ABSTRACT
This work investigated the production and quality evaluation of bio-margarine produced from palm, melon and coconut oils using L. acidophilus, L. bulgaricus and S. thermophilus starter cultures. Six samples of uninoculated margarines samples (MAG 001-MAG 006) were prepared with the following blend ratios of 90:2:8,80:4:16,70:8:22,60:10:30 and 40:30:30 of coconut, melon and palm oils. And three samples (MAG 007-009) lactic acid bacteria starter culture inoculated margarines were prepared at the ratios of 60:10:30,50:20:30 and 40:30:40 of coconut, melon and palm oils. They were inoculated with L. acidophilus, S. thermophilus and L. bulgaricus respectively. The total bacteria count, total coliform count and total fungi count of the starter culture inoculated margarine samples were determined for a period of 12 days using standard microbiological techniques. Vitamin, mineral, fatty acid profile, physicochemical properties and sensory evaluations were also determined. At day 1 storage, there were no significant growth in all the samples except in samples MAG 007-MAG 009 which ranged from 2.0 x 105 – 5.4 x 106. No significant growth was shown at day three (3) of storage in all the samples except in samples MAG 007- MAG 009 ranged from 6.0 x 105 – 7.0 x 106cfu/g. At the 6,10 and 12 days duration of storage showed no significant growth in all the bio-margarine samples except in samples MAG 007 to MAG 009 which ranged from 7.4 x 106cfu/g. Vitamin A content ranged from 96.37ug to 103.48ug.Vitamin B1 ranged from 0.0435mg/100g 0.055mg/100g, vitamin B2 ranged from 0.020mg/100g to 0.0335mg/100g.Vitamin B3 ranged from 0.16mg/100g to 0.27mg/100g, B9 ranged from 27.75mg/100g to 32.17mg/100g ,B12 ranged from 0.11mg/100g to 0.18 mg/100g.The mineral composition of the bio-margarine samples ranged as follows; iron(0.079– 0.125mg/kg), manganese (0.008-0.044 mg/kg), potassium (4.84–7.41 mg/100g), magnesium ( 0.89 -0.044 mg/kg), and soduim (0.69 – 1.03mg/100g) .The fatty acid profiles of the bio-margarine samples ranged as follows; cupric acid (0.0124-0.0144%), capric acid (0.0039-0.0065%), lauric acid (0.1585-0.2655%), myristic acid (0.0865-0.1355%), palmitic acid (5.58-8.44%), palmitoic acid (0.045-0.088%), stearic acid (1.48-2.36%), oleic acid (42.29-56.36%), linoleic acid (15.59-21.15%), linolenic acid(0.149-0.236%), aroteic acid (1.285-1.625%) and behenic acid (1.175-3.355%).The physicochemical properties of the bio-margarine samples ranged as follows; density (0.918 – 0.966g/cm3), specific gravity (0.9065 – 0.9095), Peroxide value (9.18 -9.88mEq/kg), Iodine value (98.86 – 105.23), Refractive index (1.388-1.496) and free fatty acid (2.48-3.86) mgKOH/g. Sensory scores of the bio-margarine samples ranged from 5.00 to 7.50, 5.15 to 7.00, 3.65 to 6.55, 5.15 to 7.05, 5.15 to 7.25 and 5.40 to 7.05 for appearance, taste, consistency, flavor, mouth-feel and general acceptability respectively. There were no significant differences (p < 0.05) in the mean sensory scores between the margarine inoculated with LAB isolates and the control.
TABLE OF CONTENT
Title
Page i
Certification ii
Dedication iii
Acknowledgment iv
Table
of Content v
List
of Tables x
List
of Figures xi
Abstract xii
CHAPTER 1: INTRODUCTION
1.1 Background
of the Study 1
1.2 Statement
of the Problem 4
1.3 Justification
of the Study 4
1.4 Objectives
of the Study 5
CHAPTER 2: LITERATURE
REVIEW
2.1 Oils and
Fats 7
2.1.1 Differences between oil and fat 8
2.2 Chemical
composition of fatty acids
9
2.2.1 Importance
of fat 10
2.2.2 Importance
of fats and oils for child growth and development 13
2.3 Fats
and Oils for optimum health 13
2.3.1 Basic
recommendations for fats and oils 15
2.3.2 Children
and fats 17
2.4 Coconut 18
2.4.1 Importance
of coconut
19
2.4.2 Benefits
of coconut 20
2.5 Melon 21
2.5.1 Nutritional
values of melon seed (egwusi) 21
2.5.1.1 Key benefits of melon (egwusi) Citrullus
colocynthis
21
2.6 Palm
Oil and health 25
2.6.1 Arteriosclerosis 28
2.6.2 Cancers 28
2.6.4 Palmitic
Acid 28
2.7 Probiotics
29
2.7.1 Types
of probiotics 30
2.7.2 History
of probiotics 30
2.7.3 Guidelines
for the assessment of probiotic microorganisms 32
2.7.4 Selection
of probiotic strains for human use 33
2.7.5 Health
benefits / roles of probiotics 33
2.9 Use
of Probiotics in Otherwise Healthy People 39
2.9.1 Safety
of probiotics in humans 40
2.10 How
to Improve Probiotics and its Benefits
40
2.10.1 Some probiotic rich foods 41
2.11 Lactic
Acid bacteria 44
2.11.1 Taxonomy 44
2.11.2 Streptococcus 45
2.11.3 Lactococcus 46
2.11.4 Enterococcus 47
2.11.5 Carnobacterium 47
2.11.6 Tetragenococcus 48
2.11.7 Vagococcus 49
2.11.8 Lactobacillus 49
2.12 Isolation
and Identification of Lactic Acid Bacteria 52
2.12.1 Factors influencing growth and bacteriocin production of lactic acid
bacteria 53
2.12.2 Lactic acid bacteria in food
fermentation 54
2.13 Metabolites
of Lactic Acid Bacteria Responsible for Food Preservation 59
2.13.1 Organic acids 59
2.13.2 Hydrogen peroxide 60
2.13.3 Carbon dioxide 61
2.13.4 Diacetyl 61
2.13.5 Bacteriocin 61
2.14 Mechanism
of food fermentation by lactic acid bacteria 62
2.14.1 Homolactic acid fermentation 62
2.14.2 Heterolactic acid fermentation 63
2.15 Importance
of fermentation 63
2.15.1 Nutritional benefits 63
2.15.2 Health benefits 65
CHAPTER 3: MATERIALS
AND METHODS
3.1 Sample
Collection 68
3.2 Isolation
and characterization of lactic acid bacteria 68
3.2.1 Culture media preparation 68
3.2.2 Isolation of microorganisms 68
3.2.3 Culture preservation 69
3.2.4 Characterization of isolates 69
3.2.4.1 Gram’s staining 69
3.2.5 Biochemical test 69
3.2.5.1 Catalase test 69
3.2.5.2 Methyl red test 69
3.2.5.3 Voges- proskauer (VP) test 70
3.2.5.4 Indole test 70
3.2.5.5 Citrate utilization test 70
3.3 Technological
properties of LAB Isolates (for selection of suitable
isolates) 71
3.3.1 Hydrogen
sulphide (H2S) production test 71
3.3.2 Determination of lactic acid, hydrogen
peroxide and diacetyl production
by LAB isolates. 71
3.3.2.1 Quantitative estimation of lactic acid 71
3.3.2.2 Determination of diacetyl formation 72
3.3.2.3 Quantitative estimation of hydrogen peroxide
formation 72
3.3.2.4 Acidification activity 73
3.4 Sample
Preparation 73
3.4.1 Extraction of oils 73
3.4.2.1 Inoculation of margarine with chosen lactic acid bacteria (Starter
Culture) 76
3.5 Microbiological Analysis 76
3.5.1 Total
viable count 76
3.5.2 Total
coliform count 77
3.5.3 Total
fungal count 77
3.6 Determination
of Vitamins and Minerals 77
3.6.1
Determination of vitamins 77
3.6.1.1 Determination of riboflavin (Vitamin B2) 77
3.6.1.2 Determination of thiamine (vitamin B1) 78
3.6.1.3 Determination of niacin (Vitamin B3) 79
3.6.1.4 Determination of vitamin C 80
3.6.1.5 Determination of vitamin A 81
3.6.2.1 Determination of sodium, potassium, iron,
magnesium and manganese 81
3.6.2.2 Preparation of standards for analysis of
minerals in samples 82
3.7 Determination
of Fatty Acids Profile 82
3.8 Measurement of Physicochemical
Characteristics of the Margarine Samples 83
3.8.1 Free fatty acid
83
3.8.2
Peroxide value 83
3.8.3 Iodine
value 84
3.8.4 Determination
of specific gravity 85
3.10.5 Determination
of density 85
3.8.6 Determination
of refractive index 85
3.9 Sensory
Evaluation 86
3.10
Statistical Analysis 86
CHAPTER 4: RESULTS
AND DISCUSSION
4.1 Morphological
and Biochemical Characteristics of Isolates 87
4.2 Technological
Properties of the Isolates 89
4.2.1 Qualitative determination of bile salts
hydrolases activity 94
4.2.2 Antimicrobial activity 95
4.3 Microbial
Evaluation 97
Values
are mean ± standard deviation of duplicate determination.
Means
in the same column followed by different superscripts are significantly
(p<0.05) different
|
4.4 Vitamin
Composition of the Bio-margarine Samples 104
4.5 Mineral
composition of the Bio-margarine samples 107
4.6 Fatty
Acids Profile 110
4.7 Some
Physicochemical Properties of the Bio-Margarine 114
Samples
4.8 Sensory
Evaluation of the Bio-margarine Samples 119
CHAPTER 5: CONCLUSION
AND RECOMMENDATION
5.1 Conclusion 121
5.2 Recommendations
121
References
122
Appendices 136
LIST OF TABLES
2.1: The
Approximate concentration of fatty acids in palm oils 26
2.2. Major divisions
within the genus lactobacillus based on
phenotypic
characteristics
51
2.3 Acetic
acid bacteria and its fermentable products 56
3.1: Sample
Ratios 73
4.1: Morphological and Biochemical
characteristics of the lactic acid
bacteria isolates 88
4.2: Technological properties of the Isolates 93
4.3. Qualitative determination of bile salts
hydrolases activity (Min) 95
4.4: Antimicrobial
activities of lactic acid bacteria strains 96
4.5a. Microbial
analysis of the margarine samples 99
4.5b: Microbial analysis of the margarine samples 100
4.5c: Microbial analysis of the margarine samples 101
4.5d: Microbial Analysis of the Margarine Samples 102
4.5e: Microbial
analysis of the margarine samples 103
4.6: Vitamin
composition of the margarine samples 106
4.7: Mineral
composition of the margarine samples 109
4.8: Fatty
acids profile composition of the margarine
samples 112
4.9: Physicochemical
properties of the margarine samples 118
4.10: Sensory evaluation of the margarine samples 120
LIST
OF FIGURES
3.1a Palm oil 74
3.1b Melon oil 74
3.1c Coconut oil 74
3.2a
Bio-margarine 001-003 75
3.2b
Bio-margarine 004-006 75
3.2c
Bio-margarine 007-008 75
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The
relationship between dietary fat intake and increased risk of chronic diseases
has been the subject of considerable debate since the early 1960s. Nutrition
education programs that have risen from this debate have increased awareness of
such linkages. With food fats and oils accounting for more than 50% of total
fat and 40% of saturated fat intake, the structure of fats and oils demand has
been examined in the light of this increased awareness and it was found that
the increased knowledge of the link between cholesterol and fat intake and
coronary heart diseases resulted in a 13% reduction in butter and 15% increase
in margarine intake (Brian, 1998).
Margarine
was initially developed as a butter substitute. The product was designed to
meet butter shortages caused by increasing urban populations during the
industrial revolution as well as to produce a table spread with satisfactory
keeping qualities for the armed forces (Lai et
al., 1999). However, over the
years, margarine has established its own image and is used by consumers for a
variety of purposes.
Margarine
is a water-in-oil emulsion produced from a non-dairy product created as a
substitute for butter. While originally made from animal fat in 1800s, today
the primary ingredient is vegetable oil. While butter is derived from animal
fat, margarine is made with vegetable oil(s). This difference has an impact on
taste, texture and nutrition. Since margarine primary component is vegetable
oil, it lacks the cholesterol and saturated fat found in butter (Zeratsky, 2015).
The
coconut tree (Cocos nucifera) is a
member of the family Arecaceae (palm family) and the only species of the genus
cocos. Coconuts are known for their great versatility as evidenced by many
traditional uses, ranging from food to cosmetics. They form a regular part of
the diets of many people in the tropics and subtropics. Nutritionally, per 100 gram
serving of raw coconut meat supplies a high amount of total fat (33 grams),
especially saturated fat (89% of total fat) and carbohydrates (24g).
Micronutrients in significant content include the dietary minerals manganese,
iron, phosphorus and zinc. It also contains 3.33g of protein (Paniappan, 2002).
Coconut is highly rich in fiber, vitamins and minerals. It is classified as” a
function” because it provides many health benefits beyond its nutritional
contents. Coconut is being used by confectionaries, bakeries, biscuits, ice
cream industries worldwide to enhance flavor and taste of various products
(Persley,1992). Coconut juice was found to be rich in calcium, protein and fat
(Belewo and Belewo,2007).
Melon
seeds (Yoruba egwusi) belong to the
family of unshelled melon seeds: Cucumis melo
cucurbitaceae. They are mostly cultivated in the Southern part of Nigeria
and is usually inter planted with yam and cassava where it serves as a cover
crop. It may account for up to 80% of dietary protein and it is used as the
only source of protein for some groups of people and also serves as a
substitute for meat or fish (Odibo, 1987). Melon seeds have been reported to
contain 3.3% moisture, 15.5% crude fibre, 10.3% crude protein, 8.2%
carbohydrate, 52% oil and 3.6% ash (Omafuvbe et al., 2004).
Apart
from the nutritional components of melon, it also possesses other health
benefits such as anti-cancer properties, heart health, cures kidney disease,
maintains healthy skin, helps in weight loss, has anti-aging properties,
promotes hair growth etc. (Vineetha, 2014).
Melon
seed is generally cultivated and grown in a variety of growing conditions and
climates. They are globally popular and are valued for their sensory,
nutritional and health attributes (Ogundele et
al., 2012). They are good
sources of protein, Omega 3 fatty acid (alpha linolenic acid), vitamins C, E
and A. It also contains zinc, magnesium and are used for preparing delicious
sweets and snacks (Agatemor and Mark, 2006).
Palm
oil is an edible vegetable oil derived from the mesocarp (reddish pulp) of the
fruit of the oil palms, primarily the African oil palm Elaeis guineensis. Palm oil is naturally reddish in color because
of its high beta-carotene content. It is not to be confused with palm kernel
oil derived from the same fruit (Poku, 2002) or palm kernel oil derived from
the kernel of the palm (Cocos nucifera).
The differences are in color (raw palm kernel oil lacks carotenoids and is not
red), and high in saturated fat content. Along with coconut oil, palm oil is
one of the few highly saturated vegetable fats and is semi solid at room
temperature. Palm oil is a common cooking ingredient in the tropical belt of
Africa, Southeast, Asia and parts of Brazil. It is used in the commercial food
industry in other parts of the world and is widespread because of its lower
cost and the high oxidative stability (saturation) of the refined product when
used for frying (Che Man et al., 1999).
Fats
and oils are composed of chains of molecules called fatty acids that are made
of mainly carbon atoms. Saturated fat means that the carbon double bonds have
hydrogen attached to them and this make the fat solid at room temperature and
are called oils. Fats and oils are high energy foods providing about 9 calories
per gram of fat. This is more than twice the energy content of sugars and
starches. This is the reason why some nutrition and diet authorities think that
fats will make you fat. However, it is not so when one knows how to choose them
properly (Lawrence, 2016). Fats and oils are needed for your brain and nervous
system, for energy production and for making most of the body’s vital hormones.
Children, in particular absolutely require fats and oils. Quality fats and oils
are also essential for the cell membranes. These must transport all vitamins,
minerals and hormones in and out of every one of the body cells.
Now,
bio-margarine is a margarine that is
inoculated with probiotics (Lactic acid bacteria). Probiotics are live bacteria
and yeasts that are good for your health, especially your digestive system.
Probiotics are often called “good” or “helpful” bacteria because they help keep
your gut healthy (Dilonardo, 2015). In recent years, different investigations
support the importance of probiotics as part of healthy diet for humans and
animals and as a way to provide a natural, safe and effective barrier against
microbial infections (Angmo et al., 2016; Oh Jung, 2015). According
to the definition by the World Health Organization (WHO), Probiotics are “live
microbial food supplements which, when administered in adequate amounts confer
a health benefit on the host” (FAO/WHO, 2001). Among the usually used
microorganisms, lactic acid bacteria (LAB) are regarded as a major group of
probiotic bacteria (Collins and Gibson, 1999). They are non-pathogenic,
technologically suitable for industrial processes, acid tolerance and bile
tolerance and produce antimicrobial substances (Mojgani et al., 2015). They are
classified as “generally recognized as safe” (GRAS) microorganisms because of
their long and safe use as starter cultures in fermented products (FAO/WHO, 2001).
1.2 STATEMENT
OF THE PROBLEM
There
is a great increase in consumer’s quest for varieties as well as speedy
advancement in the extent have posed serious challenges to the Nigerian food
industries. Also the cost of butter (which is made from animal fat) increases
day by day making it difficult for low income consumers to have access to these
foods, furthermore, the health risk encountered in the consumption of animal
fat which increases cholesterol in human. In line with this, there is need for
us to produce a margarine (bio-margarine)
which is enriched with probiotics using available local plant materials, since
margarine primary component is vegetable oil, it lacks the cholesterol and
saturated fats found in butter.
1.3 JUSTIFICATION OF THE STUDY
A
country blessed with natural resources like Nigeria is not expected to depend
solely on the importation of almost all the raw materials needed in food
production. Overtime, there has been a lot of improvement on product innovation
worldwide. Margarine are made from vegetable oils such as rapeseed, safflower,
olive etc which are not readily available in Nigerian, instead of importing
these raw materials owing to the high cost of importation, it is important for
us to make use of our indigenous raw material such as palm, coconut and melon
oils.
Margarine
was initially developed as a butter substitute. The product was designed to meet
butter shortages caused by increasing urban populations during the Industrial
Revolution as well as to produce a table spread with satisfactory keeping
qualities for the armed forces (Lai et al., 1999). Now bio-margarine is a margarine that is inoculated with probiotics
(Lactic acid bacteria) and are good for health especially our digestive system.
1.4 OBJECTIVES OF THE STUDY
The
main objective of the study is to produce and evaluate the quality of bio-margarine
from three different vegetable oils (palm, coconut and melon oils) using
probiotic cultures of lactic acid bacteria.
The
specific objectives of this study are;
1. To isolate and characterize lactic acid
bacteria from yoghurt.
2. To screen the lactic acid bacteria
isolates for suitability as probiotic culture.
3.
To select suitable strains of LAB as probiotic culture for margarine
production.
4. To blend the three vegetable oils
using different ratios.
5. To produce margarine samples from
blends of the oils with and without the use
of lactic acid bacteria
starters.
6. To
determine the microbial, vitamin and mineral content of the margarines produced.
7. To determine the fatty acids profiles of
the margarines.
8. To determine the physicochemical
properties of the margarine.
9. To evaluate the sensory attributes of
the bio-margarine.
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