QUALITY AND SHELF LIFE EVALUATION OF BEEF USING

Gongronema latifolium AND Ocimum basilicum EXTRACTS

By

Anthony Pius BASSEY

B. AGRIC. (ANIMAL SCIENCE) CALABAR
Matric. No: 203194

A PROJECT SUBMITTED TO THE

ANIMAL PRODUCTS AND PROCESSING UNIT
DEPARTMENT OF ANIMAL SCIENCE
FACULTY OF AGRICULTURE AND FORESTRY
UNIVERSITY OF IBADAN, IBADAN
NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

MASTER OF SCIENCE ([Link].) DEGREE IN ANIMAL SCIENCE,
UNIVERSITY OF IBADAN, IBADAN
NIGERIA

SEPTEMBER, 2018

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 CERTIFICATION

I certify that this research study was carried out by BASSEY, ANTHONY PIUS with
Matriculation Number 203194 in the Animal Products and Processing Unit, Department of Animal
Science, Faculty of Agriculture and Forestry, University of Ibadan, Ibadan, Nigeria under my
supervision.

…………………………… ……………………………
Dr Olubunmi O. Olusola DATE
B. Sc., M. Sc., Ph.D. (Ibadan), RAS
Senior Lecturer, Animal Products
Department of Animal Science
University of Ibadan
Ibadan, Nigeria.

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 DEDICATION

I dedicate this research work to Almighty God, for by His grace alone was I able to bear the load
to the end. Also to my parents, Sir/Lady Pius Etong Bassey (KSJ, JP), my sister and best friend,
Ms. Christiana Etong and to my world yet unborn.

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 ACKNOWLEDGEMENTS

My profound gratitude and obeisance go to Almighty God for making it possible to carry out this
research work amidst the twists and turns. By His grace alone were the imperfections perfected.

My heartfelt thanks and appreciation go to my amiable and illustrious supervisor, Dr Olubunmi O.

Olusola who, like a mother, contributed immensely through the course of this project. Despite her
crowded and busy schedule, she gave me the required audience needed to successfully complete
this study from constructive criticisms to proper guidance. I am elatedly humbled to be under your
tutelage Ma. For this altruism, I pray God gives you and your family the enablement to continue
to grow and soar. Amen. Also, without mincing words, my special appreciation goes to a father
figure, an astute scholar, a savvy Meat Scientist, a detailed personality and the Head of Animal
Products and Processing Unit, Prof. A. B. Omojola for his drive, positive impact, advices and
motivational speeches in and outside the lecture room. May the Almighty God bless you and your
family Sir. Amen. More so, words cannot describe the painstaking efforts of a simple, God-fearing,
altruistic, humble laboratory technologist, Mrs. Udoh. I know the adjectives to describe her person
will not fail me, but all I can say in synopsis is that, she is a caring mother and a good woman. I
pray God blesses her and her family for her daily sacrifice throughout the course of my laboratory
work. Amen. My appreciation also goes to one aunty and a PhD student I know, Mrs. Ayobami
Adeshola. I just keep smiling as I am putting this down because knowing her has not only been a
privilege but also an honour. She knows she is a good person and God will bless her accordingly
for her efforts.

To the erstwhile Head of Department, Prof Babayemi, I appreciate his efforts to lecture, guide and
counsel us, his students. To the incumbent Head of Department, Prof A. E. Salako whom I describe
as a teacher; for his passion to always breaking the most complex of “things” into their simplest
forms. As the Lord puts the heaviest loads on the strongest backs, I pray He will stand him tall
above his ilks and give him the courage to paddle this great responsibility to greater heights. Amen.
I also wish to appreciate my lecturers; Dr E. O. Ewuola, Dr. Osaiyiwu Henry, Dr O. A. Adebiyi,
Dr B. R. O. Omidiwura, Dr O. A. Ogunwole, Dr O. Odu, Dr Shokumbi and the amiable
Postgraduate Coordinator, Dr. O. Adeleye. My First Degree lecturers; Prof Isika (of blessed
memory), Prof Ausaji Ayuk, Prof Bassey Okon, Dr. Magnus Anya, Dr. Lawrence Ibom, Dr
Kennedy Oko, and referees; Dr Affiong Henry and Dr Okokon Effiong of Animal Science

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Department, University of Calabar, for the lecture sessions, advices and counseling to making me
a responsible person.

My special thanks, unfeigned loyalty and undying respect go to my parents, Sir and Lady Pius
Etong Bassey (KSJ, JP) for their sacrifice, self-denial, financial support, prayers, dedication,
inspiration, acumen and love. They taught me to always take the crookeds with the straights for in
that will my stars align. I love you both (Papa and Mama), for instilling in me the qualities to stay
positive amidst the challenges and turmoil throughout this programme. I will always remain
grateful and I pray the good Lord keeps you both for us (your family) and my unborn kids too.
Amen. A big kudos to my siblings; Mrs. Glory Matthew, Hon Bassey Pius Bassey, Mrs. Gladys
Esienita Bassey, Mr. Gregory Emeh Etong, Mrs. Rose Udeme Ekott, Ms Christiana Pius Etong
(my bestie), Mr. Patrick Etong Etong and Mr. Paul Pius Bassey. Also, my inlaws; Mr. Esienita
Bassey, Barr Udeme Ekott, Mrs. Item Bassey, Mrs Nora Etong and Mrs Patrick Etong. I appreciate
your invaluable contributions towards my academic exploits. God bless you all.

How can I forget my gorgeous nieces; Kenny Esienita, Maggie Esienita, Bright Gregory, Mirabel
Patrick and Aniebiet Gregory. My cute nephews; Blessed Bassey, Godspower Bassey, Nsikanabasi
Ekott, Pius Gregory, Henry and Harold Esienita (the twins), Winner Bassey and the little one, Etini
Ekott. I thaw in awe thinking of you guys, the reason why uncle keeps working harder to be able
to afford your future yearnings. Love always. My appreciations also go to my uncle, Engr. Luke
Edet Emeh and wife. Their children and my cousins; Mr. Etebong Emeh and family, Mrs.
Mmaobong and family, Mr. Eteka Emeh and family, Ms Queen Emeh. Family is all there is and
family is who we are.

The efforts of Ayokunle of Animal Biochemistry and Nutrition (ABN) and Tunde Dada will
always remain indelible in my heart. Giving a stranger a roof to lie under for months with no
equivocations, will always make me indebted to them both. I do not have the right words to thank
them both, but I know God will continue to bless them for everything. I also wish to thank my
amiable Pastors; Prof/Mrs. Paul Oyetaiwo and family, Mr./Mrs. Abimbola and all the members of
Salvation and Praise Assembly, Ajibode for their welfare and support, prayers and visitations. I
pray God continues to bless them according to His riches in glory. Amen. Also, my prayers go to
Kike (roommate as she fondly calls me), who gave me her place to stay for months with no second
thoughts while serving. God will bless her tremendously for everything she has done for me.

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Amen. To a buddy that I stay with, Ayomide Akinbote; Mr Tunde (caretaker), my neighbours:
Deolu and Tunde Egunjobi; Peace and Tolu; Kike and Yinka; Taiwo, Kenny, Dammy, Toby and
Omolara Gbadegesin; Juliet and Bukky; Shalom of Veterinary Medicine. Also, my mama in
Ibadan Mrs. Akinola and her children, Tolu, Anuoluwa, Timileyin for their support, humour and
care during my stay for this programme. Distance will be inevitable but individually and/or
collectively, I will always be grateful for everything you all have done for me.

I acknowledge my undergraduate colleagues/friends for their prayers and motivation; Akanimo

Ekereka, Elijah, Elijah, Testimony Ayara and Mrs. Imaobong Uffang. The colleagues I first met
in school; Iyanuoluwa (APB), Hemmings (APM), Otort Fashion (APB), Emmanuella-Michella
(crush of life, APM), Taiwo Makinde (ABN), Kunle (APM), Olutolani (ABN), Wale (ABN). Other
colleagues turned good friends; Semiu (ABG), Subair (ABN), Anjorin (ABG), Dele (APM), Josh
(APM), Stakeholda (APM), Seyi (ABN), David (ABG), Freedom (ABG) Jesse (ABG) and the
good people of Animal Biochemistry and Nutrition (ABN), Animal Breeding and Genetics (ABG),
Animal Production and Management (APM) and Animal Physiology and Bioclimatology (APB).
For the 14 inextricable faces representing the Best Unit of the Year, Animal Products and
Processing (APP); Mama Ajibade Jumoke, Amune Jeremiah, Mrs. Bukola Popoola (my personal
person), Omoyemi Ismael (my blood), Oluchi Odii, Ogunola Oluronke, Akinduro Temitope,
Bankole Oluwatosin, Ilesanmi Ifeoluwa, Bisiriyu Idris, Yetunde Agbana, Oladipupo Comfort and
Akintunde Folake. Also, some undergraduates I was privileged to meet; Victoria (400L), Titi
(400L), Ife (400L), Seyi and Daniel (500L), may God bless them all for the bond, the communion,
the great moments we have shared throughout the programme.

I really wish to, with all humility and sincerity, thank Mrs. Caroline Evans of nPower for that life-
changing call on the 6th of July, 2017. Without God using her to keep me afloat, I do not know
how this programme and research work would have fared. As God has opened the doors of
overflow for everyone I have and forgot to mention in this medium, I know she will also partake
immensely from the bounty. Amen.

Finally, I wish to thank you Lord again for all You have done for me from the first day I alighted
from the bus on Wednesday, 15th March, 2017 till this very moment I am putting this down
(Monday, 17th September, 2018). It is neither by my power nor strength but by your Grace alone.

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I solicit your sense of direction, serenity to accept the things I cannot change, courage to change
the ones I can and the wisdom to know the difference. Amen.

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 TABLE OF CONTENTS

Title Page………………………………………………………………......................................…i

Certification…………………………………………………………………………….………....ii

Dedication……………………………………………………………………………….….….....iii

Acknowledgement…………………………………………………………………………….….iv

Table of Contents……………………………………………………………………….……….viii

List of Tables……………….………………………………………………………………...…xiii

Abstract…………………………………………………………………………………….........xiv

CHAPTER ONE………………………………………………………………………………..…1

1.0 INTRODUCTION……………………………………………………………...……...….1

1.1 Objectives of the Study…………………………………………………………...…….....3

1.1.1 General objective…………………………………………………………………,,….......3

1.1.2 Specific objectives…………………………………………………………………..….....3

1.2 Significance of the study……………………………………………………….…….…....3

CHAPTER TWO……………………………………………………………………….…………5

2.0 LITERATURE REVIEW……………………...…………………………………….……5

2.1 Beef…………………………………………………………………………….………….5

2.1.1 Properties of Fresh Meat……………………………………………………….………….5

[Link] Water Holding Capacity (WHC)……………………………………………….…………5

[Link] Colour………………………………………………………………………….………….6

[Link].1 Effect of pH on Meat Colour………………………………………………….………...6

[Link] Structure, Firmness and Texture………………………………………………….……….7

2.2 Some Microorganisms Associated with Meat………………………………………….…7

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2.2.1 Salmonella spp…………………………………………………………………………….8

2.2.2 Staphylococcus spp…………………………….………………………………………….8

2.2.3 Campylobacter spp…………………………………………………………………..........8

2.2.4 Escherichia coli (E. coli)………………………………………………………………….9

2.3 Factors Influencing Growth of Microorganisms in Meat…………………………………9

2.3.1 Intrinsic factors………………………………………………………………………..…10

[Link] Moisture Content Available in the Product (denoted as aw)……………………...…...…10

[Link] The pH-value and acidity level of the product………………………....……...…………10

[Link] Nutritive value of the product……………………………………………………………11

[Link] Naturally occurring and added antimicrobials…………………………………………...11

2.3.2 Extrinsic factors………………………………………………………...………………..11

[Link] Effect of packaging materials……………………………………………………………11

[Link] Effect of Temperature/Time on Microbial Growth………………………….…………..12

2.4 Sources of Microbial Contamination……………………………………….…….……...13

2.5 Control of Microbial Contamination…………………………………………………….13

2.5.1 Good Hygiene Practices (GHPs)………………………………………………………...13

2.5.2 Hazard Analysis and Critical Control Points (HACCP)……………………………........15

[Link] Conduct a Hazard Analysis……………………………………………………….……...16

[Link] Identity the Critical Control Points (CCPs)………………………………………….......16

[Link] Establish Control Limits for each CCP Identified……………………………………….16

[Link] Establishment of Monitoring Requirements……………………………………………..16

[Link] Establishment of Corrective Actions…………………………………………………….17

[Link] Establishment of Verification Procedures……………………………………………….17

[Link] Establishment of Documents and Records………………………………………………17

2.5.3 Applying HACCP in Meat Processing…………………………………………………..17

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2.6 Preservation of Meat……………………………………………………………….…….18

2.6.1 Curing method……………………………………………………………………….......18

2.6.2 Drying method……………………………………………………………………….......19

2.6.3 Smoking method…………………………………………………………………………19

2.6.4 Freezing method……………....………………………………………………………….20

2.6.5 Cooking method………………………………………………………………………….20

2.7 Effect of spices on Meat Preservation…………………………………….......................21

2.7.1 Ocimum basilicum……………………………………………………………………….21

[Link] Antimicrobial Activity……………………………………………………………….......21

[Link] Antioxidant Activity……………………………………………………………………..23

2.7.2 Gongronema latifolium………………………………………………………………..…23

CHAPTER THREE

3.0 MATERIALS AND METHODS………………………………………………………...25

3.1 Types and Source of Study Materials…………………………………………………....25

3.1.1 Preparation of Aqueous Extracts………………………………………………………...25

3.1.2 Phytochemical analyses of test extracts………………………………………………….25

[Link] Determination of Tannin………………………….………………………….…………..25

[Link] Determination of Saponin………………………………………………………..………26

[Link] Determination of Alkaloid.…………………………………………...………………….26

[Link] Determination of Phenol…………………………………………………………………27

[Link] Determination of Flavonoid……………………………………………………………...27

3.1.3 1-diphenyl- 2-picrylhydrazyl (DPPH) Radical Scavenging Assay………………………27

3.1.4 Preparation of Meat Samples…………………………………………………………….28

3.1.5 Preparation of Curing Solution…………………………………………………………..28

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3.2 Measurement of Parameters……………………………………………………………..31

3.2.1 Determination of Cooking loss and Yield……………………………………....……….31

3.2.2 Proximate analysis……………………………………………………………………….32

3.2.3 Sensory Evaluation………………………………………………………………………32

3.3 Parameters on Storage Days……………………………………………………………..32

3.3.1 Water Holding Capacity (WHC)………………………………………………………...32

3.3.2 Measurement of Shear Force (tenderness)……………………………………………….32

3.3.3 Colour Reading…………………………………………………………………………..33

3.3.4 Assessment of Shelf-life…………………………………………………………………33

[Link] Lipid oxidation using Thiobarbituric acid-reactive substances (TBARS)……………….33

[Link] Microbial Counts Analyses………………………………………………………………33

3.3.5 Determination of pH……………………………………………………………………..34

3.4 Statistical Analysis……………………………………………………………………….34

3.5 Study Plan………………………………………………………………………………..34

CHAPTER FOUR

4.0 RESULTS……………………………………………………………………….……….35

4.1 Phytochemical constituents of test extracts (Ocimum basilicum and Gongronema

latifolium)………………………………………………………………………………..35

4.2 Mean concentration of Radical Scavenging Activity (DPPH) of test ingredients (%)….35

4.3 Physicochemical characteristics of product treated with test extracts…………………..35

4.4 Colour characteristics of raw beef after curing………………………………………….35

4.5 The pH of cure, extract and cured meat…………………………………….…………...41

4.6 Proximate Composition of smoked beef treated with Ocimum basilicum and Gongronema
latifolium extracts………………………………………………………………………..41

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4.7 Sensory characteristics of smoked beef treated with O. basilicum and G. latifolium
extracts……………………………………………………………………………….….41
4.8 Effect of O. basilicum and G. latifolium extracts on shear force (tenderness) and water
holding capacity of smoked beef during storage days……………………………….…..45

4.9 Effect of O. basilicum and G. latifolium extracts on colour of smoked beef during storage
days………………………………………………………………………………………45
4.10 Effect of O. basilicum and G. latifolium extracts on lipid oxidation and pH of smoked beef
during storage days………………………………………………………………..……...46

4.11 Effect of O. basilicum and G. latifolium extracts on microbial status of smoked beef during
storage days………………………………………………………………………………46

CHAPTER FIVE

5.0 DISCUSSION……………………………………………………………………………51

CHAPTER SIX

6.0 CONCLUSION AND RECOMMENDATION………………………………………….56

6.1 Conclusion……………………………………………………………………………….56

6.2 Recommendation………………………………………………………………………...56

REFERENCES…………………………………………………………………………………..57

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 LIST OF TABLES

Table 1: Experimental Composition of Ingredients for the study…………..….…….……29

Table 2: Composition of spices for the study……...………………………………………30

Table 3: Phytochemical constituents of test extracts (Ocimum basilicum and Gongronema

latifolium)…………………….…….………………………………..…………...37

Table 4: Mean concentration of Radical Scavenging Activity (DPPH) of O. basilicum and

G. latifolium extracts……..…………………….……………………….……..…38

Table 5: Physical characteristics of smoked beef treated with O. basilicum and G.

latifolium extracts………..……………………………….………………………39

Table 6: Colour characteristics of fresh beef after curing…………………..……………..40

Table 7: The pH of Extracts, Cure and Cured Meat before Smoking…………………..…42

Table 8: Proximate Composition of smoked beef treated with Ocimum basilicum and
Gongronema latifolium extracts (%)…………………………………..…………43

Table 9: Sensory characteristics of smoked beef treated with O. basilicum and G. latifolium
extracts…………………….……………………………………………………..44

Table 10: Effect of O. basilicum and G. latifolium extracts on shear force and Water Holding
Capacity (WHC) of smoked beef during storage days………………………..….47

Table 11: Effect of O. basilicum and G. latifolium extracts on colour of smoked beef during
storage days…………………………………………………………………........48

Table 12: Effects of O. basilicum and G. latifolium extracts on lipid oxidation and pH of
smoked beef during storage days………………………………………………....49

Table 13: Effect of O. basilicum and G. latifolium extracts on microbial status of smoked
beef (x104CFU/g)………………….…………………………………………..…50

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 ABSTRACT

Growth of pathogenic organisms, lipid oxidation, changes in colour and pH result in the
deterioration of the quality of meat and meat products thus rendering them to be unacceptable for
human consumption. The health risks however, associated with using synthetic preservatives in
keeping meat and meat products over time have resulted to seeking natural alternatives. The
objective of this project therefore was to find natural plants with antioxidants and antimicrobials
properties that could be potentially used as natural preservative in smoked beef compared with a
chemical preservative (nitrite). The inhibitory effects of Sweet basil, Ocimum basilicum extract
(OBE) and Bush buck, Gongronema latifolium extract (GLE) extracts were investigated to
evaluate the shelf life of intermediate moisture smoked beef stored at room temperature of 25oC
for 9 days of storage.

Freshly cleaned beef of 1.3kg each were allotted to four treatments in the study. 100ml of distilled
water was used to extract 10g of O. basilicum (OB) and G. latifolium (GL) leaves for T2 and T3
respectively, while 5g each of OB and GL leaves were used in 100ml of distilled water for T4.
After the aqueous extraction, treatments 1, 2, 3 and 4 were formulated with 0.5gNitrite in
27gWater, 27.5gOBE, 27.5gGLE and 13.75g each of OBE+GLE mix in a 100% composition of
ingredients respectively. Experimental design was a Completely Randomised Design.
Phytochemical and 1–diphenyl- 2-picrylhydrazyl (DPPH) analyses were carried out on the plants
to ascertain their percentage concentrations while sensory characteristics (using a 9point Hedonic
scale) and weight readings (cooking loss and yield) were taken on freshly prepared smoked beef
product on first day of preparation only. Lipid oxidation using Thiobarbituric acid-reactive
substances (TBARS), Microbial analysis, Water Holding Capacity, Shear force, Colour and pH
were determined on 0, 3, 6 and 9 days of storage.

Phytochemical results showed Gongronema latifolium having higher concentrations for saponins
(7.31), alkaloids (5.19) but lower in phenols (0.47) and flavonoids (4.01) than Ocimum basilicum
with tannin levels in trace amount in both plants. G. latifolium showed a high DPPH (radical
scavenging) potential of 56.01% compared to 49.87% analysed for O. basilicum. Cooking loss and
yield were not significantly different (p>0.05) but ranged between 50.38 – 52.30 (cooking loss)
and 47.70 – 49.62 (yield), with treatment 2 showing the highest and lowest values respectively.
Proximate composition of the freshly prepared products varied significantly (p<0.05) across the

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treatments with T2 having the lowest for moisture (29.85%), T4 with highest mean value for Crude
Protein (66.48%) while T3, T4 and T1 had the lowest values for Ether Extract (9.60), 4.40 for Ash
and 19.14 for NFE respectively. Organoleptic qualities were not significantly affected (p>0.05) by
the incorporated extracts however, T3 was assessed to be most preferred (6.45) for Overall
Acceptability closely followed by T1 (6.15) by the panelists. Treatments T2 and T3 had the highest
(6.10) and lowest (5.10) mean values for flavour, T1 and T2 were judged as highest (6.15) and
lowest (5.35) for colour while T1 and T4 had the highest (6.45) and least (5.55) ratings for taste
respectively. Water holding Capacity for products increased slightly on day 3 but treatments did
not show significance (p>0.05) except for days 0, 6 and 9 where a decrease was observed.
Treatment T2 showed higher value and T4 lower mean values across the days with no significant
difference (p>0.05) observed for shear force except on day 9. The colorimeter rating for lightness
(L) did not vary (p>0.05) across the days, while redness (a) and yellowness (b) differed in day 9
with T2 and T1 having lower (9.30) and higher (37.35) values respectively. For pH, no difference
was observed for all treatments. Total Plate Count of (log10CFU/g was below the Maximum
Permissible Limit of 7log10CFU/g although increasing values were observed as days progressed
with T3 showing highest values of 5.19 and 5.05 respectively for bacteria and fungi counts.

Inclusion of O. basilicum and G. latifolium at 10g of fresh leaves in 100ml of water showed
antimicrobial and antioxidative effects on the microbial counts and lipid oxidation, colour and pH
parameters as observed in the study, but replacing nitrite is still a work in progress. Therefore,
further study could be carried out to explore the rich potentials of both plants to help ascertain the
suitable inclusion level necessary to replace chemical preservatives in improving the quality and
shelf life of meat and meat products.

Keywords: Ocimum basilicum, Gongronema latifolium, leaf extracts, Phytochemicals, DPPH.

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