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
naturaly 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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