ABSTRACT
Fresh cow milk was purchased from Fulani hendsmen while strain of Streptococcus thermophillus coded (9) and Lactobacillus acidophilus (LAB 1 and 2) coded 10 and 13 respectively were obtained from microbiology laboratory in London Metropolis University in United Kingdom. The bacteria strains were purified from 150C to 450C on MRS medium. 400C was observed best for optimal temperature with more viable (LAB) on the milk. Raw milk, 5% skimmed milk, was homogenized and pasteurized at (630C for 30min) cooled to 450C. 500ml of treated milk was inoculated in single with 1ml of (LAB) strain and incubated at 400C or 9h. The samples was cooled and preserved for further use having commercial yoghurt as (control). Proximate, physicochemical and microbial analysis was carried out. There were significant decrease (P<0.05) in total bacteria count of fermented nono samples inoculated with Streptococcus thermophillus coded (9) and Lactobacillus acidophilus (LAB 1 and 2) coded 10 and 13 respectively after inoculation and fermentation with values (8.2 x 106cfu/ml to 7.0 x 106cfu/ml), (7.3 x 106 to 6.3 x 106cfu/ml) and (6.0 x 106 cfu/ml to 5.9 x 106 cfu/ml) respectively, while fresh milk recorded (1.60 x 104cfu/ml). Control recorded no growth on total bacteria load, total viable count, and total coliform count. The total viable count of fresh milk was (1.45 x 104 cfu/ml) while sample inoculated with Streptococcus thermophillus coded (9) and Lactobacillus acidophilus (LAB 1 and 2) coded 10 and 13 after inoculation and fermentation recorded (7.4 x 105 cfu/ml to 4.9 x 105 cfu/ml) (6.2 x 105 cfu/ml to 4.0 x 104 cfu/ml), (6.0 x 104 cfu/ml to 5.9 x 105cfu/ml) respectively.No growth of coliform on samples inoculated with Streptococcus thermophillus coded (9) and Lactobacillus acidophilus (LAB 1) coded (10) while sample inoculated with Lactobacillus acidophilus (LAB 2) coded 13 recorded counts from (3 x 104 cfu/ml to 1 x 104 cfu/ml) after inoculation and fermentation respectively, Crude protein of the samples ranged from (3.5 to 4.92%),sample coded (13) recorded the highest protein content while control was observed lowest, crude fiber of fermented nono samples and control has no significance difference (P>0.05) with the value (0.15 to 0.21%), ether extract of the samples ranged from (2.61 to 3.86%) as fermented nono samples coded (9, 13) recorded no significance difference (P>0.05) with the values (3.85 and 3.86%) respectively, Ash content of the samples ranged from (0.72 to 1.21%), carbohydrate content of the samples ranged from (9.47 to 13.09%). Energy value of the samples ranged from (89.62 to 98.52%). However, control was significantly low in crude protein, fat and ash but has highest value in carbohydrate. The sensory attributes showed that control was significantly high in mouth feel, sweet, sourness, after taste, consistency and general acceptability than other samples. This might be influence of ingredients added during production of the control.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table of Content vi
List of Tables vii
List of Figures viii
Abstract ix
CHAPTER 1:
INTRODUCTION
1.1 Background
of Study 1
1.2 Problem
Statement 4
1.3 Objective
of the Study 4
1.4 Justification
of the Study 5
CHAPTER 2: REVIEW OF RELATED LITERATURE
2.1 Traditional
Food Fermentation 6
2.2 Microorganisms
Associated with Traditional Food Fermentation 7
2.2.1 Lactic acid bacteria 7
2.2.2 Acetic
acid bacteria (AAB) 9
2.2.3 Yeasts 10
2.2.4 Moulds 12
2.3. Spontaneous
Fermentation 13
2.3.1 Back slopping fermentation 13
2.4 Starter
Cultures 14
2.5 Introduction of Bacteria capable of
producing Antimicrobial Substance
for Starter Culture 16
2.6 Development of Inoculum (Starter
culture) 17
2.6.1 Single and mixed culture fermentation 18
2.6.2 Selection of (LAB) for starter culture 19
2.6.3 Genetic improvement of starter cultures 19
2.6.4 Functional starter cultures 20
2.6.5 Multi-strain dehydrated starter culture 24
2.7 Scale
up Strategy 26
2.8 Antimicrobial
Activity 28
2.8.1 Bacteriocin production 29
2.9 Enzymatic
Activity cf Fermenting Microorganisms (Amylolytic activity) 31
2.9.1 Applications of amylases 32
2.9.2 Proteolytic
activity 32
2.9.3 Lipolytic activity 33
2.9.4 Acidification activity 33
2.10 Prospects and of future use Starter Culture for Traditional
African Food Fermentation 34
2.11. Dairy Products 35
2.11.1 Different products
from dairy foods 38
2.11.2 Cheese
production and microbial activities 41
2.12
Major roles of Microorganisms in Cheese Production 42
2.13 Yoghurt Production and the Microbial
Activities Involved 43
2.13.1 Major roles of microorganisms in yoghurt
production 43
2.14 Ice Cream Production and the Microbial
Activities Involved 44
2.15 Factors Affecting Spoilage in Dairy Products
46
2.15.1 Prevention of microbial spoilage in milk. 48
2.16 Health Benefit of
Milk to Man 49
CHAPTER 3: MATERIALS
AND METHODS
3.1 Materials 51
3.2 Preparation
of Micro Media 51
3.3 Preliminary
Test for presence Isolate (subcultured from vial to plate) 52
3.3.1 Subcultured
from plate to slant bottle 52
3.3.2 Concentration of the starter culture
determination 53
3.3.3 Gram staining 54
3.3.4 Catalase test 54
3.3.5 Indole
test 54
3.4.6 Citrate
utilization test 55
3.3.7 Hydrogen
sulphide (H2s) production test 55
3.3.8 Sugar fermentation test 56
3.4 Determination of
the Optimal Fermenting Temperature of the Single Starter Culture 56
3.4.1 Production of fermented “nono”
59
3.5 Proximate
Analysis 61
3.5.1 Determination of moisture
content 61
3.5.2 Fat determination 61
3.5.3 Determination of ash content 62
3.5.4 Determination of protein content 63
3.5.5 Determination of
total carbohydrate 64
3.6
Physicochemical Analysis 64
3.6.1 Determination of pH level of
the samples 64
3.6.2 Total solid determination 65
3.6.3 Determination of
total soluble solid 65
3.6.4 Determination of lactose 66
3.6.5 Determination of
total titratable acidity 67
3.6.6 Determination of temperature 67
3.7 Microbial
Analysis 67
3.7.1 Determination of total viable count 67
3.7.2 Total microbial count 68
3.7.3
Total coliform count 69
3.8 Sensory Evaluation 70
3.9 Statistical Analysis 70
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Preliminary Test Result 71
4.2 Identification of Coded Isolates
72
4.3 pH Values of Uninoculated
Treated Milk (control) and Inoculated Samples 73
4.4 Proximate Composition of the
Samples 87
4.5 Physichochemical Composition of
Samples 92
4.6 Microbial Analysis of Samples 96
4.7 Sensory Evaluation of Samples 103
CHAPTER 5: CONCLUSION AND RECOMENDATIONS
5.1
Conclusion 107
5.2 Recommendations
107
REFERENCES 108
APPENDIX
LIST OF TABLES
2.1: Lactic acid bacteria and
their health benefits for food
Production companies 23
2.2: Dairy products and typical types of
spoilage microorganisms or
microbial activity 40
4.1: Identification of the Coded
Isolates 72
4. 2: Physichochemical Composition of fermented Nono and commercial
Yoghurt (Control) 95
4.3: Proximate Composition of fermented Nono and commercial
Yoghurt (Control)
91
4.4: Quality
attributes of fermented Nono and commercial yoghurt (Control) 106
LIST OF FIGURES
3.1: Flow chart of
preliminary test for novel single strain starter culture 57
3.2: Flow Chart
for the Production of
fermented‘‘Nono’’ 60
4.1: pH value of inoculated and uninoculated fermenting cow milk at
150C
for 24h 74
4.2: pH value of Inoculated and uninoculated fermenting fresh cow
milk
at 250C 76
.
4.3: pH value of Inoculated and uninoculated fermenting fresh cow
milk
at 250C 78
4.4 pH
value of inoculated and uninoculated fermenting fresh cow milk
at 300C 80
4.5: pH value of inoculated and uninoculated fermenting fresh cow
milk
at 350C 82
4.6: pH value of inoculated and uninoculated fermenting fresh cow
milk
at 400C 84
4.7: pH value of inoculated and uninoculated fermenting fresh cow
milk
at 450C 86
4.8:
Total Viable Count 97
4.9: Total
Microbial Load. 99
4.10: Total Coliform count. 102
LIST OF PLATES
Plate 3.1: Preparation of starter
culture process 52
Plate 3.2:
Novel Single Strain starter culture process 58
Plate 3.3:Fermented Nono and the
control (Commercial plain yoghurt) 59
CHAPTER 1
1.0 INTRODUCTION
1.1 BACKGROUND OF
STUDY
Nono is a chance traditionally fermented Nigerian diary product produced and commercialized
by the Fulani tribe. It is used as a staple food in the West African Sub-region and the Middle
East where it is known as Dahi or Lassai (Nahar et al., 2007; Nebedum and obiakor,2007) Untreated milk spoils
within a short time due to microbial activity which gives milk a sour taste and
causes precipitation of casein, a processes known as “curdling”. Off flavours
and off odours are developed, causing serious wastage of this very important
food product. Bacterial actions in milk result in acid production which ceases
when the acid strength is increased. Subsequently, mould and yeast present in
milk flourish in the acid medium. They utilize the acid and cause chemical
changes producing an alkaline condition. Finally, bacteria infiltrate and
decompose milk constituents to by-products that are of public health
significance. Unhygienic milking conditions and uncontrolled processing methods
may create disease conditions such as tuberculosis, typhoid fever, septic sore
throat, diphtheria and scarlet-fever. (Shakuntala et al, 2008).
Thus, the
introduction of single strain starter culture for production of fermented nono
will establish improved standardized quality fermented nono, reduce wastage and
improve the availability of the milk product. Inherent microorganisms are
responsible for traditional food fermentation. Meanwhile, starter cultures are
introduced to ensure and enhance fermentation and favourable processing
conditions which can be selected to ensure desired quality (Adesulu and
Awojobi. 2014). These processes encourage development of a desirable safe
microflora, which preclude growth and sporulation of spoilage bacteria and food
borne pathogen. With the increasing demand for natural preservation techniques,
introduction of fermentable microorganisms on some foods is gaining interest
(Adesulu and Awojobi, 2014). Acid producing microorganisms show some level of
food safety by inhibiting the growth of undesirable pathogens such as Ecoli, Enterobacter agglomerans, Listeria
monocytogenes and Pseudomonas spp.The
introduction of viable lactic acid bacteria as starter cultures has gained
interest to standardize traditional fermentation steps. Many studies have focused on characterization
of these microorganisms that are involed in traditional fermentation (Owusu-Kwarteng
et al., 2012; Ekwem, 2014;
Obinna-Echem et al., 2014)
Traditionally, milk
can be fermented by natural occurring microorganisms, especially lactic acid
bacterial generally improves palatability, digestibility, and nutritive value
of milk products (Achi, 2005).Pure bacteria from wild environs of fermentable
foods display diverse metabolic activities that differ strongly from mixed
starter cultures used in many food industries
(Lionie Marine Schutte, 2013). These include the viability state and
ability to survive growth difference behaviour, abilty to survive and flourish
on a perticlar food or raw material, antimicrobial properties and flavor and
aroma attributes. Starter culture of acid
producing bacteria needs to inhibit the growth of other non acid production microorganism by production of antimicrobial substance such as bacteriocin. Ability
of bacteria to produce Lactic acid during fermentation also reduces the
level of antinutrients such as lectin, oxalate, tannins, saponins etc (Ayad et al., 2002). The amount of vitamins
can be improved in the fermented foods.
Preservative
activities of lactic acid producing microorganisms have been observed in many
fermented foods and the increase lactic acid level to 4.5 during fermentation
which killed or retard the existence of spoilage organisms, leading to food
deterioration, food infection and illness.(Schnurer and Magnusson, 2005). The
expiration period or time of the said fermented food is prolonged. Lactic acid
bacteria also produce some inhibitory properties such as bacteriocin and acetic
acid against other bacteria (Oyewole, 2012).Some bacteria capable of producing
lactic acid during fermentation have been proved with anti-tumour effects. They
as well produce some organic acids capable of killing and retard the growth of
fungal such as benzoic acid, lactic acid, sorbic acid and nucleic acid. Thus,
introduction of lactic acid bacteria as a starter culture helps inhibit and
kill the non acid tolerant microorganisms during fermentation within
environment.
Furthermore,
bacteria capable of producing organic acid (lactic acid) during fermentation
has been demonstrated to inhibit growth of gram-negative bacteria which are
highly resistant to most processing (Lionie Marine Schutte, 2013). Thus,
fermented foods can control diarrhoeal diseases in children. In recent years,
interest has increased on development of latic acid bacteria starter culture
for most traditionally fermented foods globally. Such lactic acid bacteria
starter cultures would be developed with
bacteria isolated from naturally ferments of such traditional foods. Use of
starter culture in traditionally fermented foods will help sustain uniformity,
eliminate all undesirable microorganisms and enhance shelf stability of such
product. The texture, colour, flavor, taste, aroma and even the nutritive value of
such products will be the same Such products are always reproduceable (Blandino et
al., 2003).The trust of this study is to employ novel single strain of
lactic acid bacteria as starter culture from isolates of naturally fermented
milk for traditionally fermented nono production.
1.2 PROBLEM
STATEMENT
Milk also known as Nono is one of
the most valued natural foods simply because of its high content of most
essential nutrients, it is fermented
into at least 400 different specialty products globally (Willey et al., 2008).numerous microorganisms are involved in natural fermentation of this
product. Unfortunately, there is no unified method and standard culture for
locally or traditionally fermented nono production. Resulting to continued
contamination and variations on the nutrient composition, texture, flavour, and
microbial load of the final product. Novel single strain lactic bacterial
starter culture is needed to produce fermented nono of consistent and safe quality;
and rich in probiotics for good health of consumers.
1.3 OBJECTIVE OF
THE STUDY
The main objective of the study was
the potential use of novel single strain starter culture for production of a
traditionally fermented ‘Nono’ based on lactic fermentation.
Specific Objectives of the Study are
to
i. Determine the potential
novel of single strain lactic acid bacteria starter culture for traditionally
fermentation of nono.
ii.
Produce traditionally fermented Nono using
single strain starter culture.
iii.
Determine the proximate properties of the
traditionally fermented Nono
iv.
Determine the physicochemical properties of the
traditionally fermented Nono
v.
Examine
microbial quality of the traditionally fermented Nono
vi. Determine the organoleptic characteristics
of the traditionally fermented Nono
with commercia yoghurt as control.
1.4 JUSTIFICATION OF THE STUDY
The use of novel single strain
starter culture for traditionally fermented ‘nono’ product will result in
unified product quality, preclude pathogenic microorganisms and improve high
content of probiotics .This will make commercial production of this product
more feasible and reliable with persistent unique quality and acceptable to
consumers and enable the product enter international market, which will creat
more job opportunities to the people.
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