.2 9 8 30L:70I 310.13 8.72 157.5 1 9 40L:60I 89.50 8.40 217.0 2 10 100% imported 84.0 8.83 241.0 3 11 100% Local 310.20 8.70 156.8

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3.2.4 Optimization of Process for Mixture D-optimal The process condition were optimized using DOE software in contrast with classical optimization process, which may lack the effectiveness of different combination for different parameters.

3.2.4.1 Constraints Applied for the Optimization Table 11 Constraints Applied for Optimization Name Goal Lower Upper Upper Lower Importance Limit Limit weight weight Local Maximum 30 70 1 1 3 Malt Imported Minimum 30 70 1 1 3 Malt Extract Maximum 8.4 9.21 1 1 4 Yield FAN Maximum 156.6 242 1 1 4

Polyphenol Minimum 83.5 310.2 1 1 4

3.2.4.2 Selected optimum solution Table 12 Selected Optimum Solution

Local % Imported% Extract FAN Polyphenol Desirability Optimum Yield Selection 36 64 8.8 198.9 109.8 0.566 Selected 3.2.4.3 Validation of optimal combination (Comparison of products) (With 100% Imported brewed Desirability of 0.744)

Table 13 Validation of Optimal Combination

Parameters Units 100% Imported 36% Local & 64 % Imported ADOF % 82.0 81.34 FAN Ppm 152 138 Polyphenol Ppm 112 119 Apparent Extract oP 2.11 2.24 Alc (v/v) % 5.25 5.18 Color EBC 9.0 8.0 VDK Ppm 0.15 0.221

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Sensory Analysis Smell +1(4) 0(1),+1(3) Taste +1(4) +1(4) Appearance +1(4) +1(4) A relevant research conducted by Johns and piece in 1964 has assumed that the brewing yeast is only capable of assimilating simple amino acids and peptides but not proteins. Therefore, the amount of free amino nitrogen (FAN) is important to guarantee a stable fermentation process. For conventional wort FAN levels of ~150 mg/L are reported to be necessary for a healthy fermentation (Dl Goods, 2005).The total amino acid content of wort is important in determining the extent of yeast growth, while the individual amino acid spectrum of the wort influences beer flavor. Therefore, the lower amounts of amino acids soluble in worts containing low levels of malted barley may result in fermentation difficulties (affecting the extent of yeast growth), while the altered spectrum of amino acids may result in the production of beers of a different flavor and aroma.

3.3. Malt Analysis Proteins fractions have different solubility patterns in different solvents, which is a basis for their separation into different fractions. The extraction and fractionation of cowpea seed proteins was carried by sequential extraction according to their solubility in different solvents, as described by Obatolu and Cole (2000) with slight modifications.

The malt flour sample of (2.5 g) in duplicate was put in a 50 mL screw capped falcon tube. The Extract of the sample was obtained twice with 50 mL distilled water for 30 minutes with continuous shaking. The Falcon tube was centrifuged with their contents at 3000 rpm for 15 min to separate the extract the form the residue. The clear supernatant containing water soluble proteins was determined for estimation by micro Kjeldahl.The remaining residue were obtainedsuccessively in a similar way with 5% KCl or 0.5 M NaCl solution, 70% ethanol and 0.2% NaOH solution for Globulin , Herdoin and Glutlin protein fractions ,respectively.The supernatant of each extract were separated and used for estimation of the salt (globulin), alcohol- (prolamin) or alkali- (glutelin) soluble fractions.The residue remaining after successive extractions represents the insoluble proteins.The protein contents of the four extracts of each sample and residue were estimated by the micro-Kjeldahl method.

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Soluble Protein –by Micro- Kjeldhal , Modified

 10ml of wort /extract solution/ was taken into dry Kjeldhal flask and digesting.

(The wort was preheated to evaporated the excess moisture and dry it ) the sample was digested by adding 3ml of concentrated Sulphuric acid and 10 gm. of catalyst and antifoam.

 [ the digestion ,distillation and titration completely were performed according to EBC Method 3.3.3].The Total nitrogen content was calculated using the following formula

Total ( N% ) = T x 14 x100 , V

Where

V is Volume of Extract solution and T is Volume of HCl taken during titration.

3.3.1. Total Protein content The nitrogen compounds in the Wort were digested with hot Sulphuric acid in the presence of catalyst KMNno4 to give Ammonium Sulphate. The digest was made with NaOH solution and the released Ammonia was distilled into an excess of Boric Acid solution.

1.5 gm. of finely ground sample was taken in the kjeldhal flask .10g of powder catalyst kmno4 was added in the flask.20 ml of 98% H2 SO4 was added and swirled to be mixed .It was digested at low temperature until trothing ceased .The solution was boiled until the brown color disappeared for 30 minutes .The digest was allowed to be cooled and the solid ammonium sulphate was formed .The digest was diluted with 250 ml distilled water anti-bumping agent was added and finally two layers are formed .The trap was fitted and the condenser unit was connected to flask, while the exit tube from the condenser dips below the surface of the boric acid solution. The Contents of the flask was swirled to ensure the rapid heating and mixing. The ammonia was distilled into an excess of 20 g /lit of boric solution which was about 25m and containing 0.5ml of screened catalyst.80 ml distillate was collected and titrated with standard acid to grey end point.The total nitrogen content in the dry sample was calculated using the following formula

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Total nitrogen % (m/m) = T-14*6.25/W (100-M) Where: T = standard acid required to neutralize ammonia after subtracting Reagent blank, in ml W = weight of sample taken in gram M = Moisture contents of the sample in %( m/m).

3.3.2. Moisture Content The determination of moisture contents of all malt by loss in mass of drying under specified condition.

First the clean moisture dish was dried in oven and had been put in the desiccators to make it free from moisture. Part of the sample milled for mashing was taken and weighted in a clean moisture dish in duplication form. The weighted sample was dried in oven at 105O C 3 hour for malt when the sample was dried in the oven it had been allowed to cool for 20 minutes in the desiccators at 20 OC. Then the contents of the dish were reweighted.

The moisture (M) contents were calculated as follow:-

M = %( m/m) = (W2-W1/W3-W1) *100

Where:

W1= mass of the sample container in gram

W2 = mass in gram of sample and sample container before drying

W3 = mass in gram of sample and sample container after drying

3.4 Wort Analysis

3.4.1 Extract yield by Mashing

At proper temperature ,the enzyme in the ‘malt mash’ are activated ,and the rapid conversion of starches to sugars, which begins in the malt plant ,continues .The production of wort serves as a key element in the brewing process. Before mashing, the grind will be subjected to gelatinization .After that at different mixing ratio and temperature the mashing will be take place. The extract

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or sugar content of malt was determined after mashing and Filtration. The extract or sugar content of resulting wort was determined from the specific gravity of the wort that can be measured by density meter. The extract of the wort were obtained from the specific gravity by means of table. ( Platotable ) at 20 o C.

The mashing bath was attempted at 45 oC.200 ml of water was stirred at a temperature of about 46 o C into 8 beakers with glass rod and balling was avoided. The temperature was ensured in the mash was exactly 45oC.The mash beaker was placed immediately in the mashing bath and the stirrers was set in the motions. The temperature of the mash was raised for 25 minutes to 70 o C. When the temperature is 70 o C further 100 ml water was added at 70 o C. The scarification rate from this point was measured. A drop of the mash was transferred to spot on the porcelain plate and drop of iodine Solution was added 10 minute after addition of the water. This test was repeated 5 minutes intervals until saccharification was completed or until clear yellow spot was obtained. The temperature was maintained of 70 o C for 1 hour and the mash cooled to room temperature in10 to 15 minutes. Extract contents of Wort was determined using the following formula. A) E1 (% m/m) = P (M + 800)/100-P B) E2 (% m/m) = (E1 *100)/100-M, Where: E1 = the extract content of sample in % (m/m) E2 = the extract content of dry malt in % (m/m) P = the extract content in wort % (m/m) M= the moisture contents of the malt in % (m/m) 800 is the amount of distilled water added into the mash to 100gm of malt in ml.

Calculation of Yield Y =V*D/E1*W1, Where, Y = yield V = actual volume of cold wort D = density of the cold wort.

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3.4.2. Total Polyphenol (Spectrophotometer Method-EBC Method 4.7.5) 10ml of the beer sample was pipette out and 8ml of CMC/EDTA reagent into a 25 ml volumetric flask Stopper and thoroughly mix the contents. Then adding 0.5ml ferric reagent to the measurement sample and thoroughly mix, and 0.5ml ammonia reagent and mix was done make up to 25ml (or 50ml) with water and mix. After kept the measurement sample for 10minutes measurement the absorbance was done in a 10ml cuvette using a spectrophotometer at 600nm.ensure that the solution to be measured was cleared. Blank Sample Mix 10 ml of the sample of beer and 8 ml CMC/EDTA reagent in a 25 ml or 50 ml of volumetric flask and 0.5 ml of ammonia reagent was added and mix well. Allowed to stand for 10 minutes and measured the absorbance. The content of polyphones was calculated using the formula, P (ppm) =A*820*F Where: P = polyphenol content (mg/liter), A = absorbance at 600 nm & F = dilution factor

3.4.3. Free Amino Nitrogen (FAN) FAN measurement assesses the level of amino acids, ammonia and end group amino nitrogen in brew. Total FAN indicates the bioavailability of nitrogen in beer. As a diagnostic test, low FAN measurements indicate slow or incomplete fermentation, while high FAN measurements may indicate haze issues and diacetyl formation. A beer sample was diluted to 1 up to 50 ml and standard 1 to 100 ml, 2ml of both diluted samples were pipetted out into separate test tubes. A fresh prepared 1ml of ninhydrin color reagents was added. The solution was heated at 100 o C for 16 minutes; the stopper was closed firmly to avoid evaporative losses. The solution was cooled in water bath at 20 OC for 20 minutes. A 5ml of dilution solution was added, mixed and the absorbance was measured in 10 mm cuvette against reagent blank (distilled water) prepared from 2 ml water and with color reagent at 570nm within 30 minutes. Three replicates glycin standard were checked using 2ml of diluted glycin solution. Equation 5 Fan determination used by the following formula

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FAN (mg/l) = A1.2d

A2 Where, A1 =absorbance of test solution or sample at 570nm in 10mm cell A2=Mean absorbance of standard solution at 570nm in 10mm cell d= dilution factor (50 if dilution was 1ml to 50 ml) 3.5 Design of the Experiment Experimental design selected for this study will be the statistical analysis Design and the Mixture design of D-Optimal utilized the extract contents of the wort, the Polyphenol and Free Amino Nitrogen of wort obtained from mashing of grains in congress Mash as response. The experiment was analyzed by the Design Expert software 7.0.1 and suitable model equation was gained. ANOVA analysis was used to compare the process variables and their significance to the condition that was suitable to obtain high extract contents of wort, low haziness and low cost expenditure. In this optimization process the process variable such as optimum blending ratio, is considered to extract high percentage of fermentable sugar with sound shelf life beer.

3.5.1 Physic-Chemical and Instrumental Analysis of Beer The Original gravity (OG), specific gravity (SG), color, PH, apparent extract, real extract, attenuation and alcohol content (v/v), (w/w) were measured using the Anton Paar Beer analyzer. The color, pH, VDK & polyphenol were measured as directed in the European Brewing Convention method (EBC, 1975). All the analysis was carried on the filtered sample.

3.5.2 Bitterness 10ml of the samples were filled into test tubes, which had previously been filled with 20ml 2, 2, 4-trimethylpentane ( Iso -octane) and covered. Samples were acidified with 1ml of 0.1N HCl and shaken vigorously for 20 min. The supernatant organic phase was carefully decanted into cuvette and the optical densities read at 275 nm wavelength. The optical density was expressed in terms of European Brewing Convention bitterness units as follows, Bitterness units (BU) = 50 x ABS275 (EBC, 1998).

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3.5.3 VDK 100ml of beer was added into the distillation flask and distillation started. The heating rate was carefully controlled to prevent over foaming. (The available period under gentle heating should be at least 6 min until the first drop is collected into the receiving cylinder).25 ml of the distillate was collected within 6 to 8 min and mixed thoroughly, 10ml of the distillate was pipetted out into 50ml flask with glass stopper, then 0.5ml % 0-phenylenediamine (reagent).mix was added and the flask was placed for 20-30minutes in the darkness. After that, 2ml 4N HCL (reagent) was added in both blank and sample flask and mixed thoroughly and within 20 min the sample was measured against the blank , The amount of VDK was determined by the following formula VDK, ppm= 2.7*ABS335

3.5.4 Total Polyphenol (Spectrophotometer Method-EBC Method 4.7.5) 10ml of the beer sample was pipette out and 8ml of CMC/EDTA reagent into a 25 ml volumetric flask Stopper and thoroughly mix the contents. Then adding 0.5ml ferric reagent to the measurement sample and thoroughly mix, and 0.5ml ammonia reagent and mix was done make up to 25ml (or 50ml) with water and mix. After kept the measurement sample for 10minutes measurement the absorbance was done in a 10ml cuvette using a spectrophotometer at 600nm.ensure that the solution to be measured was cleared. Blank Sample: Mix 10 ml of the sample of beer and 8 ml CMC/EDTA reagent in a 25 ml or 50 ml of volumetric flask and 0.5 ml of ammonia reagent was added and mix well. Allowed to stand for 10 minutes and measured the absorbance. Calculate the content of polyphones using the formula, P (ppm) =A*820*F Where: P = polyphenol content (mg/liter), A = absorbance at 600 nm & F = dilution factor

3.5.5 Determination of calcium ion (Ca2+) Apparatus Glass titration burette, conical flask Erlenmeyer 250 ml, volumetric pipette, 3 ml,

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10 ml, Stainless steel spatula and Magnetic stirrer,

Reagents Potassium hydroxide 8 N, EDTA 0.01 mol/l (N/50), Cal Red indicator (calcium indicator: Prepared from Calconcarboxylic acid 1 g and Potassium Sulphate 99 g) 10 ml of sample was pipetted into a conical flask. Distilled water was added until 100 ml and 3 ml of potassium hydroxide solution was added. The solution was mixed well with magnetic Stirrer for 5 minutes. Then 1 pinch of Cal-Red indicator was added and titrated with 0.01 Mol / l EDTA. The amount of calcium was determined by the following formula Calcium, ppm = 40.08*V1 *0.01*1000/ml of sample. Where, 40.08 is factor. V1 = the Volume of EDTA consumed

3.5.6. Determination of Foam Stability Value To determine the foam stability of beer using NIBEM T / TPH foam stability tester. Beer, is attemperated to 20 + 0.5 OC in a sealed container, is dispensed through foam flashing device (Flasher) in which beer is forced under Carbon dioxide pressure through an orifice. This Produces a Standard glass of beer/ foam.

3.5.7 Pasteurization The finished product were packaged in bottles to volumes of 330 ± 5 ml and pasteurized above 60 °C for about 42 to 45 minutes using the bottles’ Krones Tunnel pasteurizer at a pasteurization unit of 19 – 23 PU.

3.6. Microbiological Analysis of the Beer

3.6.1 Saccharomyces Wild Yeast Standard medium: kleyns’ agar 20 ml of fermenting beer (or 0.5 g of yeast in 20 ml of beer) was placed in a 25 ml test flask with a tight lid in a water bath at 46 ± 0.3 o C for 18 min.

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The sample was cooled immediately to room temperature and the beer was decanted leaving approx. 2 ml of Suspended yeast in the bottom of the flask. 10 ml of beer was added; 0.5 ml of 96 % ethanol was added for inhibition of bacteria. The flask was plugged with cotton and incubated at 27 o C for 48 hours. The supernatant liquid was decanted and a drop of the yeast was transferred into a Petridish, previously prepared with hardened Kleyns’ agar. The Petridish was incubated for 48 hours at 27 o C. The yeast cells were examined from Kleyns’ agar for spores under the Microscope. In bright field microscopy wild yeast spores are refracting, i.e. they become bright when the focusing is slightly altered by the fine adjustment knob. In phase contrast microscopy the spores are visible as dark particles. Spores of saccharomyces wild yeast can be distinguished from particles in the ascus by being present in numbers of 2, 3 or 4 in the ascus, and by being globe -shaped and equal of size. Spores of brewer’s yeast may interfere with the results from time to time. They can be distinguished from wild yeast spores by being large so that they fill out the whole ascue. They don’t refract light in bright field microscopy. Do not stain the spores as this is likely to cause interference by prephasial brewery yeast spores. 3.6.2 Total Count

3.6.2.1. Method

Standard medium: UBA Procedure Standard Techniques Membrane: Methods Laboratory membrane filtration or sampling through membrane filters. Filter membrane growth technique.  Sample size 100 ml  Incubation Aerobic  Time 3 days  Temperature 27 oC  Examination Colony counts under Microscopy Expression of Results: Number and type of Colonies per 100 ml of sample.

3.6.3. Lactic Acid Bacteria Membrane filtration followed by anaerobic incubation on a selective medium containing actidione .The samples were taken aseptically

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Reagents Standard medium: UBA + 10 ppm actidione Procedure Standard Techniques Membrane methods: Laboratory membrane filtration, or Sampling through membrane Filters. Filter membrane growth technique.  Sample size 100ml

 Incubation Anaerobic(CO2 - Catalysed)  Time 3 days  Temperature 27 oC  Examination: Colony counts, Microscope, Catalase and gram- reaction if necessary Procedures for Preparation of the Media  UBA MEDIUM Reagents  UBA medium  NaOH 35 % / HCl 20 % 62 g of the UBA powder (reagent 1) was suspended rapidly in 750 ml of demineralized water and mixed thoroughly. Heated with frequent agitation and boiled for 1 minute to completely dissolve the powder. While the medium was still hot, 250 ml commercial beer was added (not degassed) and mixed well. It was dispensed into the laboratory bottles, at the rate of 200 ml per bottle preferably. Autoclaved for 10 minute 121 ° C.

3.7. Sensory Analysis An organoleptic profile (aroma, taste, color, mouth feel, bitterness & foam stability) tasting are conducted on 6 beer samples i.e. 100% imported Malt brewed and (36 % Local, 64 % imported) Malt brewed with its triplicate. The sensory analysis was conducted using 3 member-trained panelists using the sensory evaluation form in appendix B. The panel judges constituted 3 males & myself who are staff and currently have been working in BGI Ethiopia Brewery having brewing experience and are familiar to beer drinking. Training in descriptive analysis of beer was conducted in three separate days for three consecutive days about the range of sensory attribute with reference beers. The performance of panelist were checked by comparing the

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sensory analysis result and instrumental result. The samples were served at 12 0C so that panel members could easily pick flavor notes. Samples were served in clean and odorless drinking glasses. All assessors had one score sheet for four samples and tasted all samples within 15 minutes. The scale was designed as three score levels for the evaluation of external appearance (foam, color intensity and turbidity), smell (hop, malt, toasted, caramel, Odour intensity), mouth feel (carbon, viscosity, astringency) and gustatory sensations (sour, bitter and sweet taste, intensity and persistence). Panelists were instructed to rinse their mouths with water before starting and between sample tasting. Parameters for evaluation are shown in the questionnaire in appendix A. The ratings of each sensory attribute were converted to numerical scores and the numerical scores were collected for statistical analyses. Only one taste panelist gave good result on the appearance but all result scores are excellent.

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Chapter -4

4. Results and Discussion

4.1. Moisture Content Comparing the moisture content of the pure local, pure imported malts with several blend ratios the blended ratio showed intermediate results between the higher imported (5.5) to lower Local (4.8) which is desirable. Considering microbiological instability, the moisture content is crucial. As the ratio of the imported malt increases it can be shown easily that the moisture content also decreases towards the standard (4.0),this indicate the imported malt is better than the local malt. This again indicates that the moisture content is directly determined the microbial infection and contamination. Thus we can conclude that the imported malt is not susceptible to be contaminated by microbes.

% of Local malt to moisture content 5.6 5.5 5.5 5.4 5.3 5.2 5.1 5 Moisture content Moisture 4.9 4.9 4.85 4.8 4.8 4.75 4.75 4.7 0 20 40 60 80 Percentage of local malt

Graph 1. % of Local Malt to moisture Content

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% of Imported malt to moisture content 5.6 5.5 5.5 5.4 5.3 5.2 5.1 5 4.9

Moisture content Moisture 4.8 4.8 4.75 4.7 4.7 4.7 4.6 4.6 4.5 0 10 20 30 40 50 60 70 80 % Imported malt

Graph 2. % of Imported Malt to moisture Content

4.2 Protein Fractionation of Malt After conducting several Fractionations of malt protein using Oswald fractionation by solubility differences of the protein types ,the prolamin(Albumin,Globulin, Prolamine and Glutlin),the haze active protein that is responsible for formation of haze by complexing with polyphenol showed significance difference between the imported and the local mal variants.The imported malt has lower Haze active protein(0.35%), comparing to the local(0.84%),this is the core result of this research which will significantly shows the difference on malts quality that determine the haziness by reacting with Polyphenol to form the protein-polyphenol complex which has a direct impact on the shelf life of beer to be produced.

Table 6 Protein Fractionation

Type of protein Protein content (%) Imported Local Albumin 3.22 4.20 Globulin 1.19 0.56 Prolamin 0.35 0.84 Gluthine 1.40 1.12

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Graph 3 % of Prolamin Content in Imported and Local Malt Prolamin content in imported & local malt 0.9 0.84 0.8 0.7 0.6 0.5 Prolamin in imported malt 0.4 0.35 Prolamin in local malt 0.3 0.2 0.1 0

4.3. Particle Size Distribution The term Non-Microbiological Particles comprise multitudes of compositional species, although they are roughly classified as proteins, usually complex with polyphenols and other molecules such as lipids, carbohydrates and/or metal ions. NMP in Beer range ‹2mm, 2-10 mm and >10mm.The important malt parameter used for NMP is particle size distribution ,the Imported Malt is better than the local which is visualized as 4.4 % than the 5.3% particles comparing to the Standard of 4%,that is crucial for colloidal stability of the beer to be produced by stokes law of Sedimentation. Particle Size Distribution of Local and Imported Table 7 Particle Size distribution

Mesh sieve size w/w of Imported w/w Assela Assela malt standard imported malt grist malt grist grist malt grist Mw 1.25 mm 1.30 4.4 % 1.54 6.1% <= 4%

Mw 1.000 mm 3.12 4.4 % 4.55 6.1% <= 4% Mw 0.50 mm 16.95 51.6% 21.55 6.1% <= 61% Mw 0.25 mm 18.85 51.6% 22.38 6.1% <= 61% Mw 0.125 mm 15.51 44.0% 14.32 35.3% <= 35% Collecting tray 43.72 35.14

4.4 Physic-Chemical, Instrumental and Microbial Analysis Results of the Finished Beer. The beer quality produced was astonishing since it conform to customer satisfaction in terms of the Ea , Alc v/v, and OG values since it takes the chosen attributes between pilsner and castle

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beer. The microbial analysis showed that the expected microbes including E.coli, LAB and Yeast are null since sterile yeast and water are used but there is too much amount of mould’s hyphae,since it is highly contaminated after pasteurization while analyzed due to cross contamination. For determination of Fusarium spp.that could be happening in barley malt that causes gushing that no chemicals were available in several chemical stores that are available.

Table 14 Physico –Chemical Analysis results of the optimized Beer

Physico -Chemical Analysis Optimized Finished Standard Beer Parameters Beer Analysis Results LL STD UL S.G 1.00872 Ea 2.24 0.5 1.5 1.5, EBC Alc v/v 5.18 1.52 4.61 5.34,ES 832 or EBC Alc w/w 4.06 1.2 3.62 4.19,ES 842 or EBC Er 4.12 1.04 3.20 3.58 ES 842 or EBC O.G 11.99 3.5 10.51 12.51ES 833 or EBC ADOF 81.34 Color 8.0 6 EBC pH 4.54 3.6 4.2 4.8 ES 830 or EBC Ca+ - Polyphenol Bitterness 14.9 13 EBC VDK 0.215 0.6 ES 843 Or EBC Foam Stability 170 Haziness,90o /25O 0.77/0.06 Microbiological Analysis E. coli 0 1 cfu / ml ES ISO 7251 0 LAB 0 - 0 0 Yeast 0

4.5 Accelerated Shelf life /Forcing Test Result After Initially Measuring the Haziness of the samples in triplicates before it was subjected to 60 o C in oven for seven days according to EBC method. The chill haze formed was determined after subtracting the amount from samples after chilled overnight. Thus, according to the result of the difference between the final and initial haze, 1.77 EBC is under the standard of EBC.We can deduce that the shelf life of beer produced will be stable with regard to chill Haze .i.e. protein- polyphenol Complex for next six up to 12 monthes which is equivalent to the

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forcing/accelerating the deterioration rate for seven days at 60 o C . Basically the chill haze is the protein amount of beer after subjected to lower temperature that retains in the beer. Due to contamination of oxygen and no stabilizer has been added to it, the haze value has showed higher value .as a recommendation if a controlled environment to minimize oxygen and highly sterile condition is applied to minimize microbial contamination the haziness increment will more hopefully decreases substantially.

Table 8 Chill Haze Measurement after Forcing Test

Trial Haze @90 Haze@90 Haze@90 Mean of Standard,EBC # Final @0 o Initial @0 Final-Haze Chill Method C(EBC) oC(EBC) Initial(EBC) Haze(EBC) 1 24.05 26.02 1.97 1.77 ≤2 2 23.10 25.00 1.99 3 24.80 26.15 1.32

4.6 Sensory Analysis An organoleptic profile (aroma, taste, color, mouth feel, bitterness & foam stability) tasting are conducted on 6 beer samples i.e. 100% imported Malt brewed and (36 % Local, 64 % imported) Malt brewed with its triplicate. The sensory analysis was conducted using 3 member-trained panelists using the sensory evaluation form in appendix B. The panel judges constituted 3 males & myself who are staff and currently have been working in BGI Ethiopia Brewery having brewing experience and are familiar to beer drinking. Training in descriptive analysis of beer was conducted in three separate days for three consecutive days about the range of sensory attribute with reference beers. The performance of panelist was checked by comparing the sensory analysis result and instrumental result. The samples were served at 12 0C so that panel members could easily pick flavor notes. Samples were served in clean and odorless drinking glasses. All assessors had one score sheet for four samples and tasted all samples within 15 minutes. The scale was designed as three score levels for the evaluation of external appearance (foam, color intensity and turbidity), smell (hop, malt, toasted, caramel, Odor intensity), mouth feel (carbon, viscosity, astringency) and gustatory sensations (sour, bitter and sweet taste, intensity and persistence). Panelists were instructed to rinse their mouths with water before starting and between sample tasting. Parameters for evaluation are shown in the questionnaire in appendix A. The ratings of each sensory attribute were converted to numerical scores and the

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numerical scores were collected for statistical analyses. Only one taste panelist gave good result on the appearance but all result scores are excellent. Table 9 sensory Analysis result Date Sample ID Appearance Smell Taste Mouth- Drinkability fullness 1 I +1(4) 0(1),+1(4) +1(4) +1(4) +1(4) II +1(4) 0(2),+1(2) +1(4) +1(4) +1(4) 2 III +1(4) 0(1),+(3) +1(4) +1(4) +1(4) IV +1(4) 0(1),+(3) +1(4) +1(4) +1(4) 3 V +1(4) 0(1),+(3) +1(4) +1(4) +1(4) VI +1(4) 0(1),+(3) +1(4) +1(4) +1(4)

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Chapter-5

5 Conclusions and Recommendations It is an obvious fact that, due to the high competition and wider customer demand that breweries are joining into the vast market that is available throughout this splendid country. The major players of the market like BGI Ethiopia, Dashen and Meta abo have faced a fierce competition from the big boys such as Heineken brewery and Bavaria who has lots of practical experiences and resources in the industry.

Beside quality issues, the near trend of marketing strategy among other things that changed the package of beer from amber bottle to clear and green bottles will definitely impacts the customer’s insight by revealing about the brightness and clarity of the beer. The Colloidal stability of beer is the most important factor in beer quality. Colloidal particles significantly shorten beer’s storage time, but importantly, also its appearance. Moreover, the despite the quality that the imported malt has surpassing the local, the huge amount of foreign exchange can be incurred by using the optimized malt ratio. Thus it is recommended to use the recipe 36% local and 64% imported malts for achieving best quality and cost effective brewery in terms of the foreign currency it incur by minimizing the full utilization of the sky touched price of imported malt .Optimization of the brewing process with respect to flavor stability requires a clear insights of the types of flavor changes during storage and the nature of the molecules involved This however may vary among beer types (e.g. pilsner beers & specialty beers) Secondly, it is essential to clarify the reaction pathways in beer leading to stalling compounds. The production process also affects the stalling of beer Knowledge of the aging phenomenon in a particular types of beer can be used to develop appropriate technological process improvements to control its particular flavor and stability.

Besides for their relevance for flavor stability, the investment costs for suggested process modifications must be evaluated and a consideration must be made between better and longer flavor stability and costs. Although few attempts are done in these areas practical experience may soon show that flavor stability is still hard to control. The obvious explanation is that the knowledge of stalling compounds is still incomplete not only concerning the number and type of compounds involved but also the reaction mechanisms.

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A further research on shelf-life of beer taking the deep kinetics knowledge is recommended for it requires a vast resource and devotion. By easily starting comparing the shelf- life of local beer the researcher can contribute for the shelf- life study.

As a Recommendation due to the vitality of the protein fractionation procedure, scarcity of the the chemicals available in the country and appropriate methods further research that lead to protein / Prolamine gene manipulation has to be conducted by local researchers. This would help as an input for further haziness study as contribution.

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References Aron, P. M., & Shell hammer, T. H. (2010). A discussion of polyphenols in beer physica And

Flavour Stability. Journal of the Institute of Brewing, 116(4), 369-380.

Adom KK, L. R,(2002),Antioxidant activity of grains. Physicochemical properties of roller

Milled Barley Brand and Flour. (J Agri Food Chem Bhatty R .S)

Bamforth C (2009). Current Perspectives on the role of Enzymes in Brewing.Journal of

Cereal Science 50(3):353-357

Briggs D.E, H .J.S., Stevens R,Young T.W(2001).Malt and Sweet Wort . Malting and Brewing

science ( Kluwer Acadamic /Plenu).

C. M., & Cochrane, M. P. (1992) .Grain Structure and Composition. Grain structure and

Composition,291- 317.EBC Analysis Committee(2010) Analytical-EBC.

Declour,J.A.,et al.(1984). Flavour and Haze Stability differences in Unhopped and hopped all

Malt Pilsner Beers brewed with Proanthocyanidin-free and with regular Malt.Duffus,

Elias H. (2016) Optimization of production of beer from maize and different barley varieties.

F G.M (2009 ) ,A Comprehensive History of Beer Brewing. In: Handbook of brewing Process,

Technology,Markets .

Giese,H., & Hopp,H. E. ( 1984).Influence of nitrogen nutrition on the amount of Hordein,

protein Z and β-amylase messenger RNA in developing endosperms of barley. Carlsberg Research Communications, 49(3), 365.

Gupta,M,A.G. N.,Gallaghar E(2010) .Barley for Brewing: Characteristic change during

malting.Brewing and Application of it is by-products.125 th anniversary review: the science of thetropical cereals sorghum, maize and rice in relation to larger Beer Brewing. (Mount Sinai School of Food Science and Environmental Health):318-328.

In:Friedman RF,Adams B(eds) The Followers of Hours.(Oxbow Books): pp 19-26

I.Hornesy . (2004).Ahistory of beer andbrewing .Royal Society of Chemistry Vol I.Journal of

Thesis Page 51

the Institute of Brewing 90(2):67-72

JR, C. G.(1992).From prehistory to history: beer in Egypt”Narziss,L(1992).Comments on

Some taste factors in beer.Brauwelt international (Germany,FR).

Nurnberg Fachverlang Hans Carl Ehrlich,P,R and A.H Ehrlich (1990).The population

explosion

Obatolu,V. A., & Cole, A. H. (2000). Functional property of complementary blends of soybean

and cowpea with malted or unmalted maize. Food Chemistry, 70 (2), 147-153.

Ogbonna,A.(2009),Beer and Health :A Review,Technical quarterly.

P.Mathias. (1959).The Brewing Industry in England.A comparision of maize,sorghum and

Barley as Brewing Adjuncts (Cambridge University Press.)

P.G.Anderson . (1989).ᵝ-glucan in Production Malting. Proceedings of 22nd Congress of

FBC.Zurich (Congress of EBC):pp.213-220.

Rabin D,F.C.(1998).The dictionary of beer and brewing .Chicago ,Fitzroy Dearbom

Publishers,2nd edn

Schmitt,M.R.,et.al(2013).”Protein mobilization and malting Specific proteinases expression

During Barley germination “Journal of cereal Science 58(2):324-332

Stratford M.(1989).Yeast flocculation: Calcium specificity.Yeast 5(6):487-496

Schonberger C,K. T, The role of hops in brewing Journal of the Institute of Brewing

J.InstBrewing.

Smith, D. B., & Lister, P. R. (1983). Gel-forming proteins in barley grain and their Relationships

with malting quality. Journal of Cereal Science, 1(4), 229-239.

Stratford, M. (1989 ). Yeast flocculation: calcium specificity. Yeast, 5(6), 487-496.

Taylor J,D.B.,Kruger J(2013).”The science of the tropical Cereals Sorghum,Maize and Rice in

relation to lager beer brewing .125 th anniversary Review J Inst Brew.

Thesis Page 52

Thompson, M. W., Strohl, W. R., & Floss, H. G. (1992). Purification and characterization of

TDP-D- glucose 4, 6-dehydratase From anthracycline- Producing

Streptomycetes.Microbiology, 138(4), 779-786.

Wardani, D. P.et al (2004). Growth and Saponin Production Of Talinum paniculatum Gaertn.

Callus culture on various addition with2, 4- dichlorophenoxy acetic acid (2, 4- D) and

kinetin. BiofarmasiJournal of Natural product Biochemistry, 2(1), 35-43.

W, Kunz (1999). Technology of Malting and Brewing .2nd edition.Berlin:VLB.

Wunderlich S.B.W, (2009), Overview of manufacturing beer: ingredients, processes and quality

Criteria.In: preedy VR (ed) Beer in health and disease prevention Elsevier, London:pp 3-

16.

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Appendix A ANOVA Table for Yield of Extract of Wort

Sum of Mean p-value Source Squares df Square F-Value prob>F Significance Model 0.28 3 0.094 2.35 0.1583 NOT Linear 5.538E- 1 5.538E- 0.014 0.9095 Mixture 004 004 AB0.12 1 0.12 2.95 0.1296 AB(A-B) 0.16 1 0.16 4.10 0.0826 Residual 0.28 7 0.040 Lack of 0.28 1 0.28 1471.26 <0.0001 YES Fit Pure 1.133E- 6 1.889E- Error 003 004 Cor Total 0.56 10 Std. Dev 0.20 R-Squared 0.5022 Mean 8.70 Adj R-Squared 0.2888

C.V % 2.29 Pred R-Squared -2.0216

PRESS 1.69 Adeq Precision 4.485

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Anova Table for FAN of Wort

Sum of Mean p-value Source squares df Square F-value prob> F Significance

Model 12399.83 2 6199.91 140.64 <0.0001 YES

Linear 11972.96 1 11972.96 271.60 <0.0001 mixture

AB426.87 1 426.87 9.68 0.0144

Residual 352.67 8 44.08

Lack of fit 350.69 2 175.34 531.34 <0.0001 YES

Pure 1.98 6 0.33 Error Cor Total 1275.5 10

St.Dev 6.64 R-Squared 0.9723

Mean 203.94 Adj R-Squared 0.9654

C.V.% 3.26 Pred R-Squared 0.9567

PRESS 551.78 Adeq Precision 24.756

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Anova Table for Polyphenol of wort

Source Sum of df Mean F-Value p-Value Significance Squares Square prob>F

Model 89931.03 2 44965.51 16.37 0.0015 YES

Linear 41957.57 1 41957.57 15.28 0.0045 Mixture

AB47973.46 1 47973.46 17.47 0.0031

Residual 21972.07 8 2746.51

Lack of Fit 5654.18 2 2827.09 1.04 0.4096 NO

Pure Error 16317.89 6 2719.65

Cor Total 1.119 10 E+005 Std.Dev 52.41 R-Squared 0.8037 Mean 161.47 Adj R-Squared 0.7546

C.V.% 32.46 Pred R-Squared 0.6082

PRESS 43847.30 Adeq Precision 8.375

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Appendix B Questionnaire for Overall Acceptability Ranking Test

Date Sample ID Appearance Smell Taste Mouth- Drinkability fullness

Notes: i. Definitions  Appearance: Color, Clarity, Foam…  Smell: Aroma of malt, hop, yeast…  Taste: sweet, bitter, sour, alkaline….

 Mouth-fullness: Ea, CO2 ii. Taste Ranking  -1 Not good  Normal  +1 Excellent

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Appendix C Malt Fractionation

Congress Mashing

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Mash Filtering

Wort Sterilization (Boiling)

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Wort Cooling

Fermentation

Filtration Yeast Addition

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Analysis

Bottling

Pasteurization

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