DEVELOPMENT OF STARTER CULTURE FOR THE
FERMENTATION OF DEHULLED MAIZE INTO NSIHO
(WHITE KENKEY)
BY
THEOPHILUS ANNAN
(10359128)
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA,
LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE AWARD OF MPHIL FOOD SCIENCE DEGREE
JULY, 2013
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DEDICATION
I dedicate this work to my lovely wife Mrs.Hellen Ama Annan, my wonderful daughters, Tamara
Millicent Naa Adoley Annan and Theodora Naa Adorkor Annan and my siblings.
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DECLARATION
I do hereby declare that this thesis is the result of my own research except for references to
works of others that have been duly cited under the supervision of Prof. Tano-Debrah and Dr.
Wisdom Amoa-Awua, and that this work either in whole or part has not been presented for
another degree elsewhere.
.................................................. .......................................
THEOPHILUS ANNAN DATE
(STUDENT)
................................................... .........................................
PROF. K. TANO-DEBRAH DATE
(SUPERVISOR)
.................................................... ...........................................
Dr. W. AMOA-AWUA DATE
(SUPERVISOR)
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ACKNOWLEDGEMENTS
This work was facilitated by financial support from the European Union under the project
African Food Tradition rEvisited by Research (AFTER) KBBE-20009-2-3-02.
I wish to sincerely thank my supervisors, Professor K. Tano-Debrah, former Head of
Department, Nutrition and Food Science, Legon and Dr. W. Amoa-Awua, Chief Research
Scientist and former Head of Food Microbiology Division, Food Research Institute, Accra, for
their constructive criticisms, guidance and mentorship which have brought this work to a
successful completion.
I am grateful to Miss Angela Adam and Mr. Alexander Appiah for helping in preparation of
microbiological media. My appreciation goes to Mr. Emmanuel Tetteh for making sure I always
had sterile Petri dishes for my analyses. I am highly indebted to Miss Kukua Amo Thompson for
her assistance and interest in this work. I am also thankful to Mr Kavi, Senior Librarian of Food
Research Institute for giving me all the articles and publications I needed for this work. I would
also thank Messr‟s Gariba and Safo for driving me to the field to collect my samples. A big
thanks to all the AFTER team members for their support. Finally, I am most grateful to my wife
for her support and encouragement.
I am above all most grateful to God almighty for giving me strength and wisdom to carry out this
work.
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ABSTRACT
Nsiho kenkey is a bland tasting stiff porridge produced from dehulled maize grains and
consumed mainly in some parts of Southern and Eastern Regions of Ghana. This study was
carried out to identify the microorganisms responsible for nsiho fermentation and to develop a
starter culture for its controlled fermentation in order to improve the safety and quality of the
product. A brief field study was carried out in two districts in the Eastern Region of Ghana to
observe the processing operations in nsiho production and obtain samples for laboratory analysis.
The population of aerobic mesophiles, lactic acid bacteria (LAB) and yeasts were enumerated on
Plate Count Agar, de Man Rogosa Sharpe Agar and Oxytetracycline Glucose Yeast Extract Agar
respectively. The species of the LAB and yeasts were tentatively identified by phenotypic
characterization based mainly on their pattern of carbohydrate assimilation and fermentation.
Lactic acid bacterria isolates were screened for rate of acidification, production of
exopolysaccharides (EPS), amylase and protease activity as well as antimicrobial activity against
some common enteric pathogens using the Agar Well Diffusion Assay. Starter culture was
developed through combination of dominant strain of lactic acid bacteria and yeasts in
production trails. Survival of four enteric pathogens (Salmonella typhimurium, NCTC 12023,
Staphylococcus aureus, NCTC 657, Vibrio cholerae NCTC 11348 and Escherichia coli. NCTC
9001) were also studied during steeping enriched with the starter cultures. Mean pH values
decreased from 5.99-3.58 units whilst Titratable acidity increased from 0.03-0.30 % during 48 h
steeping of dehulled maize grains in nsiho prodcution. Similarly, pH values decreased from 5.94-
3.50 units and Titratable acidity increased from 0.27-0.36 % of 12 h of subsequent dough
fermentation. Lactic acid bacteria population increased from a concentration of 104 to108 cfu/ml
during steeping and from 105 to108 cfu/g during the dough fermentation. Yeasts counts increased
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from 102 to 106 cfu/ml during steeping and from 103 to107 cfu/g during dough fermentation. The
lactic acid bacteria responsible for nsiho fermentation were identified to be Lactobacillus
fermentum, 47.1%, Lactobacillus brevis, 25%, Lactobacillus plantarum, 14.42%, Pediococcus
pentosaceus, 8.65% and Pediococcus acidilactici, 4.8%. The dominant yeasts species were
Saccharyomyces cerevisiae, 47.6%, Candida krusei, 29.1%, Debaryomyces spp., 15% and
Trichosporon spp., 8.3%. Isolates of Lactobacillus fermentum exhibited a faster rate of
acidification than the other LAB isolates tested. Most of the LAB isolates screened produced
EPS, a few showed amylolytic activity whilst none exhibited protease activity. There was no
interaction between the LAB isolates; however, a few of the LAB inhibited the growth of yeasts.
The use of starter culture involving combination of LAB and yeasts isolates decreased the
steeping time of the dehulled maize grains from 48 h to 24 h. The use of various combinations of
LAB and yeasts as inoculum enrichment during fermentation trials produced kenkey acceptable
to the taste panel, however, no significant differences (p<0.05) were recorded in taste, texture
and odour when compared to the spontaneous fermented nsiho. In challenge testing during
steeping of maize grains Vibrio cholerae could not be detected after 48 h whilst counts for
Salmonella typhimurium, Staphylococcus aureus and Escherichia coli were reduced by 3 log
cycles. Based on antimicrobial activity and technological properties exhibited by the isolates and
the sensory quality of nsiho, the best starter organisms selected for nsiho production consisted of
mixed cultures of Lactobacillus fermentum, Lactobacillus brevis and Saccharomyces cerevisiae.
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TABLE OF CONTENTS
DEDICATION................................................................................................................................i
DECLARATION...........................................................................................................................ii
ACKNOWLEDGEMENTS.........................................................................................................iii
ABSTRACT...................................................................................................................................iv
TABLE OF CONTENTS.............................................................................................................vi
LIST OF TABLES......................................................................................................................xiii
LIST OF FIGURES.....................................................................................................................xv
1.0 INTRODUCTION...................................................................................................................1
1.1 Maize Production and Consumption.......................................................................................1
1.2 Traditional Processing of Maize.............................................................................................1
1.2.1 Processing of Maize into Kenkey.....................................................................................2
1.3 Rationale for the study...........................................................................................................3
1.4 Main Objective........................................................................................................................4
1.4.1 Specific Objectives...........................................................................................................4
2.0 LITERATURE REVIEW.......................................................................................................5
2.1 Maize Grains...........................................................................................................................5
2.2 Some Ghanaian Traditional Food Products Made from Maize..............................................5
2.2.1 Fomfom.............................................................................................................................6
2.2.2 Kokui (Ewe kenkey).........................................................................................................6
2.2.3 Akpiti (Ga), Bamfobisi (Twi), or Amo kaklo (Ewe)........................................................8
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2.2.4 Abolo.................................................................................................................................8
2.2.5 Banku.................................................................................................................................8
2.2.6 Koko..................................................................................................................................8
2.2.7 Kenkey..............................................................................................................................9
2.2.7.1 Cleaning...................................................................................................................9
2.2.7.2 Steeping..................................................................................................................11
2.2.7.3 Nutritional Aspect of Kenkey................................................................................11
2.3 Starter Cultures.....................................................................................................................11
2.3.1 Moulds............................................................................................................................12
2.3.2 Yeasts..............................................................................................................................12
2.3.3 Bacteria...........................................................................................................................13
2.4 Attributes of Starter Culture..................................................................................................13
2.4.1 Desirable Properties of Starter Culture...........................................................................14
2.5 Starter Cultures in African Cereal Fermentation..................................................................15
2.5.1 Use of Starter Cultures in Maize Fermentation..............................................................16
2.5.2 Use of Starter Cultures in Ogi Preparation.....................................................................17
2.5.3 Use of Starter Cultures in Uji Preparation......................................................................18
2.5.4 Use of Starter Cultures in the Production of Mawe........................................................19
2.5.5 Use of Starter Cultures in Mahewu Production..............................................................19
2.6 The Role of Lactic Acid Bacteria in Fermentation...............................................................21
2.6.1 Microorganisms Involved in the Lactic Acid Fermentation...........................................22
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2.7 Methods used for the Identification of Lactic Acid Bacteria...............................................26
2.7.1 Conventional Taxonomy.................................................................................................26
2.7.2 Numerical Taxonomy.....................................................................................................26
2.7.3 DNA Based Techniques..................................................................................................27
2.8 Anti-microbial Substances Associated with Lactic Acid Bacteria.......................................28
2.8.1 Organic Acids and Low pH............................................................................................28
2.8.2 Bacteriocins.....................................................................................................................29
2.8.3 Hydrogen peroxide..........................................................................................................30
2.8.4 Carbon Dioxide...............................................................................................................31
2.8.5 Diacetyl...........................................................................................................................31
2.9 Lactic Acid Bacteria as Probiotics........................................................................................32
2.10 Prebiotics.............................................................................................................................33
2.11 Role of Yeasts in Maize Fermentation................................................................................34
2.11.1 Saccharomyces cerevisiae............................................................................................35
2.11.2 Candida krusei..............................................................................................................35
2.12 Technological properties of lactic acid bacteria.................................................................36
2.12.1 Exopolysaccharides.......................................................................................................36
2.12.2 Starch Hydrolysis..........................................................................................................37
2.12.3 Proteolysis.....................................................................................................................38
3.0 MATERIALS AND METHODS..........................................................................................39
3.1 Study Area ...........................................................................................................................39
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3.2 Study Design.........................................................................................................................39
3.2.1 Sample Collection and Preparation.................................................................................39
3.3 Chemical Analyses................................................................................................................39
3.3.1 Determination of pH.......................................................................................................39
3.3.2 Determination of Titratable Acidity................................................................................40
3.4 Microbiological Analyses.....................................................................................................40
3.4.1 Enumeration of Microorganisms....................................................................................40
3.4.1.1 Homogenization and Serial Dilution.....................................................................40
3.4.1.2 Enumeration of Aerobic Mesophiles.....................................................................40
3.4.1.3 Enumeration of Lactic Acid Bacteria....................................................................41
3.4.1.4 Enumeration of Yeasts...........................................................................................41
3.4.2 Isolation of Lactic Acid Bacteria....................................................................................41
3.4.3 Characterisation of Isolates.............................................................................................41
3.4.3.1 Microscopic Examination......................................................................................41
3.4.3.2 Gram‟s Reaction....................................................................................................42
3.4.3.3 Catalase Reaction...................................................................................................42
3.4.3.4 Oxidase Test...........................................................................................................42
3.4.3.5 Growth at Different Temperatures.........................................................................42
3.4.3.6 Gas production from Glucose................................................................................43
3.4.3.7 Salt Tolerance Test...............................................................................................43
3.4.3.8 Growth at Different pH..........................................................................................43
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3.4.4 Identification of Lactic Acid Bacteria by Carbohydrate Fermentation Profiles.............44
3.4.5 Isolation of Yeasts..........................................................................................................44
3.4.5.1 Macroscopic and Microscopic Examination..........................................................44
3.4.6 Identification of Yeasts Isolates using the ID 32 Kit......................................................44
3.5 Technological Properties of Identified Lactic Acid Bacteria...............................................45
3.5.1 Rate of Acidification of Maize Grains and Dough by LAB...........................................45
3.5.2 Production of Exopolysacchrides (EPS) by LAB Isolates..............................................45
3.5.3 Test for Amylase Secretion by LAB Isolates.................................................................46
3.5.4 Test for Protease Secretion by LAB Isolates..................................................................47
3.5.5 Antimicrobial Studies.....................................................................................................47
3.6 Development of Starter Culture............................................................................................48
3.6.1 Dehulled maize...............................................................................................................48
3.6.2 Starter Cultures...............................................................................................................48
3.6.2.1 Preparation of Inoculum........................................................................................48
3.6.3 Inoculation Trials............................................................................................................49
3.6.3.1 Fermentation with Single Starter Culture..............................................................49
3.6.3.2 Fermentation with Combined Starter Culture........................................................50
3.7 Survival of Enteric Pathogens during Steeping of Dehulled Maize.....................................50
3.8 Preparation of Nsiho in the Laboratory for Sensory Evaluation...........................................51
3.8.1 Sensory Analysis..............................................................................................................51
4.0 RESULTS...............................................................................................................................53
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4.1 Field Study............................................................................................................................53
4.2 Chemical analyses of Maize during Steeping and Dough Fermentation..............................55
4.3 Changes in Microbial Population during Steeping and Dough Fermentation of Nsiho.......57
4.3.1 Population of Aerobic Mesophiles..................................................................................57
4.3.2 Population of Lactic Acid Bacteria.................................................................................59
4.3.3 Population of Yeasts.......................................................................................................60
4.4 Characterisation and Identification of Lactic Acid Bacteria.................................................61
4.5 Characterisation and Identification of Yeasts Population.....................................................63
4.6 Technological properties of Lactic Acid Bacteria Isolates...................................................63
4.6.1 Rate of acidification by Lactic Acid bacteria Isolates....................................................63
4.6.2 Production of Exopolysaccharides (EPS) by Lactic Acid Bacteria Isolates...................68
4.6.3 Amylase Secretion by Lactic Acid Bacteria Isolates......................................................68
4.6.4 Protease Secretion by Lactic Acid Bacteria Isolates.......................................................68
4.6.5 Antimicrobial Activity of Lactic Acid Bacteria against some Common Pathogens
.....................................................................................................................................69
4.6.6 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates...............70
4.6.7 Antimicrobial Interaction between Lactic Acid Bacteria Isolates..................................71
4.7 Starter Culture Trials.............................................................................................................73
4.7.1 Changes in Microbial Population...................................................................................73
4.7.2 Acidification of Steep Maize and Fermenting Dough in Fermentation Trials with Starter
Culture...........................................................................................................................75
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4.7.3 pH Changes during Steeping and Dough Fermentation Inoculated with Combinations of
Starter Cultures..............................................................................................................77
4.7.4 Changes in Titratable Acidity during Steeping and Dough Fermentation Inoculated with
Different Combinations of Starter Cultures..................................................................80
4.7.5 Microbial Population during Steeping using Different Starter Cultures ………….......82
4.7.6 Microbial Counts during Nsiho Dough Fermentation using Different Starter Culture...84
4.8 Survival of Enteric Pathogens during the Steeping of Dehulled Maize Grains using
Different Starter Cultures...................................................................................................86
4.9 Sensory Quality of Nsiho Produced using Starter Cultures..................................................89
5.0 DISCUSSION.........................................................................................................................92
5.1 Changes in Acidity during Spontaneous Fermentation........................................................92
5.2 Role of Lactic Acid Bacteria in Nsiho Fermentation............................................................92
5.3 Role of Yeasts in Nsiho Fermentation..................................................................................95
5.4 Antimicrobial Activity of Lactic Acid Bacteria against Common Enteric Pathogens.........95
5.5 Microbial Interactions during Nsiho Fermentation..............................................................96
5.6 Technological Properties.....................................................................................................98
5.7 Selection of Starter Cultures.................................................................................................99
5.8 Survival of Enteric Pathogens during Maize Steeping Inoculated with Starter Culture.....101
6.0 CONCLUSION....................................................................................................................104
7.0 RECOMENDATIONS........................................................................................................106
REFERENCES...........................................................................................................................107
APPENDIX.................................................................................................................................121
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LIST OF TABLES
Table 2.1 Mixed starter cultures applied in the production of traditional acid- and acid-alcoholic
fermented gruels.................................................................................................................15
Table 2.2 Traditional lactic acid cereal- based fermented foods and beverages.....................20
Table 2.3 Metabolic products of lactic acid bacteria which exhibit antimicrobial properties.......32
Table 4.1 Mean pH values during the fermentation of dehulled maize into nsiho........................56
Table 4.2 Mean Titratable acidity (%) changes during the fermentation of dehulled maize into
nsiho...................................................................................................................................57
Table 4.3 Mean (cfu/ml or cfu/ g) mesophilic counts during the fermentation of dehulled maize
into nsiho............................................................................................................................58
Table 4.4 Mean LAB counts during the fermentation of dehulled maize into nsiho.....................59
Table 4.5 Mean yeast counts during the fermentation of dehulled maize into nsiho....................60
Table 4.6 Biochemical characteristics of lactic bacteria isolated from steeping water and
fermenting dough...............................................................................................................62
Table 4.7 Change in Titratable acidity during acidification of steeping water by lactic acid
bacteria...............................................................................................................................67
Table 4.8 Change in Titratable acidity during acidification of fermenting dough by lactic acid
bacteria...............................................................................................................................67
Table 4.9 Amylase, protease and exopolysaccharides (EPS) secretion by lactic acid bacteria
isolated during maize and dough fermentation..................................................................69
Table 4.10 Antimicrobial activity of lactic acid bacteria against pathogen indicator- strains.......70
Table 4.11 Antimicrobial interaction between lactic acid bacteria and yeasts isolates.................71
Table 4.12 Antimicrobial interaction between lactic acid bacteria isolates...................................72
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Table 4.13 Mean microbial counts (cfu/ml and cfu/g) for fermentation trials carried out with
starter culture.....................................................................................................................74
Table 4.14 Mean pH and Titratable acidity values for fermentation trials with starter cultures...76
Table 4.15 Percentage Titratable acidity values during steeping and dough fermentation of nsiho
using different starter culture.............................................................................................81
Table 4.16 Mean microbial counts (cfu/ml) during the steeping of dehulled maize grains using
starter cultures....................................................................................................................83
Table 4.17 Mean microbial counts (cfu/g) during nsiho dough fermentation using different starter
cultures...............................................................................................................................85
Table4.18 Count for survival of enteric pathogens inoculated into spontaneous and mixed starter
culture fermentation of steeped dehulled maize................................................................87
Table 4.19 Sensory scores for nsiho prepared by spontaneous fermentation or through the use of
a starter cultures.................................................................................................................90
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LIST OF FIGURES
Figure 2.1: Production of fomfom...................................................................................................7
Figure 2.2: Production of Ga and Fanti kenkey.............................................................................10
Figure 2.3: The path way for glucose dissimilation by homofermentative and heterofermentative
bacteria...............................................................................................................................23
Figure 4.1 Flow diagram for production of Nsiho (white kenkey)................................................54
Figures 4.2 a-c Graphs showing changes in pH during the rate of acidification of steeped
dehulled maize by lactic acid bacteria isolates..................................................................65
Figures 4.2 d-f Graphs showing changes in pH during the rate of acidification of fermenting
dough by lactic acid bacteria..............................................................................................66
Figure 4.3 Graph showing steeping with added mixed cultures....................................................78
Figure 4.4 Graph showing dough fermentation with added mixed cultures..................................79
Figure 4.5 Principal Component Analysis bi-plot (PCA)..............................................................91
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1.0 INTRODUCTION
1.1 Maize production and consumption
Maize (Zea mays) is a cereal grain which belongs to the grass family (Gramineae) and is
ranked the third most abundant cereal crop produced worldwide. Maize is the principal
cultivated cereal grown in Ghana, mainly in the middle-southern part (transitional and forest
zones), with an estimated 15% grown in the northern regions of the country. Its gross
production in 2009 was 1,620.000 metric tons with per capita consumption of 43.8 Kg/annum
and net surplus of 115,750 metric tons (Ministry of Food and Agriculture, 2010).
As a cereal crop widely used in Ghana, maize contributes significantly to the total calorie of
the Ghanaian diet, furnishing between 90-95% of the total calorie in the diet of the people in
the coastal plains. However, the consumption of maize is less extensive in the northern part of
the country where sorghum and millet are the main staples (Dovlo, 1970; Ijabadeniyi and
Omoya, 2006).
1.2 Traditional Processing of Maize
The traditional processing of maize into various fermented and semi-fermented food products
plays an important role in the food supply system of Ghana, and contributes to the curtailment
of the post harvest losses and national food delivery (Sefa –Dedeh, 1993). The maize grains,
by a variety of indigenous processes, is transformed into an intermediate or finished product
with a stable shelf-life, more digestible, improved nutritive quality and desirable organoleptic
properties. Processing may also include improvement of the protein quality of the maize
product by fortification with legumes such as cowpeas and soybeans (Plahar and Leung 1983;
Nche et al., 1994; Obiri-Danso et al., 1997; Plahar et al., 1997; Sefa-Dedeh et al., 2000).
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Traditional maize processing methods involve combination of different unit processing
operation including steeping, dehulling, roasting, milling, fermenting, aflatalization,
moulding, cooking and packaging. These skills have been developed and handed down from
one generation to the other. Processing maize into fermented dough is mainly by traditional
methodologies and technologies which utilize local raw materials and local equipment which
have strong links with rural traditional environment (Sefa-Dedeh, 1993; Afoakwa et al., 2004).
Maize processing is one of the major rural /informal food industries in Ghana serving as a
means of livelihood for a number of rural women who rely on family labour and engage in
commercial activity as a household enterprise.
1.2.1 Processing of Maize into Kenkey
There are mainly two types of kenkey, the Ga kenkey locally called Komi, and the Fanti
kenkey known as Dokono, and these differ in their organoleptic quality and the processing
procedures (Halm et al., 2004). However, there is another type of kenkey, Nsiho or Akporhi
which is produced from dehulled or polished maize, (referred to as white kenkey in the
present work), that is widely consumed in parts of Eastern, Western, Central and Volta regions
of Ghana. Nsiho production involves dehulling or polishing of maize grains which are then
steeped for 48 h and milled into a moist meal. This is then kneaded with water into dough and
left to ferment spontaneously for up to 24 h. The dough is pre cooked, moulded into balls,
wrapped in corn husk and steamed for about 1-2 h.
Traditional processing of foods such as nsiho and other types of kenkey is an important socio-
economic activity which serves as a livelihood for many families. However, many problems
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are encountered during the traditional processing of foods which need to be addressed to
improve the activities of the traditional food processors.
Problems encountered during the traditional processing of foods in Ghana include, the time
consuming nature of the process, lack of proper hygienic and sanitary practices, contamination
of products, low production efficiency, uneconomic utilization of energy sources and
unorganised and scattered nature of the areas of manufacture. Other problems include
improper and ineffective organisation of production schedules, lack of proper recruitment and
training of personnel, lack of proper specification of raw materials and rudimentary packaging
of products compromising of good manufacturing practices (Lartey, 1975; Amoa-Awua et al.,
2007). According to Halm et al., (2004), indigenous maize processing industries need the
injection of suitable scientific and technological know-how in every facet of their operations.
In addition key areas for ensuring compliance with Good Manufacturing Practice (GMP) have
been identified by Amoa-Awua et al., 2007.
1.3 RATIONALE FOR THE STUDY
With increasing urbanisation, there is a gradual shift from a traditional based society to a
modern society in several African countries. As a consequence some successful attempts have
been made towards upgrading traditional processing technologies, including the production of
Ga and Fante kenkey.
Also, a lot of literature has been published on kenkey produced from whole maize i.e Ga and
Fante kenkey based on their in-depth scientific studies. These studies have formed the basis
for continual attempts to improve and upgrade the production of quality Ga and Fante kenkey
including the use of starter culture and implementation of quality assurance measures.
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It is only currently that such detailed studies are being carried out on kenkey produced from
dehulled or polished maize and which will form a basis for upgrading the potential of nsiho. It
is in this regard that this work has been carried out to identify the dominant microorganisms
involved in nsiho fermentation with a view to develop starter cultures for the production
process.
The development of starter culture will optimise the process of nsiho fermentation and provide
a means for controlling the quality of the product leading to stable organoleptic characteristics
and also eliminate the quality risks inherent in traditional nsiho production. With the use of
starter culture, it is expected that fermentation time will be significantly reduced and quality
enhanced for mutual benefit of the producers and consumers in Ghana and the Diaspora.
1.4 MAIN OBJECTIVE
To develop a starter culture for the fermentation of dehulled maize grains into nsiho
1.4.1 SPECIFIC OBJECTIVES
To isolate, characterise and identify the lactic acid bacteria and yeast responsible for
the fermentation of dehulled maize into nsiho
Determine the technological properties of the dominant lactic acid bacteria and yeasts
involved in nsiho fermentation.
To investigate the antimicrobial property of nsiho against common enteric pathogens
during the steeping of dehulled maize grains
Develop starter culture from lactic acid bacteria and yeast for control fermentation of
dehulled maize in order to assure the quality of nsiho
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2.0 LITERATURE REVIEW
2.1 Maize Grain
Maize (Zea mays) is a major staple crop in Ghana, and it is vital to the survival and food
security of a large segment of the population, contributing over 55 % of the total energy intake
(Sefa-Dedeh and Mensah, 1989). It is also an important component of poultry and livestock
feed and to a lesser extent, a substitute in the brewing industry. Maize is an important
commodity in West Africa sub-regional trade, particularly between Ghana, Burkina Faso,
Mali, Togo and Niger through mainly informal trading.
Maize which is ranked the third most abundant cereal crop produced worldwide was originally
cultivated in Central America as far back as 4,500 BC (Mc Gee, 1984). Maize was introduced
to Ghana around the 15th century by the Portuguese (La Anyane, 1956). Nutritionally, it
contains about 9.8 % protein, 4.9 % fat, 1.4 % ash, 2 % crude fibre, and 63.6 % carbohydrate
(Alais and Linden, 1991). Like most cereals it is limiting in the amino acid lysine as well as
tryptophan and contains anti-nutrients such as phytates.
2.2 Some Ghanaian Traditional Food Products Made From Maize
Maize utilization in Ghana is mainly in the form of whole maize dough, refined maize dough,
and roasted corn or by cooking the whole maize. Whole grains may be processed into dry
grits, or roasted into a meal or fermented for use in the making of maize beverages (Sefa-
Dedeh, 1993). The bulk of the maize produced is consumed in the form of fermented products.
Fermentation of maize in Ghana can be described as an indigenous technology that has been
transferred from one generation to the other (Sefa-Dedeh, 1993). Many authors (Whitby 1968;
Dovlo 1970; Sefa- Dedeh, 1993) have reviewed that maize is processed into different forms
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such as semi solid products like banku, kenkey and liquid products like koko. The production
of banku, fomfom and koko will be described briefly but a more detailed description for
kenkey; the principal product prepared from fermented whole dough and widely consumed in
Ghana.
2.2.1 Fomfom
This product is popular in the Western region of Ghana. The indigenous procedure for
processing maize into fomfom is illustrated in Fig 2.1. Fomfom is a stiff porridge similar to
kenkey in consistency but made from dehulled maize. The maize is dehulled and milled. The
milled dough is mixed with water and an inoculum of old dough is added to shorten
fermentation period from 24 h to 6-9 h. The fermented dough is shaped into balls with holes
made in the centre. The balls are cooked twice in boiling water. After each boiling, the balls
are pounded in a mortar. After the second pounding, the mass is moulded into balls and
packaged in banana leaves ready for consumption (Johnson and Halm, 1998).
2.2.2 Kokui (Ewe kenkey)
This maize meal is made by sifting to remove some of the bran. A portion of this is gelatinised
(known as aflata) and mixed with the remaining ungelatinised portion. The mixture of aflata
and the raw dough are left to ferment for about 8 h. Balls are made from this mixture and
steamed
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Dry Maize
Washing (Optional)
Dehull, Winnow & Wash
Steep, Mill & Make Dough (Steeping for 24h)
Fermentation (8-24h)
Mould dough into balls
1st Cooking & 1st Pounding
2nd Cooking 2nd Pounding
Mould & Package using banana leaves
FOMFOM
Fig 2.1: Production of Fomfom
Source: Johnson and Halm, (1998)
.
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2.2.3 Akpiti (Ga), Bamfobisi (twi) or Amo kaklo (Ewe)
The preparation of this product, known by different names among the different ethnic groups
is similar to kokui preparation except that sugar is added to the aflata /dough mixture and is
left to ferment for 6-8 h. Balls are made from this mixture which are deep fried and served as
snacks (Whitby 1968; Dovlo 1970).
2.2.4 Abolo
Depending on the ethnic group the maize is rinsed with water, ground and sifted to remove the
bran. This is then reconstituted with water to form unfermented dough which is cooked to
gelatinise (aflta). A mixture of cooked and uncooked dough is allowed to ferment for about 8
h and depending on which tribe; it is either cooked (Ga) or steamed (Ewe) (Whitby 1968;
Dovlo 1970).
2.2.5 Banku
Banku is normally consumed along the coastal regions of Ghana. Fermented dough is made
into slurry with water and stirred till cooked into a semi- solid consistency. This is then
moulded into balls and eaten with sauce (Whitby, 1968, Owusu Ansah et al., 1980).
2.2.6. Koko
Koko is a thin gruel used by some as a weaning food for infants and consumed as breakfast
porridge all over the country. It is made by adding a large volume of water to fermented whole
maize dough to form slurry which is cooked by boiling into a thin gelatinous porridge. It can
also be prepared by adding large quantity of water to milled steeped maize. The mixture is
strained to get rid of chaff and the slurry left overnight to settle and ferment. The supernatant
water is decanted and the slurry used to prepare koko (Halm et al., 1996).
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2.2.7 Kenkey
Kenkey is a principal and popular product prepared from fermented whole maize dough. It is a
stiff gruel or dumpling from fermented maize dough which is wrapped in leaves and boiled. It
is also a sour tasting food with moisture content between 62-68 %, pH of 3.7 and a shelf life of
about 3-4 days with no refrigeration.
There are two main types of kenkey, the Ga kenkey locally called komi and the Fanti kenkey
known as dokono. The difference lies in some processing aspect such as the packaging
material for Ga kenkey which is maize husks, while that of Fanti kenkey is the plantain leaf.
Salt is added to the Fanti kenkey. Several authors (Christain, 1966, 1970; Whitby, 1968;
Dovlo, 1970; Nyarko-Mensah and Muller, 1972; Plahar and Leung, 1982; Sefa-Dedeh and
Plange, 1989; Halm et al., 1993, 1996, and 2004) have described the various traditional
methods of kenkey production. It involves various unit operations such as cleaning, steeping,
milling and doughing, dough fermentation, aflata preparation, mixing of aflata and raw
dough, moulding and packaging and cooking into kenkey (Fig 2.2).
2.2.7.1 Cleaning
The maize is cleaned by winnowing to remove chaff by stream of air, hand picking to remove
stones, damaged grains, insects, and debris and maize cobs. Sedimentation is also used where
the maize is poured into a big basin of clean water and stirred with a wooden ladle to allow
mature and quality grains to settle and less dense immature grains, insect damaged and
mouldy grains settle at the top. The latter is scooped off using baskets or sieve and discarded
or used as animal feed (Lartey, 1967, Ofosu, 1971).
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Maize kernels
Cleaning
Steeping (24-48h)
Milling
Doughing
(Moistening with water)
Fermentation (3 d) Fermentation (2d)
Pre-cooking (1/3 of dough) Pre-cooking (2/3 of dough + salt)
Mixing of precooked dough with Mixing of precooked dough
remaining fresh dough with remaining fresh dough
Moulding and packaging Moulding and packaging
(in plantain leaves) (in maize husks)
Cooking (3h) Cooking (3h)
Fanti Kenkey Ga Kenkey
Fig. 2.2: Production of Ga and Fanti kenkey
Source: Halm et al., (1996)
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2.2.7.2 Steeping
Cleaned maize grains are steeped in tap water in steeping tanks for one to three days
depending on the moisture content, variety and hardness of the maize used. Steeping allows
imbibition of water into the grains which facilitate increase of kernel size, facilitate size
reduction and condition the grain for smooth milling (Akingbala et al., 1987; Nche et al.,
1996). Lactic fermentation have been reported to begin during steeping and moulds present on
the maize grains are inhibited and disappear after steeping (Sefa –Dedeh, 1993; Halm et al.,
1993; Jespersen et al., 1994).
2.2.7.3 Nutritional Aspect of Kenkey
The nutritional value of the maize product kenkey is basically dependent on the variety of
maize from which it is made and the processing technique applied in production. Processing
methods such as soaking, packaging materials, cooking and fermentation may either reduce or
increase one nutrient depending on the nature of the nutrient been considered (Ankrah, 1972).
Calcium losses occur during kenkey production, phosphorus content, however, increases but
due to anti-nutritive factors such as phytic acid which bound this mineral, there is a reduction
in bio-availability of both phosphorus and calcium (Bediako-Amoa and Muller, 1976).
On a dry basis, the proximate composition of Ga kenkey is approximately 8.9-9.8 % protein,
1.3-3.2 % fat, 0.5-1.9 % ash, 10.6-78.6 mg/100g calcium, 202.4-213.8 mg/100g phosphorus,
6.5-12.6 mg/100g iron and 74.3-87.1 % total carbohydrates (Halm et al., 2004).
2.3 STARTER CULTURE
A starter culture may be described as a preparation containing high numbers of viable
microorganisms, which may be added to accelerate the fermentation process and to bring
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about desirable changes in a food substrate. Being adapted to the substrate; a typical starter culture
facilitates improved control of a fermentation process and predictability of its product (Holzapfel,
2007). Starter cultures are selected specifically for a substrate or raw material e.g. milk, meat,
cereals, legumes, roots and tubers, etc. Microorganisms used in starter cultures include
moulds, yeasts and bacteria.
2.3.1 Moulds
Moulds play a minor part in fermented foods of Africa. Foods such as Miso (prepared from
rice and soy beans with Aspergillus oryzae and yeasts), Tempe (prepared from soy beans with
Rhizopus oligospos) and soy sauce or Shoyu (prepared from soy beans and wheat by a mixed-
culture fermentation with Asp. oryzae, yeasts and lactobacilli) are typical examples of
traditional Asian foods produced on an industrial scale and well known worldwide (Ebine,
1989; Fukushima, 1989). In Europe, traditional mould ripened foods are mainly restricted to
blue-mould (Penicillium roqueforti) and white-mould (Penicillium camemberti) cheeses and
mould-ripened fermented sausages (Leistner, 1990).
2.3.2 Yeasts
Plant materials containing fermentable sugars provide suitable substrates for yeasts species of
Saccharomyces, Candida, Torula, Hansenula, etc. These yeasts, especially Saccharomyces,
are typically associated with spontaneous alcoholic fermentations such as African opaque
beers and palm wine. Generally, these are mixed strain fermentations, as exemplified by the
combined lactic and alcoholic fermentation of palm wines and most opaque cereal beers. For
the industrial production of Western style beers and even traditional African beers, selected
strains of Saccharomyces cerevisiae are used. Jespersen et al., (1994), isolated a mixed flora
comprising Candida, Saccharomyces, Trichosporon, Kluyveromyces, and Dabaryomyces
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species from raw maize, during steeping and early phase of fermentation of maize dough
fermentation for kenkey production.
2.3.3 Bacteria
Among the bacteria associated with food fermentation lactic acid bacteria (LAB) are of
predominant importance. This is especially true for Africa, probably the continent with the
largest spectrum and richest variety of lactic fermented foods. Lactic acid bacteria are basic
starter cultures with widespread use in the dairy industry for cheese making, cultured butter
milk, cottage cheese and cultured sour cream. It is also widely used in cereal fermentation in
Africa (Jay, 1986; Holzapfel 2002). Therefore, identifying and providing a practical means of
using appropriate starter culture is advantageous due to competitive role of microorganisms
and their metabolites in preventing growth and metabolism of unwanted microorganisms.
2.4 Attributes of Starter Culture
Starter culture improved shelf-life; enhanced inhibition or elimination of foodborne pathogens;
improved sensory quality (taste, aroma, visual appearance, texture, consistency); reduced
preparation procedures (reduction of cooking times and lower energy consumption); improved
nutritional value („upgrading‟) by degradation of antinutrition factors. It also improves protein
digestibility and bio-availability of micronutrients; biological enrichment, e.g. through the
biosynthesis of vitamins, essential amino acids and proteins, improved toxicological safety by
degradation (detoxification) of toxic components such as linamarin in cassava; and probable
degradation of mycotoxins (Holzapfel, 1997; 2002).
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2.4.1 Desirable Properties of Starter Culture
A starter culture is used to improve a fermentation process, such as lactic, alcoholic or alkaline
fermentation. The use of „back-slopping‟to start a new batch resembles the principle of starter
cultures in an empirical sense.
The primary consideration before introducing starter cultures for traditional food
fermentations is to significantly contribute to an improvement of processing conditions and
product quality with respect to:
rapid or accelerated acidification or alcohol production,
an improved and more predictable fermentation process,
desirable sensory attributes,
improved safety and a reduction of hygienic risks.
According to Holzapfel (1997), traditional procedures, back-slopping, adaptation and the
particular environment have contributed to the selection of a microbial population typical of a
particular fermentation process. This has therefore necessitated for the study of interactions
between single and mixed strain cultures from traditional products, both under defined
conditions. Food substrates, with respect to: competitive behaviour, viability and survival,
antagonism against pathogens and spoilage microbes, rates of acid or alcohol production,
sensory changes, and, also taking into account the type of primary metabolites formed (e.g.
lactic isomers), degradation of antinutrition factors.
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Table 2.1: Mixed starter cultures applied in the production of traditional acid- and acid-alcoholic
fermented gruels
Product Country Raw materials Inoculum (starter) Storage conditions Microorganisms
Mawe Benin maize previous batch active fermentation heterofermented
(sourdough) lactobacilli and yeasts
Mahewu Southern Africa maize small portion of whole- (?) mainly heterofermented
(wheat flour) lactobacilli (yeasts)
Kocho Ethiopia ensete fermenting kocho pit LAB, yeasts
( flour) (false banana)
Pito Ghana, sorghum “Inoculation belt” drying Lactobacilli, yeast
Source: Holzapfel (2000).
2.5 Starter Cultures in Africa Cereal Fermentation
The basis of the development of starter cultures is the ability to control the specific
microorganisms or the succession of microorganisms that dominate the microflora of foods
(Caplice and Fitzgerald, 1999). Adaptation to the substrate allows a typical starter culture to
gain improved control of a fermentation process, particularly the initial phase, and the
predictability of fermentation products (Holzapfel, 1997). Identifying and providing a
practical means of using starter cultures is advantageous due to the competitive role of
microorganisms and their metabolites in preventing growth and metabolism of unwanted
microorganisms. A strong starter may reduce fermentation times, minimize dry matter losses,
avoid contamination with pathogenic and toxigenic bacteria and moulds, and reduce the risk of
incidental micro-flora causing off-flavours in foods (Haard, 1999). The challenge remains
however, to improve fermentations without losing other desirable traits or introducing
accidentally, undesirable characteristics.
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Strains of lactic acid bacteria and yeasts have been successfully used as starter cultures in a
number of indigenous fermented foods. The following describes the use of starter cultures in
selected African foods.
2.5.1 Use of Starter Cultures in Maize Fermentation
Using combined inocula of Saccharomyces cerevisiae and Candida kefyr, Nyarko and Danso
(1991), found that the acceptability of fermented maize dough was significantly improved
over the non- inoculated naturally fermented maize dough. Nche et al., (1994) were able to
accelerate the fermentation of maize dough for kenkey production from two days to 24 h using
a back-slopping inoculum. Although a pH drop from 5.65 to 3.79 was achieved within 24 h,
sensory evaluation indicated poor acceptability of the textural characteristics of the dough for
kenkey production due to the omission of the soaking step practised traditionally. Maize
dough prepared from the accelerated fermented dry milled flour had inferior gelatinisation and
pasting properties compared to the traditionally prepared dough. Soaking or steeping of cereal
grains in water prior to wet milling, softens the grain endosperm. The penetration of water into
the interior of the kernels during steeping requires hours in an ambient temperature and
simultaneous fermentation occurs by fermenting microorganisms, which originate from the
surface of the kernels or other sources such as the steeping vessel (Odunfa, 1985). The
resulting starchy material from wet grinding carries the sour flavour, which has become an
essential and desired element in local foods cooked from the fermented slurry or dough
(Campbell-Platt, 1987).
Use of six strains of L. fermentum and one strain of yeast, S. cerevisiae, showed that the
typical final pH of 3.7 could be attained within 24 h of dough fermentation instead of 48 h as
observed with spontaneous dough fermentations (Halm et al, 1996). The organoleptic
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acceptability of kenkey and koko, the most popular traditional products, prepared from maize
dough fermented with starter cultures was in most cases not significantly different from those
prepared from traditional spontaneously fermented dough. Poorer textural characteristics of
kenkey produced from 24 h fermented dough with starter cultures contributed to the
unacceptability of this product by a trained panel of judges. Halm et al, (1996), also suggested
that other enzymatic and biochemical changes may be important in the spontaneous
fermentation process that contributes to the development of texture. The sour porridge, koko
was however acceptable when prepared from dough fermented with starter culture for 24 or 48
h. The authors attributed this to the less important role texture may have played in the
acceptability of koko, it being a thin slurry, in relation to the stiff dumpling-like texture of
kenkey.
2.5.2 Use of Starter Cultures in Ogi Preparation
Akinrele (1970) showed that „Ogi‟ produced from a mixed culture of Lactobacillus and
Acetobacter enriched the nutrient quality by increasing the concentrations of riboflavin and
niacin above that found in both the unfermented grain and the „Ogi‟ produced by traditional
spontaneous fermentation. A mixed culture inoculum of L. plantarum, Lactococcus lactis and
Saccharomyces rouxii was developed for cultured „Ogi‟ production, which resulted in
increased rate of souring of the dough (Banigo et al., 1974). Sanni et al., (1994), found higher
levels of ethanol in spontaneously fermented Nigerian „Ogi‟ as compared to samples using
inocula of lactic acid bacteria. Using fifty mutants from L. plantarum and seven mutants from
yeast strains selected from cultures capable of over producing lysine, twelve- and three-fold
increases, respectively, in lysine production were observed in „Ogi‟ (Odunfa et al., 1994).
Starter culture strains of lactobacilli isolated from local fermented foods with strong
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antibacterial activity were used in the production of „Dogik‟, an improved „Ogi‟ with
potentials for use in diarrhoea control (Olukoya et al., 1994). „Ogi‟ prepared by fermentation
with L. plantarum as starter culture decreased the pH from 5.9 to 3.4 within 12 h compared to
2-3 days required in the normal traditional process (Sanni et al., 1994). High increases in
levels of lysine and methionine were also observed in „Ogi‟ prepared from dehulled maize
grains inoculated with mixed starter cultures of Saccharomyces cerevisiae and Lactobacillus
brevis (Teniola and Odunfa, 2001). The best tasting „Ogi‟ comparable to traditionally
fermented „Ogi‟ was achieved with single starter cultures of L. brevis.
2.5.3 Use of Starter Cultures in Uji Preparation
The ability of pure lactic cultures isolated from naturally fermenting „Uji‟ mash and pure
cultures of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus
and Lactobacillus delbruecki to ferment „Uji‟ was investigated by Mbugua and Ledford,
(1984). They found that most bacterial strains failed to successfully ferment sterile or heat-
treated „Uji‟ slurries as evidenced by poor acid formation. They attributed this to the absence
of symbiotic relationships in sterile media, usually present in mixed bacterial populations, as
well as the destruction of thermolabile factors and changes in the isolated organisms during
the sub-culturing process. „Uji‟, fermented by mixed native „Uji‟ bacteria was more
organoleptically acceptable than isolated starter culture of L. bulgaricus or S. thermophilus. In
another study, Masha et al. (1998) compared in laboratory trials the fermentation of „Uji‟
using a starter culture of lactic acid bacteria ( L. plantarum, L. brevis, L. buchneri, L.
paracasei and Pediococcus pentosaceus), using the method of backslopping (inoculum from a
previous fermentation) and by spontaneous means at 30ºC. They found that the lowest pH of
3.5 was attained with the lactic acid bacteria starter culture fermentation while viscosity of
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„Uji‟ was only marginally affected by method of fermentation. The aroma profile of „Uji‟
fermented with lactic acid bacteria was characterised by high concentrations of acids (
hexanoic, octanoic and nonanoic) and some alcohols ( 1-propanol, 1-hexanol, 1- nonanol and
2- undecenol), spontaneously fermented samples were shown to have high concentrations of
esters ( ethyl butanoate, hexyl acetate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate and
ethyl nonanoate) other alcohols ( ethanol, 1- butanol, 3-methyl- 1-butanol and 2-methyl-1-
propanol) and acids ( acetic and heptanoic acid), while the backslopping method of
fermentation was characterised as having low concentrations of all volatiles identified.
Unfermented „Uji‟ was characterised as having mainly high levels of aldehydes (pentanal,
hexanal, heptanal, nonanal, (E)-2-heptenal and (E)-2-octenal) and other compounds (2-
heptanone, 2-pentyl furan, 1-octen-3-ol and isopropyl alcohol).
2.5.4 Use of Starter Cultures in the Production of Mawe
Single starter cultures of L. fermentum and L. brevis were found to be effective in fermenting
sterile „Mawe‟ suspensions to produce porridge with similar acidity levels as the naturally
fermented „ Mawe‟ (Hounhouigan et al., 1999). Starter cultures of only the yeasts, C. krusei
and S. cerevisiae produced „Mawe‟ with high pH (5.6 and 5.5 respectively) and low titratable
acids expressed as percentage lactic acid (0.05 and 0.06, respectively). Results of sensory
evaluation showed that the porridges produced with „Mawe‟ fermented with starter cultures
had less flavour than the traditional commercially produced „Mawe‟ porridge.
2.5.5 Use of Starter Culture in Mahewu Production
The traditional spontaneously produced „ Mahewu‟ is considered undesirable because it
involves a long fermentation time (about 36 h), proceeds too irregularly, permitting the
development of undesirable bacteria resulting in undesirable off-flavours from secondary
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fermentations (van der Merwe et al., 1964). For these reasons considerable research has been
devoted over the years to the use of starter cultures to produce „Mahewu‟ of consistently high
quality and in relatively shorter times of 8 to 12 h (Schweigart and Fellingham, 1963, van der
Merwe et al., 1964, Schweigart, 1970, van Noort and Spence, 1976, Hesseltine, 1979). The
most satisfactory acid producing starter culture was found to be L. delbruecki (van der Merwe
et al., 1964). Freeze or spray-dried „ Mahewu‟ cultures consisting mainly of L. delbruecki
could be used effectively as starter culture for bulk fermentations, however, a lag phase of 8 h
in contrast to 3 h with the use of fresh starter cultures was observed (Schweigart, 1971).
Table 2.2: Traditional Lactic Acid Cereal- Based Fermented Foods and Beverages
Product Country Cereal Nature of Fermented Product Product Use
Banku Ghana Maize, Cassava Dough Cooked Dough
Ben-saalga, koko Burkina Faso, Ghana Pearl millet Slurry Gruel
Bushera Uganda Sorghum, millet Slurry Beverage
Gowé (Sifanu) Benin Sorghum Cooked slurry Beverage
Hussuwa Sudan Sorghum Dough Dough-like food
Injera Ethiopia Tef, sorghum, corn, finger Batter Flat bread
millet, barley
Mahewu South Africa, Zimbabwe Maize Slurry Beverage
Mawé Benin,Togo Maize Dough Basis for ready-to-serve
foods
Ogi West Africa Maize,millet,sorghum Slurry Basis for ready-to-serve
foods
Poto poto Congo Maize Slurry Gruel
Togwa East Africa Maize Cooked slurry Beverage
Source: (Adams, 1998, Blandino et al., 2003)
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2.6 The Role of Lactic Acid Bacteria in Fermentation
Lactic acid bacteria have been defined as Gram positive, non- sporing, cocci, cocobacillus or
rods, dividing in one plane only with the exception of the pediococci, lacking catalase, devoid
of cytochromes, of non-aerobic habitats but aerotolerant, fastidious, acid tolerant, requiring a
fermentable carbohydrate for growth and converting glucose mainly to lactic acid or lactic
acid CO2, ethanol and/ or acetic acid (Sharpe 1979; Axelsson 1993).
Lactic acid bacteria are the most widespread of desirable microorganisms in food
fermentation. They are found in fermented cereal products, milk, cheese and fermented meats
(Campbell-Platt, 1987). Lactic acid bacteria convert the available carbohydrate to organic
acids and lower the pH of food. These acids as well as other flavour compounds which include
diacetyl, acetaldehyde and acetoin contribute to the desired taste and flavour of food
(Pederson, 1971).The low pH that is created also makes the food unfavourable for the growth
of spoilage and pathogenic bacteria (Adams and Moss, 1995).
Lactic fermentation can be divided into two broad categories distinguishable by the products
formed from glucose (Vandamme et al., Stiles and Holzapfel, 1997). These are referred to as
homofermentation and heterofermentation.
Homofermenters convert glucose 1,6-diphosphate using Embden Meyerhof (EM) pathway
(Dirar and Collins, 1972). The enzyme aldolase cleaves fructose 1,6 diphosphate between C3
and C4 to give the phosphate esters dihydroxyacetone phosphate and D-glyceraldehyde-3-
phosphate. The end product of this fermentation pathway is lactic acid (de Vries and
Stouthamer, 1968), (Figure 2.3).
The overall lactic acid fermentation pathway can be expressed as follows:
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1 Glucose + 2NAD+ + 2ADA +2Pi → 2Pyruvate + 2NADH + 2H+ +2ATP+
Pyruvate +NADH +H+ ↔ Lactate +NAD+
In heterolactic acid fermentation (Figure 2.3), the pentose phosphate pathway is used instead
of the EM pathway of glycolysis. This type of fermentation produces ethanol and carbon
dioxide in addition to lactic acid in the molar ratio 1:1:1.
The ethanol and the CO2 come from the glycolytic portion of the pathway. There two possible
ways by which ethanol is formed, (Holzapfel and Wood, 1995). Acetaldehyde formed by the
cleavage of pyruvate by pyruvate decarboxylase is reduced in the presence of alcohol
dehydrogenase to form ethanol. Ethanol can also be formed by a combination of acetyl coA
reduction to acetaldehyde followed by reduction of acetaldehyde by reduction of ethanol
dehydrogenase.
The overall reaction for the heterofermentation reaction can be expressed as follows:
Glucose +ADP +Pi → Lactic acid + Ethanol +CO2 + ATP
2.6.1 Microorganisms Involved in the Lactic Acid Fermentation
The lactic acid bacteria are rod shaped or spherical microorganisms. Their names derives from
the fact that ATP is synthesised through fermentation of carbohydrates, which yield lactic acid
as a major and sometimes the sole end –product (Vandamme et al., Stiles and Holzapfel,
1997).
The taxonomy of lactic acid bacteria is in a state of flux but the principal genera are
Lactobacillus, Pediococcus, Steptococcus, Leuconostoc, Carnobacterium, Enterococcus,
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Lactococcus, lactospaera, Oenococcus, Weissella, tetragenococcus and Vagococcus (Adams
and Nicolaides, 1997; Axelsson, 2004).
(a) (b)
Fig. 2.3: The path way for glucose dissimilation by homofermentative and heterofermentative
bacteria (Kandler, 1983)
Lactobacilli are Gram positive, catalase negative rods that often occur in chains. They can also
be coccoid. They grow poorly in air but better under reduced oxygen tension. Although those
in foods are typically microaerophilic, many true anaerobics exist. They are
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chemoorganotrophic and require complex media. Their metabolism is fermentative and at least
one of the carbon end products is lactate. Their optimum growth is 30-40 oC (Axelsson, 1993).
All members of the genera Pediococcus and Streptococcus are homofermentative, Gram
positive, catalase negatative cocci. Pediococcus divide in two planes at right angles to produce
tetrads of cells but sometimes only pairs of cells are seen. Single cell are rare and chains are
not formed. They are facultative anaerobes although some strains are inhibited on incubation
in air. They are chemoorganotrophic and require nutritionally rich media and a fermentable
carbohydrate (mainly mono- and diasaccharides). Glucose is fermented with the production of
acid but no gas. The major product is lactate. The optimum growth temperature is 25-40 oC.
Streptococci divide in one plane to produce chains (Hardie, 1986). The cells are spherical or
ovoid occurring in chains or pairs. They are facultatively anaerobic chemo-organotrophs
which require nutritionally rich media for growth and sometimes 5 % carbon dioxide. Growth
is generally restricted to 25- 45 oC (optimum 37 oC).
Leuconostoc are Gram positive, catalase negative cocci that are heterofermentative. The cells
are spherical or somewhat longer than broad when in or pairs. Sometimes short rods with
rounded ends and occurs in long chains. They grow slowly, producing small colonies that may
be slimy on media containing sucrose. They are facultative anaerobes and chemo-
organotrophic with obligate requirements for a fermentable carbohydrate as well as
nutritionally rich medium. Glucose is fermented with the production of D (-) lactate, ethanol
and gas. The optimum growth temperature is 20-30 oC (Garvie, 1986).
Carnobacteria are Gram positive, catalase negative, straight slender rods. They occur singly or
pairs and sometimes in short chains. They may or not be motile. They are non sporing
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chemoorganotroghs that are heterofermentative. They produce mainly L (+) lactate from
glucose and gas is produced by some species. They grow at 0 oC but not at 45 oC (optimum 30
oC). They differ from Lactobacilli in being unable to grow on acetate medium.
Enterococci were once a sub-group of the genus Streptococcus. The cells are spherical or
ovoid and occur in pairs or short chains. They are sometimes mobile by means of scanty
flagella. They lack obvious capsules, are facultative anaerobic and are chemoorganotrophs
with fermentative metabolism. A wide range of carbohydrates, including lactose are fermented
with the production of mainly L (+) lactic acid. Gas is not produced. They are gram positive
and grow at 10 oC and 45 oC with an optimum of 37 oC (Devriese et al., 1991).
Lactococci are Gram positive, catalase negative, non motile ovoid cells that occur singly, in
pairs or in chains. Endospores are formed. They are non motile and without capsules. They are
facultative anaerobes which can grow at 10 oC but not 45 oC with optimum temperature of 30
oC. They are chemoorganotrophs with fermentative metabolism. A number of carbohydrates
are fermented with the production of lactic acid but no gas (Schleifer et al., 1985).
Vagococci are Gram positive, non-sporing, spheres, ovals or short rods which occur singly, in
pairs or in short chains, some motile by peritrichous flagella. They are catalase negative and
chemoorganotrophic with a fermentative metabolism. They produce acid but not gas from a
number of carbohydrates. Glucose fermentation yields mainly L (+) lactate. They can grow at
10 oC but not at 45 oC with optimum growth at 25-35 oC.
Lactic acid bacteria differ with respect to the isomers of lactic acid that they produce. This is
determined by the specificity of the lactic dehydrogenases which mediate pyruvate reduction.
Some species contain only D-lactic dehydrogenase and hence form the L-isomer. Some
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species contain two lactic dehydrogenase of differing stereospecificity and form racemic lactic
acid (Vandamme et al., 1996; Stiles and Holzapfel, 1997).
2.7 Methods used for the identification of lactic acid bacteria
2.7.1Conventional Taxonomy
Lactic acid bacteria have been traditionally grouped and identified based on their biochemical,
physiological and morphological tests (Sharpe, 1979). Bottazzi (1988) classified LAB on six
physiological tests which included production of gas from glucose, hydrolysis of arginine,
growth and survival at 15, 45 48, 60 and 65 oC and tolerance of 4, 6, and 8 % NaCl. The use of
features such as the isomer of lactic acid produced, the comparative DNA base composition
and cell wall composition have been included in LAB identification schemes (Sharpe, 1979;
Kandler and Weiss, 1986). All the above features have been combined in a number of studies
for the isolation and identification of LAB from different environment (Garvie, 1984; Hastings
and Holzapfel, 1987; Schillinger and Lucke, 1987). The use of rapid identification systems,
such as the API (API systems S.A., La Balme Les Grotte, Montalieu, France) can examine
isolates using different carbohydrate fermentation characteristics.
2.7.2 Numerical Taxonomy
The application of numerical taxonomy as a technique to characterise bacteria is well
established and allows the stimulation examination of large numbers of phenotypic
characterisation test in an objective manner (Dykes, 1991). This is achieved by coding the
results of individual characteristics tests as either binary or numerical values, the combination
of which represents specific profiles characteristics of a particular strain. The principle of
numerical taxonomy has been applied to lactic acid bacteria from a number of sources
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including processed meats (Hastings and Holzapfel, 1987); wine (Dicks and Van Vuuren,
1988); Scotch whisky distilleries (Priest and Pleasant, 1988). These methods have proved to
be useful in the taxonomic status of isolates.
2.7.3 DNA Based Techniques
The analysis of the diversity of natural microbial populations relied on direct extraction,
purification and sequencing of 5S rRNA molecules from enrionmental samples (Theron and
Cloete, 2000). Determination of DNA base composition of LAB by electrophoresis is a well
known technique (Garvie, 1984). This technique has limited use in bacterial taxonomy since
the base composition of two biological unrelated organisms as determined by these methods
may be the same. However, examination of bacteria at the molecular level by DNA homology
technique is regarded as one of the most reliable indicators of natural relationships between
different strains (Dykes, 1991). This technique has widely been used for LAB taxonomy. It
lead to the creation of new genera such as Lactococus and Carnobacteria (Collins et al.,
1987).
DGGE is an electrophoretic separation method based on differences in melting behaviour of
double stranded DNA fragment (Fisher et al., 2003). DGGE exploits the fact that identical
DNA molecules, which differ by only one nucleotide within a low melting domain, will have
different melting temperatures. When separated by electrophoresis through a gradient of
increasing chemical denaturant (usually from amide and urea), the mobility of the molecule is
retarded at the concentration at which the DNA strands of low melt domain dissociate
(Webster et al., 2002). The branched structure of the single stranded moiety of the molecule
becomes entangled in the gel matrix and no further movement occurs. Complete strand
separation is prevented by the presence of a high melting domain, which is usually artificially
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created at one end of the molecule by incorporation of a GC clamp. This is accomplished
during PCR amplification using a PCR primer with a 5‟tail consisting of a sequence of 40 GC.
2.8 Anti-microbial Substances Associated with Lactic Acid Bacteria
2.8.1 Organic Acids and Low pH
Lactic acid fermentation is characterised by the accumulation of organic acids, primarily lactic
and acetic acids, and the accompanying reduction in pH (Berry et al., 1990). Acid production
is an efficient tool for inhibiting pathogenic and spoilage bacteria since organic acids produced
during fermentation has broad antibacterial activities (Piard and Desmazeaud, 1991;
Cherrington et al., 1991). Lactic acid, generated in situ, is traditionally used for improving
food safety and shelf-life (Adams and Hall, 1998). Levels and proportions of organic acids
produced depend on the species of the micro organism involved, the chemical composition of
the culture environment and the physical conditions encountered during fermentation (Fields
et al., 1981; Lindgren and Dobrogosz, 1990; Sanni, 1993).
The preservative action of acids may be partly due to the depression of internal (cytoplasmic)
pH (Russel, 1992). Undissociated acid molecules are lipophilic and pass readily through the
plasma membrane by diffusion. In the cytoplasm acid molecules dissociate into charged
anions and protons (Salmond et al., 1984). These cannot pass across the lipid bilayer and
accumulate in the cytoplasm, thus reducing the pH. The acidified cytoplasm in turn inhibits
metabolism, in particular the enzymes of metabolism (Krebs et al, 1983; Stratford and
Anslow, 1998).
A principal target for many antimicrobial compounds is the plasma membrane and an
alternative mechanism which may explain how acids act as preservatives is that they eliminate
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the proton motive gradient (Eklund, 1985). The selective permeability of the plasma
membrane to protons allows the cells to create a p H gradient and an electrical potential which
together form the proton motive force (Eklund, 1989). In the chemiosmotic theory, the energy
contained in the proton motive force is then used to drive the uptake of essential nutrients such
as amino acids (Bracey et al., 1998). The accumulation of charged acid particles in the
cytoplasm disrupts the proton motive force and prevents uptake of amino acids (Freese et al.,
1973).
2.8.2 Bacteriocins
Lactic acid bacteria are well- known for their production of antimicrobial proteins of peptides
collectively known as bacteriocins (Hugas 1998). These protein complexes (protein
aggregates, lipocarbohydrate proteins, glycoproteins) are active against Gram positive bacteria
and normally known to display a narrow range of inhibitory activity that affects closely related
species within Lactobacillaceae (Klaenhammer, 1998). Bacteriocins have been isolated from
fermented milk and dairy products (Litopoulou-Tzanetaki, 2000) and mahewu (Holzapfel,
1995).
The ability of many bacteriocins to inhibit some food-borne pathogens makes them attractive
as potential food preservation agents. The best characterised bacteriocin produced by lactic
acid bacteria is nisin. Nisin is produced by Lactococcus lactis subsp. Lactis and has been
available commercially in concentrated form since 1959 (Daeschel, 1989). Most micro-
organisms require an intact plasma membrane. Nisin is strongly attracted to phospholipids in
bacterial and liposomal membranes. Cationic nisin molecules initially interact by electrostatic
attractions with anionic membrane phospholipids. They reorient themselves in the membrane
such that they form non-selective pores (Sahl et al.,1995). The net result is that nisin makes
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the cytoplasmic membrane permeable which causes the release of accumulated amino acids
from the cells as well as membrane vesicles of sensitive bacteria by leakage (Hurst, 1981;
Kalchayanaad et al., 1994).
Nisin exhibits broad spectrum inhibitory activity against Gram positive bacteria, including
spore-forming bacteria (Klaenhammer, 1998). It inactivates thermophilic spoilage
microorganisms in canned goods (Stevens et. al., 1992; Maisnier-Patin et al., 1992). Nisin and
pediocin, a bacteriocin produced by Pediococcus species, have been shown to be effective in
controlling Listeria monocytogenes in white pickled cheese, skim milk, yoghurt and other
foods ( Montville and Winkowki 1997).
2.8.3 Hydrogen Peroxide
The antimicrobial effect of hydrogen peroxide is well documented, and it is produced by a
wide range of lactic acid bacteria which do not produce catalase but possess flavoprotein
oxidases which react with dioxygen (Condon 1987; Earnshaw 1992; De Vuyst and Vandamme
1994b). The use of oxygen as an electron acceptor is favoured as an additional energy
generated (Lindgren and Dobrogosz 1990). Because lactobacilli are catalases negative, H2O2
can accumulate in the cells and effect antimicrobial activity (Kandler and Weiss 1986). The
bacteriocidal effect is attributed to its oxidizing effect on the bacteria cell, and to the
destruction of basic molecular structure of the cell protein (Lindgren and Dobrogosz 1990).
H2O2 in some cases can be precursor for the production of potent agent such as superoxide and
hydroxyl radicals (Condon 1987).The lactoperoxidase system is an antimicrobial system that
occurs in milk oxidised by hydrogen peroxide to produce antimicrobial hypothiocynate and
thiocyanous acid. This system has been used successfully in extending the shelf life of raw
milk and cottage cheese (Earnshaw and Banks 1989).
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2.8.4 Carbon Dioxide
Carbon dioxide is produced by heterolactic fermentation and contributes to an anaerobic
environment that is toxic to most aerobic food microorganisms. Also, carbon dioxide in itself
has an antimicrobial activity because it creates partial pressure (Lindgren and Dodrogosz,
1990). Carbon dioxide is believed to accumulate in the lipid bilayer due to inhibition of
enzymatic decarboxylations causing disfunction of membrane permeability.
2.8.5 Diacetyl
Aside from being an important flavour and aroma contributor to fermented products and
especially in butter, diacetyl is an antimicrobial agent (Marshall 1987). This compound is
formed by the citrate fermentation via pyruvate and further metabolized anaerobically and
aerobically to diacetyl and acetoin (De Vuyst and Vandamme 1994). Diacetyl is an
antimicrobial agent needed in high amounts to be effective against only Gram negative
bacteria and yeasts .Inhibitory activity is by interference with arginine utilization, which is
achieved by a reaction with the arginine-binding protein of the susceptible organism (Jay and
Rivers, 1984).
Jay (1982) reported that yeast and Gram negative bacteria were more sensitive than the non-
lactic acid bacteria and Gram positive bacteria to diacetyl. Lactic acid bacteria and the
clostridia seemed to be sensitive to its inhibitory activity.
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Table 2.3: Metabolic products of lactic acid bacteria which exhibit antimicrobial properties
Product Main target organisms
Organic acids
Lactic acid Putrefactive and Gram- negative bacteria, some fungi
Acetic acid Putrefactive bacteria, clostridia, some yeasts and fungi
Hydrogen peroxide Pathogens and spoilage organisms in protein- rich foods
Low- molecular metabolites
Reuterin (3-OH-propionaldehyde) Wide spectrum of bacteria, moulds and yeasts
Diacetyl Gram- negative bacteria
Fatty acids Different bacteria
Bacteriocins
Nisin Some LAB and Gram-positive bacteria
Other Gram- positive bacteria, inhibitory spectrum accord-
ing to producer strain and bacteriocin type
Source: Holzapfel et al., (1995)
2.9 Lactic Acid as Probiotics
Probiotics are viable microbial food supplement containing single or mixed cultures of live
microorganisms that beneficially affects the host (human or animal) by enhancing or
maintaining a desirable microbial balance in the gut leading to improving the health status of
the host (Salminen et al., 1998). They act by reducing harmful enteric microorganisms to
maintain a healthy gut micro flora for potential health benefit. Metchnikoff (1908) observed
that feeding of probiotic cultures prevented or treated disease. Probiotic cultures are consumed
in foods or capsules or facilitated by ingestion prebiotics (compounds that enhance the
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proliferation of beneficial indigenous bacteria), Okamura et al.,(1986) demonstrated with
tissue culture infection assay that administration of Bifidobacterium infantis prohibited
invasion and intracellular multiplication of S. flexneri. Fuller (1992) observed that probiotic
cultures have inhibitive or antagonistic effects against almost all foodborne pathogens
including Salmonella, Shigella, E. coli, Campylobacter, Clostridium, Yersinia, Vibro and
Candida. Saavedra et al (1994) showed that supplementing infant formula with
Bifidobacterium bifidum and Streptococcus thermophilus reduced the incidence of acute
diarrhoea and rotavirus shedding infants. Bernet et al., (1994) observed that consumption of a
greater number of lactobacilli provided increased protection against cell association by
enterotoxigenic and enteropathogenic E. coli, S. typhimurium and Y. pseudotuberculosis.
Intestinally derived lactobacilli and bifidobacterium predominate as probiotics (Hughes and
Hoover, 1991) and act by establishing themselves in the human gastrointestinal tract in
proportionally high numbers, thus protecting the gut against invasive pathogenic agents that
cause foodborne intestinal infection like diarrhoea. Hence regular consumption of foods
containing probiotics has a strong potential to help maintain beneficial and stable intestinal
microflora that promotes intestinal health especially in compromised or under developed gut
flora such as the elderly, infants and patients treated with antibiotics or chemotherapy.
2.10 Prebiotics
Prebiotics are non-digestible food ingredients that have a beneficial effect on the host by
selectively stimulating growth of health-promoting bacteria (Desai et al., 2004). They are
assimilated by beneficial bacteria such as Bifidobacteria hence improving their growth activity
leading to an enhanced intestinal balance. For a food to qualify as a prebiotic, it has to be non-
digestible by human enzymes; has to undergo selective fermentation by potentially beneficial
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bacteria in the colon; should cause an alteration in the composition of the colonic microbiota
towards a healthier composition and all these changes should lead to a beneficial health to the
host (Pariyaporn et al., 2003). Examples of prebiotic substrates include inulin, lactulose,
fructo-oligosaccharides, sugar alcohols such as lactitol and xylitol (Salminen et al., 1998).
Most of these are obtained from natural sources or synthesized naturally from sucrose
(Roberfroid, 1998).
2.11 Role of Yeasts in Maize Fermentation
Yeasts are unicellular fungi as compared to moulds which are multicellular. They differ from
bacteria by their larger cell size and their oval, elongated, elliptical or spherical cell shape. By
Kurtzman (1994) definition, yeasts represent a unique group of fungi characterised by
vegetative growth that is predominantly unicellular and by the formation of sexual state which
are not enclosed in fruiting bodies. Yeasts have been traditionally classified for several years
based on their physiological, morphological and biochemical criteria. These conventional
methods are found to be laborious and time consuming as well as being unreliable in their
ability to differentiate at species level (Casey et al., 1990). To alleviate the problems
associated with the classical methods, DNA- based methods have been develop in an attempt
to simplify the identification of yeasts isolates. Nucleic acid techniques such as DNA and PCR
are sensitive and have the advantage of not been influenced by the environmental conditions
(Kurtzman 1994).
Yeasts are known to produce a wide range of compounds such as organic acids, esters,
alcohols, aldehydes, lactones and terpenes (Stam et al., 1998). Several authors (Obiri-Danso,
1994; Hayford and Jespersen, 1999; Hayford and Jakobsen, 1999; Jespersen, 2002) have
found Candida krusei and Saccharomyces cerevisiae as the dominant yeast during steeping
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and maize dough fermentation. Jespersen et al, (1994), found a mixed flora comprising
Candida, Saccharomyces, Trichoporon, Kluveromyces and Debaryomyces species from raw
maize during steeping and the early stages of fermentation. The presence of large numbers of
yeasts (Candida krusei and Saccharomyces cerevisiae) in maize dough fermentation either in
pure cultures or combination have been found to influence the organoleptic and structural
quality of maize dough (Nyarko and Obiri-Danso, 1992; Jespersen et al., 1994).
2.11.1 Saccharomyces cerevisiae
The yeast S. cerevisiae belongs to the genus Saccharomyces which is one of the largest genus
under the phylum Ascomycota. It is perhaps the most useful yeast, which has been very
instrumental in baking and brewing since ancient times. The cells of S. cerevisiae are round to
ovoid, with a diameter of 5–10 micrometres and reproduce vegetatively by multilateral
budding. All strains of S. cerevisiae can grow aerobically on sugars such as glucose, maltose,
and trehalose but not on lactose and cellobiose. (Beneke and Stevenson 1987; Kurtzman and
Fell, 1998).
Early classification allocated to genus Saccharomyces, a large number of species which
constituted a somewhat heterogenous mixture. In current classification system baker‟s yeast,
brewer‟s yeasts, wine yeasts, champagne yeasts are all considered to be strains of
Saccharomyces cerevisiae (Kurtzman and Fell, 1998).
2.11.2 Candida krusei
Candida krusei belongs to the genus Candida, a member of the group of imperfect yeasts
(anarmorphic) of the phylum Ascomycota. Vegetative reproduction is by multilateral budding
and the cells occur singly, budding and in chains. The cells are ellipsoidal and elongate, and
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pseudohyphae and septate hyphae may be present (Kurtzman and Fell, 1998). In the new
edition of “The Yeast” Candida krusei is discussed under Issatchenkia orientalis and this is
because Candida krusei is considered to represent the anamorphic form of this species, as the
type strains of both species including other isolates showed significant (93 % - 100 %) DNA
base sequence complementary. The assimilation pattern of Candida krusei is quite
homogenous and there is no difference between the two pathogenic organisms and organisms
from natural fermentation of maize dough. Candida krusei is not easily differentiated from
other phenotypically similar species such as Candida valida (Kreger van Rij, 1984;
Kurtzman and Fell, 1998).
Many authors (Halm et al., 1993; Jespersen et al; 1994; Hayford and Jakobsen 1999) have
found Candida krusei as dominant yeast in many African fermented cereals such as maize and
sorghum, root crops like cassava (Amoa-Awua et al., 1997; Oyewole, 1990) and alcoholic
beverages such as palm wine in Ghana, sorghum beer in West Africa (Nout 1980, Konlani et
al., 1996a). This yeast is able to survive in acid fermented products due to its acid resistance
nature (Spicer and Schroder, 1980).
2.12 Technological Properties of Lactic Acid Bacteria
2.12.1 Exopolysaccharides
Lactic acid bacteria strains have been documented to produce expolysacharides (EPSs) that
have received a lot of attention due to their contribution to improvement of texture and
viscosity of fermented food products (Patricia et al., 2002; Savadogo et al., 2004). The lactic
acid bacteria produce EPS probably as a protective function in their natural environment such
as against desiccation, phagocytosis, phage attack, osmotic stress, antibiotics or toxic
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compounds (Patricia et al., 2002). The EPS may have a role in cell recognition, adhesion to
surfaces and formation of biofilms that facilitate colonisation to various ecosystems. This is a
beneficial attribute for probiotics in their endeavor to colonize the GIT. Health benefits have
been attributed to some exopolysaccharides. They have been reported to possess anti-tumor,
anti-ulcer, immunomodulating and cholesterol lowering effects (De Vuyst and Degeest, 1999).
Kazitawa and Ittoh (1992) reported an increase in B-cell dependent mitogenic activity induced
by slime material products from L. lactis subsp. cremoris; water soluble EPS from kefir grains
were shown to retard tumor. Kalui et al., (2009), reported the production of EPS ranging from
298.53 to 431 mg/l by L. plantarum and L. rhamnosus isolates from ikii, a spontaneously
fermented maize porridge. Spontaneously fermented products therefore have potential and it is
therefore necessary to assess them for production of EPS with an aim of tapping into the
health benefits associated with EPS.
2.12.2 Starch Hydrolysis
Blandino et al., (2003) indicated that the level of carbohydrate, some non digestible and
oligosaccharides decrease during cereal fermentation. Amylolytic lactic acid bacteria have
been isolated from cereal fermentation in tropical climates (Ga”nzle et al., 2008, Sanni et al.,
2002). Many authors have indicated the presence of lactic acid bacteria in some African
fermented products. Olasupo et al., (1996) isolated amylolytic lactic acid bacteria from
Ghanaian kenkey (fermented maize dough) and nono (Nigeria). Agati et al., (1998), found
amylolytic L plantarium strains from retted cassava in Nigeria and Congo respectively, while
amylolytic L. fermentum (strains Ogi E1 and Mw2) were isolated from mawe and ogi in
Benin. Hounhouigan et al., (1993b) reported some amylolytic lactic acid bacteria in mawe
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from Benin whiles Johansson et al., also indicated that amylolytic lactic acid bacteria
accounted for 14 % of the total lactic acid bacteria isolated from Nigerian ogi.
2.12.3 Proteolysis
Protein degradation during cereal fermentation is among the key phenomena that affect the
overall quality of product (Ga¨nzle et al., 2008). Overall, LAB plays a minor role in protein
hydrolysis (Wieser et al., 2008). Most cereal-associated LAB do not possess extracellular
proteinase activity and prefer peptides uptake rather than amino acids transport (Thiele et al.,
2003). Thus, primary proteolysis is exerted by the cereal endogenous enzymes, which are
activated by the low pH. Further hydrolysis of peptides into amino acids is then exerted by
intracellular peptidases of LAB in a strain-specific manner: the type and amount of released
amino acids depend on the fermenting strain (Di Cagno et al., 2002). Though LAB do not
influence the overall amount of hydrolysed proteins in respect to aseptically acidified cereal
doughs, they affect the pattern of hydrolysed products, increasing the amount of dipeptides
and amino acids (Di Cagno et al., 2002; Thiele et al., 2003). Studies conducted by Nanson and
Field (1984), showed that during the fermentation of cornmeal, the concentrations of available
lysine, methionine and tryptophan increased. In another study by Hamad and Field (1979),
fermentation was observed to significantly improve the protein quality and the level of lysine
in maize, millet and sorghum. Also, the tryptophan content of ogi, a millet based porridge
increased with fermentation (Mckay and Baldwin, 1990).
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3.0 MATERIALS AND METHODS
3.1 Study Area
The study was carried out in two towns in the Asuogyaman District in the Eastern Region of
Ghana, namely, Anum and South Senchi where dehulled kenkey is widely consumed.
3.2 Study Design
A brief field study involving informal interaction and administering of semi structured
questionnaires to producers, consumers and vendors was carried out. The study also involved
observation of the processing procedures and two experienced processors were selected from
each area for collection of samples.
3.2.1 Sample Collection and Preparation
Samples were aseptically collected from four processors at the various stages of processing
maize into nsiho. The samples collected were dehulled maize, steep water at interval of 0, 24,
and 48 h of steeping and fermenting dough at 0, 4, 8 and 12 h of dough fermentation. Samples
were transported in an ice chest with ice packs to the CSIR-Food Research Institute‟s
microbiology laboratory in Accra for microbiological and chemical analyses.
3.3 Chemical Analyses
3.3.1 Determination of pH
The pH of steep water was determined directly using a pH meter (Radiometer pHM 92.
Radiometer Analytical A/S, Bagsvaerd, Denmark) after calibration using standard buffers, and
fermenting dough was determined after blending with distilled water in a ratio of 1:1.
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3.3.2 Determination of Titratable Acidity
The titratable acidity was determined by the method described by Amoa-Awua et al., (2006).
For each sample (steep water and fermenting dough) 10 ml or 10 g of sample was made up to
200 ml with distilled water and 80 ml titrated against 0.1 m NaOH using 1 % freshly prepared
phenolphthalein as indicator. One millilitre of 0.1 N NaOH was taken as equivalent to
9.008x10-3 g lactic acid.
3.4 Microbiological Analyses
3.4.1 Enumeration of Microorganisms
3.4.1.1 Homogenization and Serial Dilution
For all solid samples, ten grams (10 g)were added to 90.0 ml sterile Salt Peptone Solution
(SPS) containing 0.1 % peptone and 0.8 % NaCl, with pH adjusted to 7.2 and homogenized in
a stomacher (Lad Blender, Model 4001, Seward Medical, England), for 30 s at normal speed.
From appropriate ten-fold dilutions 1 ml aliquots of each dilution was directly inoculated into
sterile Petri dish plates and the appropriate media added for enumeration and isolation of
microorganisms. All analyses were done in duplicate.
3.4.1.2 Enumeration of Aerobic Mesophiles
Aerobic mesophiles were enumerated by the pour plate method on Plate Count Agar medium
(Oxiod CM325; Oxoid Ltd., Basingstoke, Hampshire, UK). Plates were incubated at 30°C for
72h in accordance with the Nordic Committee on Foods Analysis Method (NMKL. No. 86,
2006).
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3.4.1.3 Enumeration of Lactic Acid Bacteria
Lactic acid bacteria were enumerated by the pour plate method on deMan, Rogosa and Sharpe
(MRS, Oxoid CM361) agar (De Man et al., 1960), pH 6.2 to which has been added 0.1 %
cycloheximide supplement to inhibit yeast growth and incubated anaerobically in anaerobic jar
with anaerocult A at 30 °C for 5 d.
3.4.1.4 Enumeration of Yeasts
Yeasts and moulds were enumerated by the pour plate method on Oxytetracycline-Glucose
Yeast Extract Agar (OGYEA), (Oxoid CM545; Oxoid Ltd., Basingstoke, Hampshire, UK) to
which OGYEA supplement was added to suppress bacteria growth. The pH was adjusted to
7.0 and incubated at 25 °C for 3-5 d in accordance with ISO 7954 (1987).
3.4.2 Isolation of Lactic Acid Bacteria
About 20 colonies of lactic acid bacteria were selected from a segment of the highest dilution
or suitable MRS agar plate. The colonies were sub-cultured in MRS broth medium and
streaked repeatedly on MRS agar until pure colonies were obtained.
3.4.3 Characterisation of Isolates
3.4.3.1 Microscopic Examination
Cell shape and arrangements were determined by phase contrast microscopy and the wet
mount technique. A drop of sterile distilled water was placed on a clean slide and a small
amount of the pure culture emulsified in it. A cover slip was placed on it and examined under
the microscope using the X40 magnification and oil immersion using the X100.
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3.4.3.2 Gram’s Reaction
Gram‟s reaction was determined using 3 % freshly prepared potassium hydroxide solution as
described by Gregersen (1978). The tip of cover slip was used to pick a pure colony of LAB
onto a clean microscopic slide and a drop of 3 % KOH was placed onto the colony on the
slide. The colony was mixed thoroughly with the solution using the cover slip and drawn for
the production of slime. Formation of a slime indicated Gram negative reaction and non-slimy
reaction indicated Gram positive reaction.
3.4.3.3 Catalase Reaction
A drop of 3 % freshly prepared hydrogen peroxide solution was placed on a clean glass slide
and a single colony of the pure culture picked and emulsified in the drop of hydrogen
peroxide. This was then observed for bubbles or effervescence resulting from the liberation of
free oxygen as gas bubbles. This indicated the presence of catalase in the culture and vice
versa.
3.4.3.4 Oxidase Test
Oxidase test was done using Identification Sticks (Oxoid Ltd., Basingstoke, Hampshire, UK).
The oxidase sticks were smeared on pure colonies and observe for colour change. Purple
colouration indicated positive results.
3.4.3.5 Growth at Different Temperatures
Two tubes containing MRS broth (Oxoid CM359) were inoculated with pure colony mass of
the test organism and incubated at 10 °C and 45 °C respectively for 72-96 h. Growths were
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determined by visual turbidity after the incubation period. This was repeated for all the
isolates.
3.4.3.6 Gas Production from Glucose
The ability to ferment and produce gas from glucose was assessed in MRS basal medium to
which glucose had been added. The medium was composed of peptone 10 g, yeast extract 5 g,
tween 80 1 ml, di-potassium hydrogen phosphate 2 g, sodium acetate 5 g, tri-ammonium
citrate 2 g, MgSO4.7H2O 0.2g, MnSO4. 4H2O 0.05g, 1 litre distilled water, pH 6.5, thus
without glucose and meat extract. The basal medium was dispensed in 5 ml amounts into
testtubes containing inverted Durham tubes and sterilized by autoclaving at 121 °C for 15
minutes. The glucose was prepared as 10 % solution and sterilized by filtration and added
aseptically to the basal medium to give a final concentration of 2 % (de Man et al., 1960). The
inoculated tubes were examined for the production of gas after 3 days of incubation.
3.4.3.7 Salt Tolerance Test
Salt tolerance test was done using MRS broth (Oxoid CM359) containing 6.5 % and 18 %
(w/v) NaCl with incubation period of 5 days at 30 °C. Growths were determined by visual
turbidity after the incubation period.
3.4.3.8 Growth at Different pH
Growth in MRS broth (Oxoid CM359) with pH adjusted to 4.4 using concentrated HCl and
9.6 using 0.1 N NaOH were determined by visual turbidity after 72 h of incubation at 30 °C.
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3.4.4 Identification of Lactic Acid Bacteria by Carbohydrate Fermentation Profiles
Isolates were tentatively identified by determining their pattern of carbohydrate fermentation
using the API 50 CH kit (BioMérieux, Marcy-l‟Etoile, France) and comparing them to the API
database.
3.4.5 Isolation of Yeast
All colonies totalling about15 from a segment of the highest dilution or suitable OGYEA plate
were selected and examined by microscopy, purified by successive sub culturing in Malt
Extract Broth (Oxoid CM57) and streaked on OGYEA (Oxoid CM545) pH 7.0 until pure
colonies were obtained.
3.4.5.1 Macroscopic and Microscopic Examination
Colonies on solid media were examined macroscopically for colonial morphology.
Characteristics described included colour, surface, size, edge, and elevation. Cultures were
also microscopically observed as wet mounts for cellular morphology.
3.4.6 Identification of Yeasts Isolates using the ID 32 C Kit
Yeast isolates were grown on OGYEA (Oxoid CM0545) and incubated at 25 °C for 48 h.
About 3 pure colonies of yeast on each agar plate were suspended in an ampule of API C
medium (BioMérieux, Marcy-l‟Etoile, France) and 135 µl used to inoculate the ID 32 C strips
that consisted of 32 copules, each containing a dehydrated carbohydrate substrate. These were
then covered with the incubation lids and incubated at 30 °C for 48 h. At the end of the
incubation period, isolates were identified by determining their pattern of fermentation and
assimilation of various carbohydrates in ID 32 C galleries.
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3.5 Technological Properties of Identified Lactic Acid Bacteria
3.5.1 Rate of Acidification of Maize Grains and Dough by LAB
Duplicate trial fermentations were carried out as inocula enrichment with seven dominant
LAB cultures identified earlier during steeping of dehulled maize and dough fermentation (L.
fermentum1, L. fermentum 2, L. brevis 1, L. brevis 2, L. plantarum, P. acidilactici and P.
pentosaceous). For the preparation of the dehulled maize grains; 300 g of washed dehulled
maize were distributed into 1litre conical flask and 450 ml (1:1.5w/v) of sterile water were
used to steep the grains. The steeped grains were milled, mixed with water and kneaded into
dough. The lactic acid bacteria cultures used as inocula enrichment was prepared from a 16 h
culture incubated at 37 ºC and 100 µl of the culture was transferred into sterile SPS and
diluted to a concentration of about 106 cfu/ml. This was checked by microscopic counting
using a Thomas counting chamber and by plating out on MRS agar. Seven different batches of
dehulled maize and dough were steeped and fermented respectively, and to each batch, one
isolate was added as inoculum enrichment. The mixture was shaken to obtain uniform
distribution, and left at room temperature to ferment for 12 h. One batch of each dehulled
maize and dough was not inoculated and used as control (spontaneous fermentation). Every 4
h, 10 ml of steep water and 10 g of dough were aseptically collected for determination of pH
and titratable acidity at 0-4 h, 4-8 h and 8-12 h and 0 h, 4 h, 8 h and 12 h respectively.
3.5.2 Production of Exopolysaccharides (EPS) by LAB Isolates
Screening of isolates for EPSs production was carried out according to Guiraud (1998).
Isolates cultured on MRS agar were streaked onto LTV agar [0.5 % (w/v) tryptone (Difco), 1
% (w/v) meat extract (Fluka, Biochemika, Chemie GmbH, Buchs, Switzerland), 0.65 % (w/v)
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NaCl (Sigma), 0.8 % (w/v) potassium nitrate (Merck, KgaA), 0.8 % (w/v) sucrose (PA
Panreac Guimica SA, Barcelona, Espana), 0.1 % (v/v) Tween 80 (Merck), 1.7 % (w/v) agar
(Sigma), pH 7.1±0.2] and incubated at 30 ºC for 48 h. The colonies were tested for slime
formation using the inoculated loop method (Knoshaug et al., 2000). Isolates were considered
positive for slime production if the length of slime was above 1.5 mm. Positive isolates were
confirmed using MRS- Sucrose Broth without glucose and peptone as described by Pidoux et
al., (1990) [1 % (w/v) meat extract, 0.5 % (w/v) yeast extract (Fluka, Biochemika), 5 % (w/v)
sucrose (PA Panreac Guimica), 0.2 % (w/v) K2HPO4.3H2O (Merck), 0.5 % (w/v) sodium
acetate trihydrate (Merck), 0.2 % (w/v) triammonium citrate anhydrous (Fluka, Biochemika),
0.02 % (w/v) MgSO4.7H2O (Merck), 0.005 % (w/v) manganese (II) sulphate monohydrate
(Merck), 0.1 % (v/v) Tween 80, pH 5.0 ± 0.2)]. The isolates were cultured in MRS- sucrose
broth and incubated at 30 ºC for 24 h. A volume of 1.5 ml of the 24 h culture was centrifuged
at 4000 g for 10 min (4 ºC) and 1 ml of the supernatant put in a glass tube and an equal
volume of 95 % ethanol added. In the presence of EPSs, an opaque link is formed at the
interface. The positive isolates were noted according to the intensity of the opaque link.
3.5.3 Tests for Amylase Secretion by LAB Isolates
Purified bacterial cultures were streaked on Nutrient Agar (Oxiod CM3; Oxoid Ltd.,
Basingstoke, Hampshire, UK) containing 2 % soluble starch (with pH adjusted to 7.2) and
incubated in an anaerobic jar at 30 °C for 3 days. After incubation, the plates were flooded
with iodine solution. Production of amylase was indicated by the formation of a clear zone
around the colonies with the remaining parts of the plates staining blue-black as described by
Almeida et al., (2007). The extent of secretion was quantified by measuring the width of the
clear zone around the colony.
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3.5.4 Test for Protease Secretion by LAB Isolates
Purified bacteria cultures were streaked on Plate Count Agar (Oxiod CM325; Oxoid Ltd.,
Basingstoke, Hampshire, UK) supplemented with 0.5 % casein. The plates were incubated at
30 oC for 3 days and then flooded with 1M HCl. Protease positive was indicated by a clear
zone around the colonies as described by Almeida et al., (2007).
3.5.5 Antimicrobial Studies
The inhibitory potential of lactic acid bacteria cultures was investigated using the Agar Well
Diffusion method as described by Schillinger and Lücke (1989) and Olsen et al., (1995). The
MRS agar was poured into Petri dishes and allowed to solidify at room temperature. Circular
wells were made in the agar using a sterile cork borer (no 4). The cultures of lactic acid
bacteria isolated at different stages of steeping and dough fermentation were cultured in MRS
broth (Oxoid CM359) at 30 °C for 24 h. A volume of 0.1 ml of each test culture was
transferred into a different well and left to diffuse into the agar for approximately 4-5 h. The
plate was then overlaid with 10 ml soft Nutrient Agar (Oxoid CM3) containing 0.25m1 of 10-1
dilution of an overnight culture of the indicator pathogens: four tested strains and one negative
control, (Salmonella typhimurium, NCTC 12023, E.coli, NCTC 9001, Vibro cholerae, NCTC
11348 and Staphylococcus aureus, NCTC 657) obtained from Food Research Institute were
used, and incubated at 37 °C for 24 h followed by examination for inhibitory reactions as
described by Mante et al., (2003).
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3.6 Development of Starter Culture
3.6.1 Dehulled Maize
Dehulled maize was purchased from a local producer on the open market at Anum, in the
Eastern region. The grains were stored in a jute sack at room temperature until ready for use.
3.6.2 Starter Cultures
Two cultures of lactic acid bacteria (L. fermentum, and L.brevis; AD.4/12 and AD.12/8
respectively) and two yeast cultures (C. krusei and S. cerevisiae; AD8/10 and SS.48/5
respectively) isolated earlier from nsiho fermentation were used. The cultures were stored in
50 % glycerol at – 20 oC.
3.6.2.1 Preparation of Inoculum
Stock cultures of L. fermentum, and L. brevis (lactic acid bacteria) and C. krusei and S.
cerevisiae. (yeasts) isolated earlier in the present work were sub cultured in MRS Broth,
(MRS, Oxoid CM361) and Malt Extract Broth (Oxoid CM57) respectively and incubated at
30oC for 24 h. Dilutions were made by transferring 0.1 ml of the sub- cultures into 9 ml
growth media and incubated for 24 h at 30 oC. After incubation, l ml from each culture was
transferred into 100 ml of growth medium and incubated at 30 oC for 24 h. The cells were then
harvested by the following procedure:
1. Nine hundred microlitres of distilled water were added to 100 μl of culture to obtain 1:10
dilution
2. The concentration of cells in each dilution was determined by counting under the
microscope using the Thomas counting chamber. The concentration of cells was determined
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by multiplying the cell count in the 16 chambers by the dilution and by a factor of 104:
(microscopic count x dilution factor x 104).
3. Concentration of 107 cells /ml was obtained for LAB and 106 cells/ml were obtained for the
yeast.
4. The cultures for inoculation were then centrifuged at 4000 rpm for 15 minutes, the
supernatant discarded and the cultures re-suspended in 4 ml sterile distilled water and
centrifuged again.
5. The supernatants were again discarded and the washed cultures reconstituted in sterile
distilled water and used to inoculate the steeped maize and the dough.
3.6.3 InoculationTrials
Fermentation experiments were conducted in duplicate and results represent duplicate
measurement. Starter cultures, L. fermentum, and L. brevis and C. krusei and S. cerevisiae
were used.
3.6.3.1 Fermentation with Single Starter Culture
For each of the fermentation trails 2.5 kg of dehulled maize kernels were steeped in 5.0 litre
water in 10 L capacity plastic bowl. Either 107cfu/ml of lactic acid bacteria or 106 cfu/ml of
yeast was inoculated into the steep water or dough as single starter culture (L. fermentum,
L.brevis, C. krusei and S. cerevisiae). The dehulled maize grains were left to steep at ambient
temperature (28-30 oC) for 48 h and sampled at 0 h, 24 h, and 48 h for determination of pH,
titratable acidity and microbiological analysis. After steeping, the grains were recovered,
milled and kneaded into dough. The dough was allowed to ferment and sampled at 0, 4, 8 and
12 h for determination of pH, titratable acidity and microbiological analysis. Four batches of
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steep water and dough were inoculated and one batch was not inoculated and served as
control.
3.6.3.2 Fermentation with Combined Starter Culture
Dehulled grains of 2.5 kg batches were steeped in 5.0 L water (1:2 w/v) in 10 L capacity
plastic bowl. Eight separate batches were prepared by adding to the steep water and dough,
cultures of L. fermentum, L.brevis, C. krusei and S. cerevisiae in the following combinations:
Con (control/spontaneous): no starter culture; CK: S. cerevisiae + C. Krusei; FK: L.
fermentum + C. Krusei; FC: L. fermentum + S. cerevisiae, FCK: L. fermentum + S. cerevisiae
+ C. Krusei; FBKC: L. fermentum + L.brevis + C. krusei + S. cerevisiae; FBK: L. fermentum,
L. brevis, C. krusei and CBK: S. cerevisiae + L. brevis + C. Krusei. Yeasts cultures were
inoculated to attain concentrations of 106 cells/ml while LAB was inoculated to attain
concentrations of 107 cells/ml. Samples of steep water and fermenting dough were collected
for analyses as described above.
3.7 Survival of Enteric Pathogens during Steeping of Dehulled Maize
The ability of different enteric pathogens to survive in steeping water during steeping of
dehulled maize grains was studied by the method described by Mante et al., (2003). The
enteric pathogens used were certified cultures of Salmonella typhimurium, Escherichia coli,
Staphylococcus aureus and Vibrio cholerae, all obtained from the Microbiology laboratory,
Food Research Institute, Ghana. A pure culture of each pathogen in Nutrient Broth at a
concentration of 106cfu/ml was inoculated into fermenting maize during steeping. 10 ml was
collected at 12 h and the population of surviving pathogens enumerated by spread plate
incubated at 37 °C. Salmonella typhimurium was enumerated on XLD medium (Oxoid,
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CM469), Escherichia coli were enumerated on VRBA (Oxoid, CM107), Staphylococcus
aureus on BP medium (Oxoid) and Vibrio cholerae on TCBS Cholera medium (Oxoid,
CM333).
3.8 Preparation of Nsiho in the Laboratory for Sensory Evaluation
Steeped maize fermented with added starter cultures were milled into a smooth paste using the
disc attrition mill. 500 g of the milled maize was mixed with 500 ml of water into slurry. The
slurry was poured into boiling (500 ml) water in a pot on an electric stove and pre-cooked by
stirring for 15 min into a thick paste known as „ohu or aflata‟. The ohu was moulded into
balls of about 100 g each. Each ball was wrapped with a cleaned maize husk, packed into an
electric steamer and steamed for 30 min.
3.8.1 Sensory Analysis
The kenkey samples prepared with different starter cultures were evaluated by a sensory panel
for acceptability based on texture, aroma (odour), taste, and overall acceptability. The sensory
panel consisted of twenty untrained panellist selected from the CSIR- Food Research Institute
who were familiar with the product. The coded samples were assessed on a nine-point hedonic
scale where 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike
slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7= Like moderately, 8 = Like very
much and 9 = like extremely (Appendix 1). Data obtained was analysed using the mean and
standard deviation to assess the level of consumer acceptability of the product as well as
analysis of variance (ANOVA) to establish significant difference among the various treatment.
Multivariate data analysis (Principal Component Analysis, PCA was carried out on mean
values with Latentix Version 2.0 (Latent 5, Copenhagen, Denmark). Each variable was mean-
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centered and scaled (multiplied by the inverse of the standard deviation) to give it the same
variance. PCA was used to visualize the inter-sample and inter-variable (sensory attribute
intensities) relationships.
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4.0 RESULTS
4.1 Field Study
The field study revealed that only women were involved in the traditional production of white
kenkey. These women had little or no formal education, and were engaged in the traditional
processing as a family trade or business in which skills had been handed from one generation
to another. The production is therefore carried out at the family level involving about three to
four women on a small scale and is artisanal in nature. Production of white kenkey was the
main source of employment in these families and it was reported to be a profitable business.
At the sites visited, the women produced a minimum of 5 kg and a maximum of 10 kg of
white kenkey per batch.
The main raw material for white kenkey production is the dehulled maize, corn husk and table
salt. All these materials are purchased locally. The procedures used to produce nsiho are
shown in Figure 4.1 and appendix 3. Processing involves winnowing and sorting of the maize
to remove dust, chaff and stones. The cleaned maize is dehulled in a disk attrition mill and
steeped in water for 48 h. After steeping, the grains are washed and milled into a smooth meal.
Two different procedures were observed for further processing after production of the smooth
meal and this depended on the locality. At Senchi all the milled meal is pre-cooked for about
30-60 minutes into a thick gelatinous paste (ohu). The ohu is moulded into balls and wrapped
in a clean corn husk. The balls are packed into a pot containing small amount of water lined
with sticks and corn husk and steamed for 1-2 h.
In the other processing procedure which was observed at Anum, the milled meal is kneaded
into stiff dough and fermented spontaneously for 6-12 h. After fermentation a two-third
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portion of the dough is pre-cooked for about 30-60 minutes into ohu. The hot ohu is mixed
with the remaining one-third uncooked dough as done for Ga and Fante kenkey. The mixture
formed is moulded into balls and wrapped in clean corn husks. The balls are packed into
perforated pan and placed over a pot of boiling water and steamed for 1 to 2 h.
Maize grains
Cleaning
(Sorting, sieving, winnowing)
Dehulling
Steeping (48 h)
Washing
Milling
Kneading into dough
Fermentation (6-12 h)
Pre-cooking of 2/3rd of dough (30-60 mins) Pre-cooking of dough (30-60 min)
(ohu)
ohu is mixed with remaining 1/3rd fresh dough
Mould and package in maize husks Mould and package in maize husks
Steaming (1-2 h) Steaming (1-2 h)
Nsiho (White kenkey) Nsiho (White kenkey)
(Procedure at Anum) (Procedure at Senchi)
Fig. 4.1. Flow diagram of the production of Nsiho (white-kenkey).
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4.2 Chemical Analyses of Maize during Steeping and Dough Fermentation
The pH and titratable acidity (expressed as percentage lactic acid) from all four production
sites during steeping and dough fermentation are shown in (Tables 4.1 and 4.2). Mean pH
values during steeping decreased from between 6.05 and 5.93 at the start of steeping to
between 3.59 and 3.55 at the end of steeping. During dough fermentation, pH decreased from
an initial range of 6.02- 5.80 for freshly prepared dough to a range of 3.52-3.46 (Table 4.1).
The most drastic drop in pH was achieved during the first day of fermentation.
The titratable acidity (TTA) values during steeping and dough fermentation process are shown
in Table 4.2. The TTA percentages obtained during steeping ranged from 0.02-0.03 % from
the start of steeping to 0.27-0.32 % after 48 h of steeping. Similar results were observed for
dough fermentation with TTA increasing from between 0.25 and 0.27 % at the start of
fermentation to between 0.35 and 0.38 % at the end of fermentation. Titratable acidity
increased in samples from all four production sites with a corresponding decrease in pH (Table
4.1 and 4.2).
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Table 4.1 Mean pH values during the fermentation of dehulled maize into nsiho
Mean pH values
Sample Processor 1 Processor 2 Processor 3 Processor 4
Steep water
0 h 5.98± 0.05 6.00± 0.03 6.05± 0.03 5.93± 0.01
24 h 4.01± 0.03 4.00± 0.01 3.81± 0.10 3.90± 0.01
48 h 3.59± 0.04 3.59± 0.03 3.57± 0.03 3.55± 0.02
Fermenting dough
0 h 5.98± 0.02 6.02± 0.01 5.98± 0.01 5.80± 0.02
4 h 5.44± 0.01 5.51± 0.01 5.51± 0.06 5.46± 0.01
8 h 3.55± 0.01 3.58± 0.03 3.76± 0.03 3.57± 0.04
12 h 3.51± 0.02 3.52± 0.04 3.49± 0.01 3.46± 0.01
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Table 4. 2 Mean Titratable acidity (%) changes during the fermentation of dehulled maize into
nsiho
Mean Titratable values
Sample Processor 1 Processor 2 Processor 3 Processor 4
Steep water
0 h 0.02± 0.01 0.03± 0.01 0.03± 0.01 0.03± 0.01
24 h 0.25± 0.01 0.25± 0.01 0.27± 0.01 0.28± 0.01
48 h 0.27± 0.01 0.29± 0.01 0.30± 0.01 0.32± 0.01
Fermenting dough
0 h 0.27± 0.03 0.25± 0.02 0.26± 0.01 0.27± 0.04
4 h 0.28± 0.01 0.26± 0.03 0.28± 0.01 0.28± 0.03
8 h 0.31± 0.03 0.29± 0.03 0.31± 0.02 0.32± 0.05
12 h 0.36± 0.01 0.35± 0.04 0.35± 0.01 0.38± 0.01
4.3 Changes in Microbial Population during Steeping and Dough Fermentation of Nsiho
4.3.1 Population of Aerobic Mesophiles
The population of aerobic mesophiles during steeping and dough fermentation from the four
production sites during the production of nsiho is shown in Table 4.3. At the start of steeping
the average microbial load was about 105 cfu/ml which increased drastically to a level of 108
cfu/ml after 24 h. The same level was maintained till the end of steeping at 48 h. For the dough
fermentation the microbial population increased steadily over the 12 h of fermentation from
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the mean concentration of 106 to 108 cfu/g (Table 4.3). The microbial population consisted of
Gram positive catalase-negative rods and cocci, Gram positive catalase positive cocci and
Gram negative bacteria.
Table 4.3 Mean (cfu/ml or g) mesophilic counts during the fermentation of dehulled
maize into nsiho
Mean mesophilic counts
Sample Processor 1 Processor 2 Processor 3 Processor 4
Steep water
0 h (3.4 ±1.6)105 (4.5±0.3)104 (1.1±0.2) 106 (2.0±1.2) 106
24 h (5.5±2.4)108 (4.6± 1.7)108 (1.0± 0.2) 108 (3.0± 0.4) 108
48 h (6.1± 1.3)108 (4.8± 1.6) 108 (3.9± 0.9) 108 (9.1± 0.4) 108
Fermenting
dough
0 h (1.7± 0.6) 106 (3.0± 1.4) 106 (5.1± 0.4) 107 (2.2± 1.5) 106
4 h (7.6± 0.8) 106 (7.6± 0.8) 106 (1.1± 0.2) 108 (2.2± 0.6) 107
8 h (2.1± 0.6) 107 (7.8± 0.7) 107 (8.6± 0.4) 108 (4.8± 0.6) 108
12 h (4.9± 0.9) 108 (5.6± 0.5) 108 (1.0± 0.5) 109 (8.7± 1.0) 108
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4.3.2 Population of Lactic Acid Bacteria
Isolates on MRS which were Gram positive catalase negative rods, coccobacilli or cocci were
considered to be lactic acid becteria. They were mainly rods, oxidase negative and non-
sporing. The counts ranged from a level of 103cfu/ml and 105cfu/ml at the beginning of
steeping to 106-108 cfu/ml after 24 h. The counts obtained at three production sites reduced
from 108 to 107 cfu/ml after 48 h. A similar trend was observed in the dough fermentation
with an increase from between 105-106 cfu/ml to 108cfu/ml at the end of fermentation (Table
4.4).
Table 4. 4 Mean LAB counts during the fermentation of dehulled maize into nsiho
Mean LAB counts
Sample Processor 1 Processor 2 Processor 3 Processor 4
Steep water
0 h (1.1± 0.1) 105 (5.1± 0.6) 104 (7.2± 0.4) 104 (1.7± 0.8) 103
24 h (5.4± 1.0) 108 (2.2± 0.4) 108 (2.8± 1.3) 108 (7.7± 0.7) 106
48 h (1.8± 0.4) 107 (4.1± 0.8) 107 (8.8± 0.9) 107 (3.2± 0.9) 108
Fermenting dough
0 h (1.0± 0.1) 105 (2.31± 1.3) 106 (2.8± 1.2) 106 (2.1± 1.1) 106
4 h (6.7± 0.6) 106 (3.1± 0.9) 106 (3.2± 2.0) 107 (2.5± 1.3) 107
8 h (3.0± 0.6) 107 (1.6± 0.1) 107 (2.1± 1.5) 109 (5.0± 0.9) 107
12 h (6.3± 1.2) 108 (5.1± 1.5) 108 (2.4± 0.7) 109 (2.6± 2.1) 108
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4.3.3 Population of Yeasts
Initial counts of yeasts at the start of steeping at all production sites were at a concentration
102 cfu/ml and increased to 104-106 cfu/ml after 48 h of steeping. Likewise, during dough
fermentation yeast counts increased gradually from between 103-104 cfu/g to 106-107 cfu/g
after 12 h (Table 4.5).
Table 4.5 Mean yeast counts during the fermentation of dehulled maize into nsiho
Mean Yeast counts
Sample Processor 1 Processor 2 Processor 3 Processor 4
Steep water
0 h (4.1±0.5)102 (2.8± 0.6) 102 (1.6± 0.8) 102 (7.5± 0.6) 102
24 h (4.9± 0.3) 104 (6.9± 0.4) 104 (1.9± 1.1) 105 (9.7± 0.8) 104
48 h (1.8± 0.5) 105 (9.8± 0.1) 104 (9.1± 0.4) 105 (1.9± 0.9) 106
Fermenting dough
0 h (5.7± 0.8) 103 (5.9± 0.1) 104 (7.8± 1.1) 104 (9.1± 0.7) 103
4 h (2.5± 0.6) 105 (4.9± 0.5) 105 (1.8± 0.1) 105 (5.9± 0.5) 104
8 h (6.0± 0.3) 105 (1.0± 0.1) 106 (1.0± 0.4) 106 (4.0± 0.6) 105
12 h (1.8± 0.7) 107 (7.8± 0.6) 107 (2.7± 0.6) 106 (7.8± 0.5) 106
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4.4 Characterisation and Identification of Lactic acid Bacteria
A total number of 208 LAB colonies were isolated from steeped water and dough
fermentation. They were all Gram positive catalase negative rods, cocobacilli or lactococci
with rods being dominant. These were considered to be lactobacillus spp. Most of the isolates
were found to be heterofermentative by their ability to produce CO2 from glucose and grew at
pH 4.4 and 9.6 and 45 oC but not at 10 oC nor in 18 % NaCl concentration (Table 4.6).
The most dominant strains were heterofermentative lactobacilli and utilised galactose, D-
glucose, D-fructose, D-mannose, ribose, melibiose, saccharose, gluconate and maltose. The
rest are D-raffinose, 5-ketogluconate, D-xylose, lactose, cellobiose, esculin, trehalose, β-
gentobiose, salin, amygdalin, l-arabinose, galactose and mannitol in the API 50 CHL galleries
(Appendix 2) and were identified as Lactobacillus fermentum.
The second most dominant species were very short rods or cocobacilli and grew at pH 4.4 and
9.6 and at 45 oC but not at 10 oC and 18 % NaCl (Table 6) and were able to ferment L-
arabionose, ribose, D-xylose, galactose, D-glucose, D-frucrose, amygdaline, maltose,
melibiose, saccharose, gluconate and 2 keto-gluconate but did not utilized Glycerol, erythritol,
sorbose, rhamnose and were identified as Lacobacillus brevis.
The third most dominant species which were rods were identified as Lactobaccillus plantarum
and grew at pH 4.4 and 9.6, but not in 6.5 % and 18 % NaCl. They were able to ferment-
arabinose, ribose, galactose, D-glucose, D-fructose, D-mannose, D-turanose, mannitol,
esculin, salicin, sorbitol, maltose, lactose, cellobiose and Gluconate.
Other species identified based on carbohydrate fermentation were Pediococcus pentosaceus
8.65 % and fermented L-arabinose, ribose, galactose, D-xylose, D- fructose, D-glucose, D-
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manose, salicin, cellobiose, esculin, lactose, mellibiose, saccharose and β-gentobiose and
lastly Pediococcus acidilactici 4.81 % which fermented ribose, D-xylose, L-xylose, D-
fructose, D-glucose, D-manose but not mellibiose and sacchrarose.
Table 4.6 Biochemical characteristics of lactic bacteria isolated from steeping water
and fermenting dough
Groups
1 2 3 4 5 6 7
Cell form rods rods cocci rods rods rods cocci
Cellular
arrangement
singles
/pairs
pairs tetrad pairs singles
/pairs
pairs pairs
Gram
reaction
+
+
+
+
+
+
+
Catalase
reaction
_ _ _ _ _ _ _
Oxidase
reaction
_ _ _ _ _ _ _
Anaerobic
growth
+
+
+
+
+
+
+
CO2 from
glucose
+
+
_
+
+
+
_
Growth at
10oC
_
_
+
_ _ _ _
Growth at
45oC
+
+
+
_
_
+
+
Growth in
6.5% NaCl
_
+
+
_
_ _
+
Growth in
18% NaCl
_ _
+
_
_ _
+
Growth at
pH4.4
+
+
_
+
+
+
+
Growth at
pH 9.6
+
+
+
+
+
+
+
Isolate
identified
L. ferme-
tum 1
L.
fermen-
tum 2
P.
pento-
taseus
L.
brevis 1
L.
plan-
tarum
L.
Brevis
2
P.
acidi-
lactici
Isolate
source
AD.4/
12
SS.24/
8
AS48/
4
AD.12/
8
SD.8/
13
AD.0/
14
AS.48/
9
A=Anum, S=South Senchi, D=Dough, S=Steep water, 4-14=isolate number
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4.5 Characterisation and Identification of Yeasts Population
A total of 185 yeast colonies were isolated from steep water and fermenting dough from the
four production sites. Initial characterisation was based on colony and cell morphology and by
fermentation of sugars in API ID 32C gallaries. The most dominant yeasts (47.6 %) were
isolated from all the processing stages and utilized galactose, glucose, sucrose, raffinose,
maltose, DL-lactate, trehalose, α-metyl-D-glucoside, melibiose but could not assimilate
lactose and were identified as Saccharomyces cerevisiae. The second dominant yeast (29.1 %)
isolated utilized glucose, N-acetyl- glucosamide and DL-lactate out of the 32 carbohydrate
tested in the ID 32 galleries and was identified as Candida krusei. The third yeasts isolates
utilized D-melizitose, D-melibiose D-glucose and were identified as Debaryomyces spp. and
constituted 15 % whilst the last group Trichosporon spp. 8.3 % utilzed D-melibiose D-
glucose, mannose, maltose, etc. Debaryomyces spp. and Trichosporon spp mainly occurred
during the initial stages of dehulled maize grains steeping.
4.6 Technological properties of Lactic acid Bacteria Isolates
4.6.1 Rate of Acidification by Lactic Acid Bacteria Isolates
pH and titratable acidity were used to evaluate rate of acidification during steeping and dough
fermentation. Figures 4 a-c show the rate of acidification during steeping. At 0-4 h the rate of
acidification ranged from 0.01 to 0.03 unit with L. fermentum 2 showing the lowest rate of
acidification (Fig.4.a). At 4-8 h fermentation (Fig 4.b), change in pH ranged from between
0.32 to 0.84 units and L. fermentum 2 and L. brevis 1 recorded the highest rate of acidification
with P. pentosaceus being the least. At 8-12 h of steeping all the isolates had increased the rate
of acidification by more than 1 unit with P. pentosaceus and P. acidilactici showing the
highest pH change (Fig 4.c). Rate of acidification was faster during the fourth to the eighth
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hour, but was the least from the zero to fourth hour during the fermentations. The Titratable
acidity expressed as percentage lactic acid, increased in both steeping water and fermenting
dough (Table 4.7 & 4.8).
Figures 4 d-e show changes in the rate of acidification for the fermenting dough. Rate of
acidification increased by 1.15 to 1.20 units within the first four (0-4) hours: with L.
fermentum 2 showing the fastest rate (Fig 4.d). Rate of acidification was faster during the
fourth to the eighth hours of fermentation and was the least in the eighth to twelveth hour.
Figure 4 e shows L. fermentum 1 and L. brevis 1 as the isolates with the highest rate of
acidification.
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Fig. 4.2 a Fig. 4.2 b
Fig. 4. 2 c
Fig 4.2 a-c Rates of acidification of steeped dehulled maize by lactic acid bacteria isolates
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Fig. 4.2 d Fig. 4.2 e
Fig. 4.2 f
Fig 4.2 d-f Rates of acidification of fermenting dough by lactic acid bacteria isolates
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Table 4.7 Changes in Titratable acidity during acidification of steeping water by lactic
acid bacteria
Time of steeping
Isolates 0 h 4 h 8 h 12 h
L. fermentum 1 0.02± 0.01 a 0.17± 0.02a 0.24± 0.01a 0.32± 0.03a
L. fermentum 2 0.02± 0.01 a 0.15± 0.01a 0.22± 0.02a 0.30± 0.01a
L .brevis 1 0.03± 0.01 a 0.12± 0.01b 0.16± 0.01b 0.31 ±0.01a
L .brevis 2 0.02± 0.01 a 0.10± 0.01b 0.17± 0.02b 0.30± 0.02 a
L .plantarum 0.02± 0.01 a 0.14± 0.01a 0.18± 0.01b 0.29± 0.02a
P. acidilactici 0.01± 0.01 a 0.15± 0.01a 0.16± 0.03b 0.25± 0.02b
P. pentosaceus 0.01± 0.01 a 0.16± 0.02a 0.19± 0.01b 0.22± 0..03b
Means with same letters in a column are not significant different (p<0.05)
Table 4.8 Changes in Titratable acidity during acidification of fermenting dough by
lactic acid bacteria
Time of fermenting dough
Isolates 0 h 4 h 8 h 12 h
L. fermentum 1 0.02± 0.01a 0.19± 0.01a 0.24± 0.01a 0.36± 0.01a
L. fermentum 2 0.02± 0.01a 0.18± 0.01a 0.27± 0.01b 0.32± 0.01b
L. brevis 1 0.02± 0.01a 0.18± 0.02a 0.22± 0.01a 0.33 ±0.01b
L. brevis 2 0.02± 0.01a 0.17± 0.01a 0.21± 0.01a 0.29± 0.03 c
L. plantarum 0.02± 0.01a 0.20± 0.01a 0.25± 0.01a 0.29± 0.03c
P. acidilactici 0.02± 0.01a 0.18± 0.01a 0.23± 0.01a 0.31± 0.01b
P. pentosaceus 0.03± 0.01a 0.11± 0.01b 0.19± 0.01c 0.25± 0.01d
Means with same letters in a column are not significant different (p<0.05)
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4.6.2 Production of Exopolysaccharides (EPS) by Lactic Acid Bacteria Isolates
A total 108 lactic acid bacteria were screened for production of exopolysaccaride. The isolates
produced varied degrees of EPS ranging from 1 mm to 4 mm of slime. About 26 %
comprising of L. brevis, P.acidilactici P. pentosaceus, L. plantarum produced slime of less
than 2 mm in length, whiles about 37 % comprising of L. fermentum1, L. fermentum 2
produced slime exceeding 2 mm in length (Table 4.9).
4.6.3 Amylase Secretion by Lactic Acid Bacteria Isolates
The lactic acid bacteria isolates were compared for their ability to secrete amylase by growing
them on modified Nutrient agar containing 2 % starch. A total of 144 isolates comprising 43 L.
fermentum, 34 L. brevis, 22 L. plantarum, 13 P. acidilactici and 8 P. pentosaceus were
screened. Out of these isolates 18.24 % of L. fermentum produced clear zones ranging from
1mm to 2 mm, 10.12 % of L .brevis produced clear zones, 12.78 % of L. plantarum produced
clear zones and 8.28 % of P. pentosaceus also demostrated amylase secretion. None of the P.
acidilactici demonstrated amylolytic activity (Table 4.9).
4.6.4 Protease Secretion by Lactic Acid Bacteria Isolates
Out of the total of 108 lactic acid bacteria isolates tested for protease activity, none of them
isolates secreted protease (Table 4.9).
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Table 4.9 Amylase, protease and Exopolysaccharides (EPS) secretion by lactic acid
bacteria isolated during maize and dough fermentation.
LAB isolates Amylase Protease EPS
L. fermentum 1 + - ++
L. fermentum 2 + - ++
L.brevis 1 - - +
L.brevis 2 - - +
L.plantarum + - +
P.acidilactici - - +
P.pentosaceus + - +
-: no inhibition zone, +: 1-2mm inhibition zone, ++: 3-4mm inhibition zone, +++:5+mm
inhibition zone
4.6.5 Antimicrobial Activity of Lactic Acid Bacteria against Some Common Pathogens
Lactobacillus fermentum showed a strong antimicrobial activity against all the pathogens
tested (Salmonella typhimurium, NCTC 12023, E.coli, NCTC 9001, Vibro cholerae, NCTC
11348 and Staphylococcus aureus, NCTC 657). The strongest inhibition were aginst Vibro
cholerae and Staphylococcus aureus with inhibition zones exceeding 5 mm while Salmonella
typhimuruim and E. coli showed inhibition zones of less than 3 mm (Table 4.10). P.
acidilactici showed a weak inhibition against all the pathogens tested whilst P. pentosaceus
was only able to inhibit Staphylococcus aureus weakly. L. plantarum showed a fair inhibition
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against Staphylococcus aureus and weak inhibition towards Vibro cholerae, Salmonella
typhimurium and E.coli. Only few of the isolates prevented the growth of E. coli
Table 4.10 Antimicrobial activity of lactic acid bacteria against pathogens indicator-
strains
Tested isolates Indicator strains
E.coli Staphylococcus
aureus
Salmonella
typhimurium
Vibrio cholera
L. fermentum 1 ++ +++ ++ +++
L.fermentum 2 + ++ ++ ++
L.brevis 1 - + - ++
L.brevis 2 - - - ++
L.plantarum + ++ + +
P.acidilactici - + + -
P.pentosaceus - + - -
-: no inhibition zone, +: 1-2mm inhibition zone, ++: 3-4mm inhibition zone, +++:5mm
inhibition zone
4.6.6 Antimicrobial Interaction between Lactic Acid Bacteria and Yeasts Isolates
There was no microbial interaction between any of the lactic acid bacteria and Saccharomyces
cerevisiae or Candida krusei. However, L. fermentum 1, L. brevis 1&2 and L. plantarum
showed antagonism against Debaryomyces spp and Trichosporon spp with clear zones ranging
from 2-3 mm. P. acidilactici and P. pentosaceus showed weak inhibition against only
Debaryomyces with inhibition zone of 2 mm (Table 4.11). Debaryomyces and Trichosporon
spp were found only in the steeping water.
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Table 4.11Antimicrobial interaction between lactic acid bacteria and yeasts isolates
Indicator strains (Yeasts)
Test isolates
(LAB)
S.cerevisiae C.krusei Dabaromyces
species
Trichosporon
species
L. fermentum 1 - - ++ ++
L. fermentum 2 - - ++ ++
L.brevis 1 - - + ++
L.brevis 2 - - + +
L. plantarum - - + +
P.acidilactici - - + +
P.pentosaceus - - + +
-: no inhibition zone, +: 1-2mm inhibition zone ++: 3-4mm inhibition zone +++:5+mm
inhibition zone
4.6.7 Antimicrobial Interaction between Lactic Acid Bacteria Isolates
There was no interaction between the lactic acid bacteria isolates except for L. fermenutm1
and L. plantarum which showed weak inhibition towards P. acidilactici (Table 4. 12).
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Table 4.12 Antimicrobial interaction between lactic acid bacteria isolates
-: no inhibition zone, +: 1-2mm inhibition zone, ++: 3-4mm inhibition zone, +++:5+mm inhibition zone NA: No interaction
Indicator strains
Test isolates L. fermentum1 L. fermentum 2 L. brevis 1 L. brevis 2 L. plantarum P. acidilactici P. pentosaceus
L. fermentum 1 NA - - - - + -
L. fermentum 2 - NA - - - - -
L. brevis 1 - - NA - - - -
L. brevis 2 - - - NA + - -
L. plantarum - - - - NA + -
P. acidilactici - - - - + NA -
P. pentosaceus - - - - - - NA
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4.7 Starter Culture Trials
4.7.1 Changes in Microbial Population
Changes in the population of lactic acid bacteria and yeasts following the addition of
different starter cultures are shown Table 4.13. Isolates used for the starter culture trials
were L. fermentum and L. brevis (LAB) and S. cerevisiae and C. krusei (Yeasts).
Following the addition of the LAB isolates i.e L. fermentum and L. brevis, high counts of
lactic acid bacteria were recorded throughout compared to the spontaneous fermentation
or addition of yeasts. Addition of yeast as starter cultures did not significantly affect the
LAB counts compared to the spontaneous fermentation. The population of lactic acid
bacteria reached a final count of 109 cfu/ml in fermentation with L. fermentum and L.
brevis, but 107 cfu/ml in the fermentation with yeast and also in the spontaneous steeping
of the dehulled maize grains (Table 4.13).Yeast counts in fermentations with added S.
cerevisiae and C. krusei increased in both cases from initial levels of 105 cfu/ml to 107
cfu/ml after 24 h of steeping whilst maximum counts were 104cfu/ml and 105 cfu/ml in
spontaneous steeping and with added L. brevis or L. fermentum starter culture
respectively.
Dough fermentation to which L. fermentum and L. brevis had been added as starter
culture recorded higher LAB counts compared to the other fermentations. Also, after 8 h,
fermentation to which L. fermentum and L. brevis had been added as starter culture
attained the same level of 108 cfu/g LAB as in 12 h spontaneous in fermented dough.
Addition of C.krusei and S. cerevisiae starter cultures recorded higher yeast counts to a
level of 108 cfu/g at the end of fermentation as compared to the other fermentations (Table
4.13).
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Table 4.13 Mean microbial counts (cfu/ml and cfu/g) for fermentation trials carried
out with starter culture
Types of fermentation
Sample Ferment
ation
time (h)
spontaneous L. fermentum
L. brevis C. krusei S. cerevisiae
Steep water
LAB 0 h (4.9±2.6)104 a (2.0±0.8)107b (5.3±2.8)107 b (5.5±1.4)104 a (5.5±0.9)104 a
24 h (4.8±1.8)107 a (3.3±1.3)108 b (2.4±1.6)108 b (6.7±1.8)107 a (6.4±1.5)107 a
48 h (7.7±1.4)107 a (4.3±1.5)109 b (2.1±0.2)109 b (4.9±0.6)107 a (8.3±2.1)107 a
Yeast 0 h (5.3±1.0)102 a (3.1±0.3)102 a (6.8±0.4)102 a (4.1±1.7)105 b (3.8±1.6)105 b
24 h (5.3±1.7)104 a (1.3±0.1)105 b (5.6±0.5)104 a (3.6±0.4)107 c (4.1±0.7)107 c
48 h (1.5±0.5)105 a (4.5±0.7)105 a (2.8±1.3)105 a (5.3±2.3)107 b (8.3±1.6)107 b
Fermenting dough
LAB 0 h (5.5±2.6)105 a (3.0±0.9)107 b (2.0±4.0)107 b (3.0±4.0)106 c (2.0±3.0)106 c
4 h (4.8±0.5)106 a (8.0±0.8)107 b (5.6±0.7)107 b (8.5±1.0)106 a (6.4±1.0)106 a
8 h (8.0±0.2)106 a (3.0±0.7)108 b (5.8±2.2)108 b (6.9±2.7)107 c (6.1±1.8)107 c
12 h (2.2±0.4)108 a (1.5±0.6)109 b (1.7±0.8)109 b (5.8±1.9)108 c (4.8±0.9)108 c
Yeast 0 h (8.7±1.8)103 a (1.0±0.4)104 a (3.6±0.4)104 b (3.3±0.5)105 c (2.7±1.4)105 c
4 h (1.0±0.3)105 a (7.6±0.8)104 a (5.2±1.3)104 a (5.7±0.4)105 b (4.4±1.4)106 c
8 h (6.8±2.2)105 a (4.2±1.7)105 a (8.4±1.2)105 a (5.9±1.5)107 b (5.3±1.2)107 b
12 h (1.3±0.6)107 a (4.9±0.8)107 a (4.1±1.2)107 a (1.3±0.6)108 b (1.6±0.6)108 b
Values are means of determinations from two separate fermentation trails. Means with same letter in a row are not
significantly different (p<0.05)
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4.7.2 Acidification of Steep Maize and Fermenting Dough in Fermentation Trials
with Starter Culture
Table 4.14 shows pH of steep water and fermenting dough to which single starter cultures
were added. The pH recorded for the spontaneous (control) fermentation dropped from
6.00 at start of steeping to 3.52 after 48 h of steeping. The pH values for steeping with
starter cultures ranged from initial levels of 6.02 -5.96 to 3.56 -3.49 at the end of
steeping. Among the starter cultures, the highest drop was recorded for L. fermentum with
3.49 and the least for C. krusei being the least 3.56 (Table 4.14). Dough fermentation
showed similar results with the starter cultures reducing the pH from 6.03-5.99 at the start
of fermentation to 3.44-3.38 at the end of fermentation (Table 4.14).
Titartable acidity increased in all fermentations as expected. The TTA values recorded
during stepping ranged from 0.03-0.04 at the start of fermentation to 0.30- 0.38 after 48 h
fermentation. Also, during dough fermentation a value of 0.37-0.53 was attained at the
end of fermentation. The concentration of lactic acid was highest in both steeping water
and dough fermented with L. fermentum and lowest with spontaneous fermentation
(Table 4.14).
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Table 4.14 Mean pH and Titratable acidity values for fermentation trials with
starter culture
Types of fermentation
Sample Fermentation
time (h)
Control L. fermentum L. brevis C.krusei S. cerevisiae
Steep water
pH 0 h 6.00± 0.03 5.96± 0.01 5.96± 0.02 6.00 ±0.01 6.02 ±0.01
24 h 4.02± 0.01 3.63± 0.01 3.68 ±0.02 3.71 ±0.05 3.70 ±0.02
48 h 3.62 ±0.01 3.49 ±0.01 3.50 ±0.01 3.56 ±0.02 3.55± 0.02
Titratable
acidity
0 h 0.03± 0.01 0.04 ±0.01 0.04± 0.02 0.04± 0.01 0.03 ±0.01
24 h 0.26 ±0.01 0.31 ±0.02 0.29 ±0.01 0.28 ±0.02 0.28 ±0.01
48 h 0.30 ±0.01 0.38± 0.01 0.35 ±0.01 0.35 ±0.02 0.34 ±0.01
Fermenting dough
pH 0 h 6.02± 0.01 5.99± 0.03 6.02± 0.01 6.03 ±0.01 6.03± 0.01
4 h 5.46 ±0.01 5.28 ±0.02 5.32 ±0.01 5.40 ±0.03 5.41 ±0.01
8 h 3.72 ±0.05 3.46 ±0.04 3.53 ±0.05 3.50 ±0.01 3.49 ±0.01
12 h 3.53 ±0.03 3.38 ±0.01 3.41 ±0.02 3.41 ±0.01 3.44 ±0.01
Titratable
acidity
0 h 0.26± 0.01 0.27± 0.01 0.27± 0.01 0.27± 0.02 0.25± 0.01
4 h 0.28 ±0.02 0.32 ±0.01 0.32 ±0.01 0.30 ±0.01 0.28 ±0.01
8 h 0.32 ±0.01 0.38 ±0.01 0.37 ±0.01 0.34 ±0.01 0.33 ±0.01
12 h 0.37 ±0.01 0.49 ±0.02 0.47 ±0.03 0.47 ±0.01 0.46 ±0.02
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4.7.3 pH Changes during Steeping and Dough Fermentation Inoculated with
Combinations of Starter Cultures
The pH changes during nsiho fermentation and with combinations of starter cultures
during steeping fermentation and dough are shown in figures 4.3 and 4.4. Addition of
any of LAB and yeasts starter cultures produced lower pH values both during steeping
and dough fermentation in comparison to the spontaneous fermentation. The lowest pH
values at all stages of fermentation were recorded when all four starter cultures were used
in combination, i.e L. fermentum + L. brevis C. krusei+ S. crervisiae. At 24 h of steeping
all fermentations using starter cultures had the pH value recorded for the spoataneous
fermentation at the end of steeping i.e 48 h. Generally, the more cultures that were
combined in starter culture mix ie 2, 3, or 4 different cultures, the faster and lower the
drop in pH observed. Using a combination L. fermentum + L. brevis + C. krusei for
steeping reduced the pH more than using a combination of S. crervisiae + L. brevis + C.
krusei or L. fermentum + S. crervisiae + C. krusei. The same trend was observed during
dough fermentation. The pH ranged from between 5.98-5.93 at the start of fermentation
to between 3.44- 3.06 at the end of fermentation, while combination with L. fermentum +
L. brevis + C. krusei+ S. crervisiae ranged from 5.93 to 3.06 after 12 h (Fig4.4).
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Fig 4.3 Changes in pH during steeping of dehulled maize grains using different
starter cultures
Control (spontaneous): no starter culture added
CK: S. crervisiae + C. krusei
FK: L. fermentum + C. krusei
FC: L. fermentum + S. crervisiae
FCK: L. fermentum + S. crervisiae +C. krusei
FBKC: L. fermentum + L. brevis + C. krusei+ S. crervisiae
FBK: L. fermentum + L. brevis + C. krusei
CBK: S. crervisiae + L. brevis + C. krusei
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Fig 4.4 Changes in pH during dough fermentation using different starter cultures
Control (spontaneous): no starter culture added
CK: S. crervisiae + C. krusei
FK: L. fermentum + C. krusei
FC: L. fermentum + S. crervisiae
FCK: L. fermentum + S. crervisiae +C. krusei
FBKC: L. fermentum + L. brevis C. krusei+ S. crervisiae
FBK: L. fermentum + L. brevis + C. krusei
CBK: S. crervisiae + L. brevis + C. krusei
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4.7.4 Changes in Titratable acidity during Steeping and Dough Fermentation
Inoculated with Different Combinations of Starter Cultures
The mean total acidity during the different types of fermentations is shown in Table 4.15.
The highest value of Titratable acidity was produced by combination of all four starter
cultures, with spontaneous fermentation yielding the least in both steep water and dough
during the fermentation periods (Table 4.15). The combination of lactic acid bacteria and
yeast produced more lactic acid than only yeast combinations. With the three
combinations, L. fermentum + L. brevis + C. krusei combination produced more lactic
acid after 48 h of steeping while L. fermentum + S. crervisiae + C. krusei produced a
higher value TTA after 12 h of dough fermentation (Table 4.15).
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Table 4.15 Percentage Titratable acidity values during steeping and dough fermentation of nsiho using different starter cultures
Types of fermentation
Fermentation
time (h)
Control/spon-
taneous
CK FK FC FCK FBK CBK FBKC
Steep water
0 h 0.03± 0.00a 0.04± 0.01a 0.04± 0.01a 0.05± 0.01a 0.04 ±0.01a 0.05± 0.01a 0.04± 0.01a 0.06± 0.01b
24 h 0.25 ±0.01a 0.28 ±0.02a 0.31 ±0.01b 0.33 ±0.02b 0.30 ±0.02b 0.30 ±0.01b 0.29 ±0.01b 0.35± 0.03c
48 h 0.28± 0.01a 0.37 ±0.01b 0.39 ±0.03b 0.38 ±0.01b 0.39 ±0.04b 0.40 ±0.02c 0.37 ±0.01c 0.42± 0.02c
Fermenting dough
0 h 0.25± 0.01a 0.25± 0.00a 0.26± 0.01a 0.27± 0.02a 0.28± 0.01a 0.27± 0.01a 0.26± 0.02a 0.30± 0.01a
4 h 0.27 ±0.01a 0.30 ±0.01b 0.33 ±0.02b 0.31 ±0.01b 0.32 ±0.01b 0.32 ±0.01b 0.33 ±0.01b 0.38 ±0.01c
8 h 0.32 ±0.02a 0.35 ±0.01a 0.39 ±0.01b 0.37 ±0.01b 0.41 ±0.33b 0.42 ±0.01b 0.43 ±0.02b 0.47 ±0.01c
12 h 0.40 ±0.02a 0.49 ±0.01b 0.53 ±0.02b 0.50 ±0.04c 0.58 ±0.01c 0.57 ±0.02c 0.56 ±0.03c 0.61 ±0.02d
Means with same letters in a row are not significant different (p<0.05)
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4.7.5 Microbial Population during Steeping using Different Starter Cultures
The microbial population during the steeping of dehulled maize grains using different
starter cultures is shown in Table 4.16. With addition of any of the lactic acid bacteria the
total LAB counts obtained at the end of 48 h steeping was at a concentration of 109
cfu/ml compared to 108 cfu/ml for spontaneous fermentation during steeping. Using a
combination of L. fermentum + L. brevis C. krusei+ S. crervisiae gave the highest LAB
counts during steeping (Table 4.16). Sponatneous fermentation recorded the least LAB
counts of 108 cfu/ml at the end of steeping and was significant different from the use of
any of the starter cultures. Among the three mix starter, L. fermentum + L. brevis+ C.
krusei showed higher counts than L. fermentum + S. crervisiae+ C. krusei and S.
crervisiae + L. brevis +C. krusei ; also, in the two mix starter S. crervisiae + C. krusei
produced lower counts as compared to L. fermentum + C. krusei and L. fermentum + S.
crervisiae (Table 4.16). With yeasts counts, combinations with the two yeasts produced
higher yeasts counts of 108 cfu/ml after 48 h of steeping, whiles using a single yeast gave
a count of 107 cfu/ml compared to 105 cfu/ml for steeping without any starter culture.
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Table 4.16 Mean microbial counts (cfu/ml) during the steeping of dehulled maize grains using starter culture
Types of fermentation
Sample Fermentation
time
Control/
spontaneous
CK FK FC FCK FBK CBK FBKC
Steep
water
LAB 0 h (5.5±0.4)10
4a (6.9±0.7)105a (3.4±0.6)107b (2.1±0.6)107b (4.5±0.2)107b (6.2±0.2)107b (3.2±1.8)107b (6.0±1.3)107a
24 h (5.4±2.1)10
7a (1.5±0.3)108a (5.6±0.9)108b (7.4±2.1)108b (4.8±0.9)108b (7.5±0.4)108b (4.6±1.4)108b (8.6±1.6)107c
48 h (5.4±1.6)10
8a (1.1±0.4)109b (6.0±0.9)109b (4.7±1.1)109b (5.8±2.4)109b (6.1±1.4)109b (1.6±0.7)109a (9.0±0.6)109b
Yeast 0 h (5.7±1.5)10
3a (4.3±0.3)107b (5.4±1.3)106c (3.9±0.6)106c (7.5±2.1)107b (6.2±2.1)106c (5.2±1.9)107b (7.4±1.3)107b
24 h (8.6±1.6)104a (4.8±1.1)107b (2.9±0.2)107b (3.4±0.6)107b (7.7±1.3)107b (3.1±0.4)107b (8.1±0.7)107b (7.8±0.7)107b
48 h (6.4±1.3)105a (8.7±2.1)107b (4.6±1.9)107b (3.3±0.8)107b (9.4±1.4)107c (7.7±0.7)107b (2.0±1.5)108c (2.6±1.4)108c
Means with same letters in a row are not significant different (p<0.05)
CON: control /spontaneous fermentation (without starter culture), CK: S. cerevisiae +C. krusei, FC: L.fermentum+ S. cerevisiae, FK: L.fermentum+ C. krusei, BK: L .brevis+ C.
krusei, FCK: L.fermentum+ S. cerevisiae+ C. krusei, FBK: L.fermentum+ L. brevis+ C. krusei, FBKC: L.fermentum+ L. brevis+ C. krusei+ S. cerevisiae
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4.7.6 Microbial Counts during Nsiho Dough Fermentation using Different Starter
Culture
The microbial population during nsiho dough fermentation using different starter cultures
are shown in Table 4.17. Using different combinations of culture which included a LAB
resulted in hundred fold increase in the population of LAB at the start of dough
fermentation compared to the spontaneous fermentation. By the end of fermentation the
difference in population of LAB between these fermentations and that of spontaneous
fermentation were reduced to ten fold; 109 cfu/g compared to108 cfu/g. The use of yeasts
did not result in a higher LAB population during dough fermentation compared to
spontaneous fermentation. All combinations of starter cultures used contained yeasts and
these resulted in higher yeasts couts in all fermentations compared to the spontaneous
fermentation. Yeast counts for all fermentation containing yeast starter culture reached an
initial level of 106 cfu/g at the start of fermentation with spontaneous fermentation
reaching a value of 103 cfu/g (Table 4.17).
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Table 4.17 Mean microbial counts (cfu/g) during nsiho dough fermentation using different starter cultures
Type of fermentation
Sample Fermentation
time
Control/
spontaneous
CK FK FC FCK FBK CBK FBKC
Fermenting dough
LAB 0 h (1.7±0.2)105a (3.6±0.2)105a (2.0±0.3)107b (5.0±0.5)107b (3.0±1.7)107b (7.0±0.7)107b (4.0±1.5)107b (9.0±0.7)107b
4 h (4.3±1.3)106a (3.4±2.1)106a (7.6±1.7)107b (3.4±0.4)108c (7.0±1.1)107b (2.2±1.6)108b (9.2±2.6)107b (8.0±0.8)108c
8 h (6.1±0.9)107a (2.5±1.7)107a (3.3±1.6)108b (4.5±0.3)108b (6.4±2.8)108b (7.8±1.6)108b (3.6±0.9)108b (6.2±2.1)109c
12 h (4.4±1.7)108a (7.4±1.9)108a (2.5±1.1)109b (1.8±0.9)109b (4.2±0.2)109b (3.6±2.4)109b (2.8±0.9)109b (8.9±0.6)109b
Yeast 0 h (6.7±0.7)103a (2.2±0.9)106b (3.8±2.2)106b (4.7±0.7)106b (5.6±2.4)106b (2.1±1.2)106b (1.0±0.8)107b (8.3±0.8)106b
4 h (2.1±1.0)104a (6.2±4.2)106b (7.2±1.3)106b (8.7±0.7)106b (6.1±1.9)107c (7.2±0.8)106b (5.1±1.1)107c (3.3±2.1)107c
8 h (1.8±0.8)105a (7.9±2.1)107b (8.6±1.1)106b (1.2±0.5)107b (3.0±1.9)108b (6.7±0.7)107b (8.2±0.7)107b (3.2±0.3)108c
12 h (8.5±0.5)106a (3.3±0.6)108b (8.6±1.6)107c (9.3±1.2)107c (5.5±1.3)108b (2.3±2.0)108b (6.2±0.8)108b (5.5±1.3)108b
Means with same letters in a row are not significant different (p<0.05)
CON: control/spontaneous fermentation (without starter culture), CK: S. cerevisiae +C. krusei, FC: L. fermentum+ S. cerevisiae, FK: L. fermentum+ C. krusei, BK: L .brevis+ C.
krusei, FCK: : L.fermentum+ S. cerevisiae+ C. krusei, FBK: L. fermentum+ L. brevis+ C. krusei, FBKC: L. fermentum+ L. brevis+ C. krusei+ S. cerevisiae
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4.8 Survival of Enteric Pathogens during the Steeping of Dehulled Maize Grains
using Different Starter Cultures
The survival of four enteric pathogens during the steeping of dehulled maize grains using
different starter cultures are shown in Table 4.18. The pathogens were inoculated into
steep water at a concentration of 106 cfu/ml. The microbial numbers of Vibro cholerae
reduced steadily during steeping and was not detected after 48 h in any of the steep water
including that of the spontaneous fermentation as control. There was also a significant
reduction in microbial population of Salmonella typhimurium, E.coli and Staphylococcus
aureus during steeping in all instances, however, these pathogens were not completely
eliminated after 48 h of steeping. Apart from the use of starter culture composed of only
yeasts, there was generally a ten fold reduction in the counts of pathogens at the end of
steeping compared to spontaneous steeping. The only exception to this was the use of
Lactobacillus fermentum and Candida krusei as a combination against E. coli and
Staphylococcus aureus in which case the same concentration was recorded as in that of
the spontaneous fermentation.
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Table 4.18 Count for survival of enteric pathogens inoculated into spontaneous and mixed culture fermentation of steeped
dehulled maize
Types of fermentation
Time (h) Spontaneous CK FK FC FCK CBK FBK FBCK
Vibro cholera
0 h 7.4x105 4.7x105 8.0x105 6.6x104 7.5x105 8.3x104 6.7x105 5.7x105
12 h 5.8x104 7.3x104 6.8x104 5.8x103 6.9x103 7.9x104 3.9x104 6.8x104
24 h 8.1x103 6.7x103 2.2x103 3.5x103 1.7x103 8.9x103 8.4x103 7.5x103
36 h 7.8x102 6.3x101 4.7x102 9.7x101 8.8x102 4.9x102 5.8x102 8.4x101
48 h nd nd nd nd nd nd nd nd
Salmonella
typhimurium
0 h 8.4x105 6.8x105 6.9x105 3.7x105 5.7x105 6.3x105 5.5x105 4.8x105
12 h 7.5x105 7.1x104 6.3x105 4.6x105 5.4x104 8.3x104 5.8x104 7.2x104
24 h 5.7x104 5.5x104 7.7x103 6.2x103 7.6x103 8.2x104 6.6x103 7.3x103
36 h 2.6x104 5.4x103 6.3x103 7.0x103 6.3x103 9.3x102 5.7x103 4.0x103
48 h 2.3x103 1.2x103 3.6x102 5.1x102 8.8x102 7.3x102 6.5x102 4.9x102
nd: not detected
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Cont.
Types of fermentation
Time (h) Spontaneous CK FK FC FCK CBK FBK FBCK
E. coli
0 h 9.4x105 5.5x105 4.2x105 7.6x104 7.4x105 7.9x105 7.8x105 6.6x105
12 h 6.5x105 6.4x105 7.8x104 7.2x104 7.8x104 7.5x105 8.2x104 7.9x104
24 h 7.7x104 7.8x104 9.1x103 8.5x103 2.7x104 9.5x103 8.1x103 8.5x103
36 h 4.6x104 3.3x104 8.3x103 9.6x102 9.6x103 7.8x103 4.9x102 9.1x102
48 h 8.8x103 6.4x103 4.3x103 8.9x102 5.5x102 6.6x102 3.5x102 2.9x102
Staphylococcus
aureus
0 h 6.3x105 7.2x105 4.8x105 5.9x105 7.6x105 7.0x105 5.6x105 5.9x105
12 h 7.6x104 9.2x104 7.1x105 3.9x105 6.2x104 5.3x104 6.5x104 8.3x104
24 h 6.7x103 7.5x104 7.7x103 6.2x103 7.6x103 8.2x104 6.6x103 7.3x103
36 h 7.9x103 6.2x104 6.3x103 7.0x103 6.3x103 7.3x102 5.7x103 4.0x103
48 h 7.3x103 2.2x103 2.9x103 5.7x102 8.8x102 4.3x102 6.4x102 4.6x102
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4.9 Sensory Quality of Nsiho Produced Using Starter Cultures
The sensory scores on a 9 point hedonic scale for nsiho prepared by spontaneous or
through the use of starter culture are shown in Table 4.19. Apart from texture, the sensory
score for every attribute i.e aroma/ odour, taste and overall acceptability was between 6.0
to 7.0 which correspond to like slightly and to like moderately for every sample assessed
including the control/spontaneous sample. The score for texture of the L. brevis and the
combined yeast sample were just above scale 7 with the others being in the range of 6.0
to 7.0
A Principal Component Analysis bi-plot (PCA) used to visualise the inter sample and
sensory attributes relationship is shown in Figure 4.5. From the plot samples, S.
cerevisiae + C. krusei, L. fermentum + C. krusei, and L. brevis + C. krusei are closely
correlated in terms of aroma.
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Table 4.19 Sensory scores for nsiho prepared by spontaneous fermentation or through the use of a starter culture
Attribute Type of fermentation
Con Lb Lf CK FC FK BK FCK FBK FBKC
Aroma 6.23±0.18 6.63±0.04 6.65±0.21 6.80±0.35 6.38±0.18 6.73±0.25 6.58±0.39 6.28±0.11 6.00±0.07 6.18±0.18
Taste 6.40±0.00 6.65±0.42 6.28±0.60 6.53±0.04 6.50±0.28 6.53±0.11 6.43±0.11 6.28±0.18 6.65±0.57 6.35±0.49
Texture 6.33±0.11 7.08±0.32 6.25±0.42 7.09±0.30 6.78±0.04 6.63±0.11 6.75±0.14 6.40±0.07 6.83±0.46 6.73±0.25
Overall 6.30±0.07 6.58±0.67 6.25±0.35 6.13±0.53 6.33±0.46 6.23 ±0.53 6.30±0.14 6.00±0.00 6.73±0.67 6.28±0.18
Acceptability
Values are mean of 20 panellists on a nine point hedonic scale, 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 =
Neither like nor dislike, 6 = Like slightly, 7= Like moderately, 8 = Like very much and 9 = Like extremely
CON: control /spontaneous fermentation (without starter culture), Lb: L .brevis, Lf: L. fermentum, CK: S. cerevisiae +C. krusei, FC: L. fermentum+S.
cerevisiae, FK: L. fermentum + C. krusei, BK: L .brevis + C. krusei, FCK: L. fermentum + S. cerevisiae + C. krusei, FBK: L. fermentum + L. brevis + C. krusei
FBKC: L. fermentum + L. brevi s+ C. krusei + S. cerevisiae
No significant difference between fermentation types (p < 0.05)
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-1.5 -1 -0.5 0 0.5 1 1.5
-1.5
-1
-0.5
0
0.5
1
1.5
2
Taste
Texture
Overall Acceptability
Lb
FBK
CK
FC
FK
PC#1 (57.107%)
PCA Bi-plot [Model 1]
Odour
BK
FBKC
Con
Lf
FCK
P
C
#
2
(
3
1
.
7
2
8
%
)
F e r m e n t a t i o n t y p e s
s e n s o r y a t t r i b u t e s
Fig 4.5 Principal Component Analysis Bi plot
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5.0 DISCUSSION
5.1 Changes in Acidity during Spontaneous Fermentation
Results of acidity in the present study indicate a steady increase in titratable acidity with a
corresponding decrease in pH during steeping of maize grains and dough fermentation.
This observation could be attributable to the increased growth of lactic acid bacteria
responsible for the fermentation of the nsiho which had produced more lactic acid and
likely utilisation of free sugars by yeast and lactic acid bacteria (Efiuvwevwere and
Akona, 1995).
Similar results have been reported by many authors in the area of maize steeping and
dough fermentation (Plahar and Leung, 1982; Jespersenet al., 1994; Halm et al., 1996;
Kalui et al; 2009). Wakil and Daodu (2011); Olukoya et al., (1993); Odunfa and Adeyele,
(1985), have reported the dominance of lactic acid bacteria during spontaneous
fermentation of cereals. Wakil and Daodu (2011) observed a decrease in pH from 5.65 to
3.5 units within 24 h of fermenting with a corresponding increase in titratable acidity
from 0.2065 to 0.2650 after 48 h. The cause of the increase in acidity and consequent
drop in pH during fermentation of cereals is as a result of production of lactic acid by
fermentative organisms.
5.2 Role of Lactic Acid Bacteria in Nsiho Fermentation
The present study showed a drastic increase in the population of lactic acid bacteria
between 103 and 104cfu/ml to 108 cfu/ml within 24 h of steeping of dehulled maize grains
and from 105 cfu/g to 108 cfu/g within 12 h of dehulled dough fermentation. This
confirms that lactic acid fermentation occurs during steeping of dehulled maize grains
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and dough fermentation. The role of lactic acid fermentation in food processing is to
effect preservation by converting sugars to organic acids thus causing a reduction in pH;
by removing carbohydrates as nutrient source and by producing antimicrobial compounds
such as hydrogen peroxide, bacteriocin and other secondary metabolites. Whilst,
preservation is important, lactic acid bacteria are currently often used to provide diversity
in food supply by altering flavour, texture, and appearance of raw commodities in
desirable way (Chassy and Morphy 1993; Davidson 1993; Salovara1993).
The dominant lactic acid bacteria identified in the present work to be responsible for
nsiho fermentation was Lactobacillus fermentum which formed about half of the lactic
acid population isolated from the steep water and fermented dough. This result is in
agreement with the work of Halm et al., (1993), who found fermentation of fermented
whole maize meal during production of Ga and Fante kenkey to be dominated by a group
of obligatory heterofermentative lactobacilli consistent with Lactobacillus fermentum and
Lactobacillis reuteri in their pattern of carbohydrate fermentation. Hayford et al., (1999)
later confirmed the dominant species to be Lactobacillus fermentum using molecular
characterization. It is therefore not surprising that Lactobacillus fermentum has been
found to be responsible for the fermentation of maize into nsiho which is also a type of
kenkey produced from dehulled maize grains in the present work. Removing the testa and
the germ of the maize therefore does not affect the species of LAB which dominates its
fermentation during steeping and dough fermentation.
In Benin, Hounhouigan (1993a) had also reported Lactobacillus fermentum as the
dominant lactic acid bacteria responsible for the fermentation of maize into mawe which
also involves fermentation of partially dehulled maize grains.
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In the present study Lactobacillus plantarum and Lactobacillus brevis were also isolated
in high numbers during maize steeping and dough fermentation. The presence of
Lactobacillus plantarum in maize dough fermentation has been reported. Nche et al.,
(1994), identified Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus confuses
and Pediococcus species as the main lactic acid bacteria present in fermenting maize and
maize cowpea dough. Olasupo et al., (1997) in their studies on selected African
fermented foods obtained 48 lactobacillus isolates from kenkey which they identified as
Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus
delbruckii and Lactobacillus acidophilus. Olsen et al., (1995) found Lactobacillus
plantarum at the initial stage of kenkey fermentation where it dominated the
heterofermentative lactic acid bacteria present.
Studies on other types of fermented products reported that lactic acid bacteria were the
dominant microorganisms involved in the fermentation of gari and kenkey, with
Lactobacillus plantarum been identified most often (Amoa-Awua et al., 1996, Kostinek
et al., 2005).
Sawadogo-Lingani et al., (2007) reported Lactobacillus fermentum as the dominant lactic
acid bacteria responsible for souring of dolo, an alcoholic beverage.
Lei and Jacobson (2004), isolated W. confuses and Lactobacillus fermentum as the
dominant lactic acid bacteria for the fermentation of millet into koko from five production
sites in Northern Ghana.
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5.3 Role of Yeasts in Nsiho Fermentation
Saccharomyces cerevisiae and Candida krusei have been identified in the present work as
the dominant yeasts involved in fermentation of dehulled maize during nsiho production.
Debaryomyces and Trichosporon species were also identified at the initial stages of
steeping.
Halm et al., (1993) had previously reported Candida and Saccharomyces species as the
most frequent isolated yeast during maize steeping and dough fermentation. These
species were later confirmed by using molecular methods to be Saccharomyces cerevisiae
and Candida krusei respectively (Hayford and Jesperson, 1999). Obiri-Danso (1994)
reported S. cerevisiae and C. krusei as the yeasts species during fermentation of maize
dough in kenkey production. Jesperson et al., (1994) isolated S. cerevisiae and C. krusei
as the dominant yeast in maize dough fermentation and suggested that since yeast are
known to produce a wide range of aromatic compounds including organic acids, esters,
aldehydes, alcohols, lactones and terpenes, they are likely to influence the organoleptic
and structural quality of fermented maize dough. Hamad et al., (1992) found that
fermented sorghum dough with high numbers of C. krusei had a more pleasant aroma
than dough with less yeast.
5.4 Antimicrobial Activity of Lactic Acid Bacteria against Common Enteric
Pathogens
In the present study L. fermentum was shown to exhibit a strong antimicrobial activity
against Staphylococcus aureus and Vibrio cholerae. A similar result was observed in dolo
production by Sawadogo-Lingani et al., (2008) who reported a high level of antimicrobial
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activity by L. fermentum against Staphylococcus aureus but weak activity against E. coli
and Listeria innocua.
Other investigations have also reported antimicrobial activity of lactic acid bacteria
isolated from African fermented foods against some common enteric pathogens (Mensah
et al., 1991; Olsen et al., 1995). Savadogo et al., (2004) reported the production of a
bacteriocin by L. fermentum strain isolated from dolo in Burkina –Faso. Kostinek et al.,
(2005) reported high production of hydrogen peroxide and a bacteriocin by a
heterofermentative strain of lactic acid bacteria isolated from fermented cassava.
5.5 Microbial Interactions during Nsiho Fermentation
There were very limited antimicrobial interactions observed beween the different species
of micoorganisms isolated from nsiho. There were no interactions between the different
species of lactic acid bacteria at the different stages of steeping and dough fermentation
of the dehulled maize grains as reported for whole maize grains by Olsen et al., (1995).
Olsen et al., (1995), again reported inhibition of lactic acid bacteria cultures isolated at
the early stages of fermentation during the production of kenkey from whole maize by
lactic acid bacteria isolates from the advanced stages of fermentation. The lack of
antagonism observed in the present work between the different lactic acid bacteria could
be due to the fact that they were not specifically selected from different stages of
fermentation.
Lactic acid bacteria to lactic acid bacteria interaction can be typified by antagonism
where bacteriocin produced by one species or strain inhibits or eliminates another
species. Interaction between L. fermentum and L. plantarum against P. acidilactici, could
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be due to microbial succession that takes place during maize fermentation which leads to
selection of stable flora (Halm et al., 1993; Hayford et al., 1999). Lactic acid bacteria that
persist to the advanced stages of fermentation showed inhibition against isolates from
early stages of fermentation (Olsen et al., 1995).
There were also no antimicrobial interactions observed between the different species of
lactic acid bacteria isolates and the yeasts; Saccharomyces cerevisiae and Candida krusei
in this present study.
However, the different species of lactic acid bacteria isolated from nsiho exhibited some
degree of antagonism against Debaryomyces and Trichosporon species. These two
isolates had been isolated at the start of steeping. Elimination of yeast species by lactic
acid bacteria could be due to antimicrobial substances produced by the lactic acid bacteria
species (Adams and Nicolaides, 1997). The present results are in agreement with
investigation by Jesperson et al., (1994) who reported a mixed flora comprising of
Candida, Saccharomyces, Trichosporon and Kluveromyces from raw maize during
steeping and early stages of kenkkey fermentation. They indicated that a natural selection
was observed during steeping and maize dough fermentation with Candida krusei and
Saccharomyces cerevisiae becoming the dominant species at the end of fermentation. The
dominance of Candida krusei and Saccharomyces cerevisiae have been confirmed by
Nyarko and Obiri-Danso, (1992); Obiri-Danso (1994).
Many authors have reported the co-existence of yeast and lactic acid bacteria in different
African fermented foods (Jesperson et al., 1994; Hounhouigan et al., 1998; Oyewole and
Odunfa 1990; Jesperson 2003; Nout 1980; Halm et al., 1993; Iwuoha and Eke 1996).
Other studies described the association as symbiotic (Wood and Hodges 1985; Saunders
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et al., 1972; Gobbeti et al., 1994). They indicated that yeast selectively utilized the
suitable amino acids after proteolytic activity of L. brevis subsp. linneri CBI resulting in
an enhanced yeast growth. Halm et al., (2003) also indicated that the lactic acid produced
by lactic acid bacteria creates an acidic environment suitable for yeast growth. Gobbeti et
al., (1994) explained that growth of lactic acid bacteria could occur in the presence of
high inoculum of yeast which results in an increased amino acid synthesis and subsequent
excretion by yeast cell autolysis creating a source of free amino acid that satisfies lactic
acid requirement. Leroi and Peduox, (1993) showed that carbon dioxide; pyruvate,
propionate, acetate and succinate produced by yeast were responsible for the stimulation
of lactic acid bacteria.
5.6 Technological Properties
The different isolates of lactic acid bacteria from nsiho displayed different rates of
acidification during steeping and dough fermentation. Overall, Lactobacillus fermentum
exhibited the fastest rate of acidification in maize slurry which gives it an advantage over
the other species in selecting for a starter culture. The decreasing of pH favours the
growth of yeast and characteristic during the spontaneous fermentation of cereal based
products (Sawadogo-Lingani et al., 2007, Sulma et al., 1991; Hounhouigan et al 1996;
Lei and Jacobson, 2004).
The ability of the isolates to secret amylase was examined on modified NA containing
2% starch. Isolates of L. fermentum, L. plantarum and L. brevis demostrated amylase
activity. Many authors have indicated the presence of amylolytic lactic acid bacteria from
cereal fermented products. Olasupo et al., (1996) isolated amylolytic lactic acid bacteria
from Ghanaian kenkey (fermented maize dough) and nono (Nigeria). Agati et al., (1998),
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found amylolytic L. plantarum strains from retted cassava in Nigeria and Congo
respectively, while amylolytic L. fermentum (strains Ogi E1 and Mw2) were isolated
from mawe and ogi in Benin. Hounhouigan et al., (1993b) reported some amylolytic
lactic acid bacteria in mawe from Benin whiles Johansson et al., (1995) also indicated
that amylolytic lactic acid bacteria accounted for 14% of the total lactic acid bacteria
isolated from Nigerian ogi.
None of the lactic acid bacteria isolates showed any protease activity. According to
Wieser et al., (2008), lactic acid bacteria play a minor role in protein hydrolysis.
Most of the lactic acid bacteria isolates produced exopolysaccharides which could
contribute to improvement of texture. According to Patricia et al., (2002) and Savadogo
et al., (2004) production of microbial exopolysaccharides contribute to the improvement
of texture and viscosity of fermented food products. The formation of exopolysaccharides
by lactic acid bacteria may function in foods as viscosifying agents, stabilizers,
emulsifiers, gelling agents and water binding agents (Van-der Berg et al., 1995; De-Vuyst
et al., 2001), and may play an important role in the rheological behaviour and texture of
the product (Sikena and Oba, 1998; De-Vuyst and Diggest, 1999). Kalui et al., (2009),
assessed production of exopolysaccharides and amounts ranging from 298.53 to 431 mg/l
were produced by L. plantarum and L. rhamnosus isolate from ikii, a spontaneously
fermented maize porridge.
5.7 Selection of Starter Cultures
One of the major aims of isolating starter cultures for use in the production of fermented
foods is to ensure consistency and to preserve the unique flavour, aroma and texture
attributes of the products (Annan et al., 2003; Glover et al., 2009). In this present work
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there was a drastic increase in acidity development with the inoculation of starter cultures
to the already existing microflora during steeping and dough fermentation. The decline in
levels of pH with corresponding rise in amounts of titratable acids have been similarly
reported by many authors ( Plahar and Leung, 1982; Jesperson et al., 1994; Halm et al.,
1996; Masha et al., 1998; Nche et al., 1994; Sanni et al., 1994., Hounhouigan et al.,
1996). Fermentations involving starter cultures of lactic acid bacteria have typically been
characterised by drastic drop in pH. Masha et al., (1998), reported a decrease in pH from
over 5.0 in the unfermented sample to final pH levels of 3.5 in Uji, a maize and millet
gruel, fermented with pure cultures of lactic acid bacteria and 4.1 in spontaneous
fermented Uji. Halm et al., (1996), using six strains of L. fermentum and one strain of S.
cerevisiae, as starter culture in undehulled maize dough fermentation, observed a rapid
decrease in pH from over 5.0 at the start of steeping maize kernels to values of 3.65 to
3.81 within 24 h of dough fermentation as compared to 3.90 in spontaneous fermented
dough. In the present study, however, the rate of acidification was more drastic than those
obtained from the undehulled maize within 24 h period. Akinrele, (1970) showed that
„Ogi‟ produced from a mixed culture of Lactobacillus and Acetobacter enriched the
nutrient quality by increasing the concentrations of riboflavin and niacin above that found
in both the unfermented grain and the „Ogi‟ produced by traditional spontaneous
fermentation. A mixed culture inoculum of L. plantarum, Lactococcus lactis and
Saccharomyces rouxii was developed for cultured „Ogi‟ production, which resulted in
increased rate of souring of the dough (Banigo et al., 1974). In another study, Masha et
al. (1998) compared in laboratory trials the fermentation of „Uji‟ using a starter culture of
lactic acid bacteria ( L. plantarum, L. brevis, L. buchneri, L. paracasei and Pediococcus
pentosaceus), employing the method of backslopping (inoculum from a previous
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fermentation) and by spontaneous means at 30ºC. They found that the lowest pH of 3.5
was attained with the lactic acid bacteria starter culture fermentation. Double-strain
starter culture combinations of lactic acid bacteria and yeasts have been reported in
several studies on sourdough to produce more aroma compounds and in many cases to
improve flavour than when used individually (Martinez-Anaya et al., 1990; Hansen and
Hansen, 1994; Meignen et al., 2001). In the present study, the levels of counts of added
starter cultures were more than that of spontaneous fermentation. The observed
significant increase in cell numbers is advantageous in providing enough enzymes to
hydrolyse the polysaccharides, hence, the production of more lactic acid and subsequent
reduction in pH.
5.8 Survival of Enteric Pathogens during Maize Steeping Inoculated with Starter
Culture
Lactic acid bacteria and yeast are often inhibitory to other microorganisms and this is the
reason of their ability to improve the quality and safety of fermented foods. The general
factors that contribute to the inhibiting properties include low pH, production of organic
acids, hydrogen peroxide, antimicrobial substances, as well as oxidation-reduction
potential (Mbugua and Njenga, 1992; Kingamko et al., 1994; Tanasupawat and
Komagata, 1995; Sawadogo-Lingani et al., 2010). In the present study four enteric
pathogens (Vibrio cholerae, Salmonella typhimurium, Escherichia coli and
Staphylococcus aureus) were inoculated into steeped maize containing different
combinations of starter cultures. Generally, the concentrations of the pathogens were
reduced during the steeping process with Vibrio cholerae completely eliminated after 48
h. The reduction and elimination of the pathogens could be due to the fact that, during
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maize kernel fermentation there was a drop in pH and production of other metabolites.
Similar findings have been confirmed by other researchers of the inability of enteric
pathogens to survive in acidifying fermenting foods.
A study by Lorri and Svanberg, (1994), indicated that, a group of children fed with lactic
acid fermented gruel had a mean number of 2.1 diarrhoea episodes compared to 3.5 for
the group fed with unfermented gruel. Similarly, Adams, (1990) suggested that lactic acid
bacteria are inhibitory to many other microorganisms when they are cultured together,
and this is the basis of the extended shelf life and improved microbiological safety of
lactic-fermented foods. Lactobacillus species can produce a variety of metabolites,
including lactic and acetic acids which lower pH, that are inhibitory to competing
bacteria, including Vibrio cholera, Salmonella typhimurium, Escherichia coli and
Staphylococcus aureus (Breidt and Fleming, 1997). Mensah et al., (1990), in their study
of maize dough weaning foods prepared by mothers in a Ghanaian village found that
the extent of contamination was higher in unfermented dough (5.0 log cfu/g) than in
fermented dough (4.0 log cfu/g). They also indicated that inoculation of maize dough and
porridge with Shigella flexneri and enterotoxinogenic Escherichia coli (ETEC) showed
that if the dough was fermented, it inhibited the inoculated bacteria to a greater extent.
Even after cooking, the porridge from fermented maize dough showed some inhibition of
the inoculated bacteria (Mensah et al., 1991). Effect of lactic fermentation on survival of
enteropathogens in cereal gruels, commonly used as weaning foods, were investigated by
Kingamkono et al., (1996) who added 28 enteropathogens to cereal gruels prepared from
low tannin sorghum and inoculated them with a lactic acid starter culture. After 6 h of
fermentation, Campylobacter strains were not detectable and after 12 h, Salmonella,
Shigella and Staphylococcus strains were also not detectable. Antimicrobial activity of
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241 lactic acid bacteria belonging to Lactobacillus plantarum, Pediococcus pentosaceus,
L. fermentum/reuteri and L. brevis isolated from various processing stages of maize
(corn) dough fermentation (for kenkey) were shown to inhibit other Gram-positive and
Gram-negative bacteria (Olsen et al., 1995). In this present study, the drastic reduction of
the enteric pathogens was similar to work done by Annan-Prah and Agyeman (1997) who
showed that the populations of enterotoxigenic Staphylococcus aureus J955 and 101685,
Staphylococcus aureus and Klebsiella pneumonia were reduced by 1-3 log units in Ga
kenkey and kenkey water.
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6.0 CONCLUSION
The results of this study have confirmed the fermentation of nsiho to be lactic acid
fermentation. This occurs during both steeping of dehulled maize grains and dough
fermentation where reduction in pH and corresponding increases in titratable acidity are
observed. There is close similarity between the fermentation of whole maize grains
during the production of Ga and Fanti kenkey and the fermentation of dehulled maize
during the production of nsiho. The lactic acid fermentation that occurs in both instances
involves Lactobacillus fermentum as the dominant lactic acid bacteria and also includes
Pediococcus pentosaceus and Pediococcus acidilactici. In nsiho fermentation, however,
Lactobacillus plantarum was found to represent a substantial part of the lactic acid
bacteria population but has rarely been reported in whole maize fermentation.
Lactobacillus brevis was also found to play an important role in the fermentation of the
dehulled maize grains. Yeasts are also involved in these fermentations and
Saccharomyces cerevisiae and Candida krusei dominate the yeasts biota in both whole
maize and dehulled maize fermentation.
The different lactic acid bacteria species from nsiho demonstrated different capacities to
acidify dehulled maize grains. Overall, Lactobacillus fermentum and Lactobacillus brevis
demonstrated the fastest rate of acidification and this occurred during 0 h to 8th hours of
steeping and 4th to the 8th hours of dough fermentation.
All isolates of lactic acid bacteria produced exopolysaccharide, a few showed amylolytic
activity whilst none showed protease activity. Through the use of lactic acid bacteria and
yeasts as starter culture, a more rapid reduction in pH was achieved both during steeping
and dough fermentation. However, no significant differences were assessed in the colour,
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texture, aroma, taste and overall acceptability of nsiho through the use of the starter
cultures as inocula enrichment.
Antimicrobial activity against four enteric pathogens, Salmonella typhimurium,
Staphylococcus aureus, Vibrio cholerae and Escherichia coli was demonstrated during
steeping of dehulled maize grains and was enhanced when lactic acid bacteria isolates
were included as starter culture. Vibrio cholerae was completely eliminated while the
others were drastically reduced to a level of 102 cfu/ml after 48 h of steeping.
Based on antimicrobial activity and technological properties exhibited by the isolates and
the sensory quality of nsiho, suitable starter culture selected for nsiho production
consisted of Lactobacillus fermentum, Lactobacillus brevis and Saccharomyces
cerevisiae.
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7.0 RECOMENDATIONS
1. Molecular methods should be carried out to confirm species of the dominant lactic acid
bacteria and yeasts responsible for nsiho fermentation.
2. The use of the starter cultures for the dough fermentation should be studied.
3. The aroma profile of nsiho produced using the different starter cultures as well as
during spontaneous fermentation should be studied.
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Appendix 1
Acceptability of white kenkey (Nsiho)
Name: Date:
Please taste all samples in the order indicated on your questionnaire and rate the taste, odour, texture
and overall acceptability of each of them according to the scale below. Please rinse your mouth in-
between sample tasting.
1. ____________ 2. ____________ 3._____________ 4. ____________5. ____________
6. ____________ 7. ____________ 8._____________ 9. ____________10. ____________
Aroma/Odour:
Sample code 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
9. Like extremely
8. Like very much
7. Like moderately
6. Like slightly
5. Neither like nor dislike
4. Dislike slightly
3. Dislike moderately
2. Dislike very much
1. Dislike extremely
Taste:
Sample code 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
9. Like extremely
8. Like very much
7. Like moderately
6. Like slightly
5. Neither like nor dislike
4. Dislike slightly
3. Dislike moderately
2. Dislike very much
1. Dislike extremely
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Texture
Sample code 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
9. Like extremely
8. Like very much
7. Like moderately
6. Like slightly
5. Neither like nor dislike
4. Dislike slightly
3. Dislike moderately
2. Dislike very much
1. Dislike extremely
Overall Acceptability
Sample code 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
9. Like extremely
8. Like very much
7. Like moderately
6. Like slightly
5. Neither like nor dislike
4. Dislike slightly
3. Dislike moderately
2. Dislike very much
1. Dislike extremely
Overall liking:
Which of the samples do you like most? ______________
Why? _____________________________________________________________________
Thank you for participating
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Appendix 2
Pattern of carbohydrate utilization of the dominant lactic acid bacteria from nsiho using API 50 CHL
Isolates
Carbohydrte L.
fermentum1
L.
fermentum2
L. brevis 1 L. brevis 2 L.
plantarum
P.
acidilactici
P.
pentosaceus
Glycerol - - - - - - -
Erythritol - - - - - - -
D-arabinose - - - - - - -
L-arabinose + ? + + + - +
Ribose + + + + + + +
D-xylose ? + + + - + +
L-xylose - - - - - + ?
Adonitol - - - - - - -
β methyl-xyloside - - - - - - -
Galactose + + + + + - -
D-Glucose + + + + + + +
D-fructose + + + + + + +
D-mannose + + - - + + +
L-sorbose - - - + - - +
Rhamnose - - - - - + -
Dulcitol - - - - - - -
Inositol - - - - - - ?
Mannitol ? - - - + - ?
Sorbitol - - - - + - ?
α methyl-D-mannose - - - - ? - ?
α methyl-D-glucoside - - - - - + -
N acethyl glucosamide - - - - + - +
Amygdaline - - + ? + - +
Arbutin - - + - ? ? +
Esculin ? + ? + + + +
Salicin + + - - + + +
Cellobiose - - + - + + +
Maltose + ? + + + + ?
Lactose + + + ? + - +
Melibiose + + + + + - +
Saccharose + + + + + - +
Trehalose - + + - + + +
Inulin - - - - - - -
Melezitose - - - + + - -
D-raffinose + + ? + - - -
Amidon - - - - - - -
Glycogen - - - - - - -
Xylitol - - - - + - -
β gentiobiose + - + - - + +
D-turanose - - - + + - -
D-lyxose + + - - - - -
D-tagatose - - - - - + +
D-fucose - - - - - - -
L-fucose - - - - - - -
D-arabitol - - - - - - -
L-arabitol - - - - - -
Gluconate + + + + + - -
2 ceto-gluconate - - + + - - -
5 cetoglunate + + ? + - - -
(+) positive, (-) negative, ?
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Appendix 3
Whole grains Dehulled grains
Steeped dehulled maize Fermenting dough
Dehulling machine Milling machine
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Slurry for ohu preparation Preparation of ohu
Cooking of ohu Mixing of ohu and fresh dough
Corn husk Washed husk for wrapping ohu
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Mixture of ohu and fresh dough Moulding of ohu
Moulding of nsiho Balls of nsiho ready for steaming/cooking
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Perforator for steaming Perforator and cooking pot
Nsiho packed in cooking bowl Nsiho on fire
Ball of nsiho Cooking of nsiho
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Cooked nsiho packed in basket Prepared nsiho for hawking
Nsiho in plate Preferred fish for consuming nsiho
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