CHARACTERIZATION OF ®²-GALACTOSIDASE BY LACTIC ACID...

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  • 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

  • ii

    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.

  • iii

    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

  • iv

    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.

  • v

    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.

  • vi

    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.

  • vii

    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

    support up till your last day 2 nd

    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

  • viii

    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 effec