CHARACTERIZATION OF β-GALACTOSIDASE BY LACTIC ACID BACTERIA FROM 
MILK AND TRADITIONALLY FERMENTED MILK PRODUCTS FROM IBADAN 
 
  
BY 
 
 
PARKHA ONI AYODELE 
B.Sc. (HONS) (ZARIA), M.Sc. MICROBIOLOGY (IBADAN) 
     MATRIC NO. 84165 
 
 
  
                A Thesis in the Department of   
                      MICROBIOLOGY 
 
 
Submitted to the Faculty of Science in partial fulfillment of 
 the requirements for the award of the Degree of 
 
 
DOCTOR OF PHILOSOPHY (Ph.D) 
 
of the  
 
 
UNIVERSITY OF IBADAN, NIGERIA 
 
 
JANUARY, 2014 
 
 
 
 
 
 
 
 
 
ABSTRACT 
    Lactose intolerance (a condition in which man elucidates an immune reaction towards the presence of 
lactose due to inability to produce enzyme lactase) is a major nutritional deficiency among some adult 
consumers of milk and other dairy products worldwide. β–galactosidase hydrolysis of milk is one of 
the promising enzymatic applications in dairy industries for reducing lactose intolerance of milk 
products. However, plant and animal sources cannot meet the high demand of the enzyme in food 
industries. Hence, the aim of this study was to characterize β–galactosidase production by Lactic 
Acid Bacteria (LAB) isolated from locally fermented milk products. 
Raw milk from Sokoto Gudali was collected from Fulani settlement in Ojoo, Ibadan along with some 
fermented milk products („‟Nono‟‟ and „‟Wara‟‟). LABs were isolated from them and identified using 
conventional methods. The ability of the isolates to hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-
galactopyranoside (X-Gal) was used to screen for β–galactosidase production. Isolates with the best 
β-galactosidase production were selected. The enzyme was extracted and optimization of growth 
conditions (temperature, pH, nitrogen, carbon sources, inoculums size and inoculums age) for β-
galactosidase production was carried out using o-nitrophenyl-β-D-galactopyranoside (ONPG). The 
enzyme produced was characterized using pH, temperature, metal and non-metal ions, and inhibitors. 
Purification of the enzyme was carried out using dialysis and chromatographic methods. Hydrolytic 
effects of the purified β–galactosidase were determined in different concentrations of lactose using 
standard method.  Data were analyzed using descriptive statistics. 
The isolated bacteria were identified as Lactobacillus plantarum (G11, E13 and E36), L. brevis, L. 
casei, L. lactis, Leuconostoc lactis, Streptococcus sp, and Bacillus subtilis.  Lactobacillus plantarum 
°
(G11) had optimum growth value of 4.2 at 20 C and pH 7.0 with maximum enzyme value of 6.2U/mL 
at 30 hrs. The optimal β-galactosidase production occurred at neutral pH and 6% inoculum size. The 
best inoculums age varied between 18 hrs and 36 hrs. The best carbon source for enzyme production 
was raffinose with maximum value of 0.3U/mL while minimum activity was found in fructose with 
0.2U/mL. The best nitrogen source was NH4NO3 with maximum value of 0.5U/mL and yeast extract 
had minimum value of 0.1U/mL.  β–galactosidase activity increased with increase in molar 
concentration of the mono-valent chloride ions in which the highest was recorded in KCl at maximum 
value of 0.08U/mL while the minimum value of 0.001U/mL was obtained by NaCl at  a concentration 
of 0.2 mmol respectively. The best sulfate ion was CuSO4 with maximum activity value of 0.2 U/mL 
at 0.2 mmol concentration and minimum value of 0.007 U/mL at 0.1 mmol by ZnSO4. The best 
enzyme inhibitor was KCN with maximum activity of 0.2U/mL at 0.2 mmol.  The specific activity of 
β-galactosidase was 292.5 U/mg, 104.2 U/mg and 585.46 U/mg for G11, E13 and E36 respectively. 
ii  
 
 
The hydrolytic effects of the purified β-galactosidase showed a maximum yield of 35.8% glucose, 
19.3% galactose and 35.3% glucose and 18.5% galactose at 80% and 60% lactose concentration 
respectively.  
β-galactosidase produced by Lactobacillus plantarum strain achieved lactose hydrolysis and could be 
of potential application for production of low lactose dairy products for consumption by lactose 
intolerant people. 
 
Keywords: Lactose intolerance, Lactic acid bacteria, β-galactosidase production, Hydrolysis  
   effect,  Enzyme characterization 
Word count: 499 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ii i 
 
 
 
 ACKNOWLEDGEMENT 
 I give all glory to God almighty for His protection over me for all this while. His grace for me 
is sufficient. 
My immeasurable gratitude goes to my supervisor and role-model, Professor, A.A. 
Onilude. I feel I do not have enough words to convey my sincere gratitude for his keen interest 
and inspiring guidance at every step of my work, in particular his many pep-talks  under difficult 
conditions. I am particularly encouraged by his intellectual prowess, guidance, and 
understanding and versatility of experience in almost all areas of life. You are indeed a blessing 
to mankind. May God‟s grace be your sufficiency (Amen). I must not fail to acknowledge the 
fatherly role of Professor Oba Fagade the HOD, Microbiology Department, who took special 
interest in the success of this program. Sir, I appreciate you for your encouraging criticisms and 
monitoring roles. The encouraging words, supervisory roles and interest of Dr. (Mrs) Bukola 
Adebayo-Tayo is highly acknowledged. But for her encouraging words. I wouldn‟t have gone 
this far. Madam,I thank you greatly. I appreciate the supporting roles of Dr. Adeniyi Ogunjobi, 
morally and materially. Words cannot express my immense gratitude to you for the role you 
played in seeing me this far. I sincerely appreciate the technical assistance freely offered to me 
by Dr. (Mrs) Olubusola Odeniyi.  
I acknowledge all members of the department, both teaching and non-teaching for their words of 
encouragements at times when I look tired and worried. I wholeheartedly appreciate the technical 
staff of the following laboratories for their services rendered in terms of equipments and 
reagents:  Microbial Physiology and Biochemistry laboratory, Microbiology Department, 
Pharmaceutical Microbiology Laboratory, Multidisciplinary Central Research Laboratory 
(MCRL) University of Ibadan, NISLT Microbiology Laboratory, Samonda, Ibadan and 
Biotechnology Laboratory, FUNAAB, Abeokuta. To my colleagues Alh. Sanuth, Felicia 
Adesina, Madam  Juliana Aluko, Popoola Bukky and the rest of our senior colleagues; to you all, 
I say thank you. May your anointing never-dry. I also want to acknowledge my technical aid 
assistants:  Ademola E.A, Oluboyede  Omolola, Wahab Idris, and Olonila Omolola, Bolanle  and 
Mr E. Oluwole (LCU). I want to acknowledge my sponsor, the Federal Polytechnic Bida for 
giving me the opportunity to undertake this program. Thank you for believing and investing in 
me. 
 I remain indebted to my wife: Mrs. R.E. Ayodele and my children for being there for me, but 
without their support and understanding, I wouldn‟t have gotten anything to call success. I 
appreciate my spiritual fathers in Christ Apostolic Church: Pastors Olatunji, Ajibodun, 
Ayanlakin, Dahunsi and a host of others for their prayer support.  To my friends: Bar. P.F. 
iv  
 
 
Mokikan, Rev. G.B. Olowo, Mr. P.E. Okperhie, Ewegbemi Emmanuel and a host of others two 
numerous to mention, I appreciate you all.  
 
Parkha, Oni AYODELE 
June, 2013. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
v  
 
 
CERTIFICATION 
 
I certify that this project work was carried out by Parkha Oni, AYODELE in the Department of 
Microbiology, University of Ibadan, Nigeria under my supervision. 
 
 
 
 
 
 
 
  …………………………………………… 
    Abiodun A. Onilude 
    B.Sc, M.Sc. (Microbiology) Ife, Ph.D. Ibadan 
    Professor of Microbiology  
    University of Ibadan, Ibadan. Nigeria. 
v i 
 
 
                      
     DEDICATION 
This work is dedicated  
To my children: Seun, Muyiwa, Owotomo , Omolara and Abiodun. You are my constant source of  
joy  and praises to the almighty God. 
To  
My late mother 
Madam Felicia Tonidunni Ayodele. 
You insisted “I must be educated”. You labored hard to give me necessary financial and material 
nd
support up till your last day 2  August, 2000, when you departed dramatically in your sleep. Mama, 
thank you even though you are no longer around to see your dream come true 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vi i 
 
 
 
TABLE OF CONTENTS 
 
Content  Page 
Title  I 
Abstact  Ii 
Acknowledgement  Iv 
Certification  V 
Dedication  Vii 
List of Tables  Vii 
List of Figures  Viii 
Table of Contents  Xiii 
   
Chapter 1           Introduction     
1.1 General consideration 1 
1.2 Statement of problem  6 
1.3 Justification for the work 7 
1.4 Aims of the study 7 
1.5 Objectives of the study 8 
   
Chapter 2 Literature Review 9 
2.1 Lactose 9 
2.1.1 Production of lactose 9 
2.1.2 Probiotic effect of Lactose 12 
2.1.3  History of lactose 13 
2.1.4.1.1 Genetics of lactose intolerance 15 
2.1.5 Lactose and Human Health 16 
2.1.6 Manifestation of lactose intolerance                               17 
2.1.7 Treatment of lactose intolerance                                     17 
2.1.8 Lactose Hydrolysis Mechanisms 18 
2.1.8.1 Acid Hydrolysis of lactose 18 
2.1.8.2 Enzymatic hydrolysis of lactose 19 
2.1.8.3 Food technology 19 
(a)Reaction mechanism 21 
 (b) Oligosaccharide formation 21 
vi ii 
 
 
Content Page   
 (c) The substrate 21 
2.2 Galactosidases 25 
2.2.1 Alpha Galactosidase ( -Gal)  25 
2.2.2 Beta Galactosidase (  -Galactosidase) 26 
2.2.3 Physicochemical Properties of  -Galactosidase                 28 
2.2.4 Applications of β-galactosidase   28 
2.2.5 Commercial procedures 32 
2.3 Food grade microorganisms   33 
2.3.1 Lactic acid bacteria 34 
2.3.1.1 The genera of lactic acid bacteria 35 
2.3.2 Food grade yeasts 36 
2.3.2.1 Yeasts and their coexistence with lactic acid bacteria            37 
2.3.2.2 Influence of yeast growth on the maturation process and final 37 
quality      of cheese       
2.3.2.3 Role of yeasts in Yogurt production           37 
2.3.2.4 The importance of yeast in the treatment of whey wastes     38 
2.3.2.5 Yeast as a spoilage organism 38 
2.4 Milk   39 
2.4.1 General Overview 39 
2.4.2 Nutritional significance                                                          39 
2.4.3 Types, Sources and Production 40 
2.4.4 Nutritional Composition and Values of milk 40 
2.4.4.1 The quality of milk 41 
2.4.4.2 Processed milk products 41 
2.4.4.3 Local/indigenous milk products  41 
2.4.4.4 Fermented-milk products 42 
2.4.4.5 Other Fermented Milk Beverages 44 
2.4.4.6                       C   u  l t u  r  e d  / f  e rmented milk products 44 
2.4.4.7 Pasteurization 45 
2.4.4.8 Other processed milk products             46 
2.4.5 Ice cream             46 
2.4.5 Cheese 46 
2.4.5 Phenotypic identification of fermentative microflora             47 
ix  
 
 
Content Page   
2.4.7 Microbial   Physiology 48 
2.4.7.1 Enzyme purification    48 
2.4.7.2 Electrophoresis 48 
2.4.7.3 Gel-electrophoresis 49 
                                                                 
Chapter 3 Materials and Methods  50 
3.1 Sample Collection 50 
3.2 Media preparation 50 
3.2 Isolation and culture methods  50 
3.2.1 Isolation and enumeration of yeasts and molds 50 
3.2.2 Characterization of obtained isolates 51 
3.2.3 Microbial morphology   51 
3.2.4 Screening of Isolates for β-galactosidase Production   51 
3.3 Beta -Galactosidase   production  52 
3.3.1 Inoculum Preparation  52 
3.3.2 Determination of Inoculum Size 52 
3.3.3 Β-galactosidase production from lactic acid bacteria 52 
3.3.4 Intracellular enzyme extraction 53 
3.3.5 Enzyme assay 53 
3.3.6 Determination of Enzyme activity 53 
3.3.7  Determination of pH, lactic acid, acetic acid  54 
3.4 Determination of Growth Studies 54 
3.5 Biochemical characterization of isolates 55 
3.6 Identification of isolates 60 
3.7 Molecular analysis of Isolates 60 
3.7.1                          E   x  t r a  c  t i on of genomic DNA of LAB isolates 60 
3.7.2                          P   C  R     a  m  plification of 16S rDNA gene  61 
3.7.3                          G   e  l   e  l e  c trophoresis of 16S rDNA PCR products 62 
3.8                             S   e q  u  e  n  cing and Analysis of 16S rDNA gene  62 
3.8.1                          P   u  r i f i c  a  tion of PCR 16S rDNA gene 62 
3.8.2                          D   r  y i  n  g   of the purified 16S rDNA genes 63 
3.8.3                          S   e q  u  e  n  c ing of 16S rDNA gene   63 
3.8.4                          A   n  a  l y  s  i s of the 16S rDNA gene sequences 63 
3.9 Effect of incubation time on growth and β-galactosidase production 63 
x  
 
 
Content                           Page  
3.9.1                          M    e  a s  u  r e ment of growth with time  63 
3.10                           O   p  t i m  ization of growth conditions for β-galactosidase production       64 
3.10.1 Effect of Temperature on β-galactosidase production 64 
3.10.2   Effect of pH on β-galactosidase production 64 
3.10.3   Effect of different carbon sources on β-galactosidase production          64 
3.10.4   Effect of different Nitrogen sources on β-galactosidase production 64 
3.10.5   Effect of agitation on enzyme production 60 
3.11 Characterization of crude enzyme 65 
3.11.3                        E   f f e  c t of Inoculums size 65 
3.11.4              Effect of Inoculums age 65 
3.11.5                        E   f f e  c t of incubation period 65 
3.11.6                        E   f f e  c t of different inhibitors 66 
3.11.7                        E   f f e  c t of different cations                                                                          66 
3.11.8                        E   f f e  c t of different anions                                                                           66 
3.11.9                        E   f f e  c t of enzyme concentration 66 
3.12                           C   o  n  s truction of ONP Standard Curve                                                       67 
3.13                           I  n  t r a  cellular Enzyme extraction                                                                  67 
3.14                           P   u  r i f ication of β-galactosidase 67 
3.14.1                        A   m   m  onium Sulphate precipitation 67 
3.14.2                        D   i a  l y sis 68 
3.15                           C   o  l u  mn chromatography                                                                            68 
3.15.1                        P   r e  p  aration of resin                                                                                     68 
3.15.2                        L   o  a d  ing of the Column                                                                               68 
3.15.3                        F  r  a  c t ionation on Sephadex G-100                                                              69 
3.15.4                        F   r a  c t ion of Sephadex C-50                                                                         69 
3.16                           P   rotein Estimation                                                                                      69 
3.17. Electrophoresis separation of protein samples/SDS-PAGE 69 
3.17.1                  Electrophoretic Separation of Extracellular -galactosidase        69 
3.17.2                        G   e  l   e lectrophoresis 70 
Chapter 4  Results 73 
Chapter 5 Discussion     148 
 References 159 
 Appendices 187 
   
x i 
 
 
   
 
LIST OF TABLES  
Table  Page 
1  Species concentration of lactose in Milk of different 11 
mammals 
 2 Properties of  -galactosidase from microbial sources  20 
3 Active sites and other properties of  -galactosidase from 30 
various microbial origins  
 4 Molecular weights of  -galactosidase from bacteria sources  31 
5  Average (%) composition of milk in different species of 43 
Mammals  
6 Percentage occurrence of bacterial isolates in different  
 samples 74 
7 Biochemical test/sugar fermentation 75 
7 Phylogenetic identity of the bacteria isolates (Blast sequence 76 
match) 
8 Screening of LAB isolates for  -galactosidase production 78 
on solid agar 
9 Screening of lactic acid bacteria for lactic acid production  79 
 
 
 
 
 
 
 
 
 
 
xi i 
 
 
 
 
LIST OF FIGURES 
Figure  Page 
 
1 Molecular structure of lactose 10 
2 Lactose intolerance around the world by percentage of 14 
population  
3 Proposed mechanism of lactose hydrolysis by  - 22 
galactosidase 
4 Proposed mechanism of galactosyl transferase 23 
Reaction by  - galactosidase   
5  - galactosidase structure  26 
4.1a Extra cellular production of  - galactosidase by the   
Selected LAB Isolates 81 
4.1b intracellular  - galactosidase production potential of 81 
selected lab isolates 
4.3 Effects of incubation time on growth and  - galactosidase 83 
production by L. plantarum (G11)   
4.4a Effect of incubation time 0n growth and β-galactosidase  
production by L. plantarum E13 83 
Effect of incubation time on growth of L. plantarum subsp.  
plantarum (E36) 85 
4.4b Effects of incubation time on growth and  - galactosidase  
production by L. plantarum subsp. plantarum  87 
4.5a Effect temperature on by L. plantarum  89 
4.5b Effects of temperature on  - galactosidase production by L.  
plantarum (G11) 89 
4.6a Effect of Temperature on growth of L. plantarum (E13-) 91 
4.6b Effect of temperature on  - galactosidase production by L.  
plantarum (E 91 13)  
4.6c Effects of temperature an protein concentration by L.  
plantarum (E13) 92 
xi ii 
 
 
Figure    Page  
4.7a Effects of temperature on growth by L. plantarum subsp.  
plantarum (E36) 94 
4.7b Effect of temperature on  - galactosidase production by L-  
plantarum subsp. plantarum (E 94 36) 
4.7c Effect of temperature on protein concentration by L.  
Plantarum subsp. plantarum (E36) 95 
4.8a HEffect of p   on  - galactosidase production by L.  
plantarum (G 97 11) 
4.8b H Effect P  can  - galactosidase production by L. plantarum  
(E ) 97 13
4.8c  Effects of pH  - galactosidase production by L. plantarum  
subsp. plantarum (E 98 36) 
4.9 Effect of agitation on  - galactosidase production by the  
strains of L. plantarum  100 
4.10a Effects of different carbon sources on  - galactosidase  
production by L. plantarum (G ) 102 11
4.10b effects of different carbon source on  - galactosidase  
production by L. plantarum (E ) 102 13
4.10c Effect of different carbon sources on  - galactosidase  
production by L. plantarum subsp. plantarum (E36) 103 
4.11a Effect of Nitrogen sources on  - galactosidase production  
by L. plantarum (G 105 11) 
4.11b Effects of Nitrogen sources on  - galactosidase production  
by L. plantarum (E 105 13)  
4.11c Effect of Nitrogen sources on  - galactosidase production  
by L. plantarum subsp. plantarum (E36) 106 
4.12a Effect of inoculums size in  - galactosidase production by  
L. plantarum (G 108 11) 
4.12b Effects of inoculums size on  - galactosidase production by  
L. plantarum  (E13) 108 
4.12.c Effects of inoculums size on  - galactosidase production by  
L. plantarum subsp. plantarum (E36)  109 
xi v 
 
 
 
Figure              Page 
4.13a Effect of inoculums age on  - galactosidase production by  
L. plantarum (G ) 111 11
4.13b Effect of inoculums age on  - galactosidase production by  
L. plantarum (E ) 111 13
4.13c Effect of inoculums age on  - galactosidase production by  
L. plantarum subsp. plantarum (E ) 112 36
4.14a Effect of temperature on  - galactosidase activity produced  
by L. plantarum (G11)  114 
4.14b Effect of temperature on  - galactosidase activity produced  
by L. plantarum (E 114 13)   
4.14c Effect of temperature on  - galactosidase activity produced  
by L. plantarum subsp. plantarum (E 115 36) 
4.15a H  Effect of P on  - galactosidase activity produced by L.  
plantarum (G11) 117 
4.15b HEffect of P   can  - galactosidase activity produced by L.  
plantarum (E 117 13)  
4.15c  H Effect of P  on  - galactosidase activity produced by L.  
plantarum  subsp. plantarum(E )  118 36
4.16a Test of stability on  - galactosidase activity produced by L.  
plantarum (G ) 120 11
4.16b Test of stability on  -galactosidase produced by L.  
plantarum (E13) 120 
4.16c Test of stability on  - galactosidase produced by L.  
plantarum (E ) 121 36
4.17a Effect of different Molar concentrations of cations on  -  
galactosidase activity produced by L. plantarum (G 124 11)  
4.17b Effect of different Molar concentrations of cations on the  
activity of  - galactosidase produced by L. plantarum (E13) 124 
4.17c Effect of different Molar concentration of cations on the  
 activity of  - galactosidase produced by L. plantarum  
 subsp. plantarum (E 125 36) 
xv  
 
 
Figure   Page   
4.18a Effect of different Molar concentration of Anions on  -  
galactosidase activity produced by L. plantarum (G 128 11)  
4.18b Effects of different Molar concentration of anions on the  
activity of  - galactosidase produced by L. plantarum (E13) 128 
4.18c Effects of different Molar concentration of anions on the  
activity of  - galactosidase produced by L. plantarum (E36)  129 
4.19a Effect of different Molar concentration of inhibitors on  
activity of  - galatosidase produced by L. plantarum (G11) 131 
4.19b Effect of different Molar concentration of inhibitors on the  
activity of  - galactosidase produced by L. plantarum (E13) 131 
4.19.c Effect of different molar concentration of the activity of  -  
galactosidase produced by L. plantarum subsp. plantarum 132 
4.20a Linear weaver-Burk plot of the enzymatic activity of L.  
plantarum at different substrate concentrations. 134 
4.20b Lineweaver-Burk plot of the enzymatic activity of L.  
plantarum (E13) at different substrate concentrations  135 
4.20c Lineweaver-Burk plot of the enzymatic activity of L.  
plantarum subsp. plantarum (E36) at different substrate  
concentrations  136 
4.21a Elution profile of  -galactosidase  on Sephadex-G-100 by  
L. plantarum (G )  138 11
4.21b Elution profile of  -galactosidase by L. plantarum (E13) on  
Sephadex-G100 by L. plantarum (G 139 11)  
4.21c Elution profile of  -galactosidase by L. plantarum subsp.  
plantarum on Sephadex G-100 column chromatography  140 
4.22a Elution profile of  -galactosidase produced by L. plantarum  
(G11) on Sephadox G-50 ion-exchange chromatography 141 
4.22b Elution profile of  -galactosidase by L. plantarum (E13) on  
Sephadox G-100 column chromatography 142 
4.22c Elution profile of   -galactosidase by L. plantarum subsp.  
plantarum on ion-exchange chromatography  143 
4.24a Percentage lactose Hydrolysis by L. plantarum (G11) at  
xv i 
 
 
Figure    Page  
 different lactose concentrations  145 
4.24b Percentage Lactose Hydrolysis by L. plantarum (E13) at  
different lactose concentrations  146 
4.24.c Percentage Lactose Hydrolysis by Lactobacillus plantarum  
subsp. plantarum (E36) at different lactose concentrations. 147 
 
 
 
 
 
 
 
 
 
 
  
xv ii 
 
 
LIST OF ABBREVIATIONS 
LAB   Lactic Acid Bacteria 
BSA  Bovin Serum Albumin 
-gal  Beta-galactosidase 
-gal  alfa galactosidase 
x-gal   5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside 
LAC* P   Lactose Persistence 
LAC* R   Normal adult lactase restriction 
GOs  Galactooligosaccharides 
TOs   Transgalactosylated Oligosaccharide 
IPTG  Isopropyl--D-thiogalactosidase 
MRS  de Man, Rogosa and Sharpe 
MMRS   Modified de Man, Rogosa and Sharpe 
OD   Optical density 
ON PG  o-nitrophenyl--D-galactopyranoside 
ONP   o-nitrophenol 
PCR  Polymerase Chain Reaction 
SCP   Single Cell Protein 
SDS – PAGE  Sodium Dodecyl Sulfate Polyacrylamide Gel Electophoresis 
YEA  Yeast Extract Agar 
YEPA   Yeast Extract Peptone Agar 
 
 
 
 
 
 
 
 
 
 
 
 
xv iii 
 
 
CHAPTER ONE 
1.0    INTRODUCTION 
1.1 General consideration 
Microbial galactosidases have become a major biotechnogical tool in the 
development of viable bioprocessing for the conversion of many non-digestible 
carbohydrates in variety of natural products into utilizable forms (Vasilijevic et al., 2002). 
Glycosidases are enzymes that are capable of hydrolyzing glycoside bonds in oligo or 
polysaccharides and heteroglycosides (Jacobson, 1994). Among these group of enzymes, β-
galactosidase (or β-D-galactosidase galactohydrolase EC.3.2.1.23), is a well known 
biocatalyst in the dairy industry as well as in synthetic glycochemistry (Karasova et al., 
2002). The importance of β-galactosidase is related to its use in milk and milk derivatives 
to decrease their lactose content, solving the problem of low lactose solubility and its low 
degree of sweetness (Somkuti et al., 1998). β-Galactosidase can catalyse two basic 
reactions: the hydrolysis of β-1,4-D-glycosidic linkage of lactose and other structurally 
similar substrates, and is also responsible for the formation of galacto-oligosaccharide 
(GOS) by transgalactosylation(Iqbal et al., 2011). This hydrolysation and 
transglycosylation activity enables them to synthesize oligosaccharides from the hydrolysis 
of natural products and catalyzes the hydrolysis of lactose into galactose and glucose 
(Furlan et al., 2000). Oligosaccharides are water soluble, mildly sweet many of which are 
not digestible in the small intestine (Karasova et al., 2002). The most commonly found 
natural substrate for  -Galactosidase is lactose, the main sugar of milk and other dairy 
products (Ferguson, 1995). Some of the  -galactosidase are able to disrupt the B-1,4  
bond and form other covalent bonds like  -1,6,  -1,3 and  -,2 (Mahoney, 1998).   
These compounds are indigestible, act as dietary fibres, and promote the growth of 
bifidobacteria in the intestine (Crittenden and playne, 1996; Sako et al., 1999). Possible 
sources of the enzyme are plants, animal organs, bacteria, yeasts, fungi and moulds. 
The problems with lactose fall within three major areas: Health, Food technology, 
and Environment which collectively constitute the main interest in  -galactosidase 
production, utilization and research (Mc Feters et al., 1969). Yeasts for example, usually 
1 
 
 
utilize only the fructose, (a trisaccharide moiety) in cane and beet molasses during 
production of alcohol after hydrolysis by invertase but not the residual disaccharide (Webb 
et al., 1976). There are many reports in the literature describing enzyme processes for 
hydrolysis of lactose by exploiting enzymes extracted from a number of species of 
microorganisms especially yeasts (of the genera Kluyveromyces and Saccharomyces) and 
fungi of the general Aspergillus, Penicillum and others (Roberto et al., 1984). Lactic acid 
bacteria (LAB), which constitute a diverse group of Lactococci, Streptococci, and 
Lactobacilli have been studied intensively with respect to their enzymes for various 
different reasons especially food-related applications (Somkuti et al.,1998; Vasiljevic et al., 
2002). 
During the last two decades, for example, the application and processing of  -
galactosidase for the hydrolysis of lactose in dairy processing industries has gained 
increased attention because  -galactosidases are capable of synthesizing oligasoccharides 
during hydrolysis of natural substrates (Cortes et al., 2005). Moreover, glucose and 
galactose expands the use of lactose in food especially dairy industries, because of a 
marked increase in sweetness and solubility (Lifron et al., 2000) which enhances frequent 
addition of oligosaccharides to beverages, infant milk powder, yogurts and other dairy 
products to improve their physico-chemical attributes. The lactose hydrolyzed milk may be 
consumed directly or may be used to produce many products including dehydrated 
products, concentrates, cheeses, yogurts, butter milk especially suitable for lactose 
intolerant individuals who are lactose maldigesters as observed amongst over 75% of the 
world population (Gekas and Lopez-leiva, 1985; Roy and Gupta, 2003; Panesar et al., 
2006). Hydrolysis of Lactose result in valuable changes on dairy products with increased 
solubility, broader fermentation possibilities, more ready fermentation of carbohydrate and 
reduced lactose concentration with associated diminished possibility of lactose 
crystallization (Roberto et al ., 1984). 
Lactose hydrolysis in whey can help to solve the pollution problem caused by whey 
and whey permeates (Rachie, 1988). In the cheese industry, lactose is a waste which causes 
several economic and environmental problems (Grosova et al., 2008). The problem arose 
because lactose is associated with high Biochemical and Chemical Oxygen Demand (BOD, 
2 
 
 
COD) and because of lactose uncertain solubility (Guimares et al., 1992); the environment 
can thus be rendered inhabitable as a result of pollution. The production of microbial 
protein from whey for example reduces the BOD value of the effluent by converting 
lactose into protein. Moreso et al. (1980) reported that the alteration of Kluyeveromyces 
fragilis has the main advantage of making easier the final disposal of whey since it lowers 
the COD by more than 90%. 
The lactose-fermenting yeast Kluyveromyces maxianus and Kluyveromyces lactis 
are both important industrial yeasts in classical applications with biomass conversion, 
ethanol, enzyme and single-cell protein production (Inchaurrondo et al., 1994). 
Kluyveromyces maxianus offer great advantages such as (Belem and Lee, 1998); 
(i) Good growth yield, which has an important economic impact in the food 
industry. 
(ii) Acceptability as safe microorganism, an important technical aspect when 
considering that the fermented products have good or pharmaceutical 
applications and 
(iii) Higher  -galactosidase activity than other yeasts.  
Scopularriopsis sp (Pastor and Park 1980) and Erwinia aroida were equally 
screened for β-galactosidase production (Flores and Ighem-Alegre, 1996). The  -
galactosidase from these different sources are well characterized with regards to their 
kinetic behaviour in the hydrolytic mode (Daniela et al., 2006). They were all reported to 
cause hydrolysis of  -1, 4-glycosidic bond in lactose (4-0-  -D galactopyranosy l  -D-
glucopyranose), in different  -D-galactosides, glycopeptides, and glucolipides. 
However, there is a definite need for  -galactosidase that is stable at high and low 
temperatures and could be approved as GRAS (generally regarded as safe) for hydrolysis of 
lactose in milk and other dairy products (Kim and Raja Gospel, 2000).  -galactosidase is 
0
also known to have a stable enzymatic activity at temperatures up to 55 C, preferable as 
0 0
low as 6 C and specifically at 4 C which corresponds to refrigerating/conservation 
temperature for dairy products.  -galactosidase hydrolyzes lactose in dairy products and 
3 
 
 
milk processing at low temperature and hinders the saprophytic activity and restricts their 
proliferation (Jelen et al., 2005). These oligosaccharides are not utilized by the mouth 
microflora (Streptococcus immitants) and as such limits the formation of caries (Karasova 
et al., 2002). Many of them are not digested in the small intestine and this makes them 
suitable for use as low calorie sweeteners for consumption by individuals with diabetes. In 
recent years, the ability of oligosaccharides to promote the proliferation of bifido bacteria 
in the colon has been recognized. These disaccharides are selectively fermented by 
bifidobacteria such that the growth of undesirable bacteria is suppressed (Molder, 1994). 
Flavor is an important attribute of grape-derived alcoholic beverages and many 
Variables contribute to it (Sarova and Nikolova, 2002). Among these, the enzymatic 
activities of  -galactosidase and esterase of yeast play an important role as they are 
involved in their breakdown. Fermandez et al., (1997), have revealed the potential of 
indigenous wine yeasts to produce enzymes that improve the sensory properties of wine. 
This has encouraged studies on yeasts which possess these enzymes (). Although, many 
microbial  -galactosidase has been investigated for the aforementioned properties and 
potential beneficiary effects namely: assimilation of food containing lactose intoleranse, for 
formation of galacto-oligosaccharides during lactose hydrolysis. (Nizamudin et al., 2008); 
 -galactosidase in bacteria and in yeast is generally intracellular (Wallenfels et al., 1972). 
The major applications for Lactose hydrolysis can therefore be summaries as 
followed (Cortes et al., 2005): 
(a) Lactose hydrolysis in liquid milk improves digestibility for lactose in tolerance 
consumers 
(b) Lactose hydrolysis increases sweetness and enhances flavor 
(c) Lactose hydrolyzes milk powders for dietetic uses, especially for infants with 
temporary  -galactosidase deficiency. 
(d) In some cases, β-galactosidase used for the manufacture of cheese and yogurt can 
increase the rate of acid development and thus reduce processing time 
(e) Lactose hydrolysis in concentrated milk products (e.g. sweetened condensed milk, 
ice cream), prevents crystallization of Lactose 
4 
 
 
(f) To serve as source of functional proteins and sweet carbohydrates used as food 
ingredients in bakery and confectionary products (USA patent No. 4, 657088: Kane 
Kramer). 
In view of the very wide applications identified above, yeast and lactic acid bacteria 
(LAB) have gained great interest in the last decade because of their GRAS status and their 
long history of usage and association with milk (Kim and Raja Gospel, 2000). Amongst 
these groups, yogurt bacteria Lactobacillus bulgaricus and streptococcus thermophilus are 
already identified as the highest  -galactosidase producers (Kreft and Jelen, 2000). The 
 -galactosidase of this culture has been characterized, showing high stability and activity 
at high temperatures (Greenberg and Mahoney, 1982). As their importance in the food and 
dairy industries is already well known, and because the lactic acid and other organic acids 
produced by these bacteria act as natural preservatives as well as flavor enhancers 
(Salminen et al., 1998a). Lactic acid is an economically valuable product with different 
applications in pharmaceutics and other industrial products. Production has increased 
considerably due to the development of new applications and the current level of 
production in technology is based on microbial fermentation (Gaudreau et al., 2005). This 
has energized in the discovery of new microbial sources with novel characteristics (Datta 
and Henry, 2006), in particular its stability and ability to lasting enough to attain the desire 
effect. As in other microbial galactosidases that are thermolabile the most important 
characteristic of the enzyme is its low heat stability.  
The classification of Lactic acid bacteria was initiated in 1919 by Orla-Jesen and 
was until recently primarily based on morphological, metabolic and physiological criteria 
(Orla-Jesen, 1919). Lactic acid bacteria are Gram- positive, usually none-motile, non-
spore-forming rods or coci (Stiles et al., 1997). Nutritionally, the lactic acid bacteria are 
extremely fastidious (John Lindquist, 1998). They are chemo-organotrophic and only grow 
in complex media.A medium that will support their growth must contain fermentable 
carbohydrate and many growth factors (De Man Rogosa et al., 1960). Lactic acid bacteria 
are classified by the fermentation pathway used to ferment glucose and by their cell 
morphology.They are widespread and can be isolated from many plant and animal sources 
(John Lindquist, 1998). 
5 
 
 
Current methologies used for classification of LAB mainly rely on 16S ribosomal 
ribonucleic acid (rRNA) analysis and sequencing (Olsen et al., 1994). Based on these 
techniques, Gram-positive bacteria are divided into two groups depending on their G+C 
content (John Lindquist, 1998). The Actinomycetes have a G+C content above 50 mol% 
and contain genera such as Atopobium, Bifidobacterium, Corynbacterium and 
Propionibacterium (John Lindquist, 1998). In contrast, the Clostridium branches has a 
G+C content below 50 mol% and include the typical LAB genera Carnobacterium, 
Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus (Berthier et al., 
1999). 
Lactic acid bacteria, including Lactobacillus, Leuconostoc, Lactococcus, 
Pedicoccus and Bifidobacterium spp., are prominent members of the comensal intestinal 
flora and are the commonly studied probiotics bacteria (He et al., 2008). They cause 
reduced lactose intolerance alleviation of some diarrhoeas, lowered blood cholesterol, 
increased immune response and prevention of cancer (Marteau and Ramband, 1993, 1996; 
Gilliland, 1996 and Salminen et al., 1998a). The selection criteria for probiotic LAB 
include, human origin, safety, viability/activity in delivery vehicles, resistance to acid and 
bile, adherence to gut epithelial tissue, ability to colonize the gastro intestinal tract, 
production of antimicrobial substances, ability to stimulate a host immune response and the 
ability to influence metabolic activities such as vitamin production, cholesterol assimilation 
and lactose activity (Salminen et al., 1996).  
1.2 Statement of problem 
Lactose (or milk sugar) is a disaccharide sugar that is found exclusively in the milk 
of mammals (Nahvi et al., 2004). However, lactose is not actively absorbed in the intestine 
of all man in all regions unless it is first split into its constituent monosaccharides: glucose 
and galactose. Hydrolysis of Lactose is effected by the lactase enzyme .Many grown-up 
adults worldwide have lost the capability to synthesize β-galactosidase (or lactase) enzyme 
as this ability begins to drop between ages 2 to 5 years (McBeans et al, 1998). It is 
estimated that the phenomenon affects over 75 per cent of world adult population resulting 
in lactose intolerance or hypolactasia (Heyman, 2006). For this group of people, it is 
impossible to consume large quantities of mammalian milk or other milk products without 
6 
 
 
adequate treatment. Enzymatic hydrolysis of lactose is a preferred option because it permits 
wider operational conditions of both temperature and pH (Gekas and Lopez-leiva, 1985). 
The application of the process has thus created a potential market for the application of β-
galactosidase. Microbial β-galactosidase from organisms such as yeasts, mold and bacteria 
still remain a better source for its commercial production (Vasiljevic and Jellen, 2001). The 
use of microorganisms in food production and /or fermentation has long been known 
especially when they have a long history of safe use and without extensive purification 
(Inchurrondo et al., 1994). The bacteria species currently used by the dairy industry for β-
galactosidase enzyme production belong to the genera of Lactobacillu and Bifidobacterium 
and comprise a limited collection of strains (Heymann, 2006, He et al., 2008). Lactic acid 
bacteria have been used for centuries to produce fermented food products like pickles, 
Saussage, yogurt and cheese (Inchurrondo et al., 1994).       
1.3 Justification for the work 
Part of the federal government of Nigeria‟s policy objectives for Agricultural 
transformation Agenda is to modernize the agricultural system including the indigenization 
of the dairy industry that is presently import dependent (Adesina, 2012). As revealed lately, 
Nigeria spends over four billion dollars annually on the importation of milk and milk 
products (Adesina, 2012). To reverse this trend, steps must be taken to establish raw milk 
collection centres at some locations in dairy producing states in Nigeria such as Adamawa, 
Taraba and other states that may be identified in the Niger trough for this purpose. Milk as 
its well known is characterized by it high lactose content. The lactose content needs to be 
reduced to facilitate the utilization of milk by all consumers, due to the fact that some 
people are lactose intolerant. β-galactosidase is a well known enzyme that reduces lactose 
contents of milk and milk products. For this program to have any meaningful impact and to 
make Nigeria self sufficient in dairy products, the traditional way of milking the cows has 
to be upgraded.Among β-galactosidase-producing organisms, bacterial sources are 
preferable due to ease of fermentation, high enzyme activities and good stability (Vasijevic 
and Jelen, 2001) .Organisms so identified should have potential for high catalytic activity 
within a reasonable range of temperature and pH. Therefore, Lactic Acid Bacteria (LAB), 
which constitute a diverse group of Lactococcus, Streptococcus, and Lactocobacilli have 
7 
 
 
attracted a great deal of attentions in recent years as a source of β-galactosidase production 
for several reasons (Somkuti et al., 1998) including its long history of usage as stater 
cultures for fermentation in dairy foods and/or in food preservation for thousands of years. 
Moreso, because of its GRAS status as extensive purification of enzymes produced by 
them may not be necessary. 
1.4 Aim of the Study  
 β-galactosidase hydrolysis of milk is one of the promising  enzymatic applications in the 
dairy industries for reducing lactose intolerance of milk products.However, plant and 
animal sources cannot meet the high demand for the enzyme in food industries.Hence the 
aim of this study was to characterize β-galactosidase production by Lactic Acid 
Bacteria(LAB) isolated from locally fermented milk products. 
1.5 Objectives of study 
This study aims at achieving the following objectives: 
1. Isolation screening and identification of beta galactosidase-producing 
microorganisms associated.. 
2. Growth and nutrient utilization of the isolates in various nutrient media. 
3. Optimization of production conditions for beta galactosidase by the isolates 
4. Purification and characterization of the beta galactoside produced by the isolates. 
5. In situ application of the enzyme to different Nigerian foods. 
8 
 
 
                                                                 CHAPTER TWO 
2.0      LITERATURE REVIEW 
2.1   Lactose 
Lactose or milk sugar is a disaccharide sugar that is found exclusively in the milk of 
mammals and it is not found naturally in any other food aside from dairy products (Kemer, 
1995). It was discovered in milk by Fabriccio Batoletti (1619) and identified as a sugar by 
Carl Wilhelm Scheele Bartoletto (1780). It was isolated and descried as essential salt 
without nitrogen in (1633). It is comprised of galactose and glucose linked together by  -
D-galactoparanosyl-(14)-D-glucose. The glucose can be in either the  -pyranose form 
or the  -Pyranose form, whereas the galactose can only have the  -Pyranose form 
(Jacobson, 1994). Hence  -lactose and  -lactose refer to an anomeric form of the 
glucopyranose ring alone (Jellen, 2005). 
Lactose makes up a little less than 80% (by weight) of the solids in milk, although 
the amount varies among species and individuals (Piverlik et al., 1992). It is also found to 
be the principal component of cow‟s milk. Indeed, it is a major constituent of the milk of 
all mammals except sea lion (Kretchmer, 1993) and as a primary source of energy for the 
neonate. The human milk is said to contain the highest lactose concentration of about 7%. 
Lactose levels in commonly milked animals such as camels, goats, sheep and cows run 
between 4 and 5 percent (Webb et al., 1976). 
Because of their high nutritional value and bioactive properties, lactose products are 
increasingly used in products designed to improve health and wellness (Jellen et al., 2002). 
Lactose offers a number of nutritional benefits not found in other sugar sources (Kim et al., 
1983). 
It also generates a prolonged energy supply due to its slow hydrolysis in the body (Flatz, 
1987) and also increases the absorption of minerals such as calcium, magnesium, and zinc 
in laboratory animals and human infants (Jelen,  2005). According to the USA dairy 
council information details, lactose contributes to a healthy intestinal flora and has minimal 
effect on tooth decay compared to other sugars. 
9 
 
 
 
 
 
 
 
 
 
 
Fig 2.1: Molecular structure of lactose (Jellen, 2005). 
 
 
 
 
 
 
 
10 
 
 
Table 2.1: Species Concentration of lactose in milk of different mammals 
Species Lactose concentration %water content  
Human 7.1 87.1 
Cow 4.6 87.3 
Buffalo 4.8 82.8 
Goat 4.3 86.7 
Sheep 4.8 82.0 
 
Source: Dairyforall. com, the milk and Dairy products website 
 
 
 
 
 
 
 
 
 
 
 
 
11 
 
 
2.1.1   Production of lactose 
Several million tons of lactose are produced annually as by-product from the dairy 
industry animals.It is generally an excellent source of protein, calories in the form of fats 
and carbohydrates, vitamins and minerals (Kosikoski, 1982). Production of both pure 
lactose and lactose-containing dairy by-products, have markedly increased since the 1960s. 
For example, its bland flavour has lent to its use as a carrier and stabilizer of aromas and 
pharmaceutical products. Purified lactase can also be purchased as high calorie diet 
additives (Jellen, 2005). Lactose is little fermented by barker‟s yeast which is of advantage 
during brewing. An easily obtained source of lactase is lactaid pills which are sold over the 
counter to treat lactose intolerance (Mishkini, 1997). 
According to Pavel Jellen, in 1996 a total of 425 Kilotons of lactose were produced 
in U.S for use as infant foods, confectionery and others (bakery, dry mixes and protein 
standardization). Another 175 kilotons were produced for pharmaceuticals, fermentation 
industries, feeds and other derivatives in Germany (Sahari and Friend, 1983). Whey is 
made up of 6.5% solids of which 4.8% is lactose (Hussain, 2007). Lactose has a relatively 
low glycemic index (Buller and Grand 1990) which makes the sugar beneficial for 
diabetics. The glycemic index of carbohydrate of food refers to the increase in blood 
glucose relative to a standard such as glucose or white bread (Sahi, 1994). Food with a low 
glycemic index causes a slow and modest rise in blood glucose levels. In contrast, foods 
with high glycemic index causes a faster and higher increase in blood glucose (Arola and 
Tem, 1994). Lactose has a lower glycemic index (65) than glucose (138), honey (104) or 
sucrose (87). Diets with glycemic index not only help decrease the risk of developing 
diabetics but such diets may also have other health benefits (Cambridge history of food 
science). 
2.1.2   Probiotics effects of lactose 
By stimulating the growth of beneficial intestinal bacteria such as Bifidobacteria 
and Lactobacilli and inhibiting pathogenic bacteria, lactose contributes to a healthy 
intestinal flora (Kemer, 1995). When the ingested lactose is not absorbed in the small 
intestine but rather become fermented by lactic acid bacteria in the large intestine, it 
12 
 
 
produces unknown volumes of carbon dioxide. Common symptoms of indigested lactose 
includes nausea, cramps, bloating, flatulence and diarrhea which begins about 30mins to 
2hrs after drinking or eating foods containing lactose (Jellen, 2005). 
Lactose is not actively absorbed by the intestine unless it is first split into its two 
monosaccharide components i.e glucose and galactose (Jellen et al., 2005). The hydrolysis 
of lactose is affected by the lactose enzyme produced by the epithelial cells in the brush 
border of the small intestine (Arola and Temm, 1994). Thus, the capacity of mammals to 
digest lactose depends on the level of lactase activity in the small intestine. Infacts and 
young children digest lactose with an enzyme lactase which splits this molecule in the 
small intestine into the two absorbable simple sugars of glucose and galactose. However, 
the majority of adults has lost this ability and is lactose malabsorbers (Buller and Grand, 
1990). 
2.1.3 History of lactose 
Gastrointestinal distress in adults after milk consumption was described in ancient 
Greek and Roman texts (Cambridge History of food science) and there were isolated 
clinical reports in the late nineteenth and early twentieth centuries but the mechanisms 
controlling lactose production were disputed for many years as a result of which the 
problem was not widely studied until the development in the 1960s of new techniques to 
study enzymatic action in the intestine (Flatz, 1987). This prompted Dahlgvist (Dahlgvist et 
al., 1977) to describe the high prevalence of diminished lactase activity in healthy adults as 
a common worldwide phenomenon in adulthood. According to this finding, production of 
lactase begins to decline in most children between the ages of 2 to 5 years (around the time 
of weaning). Arola and Tam (1994) reported that most adults retain only about 10 percent 
of infant- level lactase activity. 
13 
 
 
 
Fig 2.2: Lactose intolerance around the world by percentage of population (Pavel Paul Jellen, 2008) 
14 
 
 
2.1.4 Genetics of lactose intolerance 
The mechanisms controlling lactase production were disputed for many years 
(Sahi, 1994). Some researchers, drawing on studies of gene regulation in bacteria, argued 
in the 1960‟s that lactase was a substrate-inducible enzyme; that is, that lactase production 
was believed to be stimulated by the presence of its substrate lactose (Fernendes et al., 
1987). In this view, populations that did not use milk as adults lost the ability to produce 
lactase, whereas groups that did consume milk and milk products retain lactase capability 
(Bullar and Grand, 1990). Biochemical studies cast doubts on this theory, and family 
studies have demonstrated that lactase production is controlled by an autosomal gene, 
recently located on chromosome 2 (Bullar and Grand, 1990). Persistence of lactase 
production is a dominant trait. 
 Following the terminology suggested by Gebhard Flatz (1987), the two alleles are 
designated as LAC* P for lactase persistence and LAC* R for normal adult lactase 
restriction. The LAC locus appears to be a regulatory gene that reduces lactase synthesis 
by reducing the transcription of messenger RNA (Arola and Temm, 1994). Persons 
inheriting LAC* P from both parents would have lactase persistence into adulthood. Those 
getting LAC* R alleles from both parents would display lactase restriction as adults. 
Heterozygotes would get different alleles and be LAC* P/LAC*R, but since LAC* P is 
dominant, lactase activity and ability to digest milk would persist beyond childhood 
(called lactase persistence or lactase tolerance).Many people with ancestry in Europe, 
West Asia, India, and parts of East Africa, maintain lactase production in adulthood. 
Fredrick J. Simoons (1969, 1994), had advanced the thesis that lactase persistence is 
closely linked to dairying. His culture-evolution hypothesis that groups that kept cattle and 
other milk animals would gain a selective advantage if adults retain the ability to use milk 
and milk products as food (Sahi, 1994). In many of these areas, milk from mammals such 
as cattle, goats, and sheep is used as a large source of food. It was in these regions that 
genes for life long lactase production first evolved (Fernendes et al., 1987; Sanul, 1990). 
By descent more than 70 percent of Western Europeans can drink milk as adults compared 
with less than 30 percent of people from areas of Africa, East and South eastern Asia and 
the Oceania. Of the Thai, Chinese and African American populations due to a mutation 
which is believed to have occurred between 5 and 10 thousand years ago (Sahi, 1994) and 
involves a single base change in the promoter region to prevent binding of the 
15 
 
 
transcription factors, 97, 90 and 73 percent respectively are reported to be lactase 
intolerant, whereas 84 and 96 percent of the US white and Swedish populations 
respectively are tolerant (Fernendes et al., 1987). 
The second center of adult lactase persistence lies in the land, of Arabia, the Sahara 
desert, and Eastern Sudan. In these regions  lactase persistence characterizes only nomadic 
populations who heavily dependent on camels and cattle such as Bedovin Arabs; the 
Tuareg of the Sahara, the Fulani of the West African Sahel, and the Beja and Kabbabish of 
Sudan(Fernendez et al., 1987). Low rates among Nigerian Fulani may indicate a higher 
degree of genetic mixing with other peoples in their abode than among the Fulani of 
Senegal (Sahi, 1994). 
2.1.5  Lactose and Human Health 
Recent developments in the field of microbiology has stimulated increased 
awareness and instense search for human friendly microorganisms amongst bacterial 
species, fungi and yeasts that have ability to utilize lactose. Lactose is the main 
carbohydrate in milk it is unique to the milk of mammals (Kemer, 1995). Unfortunately, 
humans cannot absorb lactose for use as a nutrient. Gurr (1987) mentioned that to be 
efficiently absorbed from the gut lactose must be digested into its constituent sugars by the 
enzyme lactase. The term lactose intolerance (LI) describes the inability of individuals to 
digest significant amount of lactose resulting from the shortage of the enzyme lactase. 
Lactase is a protein produced in the cells of the epithelium of the small intestine. It is mot 
accentuated in the Muscosa cells of the brush bother of the Jejunum (Kim et al., 1983; 
Buller and Grand, 1990; De Vresse, 2001). About 90-100 percent of Native Americans, 
Native Africans, Asians and Eskimos are lactose intolerant (Mishkin, 1997). As many as 
80 percent of African-Americans, and Mexican-Americans experience lactose intolerance. 
It is estimated that over 75 percent of adults worldwide incure lactose intolerance or 
hypolactasia, or lactase deficiency. 
The enzyme lactase is a disaccharide which acts selectively upon lactose contained 
in milk and dairy products hydrolyzing it into its two component monosaccharides, 
glucose and galactose (Ferguson, 1995). Lactase can be detected in the human fetus by the 
eighth week of gestation (Pray, 1999). Levels continue to rise during the remainder of 
16 
 
 
gestationperiod, peaking the second to third day post partum and remaining high during 
infancy. This lactase is critical in allowing the mammal to gain nutritional value from milk 
and other dairy products by allowing absorption and use of the component 
monosaccharide. Furthermore, if undigested, lactose on reaching the small or large 
intestine produces numerous troublesome symptoms that lead to abdominal pains. 
However, what happens after infancy is determined by one‟s genetic make up. If 
man possesses the more common autosoma recessive gene, lactase activity begins to drop 
between the ages of 2 and 20 years (McBean et al., 1998; Lee et al., 1998). On the other 
hand (or chromosomal) if one possesses the less common autosoma dominant gene, 
lactase activity remain high into adulthood. 
2.1.6 Manifestation of lactose intolerance 
The problems relating to the inability to digest lactose occurs in several anatomic 
locations (Fernendes et al., 1987). In the small intestine, lactose exerts an Osmotic pull 
which prevents reabsorption of water into the body. The individual then suffers from 
accompanying abdominal pain, nausea, and rumblings which can lead to decreased quality 
of life and daily activities (Ferguson, 1995). Intestinal contents moved through more 
rapidly to the large intestine. Bacteria in the large intestine ferment the lactose, producing 
hydrogen, organic acids, and carbon dioxide. Abdominal pain and bloating become 
pronounced. Eventually the patient may suffer from watery diarrhea, flatulence, 
involuntary leakage and staining of underclothing and/or other inconveniences, even from 
a competent rectal sphincter (Ferguson, 1995). However, secondary  -galactosidase 
deficiency can occur at any age. It is a transient state of  -galactosidase deficiency due to 
the damage to the intestinal mucosa where  -galactosidase is produced. 
2.1.7 Treatment of lactose intolerance 
As lactose intolerant people do not have ability to synthesize  -galactosidase 
enzymes, the treatment of milk and its derivatives with  -galactosidase is required 
(Fernendes et al., 1987). Among the enzymes hydrolases are receiving increasing 
industrial applications, especially β-galactosidase or β-D-galactoside galactohydrolase (EC 
3.2.1.23), which catalysis the hydrolysis of lactase into galactose and glucose has got 
17 
 
 
special attention (Furlan et al., 2000). The importance of β-galactosidase is related to its 
use in milk and milk derivatives to decrease their lactose content thereby solving the 
problem of low lactose solubility and degree of sweetness. Furthermore, the economic 
interest in this enzyme is related to its use in food and pharmaceutical industries because 
of the deficiency of β-galactosidase in many people (Gekas,1985).  Therefore, products 
free of lactose or low lactose content can be consumed without any problems by lactose 
intolerant people (Furlan et al., 2000). This has thus created a potential market for the 
application of  -galactosidase thereby increasing the demand for the discovery of new 
species of microorganism producing  -galactosidase with novel characteristics that will 
be of great value to the enzyme industry for different applications (Cortes et al., 2005). 
2.1.8 Lactose Hydrolysis Mechanisms 
Lactose hydrolysis can be performed in two ways namely acid hydrolysis and 
enzymatic hydrolysis (Gekas and Lopez-leiva, 1985)). 
2.1.8.1 Acid Hydrolysis of lactose 
First way of lactose hydrolysis is acid hydrolysis (Gekas and Lopez-Leiva, 1985. It 
is carried out by a homogenous reaction in acid solution or in a heterogeneous phase with 
ion exchange resins (Gekas and Lopez-Leiva, 1985). Acid hydrolysis can be performed 
under harsh conditions. For example; 80% hydrolysis may be achieved in three minutes at 
0
pH 1.2 and 150 C (Gekas and Lopez-Leiva, 1985). Although this procedure seems to be 
simple, it has several disadvantages the most important of which is protein denaturation 
due to low pH and high temperature. It causes reduction in the function of the proteins 
(Gekas and Lopez-Leiva, 1985). Thus, it prevents their uses in many products (Bury et al., 
2001). Moreover, the presence of salts in whey causes deactivation of acid and requires a 
demineralization step. Other disadvantages include off-colour and off-flavour formation. 
Due to many drawbacks of acid hydrolysis, enzymatic hydrolysis by  -galactosidase is 
the preferred method of lactose hydrolysis (Gekas and Lopez-Leiva, 1985).  
 
 
18 
 
 
2.1.8.2   Enzymatic hydrolysis of lactose 
Lactose hydrolysis can be performed either by enzymatic catalysis carried out with 
 -galactosidase enzyme that permits wider operation conditions of both temperature and 
pH (Gekas and Lopez-leiva, 1985). The enzymatic hydrolysis of lactose offers some 
benefits mainly in three areas: Health, food technology and environment (Gekas and 
Lopez-Leiva, 1985 ). 
Health: The health aspects are as described for lactose intolerance patients in the opening 
paragraph of this exposition (Furlan et al., 2000). 
2.1.8.3    Food technology 
The high lactose content in milk products like ice-cream, frozen milks, whey 
spreads and condensed milk, can lead to excessive crystallization resulting in products 
with a mealy, sandy or gritty texture (Zadow, 1993). Further more, the economic aspect of 
transport and storage require the concentration of milk whey by evaporation or 
ultrafilteraction (Zadow, 1993). The easy crystallization of lactose, however, represents a 
strong limitation in such processes (Zadow, 1993). Monosaccharides derived from 
hydrolysis are highly soluble and usually prevents the crystallization of the remaining 
lactose (Zadow, 1993). Using galactosidase to process such products could reduce lactose 
concentrations to acceptable value. This process can thus contribute significance 
improvement to technological and sensorial quality of those foods like increasing the 
digestibility, softness and creamness, etc (Zadow, 1993). Hydrolysis of lactose present in 
whey converts whey into very useful sweet syrup, which can be used in the dairy, 
confectionery, baking and soft drinks industries (Pivarinka et al., 1995). In addition, 
several studies have demonstrated the opportunity to degrade whey lactose using  -
galactosidases for manufacturing galacto-oligosaccharides (Foda and Lopez-leiva, 2000; 
Novalin et al., 2005). 
 
 
 
19 
 
 
Table 2.2: Properties of  - galactosidase from microbial sources. 
Sources pH Optimum Temperature optimum Activators Inhibitors 
0
( C) 
A niger  3.0-4.0 55-60 None None 
needed 
A. oryzea 5.0 50-55 None None 
needed 
t +2 +2 +
K.lactic 6.5-7.3 35 K , mg Ca , na  
t +2 +2 +
K. Fragilis 6.6 37 K , Mn , Ca , Na  
+2
mg  
t +
E. Coli 7.2 40 Na , K   
+2
B. subtilis 6.3 50 None Ca  
needed 
+2
B. stearothermophilus 5.8-6.4 65 Mg   
t +2
S. thermophilus 7.1 55 K , Mg   
L. bulgaricus  42.45   
C. inaegualis 6.0 30.55   
 
Source: Gekas and Lopez-Leiva 1985, Mahoney, 1985 
  
20 
 
 
Enzymologists have attributed great importance to the galacto- syltransferase 
activity of  -galactosidase to obtain the synthesis of oligosaccharides with two or more 
galactose units starting from lactose (Rojaka et al., 2003). Solutions with high lactose 
concentrations submitted to the action of  -galactosidase and thereby suffer 
transgalactosylation reaction hence producing transgalactosylated oligosaccharide (TOS). 
These products act as functional foods with several beneficial effects for their consumers 
(Ferguson, 1995). Diets enriched with TOS significantly increase the population of 
Bifidobacterium like Lactobacillus and some species of Streptococcus, with a consequence 
decrease in the concentration of putrefactive bacteria in the gut of humans and animals. It 
is also known, however, that the number and amount of oligosaccharides formed from 
lactose depends on the origin of  -galactosidase (Ferguson, 1995). Besides, TOS are 
non-carcinogenic sugars and can be widely used as additives in several infant formulations 
and in the manufacture of Candy, Pastry, etc, because of their heat stability (Ferguson, 
1995). 
This change in the intestinal flora (bifido effect) composition has been proposed to be 
responsible for the decrease of putrefactive products in the feces, lower blood cholesterol 
2+ 
content, and higher Ca absorption, a small loss of bone tissue in overiectomised rats and 
a lower incidence of Colon Cancer (  Gaudreau et al., 2005). 
(a) Reaction Mechanism  
Enzymatic hydrolysis of lactose is one of the most important biotechnological 
processes in the food industry (Furlan et al., 2000). Lactose hydrolysis mechanism was 
explained by using the enzyme  -galactosidase obtained from Escherichia coli (Jacobson 
1994).  
A double-displacement reaction mechanism was proposed in which  -
galactosidase formed and hydrolyzed a glycosyl-enzyme intermediate via carbonium ion 
galactosyl transition state (Wallfels and Malhrotra, 1961). It is proposed that the active site 
of  -galactosidase contains cysteine and histidine amino acids which function as proton 
donor and proton acceptor, respectively (Wallenfels, 1972). Cysteine amino acid contains 
the sulphydryl group acted as proton donor and histidine residues contains imidazole 
21 
 
 
group acted as neucleophile site to facilitate cleavage of the glycosidic bond, respectively, 
during the enzymatic hydrolysis procedure ( Wallenfels, 1972)  
The second reaction that occurred is called galactosyI (transgalactosyI) reaction. In 
this reaction,  galactosidase transferred the galactosyI moiety from the interdicted to an 
acceptor containing a hydroxyI group (Figure 2.4) (Mahoney, 1998). When this acceptor is 
water, free galactose is formed by hydrolysis. However, under certain conditions, other 
sugars are able to act as acceptors and give rise to oligosaccharide formation (Mahoney, 
1998). 
(b) Oligosaccharide formation 
Enzymatic hydrolysis of lactose is accompanied by galactosyI transfer of other sugars, 
thereby reducing oligosaccharides (Furlan et al., 2000). The amount and nature of the 
oligosaccharide formed by transgalactosyI reaction depends mainly on the enzyme source 
and the nature and concentration of the substrate (Gekas et al., 1985). 
(c) The substrate 
The yield of oligosaccharides can be increased by using higher substrate and/or by 
decreasing the water content (Mahoney 1998). Quantitatively, the disaccharide allocatese 
is one of the major oligosaccharides production can be reached between 30-40% of the 
total sugars present (Huber et al., 1976). However, at lower initial lactose levels, such as 
those found in milk and whey, transferase activity is reduced, where maximum 
oligosaccharide levels can reach 22 to 25% (Huber et al., 1976). 
Oligosaccharides are hydrolyzed slowly, both in vitro and in vivo (Nielsen et al., 1992). 
They can be considered to be physiologically functional foods which promote the growth 
of bifidobacteria in the colon and a wide variety of health benefits has been claimed in 
connection with this effect (Mahoney, 1998). 
 
 
 
22 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.3: Proposed mechanism of lactose hydrolysis by  - galactosidase 
(Source: Richmond et al., 1981) 
 
 
 
 
 
 
 
 
23 
 
 
 
 
 
 
 
 
Figure 2.4: Proposed mechanism of galactosyl transfer reaction by  galactosidase. 
 (Source: Richmond et al., 1981) 
 
 
 
 
 
 
24 
 
 
The mechanism for the transferase reaction is still not known but it is thought to involve a 
1, 4-linked galactose being transferred to a 1, 6-linkage (Huber et al., 1976). The result of 
oligosaccharide formation is beneficial to human health (Welttagen et al., 1998). They 
have been added to infant formula as potential bifidus factors” to promote the growth and 
the establishment of bifid bacteria in the intestine (Hsu et al., 2005). Moreover, other 
reported therapeutic benefits of oligosaccharide consumption include decreased serum 
cholesterol levels, enhanced absorption of dietary calcium and enhanced synthesis of B-
complex vitamins (Onishi et al., 1995). 
2.2 Galactosidases 
This is described as either of two types of Enzymes:  - (or alpha) galactosidase 
EC3.2.1.22, with a systematic name as:  -D-galactoside galactohydrolase; otherwise 
called melibiase (Guimares et al., 2001). It is an enzyme that hydrolytically removes 
terminal non-reducing  -D-galactose residues in  -galactosides including galactose, 
oligosaccharide, galactomannans, and galactolipids (Luonteri et al., 1998, de vries and 
viser, 2011).  
 -galactosidase EC3.2.1.23 with systematic name:  -D-galactoside galactohydrolase, 
also known as: lactose is an enzyme that removes terminal  -D-galactose residues in  -
galactosides by hydrolysis. 
2.2.1 Alpha Galactosidase ( -Gal) 
Galactosidase or melibiose (α-D-galactosidase galactohyrolase, EC 3.2.1.22) is an 
exogalactosidase that cleaves the terminal non-reducing α-1-6-linked galactose residues 
from α-D-galactosides including galactose olligosaccharrides such as melibiase, raffinose 
and stachyose and branched polysaccharides such as galactomaannans and 
galactoglucomannans (Manzanares et al., 1998, Shibuya et al., 1995). The enzyme is 
widely distributed in nature: microorganisms, plants, and mammals (Naumoff, 2004 and   
Matrai et al., 2000). They are of particular interest in view of much potential of their 
biotechnological and industrial applications. In plants, α-galactosidase has been greatly 
investigated from the seeds in relation of germination where it is involved in the 
mobilization of reserved carbohydrates like raffinose and stachyose (Mita and Steinkraus, 
25 
 
 
1975, Kang and Lee, 2001). They play a crucial role in upgrading nutritional values of 
legume-based foods: Industrially (food processing in particular), it is important in 
removing raffinase in beet sugar which inhibits normal crystallization of sucrose 
(Ohtakara and Mitsutomi, 1987). It is also used in the hydrolysis of raffinose and 
stachyose present in soybean milk (Guimaraes et al., 2001). Transglycosidase activity was 
also demonstrated in some of the α-galactosidase (Anisha et al., 2007). The 
galactooligosaccharides by the transferase action of this enzyme can be used as a Probiotic 
in functional foods. It also has interesting applications in the pulp and paper industries 
(Kang and Lee, 2001). 
2.2.2 Beta Galactosidase (  -Galactosidase) 
 -galactosidase (  -D-galactoside galactohydrolyse; E.C. 3.2.1.23), in other 
words lactase, (Yang et al., 1995) is a commercially important enzyme that catalyzes the 
hydrolysis of lactose into its constituent monosaccharides glucose and galactose (Figure 
2.5) 
The enzyme from several sources have been well characterized, especially the enzyme 
from   Escherichia coli serves as a model for the understanding of the action of the 
enzyme (Jacobson 1994). 
 
 
 
 
 
 
 
 
 
26 
 
 
 
 
 
 
 
 
 
 
Fig 2.5  -galactosidase structure (source: Yang et al., 1995) 
 
 
 
27 
 
 
2.2.3 Physicochemical Properties of  -Galactosidase 
The enzymes derived from various microbial sources have different properties, 
such as protein chain length, and the position of the active site. However, it has been found 
recently that  -galactosidase from different sources have the same amino acid residue 
and glutamic acid, as their catalytic site, as shown in Table 2.3. 
Molecular weight of  -galactosidase varies between organisms as well. E. coli  -
galactosidase has a molecular weight of 116, 353 kDa per monometer (approximately 540 
kDa per molecule). Molecular weights of other bacteria‟s  -galactosidase are presented 
in Table 2.4 
The effects of mono-and divalent cations have been well documented (Greenberg 
and Mahoney, 1982; Garman et al., 1996; Kreft and Jelen, 2000; Vasiljevic and Jelen, 
2002). Divalent cations such as magnesium and manganese cations may have a positive or 
negative effect (Garman et al., 1996). The rate of lactose hydrolysis by  -galactosidase 
+2
from each species was enhanced by Mg  (Vasiljevic and Jelen, 2002). However, the 
+ +
effect of K  and Na  differed from strain to strain. In another study, manganese was found 
to be the most effective cation followed by magnesium for the maximum  -galactosidase 
activity of Streptococcus thermophilus (Greenberg and Mahoney, 1982). 
+2)
Calcium (Ca  is known as an inhibitor of  -galactosidase (Greenberg and Mahoney, 
1982). However, all of the calcium in milk is bound to casein. As it is not free in solution, 
it does not inhibit  -galactosidase activity (Garman et al., 1996). 
2.2.4 Applications of β-galactosidase  
β-galactosidase enzyme used in the hydrolysis of lactose is one of the most 
important biotechlogical processes in the food industry. It has potentially beneficial effects 
on the assimilation of foods containing lactose, as well as the possible technological 
advantages as below: (Gekas and Lapez-leiva 1985, Jurado et al., 2002) 
1. Elimination of lactose intolerance by encouraging the utilization of lactose as an 
Energy Source 
28 
 
 
2. Formation of galacto-oligosaccharides during lactose hydrolysis to favour the growth 
of intestinal bacterial micro flora. 
3. Improvement in the technological and sensorial characteristics of foods containing 
hydrolyzed lactose from milk or whey: increased solubility, greater sweetening 
power, formation of monosaccharide which are easier to ferment certain products 
such as yoghurt and reduction of the maillard reaction. 
4. Greater biodegradability of whey.   
Many organisms have been known to have high β-galactosidase production for 
commercial use. Although, yeasts (intracellular enzyme), fungi or molds (extra cellular 
enzyme) are known to produce β-galactosidase (Gekas and Lopez-leiva, 1985). Bacterial 
sources have gained more importance and they are preferable due to ease of fermentation. 
High activities of exploited sources of β-galactosidase have been of microbial origin 
(mainly yeast and moulds) (Shibuya et al., 1995). 
 Although properties of the microbial β-galactosidase vary with the organism, 
suitable enzymes must be selected according to their application of an enzymes can be 
determine by its operational pH enzymes from yeast and bacteria (Shibuya et al., 1995). 
Acid pH enzymes from fungi and neutral-pH enzymes from yeast and permeate whereas; 
neutral-pH enzymes from yeast and bacteria are generally suitable for milk and sweet 
whey process (Mahoney, 1985) 
Fungal enzymes have pH optima in the range of 3 to 5 and are therefore, suitable 
for processing acid whey and permeate. They also have relatively high optimum 
 O O
temperature which is between 55 C -60 C. (Flores et al., 1996) However, fungal β-
galactosidase is not as pure as from a yeast source and it may contain other enzymes such 
as protease, lipase or amylase (Mahoney, 1985). As a consequence of these limitations, 
fungal applications of β-galactosidase have been limited as high acid products and 
pharmaceutical preparations (Mahoney, 1985). 
 
 
 
29 
 
 
 
Table 2.3 Active sites and other properties of  -galactosidase from various 
microbial origins. (Source: Zhou and Chen 2001).  
 
Enzyme Origin K.lactis E.coli A.niger  
Length (AA) 1025 1023 1006 
482 461 200 
Proton donor Glu Glu Glu
551 537 298
Nucleophile/base Glu Glu  Glu  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
30 
 
 
 
Table 2.4 Molecular weights of  -galactosidase from bacterial sources 
 
Organism Molecular Weight (kDa) Source 
S. solfataricus 240 Grogan (1991) 
S. thermophilus 530 Greenberg and Machoney (982) 
L.acidophilus 570 Nielsen and Gilliland (1992) 
L.lactis 500 McFEters et al., (1969) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
31 
 
 
2.2.5 Commercial procedures 
Enzymatic hydrolysis of lactose by β-galactosidase is one of the most popular 
technologies to produce lactose-reduced milk for consumption by lactose-intolerant people 
(Gekas and Lopez-leiva, 1985). Evolution of lactose-products began when the enzyme β-
galactosidase became accessible to the consumer (Kligerman, 1981). The consumer could 
add this enzyme to a glass of fresh milk to hydrolyse the lactose. After the overnight 
incubation at refrigerator temperature, lactose content is reduced by 70% or more 
depending on the enzyme amount added (Kligerman, 1981). Most lactose intolerant people 
could then consume the “lactose-reduced” milk without adverse effects (Kligerman, 
1981). 
With the developments in processing techniques, hydrolyzing lactose before 
packaging, certain dairy products have become more prevalent (Gilliland et al., 1996). 
Through the advances in research, the procedure has become more cost effective due to 
increased understanding of the process and its effects on characteristics of the product 
(Holsinger and Kligerman, 1991). With these new advancements, various types of lactose-
reduced foods such as milk. American cheese, cottage cheese and ice cream have made 
their way into this growing market (Reiter, 1991). Many research efforts have been 
focused on ways to reduce or remove the lactose in dairy products (Kocak and Zadow, 
1989). One of the more common ways to accomplish this is through the use of enzymes. 
Three techniques may be utilized to accomplish enzymatic lactose hydrolysis (Holsinger 
and Kligerman, 1991): 
1. “Single-use” or “throw-away” batch systems 
2. Recovery systems (β-galactosidase reuse systems) 
3. Immobilized enzymes which are systems where the enzyme is chemically bound to 
an inert matrix (Holsinger and Kligerman, 1991). 
The advantages of immobilized enzymes are clear: ease in enzyme recovery and 
reuse, ease in product recovery and continuous operation (Bury et al., 2001). The 
behavious of immobilized enzyme reactors have been extensively studied as a critical 
point in the industries process production (Li et al., 2007). 
32 
 
 
It has been proposed that the “single-use” enzyme batch system could be used 
conveniently during the development of new lactose reduced products, whereas the latter 
systems are better for large-scale operations (Holsinger and Kligerman, 1991). The 
method ultimately used is mainly dependent on a number of factors including pH of the 
product, maximum temperature the product will reach and how long it will remain there, 
contact time, activity of the enzyme, substrate, and cost (Zadow, 1986). 
2.3  Food grade microorganisms  
Although  -galactosidases (lactase) have been found in numerous biological 
systems, microorganisms such as yeasts, mold and bacteria groups still remain the only 
sources for its commercial production (Vasiljevic and Jelen, 2001). The use of 
microorganisms in food production is accepted when they have a long history of safe use. 
The bacterial species currently being used by the dairy industry which produce  -
galactosidase enzyme, belong to the genera of Lactobacillus and Bifidobacterium 
(Heyman et al., 2002). It comprises a limited collection of strains (Fernandez et al., 1999; 
Heyman, 2006; He et al., 2008). These bacteria have become a focus of scientific studies 
for three (3) particular reasons (Somkuli et al., 1998): (a) lactose maldigesters may 
consume some fermented dairy products with little or no adverse effects, (b) these bacteria 
are Generally Regarded As Safe (GRAS) so the  -galactosidase enzymes derived from 
them might be used without extensive purification (Vasiljevic and Jelen, 2001), (c) some 
strains have probiotic activity such as improved digestion of lactose and a suitable strain 
selection must be carried out to manufacture probiotic dairy products (Vinderola and 
Reinheimer, 2003). 
 -galactosidase enzyme therefore, is one of the glycosidases that is widely used 
in dairy industry and is produced by most lactobacilii and yeasts (Karasova et al., 2002; 
Corral et al., 2006; Nguyen et al., 2007). It has two enzyme activities: one is responsible 
for the hydrolysis of lactose and also cleaves cellobiose, cellotriose, cellotetrose and to 
certain extent cellulose and the other,  -glycosides (Troelsen, 2005; Heyman, 2006). 
However, the  -galactosidase from thermophilic Lactic acid bacteria is an intracellular 
enzyme (Kreft et al., 2000). Its release from microorganisms may be obtained either by 
33 
 
 
mechanical disruption or by chemical permeabilization of the cell membrane (Kreft et al., 
2000). The effectiveness of the various disruption methods differs for different microbial 
genera and strains. The literature tends to focus more on the distruption of yeasts with 
much less information being available on the disruption of lactobacilli (Bury et al., 2005). 
2.3.1  Lactic acid bacteria 
These are Gram-positive, usually non-motile, non-spore forming rods and cocci, 
naturally present in raw food materials and in the human gastrointestinal tract (Salminen et 
al., 1998a). They lack the ability to synthesize cytochrome and porphyrins and therefore 
cannot generate ATP by creation of a proton gradient. The lactic acid bacteria can only 
obtain ATP by fermentation, usually of sugars (Chammes et al., 2006). Since they do not 
use oxygen in their energy production, lactic acid bacteria happily grow under anaerobic 
conditions, but they can also grow in oxygen‟s presence as they are protected from oxygen 
by-products (e.g H2O2) because they have peroxidases (Kim et al., 1983). Hence they are 
referred to as aerotolerant anerobes. They differ from other organisms by their ability to 
ferment hexoses to lactic acid ( Frazir et al., 1978).  
Lactic acid bacteria can be divided into two groups based on the products produced 
from the fermentation of glucose (John Lindquist, 1998). Homofermentative Organisms 
ferment (i) glucose to two moles of lactic acid, hence lactic acid is the major product of 
this fermentation. Heterofermentative lactic acid bacteria ferment 1 mole of glucose to 1 
mole of lactic acid, 1 mole of ethanol, and 1 mole of C02 (John Lindquist, 1998).  The 
homofermenters possess the enzyme aldolase and they are able to ferment glucose more 
directly to lactic acid than the heterofermenters (John Lindquist, 1998). The 
heterofermenters use the alternative Pentose Monophosphate Pathway, converting six 
carbon sugars hexoses to five carbon sugars (pentoses) by the enzyme phosphoketolase, 
producing in the process both aldehyde and diacetyl-highly desirable aromatic and flavor-
enhancing substances (Car et al., 2002). The heterofermenters are often use in dairy 
industry because of these flavor-enhancing substances (Wood and Holzapfel, 1995 and 
Car et al., 2002). Because of the low energy yields, lactic acid bacteria often grow more 
slowly than microbes capable of respiration, and produce smaller colonies of 2-3mm. 
http://www:bact.wisc.edu/Microtextbook/rodex.  
34 
 
 
 
2.3.1.1 The genera of lactic acid bacteria 
Lactic acid bacteria are classified by the fermentation pathway used in fermenting 
glucose and by their cell morphology. Lactobacilli are rod-shaped organisms that can be 
either hetero-or homofementative (John Lindquist, 1998). Lactobacilli are more tolerant to 
acid than the other genera of lactic acid bacteria and this property makes them important in 
the final phases of many food fermentations when other organisms are inhibited by the 
low pH (Axelsson, 1998). 
Leuconostoc are ovoid cocci, often in chains (Wood and Holzaphel, 1995). All bacteria of 
this genus have a heterofermentative mode of metabolism. Pediococcus are cocci often 
found in pairs and tetrads that are strictly homofermentative (Axelsson, 1998). Their 
habitat is restricted mainly to plants. S.cerevisiae has been used as a starter culture for the 
fermentative of some sausages with great success (Wood and Holzaphel, 1995, Axelsson, 
1998, Carr et al., 2002). Streptococcus are cocci in chains that are distinguished from the 
Leuconostoc by their strictly homofermentative metabolism (Wood and Holzaphel, 1995). 
These organisms can be Isolated from oral cavities of animals, the intestinal tract, skin and 
any food that comes in contact with these environments. Enterococcus and Lactococcus 
are two recent taxonomic divisions of lactic acid bacteria (John Lindquist, 1998). They 
were created to re-organize the large and divergent Streptococcus genus into smaller, more 
related groups of bacteria. Enterolcoccus are gram-positive cocci that form pairs or chains. 
They are distributed widely in the environment particularly in faeces of vertebrates 
(Jacobson, 1994). Lactococcus includes strains that are gram-positive, spherical cells, 
occurring in pairs or chains. They have a strict homofermentative metabolism and are 
found in dairy and plant products (Wood and Holzaphel, 1995; Axelsson, 1998). 
Lactic acid bacteria have been used for centuries to produce fermented food 
products including Pickles, Sauerkraut, Sausage, Yogurt, Cheese, Butter milk and soy 
sauce (Rachi, 1988). They are widely used by man a/or in food preservation. They are 
widely used as starter cultures for fermentation in dairy, meat and other food industries. 
They remain the main factors of fermentation and protection of fermentative foods 
(Chammas et al., 2006). 
35 
 
 
These food grade bacteria can also improve the safety, shelf-life nutritional values 
as well as other qualities as enumerated above (Kim and Raja Gospel, 2000). In addition 
lactic acid bacteria can be used as cell factory for the production of food additives and as 
well function as probiotics thereby contribute to the general health of the consumer upon 
consumption (Kim and Raja Gospel, 2000).  
2.3.2 Food grade yeasts 
Yeasts have been considered the predominant microbial enzyme source for food 
applications (Ah et al., 2002). For centuries man has applied yeasts as starter cultures in 
Cheese, Bread, Wine, Beer, and other alcoholic fermentation products (Lowes et al., 
2000). Different varieties of yeasts are the world‟s premier industrial microorganisms 
which has wide exploitation in the production of food beverages and pharmaceuticals 
(Savora, 2002). Yeasts can grow at room temperature and can utilize (assimilate and/or 
ferment) a variety of carbohydrates (Nahvi and Moeini, 2004), such as pentose, hexose, 
disaccharides, and rarely, polysaccharides (Barnette et al., 1999). They are regarded as 
important component of the mioroflora of many cheese varieties because of the low pH, 
low moisture content, high salt concentration and refrigerated storage condition of these 
products (Fleet, 1990; Deveyol, 1990).  
Yeasts are widely distributed in nature in almost all niches and are therefore often 
found as contaminants, in both commercial and traditionally fermented milk (Nahvi and 
Moeini, 2004). In some circumstances, however, like in the production of Kefin and 
Kumis, where they are deliberately introduced into milk to bring about the desired aroma 
and flavour of the final product (Nahvi and Moeini, 2004).  
As is commonly observed, the dairy products are especially favourable medium for growth 
of yeast due to the acidic reaction of the medium. Another important condition for their 
development is their ability to grow at low temperature (low pH value and low moisture 
content) which are unsuitable for many other microorganisms especially bacteria (Sarova, 
2002). Cultured milk products create favourable conditions for microbial growth (Viltoen 
et al., 2003). The changes of cultured milk products caused by metabolic activity of yeasts 
depend on degradation of lactose or compounds of its hypolysis, secretion of lipolytic and 
36 
 
 
proteolytic enzymes, assimilation of organic salts and the ability to multiply at low 
0
temperature (5-10 C) during product storage (Fleet, 1992; Norvhus, 1996). 
2.3.2.1 Yeasts and their coexistence with lactic acid bacteria  
Yeasts usually adapt to coexistence with lactic acid bacteria as one of the groups 
gain the upper hand over the other or both groups grow intensively together (Ah et al., 
2002). By this process, specific interactions take place. The yeasts play favourable effects 
upon the bacteria due to the changes in pH value in the medium and the secretion of 
biologically active substances such as vitamins, enzymes, amino acids, etc. In some cases, 
the yeasts significantly increase the viability of the lactic acid bacteria (Roberto et al., 
1984). The investigations of the microflora found in the dairy drinks Kefir and Kumis 
proved the presence of lactic acid bacteria and yeasts coexisting as the later play 
stimulating effects on the bacteria (Gekas and Lopez-Leiv, 1985).   
2.3.2.2 Influence of yeast growth on the maturation process and final quality of 
cheese 
There are several mechanisms by which yeast growth is thought to influence the 
maturation process and quality of cheese (Sarova, 2002). Fermentation of residual lactose 
within the curd by species of Kluyveromyces maxianus produce secondary (flavour) 
metabolites as well as carbondioxide gas that opens up the curd texture (Rachie, 1988). 
Furthermore, the activities of extracellular proteases and lipases produced by some yeast 
species such as Candida lipolytica could alter curd flavour and texture. According to Fleet 
(1992), the release of autolytic products by yeasts can influence these cases. The high 
proteolytic and lipolytic activity of yeasts does not allow the cheese surface to form: the 
yeast may produce substances that stimulate surface growth of both lactic acid bacteria 
and aerobic bacteria (Welthagen and Viljoen, 1998). They may ferment residual lactose, 
metabolize the lactose and influence flavour formation by producting volatile acids and 
carbonyl compounds (Fleet, 1992).  
2.3.2.3 Role of yeasts in Yogurt production  
Yogurt is considered as a potential source of different metabolites of 
biotechnological interest     (Bralasiewicz et al., 2001). Hence numerous studies have been 
37 
 
 
undertaken to isolate and screen potentially important microbes in particular yeasts from 
yogurt (Bralasiewicz et al., 2001). The presence and predominance of certain yeast species 
in yogurt is associated with their chemico-physical properties including their ability to 
ferment variety of sugars, and to produce several enzymes that hydrolyze milk sugars, fats, 
and proteins (Gekas and Lopez-Leiv, 1985). Such enzymes could have multidimentional 
applications in pharmaceutical industries. β-galastosidase produced by lactose fermenting 
yeast species of Kluyveromyces group is indeed been utilized for the treatment of lactose 
intolerance (Gekas and Lopez-Leiv, 1985). Furthermore, yeast isolates from yoghurt 
shown bactericidal activity against Helicobacter pylori suggesting its potential application 
as probiotics (Ah et al., 2002).  
2.3.2.4 The importance of yeast in the treatment of whey wastes 
Whey is the aqueous fraction of milk generated as a by-product of cheese 
manufacturing (Rachie, 1988). The main solute in cheese whey is lactose, present at a 
concentration of 4.5-5%. Because of its high organic content, dumping it directly to the 
environment causes serious contamination problems (Rohm et al., 1992). As a solution, 
bioconversion of whey into Single Cell Protein (SCP) or ethanol has been adopted in 
several countries ((Nahvi and Moeini, 2004). This is achieved by employing yeasts of 
different species including Kluyveromyces, candida and Trichosporous as they are 
naturally able to metabolize lactose. Single cell protein can be used as a dietary 
supplement in feeding domestic fowl and farm animals.As they can grow at between 25-
o
30 C (Sarova and Nikolova, 2002), yeast can be exploited to manage lactose pollution. 
This attributes can be exploited to manage the biodegradable wastes of the food, and 
beverage industries that is non friendly in the environment (Nahvi and Moeini, 2004). 
2.3.2.5 Yeast as a spoilage organism  
Yeasts play a dual role: in some cheese types, they make a positive contribution to 
the development of flavour and texture during maturation; while in other varieties they can 
be regarded as spoilage organisms. The main defects caused by spoilage yeasts are fruity, 
bitter or yeasty off-flavours, gas production, discolouration changes and loss of texture 
quality due to gas-production and packaging (Seiler and Busse, 1990; Rohm et al., 1992) 
38 
 
 
contamination by yeast in yogurts is generally related to the fruits added and/or poor 
hygienic practices during packaging operations (Bralasiewicz et al., 2001).   
2.4 Milk 
2.4.1 General Overview 
Milk, according to F.A.O. (1990) is a unique biological fluid secreted by mammary 
glands of mammals for the primary purpose of nourishing their young.It is believed to be 
nature‟s most nearly perfect simple food (Ihokoronye and Ngody, 1998). Milk has been 
used as food since antiquity. Although a liquid, it contains greater amounts of solids than 
many other food materials that occur in nature in solid forms (Vedamuthu, 1983). This 
unique feature is made possible by well known physico-chemical forces in nature 
(Vedamuthu, 1979). Milk is designed to satisfy the nutrient requirement of the young and 
rapidly growing mammals.  
2.4.2 Nutritional significance 
Milk is often regarded as being nature‟s most complete food. It earns this 
reputation by providing many of the nutrients which are essential for the growth of the 
human body (Sarova, 2002). Being an excellence source of protein and having an 
abundance of vitamins and minerals, particularly calcium, milk can make a positive 
contribution to the health of a nation (Bralasiewicz et al., 2001). The realization of its 
nutritional attributes is clearly illustrated by the implementation of numerous “school milk 
programs” worldwide.Nearly every civilization has consumed cultured milk of one type or 
another and these products have been and are still of extreme importance  in the nutrition 
of people throughout the world(Anonymuos,1987). Dairy products provide about 76% of 
Calcium, 45% of Riboflavin, 25% of protein, 12% of vitamin and 10% of thianin 
proportional to the recommended dietary allowances in the human diet (Brink et al. 
,1969). In traditional milk-drinking populations milk has always been converted to 
concentrated forms that retain their feed value for a longer period (Chamberlain,1990). 
The shortage of milk and dairy products in most developing tropical countries is due 
primarily to low level of milk production and lack of effective processing and preservation 
techniques (Sarova, 2002).   
39 
 
 
2.4.3  Types, Sources and Production  
Milk is inherently a highly perishable food.Fermentation of milk occurred naturally 
in surplus milk set aside for later use, which were later observed that in some cases to 
form smooth curd with pleasing acid, odour and flavor, became putrid and cause sickness 
when consumed as such (Bulut et al., 2005). From this observation, the preparation of 
fermented or cultured milk products became an art which was handed down from one 
generation to another (Shahari and Friend, 1983). Cultured dairy products such as cheese, 
yogurt, Kefir and clabbered milk have been part of human diet since before recorded time 
(Datta et al., 2006). These are all forms of cultured, soured or fermented milk. At no point 
in 5,000 years of recorded history was there ever a time when milk and its products were 
not a significant part of the human diet (Fox and Mulrihill, 1982). This is what Sally 
Fallon (1982) in her book “Nourishing Traditions” calls scene of these highly nourishing 
foods that the human race through history to this point in time. Since fresh raw milk is 
loaded with numerous beneficial bacterial that when left to their own devices for more 
than a few hours at room temperature will begin the process of fermentation, its probably 
safe to extrapolate that early shepherds discovered that souring not only acts as a means of 
preservation but also improves its digestibility and nutrient availability (Molder, 1994). 
Milk however, is a highly perishable food and there is a high risk of transmitting 
food poising bacteria to consumers (Anon., 1978). It is stressed therefore, that milk 
processing of any kind must be done under carefully controlled hygienic conditions. 
2.4.4.     Nutritional Composition and Values of milk 
Milk contains three major proteins, the principal among them being Casein (Gill et 
al., 1994). When milk is converted into curd either by acid coagulation (e.g Lactic acid 
fermentation) or by enzyme clotting (renette curd), the solid mass is formed by Casein 
(Gill et al., 1994). The watery exudates, whey, contain the other two: lacto-talbumin and 
lactoglobulim. It is for this reason that the two are called “Whey proteins” (Fox and 
Mulvihill, 1982). 
In addition to this, the micromolecular aggregates or casein “micelles” also contain 
small ions such as Calcium, phosphate, magnesium and aerate referred to as Colloidal 
Calcium phosphate (Cap) (Christensen, 1975). According to O‟mahomy (1988), lactose is 
40 
 
 
the principal Carbohydrate of milk, and it is responsible for souring of milk and cream. Its 
value in milk may be as high as 5.5% and as low as 3.5% (Webb et al., 1976). Lactose 
occurs in true solutions in milk. It is a disaccharide made up of glucose and galactose 
linked together by a  -1, 4-linkage (Fox et al., 1982). Microorganisms capable of 
fermenting lactose produce lactic acid as a by-product (Fox et al., 1982). And to the 
cheese maker, lactose is the single most important milk component. According to Fox et 
al., (1982) it is the source of a steady rate of acid accumulation in the cheese vat which is 
absolutely necessary for curd formation, moisture expulsion, texture and flavour 
development. At present, the milk of dairy cows, sheep, goats, camels, water buffalo, 
reinder Yaks and mares is used in various parts of the world (Chirstensen, 1975). 
It is mandatory to ferment and preserve milk to avoid outbreaks of food-borne 
illness and intoxications that could be introduced through the use of raw milk in making 
(e.g. Yogurt or cheese). 
2.4.4.1   The quality of milk 
The type of animals, its quality, and its diet can lead to differences in the colour, 
flavour, and composition of milk (Gill et al., 1994). Infections in the animal which cause 
illness may be passed directly to the consumer through milk. It is therefore extremely 
important that quality-control tests are carried out to ensure that the bacterial activity in 
raw milk is of an acceptable level, and that no harmful bacteria remain in the processed 
products. 
2.4.4.2   Processed milk products 
Although Cow‟s milk is the most popular in many countries, milk can be obtained 
from many different sources. For example, milk from goats and sheep makes a substantial 
contribution to the total milk production in countries of Eastern and Southern Europe, 
Malawi and Barbadoes, whereas the water buffalo is a common source of milk in much of 
Asia. Table 2.5 illustrates some of the differences in composition between these milks. 
2.4.4.3   Local/indigenous milk products 
41 
 
 
The degree to which milk consumption and processing occurs will differ from region to 
region. It is depended upon a whole host of factors, including geographic and climatic 
conditions, availability and cost of milk, food taboos and religious restrictions where 
processing does exist many traditional techniques can be found for producing indigenous 
milk products (Cortes et al., 2005). These are more stable than raw milk and provide a 
means of preservation as well as adding variety to the diet (Kolars et al., 1984). In 
addition, the introduction of Western-style dairy products and the subsequent setting up of 
small scale dairies has provided more choice of dairy products to the consumer 
(Verdamuthu, 1978). 
 
2.4.4.4 Fermented-milk products  
Such as yogurt and soured milk contain bacteria from the Lactobacilli group (Bralasiewicz 
et al., 2001). These bacteria occur naturally in the digestive tract and have a cleansing and 
healing effect (Ah et al., 2002). Therefore, the introduction of fermented products into the 
diet can help prevent certain yeasts and bacteria which may cause illness (Cortes et al., 
2005). 
Many people suffer from a condition known as „lactose intolerance‟. This means 
they are unable to digest the milk lactose. Such people however, tolerate milk if it is 
fermented to produce foods such as yogurt. During fermentation lactic acid producing 
bacteria break down lactose, and in doing so eliminate the cause of irritation. 
 
 
 
 
 
 
 
42 
 
 
 
 
 
Table 2.5 Average (%) Composition of milk in Different Species of Mammals 
 Percentage of Composition  
Species Water Fat Protein Lactose Ash 
Ass 90.0 1.3 1.7 6.5 0.5 
Buffalo 84.2 6.6 3.9 5.2 0.8 
Camel 86.5 3.1 4.0 5.6 0.8 
Cat 84.6 3.8 9.1 4.9 0.6 
Cow 86.6 4.6 3.4 4.9 0.7 
Dog 75.4 9.6 11.2 3.1 0.7 
Elephant 67.8 19.6 3.1 8.8 0.7 
Ewe 79.4 8.6 6.7 4.3 1.0 
Goat 86.5 4.5 3.5 4.7 0.8 
Guinea pig 82.2 5.5 8.5 2.9 0.9 
Human 87.7 3.6 1.8 6.8 0.1 
Liama 86.5 3.2 3.9 5.6 0.8 
Mare 89.1 1.6 2.7 6.1 0.5 
Porpoise 41.1 45.8 11.2 1.3 0.6 
Reindeer 68.2 17.1 10.4 2.3 1.5 
Sow 89.6 4.8 1.3 3.4 0.9 
Whale 70.1 19.6 9.5 -- 1.0 
Sheep 80.71 7.9 5.23 4.81 0.9 
Horse 89.04 1.59 2.69 6.14 0.51 
Source: 2011 Dairy for all. Com, the milk and Diary products website 
43 
 
 
 
 
2.4.4.5     Other Fermented Milk Beverages 
According to official legislation, fermented products derived from milk result from 
fermentation of lactose by means of different bacterial strains (Roginsky, 1988). The most 
recognized fermented product, yogurt, is defined by Codex Alimentarius of 1992 as a 
coagulated milk product that results from fermentation of glucose to lactose by 
Lactobacillus bulgaricus and Streptococcus thermophilus (Rosodo et al., 1992). Other 
lactic acid bacteria (LAB) particularly from the general Lactobacillus, Streptococcus and 
Bifidobacterium can be combined with yogurt starters to produce fermented milks with 
specific desirable characteristics (Kolars et al., 1984).  LABS have been use for thousands 
of years to produce fermented food and milk products. 
2.4.4.6 Cultured/fermented milk products 
Yoghurt is one of the oldest fermented products that is believed to have originated 
from Bulgaria several centuries ago. It was thought to have evolved by allowing naturally-
contaminated milk to sour at a warm temperature. Yoghurt is a highly nutritious acidic 
product of milk of various forms that is of plant or animal origin (Frazier and Westhoff, 
1978). Yogurt can be unsweetened or sweetened, set, or stirred. Curd is the name given to 
a yoghurt-type product made from buffalo milk. Yoghurt according to Vanthamathu 
(1978), may be defined microbiologically as the end product of a controlled fermentation 
of high solids whole milk with a symbiotic mixture of Streptococcusthermophilus and 
Lactobacillusbulgaricus.To obtain good flavoured yoghurt, starter strains are selected with 
a view to obtaining good symbiosis (Moon andReinbold, 1976). The balance of these 
organisms in the culture is important for quality production.The milk which is the raw 
material used in the manufacturing of yoghurt may be obtained from various sources 
(Drissen et al., 1977). The distinctive flavor is contributed by blactic acid, which is 
odourless and with trace amounts of acetaldehyde, diacetyl and acetic acid, which are 
volatile and have strong odours (Verdamuthu, 1978). Apart from the plain yoghurt, fresh 
yoghurt presently marketed is sold in different forms for different applications. The 
varieties include flavoured yoghurt without fruit (like lemon and vanilla), yoghurt with 
44 
 
 
fruit purees whole or sliced fruits mixed uniformly throughout (Swiss style) or filled in the 
bottomof cup or on top of the yoghurt to mixed by the consumer (Sundae style), soft or 
hard –frozen yoghurt sticks, fluid yoghurt drinks and fruit topped pies field with yoghurt 
products are also sold for making up baking mixes, soup, salad dressing and confectionery 
(Vedamuthu and Neville, 1978).   
The principles of preservation for yoghurt are (Vedamuthu and Neville, 1978); 
 Pasteurization of the raw milk to destroy contaminating microorganisms and 
enzymes 
 An increase in acidity due to the production of lactic acid from lactose. This 
inhibits the growth of food-poisoning bacteria. 
 Storage at a low temperature to inhibit the growth of microorganisms. 
Yoghurt types and products include (Kolars et al., 1984): 
 Sweetened stirred style yogurt with fruit preparation 
 Fruit-on-the-bottom set style: fruit mixture is layered at the bottom followed by 
inoculated yogurt, incubation occurs in the sealed cups 
 Soft-serve and Hard Pack frozen yogurt 
 Probiotic yogurts: it has become quite common to add probiotic bacterial strains to 
yogur (those with proven health-promoting benefits, in addition to ST and LB). 
These could include lactobacillus acidophilus, Lactobacilus casei, or 
Bifidobacterium spp. When probiotics are added, it has also become common to 
add ingredients known as prebiotics, such as inulin, which will, after digestion, aid 
in the growth of the probiotics in the colon (Ah et al., 2002). 
 
 2.4.4.7 Pasteurization 
0
Pasteurization is carried out by heating to 65 C for a period of 30 minutes (Driessen et al., 
1977). This product was originally fermented by product of butter, but today it is more 
common to produce cultured buttermilks from skim or whole milk (Driessen et al., 1977). 
The culture most frequently used Lactococcus lactis, perhaps also subsp. Cremoris or 
0 0
diacetylactis. Milk is usually heated to 95 C and cooled to 20-25 C before the addition of 
the starter culture. Starter is added at 1-2% and the fermentation is allowed to proceed for 
45 
 
 
16-20 hours, to an acidity of 0.9% lactic acid. This product is frequently used as an 
ingredient in the baking industry, in addition to being packaged for sale in the retail trade 
(Viljoen et al., 2003). 
2.4.4.8   Other Processed milk products. 
Ice cream 
Ice cream is a frozen mixture which contains milk, sugar, fat, and optical 
thickeners (e.g. pectin or gelatin), colouring, and flavouring. It may be sweetened and 
flavoured in numerous ways with nuts, fruit pieces, and natural or artificial flavours and 
colours (Hastings et al., 1987). 
The principles of preservation are (Kreft et al., 2000);  
(a) pasteurization to destroy most micro-organism and enzymes 
(b)freezing to inhibit microbial growth 
Acidophilus milk 
Acidophilus milk is traditional milk fermented with Lactobacillus acidophilus 
(LA), which has been thought to have therapeutic benefits in the gastrointestinal tract 
(Gilliland et al., 1996). Skim or whole milk may be used. The milk is heated to high 
0
temperature, e.g., 95 C for 1 hour, to reduce the microbial load and favour the slow 
growing LA culture (Sraidhar et al., 1989).  
2.4.5 Cheese 
Cheese is a wholesome article of food, made from the curd of milk coagulated by 
rennet, separated from the whey and pressed into a solid mass (Singh and Rachie, 
1988).Cheese is a solid curd with some or all of the whey drained off, and either used in 
its present form or dried with further maturation taken place in storage (Rachie, 1989). 
The process is achieved by the combined action of lactic acid bacteria and the enzyme 
rennin. Just as cream is a concentrated form of milk fat, cheese is a concentrated form of 
milk-protein (Rachie, 1989). The curd can be formed by activities of lactic acid bacteria, 
organic acids such as lemon juice, vinegar or rennet, sometimes in the presence of heat 
(Chamberlain, 1996). The differences in cheeses that are produced in different regions 
46 
 
 
results from variations in the composition and type of milk, variations in the process, and 
the bacteria used (Chamberlain, 1996). The different cheese varieties can be classified as 
either hard or soft. They can also be classified on the basis of moisture content, into very 
hard, semi hard and soft cheese (Muir, 1997). 
In many parts of West Africa, including Nigeria, the normadic Fulani produce the 
bulk of milk from cattle and goat. Traditional cheese making as practiced by the Fulani 
house wives is probably the most important technique of preserving surplus in rural 
communities in Nigeria and in Benni republic (Aworh, 1987).”Warankasi” in Nigeria is a 
soft un-ripened cheese made from milk. It is regarded as an important source of protein as 
it ay replace meat of fish or be used in various food recipes(Aworh and Egunolety, 1985). 
2.4.6  Phenotypic identification of fermentative microflora 
This procedure is time consuming and often problematic due to ambiguous biochemical or 
physiological traits(Hasting and Holzapfel,1987; Hugas et al., 1993).In the last few years, 
the development of molecular typing methods has offered the possibility of accelerating a 
great deal of bacterial identification. Moreover, direct sampling in complex matrices, such 
as food, may avoid biases related traditional methods. Several molecular techniques have 
been applied for the identification of the main bacterial population isolated from meat 
products such as Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) of whole cell proteins(Samelis et al., 1995); hybridization with rRNA probes 
(Nissen and Dainty, 1995); restriction fragment length polymorphism analysis of 
16SrRNA gene(Sanz et al., 1998), randomly applied polymorphism DNA-PCR 
analysis(Berthier et al.,1998; Andrighetto et al.,2001); PCR-temperature gradient gel 
electrophoresis(Cocolin et al., 2000) and species-specific PCR (Yost et al., 2000). 
Species-specific PCR is a rapid and reliable molecular technique for the characterization 
of bacterial communities without colony isolation. However, the sensitivity of PCR in 
foods can be reduced due to the complexity of the food matrix and the presence of many 
PCR inhibitors. Substances which have been proven to be PCR inhibitory, such as 
Proteinase (Powell et al., 1994), heme compounds (Akane et al.,1994),chelating agents 
and proteins (Buffone et al.,1991) may be present in fermented sausage homogenates. 
Thus many sample preparation methods such as dilution, centrifugation, filteration, 
47 
 
 
aqueous two-phase systems adsorption methods, and DNA extraction have been developed 
to overcome the effects of PCR inhibitory substances (Lantz et al., 1994).      
2.4.7 Microbial   Physiology 
Growth in the microbial world is usually referred to as an orderly increase in the 
numbers of individuals and the quantity of the cellular constituents which depends on the 
ability of the cells to form new protoplasm from the available nutrients in the 
environment.In most bacteria species this process involves increase in cell mass and 
duplication of organelles, synthesis of new cell wall, partitioning of the two chromosomes, 
septum formation all of which result in binary fission. Growth can therefore be measured 
in terms of changes in cell mass and cell number. 
Physiological characterization of microbial isolates is usually carried out by the 
determination of morphological and biochemical characteristics. The use of physiological 
methods in microbial identification and characterization in combination with molecular 
identification and typing techniques yields the desired results especially in microbial 
ecosystems where the variety of the different strains is of interest. Of equal importance are 
the various responses to Temperature, acid tolerance test (pH), Oxygen potential, growth 
on different Carbon sources which are all beneficial in arriving at a complete physiological 
profile of an organism. 
2.4.7.1 Enzyme purification    
Enzymes are the catalysts of biological processes. The activities of enzymes have been 
recognized for thousands of years. Most workers have employed the principle of 
Centrifugation of microbial cultures to obtain the enzyme fractions from the culture broth 
or media. The products obtained there from are labeled as “crude enzyme” whose 
activities could be accurately characterized through progressive purification procedures.  
Some of those steps would involve ammonium sulphate precipitation, Acetone 
precipitation, Dialysis through a permeable membrane and passage through columns of 
different mesh/ pore sizes 
2.4.7.2 Electrophoresis 
48 
 
 
Electrophoresis refers to the migration of ions in an electric field, a property widely 
employed for the analytical separation of biological molecules. Proteins constitute a major 
fraction of any biological system even though its presence may either be dominant or 
sparse. To isolate these proteins they have to be in solution. Different proteins move at 
different rates as a result of their diverse charges and frictional coefficients so that the 
reading and trailing edges of the migrating protein columns of each species form separate 
moving boundries in a buffer solution. Moving boundary electrophoresis was one of the 
few powerful analytical techniques commonly employed in the early years of protein 
chemistry. However, because of its many drawbacks such as the convective mixing of the 
sample which often limits the resolution to be achieved, other techniques like zone 
electrophoresis in which the sample is constrained to move in a solid support such as filter 
papers, cellulose or a gel. Moreover the small quantity of material that is used in zone 
Electrophoresis permits the various sample components to migrate as descrete bands. 
2.4.7.3 Gel-electrophoresis 
Gel- electrophoresis is among the most powerful and most conveniently used methods for 
micro-molecular separation. The gel in common use, Polyacrilamide and Agarose, have 
pore of different molecular dimensions whose sizes can be specified. The molecular 
separations are therefore based on gel filteration as well as the electro-phoretic mobility of 
the molecules being separated. Sodium dodecyl sulfate (SDS), a detergent that is often 
used in biochemical preparations, binds quite firmly to proteins causing them to assume a 
rod-like shape. Consequently, the electrophoresis of proteins in an SDS- containing 
polyacrilamide gel, separates them in order of their molecular masses because of gel 
filtration effects. 
 
 
 
 
49 
 
 
                                                         CHAPTER THREE 
3.0    MATERIALS AND METHODS 
3.1 Sample Collection 
Twenty five packs of different brands of yogurt manufactured by three different 
companies, namely; Ice creams from Mr Biggs and Tantalizer, yogurt was collected from 
Fanyogo, Cedaa yogurt and De-real yogurt while local cheese „wara‟ were sourced and 
purchased from  Ojoo and Sasa market with different retail outlets in Ibadan.  All samples 
were transported in sterilized ice-packed containers to the laboratory and stored in deep 
freezers (4℃) till further processing.  
    3.2   Media preparation 
 For isolation of Bacteria 2g of cheese and 2 mL of milk was added to 5 mL of MRS broth 
o
and incubated anaerobically (24 hrs at 37 C).Then 5ml of each were spread onto MRS 
o
agar. Plates were incubated in anaerobic jars at 37 C for 48 hrs.  
3.2 Isolation and culture methods 
3.2.1 Isolation and enumeration of yeasts and moulds 
Content of each sachet of yogurt was uniformly mixed and a 10 mL sample was 
aseptically withdrawn and mixed in a flask containing 90 mL of 0.1% sterile peptone 
solution. Ten fold serial dilutions (Harrigan and MacCance, 1960) were made to a power 
of 10 for counting purposes. One mL of each dilution level was introduced into sterile 
Petri dishes to which 20 mL sterile molten agar (SDA, YEA and MRS) was added and the 
mixture gently swirled to ensure even distribution of the inoculum.  
50 
 
 
For enumeration of total fungal count 1 mL of each serial dilution was mixed with 
Saboraud Dextrose Agar (SDA).  The medium was allowed to solidify and then incubated 
o
at 37 C for 24-48hr (Moreira et al., 2001) 
Lactic acid bacteria were isolated on De Mann Rogosa Sharpe (MRS) agar incubated 
o
under appropriate anaerobic conditions at 37 C for 24h.  Bacterial isolates were examined 
by Gram-stain and identified by standard bacteriological and biochemical methods (Sneath 
et al., 1984).  Acid production from carbohydrates in MRS broth base was evaluated.  
Their ability to produce lactic acid was also studied by titrating 10 mL of homogenized 
samples against 0.25 mol/L NaOH using 1 mL of phenolphthalein indicator (0.5% in 
50%alcohol ). 
3.2.2   Characterisation of obtained isolates  
3.2.3   Microbial morphology  
The morphological and colonial appearance of isolates were observed and recorded. 
Colony and cultural appearances were examined with the aid of magnifying lens. The 
forms and sizes of the isolated organisms were observed. The parameters used depended 
on the shape, colour, elevation, margin, texture, size.  Microbial growth in broth culture 
tubes was also observed and examined for uniformity, granular sructure or a uniform 
growth with deposit or surface pellicle.  
The microscopic study was carried out to enhance distinct identification. This included the 
size, shape and type of cell wall staining. Photo-micrographs of important isolates were 
carried out at high magnifications (×400).   
3.2.4   Screening of Isolates for β-galactosidase Production  
In situations where N, N-dimenthyl formamide could not be readily obtained, 0.1mM 
IPTG (isopropyl-thio-β-D-galatophyranoside) was used as an inducer. 
X-gal (5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside) is a chromogenic substrate 
which has been used to screen β-galactosidase positive organisms.One milliliter(1ml) of 
culture samples was serially diluted and plated on MRS agar containing 0.01% X-gal (5-
bromo-4-chloro-3-indolyl-B-D- galactopyaranoside) and 0.1mM IPTG (Isopropyl-thio-β-
51 
 
 
o
D-galactopyranoside) as an inducer.  Plates were anaerobically incubated at 37 C for 24-
48h for colour formation. Colonies producing β-galactosidase were green (Vinderola and 
Reinheimer, 2003). 
3.3 Beta -Galactosidase   production    
3.3.1   Inoculum Preparation  
 The inoculum cultures were grown on a medium composed of modified MRS broth. 
Inoculum was prepared using 24h-old cultures on agar slants.  This was rinsed with 5ml of 
sterile distilled water and thoroughly mixed so that the cells become evenly distributed.  
Thereafter, 1ml of the bacterial cell was transferred into another sterile bottle and stained 
with a drop of crystal violet. 
3.3.2   Determination of Inoculum Size 
Single colonies of the different selected bacterial isolates were inoculated into freshly 
prepared and sterilized modified MRS broth. Each broth culture was serially diluted out in 
sterile distilled water (Harrigan and McCance, 1966) and a mL quantity of each dilution 
fold was introduced into sterile petri dishes. Sterile molten agar was added by the pour 
plate method and the dish thoroughly swirled to ensure adequate and even mixing of the 
cells. The number of cells per petri dish was counted and extrapolated to determine what 
-6 
contained in the initial culture broth. This broth is now diluted out to use at a 10 colony 
forming unit per ml of culture broth. 
3.3.3   Β-galactosidase production from lactic acid bacteria 
  Modified MRS broth (MMRS) which has the same composition with MRS broth except 
that 2% lactose was replaced with 2% glucose was used for β-galactosidase production. 
Fermentation were started with the introduction of 10% of inoculums into 150 mL culture 
medium in 250 mL Erlenmeyer flasks. This was incubated under anaerobic conditions for 
24 hours. Isolates were sub-cultured successivefully in MRS broth and again in modified 
MRS broth for the period of cultivation. Optical Density (OD600nm) of culture inoculum 
(10 mL) was measured before the inoculation into 600ml modified MRS medium at pH 
7.0.  Inoculum culture (1 mL) was diluted with peptone water (9 mL) before reading the 
OD at 600nm. 
 
52 
 
 
3. 3. 4   Intracellular enzyme extraction 
 For intracellular extraction of enzyme, 10 mL of fermented broth (MMRS) is harvested 
0
by centrifugation at 7000 rpm and 4 C for 15 min. The pellet obtained was washed with 10 
mL sodium phosphate buffer (pH 7.0). Two milliliter (2 mL) of Isoamyl alcohol was 
added into the tube containing 10 mL of fermented broth.  After gentle mixing, the content 
was put on rotary shaker and shaken at 180 rpm for 15 min. The mixture was again 
o o
incubated at 37 C for 15 mins. This solution was then centrifuged at 7000 rpm and 4 C for 
15mins. Finally the pellet was re-suspended in 9 mL 0.05M sodium phosphate buffer pH 
7.0. The supernatant was immediately transferred into eppendorf tubes for enzyme assay. 
o
Supernatant was stored at 4 C until the assay time (Barberis and Gentina, 1998).  This 
solution was labeled “Cell Free Crude Extract”. 
3.3.5   Enzyme assay 
The modified method method method method method method method method method 
method method method method described by Food Chemical Codex (1993) was used, 
-3
whereby 2.5×10  mg of the chromogenic substrate ONPG (o-nitrophenyl-β-D-
galactopyranoside), (Sigma-Aldrich) was dissolved in 0.2M sodium phosphate buffer (pH 
7.0). The crude permeabilized cell suspension (1.5 mL) was incubated with 2 mL of the 
o
above substrate (ONPG) mixture at 37 C for 15min {Miller, 1998}.  The reaction was 
terminated by the addition of 1ml of 10% cold Na2Co3.  The liberated o-nitrophenol 
(ONP) was measured spectrophotometrically at 420 nm absorbance. ONP concentration is 
calculated using an extinction coefficient.One unit enzyme activity (U) is defined as 
µmoles of o-nitrophenol formed per ml per min. Folin Lowry method was used for protein 
estimation using BSA as standard. Protein content within all culture filtrates was tested for 
by the method of Lowry et al.(1951) using bovine serum albumin as standard. The agents 
required were solution A: 1% CuSO4.5H2O, solution B: 2%Na-K-Tartarate and solution C: 
2% Na2CO3 in 0.1M.Ratio 1:1:98 of solutions A, B and C were mixed. 
3.3.6   Determination of Enzyme activity 
One unit of enzyme activity (U) was defined as the amount of enzyme that liberated 
1mMol of o-nitrophenol per minute under the assay condition (Kuby and Lard, 1953). The 
-1
results are expressed as U/ml.  Specific activity was defined (Ug ) as the number of 
enzyme units per gram of cell biomass.  The biomass concentration was measured 
-1
gravimetrically as dry cell mass (gm ) by centrifuging, 20 mL of original culture broth at 
53 
 
 
0
4000rpm for 20 min; followed by drying the cell in a pre-heated oven-dryer at 100 C 
overnight at appropriate temperature overnight. 
A volume of resuspended cells in 0.2M phosphate buffer (pH 7.0) amounting to 10-20mg 
of dry cell mass was mixed with 5 mL isoamyl alcohol and diluted up to 25 mL with 0.2M 
phosphate buffer pH 7.0. The mixture was shaken for 15 min. at room temperature to 
make the cell envelope permeable and used for the enzyme assay as stated under section 
3.3.5. 
3.3.7   Determination of pH, lactic acid, acetic acid 
3.3.7.1 Quantitative Estimation of Lactic acid: The production of Lactic acid was 
-1
determined by titrating 10 mL of the homogenized sample against 0.25mol  NaOH using 
1ml of phenolphthalein indicator (0.5% in 5% alcohol).Each milliliter of 1N NaOH is 
equivalent to 90.08mg of lactic acid. The titratable acid was calculated according to 
A.O.A.C method (1990).  
Titratable acidity = MI NaOH × N NaOH ×M.E × 100 
Volume of sample used 
    
 The lactic acid was determined in MRS broth cultures by titrating 25ml against 0.1M 
NaOH in a 100 mL conical flask, to the first trace of pink colouration.  This was repeated 
two times and the average titre value was taken. Where: MI NaOH = Volume of NaOH 
used; N NaOH = normality of NaOH solution; M.E = Equivalence factor.1ml of 0.1M 
NaOH=9.008mg lactic acid (Mante et al., 2004) 
3.4. Determination of Growth  
Modified MRS (MMRS) medium was used during growth studies for enzyme production.  
The growth studies were performed in 500 mL volume of MMRS, which includes 
calculation of change in pH, change in turbidity of medium and total β-galactosidase 
activity of the isolates and measurement of change in turbidity of medium for construction 
of growth curve of the isolates. 
The number of mole of ONP (O-nitro phenol) Liberated was determined from the standard 
curve of ONP. (One unit (U) of β-galactosidase activity was defined as the amount of 
enzyme required to release 1.0 µmol of o-nitrophenol (ONPG) per minute under standard 
54 
 
 
assay conditions. The extinction coefficient of dissolved o-nitro phenol at 420 nm was 
found to be (1.3546) mL µmol-1 cm-1. 
Each experiment was done at least twice. Then the results were taken. 
Activities were Calculated as described by Baran formula (Baran, 1996) 
                                 U/mL=             ∆A42O/min, where: 
                                                        Ԑ 
     Ԑ= (extinction coefficient): 1.3546 mL µmol-1 cml. 
The rate of absorbance change (∆A420/ mm) was calculated from linear position of the 
curve by using linear regression. 
The rate of absorbance change (∆A420nm/mm) was calculated as follows  
(∆A420/min) =∆ A420/min (Enzyme reaction) _∆A420/min (blank). 
A specific activity was calculated as follows (Baran, 1996), 
U/mg protein =   ∆A420/min 
     Ԑ (Extinction) × mg protein /mL reaction medi 
 
3.5   Biochemical characterization of isolates  
Various biochemical tests were carried out for possible identification of the isolates.  
Young cultures (24-48hours old) were used for each biochemical test inoculation except 
where otherwise stated. 
3.5.1 Catalase test 
This test detects the presence or absence of catalase in the isolates. Catalase is an enzyme 
found in most bacteria and is known to catalyze the breakdown of hydrogen peroxide 
(H2O2) to lobe oxygen as (O2) in this process. 
 2H2O2                            2H2   + 2O2 
The test was carried out using the method of Kiss (1984).  To a loopful of the culture was 
added few drops of 3% hydrogen peroxide on a clean sterile slide and observed for 
55 
 
 
bubbles.  Evolution of gas or white froth indicates catalase positive reactions while 
absence of froth indicates negative reactions. 
3.5.2 Oxidase test 
This detects the presence or absence in bacteria of certain oxides that will catalyze the 
transport of electrons between electron donors in the bacteria and redox dye.  A few drops 
of 1% aqueous solution of tetramethyl-p- phenylene-diamine hydrogen peroxide were 
added to a piece of whatman No. 1 filter paper with the aid of glass rod to moisten the 
filter paper.  The filter paper was then smeared with the different isolates at different 
positions.  A purple colour (within 5-10 seconds) was regarded as positive and a delayed 
reaction or no colouration at all was negative (Seeley and Vandemark, 1972) or 
delayed/weak positive reactions (Kics, 1984). 
3.5.3 Methyl Red Test 
Glucose phosphate peptone broth was prepared as described by Harrigan and Mc Cance 
(1976).  Ten milliliters of the broth were dispensed into screw cap tubes and sterilized by 
autoclaving.  The test organisms were then inoculated into the broth and incubated.  A few 
drops of methyl red indicator were added to the cultures after the incubation period. 
Development of red colouration was considered positive; otherwise it was negative 
(Harrigan and Mc Cance 1966). 
3.5.4  Indole Production 
Two percent (2%) (w/v) peptone broth was prepared and 10 mL was dispensed into each 
screw-capped tube and then sterilized by autoclaving.  Inoculation of the tubes with the 
o
isolates was carried out and incubated for 5-7 days at 35 C.  To 1ml of the broth culture, 1 
mL of 6% -naphtol solution and 1ml of 40% potassium hydroxide (kovac‟s reagent) was 
added in a sterile tube.  Development of red colour within 5mins was considered positive 
(an indication of indole production) while absence of such colouration indicated a negative 
result. 
3.5.4 Oxidative/fermentative test 
The medium of Hughes and Leifson (1953), containing bromothymol blue, was used.  Ten 
(10.0) mL of the solution medium was dispensed into screw-capped bottles and then 
56 
 
 
sterilized by autoclaving.  Two replicates of each tube were inoculated with each of the 
test organisms.  One of the tubes was covered with sterile paraffin and the tubes were then 
o
incubated for 5-8 days at 35 C.  Acid production is shown by colour change from blue to 
yellow.  Fermentation organisms have acid produced in both tubes while oxidative 
organism produces acid only in tube without paraffin seal. 
3.5.6 Homo fermentative/hetero fermentative test 
As obligate fermenters of carbohydrates, the lactic acid bacteria are universally able to 
ferment glucose.  These can be divided into two groups one can culture the test organism 
in a medium containing glucose and, after good growth is obtained, plunge a red-hot 
inoculating loop into the broth culture: this will cause release of CO2 which will be seen as 
an eruption of very fine bubbles rising to the surface of the broth and forming a noticeable 
ring if a test organism is heterofermentative.based on the end products formed during the 
fermentation of glucose.  Homofermentative lactic acid bacteria produce lactic acid as the 
sole or major end product, while the heterofermentative lactic acid bacteria produce 
equivalent amounts of lactic acid, CO2 and ethanol.  The hot-loop test can be used to 
differentiate these two groups.  As CO2 is very soluble in water at high pH, a Durham tube 
was unable to detect its production. If, however, the temperature of the solution is 
increased, CO2 will become insoluble and will be released in the gaseous form. 
3.5.7 Nitrate Reduction Test 
This test was carried out to investigate the ability of the bacterial isolates to reduce nitrate 
compounds to nitrite and ammonia.  Nitrate peptone water containing peptone water with 
addition of 0.1% KNO3 (potassium nitrate) was the medium used for the test. Ten milliters 
of the medium was dispensed into test tubes with inverted Durham tubes.  These were 
autoclaved, allowed to cool and inoculated with test organisms.  An uninoculated tube 
0
served as control.  The tubes were then incubated at 30 C for 5-7 days and the presence of 
nitrite was determined by adding to each tube, 0.5 ml of 1% sulphanilic acid in 5 ml acetic 
acid followed by 0.5 ml of 0.6% N, N-dimethyl 1-P-diamise dichloride in 5.0 ml acetic 
acid.  Development of red colouration gives a positive result while the presence of gas in 
Durham tubes indicates production of Nitrogen (Payne, 1973).  
3.5.8 Growth in sodium chloride (NaCl) 
57 
 
 
Growth of isolates in 0.01% (w/v) phenol red glucose peptone water (GPW) containing 
6.5% (w/v) and 4.0% (w/v) NaCl, each, was carried out and examined after 3-4days of 
o
incubation at 30 C.  Colour change from red to yellow indicates positive result, otherwise 
result is negative (Harrigan and McCance, 1966).  Uninoculated tubes served as control. 
3.5.9 Production of ammonia from arginine 
The production of ammonia from arginine was tested using the method of Doring (1988).  
A modified nutrient broth (Nutrient-arginine broth) was used.  The nutrient broth without 
arginine was used as control.  24hrs old cultures were inoculated into 4% arginine broth 
o
(5mls) in test tubes plugged with cotton wools and incubated at 30 C for 5-7 days.  Few 
drops of Nesslers reagent were then added after the incubation period to drop of the broth 
in small size test tubes.  Development of deep yellow or brown colour indicates positive 
reactions (Harrigan and McCance, 1966). 
3.5.10 Oxygen relationship of isolates 
This involves oxygen requirements of the isolates for the utilizations of compounds.  It is 
used extensively to show the decomposition of simple carbohydrates (Holding and Collee, 
1971).  10.0 ml of Yeast Extract peptone agar (YEPA) was dispensed into each test tube 
and sterilized by autoclaving.  The tubes were later inoculated with the test organisms at 
o
4 C for 7 days.  Areas of growth along the tubes were noted after the incubation period.  
Growth at the surface indicates obligate aerobes while obligate anaerobes grow at the 
bottom of the tubes.  Those along the length of the tubes are facultative anaerobes while 
microaerophilic organisms grow near the surface. 
3.5.11 Gelatin Hydrolysis 
Gelatin is a protein which can be metabolized only by those microorganisms which are 
capable of producing proteolytic enzymes that breaks it down.  When broken down, 
gelatin loses its gelling qualities.  Gelatin broth was prepared by suspending 10g of gelatin 
in 100 ml of nutrient broth to give a broth of 10% (w/v) gelatin. Nine ml of this distributed 
o
into each test tube plugged with cotton wool and  sterilized by autoclaving at 121 C for 10 
o
min.  A loopful of the isolates inoculated into the tubes and incubated at 4 C for 10days.  
Gelatin hydrolysis was tested for by cooling the tubes in freezer for about 15 minutes after 
58 
 
 
which the tubes were observed for gelatin hydrolysis.  The tubes in which gelatin 
hydrolysis did not take place had their gelatin content in solidified state, while a positive 
reaction was indicated by the gelatin remaining in the liquid state without solidification 
(Harrigan and McCance, 1966). 
3.5.12 Motility Test 
o
Test organisms were grown in sterile peptone water broth in McCartney bottles at 4 C for 
about 7-8 days. About a drop or two from the micropipette of the broth was then placed in 
a hollow in a clean cavity slide and with cover slides using Vaseline seals to place the 
cover slide in position to exclude air bubbles. The slides were then examined under the 
lower power of the microscope to observe for any possible motility of the different 
organisms on slides.  Motility was recorded as positive for any movement noticed of the 
isolates, otherwise motility was negative (Seeley and Van Demark, 1972). 
3.5.13 Starch Hydrolysis 
Starch agar plates as described by Seeley and Van Demark, 1972, were used.  One isolate 
o
was streaked on each of the plates and then incubated for 5 days at 30 C.  Starch 
hydrolysis (due to amylase activity of the isolates) was tested for by flooding the plates 
with dilute Gram‟s iodine solution.  Occurrence of clear zones  along the line of streak was 
a positive result. 
3.15.14   Voges Proskauer Test 
This is a test for the production of acetyl methyl carbinol from glucose. Cultures of methyl 
red broth were tested to know whether the isolates after producing acid from glucose are 
capable of producing acetyl methyl carbinol from the acid by adding 1.0 ml of 6% NaOH 
and 0.5 ml of 5% solution of -naphthol in absolute ethanol to 1ml of 72 hrs culture of the 
o
isolates, after 3 – 5 days of incubation at 4 C.  Appearance of a pale pink colouration 
within 5mins was recorded as positive but negative results could be left for up to 3-5hrs to 
check for slow reaction. 
3.5.15   Production of Hydrogen Sulphide 
Some bacteria are capable of decomposing sulphur compounds such as cystine or reduced 
inorganic sulphur compounds to produce hydrogen sulphide. Lead acetate paper strips in 
59 
 
 
tubes containing the thiosulphide broth (nutrient broth plus 0.01% thiosulphate) was used.  
Production and liberation of hydrogen sulphide causes blackening of the lead acetate paper 
strips. 
3.15.16     Sugar Fermentation Test 
This is a test to confirm the ability of the isolated microorganisms to utilize various sugars 
as carbon sources.  The products of sugar or carbohydrates metabolism depend largely 
upon the type of enzyme possessed or produced by the organism.  Sugar utilization is 
coupled with acid production which can be confirmed by the addition of an indicator to the 
medium.  Gas may also be formed during the reaction and this may be detected by 
inserting Durham tube in an inverted position into the reaction tube 
Preparation of Sugar Solutions 
One percent (w/v) of each of the peptone water and sugar was prepared to result in 
peptone water-sugar broth.  (Peptone 1.0% NaCl (0.1%), fermentable sugar (1.0%)).  
Phenol red (0.01% w/v) was added to the broth as indicator.  The sugars used include 
lactose, mannitol, sorbitol, dulcitol, sucrose, dextrin, maltose, glucose, saccharose, xylose, 
arabinose, inulin, raffinose, cellulose, mannose, salicin, rhamnose, fructose, sorbose, 
galactose, ribose, and inositol.           
3.6 Identification of isolates 
Results of the various biochemical tests were utilized in identifying the isolates with 
particular reference to Bergey‟s Manual of Determinative Bacteriology (Sneath, 1986). 
However some of the isolates could not be identified to the species level. The API 50 CH 
(for rods and cocci) was used for further identification of some of the isolates using the 
manufacturer ҆s online software (www.apiweb.biomerieux.com).  
   
3.7     Molecular analysis of Isolates  
3.7.1 Extraction of genomic DNA of LAB isolates 
DNA extraction from the LAB isolates was carried out using a modified GES (5 M 
guanidine thiocyanate (Fisher Scientific BPE221-250), 0.1 M EDTA Sigma E-5134), and 
0.5% N-lauroyl-sarcosine sodium salt  (Sigma L-5777) (w/v) DNA extraction method 
(Pitcher et al.,1989). Aliquots of 1.5 mL of overnight cultures grown in appropriate broth 
60 
 
 
were centrifuged (Biofuge, Heraeus, Germany) in eppendorf tubes at 13,000g for 1min. 
Pellets obtained were washed in 1 mL of ice cold lysis buffer (25mM Tris-HCl (Sigma T-
6066), 10 mM EDTA , 50 Mm sucrose (BDH GPR 302997J) pH 8.0). The pellets were re-
-1
suspended in 100 µl of lysis buffer in addition to 50 mgml  lysozyme (Sigma L-6876) and 
incubated at 37°C for 30min. volumes of 0.5 mL GES solution were added and mixed 
well. This was incubated at room temperature for 15 min. The lysate was then cooled on 
ice for 2 min and 0.5 mL of 7.5 M ammonium acetate (Fisher Scientific A3440/60) (also 
cooled on ice) was added, voltexed and incubated on ice for 10 min. 
Aliquots (0.5ml) of 24:1 chloroform: isoamylalcohol (Sigma CO549-1QT) were added, 
voltexed and centrifuged for 10 min at 13,000g. Aliquots of 800 µl of the upper phase 
were removed quantitatively and placed into a clean eppendorff tube. Cold isopropanol 
(Fisher Scientific P/7500/21) (0.54 vol) was added and mixed for 1 min. This was then 
centrifuged at 13,000g for 5min and the supernatant removed from the pellet. The pellet 
0
was washed three times in 500 µl of 70% ethanol and dried at 37 C for 15 min.Aliquots 
(50µl) of TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.5) were added and 5 µl of the 
DNA  were checked on 1% agarose (Biogene,Kimboltton,UK) gels in 200 ml 1X TAE 
(4.84 g Tris base, 1.14 g glacial acetic acid, 2 ml 0.5 M EDTA, pH 8) buffer and the DNA 
0
samples were then stored at -20 C for future use. 
3.7.2  PCR amplification of 16S rDNA gene 
The modified method of Bulut et al., 2005, was used. Amplification of 16S rDNA gene-
ITS region, was performed by using the following primers: order to determine the 
phylogenetic grouping of the six bacterial isolates,their 16S rDNA genes were amplified 
by standard PCR using universal Operon primer series :OPA1,OPA2 OPA3,OPG16 And 
OPF17 Respectively Designed To Target The Conserved Region Of Bacteria 16S rDNA 
(Macrogen Corp,9700 Great Seneca Highway,Rockville MD 20850 USA) Forward (16S 
ITS For), 5'-AGAGTTTGATCCTGGCTCAG-3'  and reverse (16S its Rev), 5'-
CAAGGCATCCACCGT-3'; 16S rDNA v3, Forward, 5'-CCTACGGGAGGCAGCAG-3' 
and 16s rDNA v3  Reverse  5'-ATTACCGCGGCTGCTGG-3'. The v3 primer was used for 
ease of sequencing of the gene, using the variable region 3 (v3), for the genetic 
identification of the isolates. 
61 
 
 
Each of the PCR was performed in a 50 µl reaction volume containing 50 ng genomic 
DNA as the template (equivalent to 1 µl), 0.2 mM- deoxynucleoside triphosphates, 
dNTPs, (Promega, U120A-U13A, Madison, WI, USA), 2.5 mM-MgCl2   10 pmo each (0.1 
µl volume) of the DNA primers in 1 X PCR buffer (Promega, UK), and 1.25 units Taq 
DNA polymerase (Promega, UK). Amplification conditions were as follows: an initial 
0
denaturation step of 5 min at 94 C, 40 amplification cycles, each consisting of 1 min 
0 0 0
denaturation at 90 C, 1 min annealing at 42 C, and 1 min elongation at 72 C. PCR 
amplifications were performed in a thermocycler (Techneprogen, Cambridge, UK). 
3.7.3 Gel electrophoresis of 16S rDNA PCR products 
Electrophoresis of the amplified 16S rDNA PCR products was performed on the Bio-rad 
contour- Clamped Homogenous Electric Field (CHEF) DRII electrophoresis cell.This was 
done through 1.5% (w/v) Agarose gel (general purpose,Biogene) in 0.5 X TAE buffer at 
84 volts for 1.5-2 hrs. A 100bp (promega G210A and 1Kb DNA ladder (promega G571A) 
were used as a molecular size markers. 
3.8 Sequencing and Analysis of 16S rDNA gene  
3.8.1 Purification of PCR 16S rDNA gene   
Seventy five microlitre of the PCR 16S rDNA amplified products were resolved in 1% 
agarose gels with the conditions earlier described .The resulting bands in agarose gel were 
carefully excised with sterile scarpels and then purified using purification kit (Wizard PCR 
preps DNA purification kit, USA). The purification process involved the introduction of 
the excised bands into 1.5 mL Eppendorf tubes and suspended in water bath that was 
0
maintained at 65 C. One mL of PCR preps purification resin was added and tubes were 
incubated for about 5 min or until the agarose gel melted completely. The DNA/ 
purification resin mix was pipette into the syringe barrel and the syringe plunger was used 
to slowly push the slurry into the attached mini column. The mini column was then 
washed with 2 mL of 80% isopropanol to remove contaminants from the DNA. The 
Eppendorff tubes, on which the columns were mounted, were then centrifuged at 10,000 x 
 0
g for 2 minutes at 4 C, to remove the remaining resin and isopropanol from the purified 
DNA in the columns. The minicolumns were transferred into new Eppendorff tubes and 
40 µl TE buffer (pH 7.5) was added into each of the columns, and left to stand for 1 min. 
62 
 
 
 0
Centrifuge the minicolumns at 10,000 x g at 4 C for 20 secs,to elute the DNA into the 
 0
centrifuge tubes. The purified DNA was kept at 4 C until use. 
3.8.2 Drying of the purified 16S rDNA genes 
To 1mlo f the purified DNA, 0.1 volume of Sodium acetate (3 M, pH 5.0) and 2.0 mL of 
0
100% ethanol were added. This was then incubated at -20 C for 1 hr. It was brought out 
 0
and left to stand at room temperature for 5 minutes, and centrifuged at 13,000 x g at 4 C 
for 45 miniutes.The liquid was removed, leaving only the DNA in the Eppendorff tubes. 
 0
The DNA was then dried at 37 C for 30 minutes. 
3.8.3 Sequencing of 16S rDNA gene   
The dry DNA samples (obtained using the v3 primer) were sequenced using a computer 
analytical sequencer (MGW-Biotech, Germany) with the v3 primer Rev, acting as the 
basis. The generated nucleotide sequences were subjected to analysis. 
3.8.4  Analysis of the 16S rDNA gene sequences  
The generated sequences of the 16S rDNA genes were subjected to alignment in the 
databases at the BLAST. Basic Local Alignment and Search Tool, website 
http://www.ncbi.nlm.nih.gov/blast. The isolates were then identified based on the result 
of the analysis. The isolates were then identified based on the result of the analysis .A 
similarity of > 98% to 16S rDNA sequences of type strains was used as the criterion for 
identification (Altschul et al., 1997). 
3.9 Effect of incubation time on growth and β-galactosidase production 
3.9.1 Measurement of growth with time  
Growth of the test organisms was determined by taking the optical density readings (OD) 
o
at 600nm.Twenty milliliter  of sample was incubated at 4 C for 15 min to stop further 
growth. Growth was measured turbidimetrically at 600nm and cells diluted with water 
when the OD was higher than 0.8.  Cell turbidity at OD600 was kept between 0.4 and 0.8 
by appropriate dilutions. pH of the medium is measured by pH meter. 
 
 
63 
 
 
3.10   Optimization of growth conditions for β-galactosidase production 
Optimization studies were carried out on the growth of the isolates to determine base 
requirements for optimal galactosidase production.  Optimization parameters will include 
the pH, optimum temperature, and effect of agitation on enzyme production 
 3.10.1   Effect of Temperature on β-galactosidase production 
Twenty milliliters (20ml) of the modified MMRS basal medium was dispensed into each 
screw-capped tube and then sterilized by autoclaving. The colour change and turbidity of 
each bottle is noted as a simple indicator of growth or no growth.  Each treatment is tested 
with triplicate bottles. 
3.10.2   Effect of pH on β-galactosidase production  
The basal MMRS medium was adjusted with appropriate quantities of Sodium dibasic 
hydrogen phosphate and monosodium dihydrogen phosphate to prepare the requied pH.  
The screw-capped bottles were placed in water bath with reciprocal shaking.  The pH 
range determined were 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 respectively while the 
0
temperature was kept constant at 37 C. 
3.10.3   Effect of different carbon sources on β-galactosidase production.  
 In` this study, galactose in the basal medium was replaced by 1% of: Raffinose, 
Melibiose, Arabinose, Xylose and Glucose separately keeping constant the rest of the 
media composition.A control experiment consisted of inoculated MRS without sugar 
additions. 
The effect of combination of raffinose (0.5%) with galactose (0.5%) and melibiose (0.5%) 
with galactose (0.5%) on -galactose production were also studied. 
3.10.4   Effect of different Nitrogen sources on β-galactosidase production. 
In this study, yeast extract and ammonium sulphate in the basal medium were replaced 
with different organic compounds (Yeast extract, peptone, malt extract, tryptone and 
ammonium nitrate) as nitrogen source.  The rest components of the media composition 
remained the same.  The concentration of organic and inorganic compounds added in the 
medium are: 01% and 0.3% respectively 
 
64 
 
 
3.10.5   Effect of agitation on enzyme production 
This was determine by agitating the culture flasks inoculated with 25 hours old test 
isolates in a rotary shaker at; 60, 80, 100, 120, 140, 160 and 180 rpm for a period of 6 
hours each after which the samples are then analysed for enzyme production total protein, 
and cell growth. 
3.11     Characterization of Crude Enzyme. 
3.11.1 Effect of temperature 
To find the optimum temperature for β-gal production, the fermentation medium after 
o
inoculation was incubated at temperatures in the range of 16 - 42 C (16, 20, 28, 37 and 
o
42 C) in an automatic incubator for 36 hrs and then check for the level of enzyme 
production at the respective temperatures.A controlled experiment consisted of an 
uninoculated broth maintained at that temperature. 
3.11.2   Effect of pH 
To investigate the effect of pH on β-gaactosidase production, the initial pH of the basal 
medium was adjusted with Na2HPO4 and NaH2PO4 in a prepared pH range of 3 – 8.0 (3.0, 
4.0, 5.0, 6.0, 7.0, and 8.0). It was then inoculated with a 16 hrs old culture and incubated at 
o
37 C for 36h.A controlled experiment consisted of an un-inoculated broth maintained at 
the test pH. 
3.11.3   Effect of Inoculums size 
To determine the effect of inoculums size on enzyme production, different inoculum levels 
are prepared as follows: 4, 6, 8, 10 and 12% (4 – 12% v/v) each of which was separately 
o
added to the fermentation medium and incubated at 37 C for 36 hrs. 
3.11.4  Effect of Inoculum age 
To determine the effect of the inoculums age on enzyme production, fermentation medium 
was inoculated with 12-24 hrs old culture at 12, 16, 20, 24, 28, and 32, 36 hrs each and 
incubated at 37ºC after which they were withdrawn and analysed for enzyme production 
and cell growth. 
3.11.5    Effect of incubation period 
To determine the optimal incubation time for maximum enzyme production, the 
fermentation medium was inoculated with 16 hrs old culture and incubated for 36 hrs: 20, 
65 
 
 
24, 28, 32, 36 hrs.  Samples were out every 4 hrs and analysed for β-galactosidase activity 
and cell growth. 
3.11.6   Effect of different inhibitors  
The inhibitors used were Ethylene diamine tetra acetic acid (EDTA), potassium cyanide 
(KCN), benzoic acid and Urea.  The stock solution of each was prepared using a range of 
concentrations (0.1mol, 0.2mol, 0.3mol, 0.4mol and 0.5mol).   From the stock, 0.1ml from 
each was added to 1ml of substrate ONPG in sodium phosphate buffer (pH 7.0) and 0.5ml 
of enzyme extract.  The reaction was stopped by the addition of 1ml of cold 10% NaCO3 
solution.   
3.11.7   Effect of different cations 
Stock solutions of different cations were prepared at different concentrations (0.1mol, 
0.2mol, 0.3mol, 0.4mol and 0.5mol). The cations used were CaCl2, MgSO4, MnSO4, 
KNO3 and NaCl.  One millilitre of each of the stock solution of the different cations was 
added to 1ml of 1% ONPG in phosphate buffer (pH 7.0).  To this mixture was added 
0.5ml of enzyme extract after which β-galactosidase activity was determined. 
3.11.8 Effect of different anions 
The anions used were NaNO3, FeSO4, FeCl2, NH4NO3 and NH4Cl.  The stock solution 
was prepared by varying the concentration from 0.1m to 0.5m.  1ml each from the stock 
solution of the different anions concentrations was added to 1ml of 1% ONPG in 
phosphate buffer (pH 7.0) and 0.5ml of enzyme extract after which the β-galactosidase 
was assayed for.  
3.11.9   Effect of enzyme concentration 
Effect of enzyme concentration on β-galactosidase activity was determined by varying the 
concentration of enzyme extract (0.5%, 1%, 1.5%, 2% and 2.5%).  This was added to 
different test tubes containing Sodium phosphate buffer (pH 7.0).  
One unit of β-galactosidase activity was defined as the amount of enzyme that librated 1µ-
mole of o-nitrophenol per minute under the assay conditions (Haider and Hussain, 2008 
b). 
66 
 
 
3.12   Construction of ONP Standard Curve 
In this assay, free ONP at different levels was used to construct the standard curve. 
0.033383g of ONP was mixed with 1ml of 0.05M phosphate buffer (pH 7.0). Final volume 
of the mixture was set at 4ml. This mixture was agitated until all the solid particles were 
0
dissolved.This mixture was transferred into a preheated water bath at 45 C. It was labeled 
as ONP stock solution. Several dilutions (0.5%, 1%, 2%, 2.5% and 3%) were carried out 
in 0.05M sodium phosphate buffer ( pH  7.0) respectively under assay conditions.  0.5 mL 
of the enzyme extract is added to 1ml of the solution and the enzyme assay is carried out. 
Absorbance was read at 420nm. 
Blank solution was prepared by mixing 3ml alcohol and 1ml sodium phosphate buffer 
0.05M (pH 7.0). For each set of dilutions of stock solution, blank solution was made. 
Absorbance values were read against the blank solutions in UV-spectrophotometer .All 
readings were carried out as duplicate experiments in three independence trials. The 
average of each set of data was plotted against respective concentrations. 
3.13   Intracellular Enzyme extraction   
Extraction was carried out by the addition of 0.2g of 1.5 mm glass beads and stirred 
vigorously in a vortex for 20 secs and placed on ice 20 secs.The harvested cells were 
suspended in 10ml of Sodium phosphate buffer and then centrifuged at 10,000 rpm for 15 
0
min. at 4 C, Sieved using watman paper and washed again with the same buffer. 
3.14   Purification of β-galactosidase  
Enzyme purification steps were sequentially carried out using Ammonium Sulphate, 
Enzyme dialysis in Viskin tubes and Column Chromatography with Sephadex resin first 
with G-100 and later with G-50 of varing sizes between  50 -100  mesh size. 
3.14.1 Ammonium Sulphate precipitation  
The crude enzyme extract was concentrated with the addition of requisite weight of 
ammonium sulphate in a “salting-out process“ up to 100% saturation ( Dixon and 
Webb,1971). Enzyme saturation was gradually achieved by using 24.3g; 28.5g and 15.7g 
to achieve 0 - 40% and 40 - 80% saturation respectively. Each quantity of ammonium 
sulphate was weighed and dissolved in 100 mL of enzyme sample with constant stirring. 
0
The mixture was maintained at 4 C overnight in a cold room. Thereafter, the solution was 
0
centrifuged at 7000 rpm for 15 min and the precipitate stored at 4 C. All precipitates were 
67 
 
 
pooled and dissolved in 0.2M sodium phosphate buffer (pH7.0) and made up to 100ml. 
The precipitated enzyme samples were introduced into dialysis tubes (Spectra/por® 
molecular porous membrane tube, Spectrum medical Industries Inc., Texas), and dialysed 
0
extensively against three changes of the phosphate buffer for 24 hrs at 4 C. The dialyzing 
0
buffer was thereafter, discarded and the enzyme stored in sterile screw capped tubes at 4 C 
for further purification by column Chromatography. 
3.14.2 Dialysis 
The precipitates for each batch were re-suspended to initial volume of culture filtrate with 
0.05M Sodium phosphate buffer pH 7.0 and dialysed in a tubular cellulose membrane 
0
against one litre of the same buffer for 24 hrs at 4 C .The fractions were pooled together 
and used as partially purified enzyme for enzyme assay. 
3.15   Column chromatography 
3.15.1   Preparation of resin 
Ten grammes of Sephadex G 100 resin was weighed into a glass beaker containing 100ml 
sterile distilled water with 0.97% sodium azide (to prevent microbial contamination) and 
200ml 0.05M phosphate buffer (pH 7.0). The gel was allowed to swell for 3days with 
0
constant gentle stirring at 4 C before loading. The gel was allowed to swell for 3 days with 
constant shaking.  
The column of Sephadex G-100 was prepared according to the method described by 
0
Olutiola and Cole (1980) and was surrounded by water jacket at 4 C. The column was 
equilibrated with 0.05M sodium phosphate buffer pH 7.0.  
3.15.2   Loading of the Column  
After the third day of soaking, the resin was loaded into a clean glass chromatography 
column (25x190mm) and allowed to compact while preventing air from being trapped 
along the column. The Sephadex was allowed to compact in liquid on top was sucked out. 
More gel slurry was introduced until the column was fully loaded to the zero point of the 
column. This was done for 48 hr before 2ml of the dialysed enzyme sample was loaded.  
The column was first eluted with the same buffer followed by a linear gradient of Sodium 
-1 -1 
Chloride from 0.1M l  to 0.5M l at a flow rate of 25ml/sec, and the eluent was collected 
in 5ml fractions. The enzyme activities and protein content in each 5ml fraction were 
determined by procedures previously stated and protein content was calculated by the 
68 
 
 
method of Lowry et al., (1951) using BSA as standard. The active fractions were pooled 
and used for electrophoretic determination  
3.15.3  Fractionation on Sephadex G-100 
The column of Sephadex G-100 was prepared and loaded according to the method 
0
described by Olutiola and Cole (1980) and was surrounded by water jacket at 4 C. The 
column was equilibrated with .05M sodium phosphate buffer pH 7.0. Two milliliter (2ml) 
of the enzyme concentrate was loaded into the column and eluted with sodium phosphate 
buffer. The eluted fractions were collected in calibrated 5ml tubes for thirty fractions. 
Each eluted fraction was analysed for β-galactosidase activity. Protein estimation was also 
carried out.  
3.15.4  Fraction of Sephadex C-50 
Fractions which showed appreciable activity after passing through Sephadex G-100 were 
pooled together and applied to the column of Sephadex C-50 ion-exchange column(1.7 x 
1.8cm) previously equilibrated with the same buffer.The flow rate was adjusted to 17.5 
ml/hr. Two milliliters (2ml) of each fraction was applied to the column and eluted with 
sodium phosphate buffer (pH 7.0). The column was first eluted with the same buffer 
-1 -1
followed by a linear gradient of Sodium Chloride from 0.1M l  to 0.5M l . The eluted 
fractions were collected as above and analysed for β-galactosidase activity .Protein 
estimation was also carried out.  
3.16  Protein Estimation 
Protein concentration was estimated according to the procedure described by Lowry et al., 
(1951) Bovin Serum Albumin was used as standard.  The reagents required were: solution 
A.  1% CuSO4. 5H20; solution B, 2% Na- K-Tartarate and solution C: 2% Na2Co3 in 0. 1m 
NaOH.  Ratio 1:1:98 of solutions: A, B and C were mixed.  Folins reagent was diluted in 
equal volumes with water.Absorbance was read at 540nm. 
3.17. Electrophoresic separation of protein samples/SDS-PAGE   
 3.17.1 Electrophoretic Separation of Extracellular -galactosidase 
Native polyacrylamide gel electrophoresis (PAGE) and denaturing sodium dodecyl 
sulfate polyacrylamide electrophoresis (SDS-PAGE) were performed on a Phast System 
Unit using precast polyacrylamide gels (Phast HGel8-25, Amersham). For SDS-PAGE,the 
0
enzyme was pre-incubated with Laemuli buffer for 5minutes at 90 C for Coomasaie blue 
0
staining and 60 C for active staining.Coomassie brilliant blue R-250 was used for 
69 
 
 
visualization of protein bands,and active staining was performed by using 4-
Methylumbelliferyl-β-D-galactosidase (MUG) as substrate which was added directly onto  
the gel.Samples that were applied to the individual lanes contained approximately 1-2µg 
of the total proteien.Isoelectric focusing(pI:  or isoelectric point) was performed in the 
range of pH 3-10 using the Multiphor System and precast dry polyacrylamide gels 
rehydrated with carrier ampholytes as recommended. The broad pI marker protein kit, pH 
3-10 was2 used to compare and determine the pI value. 
b. Stacking gel buffer 
 All the components were dissolved in 500ml distilled water in a 1 litre standard 
volumetric flask.  The pH was adjusted to 6.8 with Potassium sulphate solution.  The 
o
solution was  made up to mark with distilled water and stored at 4 C. 
c. Running gel buffer 
 1.5M Tris, 8mM EDTA, 0.4% SDS pH 8.8 water in a 1 litre standard volumetric 
flask.  The pH was adjusted to 8.8 with hydrochloric acid.  The solution was then made up 
o
to mark with distilled water and stored at 4 C. 
e. Electrode buffer: Glycine, SDS, sodium salt EDTA 
 All the components were dissolved in 4 litres distilled water with constant stirring.  
o
The solution was kept at 4 C. 
f. Sample buffer 
 6ml glycerol was mixed with 1.2ml of potassium phosphate buffer pH 6.8, 1.5g of 
sodium dodecyl sulphate (SDS), 30mg dithiothreitol (DDT) and 10mg bromophenol blue.  
Distilled water was added to make 20ml.  The mixture was dispensed into test tubes in 
o
aliquots of 1ml each and stored at 20 C until needed. 
3.17.2 Gel-electrophoresis 
The stacking gel and running gel were prepared on the day of the experiment.  The 
running gel solutions were poured into the gel assembles to a level of about 4cm below the 
maximum filling level.  Distilled water was layered on the gel surface using a Pasteur 
pipette to ensure an even surface and also to avoid evaporation of the gel while the 
polymerizing gel was allowed to polymerize for 1 – 2 hours. 
The water on the gel was poured off and the stacking gel was poured onto the polymerized 
running gel and a comb (1.5 mm thick) was gently inserted to obtain wells. 
70 
 
 
The protein samples were prepared by mixing sample buffer (in a dilution of 1:1 (v/v) with 
o
enzyme samples.  The mixtures were place in a dry bath and heated for 4 minutes at 95 C.  
Equal volume of samples was applied to the bottom of the sample wells with a Hamilton 
syringe.  Molecular weight standards (Bio Rad high and low ranges) were  applied into 
wells alongside the samples. 
Gel electrophoresis was conducted at 120volts in a vertical electrophoresis apparatus for 
about 1.5 hours or until the bromophenyl blue dye has migrated to the bottom of the gel. 
At the end of the electrophoresis, the gel was carefully removed from the glass plates and 
stained by soaking in the staining solution for 20 minutes with gentle agitation.  Excess 
stain was removed by immersing the gel for 1 hour in several changes of the destining 
solution each lasting for 15 – 20 minutes.  The gel was removed and allowed to dry. 
3.17.3  Effect of different inhibitors on purified enzyme 
The inhibitors used were Ethylene diamine tetra acetic acid (EDTA), potassium cyanide 
(KCN), benzoic acid and Urea.  The stock solution of each was prepared using different 
concentrations (0.1M, 0.2M, 0.3M, 0.4M and 0.5M).   From the stock, 0.1 mL from each 
was added to 1 mL of substrate in sodium phosphate buffer (pH 7.0) and 0.5 mL of 
enzyme extract.  The reaction was stopped by the addition of 1 mL of cold 10% NaCO3 
solution.   
3.10.2  Effect of different cations on purified enzyme 
Stock solutions of different cations were prepared at different concentrations (0.1M, 0.2M, 
0.3M, 0.4M and 0.5M). The cations used were CaCl2, MgSO4, MnSO4, KNO3 and NaCl.  
1ml of each of the stock solution of the different cations was added to 1 mL of 1% ONPG 
in phosphate buffer (pH 7.0). To this was added 0.5 mL of enzyme extract respectively 
after which β-galactosidase activity was determined. 
3.10.3 Effect of different anions on purified enzyme 
The anions used were NaNO3, FeSO4, Fecl2, NH4NO3 and NH4Cl.  The stock solution was 
prepared by varying the concentration from 0.1M to 0.5M.  1 mL each from the stock 
solution of the different anions concentrations was added to 1 mL of 1% ONPG in 
phosphate buffer (pH 7.0) and 0.5 mL of enzyme extract after which the β-galactosidase 
was assayed for.  
71 
 
 
3.10.4 Determination of percentage Lactose hydrolysis using -galactosidase obtained 
from LAB-isolates  
Determining the hydrolytic effect of purified -galactosidase. Rotary evaporator was used 
to obtain different concentrations of the raw milk. By this method, percentage milk 
concentrations of 100%, 80%, 60%, 40% and 20% respectively prepared.  To known 
volumes of the enzyme concentrate as follows: 1.5, 2.0, 2.5 and 3.0 mL respectively. The 
best volume with the highest hydrolysis was determined using Lactose hydrolysis by 
Spectrophotometry method. 
% Lactose Hydrolysis = Absorbance × Average gradient × DF 
10,000 
                                                   
 
 
 
 
 
 
                                          
 
 
 
 
 
 
 
 
 
72 
 
 
 
           CHAPTER FOUR 
4.0       RESULTS 
4.1 Sample collection and isolation of bacteria 
A total number of thirty strains of lactic acid bacteria and one strain of Bacillus were 
isolated from raw milk and dairy products. The isolates were predominantly from local 
cheese(wara). Results of biochemical characterization tests revealed that the isolates 
belong to the genera of lactic acid bacteria. The isolates were characterized and identified 
by their cultural and morphological features   as well as physiological and biochemical 
properties. The lactic acid bacteria were identified as Lactobacillus plantarum, 
Lactobacillus brevis, Lactobacillus casei, Leuconostoc lactis, Lactobacillus lactis, 
Streptococcus spp and Bacillus subtilis. Lactobacillus plantarum had the highest 
percentage of occurrence (35%), followed by L. brevis (20%) while the least is recorded in 
Streptococcus spp.  and Bacillus spp (5%)  (Table 4.1). 
All the lactic acid bacteria isolates were small colonies and the cell shape revealed  
medium short rods to long rods. Some were  cocci, non-motile, non-sporing, gram-
positive, methyl red,catalase positive, oxidase negative, nitrate reduction negative, Voges 
Proskauer tests negative and some strains hydrolysed starch while the Bacillus spp was 
catalase positive, short rods, Gram-positive, spore former. The isolates were facultative 
anaerobes and were fermentative rather than oxidative in nature (Table 4.2). 
Table 4.3 shows the result of the corfirmation of the isolates using molecular techniques. 
Lactobacillus plantarum (G11) with nucleotide length 869 displayed 99% homology and 
was identified as such. In the same sequence are isolates G12 and E13, 890 nucleotide 
length, 99% homology, E36: 890 nucleotide length, 99% homology. However, isolte F6 
had nucleotide length of 920 and 100% homology with Lactoccocus lactis while isolate 
F26 with nucleotide length 817 had 100% homology with Bacillus subtilis. 
 
 
73 
 
 
Table 4.1: Percentage Occurrence of bacterial isolates in the different samples 
Probable Organisms Number of Percentage 
 Occurrence  of  
Occurrence 
Lactobacillus plantarum 7.0 35.0 
Lactobacillus  casei 3.0 15.0 
Lactobacillus brevis 4.0 15.0 
Lactococcus lactis 2.0 10.0 
Leuconostoc lactis 3.0 15.0 
Streptococcus spp 1.0 5.0 
Bacillus subtilis 1.0 5.0 
Total 21.0 100.0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
74 
 
 
Table4.2: Biochemical Characterization of the Isolates 
F28 + + + - + + + - + + + + - + + - + rod - + - - - - - - - L. brevis 
E11 + + + - + + + - + + + + - + + - + rod - + - - - - - - - L. brevis 
G31 + + + + + + + + + - + + + + + - + cocci - + - - - - - - - Streptococcus spp 
E13 + + + + + + + + + + + + + + + - + rod - - - - - - - - - L. plantarum 
E2 + + + + + + + + + + + + - + + - + rod - - - - - - - - - L. casei 
E1 + + +w + +w - +w + + + + + - + + - + rod - - - - - - - - - L. casei 
E36 + + + + + + + + + + + + + + + - + rod - - - - - - - - - L. plantarum 
F5 + + + + + - + +w + + - + - + + - + cocci - - - - - - - - - Lactococcus lactis 
G12 + + + + + + +w - + + + + - + + - + cocci - - - - - - - - - Leuconostoc lactis 
G32 + + + + + +w + + + + + + + + + - + cocci - - - - - - - - - Streptococcus spp 
14E + + + + + + + - + + + + +w + + - + rod - - + - - - - - - L. plantarum 
E30 + + + + + - +w + + + + + - + + - + cocci - - + - - - - - - Lactococcus lactis 
F6 + + + + + +w +w - + + + + - + + - + cocci - - + - - - - - - Leuconostoc lactis 
E17 + + + + + +w +w - + + + + - + + - + cocci - -  - - - - - - Leuconostoc lactis 
F33 + + + + + + + + + + + + + + + - + rod - - + - - - - - - L. plantarum 
E22 + + + + + + +w - + + + + +w + + - + rod - - + - - - - - - L. plantarum 
G11 + + + + + + + + + + + + + + + - + rod - - + - - - - - - L. plantarum 
F26 + + + - + + + - + + + + - + + - + rod - + - - - - - - - L. brevis 
F26* + + + - + + + - + + + + - + + - + rod - + - - - - - - - L. brevis 
ME + + + + + + + +w + + + + + + + - 75 + rod - - + - - - - - - L. plantarum 
 
Isolates 
Saccharose 
Glucose 
Xylose 
Sorbitol 
Lactose 
Arabinose 
Mellibiose 
Raffinose 
Maltose 
Mannose 
Sucrose 
Fructose 
Mannitol 
Trehalose 
Ribose 
Inositol 
Gram reaction 
Shape 
Nitrate 
reduction  
Arginine 
hydrolysis 
Aesculin 
hydrolysis 
Endospore 
formation 
Oxidase 
Catalase 
Voges-
Proskauer test 
H2S 
production 
Indole Test 
Probable 
Organisms 
 
 
Table 4.3: Phylogenetic identity of the isolates (blast sequence match) 
Bacteria Isolate Length of the Best % Name of the 
division code Nucleotide phylogenetic similarity organisms 
match 
(accession no) 
Lactobacillus  G11 869 JF 965385.1 99 L. plantarum 
Lactobacillus G12 890 JF 965387.1 99 L. plantarum 
Lactobacillus E13 890 JF 757224.1 99 L. plantarum 
Lactobacillus E36 896 AB 601179.1 99 L.plantarum subsp. 
plantarum 
Lactobacillus F6 920 Jq 723699.1 100 Lactococcus lactis 
Bacillus F26 817 Jq 835018.1 100 Bacillus subtilis  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
76 
 
 
Screening of the isolates for β-galactosidase production on solid agar 
The LAB isolates were screened for β-galactosidase production on solid agar using X-gal 
(5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside) as an indicator. Table 4.4 shows the 
ability of the LAB strains to produce β-galactosidase on solid agar. The isolates which 
were able to produce β-galactosidase were observed to have green coloration after 
0
anaerobic incubation at 37 C for 36 hrs. Out of the 30 isolates screened, 12 had the ability 
to utilize X-gal within 24 hours of incubation as shown in Table 4.3. 
The isolates are classified as very good, weak or poor producers of β-galactosidase. Based 
on the result, 7 LAB strains were termed very good producers, 4 were weak producers 
while only 1 was a poor producer. However, 19 isolates were negative to β-galactosidase 
production. 
Table 4.5 shows the result of the screening of lactic acid production (Table 4.3) in MRS 
broth medium. Lactic acid production ranged from 7.134 mg/L in isolate E13 after 24 hrs 
of incubation to 15.240 mg/L after 48hrs in which highest production was recorded in 
isolate F26 at 36 hrs of incubation while isolate E13 had the least production at 24hrs of 
incubation 
 
 
 
 
 
 
 
 
 
 
 
77 
 
 
Table 4.4: Screening of lactic acid bacterial isolates for β-galactosidase Production on 
solid agar 
 
Serial Isolate Code  x-gal Reaction Serial Isolate x-gal 
No No Code  Reaction 
1 3E - 17 E39 - 
2 4E - 18 14E + 
3 E1 + 19 F5 + 
4 E2 - 20 F6 + 
5 E11 - 21 F26 + 
6 E13 + 22 F27 - 
7 E14 + 23 F28 - 
8 E17 - 24 F33 - 
9 E18 - 25 G8 + 
10 E19 - 26 G11 + 
11 E20 - 27 G12 + 
12 E22 - 28 G31 - 
13 E28 - 29 G32 - 
14 E29 - 30 G37 - 
15 E30 + 31 G38 - 
16 E36 +    
 
            + = has potential for β-galactosidase production 
- = absence of β-galactosidase production
78 
 
 
 
Table 4.3: Screening of lactic acid bacterial isolates for lactic acid production in 
broth 
  
 
 Incubation Time (hrs)/lactic acid concentration (mg/L) 
Isolates code 24 48 72 
 c  b  a
G11 9.2964  9.4584  11.1012  
 c  a  b
F26 8.9718  15.1332  11.1684  
 c  a  b
E36 9.18816  14.3226  10.9476  
 c  a  b
G12 11.4042  14.0826  10.554  
 a  b  c
F6 11.3502  10.6476  7.7184  
  c  a  b
E13 7.134  14.8092  11.6964  
 c  a  b
14E 10.9176  15.2406  11.7042  
 
Figures with different superscripts are significantly different using Duncan‟s Multiple Range Test.
79 
 
 
Screening of the selected LAB strains for Extracellular and Intracellular β-
galactosidase production using submerged fermentation under static and agitation 
 The ability of the LAB strains to produce extracellular and intracellular β- galactosidase 
using submerged fermentation under static and agitation conditions was investigated.  
Extracellular β- galactosidase production under static and agitated condition is shown in 
Figure 4.1a. β -galactosidase production ranged from 0.0172 U/ml in isolate G12 to 
0.2145U/ml in isolate 14E under static condition, while production ranged from 0.0053 
U/ml in E13 to 0.1296 U/ml in isolate 14E. Isolate 14E had the highest β-galactosidase 
production under static condition, while Isolate G12 had the least production under static 
condition.  
 Intracellular β-galactosidase production by the isolates ranged from 0.2057 in isolate F6 
to 7.8892U/ml in isolate G11 under static condition. Under agitated condition β-
galactosidase production ranged from 0.3270 U/ml in isolate F6 to 8.5612U/ml in G11. 
L.plantarum had the highest production under agitation condition as shown in Figure 4.2b. 
It was observed that L.plantarum had highest intracellular enzyme production was 
recorded under agitated condition except for isolate L.plantarum (E13) which produced 
the highest value under static condition as shown in Figure 4.1b   
 
 
 
 
 
 
 
 
 
 
 
80 
 
 
  
0.016
0.014
0.012
0.01
0.008 Static
0.006
Agitated
0.004
0.002
0
E13 F26 G12 E36 F6 G11 14E
Isolate code  
Figure 4.1a: Extracellular production of β-galactosidase by the selected LAB isolates  
0.6
0.5 Static
0.4 Agitated
0.3
0.2
0.1
0
E13 F26 G12 E36 F6 G11 14E
Isolate code
 
 
 Figure 4.1b: Intracellular β-galactosidase production potential of selected Isolates 
 
 
 
81 
 
Enzyme Activity (U/ml)
 
Effect of incubation time on growth and β- galactosidase production by the selected 
LAB strains 
 
Figure 4.2a shows the effect of varying incubation time on growth by L.plantarum (G11). 
It ranged from 1.1066 to 3.035OD600nm in which the highest growth was attained after 18 
hrs of incubation. There was a gradual increase in growth with increase in incubation time 
up to and including 30 hrs. This was followed by a sharp drop at 36hrs. Stability was 
reached at 42 hrs. 
Comparatively, Figure 4.2b, shows the effect of incubation time on growth and β-
galactosidase production by L. plantarum (G11) which ranged from 1.1066/ml to 
3.035OD600nm and 4.0566-7.6267U/ml respectively in which the highest β- galactosidase 
production was recorded between 18 and 30hrs after incubation. Growth was observed to 
increase gradually from inception up to 12 hours of incubation and attained peak   after 30 
hours of incubation and thereafter entered a sharp decline as incubation progresses. 
Similar trend was observed in β-galactosidase production, showing a correlation between 
peak microbial growth and maximum enzyme production. The exponential phase lasted 
for 6 hours and then entered progressive increase in enzyme activity for another 18 hours 
of incubation as shown in the figure. 
 
 
 
 
 
 
 
 
82 
 
 
3.5
3
2.5
2 OD
1.5
1
0.5
0
0 6 12 18 24 30 36 42
incubation time(hrs)
 
Figure 4.2a:Effect of incubation time on growth of isolate G11 
 
 
 
9
8
7
6
5
4
3
2
1
0
0 6 12 18 24 30 36 42
Incubation Time (hrs)
 
 
Figure 4.2b: Effect of incubation time on β-D-Galactosidase production by the isolate 
G11  
 
 
 
 
 
83 
 
Absorbance
Enzyme activity (U/ml)
 
Figure 4.3a shows the effect of incubation time on growth by L.plantarum isolate E13. 
Rapid growth was observed in the first 6hrs after which was followed by a gradual slow 
down.Growth ranged from 0.5706-0.8090OD600nm in which the highest growth (1.889 
OD600nm) was attained at 24hrs of incubation.This was followed by a gradual decline in 
growth which eventually resulted into a sharp decline at 36hrs when no further increase in 
growth take place again even with increase in incubation time. 
Figure 4.3b shows the effect of incubation time on β-galactosidase production by 
L.plantarum G11.It shows a correlation between growth and β-galactosidase 
th
production.Production was rapid during the early parts until the 6  hr which was followed 
th th
by a gradual decline between the 6  and the 12  hrs.Therafter, production gradually 
th
increases up till the 30  hr when maximum β-galactosidase production (4.953 U/ml) was 
attained. This was followed by a sharp decline in production with increase in incubation 
time   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
84 
 
 
 
3.5
3
2.5
2
1.5
OD
1
0.5
0
0 6 12 18 24 30 36 42
Incubation Time (hrs)
 
 
Figure 4. 3a:Effect of incubation time on growth of L.plantarum isolate E13 
 
9
8
7 Enz U/ml
6
5
4
3
2
1
0
0 6 12 18 24 30 36 42
Incubation Time (hrs)  
 
Figure 4.3b: Effect of incubation time on β-D-Galactosidase production by 
L.plantarum isolate E13  
 
85 
 
Enzyme Activty (U/ml) 
Absorbance
 
Effect of incubation time on growth and enzyme production by L.plantarum 
subsp.plantarum 
 Figure 4.4a shows the growth of L.plantarum subsp.plantarumincubated at different time 
intervals (hrs).Growth ranged from 1.5542 to 3.3242600nm in which the highest was 
recorded at 30 hrs of incubation beyond which drop in growth was observed. 
The figure also shows that β- galactosidase production byL.plantarum subsp.plantarum 
has two log phases in which the first lasted for 6hrs during which there was gradual 
increase in production. After 6hrs, a momentary lag phase was observed before the second 
exponential phase was attained between 12-18hrs of incubation which gradually entered 
into the stationary phase between 18-30hrs.This was then followed by a sharp drop at 
36hrs and stability was reached at 42hrs. 
Figure 4.4b shows a correlation between growth and and β- galactosidase production by L. 
plantarum subsp.plantarum where increase in growth also results in corresponding 
increase in production. β- galactosidase production by  L.plantarum subsp.plantarum  
ranged from 2.9496 to 9.3708 U/ml within the time of incubation in which the highest was 
recorded at 30hrs. It shows that β- galactosidase production by L. plantarum 
subsp.plantarum   started shortly after incubation and reached peak production of 9.371 
U/ml at 30hrs beyond which drop in production was observed as incubation time 
continued.  
 
 
 
86 
 
 
3.5
3
2.5
2
OD
1.5
1
0.5
0
0 6 12 18 24 30 36 42
Incubation Time (hours)  
 
 Figure 4.4a: Effect of incubation time on growth of L. plantarum subsp. plantarum 
(E36) 
12
10
8
6 Enz U/ml
4
2
0
0 6 12 18 24 30 36 42
Incubation Time (hrs)  
 
 
Figure 4.4b: Effect of incubation time on β-galactosidase Production by L.plantarum 
subsp.plantarum(E36) 
Effect of temperature on growth and β-galactosidase production by the selected LAB 
isolates 
87 
 
Absorbance (O.D)
Enzyme activity (U/ml)
 
 Figure 4.5a shows the effect of temperature on growth byL.plantarum(G11). Gradual 
0
increase in growth was observed between 20-30 C.Growth ranged from 0.7733 to 1.0568 
0
OD600nm. The highest growth was recorded at 40 C. A gradual decrease in growth was 
0
observed in L.plantarumG11with increase in temperature above 35 C. Maximum growth 
0 0
was best between the temperature of 30 C and 40 C with a peak absorbance of 1.0569. 
This was followed by a sharp drop at 40hrs. 
Figure 4.5b shows the effect of temperature on β-galactosidase production by 
L.plantarum.G11 It was observed that enzyme production ranged from 3.6752 to 4.919 
0 0
U/ml. Temperature of 20 C to 35 C favored β-glactosidase production with optimal yield 
0
of 4.919 U/ml at 35 C.  β-galactosidase production  was maintained within the  
0 0
temperature range of 30 - 35 C and at 35 C as the temperature increases there was 
reduction in β-galactosidase production which is an  indication  that β-galactosidase  
produced by this species of L.plantarum is temperature sensitive, stable at low temperature 
and therefore thermo-labile. Fig 4.5c shows the effect of temperature on protein 
0
production by L. plantarum (G11). It shows highest production at 25 C followed by a 
0
sharp drop at 30 C. Thereafter, a gradual a decrease in production a temperature increases. 
0
Stability was reached at 45 C. 
Effect of temperature on growth and β-galactosidase production byL.plantarum (E13) was 
investigated as shown in Figure 4.6a. Growth ranged from 0.825 to 1.3483 OD600nm in 
0
which the highest growth was recorded at 35 C, the results showed that L.plantarum (E13) 
maintained gradual growth with increase in temperature. A sharp increase in growth 
0 0
activities was observed from 25 C to 30 C (a short exponential phase) which gradually 
0
enter into a stationary/growth phase between temperature of 30 and 35 C. Peak absorbance 
0 0
values of 1.348was supported by 35 C.Further increase in temperature (above 35 C) did 
not support further increase in growth of the isolate.    
Figure 4.6b shows the effect of varying temperatures on β- galactosidase production by 
L.plantarum(E13). From the result, it was observed that β- galactosidase production 
0
ranged from 1.9066 to 2.220U/ml. The results indicated that temperature of 20 C best  
 
 
88 
 
 
1.2
1
0.8
0.6
0.4
0.2
0
20 25 30 35 40 45
Temperature (oC )
 
Figure 4.5a: Effect of temperature on growth of L.plantarum(G11). 
6
5
Enz U/ml
4
3
2
1
0
20 25 30 35 40 45
Temperature (0C)
 
Figure 4.5b: Effect of temperature on β-galactosidase production by 
L.plantarum(G11). 
 
89 
 
Absorbance (OD600nm )
Emxzyme Activity ( U/ml)
 
4.5
4
3.5
Protein
3
2.5
2
1.5
1
0.5
0
20 25 30 35 40 45
Temperature (°C)  
Figure 4.5c: Effect of Temperature on protein production by L. 
plantarum (G11). 
 
 
 
 
 
 
 
 
 
90 
 
protein (mg/ml)
 
1.6
1.4
1.2
1
0.8
OD
0.6
0.4
0.2
0
20 25 30 35 40 45
Temperature (°C)  
Figure 4.6a:  Effect of temperature on growth of L. plantarum (E13). 
8
7
6
5
4
3
2
1
0
20 25 30 35 40 45
Temperature (oC )  
 Figure 4.6b: Effect of temperature on β- galactosidase production by L. 
plantarum                 (E13). 
91 
 
Absorbance (OD600nm )
Enzyme activity (U/ml)
 
16
14
12
10
8
Protein
6
4
2
0
20 25 30 35 40 45
Temperature (°C)  
Figure 4.6c: Effect of temperature on protein synthesis by L. plantarum (E13). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
92 
 
protein (mg/ml)
 
supported β- galactosidase production byL.plantarum (E13). β-galactosidase production 
0 0
was highestbetween  25 C and 35 C, thereafter, a gradual decline was observed between 
0 0
35 C and 40 C. Further increase in temperature resulted in decrease in β- galactosidase 
production. 
Figure 4.7a shows the effect of varying temperatures on growth by L.plantarum 
subsp.plantarum. It shows that there was a sharp decline in growth at low temperature 
0 0
from 20 C-25 C. There was a gradual increase in growth with increase in 
0
temperature.Steady growth was maintained between 25-40 C  
0
Figure 4.7bshows that low temperature of 20 C supported highest β-galactosidase 
production which ranged from 4.4385 to 7.3176U/ml in which the highest was recorded at 
0
35 C.A gradual drop in β-galactosidase production then follows. Thereafter production 
declined gradually with increase in temperature. This showed that β-galactosidase from L. 
plantarum subsp. plantarum (E36) is thermo labile.   
Figure 4.7c shows the effect of varying temperature on protein profile of L. plantarum 
subsp. plantarum. The results indicated that the protein production was maximally 
0
supported by low temperature up to 25 C with peak production value of 3.371 mg/ml. 
0
Further temperature increase resulted into sharp decrease (4.430 mg/ml at 30 C) in 
enzyme production. Gradual reduction in protein level was observed as the temperature 
increases. This showed that high temperature was not favorable to protein stability by L. 
plantarum subsp. plantarum. 
 
 
 
 
 
 
 
 
 
 
 
93 
 
 
 
 
 
2.5
OD
2
1.5
1
0.5
0
20 25 30 35 40 45
Temperature (°C)
 
Figure 4.7a:  Effect of Temperature on Growth for L. plantarum 
subsp.plantarum (E36) 
9
8 Enz U/ml
7
6
5
4
3
2
1
0
20 25 30 35 40 45
Temperature (0C)
 
 
Figure 4.7b: Effect of Temperature on β-galactosidase production for L. 
plantarum subsp.plantarum (E36). 
 
 
 
94 
 
Absorbance (OD600nm )
Enzyme Activity (U/ml)
 
10
9
8 Protein
7
6
5
4
3
2
1
0
20 25 30 35 40 45
Temperature (0C)
 
Figure 4.7c: Effect of temperature on protein profile of L. plantarum   
subsp.plantarum 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
95 
 
protein (mg/ml)
 
Effect of pH on β- galactosidase productionby the selected isolates in an optimised 
medium 
The effect of pH on β-galactosidase production by the three L .plantarum.Strains were 
investigated Figure 4.8a.Shows the effect of pH on β-galactosidase production by L. 
plantarum (G11). Production ranged from 0.1545 U/ml to 0.5729 U/ml.The highest β-
galactosidase production (0.5627U/ml) was observed to occur at pH 7.5 Thereafter,a 
gradual decrease in β-galactosidase production was observed as pH increases. 
Figure 4.8b profiles β-galactosidase production by L. plantarum (E13). Peak β-
galactosidase production (0.4679U/ml)was attained at pH 7.0. Thereafter, production 
gradually declined as pH increases. 
Figure 4.8c shows β-galactosidase production activity by L. plantarum subsp. plantarum 
(E36) in which pH 7.0 supported highest β-galactosidaseproduction (0.5729U/ml) 
thereafter production decrease sharply with increase in pH.Production activity by this 
strain follow similar trend as observed in strain (E13). 
Out of all pH regimes tested for, pH 5 least supported β-galactosidase production.  A 
neutral to alkaline broth environment favored   β-galactosidase production by all the 
strains. 
  
 
 
 
 
 
 
96 
 
 
0.25
0.2
0.15
0.1
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8 pH   
Figure 4.8a: Effect of pH on β-galactosidase production byL.plantarum (G11) 
0.25
0.2
0.15
0.1
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8
pH  
 
              Figure 4.8b: Effect of pH on β-galactosidase production by 
L.plantarum(E13). 
 
 
 
 
 
 
97 
 
Enzyme Activity (U/ml)
Enzyme Activity (U/ml)
 
 
 
 
0.25
0.2
0.15
E36
0.1
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8
pH
 
Figure 4.8c: Effect of pH on β-galactosidase production by L. plantarum subsp. 
plantarum (E36). 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
98 
 
Enzyme Activity (U/ml)
 
Effect of agitation speed on β-galactosidase production. 
The effect of agitation on β-galactosidase production by the selected LAB isolates was 
investigated. Figure 4.9 shows the effect of agitation on β -galactosidase production by the 
isolates. β-galactosidase production by the different isolates ranged from 0.004 U/ml (0 
rpm) to  0.186 U/ml (100rpm).The result showed that highest production was favoured at 
speeds less than 100rpm(between 60-120 rpm) rather than at higher speeds as shown by L. 
plantarum. (G11), L. plantarum. (E13) and L. plantarum subsp. plantarum (E36) 
respectively (Table 4).The speed of agitation increased enzyme production to a maximum 
limit of 60rpm by L. plantarum ( G11). There was no further increase in production even 
with increase in the agitation speed. The effect of agitation on β- galactosidase production 
by L .plantarum (G11) showed that 60rpm supported maximum production of 0.184 U/ml. 
For L. plantarum (E13) agitation speed of 120 rpm give maximum production of 0.182 
U/ml. Further increase in agitation speed has no effect on production of β-galactosidase. 
Increase in the speed of agitation for β-galactosidase production does not result into 
changes in the amount of production.Observations of the three strains of plantarum shows 
that each of the strains has different agitation speeds for β-galactosidase production. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
99 
 
 
 
 
0.25
0.2
0.15
G11
E13
0.1 E36
0.05
0
0 60 80 100 120 140 160 180 200
speed (rpm)
 
 
        Figure 4.9: Effect of agitation on β-galactosidase production by LAB isolates. 
 
 
 
 
 
 
 
 
 
100 
 
Enzyme Activiy (U/ml)
 
 
Effect of carbon sources on β-galactosidaseproduction. 
Figures 4.10a - 4.10d show the effect of different carbon sources on β-galactosidase 
production by the selected LAB isolates. All the fermentable sugars supported β-
galactosidase production in all the selected isolates. Glucose, Galactose, Maltose and 
Mellibiose maximally enhanced β-galactosidase production. For L.plantarum (G11), 
Raffinose was found to be the best carbon source for β-galactosidase production having 
the highest enzyme activity of 0.305U/ml. This was followed in decreasing orders by 
mellibiose, galactose and saccharose. The least supporting carbon source was found to be 
fructose(0.1692U/ml) for the single sugars, while in the case of combined sugars, maltose 
+ galactose(0.2959U/ml) was 14.47% better than galactose and 0.74% than maltose as 
single sugars respectively (Figure 4.10a). 
For L. plantarum (E13), all carbon sources tested poorly on average. Only mellibiose, 
(0.2786U/ml) showed significant effectiveness. Galactose (0.2698U/ml) and maltose 
(0.2786U/ml) appreciably supported β-galactosidase production while glucose and 
trehalose either had close to zero or very low activity among the single sugars. The same 
trend of activity was observed in the case of combined sugars where maltose + Galactose 
combination was 11.65% and 5.47% better than Raffinose + Galactose and Trehalose + 
galactose combinations respectively (Figure 4.10b).     
All sugar combinations specifically raffinose + galactose and Trehalose + Galactose 
performed appreciably well at 4.63% and 1.11% higher than Mellibiose as a single sugar 
respectively. Combining effect of the sugars showed that Galactose and Maltose are the 
best carbon source for L. plantarum (G11) and L. plantarum (E13). As for L. plantarum 
subsp. plantarum melibiose, galactose and saccharose at 0.2982U/ml,0.2827U/ml and 
0.2689U/ml respectively best supported β- galactosidase production as single sugars while 
raffinose and galactose best supported β- galactosidase production as combined sugars 
(Fig.4.10a). 
 
 
 
101 
 
 
0.35
0.3
0.25
G11
0.2
0.15
0.1
0.05
0
Figure 4.10a: Effect of different carbon sources onβ-galactosidase production by 
L.plantarum. (G11). 
0.35
0.3 E…
0.25
0.2
0.15
0.1
0.05
0
Carbon sources
 
Figure 4.10b: Effect of different carbon sources onβ- galactosidase production by L. 
plantarum (E13) 
 
 
102 
 
Enzyme activity(U/ml) Enzyme activity (U/ml)
 
0.35
0.3 E36
0.25
0.2
0.15
0.1
0.05
0
 
Figure 4.10c: Effect of different carbon sources on Growth β- galactosidase 
production by L. plantarum subsp. plantarum (E36) 
0.35
0.3
0.25
0.2
0.15
0.1
G11
0.05
E33
0
E36
 
Figure 4.10d: Effect of different carbon sources on β- galactosidase production by 
LAB isolates.  
103 
 
Enzyme activity (U/ml) Enzyme activity (U/ml)
 
Effect of organic and inorganic nitrogen sources on β-galactosidaseproduction 
The effect of organic and inorganic nitrogen sources on β-galactosidase production by 
Lactobacillus plantarum strains was investigated (Figure 4.11). Nine (organic and inorganic) 
nitrogen sources were used. The results are as shown in the following figures, it was observed 
that casein and yeast extracts as organic nitrogen sources were the best enhancer for β-
galactosidase production in both L.plantarum. (G11) and L. plantarum (E13) whereas all the 
inorganic nitrogen sources best supported β- galactosidase production with Potassium nitrate 
and Ammoniumnitrate salts recording the highest production of 0.2806U/ml and 0.2619U/ml 
respectively by L. plantarum (G11).  
Also β- galactosidase production by L. plantarum (G11) was enhanced in broth augmented 
with malt extract, yeast extract, casein, urea and ammonium salts. A similar production trend 
was also observed with L.plantarum (E13) reaching peak production of 0.45583U/ml with 
ammonium nitrate. However, almost all the salts performed poorly in the case of 
L.plantarumsubsp. plantarum (E36) as none of the organic salts significantly boosted β-
galactosidase production except ammonium nitrate. It was observed from fig. 4.10c that all the 
salts performed poorly in supporting β-galactosidase production by L plantarum   subsp. 
plantarum (E36) just as observed with L. plantarum (E13). 
The profile of the effect of the salts on β- galactosidase productionby the three strains tested 
isbest represented in Figure 4.11d. 
 
 
 
 
 
 
 
 
 
 
 
104 
 
 
 
0.35
G11
0.3
0.25
0.2
0.15
0.1
0.05
0
 
Figure 4.11a: Effect of different nitrogen sources on β- galactosidase production by 
L. plantarum (G11). 
0.6
0.5 E13
0.4
0.3
0.2
0.1
0
 
   Figure 4.11b: Effect of different nitrogen sources on β- galactosidase production by 
L. plantarum (E13).  
 
105 
 
Enzyme activity (U/ml)
Enzyme activity (U/ml)
 
0.25
E36
0.2
0.15
0.1
0.05
0
Figure 4.11c: Effect of different nitrogen sources on β- galactosidase production L. 
plantarum subsp. plantarum (E36) 
0.6
0.5
0.4
0.3
G11
0.2 E13
E36
0.1
0
 
Figure 4.10d: Effect of different nitrogen sources on β- galactosidase production by 
selected LAB isolates. 
 
 
 
 
 
106 
 
Enzyme activity (U/ml) Enzyme activity (U/ml)
 
 
Effect of inoculum size on β-galactosidase production activity by selected LAB 
isolates. 
Figure 4.12a: is showing the effect of different inoculum sizes on β-galactosidase 
production.As shown in the figures, the inoculum sizes tested  ranged from 4 – 12%  as to 
determine the best inoculum size that best augment isolate growth and β-galactosidase 
production by LAB strains (Table 13) under optimised conditions. Inoculum size 6% best 
supported enzyme production for all isolates. Growth and enzyme activity was best 
supported by low (%)  inoculum size attaining peak activities at 6%.This was followed in 
decreasing orders by sizes 8%,10% and 12% respectively.This trend were best 
demonstrated by L. plantarum (E13) and L. plantarum subsp. plantarum (E36) 12% 
increase in inoculum level have no effect on β-galactosidase production by the LAB 
isolates. 
Figure 4.12b profiles the effect of inoculum size on β-galactosidase production by by L. 
plantarum (E13). It shows a gradual decrease in β-galactosidase production as percentage 
inoculums size increases. 
 Figure 4.12c shows the effect of inoculums size on β-galactosidase production by L. 
plantarum   subsp. plantarum (E36). Six percent 6% inoculums size supported highest β-
galactosidase production, Thereafter, gradual  decrease in production was observed as 
percentage inoculums size increases. 
 
 
 
 
 
 
 
 
107 
 
 
 
5
4.5
4
3.5
3
2.5 G11
2
1.5
1
0.5
0
4% 6% 8% 10% 12%
Inoculum size  
 
Figure 4.12a:  Effect of inoculums size on β-galactosidase production by L. plantarum 
(G11). 
 
14
12
10
8
6
E13
4
2
0
4% 6% 8% 10% 12%
Inoculum Size 
 
Figure 4.12b:  Effect of inoculum size on β-galactosidase production by L. plantarum 
(E13). 
 
 
108 
 
Enzyme activity (U/ml) Enzyme activity (U/ml)
 
9
8
7
6
5
4
E36
3
2
1
0
4% 6% 8% 10% 12%
inoculum size 
Figure 4.12c: Effect of inoculum size on β-galactosidase production by L .plantarum 
subsp. plantarum (E36)  
 
 
 
 
 
 
 
 
 
 
 
 
109 
 
Enzyme activity (U/ml)
 
Effect of inoculum age on β-galactosidase production  
The effect of inoculum age on enzyme production was determined as shown in Figure 
4.13a. The age of the cultures ranged from 6 – 42 hours to determine the best inoculums 
age that was suitable for peak β-galactosidase production. It was observed that β-
galactosidase prodction ranged from 0.0851U/ml to 0.2965U/ml for all the LAB isolates.  
Figure 4.13a shows the effect of varying inoculums age on β-galactosidase production. 
There was gradual increase in inoculum age from 6-18 hrs. The highest β-galactosidase 
amount (0.2697U/ml) was achieved at 18hrs. There was reduced production at 24hrs 
which immediately attained another peak(0.2614U/ml) again at 30hrs. This was followed 
by a gradual decrease in β-galactosidase production with increase in time. Therefore L. 
plantarum (G11) attained maximum production with 18hrs old inoculums.  
Figure 4.13b shows the effect of varying inoculums age on β-galactosidase production by 
L. plantarum (E13). It shows high activity at 6hrs (0.2916U/ml) which gradually decreases 
with increase in time until reaching peak production (0.2965U/ml) at 24hrs. This was 
followed by decrease in production with increase in time.stability was reached at 42 hrs. 
The best inoculums age for β-galactosidase production was 24hrs old. 
Figure 4.13c shows the effect of varying inoculum age on β-galactosidase production by L. 
plantarum subsp. plantarum (E36). It shows minimal β-galactosidase activity at 6hrs and 
12hrs old inoculum even though what was produced at 6hrs was higher than at 12hrs. 
However, a sharp increase was observed at 18hrs (0.2619U/ml) with minimal decrease in 
activity at 24hrs but attained peak production with 30hrs old inoculums. Therafter, gradual 
decrease in production with increase in time was observed.Stability was achieved with 
42hrs inoculum.  
 
 
 
110 
 
 
5
G11
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
6 12 18 24 30 36 42
Inoculum Age (hrs)
 
Figure 4.13a: Effect of inoculum age on β-galactosidase production at different time 
intervals for L. plantarum (G11) 
6
5
4
3
2 E13
1
0
6 12 18 24 30 36 42
Inoculum Age (hrs)
 
Figure 4.13b: Effect of inoculum age on β-galactosidase production at different time 
intervals for L. plantarum (E13). 
111 
 
Enzyme activity (U/ml)
Enzyme activity (U/ml)
 
5
4.5
4
3.5
3
2.5
2 E36
1.5
1
0.5
0
6 12 18 24 30 36 42
Inoculum Age (hrs)
 
Figure 4.13c: Effect of inoculum age on β-galactosidase production at different time 
intervals by L. plantarum subsp. plantarum (E36)  
 
 
 
 
 
 
 
 
 
 
 
112 
 
Enzyme activity (U/ml)
 
Enzyme characterization 
Effect of temperature on enzyme activity 
The effect of varying temperature on the activity of β-galactosidase by the selected LAB 
isolates was determined. Figure 4.14a shows the effect of temperature on L.plantarum 
0 0
within the range of 15 C -50 C on β-galactosidase activity. β-galactosidase activity ranged 
from 0.00135U/ml-0.1225Uml. It shows that the lowest activity (0.00135 U/ml) was 
0
recorded by L.plantarum(G11) at 15 C in which the highest activity (0.1225U/ml) was 
0
recorded at 45 C. A gradual increase in β-galactosidase activity was observed between 
0 0
15 C and 20 C. This was displayed better temperature stability than others as relative 
0
stability followed by a sharp drop at 250C followed by a stationary phase up to 30 C. 
Another gradual increase in β-galactosidase activity now follows attaning a peak 
0
(0.1253U/ml) as stability reaches 50 C. 
 
In figure 4.14b β-galactosidase production by L.plantarum (E13) shows better temperature 
0 0
stability between 20 C- 45 C. A sharp increase in β-galactosidase production (0.04711 -
0 0
0.1285 U/ml)was observed from 15 C-20 C. This was followed by a gradual drop in β-
0
galactosidase activity with increase in incubating temperature up to and including 30 C. 
0
This was followed by a sharp increase at 35 C. Increase in β-galactosidase activity 
increases gradually with increase in temperature until peak activity (0.1536U/ml) attained 
0 0
at 40 C. A drop in activity was observed at 50 C. 
Figure 4.14c Profiles of β-galactosidase activity by L. plantarum subsp. plantarum (E36) 
within the temperature ranged tested. Gradual increase in β-galactosidase activity was 
0 0
observed between 15 C-30 C as β-galactosidase activity increases with with temperature. 
0
Maximum β-galactosidase activity (0.1681U/ml) was obtained at 30 C. This followed by a 
0 0
sharp drop at 35 C. Another sharp increase immediately occurred at 40 C, attaining peak 
0 0
activity (0.1328U/ml) at 45 C before finally recording another sharp drop at 50 C 
The characteristic behavior of these isolates and β-galactosidase production by each 
showed that L. plantarum (G11) has thermophilic properties by supporting optimal β-
0
galactosidase activity at 45 C while L. plantarum subsp. plantarum (E36) can be said to be 
0
mesophilic as temperature of 30 C supported maximum β-galactosidase activity of 
0.168U/ml 
 
113 
 
 
 
 
 
0.14
0.12 G11
0.1
0.08
0.06
0.04
0.02
0
15 20 25 28 30 35 40 45 50
Temp (0c)  
       Figure 4.14a: Effect of temperature on β-galactosidase activity by (G11) L. 
plantarum 
0.18
E13
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
15 20 25 28 30 35 40 45 50
Temp (0c)
 
Figure 4.14b: Effect of temperature on β-galactosidase activity by L. plantarum (E13) 
 
 
 
114 
 
Enzyme activity(U/ml)
Enzyme activity (U/ml)
 
 
0.2
E36
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
15 20 25 28 30 35 40 45 50
Temperature
Figure 4.14c: Effect of temperature on β-galactosidase activity by L. plantarum subsp. 
plantarum     (E36)  
 
 
 
 
 
 
 
 
 
 
 
 
115 
 
Enzyme activity (U/ml)
 
Effect of pH on β-galactosidase activity  
Figure 4.15a shows the effect of varying pH on β-galactosidase activity as expressed by 
the selected LAB Isolates within pH range of 5.0 and 8.0. The highest β-D-galactosidase 
activity of 0.182 U/ml was produced by L. plantarum (G11) at pH 6.0.Highest β-
galactosidase activity was observed at pH 5.0. It shows a gradual drop at5.5 which 
gradually increased to an initial peak activity (0.1825U/ml) at pH 6.0. A gradual drop in 
activity was observed at pH 6.5 which remain sustained up to and including pH 7.0. This 
was followed by a sharp drop at pH 7.3 followed by a sharp increase at pH 7.5. Highest β-
galactosidase activity (0.1903U/ml) was recorded at pH. Hence L. plantarum is pH 
tolerant. 
Figure 4.15b shows the profile effect of pH on the activity of β-galactosidase. At pH 5.0, 
very high β-galactosidase activity (0.1612U/ml) was observed followed by a sharp drop at 
pH 5.5. A gradual increase in activity was again seen at pH 6.0 attaining maximum β-
galactosidase activity (0.2265U/ml) at pH 7, that is, it expressed peak activity at neutral 
pH. 
Figure 4.15c shows the effect of pH on the activity of β-galactosidase produced by 
L.plantarum subsp.plantarum (E36). High β-galactosidase activity(0.1659U/ml)was 
observed at pH 5.0 which shows a sharp decline at pH 5.5. A gradual increase in activity 
was observed up to and including pH 7.0. This was followed by a gradual decrease in β-
galactosidase activity until maximum activity was attained (0.1903U/ml) at pH 8.0. 
 
 
 
 
 
 
 
 
 
116 
 
 
 
0.25
0.2
0.15
G11
0.1
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8
pH  
Figure 4.15a: Effect of pH on β-galactosidase production by L. plantarum (G11). 
 
 
 
0.25
0.2
0.15
0.1 E13
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8
pH  
Figure 4.15b: Effect of pH on β-galactosidase production by L .plantarum (E13). 
 
 
 
 
 
 
117 
 
Enzyme Activity (U/ml) Enzyme Activity (U/ml)
 
 
0.25
0.2
0.15
0.1
E36
0.05
0
5 5.5 5.8 6 6.5 6.8 7 7.3 7.5 7.8 8
pH
Figure 4.15c: Effect of pH on β-galactosidase production by L. plantarum subsp. 
plantarum (E36)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
118 
 
Enzyme Activity (U/ml)
 
Stability of β-galactosidase activity at different temperatures 
 Stability of β-galactosidase activity at different temperature was investigated using 
0 0
temperature range from 20 C - 75 C as shown in Figure 4.16a. It is observed that the 
temperature profiles of the hydrolytic activity of LAB β-galactosidase produced by the 
different isolates. It shows that L. plantarum (G11) has its highest activity with ONPG as 
0
substrate at 25 C (0.066U/mg). This was followed a gradual decrease in activity with 
increase in temperature. 
A figure 4.16b show that L.plantarum (E13) has low hydrolytic activity at ordinary 
0
temperature.Sharp increase in activity was observed at 40 C reaching maximum 
0 0
(0.032U/mg)at 45 C. This was followed by a sharp drop in activity at 50 C. A gradual 
0
decrease in activity was observed as temperature increases.Stability was achieved at 75 C. 
Figure 4.16c shows the effect of temperature on the hydrolytic capability of L. plantarum 
subsp. plantarum (E36) at different temperature regimes. A sharp drop in activity was 
0 0
observed at 20 C with minimal increase in activity at 30 C. This was followed by a sharp 
0
increase in activity (0.035U/mg) at 40 C. A further drop in activity was again observed at 
0 0
45 C followed by another sharp increase in activity at 50 C. Gradual decrease in activity 
0
then follows as the temperature increases. Stability was achieved at 75 C. 
 
 
 
 
 
 
 
 
 
119 
 
 
0.07
0.06
0.05
0.04
0.03
G11
0.02
0.01
0
20 25 30 35 40 45 50 55 60 65 70 75
Temperature (°C)
 
Figure 4.16a: Stability of β- galactosidase produced by L. plantarum (G11) at 
different temperatures 
0.08
0.07
0.06
0.05
0.04
0.03
0.02 E13
0.01
0
20 25 30 35 40 45 50 55 60 65 70 75
Temperature (°C)
 
Figure 4.16b: Stability of β- galactosidase produced by L. plantarum (E13) at 
different temperatures 
 
 
 
 
 
 
 
120 
 
Enzyme activity (U/ml)
Enzyme activity (U/ml)
 
0.09
0.08
0.07
0.06
0.05
0.04
0.03 E36
0.02
0.01
0
20 25 30 35 40 45 50 55 60 65 70 75
Temperature (°C)
 
Figure 4.16c: Stability of β- galactosidase produced byL.plantarum 
subsp.plantarum (E36) at different temperatures. 
 
 
 
 
 
 
 
 
 
 
 
 
121 
 
Enzyme activity (U/ml)
 
Effect of molar concentrations of cations on β-galactosidase activity 
Figure 4.17a shows the effect of different concentrations of cations on β-galactosidase 
activity produced by L. plantarum (G11). For KCl high activity was observed at 0.1molar 
concentration (0.0643U/ml). This was folloed by gradual decrease at 0.2mM. A maginal 
increase in activity was again observed as molar concentration again increases until peak 
activity was attained (0.0718U/ml) at 0.4mM. Thesame trend in activity was also observed 
with CaCl2where β-galactosidase activity increases with increase in molar concentration. 
A sharp increase in activity was observed from 0.1-0.2molar concentration. There was 
another drop in activity 0.2-0.3mol. This was followed by a gradual increase in activity up 
to 0.5mol with peak activity of 0.0056U/ml.Molar concentration of 0.3-0.4mol was best 
for NaCl with the best activity of 0.0188U/ml at 0.4 molar concentration.NH4Cl displays 
gradual increase in β-galactosidase activity as molar concentration increases. Highest 
activity (0.0319U/ml) was recorded at 0.2mol. Thereafter, gradual decrease in activity was 
observed as molar concentration increases.MgSO4showed supportive activity in all 
concentration though with decreasing activity as concentration increases. MnSO4 shows 
gradual increase in activity from 0.1-0.3molar concentration with peak activity of 
0.0282U/ml. Thereafter, gradual decrease in activity with increase in concentration was 
observed. 
Figure 4.17b profiles the effect of different molar concentrations of cations on β-
galactosidase activity produced by L. plantarum (E13). The different concentration of KCl 
followed the same trend as observed by L. plantarum (G11). Highest activity 
(0.0625U/ml) was observed at 0.3molar concentration.This was followed by a sharp drop 
at 0.4 molar concentration with peak increase (0.0548U/ml) at 0.5molar concentration. 
The highest activity by β-galactosidase activity against different molar concentration of 
CaCl2was recorded at 0.1 molar concentrations (0.0529U/ml). This was followed by a 
gradual decline in activity as concentration increases. With different concentration of 
NaCl salt, 0.4 molar concentrations was observed to produced the highest activity 
(0.0445U/ml). Although, 0.1molar concentration produced high activity but a gradual drop 
in activity was observed with increase in molar concentration.NH4Cl salt also produce 
high activity at 0.1molar concentration(0.0367U/ml). However, gradual decrease in 
activity was observed as concentration increases.A sharp drop was produced at 0.4 molar 
122 
 
 
concentrations. This was followed by a sharp increase in activity at 0.5molar 
concentration. Different concentration of MgSO4shows that 0.2mol. has the best activity 
forβ-galactosidase. Gradual decrease in activity was observed as concentration increases. 
MnSO4 produced maximum activity at a concentration 0.2mM.Thereafter,gradual decrease 
in activity was observed as concentration inceases. 
Figure 4.17c shows the effect of different molar concentrations of cations on β-
galactosidase activity produced by L. plantarum subsp. plantarum (E36) 0.2mM of KCl 
produced the highest activity (0.0831U/ml). This was followed by a gradual decrease in 
activity at 0.3mM.A sharp drop in activity was observed at 0.4Mmol which was followed 
by a gradual increase at 0.5mM.CaCl salt has best activity at 0.1mM 
(0.0507U/ml)followed by a gradual drop at 0.2mM. There was another gradual increase at 
0.3molar concentration which there after entered into gradual decrease as molar 
concentration increases.As for NaCl,0.2mM produced highest activity (0.0186U/ml) 
followed by gradual decrease as molar concentration increases.The best activity for 
different concentrations of NH4Cl (0.0288U/ml) was produced at 0.4molar 
concentration.There was however an appreciable activity at all concentrations.The best 
activity recorded for magnesium sulphate was at 0.3Mm (0.0286U/ml).All other 
concentrations performed poorly. MnSO4was best at 0.1molar concentration 
(0.0163U/ml).These was followed by gradual decrease in activity as concentration 
increases. 
 
 
 
 
 
 
 
 
123 
 
 
 
0.1
0.08 Kcl
0.06 Cacl
0.04 Nacl
0.02 NH4Cl
0 MgSO4
0.1 0.2 0.3 0.4 0.5
MnSO4
Molar concentration(mol/dm3)
 
Figure 4.17a: Effect of different molar concentrations of cations on β-galactosidase 
activity by L. plantarum (G11). 
 
0.09
0.08
0.07
0.06
Kcl
0.05
Cacl
0.04
0.03 Nacl
0.02 NH4Cl
0.01 MgSO4
0 MnSO4
0.1 0.2 0.3 0.4 0.5
Molar concentration (mol/dm3)
 
Figure 4.17b: Effect of different molar concentrations of cations on β-galactosidase 
activity by L. plantarum (E13). 
 
 
 
 
124 
 
Enzyme activity (U/ml) Enzyme activity(U/ml)
 
0.09
0.08
0.07
0.06
Kcl
0.05 Cacl
0.04 Nacl
0.03 NH4Cl
0.02 MgSO4
0.01 MnSO4
0
0.1 0.2 0.3 0.4 0.5
Molar concentration (mol/dm3)
 
Figure 4.17c: Effect of different molar concentrations of cations on β-galactosidase 
activity by L. plantarum subsp. plantarum (E36)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
125 
 
Enzyme activity(U/ml)
 
Effect of varying molar concentrations of anions on β-galactosidase activity. 
The effect of Anions on β-galactosidase activity using varying molar concentrations on 
selected bacterial isolates was determined as shown in figure 4.18a- 4.18c. The anions 
tested include copper sulphate, sodium nitrate, potassium nitrate and zinc sulphate with 
molar concentration ranging from (0.1-0.5M). β-galactosidase activity ranged from lowest 
activity of 0.00698 U/ml ( in 0.1M) to optimum activity of 0.19031 U/ml (in 0.3M). β-
galactosidase activity increases as molar concentration of the anions increases and 
thereafter declined.  
Figure 4.18a shows the effect of molar concentrations of anion salts on β-galactosidase 
activity produced by L.plantarum (G11). From the figure β-galactosidase activity 
increases with increase in molar concentrarion of sulphate ions of copper. Gradual 
increase is observed from 0.1-0.3Mmol with peak activity (0.19303U/ml) at 0.3Mmol. 
This was followed by a gradual decrease in activity as concentration increases. As for the 
sodium nitrate salts, asharp increase at 0.2Mmol was observed. This was followed by a 
gradual decrease in activity withanother increase at 0.5Mmol concentration. KNO3 salt had 
supportive activity in all concentrations. Highest activity was recorded at 0.1Mmol. This 
was followed by a gradual decrease as concentration increases except at 0.4molar 
concentration when marginal increase was observed. Different concentration of ZnSO4 
solutions show increase activity as concentration increases with peak activity at 0.2Mmol 
(0.0294U/ml). A shrp drop in activity was observed at 0.3Mmol, followed by a gradual 
increase in activity with increase in concentration.  
Figure 4.18b profiles the effect of molar concentrations of anions on β-galactosidase 
activity produced by L .plantarum (E13). The figure reviewed a gradual increase in β-
galactosidase activity as CUSO4molar concentrations increases. The highest activity was 
observed at 0.5molar concentration. With different concentration NaNO3, highest activity 
was attained at 0.1 molar concentrations. However, there was enhanced performance at all 
concentrations. KNO3 has it highest activity at 0.1Mmol. This was followed by a gradual 
drop in activity as molar concentration increases. The same trend in activity was observed 
with similar concentrations of ZnSO4 salt. The highest activity was recorded at 0.1Mmol. 
126 
 
 
Figure 4.18c shows the effect of molar concentrations of the different anion salts on β-
galactosidase activity produced by L. plantarum subsp. plantarum (E36). CUSO4 shows 
supportive activity on β-galactosidase activity at all concentration tested. The highest 
avtivity (0.1559U/ml) was recorded at 0.3Mmol. Marginal drop in activity was observed 
0.4molar concentration followed by gradual increase at 0.5Mmol. The 
NaNO3performedpoorly in all concentration tested except for 0.1 and 0.2 molar 
concentrations. Solutions of KNO3 performed relatively better than that of NaNO3. Sharp 
increase in activity was observed at 0.2molar concentration (0.0833U/ml).  This was 
followed by a gradual decrease in activity. Stability was achieved with higher 
concentrations. ZnSO4 cocentrations performed poorly except at 0.2Mmol where the 
highest activity (0.1002U/ml) was achieved. 
 
 
 
 
 
 
 
 
 
 
127 
 
 
0.2
0.18
0.16
0.14
0.12
CuSO4
0.1
NaNO3
0.08
0.06 KNO3
0.04 ZnSO4
0.02
0
0.1 0.2 0.3 0.4 0.5
Molar Concentration (mol/dm3)
 
Figure 4.18a: Effect of different molar concentrations of anions on β-galactosidase 
activity produced by L. plantarum (G11).  
 
0.25
0.2
0.15
CuSO4
0.1 NaNO3
KNO3
0.05
ZnSO4
0
0.1 0.2 0.3 0.4 0.5
Molar Concentration (mol/dm3)
 
Figure 4.18b: Effect of different molar concentrations of anions β-galactosidase 
activity produced by L. plantarum (E13). 
 
 
128 
 
Enzyme activity (U/ml) Enzyme  activity(U/ml)
 
 
0.18
0.16
0.14
0.12
0.1 0.1
0.2
0.08
0.3
0.06 0.4
0.04 0.5
0.02
0
CuSO4 NaNO3 KNO3 ZnSO4
Molar Concentation(mol/dm3)
 
Figure 4.18c: Effect of different molar concentrations of anions on β-galactosidase 
activity produced by L . plantarum subsp. plantarum (E36). 
 
 
 
 
 
 
 
 
 
 
 
129 
 
Enzyme activity(U/ml)
 
Effect of different molar concentrations of inhibitors of β-galactosidase activity. 
Figures 4.19-4.19c shows the effect ofvarying molar concentration (0.1-0.5M) of selected 
Inhibitors on β-galactosidase activity produced by the selected LAB isolates. Inhibitors 
tested on the β-galactosidase activity were Urea, EDTA and Potassium cyanide. β-
galactosidase activity was found to range from  0.00966 U/ml (0.3M) to 0.17444 U/ml 
(0.2M).  
Figure 4.19a shows the effect of different molar concentration of different inhibitors on β-
galactosidase activity produced by L. plantarum (G11). Activity of inhibitors shows sharp 
increase from the lowest concentration of 0.1-0.2Mmol with marginal activity 0.0454U/ml 
for urea. The highest activity (0.0725U/ml) was obtained at 0.4Mmol, this was followed 
by a gradual decrease in activity. EDTA shows supportive activity at all concentrations 
except at 0.2 molar concentrations where a sharp drop was observed. This was followed 
by a gradual increase in activity with maximum (0.1587U/ml) at 0.5Mmol. KCN also gave 
enhanced activity at all concentrations attaining highest activity (0.1744U/ml) at 0.2Mmol. 
This was followed by a gradual decrease in activity as molar concentration increases.  
Figure4.19b profiles the effect of inhibitors on β-galactosidase activity produced by L 
.plantarum (E13). Urea shows high activity (0.0762U/ml) at 0.1Mmoland then followed 
by gradual drop at 0.2molar concentration and another sharp drop 0.3Mmol was observed 
followed by a sharp increase (0.0953U/ml) was observed as the highest activity by urea. 
The highest activity by EDTA(0.0887U/ml) was attained 0.3 molar concentration, this was 
followed by a sharp drop in activity at 0.4Mmol which was followed by increase  with 
increase in concentration. KCN shows supportive activity at all concentration tested. 
Highest activity (0.1692U/ml) was attained at 0.2Mmol. 
Figure 4.19c shows the effect of different molar concentrations of the inhibitors onβ-
galactosidase activity produced by L.plantarum subsp. Plantarum. It shows that different 
concentrations of urea have low activity at low concentrations. The highest activity 
(0.0599U/ml) was recorded at 0.3 molar concentrations. This was followed by agradual 
decrese in activity with increase in concentration.EDTA produced the highest activity 
(0.0857U/ml) at 0.2 molar concentration. This was followed by a gradual decrease up to 
but including 0.4Mmol.  Another peak activity was observed at 0.5 molar concentrations.  
130 
 
 
 
 
 
0.2
0.18
0.16
0.14
0.12
0.1 UREA
0.08
EDTA
0.06
KCN
0.04
0.02
0
0.1 0.2 0.3 0.4 0.5
Molar Concentration (mol/dm3)
 
Figure 4.19a: Effect of different molar concentrations of inhibitors on β-galactosidase 
activity produced by L. plantarum (G11) 
0.18
0.16
0.14
0.12
0.1
UREA
0.08
0.06 EDTA
0.04 KCN
0.02
0
0.1 0.2 0.3 0.4 0.5
Molar concentration (mol/dm3)
 
Figure 4.19b: Effect of different molar concentrations of Inhibitors on β-
galactosidase activity produced by L. plantarum (E13). 
 
131 
 
Enzyme activity(U/ml) Enzyme activity(U/ml)
 
 
0.12
0.1
0.08
0.06 UREA
EDTA
KCN
0.04
0.02
0
0.1 0.2 0.3 0.4 0.5
Molar conccentration (mol/dm3)
 
Figure 4.19c: Effect of different molar concentrations of inhibitors on β-galactosidase 
activity produced L. plantarum subsp. plantarum (E36). 
 
 
 
 
 
 
 
 
132 
 
Enzyme activity(U/ml)
 
KCN gave a supportive activity at concentrations tested. However, the best concentration 
for β-galactosidase activity is 0.5Mmol with equivalent activity of 0.1113U/ml. 
Effect of different Substrates and Enzyme concentration  
The Michaelis constant (Km) of the purified enzyme was determined using different 
concentrations of the enzyme and the substrate. Values were obtained from double 
reciprocal plotting as described by Lineweaver and Burk (1934). The enzymatic kinetics 
of the LAB isolates was outlined in Figures 4.20 through. The Michaelis constant for L. 
plantarum (G11) β-galactosidase was determined. Vmax was found to be 349.8572nkat or 
0.35nkat and Kmax to be 2.887mg/100ml.Michaelis constant for L. plantarum (E13) β-
galactosidase was found to be 63.784nkat and Vmax of 1.73882mg/100ml. L. plantarum 
subsp plantarum E36 has Vmax of 40.03894µg/sec and Kmax of 2.57753mg/100ml (Figure 
4.20). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
133 
 
 
 
1/V 3
y = 0.928x + 0.346
R² = 0.999
2
1
0
-5 -4 -3 -2 -1 0 1 2 3
1/V
-1 Linear (1/V)
-2
-3
-4
 
Figure 4.20a: Lineweaver-Burk plot of the enzymatic activity of isolate G11 at 
different Substrate Concentrations 
In mM concentrations, the constant (0.003684uM, same as 3.684nKat) will be used to multiply 
absorbances equivalent to mg concentrations 
Vmax = 349.8572nKat 
Kmax = 2.887mg/ml 
 
134 
 
 
1/V y = 0.559x + 0.972
R² = 0.996
2.5
2
1.5
1
0.5
0
-4 -3 -2 -1 0 1 2 3
-0.5
-1
-1.5
 
 
Figure 4.20b: Lineweaver-Burk plot of the enzymatic activity of isolate E13 at 
different Substrate Concentrations 
 In mM concentrations, the c onstant (0.003684uM, sam e as 3.684nKat) will be used to multiply 
absorbances equivalent to mg concentrations 
Vmax is equal to 63.784nKat 
Kmax is 1.73882 mg/100ml   
  
 
 
135 
 
 
1/V
2.5
y = 0.445x + 1.147
R² = 0.937
2
1.5
1
0.5
0
-4 -3 -2 -1 0 1 2 3
-0.5
-1
 
Figure 4.20c: Lineweaver-Burk plot of the enzymatic activity of isolate E36 at 
different Substrate Concentrations 
In mM concentrations, the constant (0.003684uM, same as 3.684nKat) will be used to multiply  
absorbance equivalent to mg concentrations 
Vmax is equal to 40.03894nKat 
Kmax is = 2.57753 mg/100ml 
 
 
 
 
 
136 
 
 
Fractionation patterns of β-galactosidase produced by selected LAB isolates on 
Sephadex G-100 column Chromatography. 
Figures 4.21a shows varied activity peaks from the fractions collected. Figure 4.21b 
showed the two β-galactosidase activity peaks of Lactobacillus plantarum isolate G11. 
The first major activity peak recorded was from fractions 9-12 which revealed an activity 
equivalent to 0.0304nMgalactose unit/ml/sec and a corresponding protein peak of 
1.599mg .The second high peak recorded from fractions 24-27 yielded 0.0313nM 
galactose unit/ml/sec and 1.679mg protein respectively. Figure 4.21c shows the 
purification pattern of L.plantarum (E13) β-galactosidase. The two distinct peaks recorded 
were from fraction numbers 8-13 and 19-23 respectively. The first major peak revealed an 
activity of 0.0296nM galactose unit/ml/sec and a corresponding protein peak of 1.642 mg 
while the second activity peak revealed an activity equivalent of 0.0057nM galactose 
unit/ml/sec corresponding to 1.366mg of protein. Figure 4.21d shows the characteristic 
patterns of β-galactosidase peaks from isolate E36.Peak values from fractions: 12-14 and 
20-24 respectively were determined. First peak enzyme activity was 0.2802 nMgalactose 
unit/ml/sec corresponding to peak protein value of 1.576mg, while fraction points 23 to 27 
produced peak enzyme value of 0.2628nM galactose unit/ml/sec and corresponding 
protein value of 1.9503 mg protein. 
 
 
 
 
 
 
 
 
 
137 
 
 
 
 
0.6 1.2
A
B
0.5 1
0.4 0.8
0.3 0.6
0.2 0.4
0.1 0.2
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction
 
Figure 4.21a:  Separation of High molecular weight proteins of L. plantarum isolate G11 
using Sephadex G-100 and  the enzymic activity of the eluants/fractions towards ONPG 
 
 
 
138 
 
Enzyme Activity
 
3.5 1
Aa
0.9
3
0.8
2.5 0.7
Ab
0.6
2
0.5
1.5
0.4
1 0.3
0.2
0.5
0.1
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction
 
Figure 4.21b: Separation of High molecular weight proteins (Fractions 3-11) of L. 
plantarum isolate G11 using sephadex G-50 by ion-exhcnage and  the enzymic 
activity of the eluants/fractions towards ONPG 
 
 
 
 
 
 
 
 
 
 
139 
 
Enzyme Activity
 
0.5 A 1.2
0.45 B
1
0.4
0.35
0.8
0.3
0.25 0.6
0.2
0.4
0.15
0.1
0.2
0.05
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction
 
 
Figure 4.21c: Separation of proteins of L. plantarum isolate E13 using sephadex G-
100 by ion-exhcnage and the enzymic activity of the eluants/fractions towards ONPG 
 
 
 
 
 
 
 
 
 
 
140 
 
Enzyme Activity
Protein
 
0.5 1.2
Enzymatic activity Bb
0.45 Protein
Ba 1
0.4
0.35
0.8
0.3
0.25 0.6
0.2
0.4
0.15
0.1
0.2
0.05
0 0
1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930
Fraction No
 
Figure 4.22a: Separation of Low molecular weight proteins (Fractions 5-11) of L. 
plantarumisolate E13 using sephadex G-50 by ion exchange and the enzymic activity 
of the eluants/fractions towards ONPG.  
 
 
 
 
 
 
 
 
 
 
141 
 
Enzyme Activity (U/ml)
Protein content (mg/ml)
 
0.9 Bb Enzyme Activity 0.45
Protein
0.8 Ab 0.4
0.7 0.35
0.6 0.3
0.5 0.25
0.4 0.2
0.3 0.15
0.2 0.1
0.1 0.05
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction No
 
 
Figure 4.22b:Separation of High molecular weight proteins (Fractions 5-11) of L. 
plantarum isolate E13 by ion-exchange using sephadex G-50 and the enzymic activity 
of the eluants/fractions towards ONPG 
 
 
 
 
 
 
 
 
 
 
142 
 
Enzyme Activity (U/ml)
Protein content (mg/ml)
 
 
0.7 1
Bb
Protein
Ba 0.9
0.6
Enzymatic activity 0.8
0.5 0.7
0.6
0.4
0.5
0.3
0.4
0.2 0.3
0.2
0.1
0.1
0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fraction No  
Figure 4.22c:Separation of Low molecular weight proteins (Fractions 17-24) of L. 
plantarum isolate E13 by ion-exchange using sephadex G-50 and the enzymic activity 
of the eluants/fractions towards ONPG. 
 
 
 
 
 
 
 
 
 
 
 
143 
 
Enzyme Activity (U/ml)
Protein content (mg/ml)
 
Result of hydrolysis experiment 
Figure 4.23a shows the percentage lactose hydrolysis by β-galactosidase produced by 
L.plantarum (G11) at different lactose concentrations. It shows a gradual increase in both 
glucose and galactose hydrolysis with increase in lactose concentration up to 40%. The 
highest % hydrolysis (35.8) for glucose was attained at 80% galactose and 19.3 glucose 
respectively. A sharp decline in glucose hydrolysis at 80% lactose concentration while 
steady galactose hydrolysis was maintained eith increase in concentration.  
Figure 4.23b shows the hydrolytic effect of β-galactosidase produced by L.plantarum 
(E13) at different lactose concentrations. It shows a sharp increase in glucose hydrolysis at 
40%. The highest hydrolytic activity (34.6% hydrolysis) was attained at 60% lactose 
concentration. This was followed with gradual decrease as lactose concentrations 
increases. A similar trend in activity was observed with respect to galactose hydrolysis. 
The highest concentration (20.2% hydrolysis) was achieved at 60% lactose concentration 
which equally decreases with incease lactose concentration. 
Figure 4.23c profiles the hydrolytic effect of β-galactosidase produced byL.plantarum 
subsp. plantarum(E36) in which % hydrolysis ranged 13.5- 31.3%. The figure shows a 
gradual increase in glucose hydrolysis from 0-40% followed by a marginal decline before 
attaining maximum hydrolysis of 31.3% at 60% lactose concentration. Gradual decrease in 
glucose hydrolysis as lactose concentration increase. Similar pattern in hydrolytic was 
observed for galactose with maximum % hydrolysis (16.4%) observed at 60% lactose 
concentration. This was followed by gradual decrease in hydrolysis as lactose 
concentration increases. 
 
 
 
 
 
 
144 
 
 
35
30
25
20
gluc 
15
galac
10
5
0
0 20 40 60 80 100
% Lactose concentration 
 
Figure 4. 23a: Percentage Lactose Hydrolysis by Lactobacillus plantarum G11 at 
different Lactose concentrations. 
 
 
 
145 
 
% Hydrolysis
 
40
35
30
25
20
gluc
15
galac
10
5
0
0 20 40 60 80 100
% Lactose concentration
 
Figure 4.23b: Percentage Lactose Hydrolysis by Lactobacillus plantarum E13 at 
different Lactose concentrations 
 
 
 
 
 
 
 
 
146 
 
% Hydrolysis
 
35
30
25
20
gluc 
15
galac
10
5
0
0 20 40 60 80 100
% Lactose concentration 
 
 
 
Figure  4.23c: Percentage Lactose Hydrolysis by Lactobacillus plantarum  subspp 
plantarum E36 at different Lactose concentrations. 
 
 
 
 
 
 
 
 
 
 
 
147 
 
% Hydrolysis
 
 
 
CHAPTER FIVE 
5.0      DISCUSSION 
 
Thirty one bacterial isolates were obtained from milk and traditionally fermented milk 
products. Nineteen out of these tested Gram-positive, Catalase-negative, oxidase-negative 
and were either rod-shaped or cocci. They were, therefore, classified as lactic acid bacteria 
based on their morphological, metabolic and physiological criteria (Holzapfel, 1995;  
Axelsson, 1998; Car et al., 2002). Their cultural and biochemical properties agreed with 
the descriptions of Schlegel (1997) and conformed with Bergey‟s Manual of Systematic 
Bacteriology (Sneath, 1986). 
The most predominant LAB species isolated from milk and locally fermented dairy 
products were Lactobacillus lactis, L.bulgaricus, L.casei, L.fermentum, L. plantarum, L. 
delbrueckii, L. brevis and Leuconostoc mesenteroides. This agreed with the findings of 
Car et al. (2002). Lactic acid bacteria are indigenous to milk and milk products. They are 
hardly found in air or soil (Schlegel, 1997). Their main function in the fermentation of 
dairy products is to perform acidification of the product by fermentation of lactose into 
lactic acid (Solem et al., 2008). Lactic acid bacteria as a group play significant role in food 
fermentation processes because of their ability to produce high level of lactic acid as well 
as been able to survive under high acidic conditions (Wood, 1977). Their usefulness in 
dairy industry have been widely reported (Shah and Jelen, 1991; 1995; Somkuti et al., 
1998; Chammas et al. 1999; 2006,) and have been grouped as either homofermentative or 
heterofermentative (Shuler et al., 2003) based on the end products of their fermentation. 
These parameters are often employed in dairy industry because of the flavour-enhancing 
aldehyde and diacetyl they produce which are regarded as highly desirable aromatic 
substances. Various metabolic and enzymatic activities of LAB lead to the production of 
volatile substances which contribute to the flavor, aroma and in some cases, texture 
development in fermented dairy products (Sneath, 2002).  
Lactic acid bacteria constitute an important group of organisms with beneficial 
attributes that have been extensively exploited in the preservation and processing of foods 
such as milk, meat and vegetables. For these purposes, they have a widespread use for 
their enhancement of nutritional values of foods, retardation of spoilage and reduction of 
148 
 
 
contamination and lots of other beneficial effects in human health (Mckay and Baldwin, 
1990). Lactic acid bacteria which constitute a diverse group of lactococci, streptococci and 
lactobacilli have been studied intensively with respect to their enzymes for various reasons 
including their generally recognized as safe (GRAS) status (Stiles and Holzapfel,1997). 
Previous works on the characterization of enzyme status produced by lactic acid bacteria 
in the treatment of health- related problems used emerging. Greenberg and Mohoney 
(1982) had characterized β-galactosidase of yogurt mixed culture consisting L.delbrueckii 
subsp. Bulgaricusand found that they display high activity and stability at temperature 
0
above 50 C and had inferred that such conditions can enhance the rate of lactose 
hydrolysis.From literature, there is scarce information on previous works in respect of the 
characterization of  -galactosidase produced by any group of lactic acid bacteria that is 
indigenous to any of our locally fermented foods.  
Lactobacillus plantarum have been the most predominant lactic acid bacteria 
species implicated in the fermentation of food preparations of different plant origins 
(Daeschel et al., 1987). Lactobacillus plantarum requires less complex nutritional 
requirements compared to other Lactobacillus species (Hammes et al., 1992). 
Lactobacillus plantarum is a versatile lactic acid bacterium which is encountered in a 
range of environmental niches including dairy, meat and especially vegetable fermentation 
(DeVries et al., 2006). 
The different isolates of lactic acid bacteria obtained for this work is an indication 
of the diversity of the group that are indigenous to milk and locally fermented milk 
products. The PCR specific primers were able to identify the selected species of lactic acid 
bacteria. Blast search analysis using whole 16S rDNA sequence resulted in the 
identification of the species above 99% level which is close to the considered cut-off value 
indicating species identity (Stackebrandt and Goebel, 1994). Hence, the genetic method of 
characterization of the isolates correlated with their biochemical classification.  
More recently, thermophilic bacteria have become an object of interest for the 
commercial production of  -galactosidase (Petzel-bauer et al., 1999). Among these, 
special attention has been paid to lactic acid bacteria (LAB) because of their GRAS status 
(Stiles and Holzapfel, 1997). Their potential uses as a source of enzyme especially  -
galactosidase have been shown to be promising (Murad, 1998) 
149 
 
 
A number of bacteria have been considered as potential  -galactosidase sources such as 
L. lactis, L. acidophilus, L. bulgaricus (Bury and Jelen, 2000; Vasiljevic and Jelen, 2001; 
Guimondeetal 2002; Akolkar et al., 2005).  
Of the nineteen lactic acid bacteria screened for  -galactosidase production, 35% 
were identified as Lactobacillus plantarum, 15% L. brevis, 15% L. casei, 15% L. lactis, 
10% are Leuconostoc lactis, 5% as Streptococcus spp. And another 5% as Bacillus 
subtilis. Six of the Lactobacillus plantarum strains showed high production of  -
galactosidase under standard assay conditions and were selected for further studies.  
Most procedures for assaying  -galactosidase are chromogenic galactosidase. As 
Wallenfels and Weil (1972) summarized, o-nitrophenyl-B-D-galactopyranoside(ONPG) 
was described as a sensitive and conversant assay that have been widely accepted and used 
most extensively. 
However, 5-bromo-4-chloro-30indolyl-B-D-galactopra-noside (or X-Gal) was used 
as substrate for detecting  -galactosidase activity. It is generally used to screen for  -
galactosidase activity on solid mediums. Favier et al.,1996, first reported a method of 
detecting bacteria strains with β-galactosidase activity by use of X-gal.He observed that 
lactic acid bacteria colonies growing on X-gal medium shows green color and are regarded 
as bacteria with β-galactosidase enzyme (Favier et al. ,1996).Greenish color was accepted 
as positive  -galactosidase activity. In order to extract intracellular products it is required 
to select suitable methods to breakdown cellular structures.  
It is well known that  -galactosidase from lactic acid bacteria is an intracellular 
enzyme, and it is not released to the outside of cells under conventional fermentation 
conditions (Bury et al., 2001) Therefore, one should consider several factors during 
selection of suitable processes. In this study, Isoamyl alcohol and bead meals were used 
under appropriate laboratory conditionsfor cell disruption and  -galactosidase extraction. 
Almost all the cell-free extracts of Lactobacillus species show high  -galactosidase 
activity in which L. plantarum(G11) produced the highest initial activity of 7.889  /ml 
while the lowest activity (0.2057  /ml) was recorded by isolate F6.  
During the study of growth characteristics and  -galactosidase production, the 
different Lactobacillus plantarum strains G11, E13 and E36 were subjected to 
150 
 
 
fermentations. Obtained data   revealed by all the strains under study shows positive 
correlation between growth and  -galactosidase activity. It was further observed that 
maximum total  -galactosidase activity corresponds to the late stationary phase of 
growth for the three strains. This is in line with the findings of Rojaka et al.(2003),that 
there is a proportional relationship presenting growth-associated product formation 
kinetics. A decline in total enzyme activity was observed after the late stationary phase 
during the growth experiments. The decline in total enzyme activity could be considered 
to result from inhibition of cellular functions due to high pH, depletion of a nutritional 
factor from the growth medium, deactivation of the enzyme due to low pH catabolite 
repression or/and inducer exclusion. (Firat, 2004). Lactic acid bacteria maintain a 
cytoplasm that is more alkaline than the growth medium which becomes acidified during 
growth by secretion of lactic acid (Beshkova et al., 2002). Hence, any decrease in 
cytoplasmic pH below a certain level inhibits cellular functions and the intracellular 
enzymes can be deactivated (Kashket, 1987). In this study, specific growth rates for the 
-1 -1
respective isolates as determined from the growth curves were 0.170h , 0.083h  and 
-1
0.250h  respectively for L. plantarum  (G11), L. Plantarum (E13) and L. plantarum 
subsp. plantarum(E36) respectively. These figures were lower when compared to a study 
performed by El Demardash et al.(2006) using Streptococcus thermophilus. (EL 
Demarclash et al . 2006).  
Highest enzyme production was achieved at 24 hrs for strains G11, E13 and 30 hrs 
for E36 as seen in the figures. As stated earlier, positive correlation between growth and 
enzyme production was observed. The incubation time of 24hrs correspond to the 
stationary growth phase. This has been stated by many researchers which confirm our 
findings (Rojakat et al., 2003).  
In the comparison of the agitation speed on  - galactosidase enzyme production, 
definite differences were observed between the strains. For example Lactobacillus 
plantarum (strain G11) agitation at a speed of 60rpm promoted  - galactosidase 
production (0.184U/ml) more than with no agitation (0.004U/ml) (control). However, for 
L. plantarum strains E13, agitation speed of 120rpm supported maximum activity of 
0.184U/ml. While agitation speed of 100 rpm supported maximum activity of 0.186u/ml 
forL. plantarum strain E36. This observation is said to be closely related to the type of 
151 
 
 
strain and its tolerance to oxygen which is transferred by means of agitation (Beshkova et 
al., 2002). As it is very well known, most lactic acid bacteria are facultative anaerobes 
with preference for anaerobic conditions. They can, therefore, be differentiated on this 
basis indeed the effect of agitation cannot be only related to the effect of oxygen. It is also 
closely related to the nutrient mass transfer which is enhanced with agitation (Beshkova et 
al., 2002). This phenomenon has to do with the nutrient requirements of each strain and 
the more fastidious characters among them.  
During the optimization of the culture medium for  - galactosidase production, 
o o
the organisms were grown in a temperature range of between 20 C to 45 C. Lactobacillus 
o
plantarum (strain G11) produced maximum initial  - galactosidase (2.952U/ml) at 20 C 
followed by a significant decline in enzyme production with further increase in the 
incubation temperature (figures 4.7a, b,and c ). The same trend in the production of  - 
galactosidase by L. plantarum strain E13 and L. plantarum subsp. plantarum (strain E36) 
o
were observed with their maximum production (3.947 U/ml) at 25 C and (4.391 U/ml) at 
o
20 C respectively. These results are in contrast to the findings of Hsu et al. (2005) who 
observed that the activity of  - galactosidase by Bifidobacteria increased as the 
o o
cultivation temperature increased from 22 C to 37 C. Further increases in the cultivation 
temperature led to a reduction in the final viable population. It also differs from those of 
Friedurek and Szczodrak (1994) as well as Smith et al. (1985)-which demonstrated that 
0
the highest  - galactosidase production by B. longum was obtained at 37 C. The 
differences could result from the organisms under study being totally mesosphilic but 
partially psychrotolerants.  
The effect of initial pH on  - galactosidase production by the isolates under study 
were determined during the optimization studies. A pH range of 5.0 to 8.0 was used. It 
was found that the highest  - galactosidase production (9.379 u/ml) by L. plantarum 
(strain G11) was at pH 7.5, while pH 6.0 was best (7.799 U/ml) for L. plantarum (strain 
E13) and pH 7.0 for Lactobacillus plantarum subsp plantarum (strain E36) with an optimum 
of 9.549 U/ml .The effect of the initial pH of the medium on enzyme production by S. 
thermophilus was studied over a pH range of 4.0 to 9.0 by Ramana Rao and Dutta (1977) 
and they reported that maximum enzyme production was observed between pH 6.5 and 
152 
 
 
7.5. Sridhar and Dutta (1991) worked on the production of  - galactosidase from 
Streptococcus cremoris on whey. They reported that the optimum pH was between 6.5 and 
7.0. The result presented by Murad (1998) showed that the highest enzyme production by 
L. bulgarious was obtained at pH 5 while Hsu et al, (2005) reported the optimum initial 
pH for  - galactosidase production by Bifidobacterium sp to be 6.5. It is obvious that 
there is a great similarity in the effect of optimum initial pH (6.0-7.5) on the production of 
 - galactosidase by the strains under study and those reported previously by Ramana Rao 
and Dutta (1977), Sridhar and Dutta (1991) and Hsu et al. (2005).  
The effect of carbon source was determined on the production of  - galactosidase 
by the isolates in the cultivation medium. Ten sugars were employed in this experiment by 
using them singly or in combinations. For Lactobacillus plantarum strain G11, Raffinose 
supported the optimum production (5.088 U/ml) of  - galactosidase as a single sugar 
while mellibiose supported the highest  - galactosidase production for Lactobacillus 
plantarum strain E13 and Lactobacillus plantarum subsp plantarum strain E36. In a study 
carried out by Vasiljevic and Jelen (2001), the highest  - galactosidase activity was 
obtained when unsupplemented skin milk was used as cultivation media (Vasiljevic and 
Jelen, 2001).  
In a study carried out by Hickey et al. (1986), they observed a significant decline 
in  - galactosidase activity of several Lactobacillus bulgaricus strains upon addition of 
small amounts of glucose to the growing culture (Hikey et al., 1986). In another study 
carried out by Smart et al. (1993), they found that 19 out of 21 Lactobacillus strains 
contain very low and non-detectable  - galactosidase activity when cultivated on media 
containing glucose or glucose + lactose (Smart et al., 1993). Vasiljevic and Jelen explored 
this by partial repression of the lac operon caused by glucose presence in MRS. which 
resulted in lower  - galactosidase activity of the culture. The moderate amount of  - 
galactosidase activity obtained in this study was comparable to many studies cited in 
literature (Montanari et al., 2000; Gaudreauer et al., 2005) and are very promising for 
industrial applications.  
The effect of nitrogen sources on  - galactosidase production was also 
determined. It was found that (NH4)2 NO3 and KNO3 were best for all the Lactobacillus 
153 
 
 
plantarum under study. Potassium Nitrate supported the optimum  - galactosidase 
production (4.676u/ml) Lactobacillus plantarum strain G11 while Ammonium nitrated was 
best for (7.597U/ml) Lactobacillus plantarum strain E13 and Lactobacillus plantarum 
subsp plantarum (Srain E36). This agreed with what was reported by Murad (1998) the 
highest production of  -galactosidase by L. bulgarcius was obtained using (NH4)2HPO4. 
He further detected that no activity in the presence of (NH4)2SO4. This fact was confirmed 
by Selvin and EL- Divary (1985) and Fiedurek and Szczodrak (1994) who reported that 
some salts such as (NH4)2SO4 showed an inhibiting effect on  - galactosidase produced 
by K. fragilis. On the contrary, Rammana Rao and and Dutta (1997) found that the best 
nitrogen source for the production of  - galactosidase by S. thermophilus was peptone, 
followed by ammonium sulphate. Furthermore, Joker and Karbassi (2009) maximized  -
galactosidase production by L. delbruekii when grown in permeate-based medium 
enriched with a combination of yeast extract, whey powder and wheat steep liquor as 
organic nitrogen sources. 
Hydrogen ion concentration (pH) is known to influence the velocity of enzymes-catalyzed 
reactions. The effect of pH on β-galactrosidase activity produced by the three 
Lactobacillus plantarum strains under investigation showed that  optimum pH was 
attained between pH 6.0 and 7.0. β–galactosidase from the three Lactobacillus plantarum 
strains showed above 80% activity (an average) between the pH ranges. The enzyme was 
essentially stable (83-95% over the pH range tested from the data, it was revealed that 
isolate G11 had maximum residual activity of 94.8% at pH 7.5 while Lactobacillus 
plantarum strains E13 and E36 showed highest residual activity of 117.5% and 83.6% 
respectively at pH 7.0. These findings are in accordance with several earlier studies in 
literature. In a study performed by Greenberg and Mahoney (1982), optimum pH for β-
galactrosidase from S. thermophilus was found to be 7.0. The same findings were reported 
in another study by Kreft and Jelem (2000) 
The effect of temperature on activity and stability of β-galactosidase produced from the 
Lactobacillus plantarum strains under study was determined between the temperature 
0 0
range of 20 C to 75 C β-galactosidase from Lactobacillus plantarum strain G11 and E13 
0
were found to have optimum temperature of 20 C,Lactobacillus plantarum strain G11 and 
Lactobacillus plantarum strain E13 retained over 50% of their activity between the 
154 
 
 
temperature range of 40-600C while Lactobacillus plantarum subs.plantarum (strain E36) 
0
showed optimum activity only at temperature extremes and at 35 C. This finding is in 
contrast to the findings of Shah and Jelen (1991) in which they observed optimum 
temperature of β-galactosidase from L.delbruekii subsp bulgaricus 11842 to be between 
0
45-50 C. Thermal stability is the ability of an enzyme to resist thermal unfolding in the 
absence of substrates (Bhatti et al., 2006). The inactivation of enzyme during heating 
occurs mostly during the initial stages of mild heating (Yemenicioghi et al., 1999; 
Rodriguez-Lopez et al., 1999; Yemencoglu et al., 2002) and is generally attributed to 
conformational changes in the enzymes active site  (Rodriguez-Lopez et al., 1999). 
 
   The effect of both monovalent and divalent metal ions on the activity of β-galactosidase 
was determined. The cations tested are Monovalent chloride salts of Potassium, Calcium, 
2+ 2+ 2+ 2+
Sodium Ammonium and Sulphate salts of Mg , Mn , Cu  and Zn . The anions tested 
are CuS04, NaN03, KN03 and MnS04. The monovalent chloride salts increased β-
galactrosidase activity with increase in  concentrations up to 0.4mmol for all the three 
Lactobacillus plantarum strains. Reduced activity was observed when the molar 
concentration was increased above 0.4mmol. Ammonium and Potassium salts have the 
best activity on β-galactosidase. Among the anions, ZnS04 produced the best optima for 
Lactobacillus plantarum (strain G11) for all concentrations, CuS04 had optimum activity 
(0.1903U/ml) at 0.03mmol while KN03 as best at 0.1mmol. In the case of lactobacillus 
plantarum (strain E13), CuS04 at 0.2mmol was best (0.2032h/ml), KN03 was best at 0.1mm 
(0.1033µ/ml) and CuS04 had optimum activity at 0.4mmol (0.1559µ/ml). This result that 
the addition of monovalent cations in appropriate concentration, show positive effects on 
2+ 2+
β-galactosidase activity. The divalent ions, Mg  and Mn  showed strong activation of β-
galactosidase activity.  
Our observation of the effects of the various cations are in agreement with the reports on 
the requirements for movalent and divalent metal ions for optimal activity and stability for 
a number of different β-galactosidases (Nakayima and Amachi, 1999; Nguyen et al., 2006; 
2+
Nguyen et al., 2007a). Ca  is a known inlibitor of some β-galactosidase (German et al., 
2+ 2+ 2+
1996). Igbal et al.,(2011) observed that 10mmol of Cu , Zn  and Fe  inactivated β-
2+
galactosidase activity  by 100%, 94% and 66% respectively. Cu  was reported to have 
strong inhibitory effect on β-galactosidase activity even at low concentrations. In fact the 
155 
 
 
effects of these ions were observed to be concentration- dependent. In various studies 
reported in literature, different results were obtained depending on the ion used and the 
source of β-galactosidase. The effect of monovalent cations (K+ and Na+) varies with the 
enzymes from different sources (Jurado et al., 2002). For the enzymes from Streptococcus 
thermophilus and with ONPG as substrate, no effect was found in one case (Somkuti and 
Steinberg, 1979). However, slight activation was observed in another (Rao and Dutta, 
2+
1981). Similarly, presence or absence of Mg  had been shown to display different effects. 
In a study by Bhowmik et al. (1987), β-galactosidase from L .acidophilus was stimulated 
2+
with Mg  whereas in another study  by Itoh et al. (1992) it had no  effect in the case of 
L.kefironofaciens (Itoh  et al., 1992).  
The results of the purification of β-galactosidase from Lactobacillus plantarum strain G11, 
Lactobacillus plantarum E13, and Lactobacillus plantarum subsp plantarum showed that 
the elution profile display two β-galactosidase activity peaks with the first peak 
representing high molecular weight filtrates and the second peak representing low 
molecular weight proteins. Enzymes purity and efficiency were estimated by 
polyacrylamide gel electrophorensis under non-denaturing conditions. The specific 
activity of β-galactosidase obtained from the Lactobacillus plantarum strains are 
292.5mol/mg, for strain G11, 104.21mol/mg for strain E13 and 585.46 mol/mg for strain E36 
respectively. This result indicated that β-galactosidase from Lactobacillu splantarum 
strains has high affinity for lactose. In this study, Kmax and Vmax values were calculated. 
Vmax represents the rate at which substrates are converted to products while Kmax (or 
Michealis constant) is the concentration that will make the enzyme act at its maximum 
velocity. Hence the Vmax for L.plantarum strain G11 was 349.857 nkat (08 0.35Ukat) while 
the Kmax was 2.89mg/100ml. and Kmax of 1.739mg/100ml. for L. plantarum (E13). L. 
plantarum subsp plantarum strain E36 has it Vmax as 40.039 nkat and Kmax of 
2.578mg/100ml-respectively. 
Shegel (1975) summarized the importance of Kmax and Vmax as numerically equivalent 
to the substrate concentration that yields half-maximal velocity. Therefore, the numerical 
value of Kmax is of interest for many reasons but most importantly, since Kmax is a constant 
for a given enzyme, its numerical values provides a means of comparative enzymes from 
different organisms or from different tissues or the same organisms. In this way it can be 
156 
 
 
determined whether enzymes A is identical to enzyme B, or whether there are different 
proteins that catalyze the same reaction. 
 The application of the purified β-galactosidase enzyme on the different 
concentrations of Lactose shows a maximum percentage hydrolysis of 35.8% glucose and 
19.35% galactose was achieved when 2.5ml of the enzyme was applied to 10ml of 80% 
lactose concentration. Furthermore a 35.35 glucose and 18%   galactose was achieved 
when the same amount of β-galactosidase enzyme was introduced to the same volume of 
60% lactose concentration. 
 Much research is focused upon microorganisms that produce beta-galactosidases 
with improved quality for production of galacto-oligosachridases. Hsu et al. (2007) have 
studied the production of GOs by transgalactosylation using β-galactosidase of β-longum 
BCRC 15708. Two types of GOs, tri-and tetrasaccharides, were formed after β-
galastosidase action on 40% lactose. Trisaccharides were the major type of GOs formed in 
general, an increase of the initial lactose concentration in the reaction mixture resulted in a 
higher GOs production A higher yield of 32.5% (w/w) GOs could be achieved from 40% 
0
lactose solution at 45 C. 
 In 2008, synthesis of GOs from 36% lactose using a recombinant beta-
galactrosidase of β infants in Pichia pestoris was investigated. The trangalactosylation 
ratio reached up to 25.2% with 83.1% conversion of initial lactose and 8.76% 
monosaccharides. 
 Igbal et al. (2010) have observed that a recombinant beta-galactrosidase from 
Lactobacillumsplantarum has a high transgalactosylation activity and was used for the 
synthesis of prebiotic Gos. The maximum GOs yield was 41% (w/w) of total sugars at 
85% lactose conversion. 
Conclusion β-gal‟s capability has been realized by the plethora of products like biosensors, 
digestive supplements. Etc. thus research and development of beta-galastosidase founds 
applications in several industries.             
 
 
 
 
 
157 
 
 
 
 
CONCLUSION AND RECOMMENDATION 
In this study, L. plantarum strains were successfully isolated and identified from raw milk 
and fermented milk products. The LAB isolates successfully produced β-galactosidase 
enzyme which was able to hydrolyse lactose sugar to simple sugars (glucose and 
galactose) in known concentration samples of milk.Percentage lactose hydrolysis which 
ranged between 31.3%- 35.8%  was achieved .This compared favourably with results  by 
other species of Lactobacillus plantarum from other regions of Asia. Therefore β-
galactosidase from these strains can be subjected to further screening and future 
development and possibly deployed in the hydrolysis of milk and milk products for 
possible.utilization by lactose- intolerant people  
β-galactosidase enzyme is a very important product to dairy industry and very costly to 
acquired. It is therefore recommended   that more efforts be made in embarking on further 
search and screening for better β-galactosidase- producing species. Other β-galactosidase 
producing microorganisms should also be identified for deployment and application in our 
home grown dairy industries. This will, however, require financial support either 
governmental or by private organizations interms of sponsorship, laboratory equipments 
and chemical reagents. 
 
 
 
 
 
 
 
 
 
158 
 
 
 
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APPENDIX I 
 Table 4.1: Percentage of Occurrence 
Probable Organisms Number of Occurrence  Percentage of Occurrence 
Lactobacillus plantarum 7.0 35.0 
Lactobacillus  casei 3.0 15.0 
Lactobacillus brevis 4.0 20.0 
Lactococcus lactis 2.0 10.0 
Leuconostoc lactis 3.0 15.0 
Streptococcus spp 1.0 5.0 
Total 20.0 100.0 
 
 
Table 4.10: Growth of G11 in MMRS broth at different pH 
pH Absorbance OD600 Protein 
 f g  f
5.0 2.6422  1.061  1.096  
 d f  e
5.5 2.9053  1.183  1.128  
 e e  d
6.0 2.7792  1.675  1.153  
 g c  c
6.5 2.5955  1.780  1.553  
 b b  a
7.0 4.2653  2.909  1.935  
 a a  b
7.5 9.3790  3.662  1.902  
 c d  g
8.0 4.2099  1.694  0.329  
 
 
Table 4.11: Growth of E13 in MMRS broth at different pH 
 
 pH Absorbance OD600 Protein 
5.0 ±  g  g  g2.7572 0.5  1.541±0.2  0.902±0.2  
 5.5  f  f  e3.4523±0.5  1.546±0.2  0.988±0.2  
6.0  a  a  c7.7986±1.0  2.943±0.5  1.410±0.5  
 
6.5 ±1.0 b ±0.5 b ±0.5 a7.1638  2.923  1.818  
7.0 
 4.8051±0.5
 c ±0.5 c  b 2.774  1.599±0.7  
7.5  d  d  d4.8013±0.5  1.843±0.3  1.311±0.7  
8.0  e  e  f 4.1525±0.5  1.633±0.3  0.932±0.2  
187 
 
 
Table 4.12: Growth of E36 in MMRS broth at different pH 
pH Absorbance OD600 Protein 
5.0  e  f  c2.6212±1.0  1.310±0.2  1.420±0.5  
5.5  c3.8533±1.0  1.990±0.5 b  b 1.804v0.5  
6.0  d  d  e3.0793±1.0  1.663±0.5  1.081±0.5  
6.5 ±1.0 f  e  f2.5262  1.497±0.5  0.909±0.2  
7.0 ±2.0 a  a  a9.5492  4.214±1.0  3.578±1.0  
7.5 ±  b  c  d4.7949 2.0  1.887±0.2  1.223±0.5  
8.0 2.3529±1.0g  g  g 1.181±0.2  0.686±0.2  
 
 
Table4.7:    Enzyme Activity      Isolate G11 
Time (hrs) Absorbance  OD600 
h  h
0 0  0  
6 2.434±1.0 f 0.664±0.2 e 
12  c  d3.469±1.0  1.401±0.5  
18 4.495±2.0 a  c 1.626±0.5  
24  d  b3.138±1.0  1.772±0.5  
30  b  a4.357±2.0  1.821±0.5  
36 2.888±1.0 e  f 0.654±0.2  
42 ±0.5 g  g1.685  0.554±0.2  
Growth Analysis of Isolate G11 in MMRS incubated at 37°C. The following parameters were 
evaluated; OD600, total enzyme activity.    
 
                                                               Table 4.8:    Enzyme Activity   Isolate E13 
Time (hrs) Absorbance  OD600 
 h  h
0 0  0  
6  d3.954±0.5  1.026±0.2 e 
12  e  d3.442±0.5  1.111±0.2  
18  c  b4.008±1.0  1.643±0.5  
24  b  a4.942±1.0  1.889±0.5  
30  a  c4.953±1.0  1.579±0.5  
36  f  f3.253±0.5  0.934±0.2  
42  g  g1.964±0.2  0.912±0.2  
Growth Analysis of Isolate E13 in MMRS incubated at 37°C. The following parameters were 
evaluated; OD600, total enzyme activity.    
                      
 
188 
 
 
                                         Table 4.9:    Enzyme Activity    Isolate E36 
Time (hrs) Absorbance  OD600 
 h  h
0 0  0  
6 1.769±0.5 f  d 1.325±0.3  
12  g  f1.418±0.5  0.872±0.3  
18 ±1.0 c ±0.1 c5.308  1.889  
24 ±1.0 b ±0.5 b5.512  1.931  
30 ±0.5 a6.223  1.995±0.5 a 
36  d  e2.986±0.5  0.989±0.3  
42  e2.803±0.5  0.380±0.1 g 
Growth Analysis of Isolate E36 in MMRS was read at 37°C. The following parameters were 
evaluated; OD600, total enzyme activity.    
SUGAR UTILISATION 
Table 4.13  Raffinose utilization by G11 
Concentration Absorbance OD600 Protein 
0.5  e  a  d3.2628±0.5  0.813±0.2  0.612±0.1  
1.0 0.5344±0.1 f  b  a 0.570±0.2  0.863±0.2  
1.5  b  c  c7.2118±2.0  0.487±0.1  0.650±0.2  
2.0  c  d5.2671±1.0  0.426±0.1  0.764±0.2 b 
2.5  d4.2513±1.0  0.360±0.1 e ±0.1 d 0.612  
3.0  a8.1530±2.0  0.327±0.2 f 0.541±0.2 f 
 
Table 4.14  Maltose + Galactose Utilization by G11 
Concentration Absorbance OD600 Protein 
0.5 5.0863±1.0 c  c  e 0.565±0.1  0.567±0.1  
1.0  e4.0862±1.0  0.774±0.2 a  a 0.770±0.1  
1.5  d4.9882±1.0  0.623±0.2 b  b 0.716±0.2  
2.0 ±2.0 b ±0.1 d  c5.8198  0.487  0.695±0.2  
2.5 3.5846±0.5 f  e  d 0.264±0.1  0.637±0.1  
3.0  a  f  f7.7528±2.0  0.209±0.05  0.496±0.1  
 
 
 
 
 
189 
 
 
Table 4.15  Mellibiose Utilization by E13 
Concentration Absorbance OD600 Protein 
0.5  a  f  d6.4094±1.0  0.248±0.05  0.664±0.1  
1.0 ±1.0 b ±0.05 e ±0.1 e5.7228  0.289  0.652  
1.5 ±0.5 f d  c3.3327  0.428±0.1  0.747±0.2  
2.0 4.8007±0.5d b  a 0.485±0.2  0.929±0.1  
2.5 ±0.5e ±0.2 a ±0.2 b4.1898  0.520  0.824  
3.0  4.9300 ±1.0c ±0.1 c  f 0.443  0.590±0.1  
 
Table 4.16  Maltose + Galactose Utilization by E13  
Concentration Absorbance OD600 Protein 
0.5  c  c  d4.8692±0.5  1.009±0.5  0.810±0.2  
1.0  b6.2422±0.5  1.334±0.5 b  a 2.806±0.5  
1.5  a  a  b6.6428±1.0  1.890±.0.5  1.162±0.5  
2.0  d  d  f4.4923±1.0  0.312±0.05  0.109±0.05  
2.5  e  e  c2.4667±0.2  0.278±0.05  0.824±0.2  
3.0 0.7923±0.2 f  e  e 0.277±0.05  0.757±0.2  
 
Table 4.17  Mellibiose Utilization by E36 
Concentration Absorbance OD600 Protein 
0.5 ±0.5 a ±0.1 f  a7.3304  0.499  0.938±0.2  
1.0 6.1876±0.5 d  e  c 0.854±0.2  0.866±0.2  
1.5  b  d  d7.0874±0.5  0.883±0.2  0.788±0.1  
2.0 ±0.1 e  c  f4.4650  1.457±0.5  0.542±0.1  
2.5  f  3.3185±0.1  1.496 ±0.5b  ±0.1e 0.677  
3.0  c  6.4490±0.5  1.749 ±0.5a  b 0.877±0.2  
 
Table 4.18  Trehalose + Galactose Utilization by E36  
Concentration Absorbance OD600 Protein 
0.5  a  f  a7.5984±2.0  0.595±0.1  0.798±0.2  
1.0  c5.8598±2.0  0.865±0.2 e  c 0.445±0.1  
1.5 5.1163±1.0 d  d  b 0.921±0.1  0.489±0.2  
2.0  f  c  d4.7607±1.0  0.942±0.2  0.374±0.1  
2.5  e  b  e5.0930 ±1.0  0.961±0.2  0.365±0.1  
3.0  ±2.0b  a6.1552  1.061±0.5  0.314±0.1 f 
 
                                     
 
 
190 
 
 
Table 1: Effects of varying Substrate Concentrations on β -D-Galactosidase Activity. 
-1
Substrate Enzyme Activity (U ml ) 
Concentration (%) 
G11 E13 E36 
0.5 d b f0.4534±0.1  0.4796±0.2  0.5014±0.1  
1.0 b c e0.7924±0.1  0.4163±0.2  0.5904±0.1  
1.5 1.0262±0.2a 0.5452±0.1a d 0.6741±0.2  
2.0 f e c0.0522±0.01  0.2713±0.1  0.7308±0.1  
2.5 e f b0.4126±0.1  0.1995±0.05  0.8112±0.2  
3.0 c d a0.7724±0.2  0.3475±0.1  0.9835±0.2  
 
                               
Table 2: Effect of Varying Enzyme Concentration on β -D-Galactosidase Activity. 
-1
Enzyme Enzyme Activity (U ml ) 
Concentration (%) 
G11 E13 E36 
0.5 c e e0.0327±0.01  0.02464±0.01  0.04321±0.01  
1.0 e c f0.01995±0.01  0.03123±0.01  0.03382±0.01  
1.5 0.04604±0.02b d d 0.02794±0.01  0.05573±0.01  
2.0 0.02453±0.01d 0.0545±0.02a a 0.09385±0.03  
2.5 a b0.06764±0.02  0.03751±0.02  0.07009±0.02b 
3.0 0.02458±0.01d e 0.02492±0.01  0.06128±0.02c 
 
                                           
Table 3: Effect of Thermal Stability on β-D-Galactosidase Activity 
O -1
Temperature ( C) Enzyme Activity (U ml ) 
 G11 E13 E36 
20 c b b0.2839±0.1  0.3188±0.1  0.3473±0.1  
25 0.6641±0.2a i0.0496±0.02  0.0197±0.01g 
30 f h0.1579±0.05  0.0641±0.02  0.0012±0.001h 
35 0.0194±0.05j f0.0938±0.05  0.0777±0.01d 
40 d0.1697±0.05  0.0818±0.05g g0.0078±0.001  
45 g b c0.1428±0.05  0.3200±0.1  0.1326±0.01  
50 e d g0.1606±0.05  0.1177±0.02  0.0066±0.001  
55 ±0.05i e h0.0859  0.1006±0.02  0.0018±0.001  
60 d c e0.1730±0.05  0.1805±0.02  0.0644±0.01  
65 h0.1025±0.02  0.0839±0.02g f0.0499±0.01  
70 g e d0.1498±0.02  0.1022±0.02  0.0731±0.01  
75 0.4611±0.1b 0.4811±0.1a a0.4304±0.1  
191 
 
 
 
                                               Table 5: Effect of Agitation on β-D-Galactosidase Activity. 
-1
Agitation  Enzyme Activity (U ml ) 
Speed (rpm) 
 G11 E13 E36 
0 f f e0.00624±0.001  0.00476±0.001  0.0055±0.001  
60 0.18419±0.05a de a 0.15433±0.02  0.18227±0.005  
80 0.13919±0.05e e c 0.15061±0.02  0.15455±0.02  
100 ±0.05c ±0.02c a0.16512  0.16994  0.18604±0.05  
120 b a b0.17509±0.02  0.18191±0.05  0.17786±0.02  
140 0.16499±0.02c ab 0.17668±0.05  0.17369±0.02b 
160 d b c0.14025±0.01  0.17273±0.05  0.15303±0.02  
180 c b d0.16009±0.05  0.1744±0.05  0.09167±0.01  
200 ±0.05c ±0.02d ±0.05bc0.16841  0.15755  0.15889  
 
 
 
 
                                                Table 6: Effect of Temperature on β-D-Galactosidase Activity. 
O -1
Temperature ( C) Enzyme Activity (U ml ) 
 G11 E13 E36 
15 f0.00135±0.001  0.04711±0.01f 0.06658±0.01f 
20 b c e0.10843±0.01  0.12851±0.05  0.09661±0.01  
25 c d d0.07578±0.01  0.11564±0.05  0.11099±0.05  
28 d c c0.06656±0.02  0.12272±0.05  0.12812±0.05  
30 0.10168±0.02b e a0.06649±0.02  0.16806±0.05  
35 a ab g0.12274±0.02  0.14587±0.05  0.00511±0.001  
40 a a d0.12528±0.02  0.15356±0.05  0.11377±0.05  
45 e b b0.00332±0.001  0.14017±0.05  0.13276±0.05  
50 0.00135±0.001f 0.00421±0.001g h0.00396±0.001  
 
  
192 
 
 
Table 7:  Effect of pH on β-D-Galactosidase Activity 
-1
pH Enzyme Activity (U ml ) 
 G11 E13 E36 
5.0 0.1349±0.05f 0.1617±0.02 c  c 0.1658±0.05  
5.5 g  f  g0.1016±0.05  0.0908±0.02  0.0848±0.05  
5.8 0.1178±0.05g  d  d 0.1524±0.02  0.1574±0.07  
6.0 b0.1825±0.02  0.1926±0.05b ±0.07de 0.1491  
6.5 e  e  d0.1453±0.02  0.1482±0.05  0.1551±0.07  
6.8 d  d  e0.1509±0.05  0.1527±0.05  0.1438±0.05  
7.0 d a0.1520±0.5  0.2264±0.05  0.1854±0.05 b 
7.3 0.0815±0.02h  d  c 0.1518±0.05  0.1624±0.05  
7.5 c0.1730±0.05  0.1486±0.02 e ±0.05 f 0.1389  
7.8 e a  c0.1423±0.05  0.2187±0.02  0.1608±0.02  
8.0 0.2051±0.05a ±0.05 c  a 0.1695  0.1903±0.02  
 
 
 
 
  
193 
 
 
APPENDIX II 
Composition of MRS both used for the Cultivation of LAB  
Peptone from Casein    10g  
Meat extract    8g 
Yeast extract     4g  
K2HP04               2g 
D(+) glucose     1g 
Tween ® 80     5.0g 
Sodium acetate      5.0g 
di-Ammonium citrate   2.0g  
MgSO4 7 H20    0.2g 
Mn S04 4H20    0.04g 
Water      1000ml 
pH was measured before autoclaving  
 
Modified MRs broth  
All components are same with MRS broth except that 2% lactose was replaced with 2% 
glucose.  
MRS Agar: All components are same with MRS broth except that 15g agar per Liter of 
de-mineralized water was added to MRS broth solution. It was boiled and autoclaved  
Preparation of o-nitrophenol--D-galactopyranoside (ONPG) Solution        
ONPG solutions were prepared by the addtion of solid ONPG to appropriate buffer 
solution (0.05M sodium phosphate solution) under the assay conditions. ONPG has low 
solubility, therefore, it should be properly mixed. The solution should be freshly prepared 
and used.  
Molecular weight of ONPG= 301.26g/L. 
  
194 
 
 
Sodium Carbonate 
0.05m is obtained by dissolving 5.3g in 1000ml deionised water.  
MNa2C03 = 106g/L. 
 
1
y = 0.024x + 0.611
R² = 0.983
0.8
0.6
Series1
Linear (Series1)
0.4
Linear (Series1)
Linear (Series1)
0.2
0
-35 -30 -25 -20 -15 -10 -5 0 5 10 15
ONPG STANDARD CURVE 
-0.2
195 
 
 
LIST OF CHEMICALS 
 
Chemicals  Supplier  
Tween 80 BDH 
5-bromo-4 chloro-3-Indolyl--D- Sigma  
galactopyranoside (x-gal). Sigma  
o-nitrophenyl--D-galactopyranoside Sigma  
(ONPG)  Sigma 
Isoamyl alcohol  Sigma 
Sodium chloride   
Magnesium sulfate   
Ammonium sulfate  
Potassium Nitrate  
di-sodium Hydrogen sulphate  
Sodium di-hydrogen sulphate   
Manganese sulfate   
Potassium di-hydrogen sulfate  
Sodium carbonate   
Ammonium chloride   
Ammonium nitrate   
MRS agar  
MRS broth  Lab M Limited 
Yeast extract Lab M Limited 
Meat extract Lab M Limited 
Peptone from casein Lab M Limited 
Lactose  Lab M Limited 
Lab M Limited 
 
 
3.5.4  
Production of ammonia from peptone 
Some bacteria are able to produce deaminase enzymes which enable them to break down peptone 
or other proteins to release ammonia. This is termed deamination. 
Deaminase medium 
 
 
 
196 
 
 
Peptone water   APPENDIX     III      
     
Peptone                       1.0g 
Sodium chloride          0.1% 
Fermentable sugar       1.0% 
Distilled water             100ml 
Nitrate medium 
Composition and preparation 
1. Peptone water plus  0.1% potassium nitrate 
2. Distribute the medium into tubes, each  with an inverted Durham tube, and sterilize at 
0
121 C for 15mins and allowed to cool before inocubation with the isolates. Uninoculated 
tudes served as control. 
0
Tubes were incubated at 30 C for 4days. The ability of the isolates to reduce nitrate to 
nitrite,ammonia or free nitrogen was determined by adding  to each tube 0.5ml of 0.6% 
dimethyl-naphthy lamine in 5.0ml acetic acid. The development of a red coloration indicated a 
positive result and the presence of gas in the Durham tube indicated the production of nitrogen 
gas. 
Com position                                                 growth in  4% NaCl 
                                                        MRS broth containing 4% Sodium Chloride is used  
Arginine broth 
For the Identification of some Streptococci (enterococci) and gram negative rods. 
Composition 
Tryptone                    0.5g 
Yeast extract              0.2g 
K2PO4                         0.2g 
L arginine monohydrate 0.3g 
Dextrose                         50mg 
Distilled water                 200ml 
 Preparation:  1.  Suspend the component in the water and heat to dissolve totally. 
 02. Sterilize at 121 C for 15mins 
197 
 
 
Yeast  Extract Peptone Agar 
Composition 
1. Yeast extract      20g 
2. Peptone              5g 
3. Agar                   20g 
4. Distilled water   500ml 
Nutrient gelatin (broth) 
Composition  
1. Nutrient broth containing 10-15% gelatin  
 02. This is sterilized at 121 C for 15mins 
Nutrient broth 
Peptone from meat        5.0g 
Meat extracts                   3.0g 
0
Suspend 8g in 1litre of deminiralized water, dispense into small containers, autoclave at 121 C for 
15mins. 
0
pH 7 0±0.2 at 25 C 
0.1% peptone water :  
Dissolve 1g of peptone in 100ml of distilled water 
0
Autoclave at 121 C for 15mins 
Casein hydrolysis medium  
For detecting casein-hydrolysis organisms 
Composition 
Skim milk        1part 
Nutrient broth (pH  7.4)  1part  
Agar              1% 
Dialyse the skim milk solution to rid it of lactose 
Add the component together and steam to dissolve 
0
Autoclave at 121 C for 15mins 
198