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Title of Thesis

Tariq Masud
Institute/University/Department Details
Department of Biological Sciences/ Quaid-i-Azam University Islamabad
Biological Sciences
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
lactic acid bacteria, yoghurt, lactococcus phasges, dahi, lactic count, titratable acidity, lactococcus lactis, lyophilized cultures, bacteriophage infection, s. thermophilus, l. delbrueckii

The first part of the project deals with the evaluation of wild strains of Lactic Acid Bacteria for the selection of starter culture for yoghurt making. A total of fifty indigenous dahi samples collected randomly from local markets of Rawalpindi and Islamabad were subjected to bacteriological and chemical examinations. Mean lactic count, titratable acidity and pH recorded were log 7.53/ml, 1.16% and 4.12 respectively. The following lactic acid bacteria were identified and their incidence is given in parentheses: L. delbureckii ssp. bulgaricus (86%), S. thermophilus (80%), S. lactis (74%), L. helveticus (34%), S. cremoris (30%), L. casei (20%) and L. acidophilus (14%) respectively. The strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, selected on the basis of culture activity test were further examined for their optimum growth temperature, generation time and titratable acidity. The results demonstrated that the generation time of S. thermophilus was relatively high as compared to L. delbrueckii ssp. bulgaricus strains. It was also noted that there was a variation among each strain for these tested parameters. Later on, two strains each of high (H) and weak (w) lactic acid producer of S. thermophilus and L. delbrueckii ssp. bulgaricus were evaluated for their symbiotic association at the recommended conditions of yoghurt making in various ratio€™s. It was observed that mixed culture of S. thermophilus and L. delbrueckii ssp. bulgaricus produced more lactic acid as compared to individual strain. Moreover, it was observed that 1:2 ratio of S. thermophilus and L. delbrueckii ssp. bulgaricus showed maximum acid production in all the tested combinations. Finally these selected combinations of 1:2 ratio of S. thermophilus and L. delbrueckii ssp. bulgaricus were again evaluated both chemically and organoleptically. It was found that chemical properties effect the organoleptic properties of the final product. The combination of S. thermophilus (H) and L. delbrueckii ssp. bulgaricus (w) was liked more as compared to other tested combinations. These strains were further tested for their survival rate during freeze-drying by using different cryoprotective agents viz skim milk, β-glycerophosphate, Na-glutamate, mannitol and sorbitol. There was non significant difference between log viable counts of these indigenous strains before and after freeze-drying for 12 months of storage at 0ºC in all the cryoprotective agents except sorbitol. These data indicate that these wild strains of S. thermophilus and L. delbrueckii ssp. bulgaricus can produce a good quality yoghurt on the basis of these observations recorded.

In part II, whey samples of indigenous dahi were also examined for the presence of virulent phages. A lytic bacteriophage Tk1 of Lactococcus lactis was isolated and was then characterized on the basis of their plaque morphology, lytic activity, latent period, burst size and phage morphology. In a subsequent experiment, variation was also detected among other isolated lactococcal phages for these parameters. All these phages were classified into group B of Bradley. The results of further study revealed that the bacteriophages belonging to this geographical region are different from the bacteriophages those reported in other countries i.e. Germany and Canada on molecular characterization.

The present study has provided some excellent understanding about the yoghurt starter culture and lactococcal phages in this region. This might help to create a base-line for research and development regarding scientific production of yoghurt, cheese and allied products which lead in acquisition of self sufficiency in the production and use of this vital element of the dairy industry in future.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
201.01 KB
2 1 Introduction 1
76.47 KB
3 2 Literature Review 6
684.24 KB
  2.1 Fermentation 6
  2.2 Nomenclature Of Fermented Milk 7
  2.3 Starters For Milk Fermentation 8
  2.4 Biochemical Composition Of Dahi 9
  2.5 Characteristics Of Yoghurt Culture 12
  2.6 Selection Of Yoghurt Culture 15
  2.7 Freeze Drying Of Cultures 16
  2.8 The Process 16
  2.9 Growth Medium 16
  2.10 Growth Conditions 17
  2.11 Harvesting Of Bacterial Cells 17
  2.12 Effect Of Cryoprotectives 18
  2.13 Dehydration And Rehydration Of Lyophilized Cultures 18
  2.14 Survival Of Lab In Different Cryoprotectives 19
  2.15 Bacteriophage Infection 21
  2.16 Isolation. Classification And Morphology Of Phages 22
  2.17 The Adsorption Of Phage Particles 225
  2.18 Temperature 26
  2.19 Lysogeny 26
  2.20 Mechanism Of Phage Resistance 27
  2.21 Plasmid Based Resistance 29
4 3 Materials And Methods 32
1549.2 KB
  3.1 Dahi Analysis 32
  3.2 Microbial Analysis 32
  3.3 Chemical Analysis 32
  3.4 Screening Of Isolates For Sufficient Acid Production 33
  3.5 Optimum Growth Temperature 33
  3.6 Cultures 34
  3.7 Heat Treatment Of Milk 34
  3.8 Preparation Of Yoghurt By Using Different Ratio €™s Of Starter Culture 34
  3.9 Preparation Of Yoghurt By Selected Combinations 34
  3.10 Culture Preparation 35
  3.11 Harvesting 35
  3.12 Suspension Of Cells 35
  3.13 Freezing Of Cell Suspensions 35
  3.14 Storage And Survival Rate 36
  3.15 Origin Of Bacteriophages 36
  3.16 Determination Of Phage Sensitivity 36
  3.17 Isolation And Growth Of Phages 37
  3.18 Determination Of Lytic Activity 37
  3.19 One Step Growth Experiment 37
  3.20 Phage Concentration And Purification 37
  3.21 Electron Microscopy Of Phages 38
  3.22 Stability Of Baceriophages 38
  3.23 Bacterial Strain, Phages And Growth Media 38
  3.24 SDS-Polyacrylamide Gel Electrophoresis (Page ) 38
  3.25 Generation Of Polyclonal Antisera 39
  3.26 Western Blotting 39
  3.27 Restriction Enzyme Analysis 39
5 4 Results 41
664.84 KB
  4.1 Dahi Analysis 41
  4.2 Incidence Rate Of Lactic Acid And Bacteria 41
  4.3 Culture Activity 42
  4.4 Comparison Among Strains Of S. Thermophilus And L. Delbrueckii .Ssp Bulgaricus For Their Developed Acidity 42
  4.5 Maximum Growth Conditions 43
  4.6 Growth Patterns At Optimum Temperatures 43
  4.7 Pattern Of Acid Production 44
  4.8 Symbiotic Relationship Between The Selected Strains Of S. Thermophilus .And L. Delbureckii Ssp. Bulgaricus At 42°C 44
  4.9 Chemical Composition Of Yoghurt Prepared From The Best Composition Of S. Thermophilus And L. Delbureckii Ssp. Bulgaricus 44
  4.10 Organoleptic Evaluation Of Yoghurt 45
  4.11 Viability Of Indigenous Strains 45
  4.12 Effect Of Selected Cryo-Protectives For 12 Months Stored At O°C 46
  4.13 Origin Of Phages 47
  4.14 Host Specificity 47
  4.15 Lytic Activity 47
  4.16 One-Step Growth Curve 47
  4.17 Morphology 47
  4.18 Isolation Of Bacteriophages 48
  4.19 Plaque Morphology 48
  4.20 Single Step Growth Curve 48
  4.21 Stability Of Growth Curve 48
  4.22 Phage Morphology 49
  4.23 Sds-Page Analysis Of Bacteriophage Lysates 49
  4.24 Western Blot Analysis 50
  4.25 Dna Endonuclease Restriction Analysis 51
6 5 Discussion 79
1267.73 KB
  5.1 Literature Cited 99
  5.2 Appendix A: Protocols For Various Tests