1
EFFECTIVE USE OF A TROPICAL HOP NAMED BITTER
LEAF [VERNONIA AMYGDALINA] EXTRACT AS A MEANS
OF EXTENDING THE SHELF – LIFE OF
LOCALLY BREWED MILLET BEER.
BY
OKOYE NANCY IJEOMA
REG №: BC ∕2006 ∕089
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF NATURAL SCIENCE
CARITAS UNIVERSITY, AMORJI–NIKE EMENE,
ENUGU STATE.
AUGUST 2010.
2
TITLE PAGE.
EFFECTIVE USE OF A TROPICAL HOP NAMED BITTER LEAF
[VERNONIA AMYGDALINA] EXTRACT AS A MEANS OF EXTENDING
THE SHELF – LIFE OF
LOCALLY BREWED MILLET BEER.
BY
OKOYE NANCY IJEOMA
REG NO: BC ∕ 2006 ∕ 089.
A PROJECT SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY,
IN
PARTIAL
FULFILLMENT
OF
THE
REQUIREMENT
FOR
AWARD OF BACHELOR OF SCIENCE
(B.SC) DEGREE IN BIOCHEMISTRY
FACULTY OF NATURAL SCIENCE
CARITAS UNIVERSITY, AMORJI – NIKE, EMENE.
ENUGU STATE.
AUGUST 2010.
THE
3
CERTIFICATION PAGE.
This project work done by Okoye Nancy Ijeoma has been approved as
meeting the requirement of the department of Biochemistry, Caritas university,
Amorji-Nike Enugu for the Award of Bachelor Degree of science (B.SC).
--------------------------------------Mrs. Oluchi Ajemba
------------------------------DATE.
Project Supervisor.
--------------------------------------Mr. Moses Ezenwali
-------------------------------DATE.
HOD, Biochemistry Department
Caritas University Enugu.
--------------------------------------External Examiner.
------------------------------DATE.
4
DEDICATION.
I cheerfully dedicate this project work first to the Almighty God for showering
me with his boundless love and gratitude through my research programme and to
all the uncelebrated researchers of this noble department of Biochemistry, that God
will bless them all, Also to my lovely parent Mr. and
Mrs. Reginald and my
sisters for their great support through my research work that God in his infinite
mercy will blessed them abundantly.
5
ACKNOWLEDGMENT.
I Thank the Almighty God who has been with me throughout this my research
programme in an unimaginable way; and was and will remain my source of
inspiration.
All thanks and gratitude go to my amiable and honourable supervisor; Mrs. Oluchi
Ajemba
for directing and puting me through in different areas needed for this
project work to be a success and to have met its set standard, Madam, God will
bless you tremendously in Jesus name.Despite her tight schedule, she never failed
to attending to me, Thank you Madam
And my unreserved appreciation goes to my humble Head of department Mr.
Moses Ezenwali and my wonderful lecturers Dr. Ikpe, Mr. Yusof Onmeh, Mr.
Peter, Dr. Ishiwu, Mr. Ugwudike, for the opportunity they gave to us in acquiring
the necessary knowledge for my career.
Also, I won’t forget my lovely classmate who encouraged me through this write up and made me believe that winners never quit. Thanks I appreciate.
In addition, I will not my pen without thanking my parent and my family
members(The Okoyes),especially Brother Arinze who has been a great support to
me through out my project work, that God Almighty will bless them in
JesusName, Amen.
6
ABSRACT
This local beer is brewed from a cereal grain called millet,and it is brewed with
bitter leave extract acting as hop substitute to see if it can increase the shelf – life
of this beer.The production process include malting, mashing, fermentation, and
maturation.The microorganisms associated with fermentation include Saccharomyces
Cerevisiae and Saccharomyces Charelienia.These yeast hasten fermentation by
converting the sugar in the beer to alcohol. Kunu is an indigenous alcoholic
beverages that is traditionally brewed with out hops,and because of this it has a
shot shelf life as compared with that brewed with the tropical hops.There
is a
significant [P≥ 0.05] difference in chemical properties of millet with hops and
without hops.Millet with hops is the best with alcoholic content of 0.325 ± 0.004,
total acidity of 0.645 ± 0.011(% lactic acid),fixed acidity of 0.041± 0.002, specific
gravity of 1.199±0.015 and pH of 4.03± 0.02.But without hops we have an
alcoholic content of 0.316± 0.005, total acidity(lactic acid) of 0.427± 0.002, fixed
acidity of 0.034± 0.005,specific gravity of 1.049± 0.007, and pH of 3.32±0.02.From
the result the beer without hops is the lowest in acceptance of chemical properties
so it is advisable that the local millet beer be brewed with the tropical hops
(Vernonia Amygdalina).
7
TABLE OF CONTENT
Title page----------------------------------------------------------------Certification page-------------------------------------------------------Dedication-----------------------------------------------------------------Acknowledgement--------------------------------------------------------Abstract-------------------------------------------------------------------Table of content------------------------------------------------------List of tables----------------------------------------------------------List of figure---------------------------------------------------------
CHAPTER ONE
1.0 Introduction------------------------------------------------------------------------------1.1 Aim of study / Project------------------------------------------------------------------
CHAPTER TWO
8
2.0 Literature Review----------------------------------------------------------------------2.1 General Description of Cereal Crops------------------------------------------------2.2 Origin of Millet and its distribution-----------------------------------------------2.2.1 Botanical [Scientific classification]------------------------------------------------2.2.2 Diseases and Pest of Pearl Millets---------------------------------------------2.2.3 Grain Composition and Nutritive value----------------------------------------2.2.4 Other Uses of Pearl Millet------------------------------------------------------2.3 Origin and Distribution of Sorghum-----------------------------------------------2.3.1 Botanical[Scientific Classification]------------------------------------------------2.3.2 Grain Composition of Sorghum--------------------------------------------------2.3.3 Other Uses of Sorghum-----------------------------------------------------------2.4
Hops-------------------------------------------------------------------------------------
2.4.1 Other Uses of Hops--------------------------------------------------------------2.5
Bitter leaves Juice and its role-----------------------------------------------------
2.5.1 Botanic Classification of Bitter leaves-------------------------------------------2.5.2 Composition of Bitter leaves--------------------------------------------------------
9
2.6
Yeast--------------------------------------------------------------------------------------
2.6.1 Origin and Distribution of Yeast-------------------------------------------------2.6.2 Composition of Yeast-------------------------------------------------------------2.6.3 How Yeast is Prepared-------------------------------------------------------------2.7
Water-------------------------------------------------------------------------------------
2.8
Clarifying agent-----------------------------------------------------------------------
2.9
Adjuncts---------------------------------------------------------------------------------
2.10 Biochemistry of brewing Process-----------------------------------------------2.10.1 Biochemistry of Malting-----------------------------------------------------------2.10.2 Biochemistry of Boiling-----------------------------------------------------------2.10.3 Biochemistry of Mashing----------------------------------------------------------2.10.4 Biochemistry of Souring-----------------------------------------------------------2.10.5 Biochemistry of Fermentation----------------------------------------------------2.11
Straining--------------------------------------------------------------------------------
2.12 Properties of Beer------------------------------------------------------------------2.12.1 Processes Involved in Beer Production-----------------------------------------
10
2.12.2 Lagering-------------------------------------------------------------------------------2.12.3 Types of Beer-----------------------------------------------------------------------2.13 Preparation of Local Beer-----------------------------------------------------------2.13.1 Factors Affecting both Local and Modern Beer-----------------------------2.13.2 Alcoholic Content of Beer------------------------------------------------------2.13.3 Differences Between Local and Modern Beer--------------------------------2.14
Packaging and Distribution--------------------------------------------------------
2.14.1 Spoilage-------------------------------------------------------------------------------2.15
Importance of fermented Alcoholic Beverages---------------------------------
CHAPTER THREE
3.0 Materials and methods ----------------------------------------------------------------3.0.1 Materials -------------------------------------------------------------------------------3.0.1.1 Chemicals and Reagents ----------------------------------------------------------3.0.1.2 Glass wares and equipments -----------------------------------------------------3.1 Preparation of samples ----------------------------------------------------------------3.1.1 Methods ----------------------------------------------------------------------------------
11
3.1.1.1 Ingredients ----------------------------------------------------------------------------3.1.2 Method used to prepared local millet beer ----------------------------------------3.1.2.1 Method used to prepared aqueous extract of bitter leaf ----------------------3.1.3 Method of chemical analysis ------------------------------------------------------3.1.4 Total acidity ---------------------------------------------------------------------------3.1.5 Fixed acidity --------------------------------------------------------------------------3.1.6 Volatile acidity-------------------------------------------------------------------------3.2 pH determination-----------------------------------------------------------------------3.3 Specific gravity --------------------------------------------------------------------------3.4 Determination of Alcohol content --------------------------------------------------3.5 Total dissolved solid (TDS).----------------------------------------------------------3.6 Total suspended solid (TSS).---------------------------------------------------------3.7 Microbial Count -----------------------------------------------------------------------3.7.1 Procedure for culturing in the plate.----------------------------------------------3.7.2 Procedure for identification of the organisms -----------------------------------
CHAPTER
FOUR
12
4.0 Results-------------------------------------------------------------------------------------4.1 Some Chemical Parameters-----------------------------------------------------------4.2 Total Acidity for local Millet Beer--------------------------------------------------4.3 Fixed acidity for local Millet Beer ----------------------------------------------------4.4 Volatile Acidity -------------------------------------------------------------------------4.5 Specific Gravity of Millet Beer------------------------------------------------------4.6 pH value for local millet beer -----------------------------------------------------4.7 Total dissolved solid.------------------------------------------------------------------4.8 Total suspended solid.------------------------------------------------------------------4.9 Ethanol Content for Bench Stored Millet-----------------------------------------4.10 Microbial Count for Bench Millet Beer-----------------------------------------4.10.1 Microbial Count for Freezed Millet Beer-------------------------------------4.11 General Discussion of Results.-------------------------------------------------------
CHAPTER FIVE
5.0 Disadvantages Associated with Over Consumption of Brewed Beer-----------
13
5.0.1 Advantages associated with consumption of brewed beer---------------------5.1 Conclusion-------------------------------------------------------------------------------5.2 Recommendation------------------------------------------------------------------------5.3 Limitations of Study-------------------------------------------------------------------5.4 References--------------------------------------------------------------------------------5.5 Appendix A -----------------------------------------------------------------------------5.6 Appendix B -----------------------------------------------------------------------------5.7 Appendix C ------------------------------------------------------------------------------5.8 Appendix D -----------------------------------------------------------------------------5.9 Appendix E -------------------------------------------------------------------------------
LIST OF TABLES
Table 1:Some Chemical Parameters on Local Millet Beer-----------------------------
14
Table 2: Total Acidity for local Millet Beer---------------------------------------------Table 3:Fixed Acidity for local millet beer. --------------------------------------------Table 4:Specific Gravity of Millet Beer--------------------------------------------------Table 5:pH value.---------------------------------------------------------------------------Table 6:Total Dissolved solid (TDS).----------------------------------------------------Table 7:Total Suspended solid (TSS).----------------------------------------------------Table 8:Ethanol Content of Millet Beer---------------------------------------------------Table 9: Microbial Count for Bench Stored Millet Beer-------------------------------Table 10: Microbial Count for Freezed Millet Beer------------------------------------Table 11:Total Microbial Count for both bench and Freezed Millet Beer ------------
LIST
OF FIGURES
Fig.1: A Scheme of the Brewing Process-----------------------------------------------Fig.2: Flow Sheet of home Brewed Products---------------------------------------------
15
Fig 3: A Schematic Representation of Glucose Degradation
Under Aerobic and Anaerobic Condition------------------------------------------------Fig 4 : The Glycolytic Pathway ------------------------------------------------------------Fig 5:The Pathway of Carbon atoms in Glycolysis and alcoholic Fermention---Fig 6:Fermentation Scheme-----------------------------------------------------------------Fig 7: Flow Sheet for the Local Production of Millet Beer-----------------------------
16
CHAPTER ONE
1.0 INTRODUCTION.
Beer, alcoholic brewery made from a Cerael grains known as barley and Millet.A
process called Fermentation in which Microscopic fungi known as yeast is used to
consume sugers in the grains to make Beers,converting them to alcohol and
carbondioxide gas.This chemical process typically produce beers with an adequate
level of alcohol content of about 2-6%.Over an aggregate of 70 types of beer are
available in the modern worlds todays.Each style of beer derives its unique
characteristics and differences in its brewing process.Four basic ingredients are used
in brewing,which includes;grains, hops,[bitterleaves as a hop’s substitute]Yeast and
water,Grains like Millet contain the natural sugers required for fermentation.It also
provides beer with flavour,colour,body and texture.Hops are small,green,coneshaped flowers from the hop plant,a vine related to the nettle plant over 50 varieties
of hops are grown throughout the world, mostly in Europe,Australia and North
America.But the hops we want to used is bitterleave acting as hop’s substitute.It is
mostly grown here in Africa.Hops provides the Beer with spicy, bitter flavour and
contribute natural substances that prevent bacteria from spoiling beer. Two species
of Yeast ;Saccharomyces cerevisiae and Saccharomyces uvarum and baker’s
yeast.Each yeast species is used in a slightly different method of fermentation and
produces a distinct type of beer. Throughout history, where ever Cereal grains were
grown, Humans made beer with beverage for them. They used Barley in Egypt, Millet
17
and Sorghum in other parts of Africa, beer brewing is a major industrial revolution
world wide. In 2002,In the United States,1800brewers,produced almost 175 millions
barrels of beer.Beer is deeply interlined in the fabric of Society, from the Economic
to the intangible locally owned breweries deliver social values better than global
companies. Since early times beer which is the products of brewing processes has
played an important role in our society, It bring people together in local pubs and
breweries generating Camaraderie within Communities. As technology has progress
brewing techniques have been developed and refined. As a result, we are currently
consuming the finest beers ever produced. This is due to our increased knowledge in
the fields of Biotechnology, Microbiology, and Chemistry.
1.1 AIM OF STUDY / PROJECT
The aim of study is primarily to have a capture of the effect use of a tropical hop named
bitter leaf(Vernonia Amygdalina) extract as a means of extending the shelf–life of a
locally millet beer.In the course of achieving this goal, the ingredients used in brewing
industries are taken into consideration together with various analysis carried on beer
samples.Finally, beer should be of an adequate composition and texture in order to satisfy
consumers need and sensitivity on the disadvantages of beer abuse to the health and
body.
18
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 GENERAL DESCRIPTION OF CEREALS CROP
Cereals are of various species and belong to the grass family cultivated for their seeds,
which is used as food (Redmond, WA 2007). Although the cereals proper do not belong
to any particular class of grasses, the use of particular species as bread plants seems to
have been determined chiefly by the superior size of the seeds or by the ease of procuring
it insufficient quantity and of freeing it from it edible covering (Nduka 1995). The most
extensively cultivated grain is barley and different kinds of millet. The cereals grains are
the stable food for the people living in the tropics, providing them with about 75% of
their total carbohydrate and 67% of their total protein intake. The grains are eaten in
many ways, sometimes as paste, roast, and porridge. Other preparations of the seeds are
often milled and others processed into flour starch, brain oil breakfast or dinner and beer.
With respect to the comparative structure, there are a few important features that cereals
grain has in common. All the cereal grains are plant seeds and therefore contain a large
central located starchy endosperm which is also rich in proteins, a protective outer coat
consisting of two or three layers of fibrous tissue and an embryo or germ usually located
near the bottom of the seeds. Millet and sorghum constituted the key substrates used by
African Breweries in the earliest centuries of indigenous beer production. Indigenous to
Africa; there were the only grains available for brewing until the introduction of maize by
the Portuguese in about the 16th century (Bryant 1948). As maize cultivation spread,
19
some brewers did incorporate maize into their brewing recipes. Since it does not malt as
well as millet or sorghum(Bantu Beer unit 1971).Scientific classification, cereals belong
to the family Poacea (formerly Gramineae), Barley in the genus Hordenm and millet is
classified in the genus Setatia, Elensine, Panicum, and Pennisetum. (Cereals Microsoft
Student Encarta 2008).
2.2 ORIGIN OF PEARL MILLET AND ITS DISTRIBUTION
The origin of this particular millet has been traced to tropical Africa. It is a widely grown
type of millet grown in Africa, Indian sub continent since pre-historical time. The earliest
archaeological record in Indian date to 2000 BC, so domestication in Africa must have
taken place earlier. The center of diversity for the crop is in Sahel Zone of West Africa,
cultivation subsequently spread to East and Southern Africa and Southern Asia.
Pearl millet is well adapted to production system, characterized by drought, low soil
fertility and high temperature. It performs well in soil with high salinity or low pH.
Because of its tolerance to difficult growing conditions, it can be grown in areas where
other cereal crop such as maize or wheat will not survive. Today pearl millet is grown on
over 260000km world wide, it accounts for approximately 50% of the total world
production of millet.
Specialized archaeologists called palaeothnobotanists, relying on data such as the relative
abundance of charred grains found in archaeological sites, hypothesize that the
cultivation of millets was of greater prevalence in prehistory than rice, especially in
20
northern China and Korea. It was millets, rather than rice, that formed important parts of
the prehistoric diet in Chinese Neolithic and Korean Mumun societies. Broomcorn
(Panicum miliaceum) and foxtail millet were important crops beginning in the Early
Neolithic of China. For example, some of the earliest evidence of millet cultivation in
China was found at Cishan (north) and Hemudu (south). Cishan dates for common millet
husk phytoliths and bimolecular components have been identified around 8300–6700 BC
in storage pits along with remains of pit-houses, pottery, and stone tools related to millet
cultivation. Evidence at Cishan for foxtail millet dates back to around 6500 BC. A 4,000year-old well-preserved bowl containing well-preserved noodles made from foxtail millet
and
broomcorn
millet
was
found
at
the
Lajia
archaeological
site
in
China.Palaeoethnobotanists have found evidence of the cultivation of millet in the
Korean Peninsula dating to the Middle Jeulmun pottery period (c. 3500–2000 BC)
(Crawford 1992; Crawford and Lee 2003). Millet continued to be an important element in
the intensive, multi-cropping agriculture of the Mumun pottery period (c. 1500–300 BC)
in Korea (Crawford and Lee 2003). Millets and their wild ancestors such as barnyard
grass and panic grass were also cultivated in Japan during the Jomon period some time
after 4000 BC (Crawford 1983, 1992).
Millet made its way from China to the Black Sea region of Europe by 5000 BC.The
cultivation of common millet as the earliest dry crop in East Asia has been attributed to
its resistance to drought and this has been suggested to have aided its spread.
21
MILLET.
MILLET OUTPUT IN 2005
Top ten millet producers — 2007
22
Production
Country
Footnote
(Tones)
India
10,610,000
*
Nigeria
7,700,000
*
Niger
2,781,928
China
2,101,000
F
Burkina
1,104,010
Faso
Mali
1,074,440
F
Sudan
792,000
*
Uganda
732,000
Chad
550,000
*
Ethiopia
500,000
F
31,875,597
A
World
23
2.2.1 BOTANICAL[Scientific Classification]of Pearl Millet
Kingdom- Plantae
Division-Magnoliophyta
Class-Liliopsida
Order- Poales
Family- Pamcoidee
Genus- Pennitetom
Species- P. glacom
Source- [www.wiki. org/millet]
2.2.2 DISEASES AND PEST OF PEARL MILLET
The two major pest of pearl millet are grasshopper and army worm. The other insects
and pest that affect the production of pearl millet are clinch and bugs.
Common disease of pearl millet that affect the proper production of pearl millet are
mildew, bacteria blight, kernel smot, and leaf sports .
24
2.2.3 GRAIN COMPOSITION AND NUTRITIVE VALUE OF MILLET
Pearl millet is high in protein content and starchy components which serves as energy
foods. The protein content quality vary greatly among the different types but they are
generally low in lysine amino acids and must be used along with animal products or fish
to balance the protein deit (MHEST, 1987). The millet seeds are important source of food
in the dry regions of Africa and Asia. People in these regions grind the seeds into flour
for flat breads and then fried cakes or they use the seeds in making porridges (World
Book Encyclopedia, 1996).
 NUTRITIVE VALUE OF MILLET GRAIN
Starch – 55-65%
Fat- 4%
Protein
2.2.4 OTHER USES OF PEARL MILLET
In its traditional growing areas in India and many African Countries, pearl millet is the
basic stable for households in the poorest countries and among the poorest people. The
grain is consumed in the form of leavened or unleavened bread, porridge, boiled or
steamed food and (alcoholic) breverages. In the sahel and elsewhere in west Africa, pearl
millet is an important ingredient of Couscous. Much of the grain is currently used to feed
birds, particularly poultry and game birds for recreational hunting, such as bobwhite
25
quail, turkey, and dove. When used to feed hens, the eggs have a higher concentration of
the healthier Omega3-fatty acids.
It is also used for ethanol production due to its rapid fermentation rate and the grain
showing promise as an economical feedback for the production.
It serves in many brewed industries as a starting material for brewed products. It is used
to produce alcoholic brewed product.
2.3
ORIGIN AND DISTRIBUTION OF SORGHUM
The origin of Sorghum is Africa. Sorghum is a tropical plant belonging to the family
Poacea, it is one of the most important crops in Africa, Asia, and Latin America. Several
varieties are released every year by sorghum breeders. Sorghum are grown for forage,
syrup, grain and broom fiber. The common varieties have thick, solid stalk and look like
maize plants but their flowers grow in branch clusters at the tip of the stem. All sorghum
fall into four main groups namely
1: Grain sorghum
2 Sweet sorghum
3 Grassy sorghum
4 Broom corn
26
Broom sorghum are grown especially for their round starchy seeds, they are the world’s
fourth most important cereals after rice, wheat and maize. Sorghum is known as ‘Milo’ in
the United States, ‘Guinea corn’, ‘Burra’ in Sudan. The crop is drought resistant
(MHEST 1987) (Mc Graw Hill Encyclopedia of science and Technology). Outside
Africa, Sorghum is not used for brewing except in the United states where it is
occasionally used as an adjunct (Okafor, 1987). More than 7000 sorghum varieties have
been identified (Keangama and Romi, 2006). In 1805 person’s suggested the name
sorghum Vulgare for Heleus sorghum (L). In 1961 Clayton proposed the name sorghum
bicolor (L). Monch as the name for the commonly cultivated sorghum and this is
currently the name accepted ( DOGGETE, 1988). Sorghum is thought to be 200million
years old (Paterson al; 2003). It is believedto originate from Africa precisely Ethiopia
(ICRISAT, 2005). From there it was distributed among the trade and shipping rate around
the African continent and through the middle east of Indian 300years ago, it is then
journeyed along the silk route into China,. Sorghum was first taken to North America in
1700-1800s through the slave trade from West Africa. It was re-introduced in Africa in
the late 19th century for commercial cultivation and spread to South America and
Australia.
27
SORGHUM.
28
2.3.1 BOTANICAL [Scientific Classification]
Kingdom-Plantea
Division-Magnoliophyta
Class-Liliopsida
Order-Poales
Family-Poacea
Genus-Sorghum
Species-S.Bicolor[Encarta, 2006].
2.3.2 . GRAIN COMPOSITION AND NUTRITIVE VALUE OF SORGHUM
Starch is the main component of sorghum grain followed by protein.The average
energetic
value
of
whole
sorghum
grain
floor
is
356Kcal/100g[BSTID-
NRC,1996].Sorghum contains non-starch polysaccharides in proportion ranging from 2
to 7% pending on the variety[Knudsen and Monck,1985]Verbrugen et al;1973].
29
2.3.3 OTHER USES OF SORGHUM GRAIN
In United State,Australia and other developed countries it is grown as animal feed.Some
species of sorghum contain Hydrogen cyanide,hardening and nitrates lethal to grazing
animals in the early stages of the plant growth.Stressed plants, even at later stages of
growth, can also contain toxic levels of cyanide[Okafor,N.1980].In India, and other
places,sweet sorghum stalks are used for producing biofuel by squeezing the juice and
then fermented into ethanol[Texas A.and M. University].In the United state it is currently
running trials to produce the best varieties for ethanol production from sorghum leaves
and stalks[Watson et al 1983].In Africa and Asia it is grown both for food and animal
feed.It is known that more than 300millions people from developing countries essentially
rely on sorghum as source of energy [Godwing and Gray, 2000].
2.4
HOPS
There are cones which are also known as flowers of the female hops vine.The cones
contains a soft resin called Alpha acid that gives beer a product of brewing its bitterness.
Hops also helps to spoilage bacteria in order to maintain flavor stability and to retain the
head of the beer. There are many different varieties of hops. They are selected from the
rest for their unique bitterness in flavor or aroma.Hops are broadly classified into two
groups namely ;
30
-Noble hops and
-Highalpha hops.
Noble hops are low in bitterness and have a pleasant aroma and flavor meanwhile Highalpha hops have high bitterness but a less pleasant aroma. The high –alpha hops can be
used more economically due to the fewer amounts required. However, the flavor of the
beer is slightly scarified.
Years ago, the recipes for beer would call for hops measured in Ounces. Since the
bitterness for each type of hop was not accounted, the beer’s flavor was inconsistent from
batch to batch. Today, the bitterness for each type of hop is rated by Alpha acid units
[AAU].It is calculated by the alpha acid percentages in the hops multiplied by the weight
31
of the hops in Ounces. Consistent control of the bitterness of the beer is achievable. Since
hops are plants, they can be dried and compressed called Whole hops, palletized hops are
ground into a powder and then pressed into Pellets for easier use and storage. The
negative aspect of hops is that they are easily perishable. Heat and Oxygen can
deteriorate hops in a matter of weeks if not stored and handled properly. Palletized hops
can be stored at room temperature only if they are vacuumed packed or packed with
Nitrogen. Hops that are not package in this manner must be stored, refrigerated or frozen.
2.4.1 OTHER USES OF HOPS
Hops assist in precipitation of protein during worth boiling through the action of hop
tanning. It also increase the shelf-life of beer by being antiseptic against beer spoilage
bacteria such as Sarcina and Pediococcus Spp. Hops gives the beer a unique bitterness in
flavor or aroma due to its composition of Resin responsible for bitterness due to the
presence of Iso-alpha acids and essential oils responsible for the aroma .Hops help to
maintain flavor stability and to retain the head of the beer. It inhibit the growth of certain
beer spoilage bacteria.
2.5 BITTER LEAVE AND ITS ROLES
The origin of bitter leave was trace to Africa, precisely in Nigeria .Its Scientific name is
Vernonia amygdalina is a shrub or small tree of 2 – 5 m with petiolate leaf of about 6 mm
32
diameter and elliptic shape. The leaves are green with a characteristic odour and a bitter
taste (Anonymous, 1999). No seeds are produced and the tree has therefore to be
distributed through cutting (Anonymous, 2000).
ECOLOGY
Its Grows under a range of ecological zones in Africa and produces large mass of forage
and is drought tolerant (Hutchioson and Dalziel, 1963 cited by Bonsi et al., 1995a). There
are about 200 species of Vernonia
MAJOR USES AND FUNCTIONS
The leaves are used for human consumption and washed before eating to get rid of the
bitter taste. They are used as vegetable and stimulate the digestive system, as well as they
reduce fever. Furthermore, are they used as local medicine against leech, which are
transmitting bilharziose. Free living chimpanzees eat the leaves, if they have attacked by
parasites. Vernonia amygdalina is also used, instead of hops to make beer in Nigeria
(Anonymous, 2000). Furthermore, is Vernonina amygdalina found in homes in villages
as fence post and pot
herbs(Anonymous,
1999)
33
BITTER LEAVE
2.5.1
VERNONIA AMYGDALINA
BOTANICAL [Scientific Classification] of bitter leaf
Scientific name; Vernonia amygdalina
Common name;
Ewuro (Ibadan, Nigeria)
Etidot (Cross River State of Nigeria)
Bitter leaf
34
Origin; Nigeria
2.5.2 COMPOSITION OF BITTER LEAVE.
Vernonia amygdalina has been observed to be eaten by goats in Central Zone of Delta
State, Nigeria. However, in general has there been found, that Vernonia amygdalina have
an astringent taste, which affects its intake (Bonsi et al., 1995a). The bitter taste is due to
anti-nutritional factors such as alkaloids, saponins, tannins and glycosides (Buttler and
Bailey, 1973; Ologunde et al., 1992 cited by Bonsi et al. 1995a; Anonymous, 1999). It
has been tried to mix Vernonia with molasses to make it more palatable, but 6.6 % of DM
intake had to be added to improve the intake of Vernonia. During the dry period Dairy
farmers from Southern Ethiopia feed boiled Vernonia, since the boiling decreases the
content of secondary plant compounds and makes the feed more palatable. Vernonia
amygdalina has also been fed to broilers, where it was able to replace 300 g kg-1 of
maize-based diet without affecting feed intake, body weight gain and feed efficiency
(Teguia et al., 1993 cited by Bonsi et al., 1995a).
2.6
YEAST
Yeast is the most important ingredient in the beer brewing. It is a single unicellular
fungi, belonging to the fungus family. Yeast is a living creature metabolizing,
reproducing, and living off the ingredience in the beer. It is responsible for the conversion
of sugar to alcohol and carbondioxide in the fermentation stage. Yeast is also final
35
component that determines the flavour of the beer. Yeast are placed within the fungi
family because they lack photosynthetic pigment (chlorophyll). Yeast are fungi that live
either as parasite or saprophytes on food (Daven port, 980). Yeast metabolizes the organs
extracted from grains, which produces alcohol and Co2 and thereby turns wort into beer (
Hammond et al 1995). There are thousands of varieties and strains of yeast. Even in the
air, wild yeast is floating around ready to contaminate. Only cultivated strains of yeast
should be utilized in the brewing of beer . If other yeast co taminate the beer, the results
can be over carbination, stange flavors and
all kinds of fermentation peculiarities.
Therefore, picking the right yeast for the desired beer is absolute critical. The two main
varieties of yeast used for beer brewing are top-fermenting yeast and bottom fermenting
yeast.
 Saccharomyces Cerevisiae (top-fermenting yeast).
 Saccharomyces Uvarum ( bottom-fermenting yeast). The names of
both are
descriptive of where fermentation takes place in the wort when the yeast is used.
The top-fermenting yeast is similar to the yeast for baking bread. It is applied for
making ales and stouts. The bottom-fermenting yeast is utilized for priduction of
lager and steam beer. The dominant types of yeast used to make beer are ale
yeast (S. Cerevisiae) and lager yeast (S. Uvarum].
36
2.6.1 ORIGIN AND DISTRIBUTION OF YEAST
Yeast,are microscopic,one–cell fungi important for the ability to ferment
carbohydrates in various substances (Boze et al 1991).Yeast in general are
widespread in nature, occuring in the soil and on plants. Most cultivated yeast
belong to genus Saccharomyces. Those known as brewer’s yeast are strains of S.
Cerevisiae,yeasts have interacted with man from time immemorial from the time
when they first learnt that fruit juices developed into intoxicating drinks and
that the dough produced from his ground cereal can be leavened, although
he did not understand these two phenomenal (Boze et al 1911).Today,yeast is
produced and used in all the six continents of the world from the single
most produced micro-organism in terms of weight.The purpose for which
yeast is used and the types of yeast employed for each purpose are
related.Of these, the production of baker’s yeast has received the greatest
attention,followed by food and fodder yeast(Caron C.1991).Due to recent
interest in the production of single cell proteins,food and fodder yeast may
become as important as baker’s yeast in terms of total quantity produced.
2.6.2 COMPOSITION OF YEAST.
Yeast are unicellular fungi,which pocesses a rigid cell wall and have
nucleus.The shapes of yeast cells vary from oval,cylindrical,circular,or triangular.
Their sizes are highly variable,vegetative propagation is by budding,fission
37
and fusion.The yeast composed of a nucleus,mitochondria,golgi complex,
endoplasmic reticulum and cell wall.
 Nucleus;
The nucleus controls all the cellular activities.This is accomplished through
DNA molecules stored in the nucleus in element called chromosome.The
DNA contains the coding for all protein production for the cell.The nucleus
is roughly spherical and about 2 micrometers in diameter.
 Mitochondria
The mitochondria are the cell’s power house. When Oxygen is sufficient and
fermentable sugar are available, the mitochondria are used to completely break
down the sugar source and form the maximum amount of ATP.
 Golgi complex
The golgi complex is responsible for packaging materials – readying it for
transport in the cell or storage.
 Endoplasmic Reticulum.
This cell organelle is the cell’s
“highway” system. Materials
are transported
throughout the yeast cell through this intercellular system.
2.6.3 HOW YEAST IS PREPARED
30g of dry baker’s yeast is weighed and dissolved in 150mls of distilled water at 50⁰c
temperature. The mixture is stirred thoroughly in order to get a homogenous mixture. The
38
the yeast is smear in the bucket where the beer will be fermented and the boiled wort
pour on it and left for 3 days for fermentation.
2.7 WATER
Among these ingredient, the most essential ingredient is H₂O. H₂O is known as the most
important raw material needed for brewing to take place. It is needed as the solvent for
this process or a medium for the brewing process to take place. Majority of the
biochemical processes take place in the presence of H₂O. The mineral, ionic content and
the pH
of water has profound effects on the type of beer produced. Some ions are
undesirable in brewing. For example, nitrates slow down fermentation while ion destroys
the colloidal stability or the beer (Hongh, J.S 1995). In general, calcium is a trace element
found in H₂o and is necessary for the complete yeast life cycle, which is also part of the
ingredients involved, also calcium ions head to a better flavor than magnesium and
sodium ions. The pH of the water and that of malt extract produced with it control the
various enzyme systems. In malt, the degree of extraction of soluble materials from the
malt, the solution of tannins and other coloring components, isomerization rate of hop
humucone and the stability of the beer itself and foam on it (Hongh. J.S. 1985). Water is
so important that the natural water available in great brewing centres of the world land
special character to beers peculiar to these hunters (Okafor, N 1980). Water low in
mineral such as that of pilsen is suitable for the prodution of a pale, light colored beer.
Water of a compositions ideal for brewing may not always be naturally available (Hongh
39
J.S 1985) if the production is of a pale beer without too heavy taste of hops, and the H₂O
is rich in carbonates than it is treated in one of the following ways.
Water may be brutalized by addition of calcium sulphate (Okafor N. 1980).
Ca(HCO₃) + 2Ca (SO₄) – 2Ca⁺⁺+ 2H₂SO₄ + 4CO₂.
The water may be decarbonated by boiling.
The H₂O may be improved by ion exchange.
Beer is compiled mostly of water (Microsoft Encarta beer 2008). Different regions have
water with different mineral components as a result of different regions were originally
better suited to making certain types of beer, thus giving them a regional character
(Hongh et al 1985). For example, Dublin has hard water well suited in making stout, such
as Guinness.(Okafor, et al 1985); while pilsen has soft water well suited in making pale
lager such as pilsher urquel.
2.8 CLARIFYING AGENT
Some brewers add one or more clarifying agents to beer, which typically precipitate out
of the beer along with protein solids and are found only in trace amounts in the finished
products (Okafor et al 1980).Clarification of beer is done through sedimentation of
suspended yeasts
and pectin substances by the action of
the
coagulating
substances(like proteins e.g albumen,couagen,blood,mineral dispersion e.g Koalin and
bantonite and carbohydrate e.g alginates).Clarification helps clear the beer from all its
40
solute.
2.9 ADJUNCTS
Starchy,materials or sugary materials such as flours of unmalted cereal grains like
barley,maize,flours of potato, cane suger or invert sugar solution or the supernatant
from the millet sediments are introduced,because the six-row barley varieties
produced malt that had more diastolic power(that is amylase)than was required to
hydrolyze the starch in the malt(Amerine et al 1972).Starchy adjuncts which usually
contain little protein contribute,after their hydrolysis,to fermentable sugars.Which in
turn increase the alcoholic content of the beverages,thus bring down the brewing
cost because they are much cheaper than malt.
2.10 BIOCHEMISTRY OF BREWING PROCESS
The first step in brewing, called malting,involves steeping the grain in H₂O for
several days
until it begins to germinate or sprout(Amerine et al 1987).During
germination enzymes within the grains convert the hard,starchy interior of a grain
to a sugar called maltose(Okafor N.1985).At this point,the grain called malt,after
several days,when the majority of the starch has been converted to sugar,the malt
is hazed and dried.This process called Kilning stops the malt from germinating any
further.A portion of the malt may be roasted to produced other varieties of beer.
After kilning, the dried malt is processed in a mill,which cracks the husks(Amerine
et al 1972).Hot water is then added when the crack malt is transfer to a
41
container called Mash,this process called mashing,and breakdown complex sugar in
the grain and releases them in the H₂O,producing sweet liquid called wort.The
temperature and mashing time affect the body and flavour of the finished beer.
In the next step called brewing,the wort is transferred to a large brewed kettle and
boiled for up to two hours,boiling sterilizes wort to kill any bacteria that may
spoil wort during fermentation.During this stage, hops are added to wort to provide
a spicy flavour and bitterness that balance the sweetness of the wort(Hammond et
al 1973).This enables the bitter oil in the hops to fully infuse into the wort
Grains → malt bin → mash tub →filtering tank.
↓
Aging → Fermenting → Starter ← Brew kettle(Hops added).
Tank
tank
tank
Fermenting tank → Racking → to market.
↓
↓
↑
∕
Canning Boiling.
↓
↓
∕
Pasteurizer.―――――∕
FIG 1;A Scheme of brewing process.
42
2.10.1 BIOCHEMISTRY OF MALTING
Malting is the first step in brewing, it involves steeping the cereals in water, such
as barley,sorghum,millet,maize.In water the cereal’s enzyme is activated.The cereals
is remove from water and spread them in a controlled condition to germinate.
After germination which takes place for 2 to 3 days heat is applied to the malted
grains so as to reduce water content in the germinated cereal.At times Gibrellines
a growth hormone is added to enhance malting.
The primary aim of malting is to promote the development of hydrolytic enzymes
that are not active in raw seed (Dewer et al;1997).The chemistry there is that
during germination cell walls are broken down and starch is released,but these
starch can not be directly broken down into pyruvate as in respiration,therefore
these enzymes are induced to convert the starch to glucose.The enzymes are
alpha- amylase and beta amylase.Also as millet germinates,it produces diastatic and
proteolytic enzymes necessary to breakdown grains substrates into sugar and
nitrogen compounds,which support yeast growth and alcohol fermentation.The
diastatic enzymes in millet malt include alpha and beta amylases,which are the
major sources of diastatic activity and also limit dextrinase and maltase(alpha –
glucosidase).In contrast to malted barley, the alpha- amylases predominate in millet
malt,accounting for about 75% of amylolytic activity(Noveille,1968).The alphaamylases are all formed during germination while the beta-amylases increases from
levels present in the original grain.
43
Maltase present millet malt does not increase during malting. Protein breakdown
also occurs
during malting as grain structure changes to produce roots and
shoots.The proteolytic activity of millet increases abot 10-fold during germination.
2.10.2 BIOCHEMISTRY OF BOILING
Dilution of the sour with water and addition of a cereal adjunt raises the pH of
the brewed to about 3,7-4(Noveille, 1968).Boiling the entire mixture for two hours
gelatinizes the grain starches from both the malt and the starchy ajunct. The heat
combines with the high acidity to soften the imprisoning protein matrix, allowing the
starch granules to break free. They burst open producing a thick tangle of starch polymers
amylose and amylopectin (BBU 1971). Boiling also decontaminates the brew, destroying
all micro organisms in the vegetative form. Since this include the lactobacilli, boiling
arrests lactic acid formation (Haggblade, 1984). The extreme heat also destroys the malt
enzymes, thereby stopping the thining acttion of the malt enzymes on their own substrate.
Boiling, in addition affect the taste of flavour of the product given it a cooked taste.
2.10.3 BIOCHEMISTRY OF MASHING
Mashing is one of the chemical processes in brewing, et involves mixing together in a
mash –tun, the malted grains, adjuncts and water at a regulated temperature and pH. After
the boiled mixture has been cooled to 60⁰c and additional malt is added, the condition are
now favourable for the convention of starch to sugars (Noveillie, 1968). When additional
malt is added, soluble sugars, free amino nitrogen, protein, proteinases, starch and other
44
diastatic enzymes are being added to the mash the amylolytic enzymes present in the
malt, Alpha and Beta amylases etc, breakdown the mass of interwined starch polymers,
thining the mashing and producing sugar we can subsequently be converted to alcohol.
While some saccharificational during this stage is due to the action of molds present in
the malt, research has shown that the molds alone are insufficient to convert the starches
(Platt,1964, Noviellie, 1968). Rather the major conversion is effected by the malt
enzymes. Insufficient acidity at this stage results in both too much thinning and too much
alcohol production (BBU, 1971).
2.10.4 BIOCHEMISTRY OF SOURING
The Sour taste in beer is as a result of lactic acid present in the beer souration is a special
type of fermentation by lactobacillus species i.e the lactobacillus convert pyruvate from
glucose as a result of glycolysis to lactic acid which gives the beer its sour taste.
2.10.5 BIOCHEMISTRY OF FERMENTATION
According to websters dictionary (1996), fermentation is the gradual decomposition of
organic compound induced by the action of living organisms, by enzymes or by chemical
agents. It is specifically the conversion of glucose into ethyl alcohol through the action of
zymase (mostly found in yeast). Alais and linden (1999), defined it as a group of
chemical changes caused by living micro organisms acting on organic materials. Also,
Northcroft (1986), defined it as a process in which a selected, stabilized organism is
grown in or more of the following end products; a liquid (wine, beer), a chemical
45
(butanol, alcohol) or a solid (yeast, cheese curds). Lehninger 1972, defined fermentation
as processes by which many organisms extract chemical energy from glucose or other
fuels in the absence of molecular oxygen. Also fermentation according to pederson,
(1978), are complex alteration of food brought about by the catalytic action of either
enzymes produced by micro organisms or enzymes indigenous to the food. The main aim
of fermentation is to break down glucose to form organic compounds. Of the many kinds
of glucose fermentation to closely related types predominate.
They are:
I.
Ho
molactic fermentation and
II.
Alc
oholic fermentation.
These occur in the absence of oxygen (Letininger, 1972). In the Homolactic fermentation,
the six- carbon sugar (glucose) is degraded to two molecules of the three- carbon lactic
acid as the sole end- product. This type of glucose break down occurs in many organisms
and in the cells of most higher animals including mammals. It is usually called
“Glycolysis”, which means the dissolution of sugar. In alcoholic fermentate, the sixcarbon glucose molecules is degraded in two molecules of the two- carbon ethand (C₂H
OH) and two molecules of CO₂. Alcoholic fermentation occurs by the same enzymatic
pathway, as glycolysis but requires two additional enzymatic steps which break down
both three- carbon fragments resulting from glucose cleavage into ethanol and CO₂. In
46
the presence of O₂, these three- carbon fragments go into the TCA cycle (tri-carboxylic
acid cycle) to generate reducing equivalents, which are used in energy generation in the
electron transport chain (respiration).
Glucose
↓
↓
Anaerobic
Aerobic condition.
↓
Condition
↓
↓
Glycolysis
Respiration
Alcoholic
(Homolactic)
Fermentation.
(Fermentation)
↓
↓
(CO₂ + H₂O)
Ethanol + CO₂.
Lactic acid.
Fig 2:A schematic representation of glucose degradation under aerobic and
anaerobic condition.
Glucose
↓
Glucose-6-phosphate.
↑ ↓
Fructose-6-phosphate.
47
↓
Fructose-1,6-diphosphate.
↓
↓
Dihydroxyacetone → Glyceraldehyde – 3- phosphate.
Phosphate
←
↓
1,3- diphosphoglycerate (2).
↑ ↓
3- phosphoglycerate (2).
↑ ↓
2- phosphoglycerate (2).
↑ ↓
Phosphoenol pyruvate (2).
↓
Pyruvate (2).
↓
Lactate or lactic acid (2).

Fig
:3 The glycolytic pathway.(Lehninger,1972).

Gly
colysis : Glucose → 2 Lactic acid
48
OH H OH OH
|
|
|
|
HO- C – C – C – C – C – CH₂OH.
|
|
|
|
H OH H H
↓
CH₃ + CH₃
|
|
CHOH CHOH
|
|
COOH COOH Lactic acid.

Alc
oholic fermentation :Glucose → Ethanol + CO₂.
OH H OH OH
|
|
|
|
HOCH₂ - C – C – C – C - CH₂OH
|
|
|
|
H OH H H
↓
↓
CH₃
CH₃CH₂OH + CO₂.
|
+CO₂.
49
CH₂OH

Fig.
4 The pathway of carbon atoms in glycolysis and alcoholic fermentation.
During anaerobic fermentation which is under alcoholic fermentation, the anaerobic
meteabolism of glucose does not require the mitochondria. Glucose
is
converted
entirely to CO₂. As a result, much less ATP is produced per mole of glucose (Harvey et
al, 1995). Yeast, for example, ferment glucose anaerobically to two ethanol and two CO₂
molecules; the net production is only two CO₂ molecules; the net production is only two
ATP molecules per glucose molecules. Recall that during the initial stage of glycolysis
(that is during the conversion of glucose to pyruvate), two NAD molecules are reduced to
NADH from the initial stages in reoxidising it to NAD (Harvey, 1995).
Anaerobic metabolism over all reaction equation.
Glucose + 2ANP + 2Pi ―2 Lactate + 2ATP.
Alcoholic fermentation rxn
Glucose + 2ADP + 2Pi ―2ethanol + 2CO₂ + 2ATP.
2.11
STRIANING
50
The factory brewers strain their mash subsequently referred to as wort in final preparation
of the substrate for alcohlic fermentation. They strain at 60⁰c rather than 30⁰c in order to
take advantages of the protective action of the temperature in preventing possible
contamination of the brew by micro organisms present on the strainers(BBU, 1971).
Straining removes up to 30% of total solids. This results in a loss of protein in the beer as
the solids contain protein.
2.12 PROPERTIES OF BEER
All beer can be further described by three additional variables, colors, bitterness, and
gravity (Microsoft Britannica 2008). Color is determined by the type and amount of
malt used to make the beer. For example, liquidity roasted malt produces a light- colored
beer while beer made only with barley that has been dark roasted is nearly black in
color.Bitterness depends on the type and amount of grain and hops used in the beers
production. Beer made with wheat, for example are generally less bitter than beers made
with dark- roasted barley(Aniche, G.N. 1982). The mineral content of the brewing H₂O
can also accentuate hop bitterness. Beer made with water that has a high level of
dissolved minerals is bitter than beer with water low in minerals. Gravity refers to the
beer’s density or the amount of dissolved sugars in the wort.
Yeast consume this sugars to produce alcohol during fermentation, so elevated sugar
levels translate into a higher alcohol content in finished beer (Aniche G.N 1982)
2.12.1 PROCESSES INVOLVED IN BEER PRODUCTION
51
In the brewing schematic, 6 processes are involved:
Mash tun
In the mash tun, ingredients for wort are mixed according to the recipe. The product
which leaves this unit is called Mash. The mash produced is sent to kettle where it is
treated with H₂O.
Kettle
The brew kettle is the apparatus where the hot H₂O and mash are combined and heated.
This unit is strategically placed below the mash tun. So that the wort can flow by gravity
to the vessel. Stainless steel is the materials from which it is made from here the slurry
moves to the heat exchanger.
Heat exchanger
A heat exchanger is necessary to cool the mixture to 70⁰c before it enters the fermenters,
yeast can not function if the temperature becomes too high. A counter current heat
exchanger works best for maximizing the amount of heat transfer that occurs.
Primary fermenter
The fermenter is where the most important step takes place.The production of the
ethanol
usually takes approximately 8-10 hours for fermentation
hydrometer is used
to
start.A
daily to measure the specific gravity of the fermenting
solution(which lead to a measure of ethanol content).Once the desire ethanol is
reached the beer is chilled by means of a cooling plate.
Cooling plate
52
Within the fermenter the beer is then sent to the aging tank where it matures.The
fermenter is then cleaned for one-half hours using a Iso degree celsuis caustic
solution.
Ageing Tank
The ageing tank allows the beer to age at cool temperature(4-15⁰C)for a week to
months, thereby allowing the beer to develop more carbonation and mellow.In
addition,”harsh flavour notes”subside and yeast fall to the bottom of the tank
(Home beer making)
2.12.2 Lagering
At the end of the primary fermention above the beer,known as green ‘beer’ is
harsh and bitter (Aniche G. N 1983).It has a yeast taste arising probably from
higher alcohols and aldehydes.Traditonally ale and lagers have been differentiated
as being either a top fermented or bottom fermented, going by home breweries
and microbrewers alike keep pushing the envelope of the craft.The main
differences between the two is lager yeasts ability to process raffinose.(Flickinger
et al 1999).Raffinose is a trisaccharide compiled of galactose, fructose and
glucose.During lagering ,secondary fermentation occurs.Yeast are sometimes added
to induce this secondary fermentation,followed by the rate of CO₂ escape from a
safety valve (Aniche,G .N 1982).This, the method used to reduce lagering time,beer
is stop at high temperature to drive volatile compound such as H₂S and
53
acetaldehyde(Aniche G.N 1982). Lagering gives the beer its final desirable
chemical properly but it is hazy due to protein-tannin complexes and yeast cells.
2.12.3 TYPES OF BEER.
Malt beer
A special kind of beer that
varies considerably among brands.Some are quite
‘hoppy’,others are only mildly.Their essential characteristic is that they have higher
alcoholic content than in other beers.
Lager
Generally understood to be a light bright sparkling beer but today all malt
beverages are lagered.
Ale
It is an aromatic,golden, fuller bodied and bitterer malt beverages with a slightly
higher alcoholic content than beer.All ale are ‘top fermenting brews’.
Stout
It is darker ale with a malter flavour.It is sweeter than ale and has a strong
character.
Pito
It is a similar product to Burukutu in its processing but it differs in its main
ingredient for brewing since maize, millet or a combination of the two is
used(Ekundayo 1980).Pito is considered more similar to kaffir beer both in its
54
processing and complex microbial requirements,which may lead to a similar
industrial production.
2.13 PREPARATION OF LOCAL BEER.
The common grain used here is millet or sorghim, but millet grain was used. It is steeped
in water for 11hours. After this period the cereals are collected,and drained to liberate
excess water. The grain was spread out on the rice bag, a cool area was choosen for this,
and the grain was covered with nylon to prevent it from drying and also to prevent flies
from peaching on it. Then it was left for 2-3days to germinate. After 3days the
germinated grains is know as malted grains. So the malted grain is sun-dried, by
spreading them in a clean rice bag under the sun without nylon for 2days. The grain is
sun-dried in order to reduce moisture content. The dried malted grains were milled to a
fine dusty powdered form. 750g of the malted grains were steeped in 3.3liters of H₂O
stirred and then allowed to settle. The supernatant was drained a bit and the remaining
supernatant and sediments boiled for 25minutes. After this, the unboiled supernatant was
added and the mixture (now called wort) was allowed to sour over night. The next day,
the sour wort was filtered with a fine sieve. The filtrate was boiled and and 200mls of
bitter leaf extract was added (this is the 2nd boiling ) for 2hrs, the boiled lasted. The
boiled wort was brought down and allowed to cool to 70⁰C. The activated baker’s yeast
was smeared on the bucket that will be used as the fermenting bin. The cool wort was
filtered again using a muslin cloth into the fermenting bin. 130mls of the baker’s yeast
55
was added and fermented allowed for 3days. The next day the drink was ready and
packaged into plastic bottle and refrigerated.
Malting :- Steep millet grain in plastic bucket (11 hours).
↓
Drain the water.
↓
Spread the drain grain on a rice bag and cover with nylon for
germination to take place (2-3 days).
↓
Spread the germinated grain under the sun to dry with out nylon for 2 days.
↓
The dried malted grains were milled to a fine dusty powder with the
machine.
↓
Mixed milled grain with water allow it to settle then separate the sediment
from the supernatant and boiled the remainig supernatant and the sediment for
25 minutes.
↓
The boiled solution is mixed with the unboiled one (wort),the wort is allow to
sour over night.
56
↓
The sour wort is filter and boiled with hops for 2 hours.
↓
Allowed to cool( 70⁰C)
↓
The cool wort was fitered again using muslin cloth in to the fermenting bin for
fermentation to take place for three days.
↓
After 3-days the drink is ready and serve or bottle.
Fig 6: Flow – sheet for the production local millet beer.
2.13.1 Factors Affecting Both Local And Modern Beer.
One of the factors is pH. PH is the degree of alkalinity or acidity of a substance or
medium. Enzymes activities are highly dependent on pH,that is, some enzymes are more
active in an alkaline condition while some are active in acidic condition. Since brewing
involves the actions of enzymes. There pH affect it greately.
Some of the enzymes involved in brewing such as alpha-amylase,beta-amylase,protease
and yeast have their various optimum pH , therefore if the time for a particular
enzymes to act is due and the pH is not optimum definitely the brewing process will
be affected.In the same manner temperature defined as the degree of hotness or coldness
57
of a substances or medium affects brewing process because the various enzymes involved
in brewing have their own conducive temperature at which there are perfectly active.But
any temperature that fall will affect the brewing process.
2.13.2 ALCOHOLIC CONTENT OF BEER.
The bases of alcoholic drinks, ethanol or alcohol is a simple substance containing
carbons, hydrogen and oxygen.In the body it acts in two distinct ways.
 It act as food which is broken down in the body to give energy.Alcohol have a
higher calorific value than fat and carbohydrates.It is absorbed by the body
without digestion(The Koronye and Ngoddy, 1985).
 It also acts as drug which affect the central nervous system(Olutayo, 1992).The
effect on the central nervous system varies from mild stimulation,when consumed
in small amount.It may lead to loss of co-ordination and even death when large
amount is consumed.The standard ethanol content of beer ranges from 3.3% to
7.5% but for local african beers, the value ranges from 1.5% to 1.7%(Kolawale;
et al).
2.13.3 DIFFERENCES BETWEEN LOCAL AND MODERN BEER.
 In local beer during brewing adjuncts and hops are not added during mashing
(Asiedu, 1989).But in modern beer brewing,adjunct and hops are added during
58
mashing process.
 In local brewing different microorganism adjuncts are allowed to act on the
wort during the fermentation process,but in morden brewing it is only a selected
strain of yeast that is allowed to ferment the wort.Other microorganism are
considered as contaminants or infections.
 In local beer brewing any source of H₂O can be used in modern only brew H₂O is
used,that is dechlorinated H₂O.
 In local beer souration occurs as one of the fermentation which gives the beer sour
taste,but in modern beer souration does not occur, because the Lactobacillus
species is considered as contaminants.
 Local beers have short shelf life of one or two days, no preservation method yet
discovered but in modern beers have long shelf-life because preservates are added
to prevent spoilage by micro-organism.
 Local beers are thick in texture and are known to contain alot of solute but In
morden beer it is watery in texture and no solute is seen.The beer is clarified by
clarifying agent.
2.14
PACKAGING AND DISTRIBUTION.
Because most of the brewer do not pasteurize their beer,alcoholic fermentation
proceeds during packaging and distribution.When the beer is ready it is canned or
59
bottle and then distributed to the consumers who are the customers in market.
2.14.1 SPOILAGE.
Local
beer spoils in 1-5 days mainly because of the microbial contamination
introduced into the wort by the convertion malt. The low temperature during alcohol
fermentation
and distribution, favour
pasteurization would
the growth
of
contaminants.While
improve the keeping quantity of the beer by destroying
spoilage microbes,no such process is applied on a large scale for convertional
millet beer.Early attempts on pasteurization failed because they led to an
unacceptable increase in beer viscosity by futher gelatinizing the starch
and
eliminating amylolytic enzymes.It also eliminated its characteristic effervescence by
killing the active yeast (Noviellie, 1968; de Schaepdrigier,1988).
2.15 IMPORTANCE OF FERMENTED ALCOHOL BEVERAGES.
The fact that the fermentive microorganism involved in the production of alcoholic
beverages are able to synthesize, from the ingredients of the drinks,certain important
nutrients required by humans, make alcoholic beverages important (Adeniyi,1991).For
example, a lot of vitamins may be synthesized or accumulated in the cells of brewer
yeasts
when
substances
such
as
malt
sprouts
and
other
substances
are
fermented(Pederson, 1979).Alcohol has food value,but its main importance in nutrition
is the effect of high alcohol
consumption upon the intake of other essential
60
nutrients, in particular enzymes. Alcohol also acts as a sedative to the
central
nervous system.While acting as such,it can provide full utilizable energy which can
be transformed into energy rich phosphates(Adeniyi,1991) .
Alcohol having a little amount of tannin helps to dialate the vein for easy flow of blood
or inorder to ease the flow of blood round the body but must be taken in little quantity but
if taken in large quantity, it will be very lethal to the system for it may lead to death.
CHAPTER THREE
3.0
MATERIALS AND METHODS
3.0.1
3.0.1.1
MATERIALS
CHEMICALS AND REAGENTS.
 1% Phenolphthalein indicator
 0.1m NaoH
 Distilled H₂O
 Mackonkey agar,Blood agar and chocolate agar.
 Sterilization tablets
 3% Hydrogen peroxide
 2ml Glucose phosphate peptone H₂O.
3.0.1.2
GLASS WARES AND EQUIPMENTS.
61
 50ml Burette, 25ml pipette, 25ml measuring cylinder and 500ml
measuring cylinder.
 Boiling tubes.
 PH meter
 Beakers.
 Conical flasks.
 Dropping pipette.
 Whatmann indicator paper,universal indicator paper.
 Hot plate.
 Dessicator.
 Oven.
 Refractometer.
 Hydrometer.
 Analytical weighing balance,digital weighing balance and kitchen
scale.
 Petri-dishes.
 Wire loop, burnsen burner.
 Plastic buckets and plastic bottles.
 Volumetric flask.
 Refrigerator/freezer.
 Retort stand and clamp.
62
 Funnel and sieve.
 Stove and cooking pots.
 Wooden stirer.
 Pastuer’s pipette.
 Density bottle.
 Muslin cloth.
 Spartular.
 Rice bag.
 Blender.
3.1 PREPARATION OF SAMPLE.
The millet grain for the preparation of traditional millet beer was purchase at
ogbete market,Holy ghost,Enugu state and 2kilogram was measured using the
kitchen scale. Also bitter leaf.baker’s yeast were also purchased at thesame place.
The already prepared kunu added to the kunu during it production to induced
fermentation was purchase at 82division Abakpa,Enugu state.
3.1.1 METHODS.
The method used in preparation of this traditional millet beer was adopted from beer
production(Okafor
2008).Also some ideas
were
borrowed
from
Hausa women
63
resident at barracks in Enugu state.
3.1.1.1 INGREDIENTS.
The main ingredients used are; 750g of millet,0.5litre of already prepared kunu,30g
of baker’s yeast,500g of bitter leaf.
3.1.2 METHOD OF PREPARATION.
The millet grain was purchased and
2 kilogram was weigh using a weighing
balance.After which it is washed and steeped in water using a plastic bucket for
11hours.After 11hours the grain is removed and the water is drain from the
grains,then the grain is spread on the rice bag,a cool area
was choosen for
this,and it is covered nylon in order to prevent the grain from getting dry and
also preventing flies from peaching on it.It is then left for 2-3 days for
germination to take place.After 3 days the germinated grains are known as malted
grains.The malted grains is sun-dried to reduce moisture content.After 2 days of
sun-drying the malted grains are milled to a fine dusty powdered form. 750g of
the milled grain were steep in 3.3litres of H₂O, stire using the wooden spoon and
allow to settle.After some time the supernatant was drained a bit and the
remaining supernatant and sediments boiled for 25minutes.After this,the unboiled
supernatant was added and the mixture(now called wort) was allowed to sour over
64
night. After souring the wort is filtered with a fine sieve.The filtrate was boiled
and 200mls of bitter leaf extract was added (second boiling) which lasted for
2hours.The boiled wort was brought down and allowed to cool to 70⁰C.The
already prepared baker’s yeast was smeared on the bucket that will be used as
the fermenting bin.The cool wort was filtered again using a muslin cloth into the
fermenting bin.130mls of the baker’s yeast was added and the bucket is close and
allowed for 3days for fermetation to take place.After 3days the drink is ready
and packaged into plastic bottles and refrigerated.Another drink is prepared in the
same manner but this time without hops.
3.1.2.1
METHOD USED TO PREPARED AQUEOUS EXTRACT OF
BITTER LEAF.
500g of bitter leaf was washed and weighed.The bitter leaf was grinded using a
blender,then 1litre of distilled H₂O was poured into the grinded bitter leaf in a
bucket and left for about 2hours.After 2hours,a muslin cloth was used for the
extraction.The filtrate(aqueous extract) was bottled in plastic bottles and stored in
the refrigerator,ready to be used as hops substitute for brewing.
3.1.3 METHOD OF CHEMICAL ANALYSIS.
The methods used for the analysis were adopted from Haddad et al (1978).
3.1.4 TOTAL ACIDITY
65
To 200mls of boiling distilled H₂O in a 500mls Erlenmeyer flask was added 1mls
of a 1% phenolphttalein indicator.The solution was titrated with 0.1m,Sodium
hydroxide solution to a faint but definite pink colour;5mls of the sample was
titrated to a pink colour with the 0.1m sodium hydroxide,using 3drops of
1%
phenolphthalein as indicator.The total acidity is expressed as lactic acid as shown
in Appendix B.
3.1.5 FIXED ACIDITY.
25mls of the samples were place in boiling tubes and evaporated carefully on a
hot plate until the volume reduc to 5mls.Then 25mls of hot distilled H₂O was
added and the solution again evaporated to a final volume of 5mls.This
process was repeated once more both the one with hop and without hops was
carried out.The residue was allowed to cool,then diluted to 50mls with distilled
H₂O;3 drops of 1% phenolphthalein or thymol blue was added and finally,the
samples were titrated
with standardized concentrated 0.1N sodium hydroxide
until a colour change was observed.Fixed acidity was calculated as shown in
Appendix B.
3.1.6 VOLATILE ACIDITY.
66
This is calculated by substacting the fixed acidity from the total acidity as
shown in appendix B.Both aciditityes were expressed on the same scale.
3.2 pH DETERMINATION.
The pH readings for the two sampls(the one with hops and the one without
hops),were checked using a pH meter.
3.3
SPECIFIC GRAVITY.
The Refractometer was used to checked the Brix level before and after
fermentation.Before using the Refractometer you first collect distilled water
using a dry applicator and place 3 drops of the sample and make it read zero
level,then wipe and do the same but this time make use of the sample,(the one
with hops and the one without hops).After getting the reading,you multiply the
Brix level(the reading) to 4 to get your specific gravity.
Brix level x 4 = Specific gravity.
OR
The second method which involve, density of sample divided by density of
H₂O which is equal to x divided by 0.998.
Specific gravity = Density of sample = x
----------------------Density of H₂O
----0.998
67
Density = mass of the sample
------------------------------Volume of the density bottle
3.4 DETERMINATION OF ALCOHOL CONTENT.
The refractometer method was used in determining the alcohol content.400mls
of
the sample were poured into a 500ml- measuring cylinder containing the sample
and the specific gravity was read off. A clean dry applicator was used to place 3
drops of the sample on the prism of the refractometer and the value of the refractive index
taken.Finding the differences between the specific gravity,the alcohol content value is
gotten.To
this differences, 0.5 was added to make up for the difference in
temperature.With the value gotten, the alcohol conent was read off from a table.This is
done on both sample (with hops and with out hops).
3.5 TOTAL DISSOLVED SOLID (TDS).
A porcelain dish was dried in the oven at 105⁰C for one hour and placed in a
dessicator.500 mls of sample was filtered using 0.45 micro-membrane filter paper
into a pre-weighed porcelain dish and the filtrate was put in a weighed porcelain
dish and evaporated to dryness in the oven.The porcelain dish was dried in the
oven at about 105⁰C for one hour and cooled in a dessicator for one hour and
68
re-weighed.TDS was then calculated as shown in the Appendix B.This method is
carried on the two sample(the millet beer with hops and the one without hops)
3.6 TOTAL SUSPENDED SOLID(TSS).
A membrane filter paper was faintly marked on the edge with alphabets.The filter
paper was dried in the oven at 105⁰C for one hour and placed in a clean
dessicator for one hour.The filter paper was weighed and the millipore filtration
apparatus
was
connected
and
flused
for
five
minutes
to
get
rid
of
debris.Membrane filter paper was placed in the membrane filter holder and soaked
with clean distilled water free of solids.This was
tightened with the millipore
spanners.Five litres of water sample was flowed through the membrane filter and
pressure was maintained at 40 pounds per square inch.The set was uncoupled at
the end of filtration and the membrane filter paper placed inside clean watch
glass and sealed.The used membrane filter paper was dried in the drying oven at
105°C for one hour placed in clean dessicator for one hour and re-weighed.Total
suspended solids was estimated as shown in Appendix B.This is carried out the
two samples(the beer with hop and the one without hops).
3.7 MICROBIAL COUNT.
The media of choice were chocolate agar, blood agar, and mackonkey agar.The
chocolate agar plate was used to enhance the growth of fastidious organisms.The
blood agar plate is an enriched medium, which supports the growth of common
69
bac teria.Mackonkey agar is a differntial medium used in the isolation of lactose
fermenters through it grows non-lactose fermenter.
3.7.1 PROCEDURE FOR CULTURING THE PLATES.
The wire loop is sterilize using the flame from a bunsen burner and the samples
were cultured aseptically with wire loop on the prepared plate then incubated at
37⁰C,for 24hours,then after 24 hours the plate were read for growth of organism.
3.7.2 PROCEDURE FOR IDENTIFICATION OF THE ORGANISMS.
Apart from the physical appearance and morphology of the organism,the following
tests where carried out to confirm their presence.
 Catalase tests:
1.Materials: 3% hydrogen peroxide,pasteur’s pipette,slide and 18-20
hour
old
culture.
2. Slide Method:
- Two drops of hydrogen were dropped on a clean grease free
slide
A and B.
-The test organism was transferred using a glass rod
to slide A while
the control was dropped on B.Presence of bubbling gas or effervescence indicates
a positive reaction.Therewas no bubbling gas which confirms the presence of
Streptococcus
pyogenes.This test distinguishes between Streptococcus pyogenes and
70
Staphylococcus species.
 Methyl Red (M-R)And Vogues Proskauer(V-P) Test.
1.Materials:Test tube of glucose phosphate peptone water (2mls),methyl red
indicator.Bacterial culture,pasteur pipette, Barrits reagents (1ml of 40% KOH
and 3ml of 5% alcoholic alpha-naphtol).
2.Procedure:
- 2ml of sterile glucose phosphate peptone water was inoculated with the
Bacteria culture in duplicate(A and B)and incubated at 37⁰C for 48hours
-
To tube A,1-4 drops of methyl red reagent was drop using pasteur’s
Pipette.
- Then mixed and colour change is observed.Bright red and yellow colour
indicate positive and negative M-R inference respectively.
- To tube B,1ml of 40% KOH and 3ml of 5% alcoholic alpha
naphtol.Mixture was shaken thoroughly an colour formation observed.
A pick colour shows a positive V-P reaction E.coli is M-R positive and V-P
negative.The test is the identification of members of enterobacteria such as Ecoli and enterobacter.
 Identification of Aspergillus Species.
Materials:- culture,cotton blue,microscope,slide,cover slip,dissecting needle.
1. From the culture a small portion of the mycelial growth using a pair of
71
sterile dissecting needles wer collected and placed a drop of lactophenol
cotton blue placed on the slide.
2. Using the dissecting needles the mycelial growth was carefully placed on the
slide.
3. The preparation was covered with a cover slip gently avoiding air bubbles.
4. The slides were examined under the microscope with x10 and x40
objectives and the observation drawn.The mycelial growth had long foot
cells,long condiophores,one big board vessicle on which carried the
condia.This conforms the morphology of aspergillus species according to
Ogbulie et. A I (1998).
CHAPTER FOUR.
4.0 RESULTS
4.1 SOME CHEMICAL PARAMETERS OF THE LOCAL PRODUCED
MILLET BEER WITH HOPS AND WITHOUT HOPS ON THREE
DIFFERENT DAYS OF PREPARATION.
Table,1:Shows the reading gotten on
pH,total acidity,fixed acidity,volatile
acidity,TDS,TSS and specific gravity on three differnt days of preparation.
Sample
pH
Total
Fixed
Volatile
TDS
TSS
Specific
millet
Acidity
Acidity
acidity
(ppm)
(ppm)
gravity
beer
g|100ml
g|100ml
g|100ml
E.C
%ᶹ∕ᶹ
72
With
4.03
0.645
0.041
0.604
1.3x10⁵ 1.4x10⁵ 1.199
0.325
hops
0.02
0.011
0.002
0.008
5.5x10⁷ 1x10⁶
0.015
0.004
With
3.32
0.427
0.034
0.393
1.2x10⁵ 3.5x10⁵ 1.049
0.316
out
0.02
0.002
0.005
0.003
2.5x10⁷ 1x10⁶
0.007
0.005
hops
Values are means for triplicate determinations ± Standard deviations.
Table 1:showed the pH values of millet beer with hops(4.03),and without hops
(3.32),Total
acidity
for a local millet beer
hops(0.427),Fixed acidity
hops(0.034),Volatile
acidity
of
with
hops(0.645),and without
millet beer with hops(0.041),and
of
millet beer with hops(0.604),and
without
without
hops(0.393),TDS of millet beer both with hops and without hops respectively as (1.3
x 10⁵),(1.23 x 10⁵),TSS of millet beer with hops as (1.41 x 10⁵),and without hops as
(3.55 x 10⁵),Specific gravity with hops (1.199),and without hops (1.049),and that of
Ethanol content with hops as (0.325%v∕v),without hops as (0.316%v∕v).
4.2 TOTAL ACIDITY FOR LOCAL MILLET BEER.
Result of total acidity of local millet beer with hops and without hops as shown
in Table 2.
 WITH HOPS
73
1st value 2nd value 3rd value MEAN
DAY 1
0.66
0.66
0.645
0.655
DAY 3
0.675
0.645
0.63
0.65
DAY 5
0.615
0.645
0.63
0.63
MILLET
BEER
DAY 1
DAY 3
DAY 5
MEAN
with hops
0.655
0.65
0.63
0.645
STDEV
0.007071
0.018708
0.012247
STDEV
0.010801
 WITH OUT HOPS.
DAY 1
DA Y 3
DAY 5
with out
hops
1st value 2nd value 3rd value MEAN
0.4275
0.4245
0.426
0.426
0.4275
0.4245
0.4215
0.4245
0.432
0.429
0.426
0.429
MILLET
BEER
DAY 1
DAY 3
DAY 5
MEAN
0.426
0.4245
0.429
0.4265
STDEV
0.001225
0.002449
0.002449
STDEV
0.001871
 Total acidity.
Millet with hops
= 0.645± 0.011.
Millet with out hops = 0.427 ± 0.002.
Table 2,shows the value of the total acidity of local millet beer,(both with
hops and without hops),from day 1 to day 5 (which comprises of the sum total
value of mean and stardard deviation from day 1,day 3, and day 5) respectively
as (0.655,0.65,0.63) 0.645 for
millet with
hops, and (0.426,0.4245,0.429)
0.4265 for millet with out hops respectively.From the table above the total mean
for the total acidity of the local prepared millet beer with hops is higher than that
74
with out hops,this is also thesame with the standard deviation.
4.3
FIXED ACIDITY FOR LOCAL MILLET BEER.
Result for fixed acidity of locally prepared millet beer both hops and without hops
are shown in Table 3.
 WITH HOPS.
1st value 2nd value 3rd value MEAN
DAY 1
0.039
0.036
0.042
0.039
DAY 3
0.042
0.0435
0.045
0.0435
DAY 5
0.045
0.039
0.036
0.04
MILLET
BEER
DAY 1
DAY 2
DAY 3
MEAN
with hops
0.039
0.0435
0.04 0.040833
STDEV
0.002449
0.001225
0.003742
STDEV
0.001929
 WITH OUT HOPS.
DAY 1
DAY 3
DAY 5
with out
hops
1st value 2nd value 3rd value MEAN
0.03
0.033
0.033
0.032
0.03
0.0294
0.027
0.0288
0.033
0.03
0.057
0.04
MILLET
BEER
DAY 1
DAY 3
DAY 5
MEAN
0.032
0.0288
0.04
0.0336
 Fixed acidity.
With hops
= 0.041 ± 0.002.
STDEV
0.001414
0.001296
0.012083
STDEV
0.00471
75
With out hops = 0.034 ± 0.005.
Table 3, shows the value f or the fixed acidity of local prepared millet beer (with
hops and without hops)from day1,day 3 and day 5,respectively. It also comprises the
mean with standard deviation from day 1 to day 5 both with hops and without hops
respectively.The mean gotten from day 1,day 3 and day5 are 0.039,0.043,0.04 plus the
total mean for the three days, 0.040833 which is approximately 0.041 for millet beer
with hops,and the mean for millet beer without hops are 0.032,0.029,0.04 plus the total
mean for the three days,0.0336 ≈ 0.034.From the table above the total mean for the fixed
acidity of local prepared millet beer with hops is higher than the one without hops but
the opposite is that with the standard deviation.
4.4 VOLATILE ACIDITY.
The volatile acidity involves the calculation by substrating the fixed acidity from the
total acidity expressed as:
VẠ = TẠ - FẠ
Where
TẠ
=Total acidity
FẠ
= Fixed acidity
VẠ
= Volatile acidity
With Hops.
Total acidity and Fixed acidity (Mean) = 0.645 – 0.041 = 0.604
76
Total acidity and Fixed acidity(STDEV) = 0.011 – 0.002 = 0.008
 Volatile acidity = 0.604 ± 0.008
 With out Hops.
Total acidity and Fixed acidity (Mean) = 0.427 – 0.034 = 0.393.
Total acidity and Fixed acidity(STDEV) = 0.002 – 0.005 = -0.003.
 Volatile acidity = 0.393 ± 0.003.
4.5 SPECIFIC GRAVITY.
Results of specific gravity for local prepared millet beer,both With hops and with
out hops for day 1,day 3 and day 5,as shown in Table 4.
 Table 4:Specific gravity for local prepared millet beer
DAY 1
DAY 3
DAY 5
MEAN
STDEV
with hops
1.22
1.19
1.188 1.199333 0.014636
with out
hops
with hops
with out
hops
1.058
1.048
SPECIFIC GRAVITY
1.199
±
±
1.049
1.04 1.048667 0.007364
0.015
0.007
Table 4,shows the specific gravity value taken for three days of a locally
prepared beer bor both with hops and with out hops.From the Table above,
the mean and the standard deviation value for beer with hops is higher than
77
that with out hops.
4.6 pH VALUE FOR A LOCAL MILLET BEER.
Results of local prepared millet beer both with hops and without hops for three
days as shown in Table 5.
 Table 5:pH value for local prepared millet beer.
DAY 1
DAY 3
DAY 5
with hops
4.06
4.04
with out
hops
with hops
with out
MEAN
STDEV
4 4.033333 0.024944
DAY 1
DAY 3
DAY 5
MEAN
STDEV
3.28
3.31
3.28 3.324641 0.022361
PH
value
±
4.03
±
3.32
0.02
0.02
Table 5,shows the pH value for day 1,day 3, and day 5 as 4.06,4.04,4.0
respectively and the mean for the three days as 4.03, for millet beer with
hops,and 3.28, 3.31, 3.28 respectively and the mean as 3.32 for millet beer
without hops.The mean value for the beer with hops is higher than that with
out hops,mean while their standard deviation of both samples are equal to
each other.
4.7
TOTAL DISSOLVED SOLID (TDS).
Results of local prepared beer as shown in Table 6.
 Table 6:Total dissolve solid of local prepared millet beer.
78
DAY 1
DAY 5
MEAN
STDEV
with hops 0.0000125 0.0000126 0.00001255 0.00000055
with out
hops
0.000012 0.0000125 0.00001225 0.00000025
MILLET
BEER
±
with hops 1.25 x 10⁵
±
with out 1.23 x 10⁵
hops
5.5 x 10⁷
2.5 x 10⁷
Table 6,shows the total dissolve solid of locally produce millet beer and
the mean with the standard deviation for both the one with hops and with
out hops.From the Table above the mean and the standard deviation for the
beer with hops is higher than that with out hop.
4.8 TOTAL SUSPENDED SOLID (TSS).
Results for total suspended solid for local prepared beer as shown in Table 7.
 Table 7:Total suspended solid of local produced beer.
DAY 1
DAY 5
MEAN
STDEV
with hops 0.000014 0.0000141 0.00001405 0.000001
with out
0.000035 0.000036 0.0000355 0.000001
hops
MILLET
BEER
±
with hops 1.41 X 10⁵
±
with out 3.55 X 10⁵
hops
1 X 10⁶
1 X 10⁶
79
Table 7,shows the Total suspended solid of a locally produced beer with
hops and wihout hops as 1.41 x 10⁵, 3.55 x 10⁵ respectively.
4.9 ETHANOL CONTENT.
Results for the ethanol content for a locally prepared beer from day 1,day 3 and
day 5 as shown in Table 8.
 Table 8:Ethanol content of locally prepared millet beer.
with hops
with out
hops
with hops
with out
hops
DAY 1
DAY 3
DAY 5
MEAN
STDEV
0.319
0.325
0.33 0.324667 0.004497
0.31
0.318
0.321 0.316333 0.004643
MILLET
BEER
±
0.325
±
0.316
0.004
0.005
Table 8,shows the Ethanol content of a locally produced beer with hops and
without hops for day 1,day 3, and day 5 respectively as(0.319,0.325,0.33), and
(0.31,0.318,0.321) respectively,and the mean values gotten for the three days,with
hops and without hops as 0.325 and 0,316 respectively.
4.10 MICROBIAL COUNT FOR BENCH STORED MILLET.
Result of some microbial count with hops and without hops for bench stored
millet beer as shown in Table 9.
80
Table 9: Some microbial counts for Bench stored millet beer with hops.
WITH HOPS.
Days
Microoganism
0
1
2
3
4
5
6
7
8
Streptococcus
-
-
-
-
-
-
-
-
-
E .Coli
-
-
-
-
-
-
-
-
-
Aspergillus
-
-
-
-
+
+
+
+
+++
pyogenes
WITHOUT HOPS.
Days
Microorganism
0
1
2
3
4
5
6
7
8
Streptococcus
-
-
-
-
-
-
-
+
++
E . Coli
-
-
-
-
-
-
-
-
-
Aspergillus
-
-
+
+
+
+
+
+
pyogenes
++++
Note: + → Present (Positive).
- → Absent (Negative).
Table 9,shows some microbial count for bench stored millet beer both with hops
and without hops from day 0 to 8.No growth was seen on the other
organism,except Aspergillus microrganism that was notice from the 4th day to the
8th day with hops and from the 2nd day to 8th days without hops(Aspergillus),
81
and from day 7 to day 8(Streptococcus pyogenes).This indicated that our hops
substitute (bitter leaf juice) have some phytochemicals in common with the normal
hops which helps to increase the shelf- life of beer.
4.10.1 MICROBIAL COUNT FREEZED MILLET BEER.
Result of some microbial count of freezed millet beer with hops and with out
hops as shown in Table 10
WITH HOPS.
DAYS
Microorganism
0
1
2
3
4
5
6
7
8
Streptococcus
-
-
-
-
-
-
-
-
-
E .Coli
-
-
-
-
-
-
-
-
-
Aspergillus
-
-
-
-
-
-
-
-
-
pyogenes
WITHOUT HOPS.
DAYS
Microorganism
0
1
2
3
4
5
6
7
8
Streptococcus
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
-
pyogenes
E .Coli
82
Aspergillus
-
-
-
-
-
Table 10,shows some microbial growth in the
-
+
+
+ +
beer without hops and this
microorganisms are seen from day 7 to day 8(Streptococcus pyogenes),and from
day 6
to
day
8 for (Aspergillus).But
with hops
no
growth
was
of
any
microorganisms was seen from day 1 to day 8.
4.10.2 GENERAL DISCUSSION OF RESULTS
Table 1:The shows the summary of the eight tables which involves pH values,Total
acidity,Fixed acidity,Volatile acidity, TDS,TSS,Specific gravity and Ethanol content
of both brewed millet beer with hops and without hops.Table 1 summarised both
mean value and standard deviation of the two sample (with hops and without
hops).
Table 2:Showed the total acidity for locally made millet beer. The total acidity
value for millet beer with hops for day 1 (0.655g ∕100ml),day 3 (0.65g ∕100ml),and
day 5 as (0.63), shows a decrease indicating that the hops substitute has some
effect on the beer.From the table above there was a significant increase (P≤ 0.05)
between the beer with hops and that without hops respectively.From day1, 3. And
5, we have 0.645g ∕ 100ml with hops and 0.427g ∕ 100ml without hops.This shows
83
that the one with hops is deteriorated.Thus there are more acid in the beer with
hops, than that without hops.
Table 3: Shows the fixed acidity for a locally produced beer.The fixed acidity for
the beer with hops is higher than that with hops.From day 1,3 and 5 with hops we
have 0.041, while without hops we have 0.034 which means there is a significant
increase of (P≤ 0.07).
Table 4:shows that the specific gravity of millet beer with hops is higher than the
one without hops.The specific gravity for the locally brewed beer with hops when
compare with that with no hops there is significant differences of (P≤ 0.05).
Table 5:The pH of the beer with hops is higher than that with no hops due to
the high lactic acid production in the one with hops,which is not in line with the
pH value given by Okafor,(1987) for millet beer.
Table 9. shows the microbial count for bench stored millet beer.from day 1 to 3,
no growth was observed in Streptococcus pyogens but from the 4th day to the 8th
day growth was seen on Aspergillus for the beer with hops.but that without hops
growth was seen on Streptococcus pyogens on the 7th day and from 2nd day to the
8th day growth was seen on Aspergillus.It was heavier on the 8th day.
Table 10. Shows the microbial count for the freezed millet beer. from day 1 to 8,no
growth
hops.But
was observed
in
Streptococcus
pyogens, E.Coli, and Aspergillus
with
84
CHAPTER FIVE.
DISADVANTAGES ASSOCIATED WITH OVER CONSUMPTION OF
BREWED PRODUCT.
 Over consumption of brewed product can be very fatal to health,due to the
present of tannin in red wine although in small quantity but if consumed in
large quantity might lead to heart diseases which will affect the rate of
respiration of that individual.
 Also over consumption of this brewed product will affect the brain since
the main food for the brain is Glucose and this glucose has ben fermented,
making the consumption of too much of this brewed product very deadly to
the brain cell for it even to the damage of brain cell by reducing the rate
of respiration.
85
 Also over consumption of this brewed product can lead to sterility due to the
presence of tannin in red wine.
 Over consumptin of brewed products liver damage for it over work the
liver due the ethanol content in the beer.etanol related high levels of NADH
+ H and acetyl-CoA in the liver lead to increase synthesis of neutral fats
and cholesterol.However, since the export of these in the form of VLDLS is
reduced due to alcohol,storage of lipids occurs(fatty liver),which is not
healthy at all.This increase in fat content of the liver is initially
reversible.However, in chronic alcoholism the hepatocytes are increasingly
replaced by connective tissue.When liver finally reaches an irreversible
stage,characterized by progressive lose of liver functions.
 Over consumption of alcoholic product hsa a very drastic effect in human
life for it lead to paralysis,heart failure,sterility,brain failure and even make
the individuals involved to caused nuisance to the society.
 Since locally made millet beer with bitterleaf extract acting as hops substitute
have not been well mordify so as to eliminate or have more effect on
fungi and other microorganisms so as to increase the shelf –life of the
beer,it is advisable that abstinence is the best option until every thing is
corrected,If at all any beer should be consumed the one without hops is
preferable but should be consumed immediately after production because it
has no phytochemicals to preserve it for a period of time and so if left for
a long period of time it will serve as a medium for the growth of
86
microorganism,which when consumed it will cause problem to the person’s
health.
5.0.1 ADVANTAGES OF BREWED PRODUCT.
 Alcoholic drink (brewed product) when consumed in a very little quantity
help to dialate the vein to ease the flow of blood round the body.
 Also a little consumption of brewed product help to produce fatty cell under
the skin hence creating an insulator to the body.
 Also locally made millet beer is very healthy to consumed immediately
after production (the one without hops).
5.1 CONCLUSION.
In conclusion, it may be said that Abstinence is the best option to achieve a
healthy liver, heart, lungs,brain infact a healthy being.And also that the locally
produced beer is more preferable to health when consumped immediately after
production for it does not contain tannin compound which is present in morden
beer(red wine),high alcoholic level which is also present in morden beer. Also
87
locally made millet beer with hops prove to have a longer shelf life than that
without hops,this explain that the bitter leaf extract which act in the place of hops
as hop substitute can prevent the growth of microoganism which may deteriorate
the beer,although it has just little effect on the fungus which may caused some
problms when consumed.In ordered to achieve our aim the bitter leaf extract
should be well mordify in such a way that it will increase the shelf-life of this
locally prepared millet beer for than more eight days without experiencing any
growth of spoilage bacteria or microorganism so as to serve the less priviledge as
their own alcoholic beer.
5.2 RECOMMENDATION.
From the results of my research program, I recommend the following:
 That the hops substitute (bitter leaf extract) be more mordify in such a way
that so as to increase the shelf – life of the locally made millet beer,so that it
may act as the morden hops and even better than the morden hops,so that
the less priviledge will have their own beer.
 That the local millet beer be well mordified as to serve to the less
priviledge as their own alcoholic drink.
 That more microorganism causing the deterioration of the local made millet
beer be identified and solution on how to prevent such growth.
88
 That the method of preparation of this local millet beer be mordified so as
to prevent the introduction of microorganism which renders the drink unsafe
for human consumption.
 That the organic acids contained in the beer be found and that bitter leaf
extract be well more mordified in such a way it is going to have effect on
fungi organisms.
 I also recommend that abstinence from all these brewed product especially
the modrn ones is the best option for a good health and also these
brewed product should be kept out of reach to children and should not be
recommended for children under - aged.
5.3 LsIMITATIONS OF STUDY.
 Due to the high amount of solids in this locally made beer especially that
without hops,finding the total acidity was cumbersome and time taken.
 Since we are researching on how to increase the shelf – life of this locally
made millet beer by using bitterleaf as hops substitute,finding the effect of
this hops on the beer was strainous and tasking.
89
REFERENCES.
Alais,C. & Linden, G.(1999). Food Biochemistry.Great Britain:Aspen Publisher Inc.
Amerine, M. A., Berg, H. W., & Cruess, W. V. (1972). The Technology of Wine
Making. USA Westport: Avi Publications.
Anonymous.(1989). .Approved methods of the American Association of Cereals.
USA.
Arnold, P. (2000).Origin and History of Beer and Brewing. From Prehistoric
Times to the Beginning of Brewing Science and Technology.
Asiedu, J. J. (1989). Processing Tropical Crops. Nigeria:Macmillian Education Ltd.
Ault, R. G., & Newton, R. (1971). Modern Brewing Technology.London,UK:Findly
Macmillian Press.
Bantu Beer Unit. (1971). Course delivered at the Pretoria College for Advanced
Technical Education.South Africa:Pretoria.
90
Barth, I. (1967). Economic Spheres in Darfur:Themes in Economic
Anthropology (R. Ford ed). pp.149-189. London:Tanistock.
Bryant, A. T. (1948). The Zulu people as they were before the white man
came.New York: Negro Universities Press.
Carr, J .G .(1969). Biolological Principles in fermentation. London: Heinemann
Educational Books Ltd.
Cavalieri, D., Mchovern, P. E., Harti. D. L., Martimer, R. & Poisineli, M.
(2003). Evidence for S.Cerevisiae fermentation in ancient wine. Journal
of Molecular Evolution. pp. 226-232. Germany:Berlin.
Christopher, B., & Aaron C. (1986). Introduction to Biochemical Engineering. p. 443.
Troy:Rensselaer Polytechnic Institute.
Deschaepdrijiver, B. et al.(1988). Reports on Vitamin C enriched Sorghum
Beer. p. 83. South Africa:Pretoria.
Dickinson, J. R. (1999).Carbon Metabolism.In the metabolism and molecular
physiology of Saccharomyces Cerevisiae. pp. 721-725.France: Paris.
Edward, H. V., Frank, H. S., Henry, G. L. & Adolph, J. M. (1977). Practical
Brewing( 4th Edition). London and New York: Macmillian Press.
Ekundayo, J. A. (1969). Production of Pito. A Nigerian Fermented Beverage. pp. 217225.Nigeria:Ibadan.
Ettasoe, C. L. & Wills, J. B.(1972). Pearl Millet and Sorghum in Leaky. Food
91
Crops of Lowland Tropics. Great Britain: Oxford University Press.
Flickinger, M. C., & Drew, S. W. (1999). Encyclopeida of Bioprocess TechnologyFermentation, Biocatalysis, and Bioseparation. USA: New York.
Gutcho, M. H. (1976). Alcoholic Beverages Processes. London: Noyes
Press.
Grossman, H. J.(1995). Beer in Encylopedia Americana. pp. 132-138. USA:New York.
Haddad, P. R., Sterns, M. & Warclaw, J. (1978). Analysis of Wine,an
undergraduate project. pp. 87-89.
Hammond, J. M., & Bamforth, C. W. (1993). Progress in the Development of
New Barley, Hop and Yeast variants for malting and brewing:
Biotechnology and Genetic Engineering.
Hough, J. S.(1985). The Biotechnology of Malting and Brewing.
UK: Cambridge University Press.
Kent, N. I. (1983). Technology of Cereals.An introduction for students of Food
Science and Agriculture. (3rd Edition). London:Pergramon Press Ltd.
Kneen, E. (1944). A Comparative Study of the Development of Amylases of
Germinated Cereals. pp. 304-314.
Lerio, F., & Pidoux, M. (1993). Characterizaion of jnteractions between
Lactobacillus Higardii and Saccharomyces Horentinus Isolated from
Sugary Kafir grains. pp. 54-60.
Lewington, A. (1998). Plants for People:the Natural history Museum
London:Cromwell Road.
92
Okafor, N.(1990).Traditional Alcoholic Beverages of Tropical Africa.Strategies
for scale-up process Biochemistry. pp. 213-220. Nigeria:Ibadan.
Paul, V. N. (1985). Taxonomy and Phytogenesis of Cereals.Germany:Berlin.
APPENDIX A
PREPARATION OF REAGENTS
1% Phenolphthalein
1.0g of phenolphthalein powder was weighed using an analytical weighing balance
and dissolved in 100ml ethanol in a cylinder. It was stirred thoroughly in order to
dissolve the salt.
0.1m NaoH
4g of NaoH was dissolved in some quantity of distilled water in a beaker and then
transferred to a 500ml volumetric flask and made up to the mark with distilled water.
93
APPENDIX B
FORMULAR AND CALCULATIONS
Total acidity
T.A (g/ 100ml) = 0.075 x M x 100 x W2
Vml
Where Mı
=
V2
=
Vı =
0.075 =
Molarity of the NaoH
Titre value
Volume of the sample (5ml)
Equivalent weight for Tartaric acid
The total acidity was expressed as tartaric and then converted to lactic by multiplying
with 0.0833 as given in the conversion table. See appendix C.
Volatile acidity
The volatile acidity was calculated by subtracting the fixed acidity from the total acidity
expressed on the same scale ie. VA = TA – FA. VA = (g/100ml)
94
Where
TA
=
Total acidity
FA =
Fixed acidity
VA =
Volatile Acidity.
Total dissolved solid (TDS)
TDS (ppm) = W2- Wı(g) x 106
Vml
Where
Wı =
Weight of empty crucible
W2
=
Weight of crucible + sample after drying.
V
=
Volume of sample evaporated
Total suspended solids (TSS)
TSS (ppm) = Wı- W2(g) x 106
Vml
Where
W1 = Weight of filter paper
W2 =
V
=
Weight of filter paper + residue
Volume of filtered sample
Fixed acidity
F. A. g/100ml =
V1 x M1 x 0.075 x 100
V2
95
Where
V1 = Volume of NaOH used
V2 = Volume of sample (25ml)
M1 =
0.075 =
Molarity of NaOH
Equivalent weight for Tartaric acid
Converted to Lactic by multiplication with 0.0833. See appendix C.
APPENDIX C
TOTAL ACIDITY CONVERSION TABLE
Expressed as
Tartari
Malic
Cutric
Lactic
Sulfuric
Acetic
c
Tartaric
1.00
0.893
0.893
1.200
0.655
0.800
Malic
1.114
1.000
0.988
1.342
0.732
0.096
Cutric
1.172
1.047
1.000
1.406
0.766
0.938
Lactic
0.833
0.744
0.711
1.000
0.544
0.667
Sulfuric
1.531
1.367
1.306
1.837
1.000
1.225
96
Acetic
1.250
1.117
1.067
1.500
0.817
1.000
Adapted from Hadded et al (1978)
APPENDIX E
STATISTICAL ANALYSIS
Student’s T –distribution table was used as adopted from steel and torrie (1960); Pearson
Hartley (1996). T- test of significance was taken at 95% confidence level of probability
(p< 0.05). The significance difference between two non-paired independent variables was
computed using the formula:
t
=
x1 – x2 against α = [(n1 + n2) -2]
where
t
=
calculated t-value
x1 and x2
= different means of two non-paired independent variables,
SEM
=
α =
n1 and n2
standard error of the mean differences
Degree of freedom
=
size of varible, this time, the number of determinations for each
variable.
97
From the above formular,
SEM= √ SD12 + SD22
–
–
n1
+
n2
where
SD1 and SD2 = the standard deviations of maens of two independent variables
Thus,
t =
x1 – x2
√ SD1² + SD2²
n1
against = [(n1 + n2) -2)
n2
Note, two means are significantly different if the calculated “t” is greater or equal, i.e. (
t> t α ) to t- distribution table.