ABSTRACT
This study was aimed at establishing the antimicrobial activity of lactic acid bacteria (LAB) on some food spoilage and pathogenic organisms. Serial dilutions of fermented maize and sorghum samples were performed and 0.1ml of the appropriate dilutions were inoculated on de Man Rogosa Sharpe medium for the isolation of lactic acid bacteria. Lactococcus and Lactobacillus species were isolated, identified and characterized. The test organisms were confirmed by biochemical tests to be Pseudomonas sp., Escherichia coli, Serratia marcescens, Bacillus sp. and Staphylococcus sp. The two LAB isolates were further confirmed molecularly to be Lactococcus specie 104.6 and Lactobacillus fermentum strain SN5. Using the agar well diffusion method, the two isolates inhibited the growth of all the test organisms except Pseudomonas aeruginosa which showed no zone of inhibition. The inhibitory activity of Lactococcus sp. 104.6 was the highest, 12.7±2.31 mm on S. marcescens, 11±1.00 mm on Staphylococcus sp. 9.6±1.52 mm on E. coli with the least inhibitory activity 8.3±1.52 mm on Bacillus sp. The highest inhibitory activity of Lactobacillus fermentum was also observed as 17.0±1.00mm on S. marcescens, followed by 14.3±1.15 mm on E. coli, 9.0±1.73 mm on Bacillus sp. with the least inhibitory activity 11±1.00mm on Staphylococcus sp. The co-culturing of lactic acid bacteria and test organisms indicated gradual reduction in the number of test organisms during the period of monitoring. The GC-MS analysis of Lactobacillus fermentum showed five volatile organic acids namely, acetic acid, propionic acid, isobutyric, butyric acid and isovaleric acid. The results of this study suggest that Lactococcus species and Lactobacillus fermentum can be used as biopreservatives in food industries rather than the synthetic chemical preservatives which is commonly used.
TABLE OF CONTENTS
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Title Page
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i
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Declaration
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ii
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Certification
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iii
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Dedication
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iv
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Acknowledgements
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v
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Table of Contents
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vi
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List of Tables
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x
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List of Figures
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xi
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List of Plate
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xii
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Abstract
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xiii
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CHAPTER 1: INTRODUCTION
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1.1 Background of the Study
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1
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1.2 Problem Statement
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2
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1.3 Justification
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3
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1.4 General Objective
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3
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1.4.1 Specific Objectives
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3
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CHAPTER
2: LITERATURE REVIEW
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2.1 Lactic
Acid Bacteria
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4
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2.2 Lactic
Acid Bacteria and Food Fermentation
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5
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2.3 Inhibition of Foodborne Pathogens by Lactic Acid Bacteria
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6
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2.3.1.
Organic acids
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7
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2.3.2.
Diacetyl
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8
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2.3.3 Hydrogen peroxide
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9
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2.3.4 Carbon dioxide
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9
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2.3.5
Reuterin
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10
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2.3.6
Bacteriocin
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10
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2.4 Lactic Acid Bacteria as Biopreservatives
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12
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2.5 Application of LAB as
Biopreservatives
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13
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2.5.1 Starter cultures for
fermented foods
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13
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2.5.2 Adjunct cultures
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13
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2.5.3 Bio-protective cultures
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14
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2.5.3 Probiotic cultures
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15
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2.6 Food Spoilage Organisms and Pathogens
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16
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2.6.1 Pseudomonas
species
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16
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2.6.2 Serratia marcescens
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17
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2.6.3 Escherichia coli
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18
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2.6.4 Staphylococcus aureus
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19
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2.6.5 Bacillus cereus
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20
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CHAPTER 3: MATERIALS AND METHOD
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3.1 Sample Collection
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21
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3.2 Collection of Test Organisms
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21
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3.3 Isolation and Characterization of Lactic
Acid Bacteria
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21
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3.3.1 Isolation of the lactic acid bacteria
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21
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3.3.2 Characterization of the Isolates
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22
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3.4 Antimicrobial Activity of the Isolates
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23
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3.4.1 Antimicrobial activity of isolates using
agar well diffusion method
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23
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3.4.2 Antimicrobial assay using the co-culturing
method
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23
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3.5 Molecular Identification of Isolates
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24
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3.5.1 DNA extraction
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24
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3.5.2 16s rRNA amplification
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24
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3.5.3 Sequencing
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25
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3.6 Analysis for the Volatile Organic Acids
Produced by the Isolates
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25
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3.7 Statistical Analysis
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25
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CHAPTER 4: RESULTS AND DISCUSSION
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4.1 Results
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26
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4.1.1 Identification of
isolates by biochemical tests
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26
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4.1.2 Growth characteristics of the bacterial
isolates at various temperatures, pH
and salt concentrations
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28
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4.1.3 Antimicrobial activity of the lab isolates
using agar well diffusion method
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30
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4.1.4 Antimicrobial activity of the LAB isolates
in co-culture with Staphylococcus specie at 37°C
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32
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4.1.5 Antimicrobial
activity of the LAB isolates in co-culture with Pseudomonas specie at 37°C
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34
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4.1.6 Antimicrobial
activity of the LAB isolates in co-culture with Serratia marcescens
at 37°C
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36
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4.1.7 Antimicrobial
activity of the LAB isolates in co-culture with Escherichia coli, at 37°C
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38
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4.1.8 Antimicrobial
Activity of the LAB Isolates in Co-culture with Bacillus specie
at 37°C
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40
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4.1.9 Changes in the titrable acidity of the
medium during separate culturing and
co-culturing of the LAB isolates and staphylococcus
specie at 37°C
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42
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4.1.10 Changes in the titrable acidity of the
medium during separate culturing and
co-culturing of the LAB and Pseudomonas
specie at 37°C.
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44
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4.1.11 Changes in the titrable Acidity of the
Medium During Separate Culturing and
Co-Culturing of the LAB and Serratia
marcescens at 37°C
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46
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4.1.12 Changes in the titrable acidity of the
medium during separate culturing and
co-culturing of the LAB and Escherichia coli at 37°C
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48
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4.1.13 Changes in the titrable acidity of the
medium during separate culturing and
co-culturing of the LAB and Bacillus
specie at 37°C.
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50
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4.1.14 Agarose Gel electrophoresis of the 16S rRNA
gene of the isolates
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52
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4.1.15 Gas chromatogram- mass spectrophotometer analysis
for the volatile organic
acids produced by the isolates
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56
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4.2 Discussion
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58
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4.2.1 Identification and
characterization of isolates
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58
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4.2.2 Antimicrobial activity of
the LAB isolates
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58
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4.2.3 Molecular
identification of the LAB isolates
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61
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4.2.3 Gas
chromatogram of volatile organic acids produced by Lactococcus specie 104.6
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61
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CHAPTER 5: CONCLUSION AND RECOMMENDATIONS
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5.1 Conclusion
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62
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5.2 Recommendation
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62
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References
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63
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LIST OF TABLES
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Page
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4.1
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Biochemical Test
Identification of Isolates
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27
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4.2
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Growth characteristics
of the bacterial isolates at various temperatures, pH and salt concentrations
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29
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4.3
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Mean diameter zone of
inhibition (mm) produced by the LAB isolates against different test organisms
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31
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4.4
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Identification of the
isolates By 16SRNA sequencing
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54
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LIST OF FIGURES
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Page
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4.1
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Survival of Staphylococcus
sp, Lactococcus sp. 104.6
and Lactobacillus fermentum during separate culturing and culturing in
a mixed population at 37°C
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33
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4.2
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Survival of Pseudomonas sp, Lactococcus sp. 104.6 and Lactobacillus
fermentum during separate culturing
and culturing in a mixed population at 37°C
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35
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4.3
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Survival of Serratia marcescens, Lactococcus sp. and L. fermentum during separate culturing
and culturing in a mixed population at 37°C.
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37
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4.4
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Survival of Escherichia coli, Lactococcus sp.
and Lactobacillus fermentum during separate culturing
and culturing in a mixed population at 37°C.
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39
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4.5
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Survival Bacillus sp., Lactococcus sp. and L. fermentum during separate culturing
and culturing in a mixed population at 37°C
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41
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4.6
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Changes in the titrable
acidity of the medium during separate culturing and co-culturing of the Lactococcus
sp. 104.6, L.
fermentum and Staphylococcus sp. at 37°C
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43
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4.7
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Changes in the titratable acidity of the medium during separate
culturing and co-culturing of the Lactococcus sp., L. fermentum and Pseudomonas
sp at 37˚C
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45
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4.8
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Changes in the titratable acidity of the medium during separate
culturing and co-culturing of Lactococcus sp., L. fermentum and Serratia marscesens at
37°C
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47
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4.9
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Changes in the titrable acidity of the medium during separate culturing
and co-culturing of Lactococcus sp
104.6, L. fermentum and E. coli at
37°C
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49
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4.10
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Changes in the titrable
acidity of the medium during separate culturing and co-culturing of Lactococcus sp., L. fermentum and
Bacillus sp. at
37°C
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51
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4.11
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Gas Chromatogram of
Volatile Organic acids produced by Lactococcus specie 104.6
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57
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LIST OF PLATE
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Page
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4.1
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Agarose gel
electrophoresis of the 16S rRNA gene of the Lactococcus specie 104.6
(Lane A), Lactobacillus fermentum strain SN_5 (Lane C). Lane N
represents the 100 bp molecular ladder
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53
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CHAPTER
1
INTRODUCTION
1.1 BACKGROUND
INFORMATION
In Africa, majority of the foods consumed are
fermented. The fermentation of such foods is usually carried out by few groups
of bacteria and yeasts. The lactic acid bacteria are involved in fermentation
of many African foods and beverages (Wassie and Wassie, 2016). Lactic acid
bacteria (LAB) are a group of Gram positive, non-spore forming, anaerobic or
facultative aerobic cocci or rods, which produce lactic acid as one of their
major fermentation products of the carbohydrates metabolism (Hayek and Ibrahim,
2013). LAB through their fermentative ability enhances the shelf life of the fermented
product (Sharma et al., 2012). They
produce varieties of antimicrobial metabolites during the process of
fermentation such as lactic acid, acetic acid, ethanol, hydrogen peroxide,
diacetyl and others. (Diop et al.,
2010). These metabolites produced confer preservative ability on them as a
natural competitive means to overcome other microorganisms sharing the same
niche (Oliveira et al., 2008).
Nowadays consumers prefer food with few or no chemical preservatives. As a
result, interests in LAB have increased over period because of their safe
association with fermented foods which are consumed by humans. Attention has
focused on the ability of LAB to produce proteinaceous substances known as
bacteriocins that inhibit the growth of pathogens such as Listeria, Clostridium, Staphylococcus, Bacillus spp. and Enterococcus spp.
Intensive study of bacteriocins produced by LAB are being carried out because
of their antagonistic activities against food-borne bacteria. Bacteriocin
producing strains of LAB may be of great significant in competing with other
organisms in intestine. They are made up of a biologically active protein
moiety which have a bactericidal mode of action and are specific while
attaching to cell receptors. Lactic acid bacteria thus enhance food safety,
improve organoleptic attributes, enrich nutrients and increases overall health
benefits (Steele et al., 2013).
Most
food spoilage meant for human consumption is caused by microorganisms. Fungi
and bacteria when given access to foods that are unprotected rapidly increase
in population, producing distasteful chemicals and toxins in some cases (Pitt
and Hocking, 2009). Due to the
ubiquitous nature of microbes, they have such enormous populations, and are
often dispersed as spores in air, water, or soil thereby they colonizing
unprotected foods. Humans developed two main strategies in order to prevent
microbial food spoilage; reducing their
access to the susceptible foodstuffs and
inhibiting growth thereby limiting population size by creating an unfavourable
environment (Rawat, 2015). Both fermented and unfermented food products are
known to be susceptible to spoilage during storage. Their spoilage during
storage is said to be due to the presence of the spoilage microbes and the
enzymes they produce, which breakdown the food/food product into entirely
different substances leading to changes in their organoleptic properties
(Fadahunsi et al., 2013). Although
starter cultures have been widely used in some fermentation of foods to enforce
uniformity in their composition, reduced shelf life still pose as a major
problem. The consumption of products contaminated with spoilage and pathogenic
microorganisms could serve as a potential health threat hence the need to
control microbial contamination of foods.
1.2 PROBLEM STATEMENT
These days, consumers are concerned about foods
preserved with chemical substance which may leave chemical residues in their
bodies after consumption and cause adverse health effects when accumulated.
However, they now rely on less processed foods which may harbour microorganisms
which can cause food spoilage or food-borne diseases.
1.3 JUSTIFICATION
The solution to food preservation challenges can be
the use of antimicrobial metabolites produced by microorganisms with the status
generally recognized as safe (GRAS) of which lactic acid bacteria is one. Most
researchers have reported the antimicrobial potential of lactic acid bacteria
using their cell free supernatant/bacteriocin (Adebayo and Aderiye, 2007;
Sharaf and Al Habi, 2011). This work focused on the evaluation of the strains
that produce antimicrobial metabolites and have wide spectrum activity on food
pathogens so they can be useful in food preservation in food industries.
1.4 GENERAL
OBJECTIVE
To characterize strains of Lactic acid bacteria
isolated from some fermented foods consumed in our localities and to evaluate
their antimicrobial activity on some food spoilage microorganisms and
food-borne pathogens.
1.4.1
Specific
objectives
1. To
isolate and identify phenotypically, biochemically and molecularly, Lactic acid
bacteria from fermented cereals, namely; maize and sorghum.
2. To
determine the growth characteristics of the Lactic acid bacteria isolates in
various conditions.
3. To
determine the antimicrobial activities of the LAB on some food spoilage
microorganisms and food-borne pathogens using agar well diffusion and
co-culturing method.
4. To
identify the organic acids apart from lactic acid produced by the strains using
GC-MS analysis.
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