Effect of enriching yoghurt with Moringa oleifera on the Physicochemical,

Functional and Microbiological properties

By
MEMORY MAREBA
C18132953N

A project submitted in partial fulfilment of the Bachelor of Science

Honours Degree in Food Science and Technology

CHINHOYI UNIVERSITY OF TECHNOLOGY

School of Agricultural Sciences and Technology

Department of Food Science and Technology
June 2022
 DECLARATION

I, MEMORY MAREBA, declare that this is my original work. This project has not been

submitted to any university or any other institute for any other degree.

Signature.................................................... Date........................

ii
 APPROVAL

This dissertation entitled ‘Effect of enriching yoghurt with Moringa oleifera on the

physicochemical, functional and microbiological properties’ meets the regulation governing

the award of the Bachelor of Science degree in Food Science and Technology at Chinhoyi

University of Technology.

Signature………………………… Date……………

iii
 ACKNOWLEDGMENT

I humbly thank my Heavenly Father who made the completion of this project a success. It

was my great pleasure and honour to be given an opportunity to study at Chinhoyi University

of Technology. This research gave me the exposure and experience I need to enter the

corporate world.

I’m deeply grateful to my supervisors, Dr. Faith Manditsera and Ms. Kudakwashe Chiwaya

for their tireless, consistent guidance, scientific support and encouragement throughout my

research, particularly during thesis development and writing of this work.

Deserving no less gratitude is the technician of the Department Food Science and

Technology, Chinhoyi University of Technology, Mr. Masheka for his technical help during

the laboratory analyses.

Lastly, I give thanks to my family and siblings with bottomless gratitude for their prayers,

love, support and their belief in me.

iv
 DEDICATION

This project is dedicated to my family, my parents and siblings, I LOVE YOU.

v
 ABSTRACT

Upsurge in the health conscious population has resulted in a gap in the functional food sector.

There is an acute rise in the demand for products with enhanced antioxidant properties.

Moringa oleifera seed contain various bioactive compounds with antioxidant activities, and

fermented dairy products have potential to be incorporated with fruits or plants to enhance

their antioxidant potential. The study aimed to evaluate the effect of enriching yoghurt with

Moringa oleifera on the physicochemical, functional and microbiological properties. Yoghurt

was incorporated with Moringa seed powder (MSP) at concentrations 0%, 0.25%, 0.5% and

1%. The samples were stored at 4ºC (chilled storage) and analyzed. Addition of MSP to

yoghurt significantly improved the rate of fermentation by promoting growth of LAB. MSP

increased WHC % up to (94± 0.05). Titratable acidity was increased up to (1.3± 0.05) whilst

pH decreased to 3.9± 0.03). Radical-scavenging activity of MSP enriched yoghurt increased

up to 84.7% in a dose-dependent manner during the 10 days of cold storage whilst total

phenolic content increased to (43.3mgGAE/g± 0.05). MSP increased the viability of LAB up

to10.4±0.03 logcfu/ml. Sensory testing showed that the addition of 0.5% and 0.25% MSP to

yoghurt did not negatively influence the overall acceptability of the product, compared to the

control. The addition of MSP to yoghurt increased the LAB count, AOA, TPC and improved

the physicochemical properties of yoghurt. MSP can be used in yoghurt as a functional

ingredient. The best yoghurt sample was obtained at 0.5% of MSP, it showed enhanced

physicochemical properties, AOA and TPC without negatively affecting the sensorial

properties.

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Contents
DECLARATION……………………………………………………………………………i
APPROVAL…………………………………………………………………………………ii
ACKNOWLEDGMENT…………………………………………………………………... iii
DEDICATION………………………………………………………………………………iv
ABSTRACT………………………………………………………………………………......v
List of tables…………………………………………………………………………………. ix
Table of figures……………………………………………………………………………......x
CHAPTER 1.............................................................................................................................1
1.1. Background of the study.....................................................................................................1
1.2.1 Statement of the problem..................................................................................................3
1.2.2 Project justification...........................................................................................................3
1.3. Objectives of the study........................................................................................................4
1.3.1. Main objective..................................................................................................................4
1.3.2 Specific objectives............................................................................................................4
1.4 Research hypothesis.............................................................................................................4
CHAPTER 2-LITERATURE REVIEW................................................................................5
2.1. Functional foods..................................................................................................................5
2.2. Enriched Yoghurt................................................................................................................6
2.3. Physicochemical properties of yoghurt...............................................................................6
2.4. Starter Culture.....................................................................................................................7
2.5. Moringa oleifera plant.........................................................................................................8
2.6. Traditional and modern use of Moringa oleifera..............................................................10
2.7. Previous studies of Moringa oleifera seeds.......................................................................11
2.8. Phytochemicals in Moringa oleifera seeds.......................................................................11
2.8.1 Flavonoids.......................................................................................................................11
2.8.2 Phenolic compounds.......................................................................................................12
2.9. Oxidative stress.................................................................................................................13
2.1.0. The principle of antioxidants.........................................................................................14
2.1.1. DPPH assay....................................................................................................................16
CHAPTER 3-METHODOLOGY.........................................................................................19
vii
3.1. Research Design................................................................................................................19
3.2. Sampling Method..............................................................................................................19
3.3. Preparation of Moringa seed.............................................................................................19
3.4. Preparation of stirred yoghurt enriched with Moringa oleifera........................................20
3.5. Extraction of Moringa seed powder and yoghurt samples................................................21
3.6. Determination of Antioxidant activity..............................................................................21
3.7. Determination of Total Polyphenol Content (TPC)..........................................................22
3.8. Determination of the presence of Flavonoids...................................................................23
3.9. Determination of Physicochemical characteristics...........................................................23
3.9.1. Measuring pH.................................................................................................................23
3.9.2. Titratable acid.................................................................................................................23
3.9.3. Water holding capacity..................................................................................................24
3.1.0. Microbiological analysis................................................................................................24
3.1.0.1. Lactic Acid Bacteria Count (LAB)………………………………………………….24
3.1.0.2. Total yeast and moulds………………………………………………………………25
3.1.01.3. Total coliforms……………………………………………………….…………….25
3.1.1. Sensory evaluation.........................................................................................................25
3.1.2. Data Analysis.................................................................................................................25
CHAPTER 4- RESULTS AND DISCUSSION....................................................................26
4.1. Antioxidant activity and Total Phenolic Content for Moringa oleifera seeds..................26
4.2. Qualitative test on Moringa seed powder and yoghurt samples for flavonoids................27
4.3. TPC and AOA of yoghurt enriched with Moringa seed powder......................................28
4.4. pH and Titratable acidity of yoghurt samples...................................................................30
4.5. Water holding capacity of yoghurt samples......................................................................32
4.6. Sensory evaluation of yoghurt samples.............................................................................36
CHAPTER 5...........................................................................................................................38
5.1. Conclusion.........................................................................................................................38
5.2.Recommendations..............................................................................................................38
REFERENCES.......................................................................................................................40
APPENDICES........................................................................................................................45

viii
List of tables
Table 1.Formulation of enriched yoghurt with Moringa oleifera seed powder.......................19
Table 2.Total phenolic content and DPPH radical scavenging % for Moringa oleifera seed
powder......................................................................................................................................24
Table 3.Flavonoids screening of Moringa oleifera seed powder and yoghurt samples...........25
Table 4.Microbial analysis of yoghurt produced with different concentration of Moringa
oleifera seed powder.............................................................................................................. 32

ix
Table of figures
Figure 1.The main areas in Zimbabwe where Moringa trees are grown...................................9
Figure 2.Images of Moringa oleifera tree, leaves, seeds and flowers......................................10
Figure 3.General pathways in a free radical chain oxidation and the action of antioxidants.. 16
Figure 4.Chemical reaction of DPPH with antioxidant compound RH...................................18
Figure 5 Experimental design..................................................................................................19
Figure 6.Total Phenolic Content..............................................................................................27
Figure 7.Antioxidant activity...................................................................................................27
Figure 8. pH of yoghurt samples..............................................................................................29
Figure 9.Titratable acidity of yoghurt samples........................................................................29
Figure 10.Water holding capacity of yoghurt samples............................................................31
Figure 11.Sensory evaluation of yoghurt samples...................................................................35

x
 CHAPTER 1

1.1. Background of the study

Recent technical advancements, combined with an increase in the number of people who are

concerned about their health, have sparked a surge in the production of innovative functional

foods. Functional foods are whole or modified foods that contain physiologically active

components that give health benefits in addition to meeting nutritional requirements. When

ingested at appropriate amounts as part of a diversified diet on a regular basis, they reduce the

risk of diseases and improve an individual's health (Megh & Hafiz, 2020). The term

"functional food" was coined in Japan, and the necessity for it arose from the rise in health-

care costs during WWII. Functional food is distinct from nutraceuticals, pharmafood, and

medifood, and it does not include dietary supplements. It falls under the category of nutrition

rather than pharmacology. Instead of preventing disease, the aim of functional foods is to

reduce the likelihood of it occurring (Arfouri, 2020).

Yoghurt is made and consumed massively all around the world. It is well-known for its great

nutritional value and health-beneficial properties. However, bioactive substances are often

lacking in dairy products, including yoghurt. To compensate for this lack, plant extracts and

powders as a source of phenolic compounds and bio-flavonoids must be added. The increase

in consumer demand for functional foods has resulted in a paradigm shift away from typical

dairy products and toward those rich in bioactive substances (Irena et al., 2022). Yoghurt

fortification using plant extracts or powders improves not just its functionality but also its

qualitative attributes (Idris et al., 2016). Flavonoids have a high antioxidant activity and are

more effective at preventing the harmful effects of free radicals (Cantuti-Castelvetri et al.,

2000). Phenolic acids also exhibit high antioxidant activity. They are able to bind metal ions

that contribute to the formation of free radicals. (Edith et al., 2018). ROS react with DNA,

1
lipids, protein, carbohydrates, and other cell molecules resulting in gene mutations. Mutation

in genes promotes conditions like aging, cancer, cardiovascular diseases, diabetes mellitus

and other cell degenerative disorders (Megh & Hafiz, 2020). Regulation of oxidative stress at

cellular levels is mandatory to prevent development of these degenerative disorders. Intake of

foods rich in polyphenols is a promising practical way to minimize damaging effects related

to reactive free radicals (Megh & Hafiz, 2020).

There is a knowledge gap in the potential uses of Moringa oleifera and its use in food

fortification. Moringa is under exploited, it can be used to make foods with improved

nutritional and health benefits (Shockery et al., 2017). It belongs to the Moringaceae family,

which includes 13 plant species. Moringa oleifera seeds takes only six months to grow from

seed sowing to harvest, which is a relatively short period of time. Even under drought

conditions, the plant grows quickly, allowing it to endure a wide range of soil and rainfall

conditions and hence be available throughout the year. Polyphenols (4581–4953 mg/100g)

are abundant in Moringa oleifera seeds, making them a rich source of bioactive compounds.

(Akinyeye et al., 2014). An antioxidant is a natural or synthetic substance which is

responsible for inhibiting cell damage by free radicals produced in the oxidation process

(Akinyeye et al, 2014). Phytochemicals like alkaloids, glycerides, flavonoids, steroids,

saponins and tannins are present in Moringa oleifera seed in large amounts despite of the

solvent used in extraction (Shirazi et al., 2014). The aim of this study was to make yoghurt

with dry Moringa seed powder and investigate how varied Moringa leaf powder

concentrations affected the physicochemical, functional, and microbiological aspects of the

yoghurt.

1.2.1 Statement of the problem

The production of free radicals is an integral part of human metabolism. Reactive oxygen

species (ROS) by-products are produced continuously due to aerobic metabolism where

2
molecular oxygen is reduced by oxidative phosphorylation that takes place in mitochondria.

ROS react with DNA, lipids, protein, carbohydrates, and other cell molecules causing gene

mutations which promote conditions like cancer, cardiovascular diseases, diabetes mellitus

and other cell degenerative disorders (Megh & Hafiz, 2020). There has been a rise in the non-

communicable diseases (NCDs) resulting in an escalation in the health conscious population.

Nowadays, consumers prefer healthy foods beyond basic nutrition. The upsurge in the

demand for functional foods has left a huge gap on the dairy fermented products. Yoghurts

have a limited content of bioactive compounds (Irena et al., 2022). There is a limited research

on Moringa oleifera seed as a functional ingredient in dairy products. Moringa oleifera seed

has high amounts of polyphenols which include flavonoids, alkaloids and phenolic acids.

Intake of foods rich in polyphenols is a promising practical way to minimize damaging

effects related to ROS. Polyphenols have high antioxidant capacity; they play major roles in

inhibiting the formation of ROS. In this research an investigation will be done on the yoghurt

enriched with Moringa oleifera, on its total polyphenols content and antioxidant activity.

1.2.2 Project justification

Moringa oleifera is a plant that is native to Africa and can be easily grown even in hot, dry

climates with poor soil. As a result, most people can benefit from it because it is widely

available. The researcher hopes to shed light on the currently under-utilized Moringa oleifera

seed, which is high in polyphenols (range of 4581–4953 mg/100 g). Polyphenols have a high

antioxidant activity and are highly effective at preventing the harmful effects of free radicals.

The developed innovative functional yoghurt would give a therapeutic strategy to minimize

the chances of non-communicable illnesses like cancer, cardiovascular diseases, diabetes

mellitus and other cell degenerative disorders caused by reactive oxygen species (Megh &

Hafiz, 2020). The other benefit from the study, would be the enhancement of the

physicochemical and functional properties of yoghurt.

3
1.3. Objectives of the study

1.3.1. Main objective

To determine the effect of enriching yoghurt with Moringa oleifera on the physicochemical,

functional and microbiological properties.

1.3.2 Specific objectives

1. To determine antioxidant activity, total phenolic content and presence of flavonoids in

Moringa oleifera seed powder.

2. To determine antioxidant activity, total phenolic content and presence of flavonoids in

Moringa oleifera enriched yoghurt.

3. To determine physicochemical properties of Moringa oleifera enriched yoghurt (pH,

titratable acidity and water holding capacity).

4. To assess microbial properties of Moringa oleifera enriched yoghurt.

5. To determine acceptability of Moringa oleifera enriched yoghurt.

1.4. Research hypothesis

Ho-Addition of Moringa oleifera to the yoghurt has no effect on its antioxidant activity, total

phenolic content, physicochemical properties and microbiological properties

H1-Addition of Moringa oleifera to the yoghurt has an effect on its antioxidant activity, total

phenolic content, physicochemical properties and microbiological properties

4
CHAPTER 2-LITERATURE REVIEW

2.1. Functional foods

The global functional food market has recently experienced a significant growth. The

growing awareness of the link between health and nutrition has had a significant impact on

consumer nutrition behavior, resulting in the emergence of the functional food concept.

Nowadays, customers place a higher value on their health, resulting in a higher demand for

functional products as opposed to basic ones (Hsieh & Ofori, 2007). In Japan, the term

"functional food" was coined. It was inspired by the Japanese Food for Specified Health

(FOSHU) and has since spread around the globe (Ashwell, 2002). Functional foods are

described as whole or modified foods that contain physiologically active components that

give health benefits in addition to meeting nutritional requirements. They are ingested as part

of one's diet and are neither a food nor a supplement (Megh & Hafiz, 2020).

Functional foods include; a whole, natural food e.g. blue berries containing polyphenols, a

processed food with added components e.g. calcium-enriched fruit juice, a processed food

from which a component(s) has been removed e.g. low fat margarine and a food where a

component(s) has been modified e.g. lactose free ice-cream (Aryana et al., 2017). Functional

foods can mitigate the risks of diseases which include cardiovascular problems, diabetes,

obesity, osteoporosis, and cancer due to the presence of bioactive compounds like

carotenoids, dietary fiber, phenolic acids, flavonoids, plant sterol and stanols (Megh & Hafiz,

2020). Recently, several researches concluded that enriching yoghurt using natural resources

particularly plants and fruits rich in polyphenols, could improve not only the quality attributes

but also the functionality of yoghurt with minimal adverse effects (Hsieh & Ofori, 2007).

2.2. Enriched Yoghurt

Yoghurt is one of the most popular dairy products in the world, owing to its inherent benefits.

It was first developed by nomadic herders in Asia, Southern and Eastern Europe. Yoghurt is a

5
Turkish term which refers to a fermented milk product. It is limited in bioactive compounds

like polyphenols, hence the need for fortification with polyphenol-rich plants (Adepoju &

Selezneva, 2020).

Polyphenols' hydroxyl substituents and aromatic structures, which can scavenge free radicals,

give them excellent antioxidant activity. Many studies on the addition of plants and fruits to

yoghurt to improve its functionality have recently been conducted. The addition of green,

white, and black tea to yoghurt improved the antioxidant activity and structural stability of

the yoghurt (Kim et al.,2019). Apple polyphenol increased the growth of lactic acid bacteria

in yoghurt, which was advantageous for pre-fermentation. Pomegranate, sweet cherries, and

grapes, which are high in phenolic compounds, increased the antioxidant activity and

polyphenol content of yoghurt when added to it (Kim et al.,2019).

Moringa oleifera seeds, on the other hand, have not been studied as a yoghurt ingredient.

Moringa seeds, for example, are rich in polyphenols and have high antioxidant activity, and

the plant is widely distributed throughout Zimbabwe (Gadzirai et al.,2013).

2.3. Physicochemical properties of yoghurt

The quality of yoghurt is related to its physicochemical properties which include the pH,

titratable acidity, syneresis and water holding capacity. The pH of a solution correlates with

the hydrogen ion activity and is measured by the use of a digital pH. Yoghurt is made by

combining the starter cultures of Streptococcus thermophilus and Lactobacillus bulgaricus in

a 1:1 ratio (Deshwal et al., 2021). Since Streptococcus thermophilus can tolerate oxygen, it

converts lactose to lactic acid, lowering the pH to 5.2 from 7. Lactobacillus bulgaricus

dominates growth at pH 4.4. Fermentation is stopped by rapidly cooling the yoghurt to 4°C.

To avoid an unpleasant acidic flavor, commercial yoghurt should have an acidic condition of

7.0–9.0 mg/g lactic acidity and a pH of 4.0–4.4. The acid-tolerant Lactobacillus bulgaricus

6
ferments slowly during refrigerated storage, leading to the well-known phenomena of post-

acidification, which is an undesirable process (Maha et al., 2021).

Syneresis is the separation of whey caused by the shrinkage of a protein gel, and it is a

common problem in yoghurt. Centrifugation and drainage procedures can be used to

determine it. The ability of yoghurt to keep its whey with the application of force or

centrifugation is known as water holding capacity, and it is linked to syneresis (Deshwal et

al., 2021). The physicochemical qualities of yoghurt are improved by adding plants high in

polyphenols, such as Moringa oleifera seed. Polyphenols have a strong affinity for proteins

and can form complexes with them, such as casein (milk protein). This compound has the

potential to improve the physicochemical qualities of yoghurt (Kim et al.,2019).

2.4. Starter Culture

One of the most important variables in determining the quality of yoghurt is the starter

culture. This phrase refers to the microorganisms (bacteria) that are employed to ferment a

certain cultured product, such as cheese, yoghurt, or kefir. The microorganism selected for

the particular purpose must have the desired effect on the product. The starter culture for

yoghurt manufacturing must be able to ferment lactose to form lactic acid, a small quantity of

carbon dioxide, and the desired flavor and aroma (Olson et al. 2010). Bacteriophages are a

type of virus that can inactivate starting cultures. The action of these bacteriophages is

responsible for a wide range of yoghurt production difficulties. They are the most common

cause of incubations that last a long time or never cease. Despite the fact that huge facilities

are in place to inspect incoming milk, bacteriophage, also known as phage, is always a risk.

Poor cleanliness and a lack of basic housekeeping increases the chance of it being found in

the drains and floor gullies of a dairy factory producing any cultured product. Antibiotics can

inactivate the starting culture. These can be found in the milk of cows who have been given

antibiotics (Al-Ahwal et al., 2010).

7
 Streptococcus thermophilus is some gram-positive bacteria that is non spore forming and

facultatively anaerobic. When cultivated in liquid media, its cells have a spherical form (0.7-

0.9 m diameter) and appear in pairs, with lengthy chains of 10-20 cells in milk. Its optimum

temperature is between 42 and 45 degrees Celsius. They are heterotrophic and often sensitive,

requiring simple carbohydrates as a source of energy and preformed amino acids as a supply

of nitrogen. Lactose is fermented homofermentatively, yielding L (+) lactic acid as the main

product (Leopodini et al., 2011).

Lactobacillus bulgaricus, on the other hand, is gram-positive. It grows well at 45 degrees

Celsius. Its fundamental metabolism is homo-fermentative, producing D (-) lactic acid in

milk at a concentration of 1.7-2.1%. Although lactic acid is the most prominent end product,

secondary end products such acetaldehyde, acetone, acetoin, and di-acetyl can also be formed

in very small amounts. Lactobacillus bulgaricus can ferment lactose, fructose, and glucose,

just like Streptococcus thermophilus, and some strains can also ferment galactose (Robinson,

2000). Polyphenols increase LAB growth while simultaneously preventing spoilage by

inhibiting the growth of spoilage bacteria like as yeasts and molds during fermentation, hence

addition of polyphenol-rich plants like Moringa oleifera in yoghurt is beneficial (Zhang et al.,

2019).

2.5. Moringa oleifera plant

Moringa oleifera is grown in a variety of production systems in Zimbabwe, and it has

naturalized in a number of regions, including Zimbabwe's four districts: Binga, Bindura,

Mutoko, and Shamva. Moringa oleifera is an umbrella-shaped tree that grows in hot, dry

climates with poor soil. It belongs to the Moringaceae family and goes by several names,

including horseradish tree (because the roots taste like horseradish), ben oil tree (the seeds are

high in behenic acid), drumstick tree (the seedpods are long and slender), and miracle tree

(because of its therapeutic properties) (Olson & Fahey, 2011).

8
The genus Moringa has thirteen species and Moringa oleiferas is the most common one. The

other species include M. hildebrandtii, M. longituba, M. pygmaea, M. rivae, M. ruspoliana,

M. ovalifolia, M. peregrine, M. concanensis, M. drouhardii, M. arborea, M. borziana and M.

stenopetala. These species can be found all over Africa (Anzano et al., 2021).

Moringa oleifera is a slender tree with umbrella-shaped branches that grows to around 10m

tall. It has pale green leaves with multiple little leaflets (1.3–2 cm long) that are 30–60 cm

long. Flowers are fragrant and creamy-white in color (diameter of 2.5 cm). When dry, the

pods have a pendulous form and are brown in color. There are approximately 20 seeds

implanted in the pith. The seeds have three papery wings and are dark brown in color (Olson

& Fahey, 2011).

Figure 1.The main areas in Zimbabwe where Moringa trees are grown (Gadzirayi et
al.,2013)

9
Figure 2. Images of Moringa oleifera tree, leaves, seeds and flowers (Anzano et al,.2021)

2.6. Traditional and modern use of Moringa oleifera

All parts of Moringa oleifera have been found to be medicinal in recent studies. The

hypoglycemic effects of phenolic glycosides isolated from Moringa oleifera seeds were tested

on HepG2 cells and STZ-induced mice. The results showed that STZ-induced mice had

significantly lower blood glucose levels, indicating that the molecule could be employed as a

safe hypoglycemic medication (Megh & Hafiz, 2020). Moringa oleifera has long been known

in most African countries for its various nutritional and medicinal benefits. The

pharmacological activities of Moringa oleifera are due to the diverse phytochemicals present

in the plant, such as flavonoids, alkaloids, saponins, saccharides, glucosinolates, tannins,

phenolic acids, and nitrile glycosides. Moringa oleifera has anti-inflammatory, anti-cancer,

antioxidant, antibacterial, cardio protecting, anti-hypertensive, hypoglycemic, and antiulcer

effects in its leaves, roots, and seeds (Bing-Xu et al, .2019).

According to recent research, all parts of Moringa oleifera are very rich in vitamin such as

essential amino acids, proteins, minerals, and vitamins, and are thus useful to prevent

10
malnutrition (Anzano et al., 2021). Moringa oleifera leaves and petals were historically

rubbed on the temples to relieve headaches. Eyes were also treated with the juice of the

leaves mixed with honey (Anzano et al., 2021).

2.7. Previous studies of Moringa oleifera seeds

Wheat biscuits or cookies have also been made with Moringa seeds. Moringa seed powder

(MSP) at a 20% concentration produced wheat cookies with a surface breaking pattern and

color identical to the control. It can be used to boost the nutritional value of bakery items like

cookies by up to 9% (depending on weight of wheat flour) without affecting their sensory

attributes (Maryam et al., 2020).

MSP has been reported to boost the nutritional value of bread made using wheat flour alone

or in combination with other flours (Rahmani et al., 2021). For example, using wheat flour

enriched with 5% MSP increased the protein and crude fibre content of bread by about 54%

and 56%, respectively (Rahmani et al., 2021). The bread samples with 5% MSP and 95%

wheat flour had the best sensory characteristics, and the proximate analysis revealed that

MSP addition significantly increased the protein (8.55 to 13.46 %), ash (0.63 to 1.76 %), fat

(7.31 to 15.75 %), and fiber (0.08 to 0.62 %) content (Shockery et al.,2017).

MSP has not been employed in the fortification of dairy products like yoghurt; instead,

Moringa leaf powder and extracts have been used, which have an undesirable green color.

The use of MSP can help to overcome the problem of color change (Shockery et al.,2017).

2.8. Phytochemicals in Moringa oleifera seeds

Phytochemicals such as alkaloids, glycerides, flavonoids, steroids, saponins, and tannins are

present in large concentrations in Moringa oleifera seed, regardless of the solvent used in

extraction (Akinyeye et al., 2014). Water, ethanol, and methanol are the most commonly used

solvents. Water is the most commonly employed solvent in the extraction of phytochemicals

11
from seeds because it is non-toxic and produces easy-to-handle extracts, although it is

ineffective (Kayshap et al., 2020). Other organic solvents, such as ethanol, acetone, and

methanol, are more efficient at releasing phytochemicals.

2.8.1 Flavonoids

Flavonoids are glycosylated derivatives that are water soluble and found in most plants. They

have a role in the formation of vibrant colors like blue, scarlet, and orange. They can be

found in several sections of medicinal plants, including the leaves, flowers, fruits, and seeds

(Gale, 2001). Flavonoids have high antioxidant activity. They work as oxygen quenchers in

the singlet and triplet ranges, as well as enzyme inhibitors, synergists, and peroxide

decomposers. Most flavonoids are more effective than vitamin E and vitamin C at mitigating

the harmful effects of reactive oxygen species (ROS). Flavonoids help the body metabolize

vitamin C and keep capillary walls healthy (Canturi-Castelvetri et al., 2000).

Flavonoids are all phenolic in nature and structurally originated from flavone, the parent

substance. Aurones, flavanones, isoflavones, betalains, anthocyanins, leucoanthocyanidins,

flavonols, flavones, glycoflavonesbiflavonyls, and chalcones are the ten classes of flavonoids

(Emmanuel et al., 2014). Flavonoids have anti-ulcerative, anti-inflammatory, antioxidant,

anti-tumoural, anti-allergic, and anti-hepatotoxic effects. The antioxidant and free radical

scavenging characteristics of flavonoids are responsible for these biological actions. The

antioxidant effects of flavonoids vary depending on the functional groups of various

flavonoids (Kayshap et al.,2022). Plants make flavonoids from the amino acids phenylalanine

and malonate. Fifteen carbon atoms are grouped in three rings in the basic flavonoid structure

(Olowule et al., 2013).

12
2.8.2 Phenolic compounds

Antioxidant capabilities of phenolic acids can be exhibited in a variety of ways. Phenolics

include hydroxyl groups, which are efficient proton or hydrogen donors. In a termination

reaction, they give protons to reactive oxygen and nitrogen species, preventing radical

production. The phenolic acids produce a radical form that is chemically more stable than the

first radical after contact with the first reactive species (Fatima et al.,2016).

Phenolic acids can produce free radicals, which are stabilized by electron delocalization in

the benzene ring. These radicals have the ability to alter radical-mediated oxidation

processes. Metal ions that contribute to the production of free radicals can be bound by

phenolic acids. Phenolic structures are amphoteric, which allows them to interact with other

molecules and proteins because of this characteristic (Halliwell & Gutteridge, 1984). The

hydrophobic benzenoic rings and the phenolic hydroxyl groups capable of hydrogen bonding

give them their amphoteric character. As a result, phenolic acids can block several enzymes

involved in radical generation, such as cytochrome P450 isoforms, lipoxygenases,

cyclooxygenase, and xanthine oxidase, making them antioxidants. Other antioxidants

including ascorbic acid, -carotene, and -tocopherol function in tandem with phenolic acids

(Fatima et al., 2016).

2.9. Oxidative stress

In its natural state, oxygen exists as dioxygen, which is a bi-radical. A bi-radical is made up

of two unpaired electrons with opposite spins (Dringen et al.,2000). The formation of free

radicals is an essential aspect of human metabolism, ROS by-products are constantly formed

as a result of aerobic metabolism, in which molecular oxygen is reduced by oxidative

phosphorylation in mitochondria. Free radicals are chemical species that are extremely

reactive and can exist on their own (Hallowell &Gutteridge, 1984).

13
The imbalance between the generation of free radicals and their neutralization by antioxidants

in the body is referred to as oxidative stress. This causes biological molecules to be

destroyed, mitochondrial function to be impaired, and the immune system to be

compromised, putting the body vulnerable to disease. The nervous system (brain, spinal

nerves) is particularly vulnerable to ROS production because it absorbs a large portion of

total body oxygen and has relatively low concentrations of antioxidant enzymes (superoxide

dismutase, catalase, and glutathione reductase) compared to other parts or systems in the

body. Lipid peroxidation is prevalent with unsaturated fatty acids. High amounts of iron

enhance the generation of ROS via iron catalysis (Dringenet al., 2000).

ROS cause gene mutations when they interact with DNA, lipids, protein, carbohydrates, and

other cell components. Aging, cancer, cardiovascular disease, diabetes, and other cell

degenerative disorders are all caused by gene mutations (Megh & Hafiz, 2020). To avoid the

development of these degenerative illnesses, oxidative stress must be controlled at the

cellular level. Polyphenol-rich foods are a promising strategy to reduce the harmful effects of

reactive free radicals in the body. Polyphenols are secondary metabolites that occur naturally

in medicinal plants and have a high antioxidant activity. Free radicals are scavenged and

absorbed, metal ions are chelated, and oxidase enzyme is inhibited by the polyphenols’

activity. In comparison to vitamin C, polyphenols are more effective at scavenging free

radicals (Megh & Hafiz, 2020).

2.1.0. The principle of antioxidants

Oxidation is the process of electrons being transferred from one atom to the next, resulting in

the molecule losing an electron and becoming oxidized. Oxidation occurs during aerobic

respiration, resulting in the formation of free radicals such as reactive oxygen species (ROS).

Antioxidants are substances or systems that can delay or prevent autoxidation by delaying or

stopping the generation of free radicals (Yehye et al., 2015). Natural and synthetic

14
antioxidants are the two main forms of antioxidants. Synthetic antioxidants are chemicals

formed through chemical methods, whereas natural antioxidants come from plants.

The initial, propagation, branching, and termination of free radicals are phases in the

antioxidant process. Antioxidants are classified as hydrophilic (vitamin C and polyphenolic

compounds) or lipophilic (antioxidants that are insoluble in water) (vitamin E and

carotenoids). Hydrophilic antioxidants are eliminated in the urine rather than being kept in

the body, but lipophilic antioxidants quickly diffuse into the lipoprotein cell membrane

(Huang et al., 2005).

Some of the endogenous antioxidants that predominantly fight against ROS are catalase

(CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD). Superoxide

anions and peroxides can be removed by these antioxidant enzymes. Ascorbate, -tocopherol,

glutathione (GSH), albumin, -carotene, uric acid, bilirubin, and flavonoids are all free radical

scavengers. They do not have any enzymes in them (Prior & Cao, 1984).

Antioxidants can also be grouped according to their mechanism; they can be either

“preventive” or “chain breaking”. Chain breaking antioxidants retard the formation of free

radicals from their unstable precursors (initiation) whilst preventive antioxidants interrupt the

radical chain reaction (propagation and branching) (Barclay et al., 2000). A chain-breaking

antioxidant readily donates its hydrogen atom to the free radical at a faster rate than the free

radical reacts with substrate. The radical which is formed after an antioxidant reacts with the

initial radical is very stable and is unable to continue the autoxidation of the chain. After this

mechanism of action, preceding antioxidants follow the hydrogen atom transfer (HAT)

mechanism (Barclay et al., 2000).

Antioxidant capacity assays are divided into mainly of two categories which are: (1)

hydrogen atom transfer (HAT) reaction based assays and (2) single electron transfer (SET)

15
based assays. The basis of SET is the redox reaction with an oxidant (Leopodini et al.,2011).

The oxidant can also be used to indicate when the reaction is completed. A natural radical

generator, an oxidizable molecule probe and an antioxidant are used in HAT-based

techniques. HAT and SET-based assays measure the oxidant’s radical scavenging capacity

rather than the sample’s preventative antioxidant capacity of a sample (Huang et al., 2005).

Figure 3. General pathways in a free radical chain oxidation and the action of antioxidants
(inhibitors). ROO˙ = free radical; AH = antioxidant (Huang et al., 2009).

16
2.1.1. DPPH assay

The DPPH assay method was originated by Blois (1958) in order to determine the antioxidant

activity using α-diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M=394.33). The assay

focuses on measuring the radical scavenging capacity of antioxidants on it. The lone electron

of nitrogen atom in DPPH receives a proton or hydrogen atom from antioxidants to the

corresponding hydrazine (Gjorgievski et al, 2015).

In general, DPPH is a stable free radical. Its stability is attributed to an electron's

delocalization over the whole molecule, which prevents the molecules from dimerizing like

most other free radicals. The DPPH's deep violet color is due to delocalization, and the color

is lost in its reduced form. The wavelength of DPPH absorption in ethanol solution is usually

about 520 nm (Sagar & Singh, 2011).

The antioxidant combines with the DPPH radical to produce the reduced DPPH form, and a

free radical. The second radical is less unstable than the first (Weststrate et al., 2002). The

DPPH assay is a common method for determining antioxidant activity in foods such as

cereals, vegetables, and herbs in a variety of solvent systems such as ethanol, aqueous

acetone, methanol, aqueous alcohol, and benzene (Yu, 2001). It is a good method for testing

cysteine, glutathione, ascorbic acid, and other antioxidants. This method of measuring

antioxidant activity can be used to compare results with other methods. Even with weak

antioxidants, DPPH can respond. The DPPH assay method can be used to analyze both

hydrophilic and lipophilic compounds in aqueous or nonpolar organic solvents (Huang et

al.,2005).

17
Figure 4. Chemical reaction of DPPH with antioxidant compound RH (Sagar & Singh.,
2011).

18
CHAPTER 3-METHODOLOGY

3.1. Research Design

The study took place between February and May of 2022. The experiments were carried out

in the Food Science Laboratory and the Chemistry Laboratory at Chinhoyi University of

Technology. Antioxidant activity, total phenolic content, presence of flavonoids, titratable

acid, pH, water holding capacity, sensory evaluation, and microbiological analysis (TCC,

LAB and yeast and moulds) were among the tests performed in this study over a period of

two weeks at 4°C storage.

3.2. Sampling Method

The convenience and simple random sampling techniques were used in this study. Moringa

oleifera seeds were collected from Domboshava District using simple random sampling. This

study used convenience sampling to get full cream UHT milk from a nearby supermarket in

Chinhoyi, Harare. Purposive sampling was done to determine yoghurt storage temperatures

(4ºC) and Moringa oleifera concentrations (0 %, 0.25 %, 0.50 %, and 1 %) in the yoghurt.

Each of the target samples had 3 triplicate samples analyzed.

3.3. Preparation of Moringa seed

The Moringa oleifera seeds were manually de-husked. The seeds were then ground into

powder using a pestle and mortar. Sieving was done through 500 micrometers to produce fine

powder. The Moringa oleifera seed powder was then packed into polythene bags and stored

in a dark, cool place.

19
Figure 5 Experimental design

3.4. Preparation of stirred yoghurt enriched with Moringa oleifera

Table 1 Formulation of enriched yoghurt with Moringa oleifera seed powder

20
Samples Yoghurt % M. oleifera seed powder % The

T0 100 0 standardised

T1 99.75 0.25 milk was heated

T2 99.5 0.50 to 42°C, which

T3 99.0 1.0 is the optimal

temperature for lactic acid bacteria growth (LAB). Sugar (9%) and Lactobacillus bulgaricus

and Streptococcus thermophilus commercial probiotic culture (2%) were added. Fermentation

was carried out in the incubator for around 5 hours at 42°C until a pH of 4.5 was reached.

Fermentation promotes the formation of lactic acid, which causes milk to curd. To stop

fermentation, the yoghurt was stirred and stored at 4°C in the refrigerator. Table 1 shows the

percentages of Moringa oleifera blended with yoghurt at 0%, 0.25%, 0.5%, and 1%. The pH,

water holding capacity, titratable acid, antioxidant activity, and total phenolic content were

then determined during a 14-day period. The microbial analysis was done in 10 days. All

Moringa oleifera and yoghurt analysis were done in triplicate.

3.5. Extraction of Moringa seed powder and yoghurt samples

Polyphenols were isolated from Moringa oleifera seed powder and yoghurt samples using the

method described by Unuigbe (2014). Approximately 10g of each sample was extracted in

100 mL of aqueous methanolic solution (75%) in a beaker wrapped in aluminum foil with

100 mL and stirred for 15 minutes with a shaker. The mixture was then centrifuged for 50

minutes at 3200 rpm at 4 ℃. Whatman No.1 was used to filter the resultant supernatants, and

the collected methanolic extracts were used in the TPC and AOA assays.

3.6. Determination of Antioxidant activity

The DPPH assay method, as modified by Shiraz (2014), was used to determine the free

radical scavenging activities of the sample extracts. A 0.1 mM DPPH solution in methanol

21
was prepared. In a dark environment, 3 mL of various concentrations (20, 40, 60, 80, and 100

g/ml) of extracts were added to 1 ml of 0.1 mM DPPH solution. The samples were incubated

in the dark for 30 minutes before being measured at 517 nm for absorbance. For statistical

analysis, all tests were carried out in triplicate. Similar tests were carried out using gallic acid

as a reference material, in which 1 mL of 0.1 mM DPPH solution was added to 3 mL of

various concentrations (20, 40, 60, 80, and 100 g/ml) of the gallic acid to be tested in. All of

the tests were carried out in the dark. After 30 minutes of incubation in the dark, the

absorbance was measured at 517 nm. The relative scavenging activity was calculated as the

percentage decrease in absorbance of the sample compared to the control.

Percentage of DPPH scavenging activity to be determined as follow:

DPPH Scavenging activity % = Absorbance of control – Absorbance of sample x 100

Absorbance of control

3.7. Determination of Total Polyphenol Content (TPC)

A 1 % solution of Gallic acid (10 mg/ml) was made by dissolving around 1 g of Gallic acid in

100 ml of methanol. Using the Folin-Ciocalteu technique, the total phenolic content of

Moringa oleifera seed powder and yoghurt samples was measured (Unuigbe, 2014). The

dilutions of (0.1, 0.5, 1.0, 2.5, and 5 mg/ml) in methanol from the standard 1 solution of

gallic acid were used to produce a standard gallic acid curve. Each of these dilutions was

mixed with 500 µl of water before adding 100 µl of Folin-Ciocalteu reagent and letting it sit

for 6 minutes. After that, 500 µl of distilled water and 1 ml of 7% sodium carbonate were

added to the reaction mixture. A spectrophotometer was used to measure the absorbance after

90 minutes at 760 nm. The same procedure was done in determining the TPC of Moringa

seed powder and yoghurt samples. Gallic acid equivalents (mgGAE/g) were used to quantify

the total phenolic content of the samples. All of the tests were carried out in triplicate

22
3.8. Determination of the presence of Flavonoids

The total flavonoids were determined using methanolic extracts. To 1mL of extracts, 5 drops

of sodium hydroxide were added. The solution turned to an intense yellow colour. Around 3

drops of 2M hydrochloric acid were added. The presence of flavonoids was indicated by a

shift in color from yellow to colorless after addition of the acid.

3.9. Determination of Physicochemical characteristics

3.9.1. Measuring pH

The pH of a solution is directly linked to the hydrogen ion activity and was determined by the

use of a digital pH meter which was recalibrated using buffers pH 4 and 7. The electrodes

were dipped into each of the samples until stable readings were obtained (AOAC, 2012).

3.9.2. Titratable acid

The AOAC (2012) procedures were used to determine titratable acidity. In a conical flask,

10g of material was weighed and 10ml of distilled water was added. Approximately 3 drops

of phenolphthalein indicator were added. Until the end point was attained, each sample was

titrated against a standardized 0.1mol NaOH (turns to a pale pink colour).

Titratable acidity was calculated using the following equation:

% Titratable acidity = Average volume of NaOH (ml) x 0.1 x 90 x 100

Sample weight (g) x 1000

Where, 0.1 = molarity of NaOH and

90 = molecular weight of lactic acid

23
3.9.3. Water holding capacity

The WHC of yoghurt samples was measured as described by Sagar & Singh (2011) with

slight modifications. Around 12ml of each sample was centrifuged at 3200rpm for 45 min,

the supernatant was collected from the residual and weighed separately.

WHC % = (Volume of sample – Volume of supernatant) x 100

Volume of sample

3.1.0. Microbiological analysis

3.1.0.1. Lactic acid bacteria count

Lactic acid bacteria (LAB) was enumerated using De Man, Rogosa, and Sharpe Agar (MRS

agar). It was made according to the manufacturer's instructions, which included suspending

the media in distilled water and warming it. The agar was gradually heated to boiling point to

dissolve it, and then autoclaved for 15 minutes at 121°C followed by immediate cooling.

Around 1ml of each sample was suspended in 9ml of buffered peptone water and serially

diluted. Up to 10^3 dilutions were formed. Each petri dish received 1ml of each sample,

which was transferred aseptically. Around 10-15mls of MRS were poured into the container.

The plates were swirled, set, and incubated for 48 hours at 37°C (Rahman et al.,2021).

3.1.0.2. Total yeast and molds

Total yeast and molds serial dilutions in ratios of 1:10 should were prepared using peptone

water. Around 0.1ml of the inoculum from each of the dilutions was picked and spread gently

on already sterilized Potato Dextrose Agar plates. The plates were incubated at 200C for

48hrs. The plates were then examined for colonies appearing on the medium, which were

then counted.

24
3.1.0.3. Total coliforms

VRBA was used to count the number of coliforms. Peptone water was used to make serial

dilutions at 1:10 ratios. Each dilution yielded 0.1ml of inoculum, which was gently placed

over already sterilized Violet Red Bile Agar (VRBA). The plates were to be incubated for 24

hours at 37°C. Colonies that appeared on the medium were counted after the plates were

examined.

CFUg − 1 = Number of colonies x Volume of dilute suspension

Dilution factor

3.1.1. Sensory evaluation

Fifteen semi-trained panelists from Chinhoyi University of technology evaluated the yoghurt

samples that had been stored overnight in the refrigerator. The acceptability test on

appearance, taste, flavour, consistency, and overall acceptability was conducted on a 9-point

hedonic scale: 9 = extremely desirable and 1 = extremely undesirable.

3.1.2. Data Analysis

Statistical analyses to determine the correlation coefficient or significant difference at 95%

were carried using SPSS version 20 software.

25
CHAPTER 4- RESULTS AND DISCUSSION

4.1. Antioxidant activity and Total Phenolic Content for Moringa oleifera seeds

Table 2.Total phenolic content and DPPH radical scavenging % for Moringa oleifera seed
powder

Moringa oleifera seed

DPPH radical scavenging % 45.8±0.05
Total Phenolic Content mgGAE/g 89±0.5

Table 2. represent the phenolic content and antioxidant properties of Moringa oleifera seed.

The total phenols content (mg gallic acid equivalents GAE/g) was 45.8±0.05 and the DPPH

radical scavenging % was 89±0.5. The results correlates with a research done by (Izza

et.al.,2018) on the Moringa oleifera seeds which indicated that the TPC of Moringa oleifera

seeds ranges from 6.70 mgGAE/g to 49.78 mgGAE/g. The amount of TPC mainly depends

on the extraction solvent and extraction time. The high antioxidant activity is due to the

presence of phenolic compounds which exhibit high antioxidant activity (Izza et.al.,2018).

The data in the table indicates that Moringa oleifera seed is rich in polyphenols and has high

antioxidant activity.

26
4.2. Qualitative test on Moringa seed powder and yoghurt samples for flavonoids

Table 3.Flavonoids screening of Moringa oleifera seed powder and yoghurt samples

Sample Flavonoids
Moringa seed powder (MSP) +
T0 (0%) -
T1 (0.25%) +
T2 (0.5%) +
T3 (1%) +
+ represents the presents of flavonoids
- represents the absence of flavonoids

Table 3. Data shows that flavonoids are present in all samples except sample T0 (0%).

Moringa oleifera seeds are a rich source of bioactive compounds i.e. polyphenols (range of

4581–4953 mg/100g) (Edith et al., 2018). The bioactive compounds include flavonoids which

are present in large amounts despite of the solvent used in extraction (Akinyeye et al., 2014).

This explains why there was a positive result for Moringa oleifera seed and the yoghurt

samples incorporated with the Moringa. Yoghurt is generally deficient in bioactive

compounds including polyphenols i.e. flavonoids. (Irena et al., 2022). The addition of plant

extracts as a source of phenolic compounds and flavonoids is necessary to overcome this

lack. Flavonoids are all phenolic in nature and structurally originated from flavone, the parent

substance. Aurones, flavanones, isoflavones, betalains, anthocyanins, leucoanthocyanidins,

flavonols, flavones, glycoflavonesbiflavonyls, and chalcones are the ten classes of flavonoids

(Emmanuel et al., 2014). Most flavonoids are more effective than vitamin E and vitamin C at

mitigating the harmful effects of reactive oxygen species (ROS). Flavonoids help the body

metabolize vitamin C and keep capillary walls healthy (Canturi-Castelvetri et al., 2000).

27
4.3. Total Phenolic Content (TPC) and Antioxidant activity (AOA) of yoghurt enriched

with Moringa seed powder during refrigerated storage.

Total Phenolic Content mgGAE/g

TPC AOA
50 100
45 90
40

DPPH Radical scavenging %

80
35 70
30 60
25 50
20 40
15 30
10 20
5 10
0 0
0 5 10 0 5 10
Storage (days) Storage (days)

T0(0%) T1(0.25%) T2(0.5%) T3(1%) T0(0%) T1(0.25%) T2(0.5%) T3(1%)

Figure 6. Total Phenolic Content Figure 7. Antioxidant activity

The antioxidant activity of yoghurt samples was determined using the DPPH assay method.

Yoghurt samples enriched with Moringa seed powder (MSP) indicated a significantly higher

total phenolic content and antioxidant activity, compared to the control yoghurt during the

storage period (10days). The antioxidant activity and total phenolic content of yoghurt

samples enriched with MSP showed a concentration-dependent pattern (p<0.05). In

particular, treatment T3 (1%), the one which had the highest percentage of Moringa seed

powder had the highest antioxidant activity % (87.4 ± 0.5), compared to treatment T0 (0%)

the control yoghurt (18.9 ± 0.5) at day 0 of storage. Treatment T3 (1%) had the highest TPC

(43.3mgGAE/g± 0.05) as well, compared to treatment T0 (0%) (2.9mgGAE/g ± 0.02) at day

0 of storage. These data show that Moringa is a very good source of polyphenols which has

high antioxidant activity. Moringa oleifera seeds are an excellent source of bioactive

28
compounds i.e. polyphenols (Leopodini et al., 2011). The current findings are related to those

reported by (Zhang et al.,2019) which showed that the addition of Moringa oleifera leaf

extract increased TPC in fortified yoghurt as compared to natural yoghurt and therefore

showed as higher AOA.

Accordingly, there was a certain decrease in AOA and TPC observed in all yoghurt samples

during the storage period (10 days). This was probably a result of the degradation of the

phenolic compounds by the effect of lactic acid bacteria. Polyphenols have a very high

affinity to proteins. Their interactions with proteins released by syneresis or peptides

originating from proteolytic processes during storage, results in the formation of polyphenol–

protein complexes which do not enter DPPH (Zhang et al.,2019).

29
4.4. pH and Titratable acidity of yoghurt samples

pH TA
1.4
5
4.5 1.2
4

Titratable acid (%)

1
3.5
0.8
3
2.5 0.6
pH

2 0.4
1.5
0.2
1
0.5 0
Day 0 Day 7 Day 14
0
0 7 14 Storage (days)
Storage (days)
T0(0%) T1(0.25%) T2(0.5%) T3(1%)

T0(0%) T1(0.25%) T2(0.5%) T3(1%)

Figure 8 pH of yoghurt samples Figure 9 Titratable acidity of yoghurt

samples

The pH of a sample correlates with the hydrogen ion activity. Yoghurt samples enriched with

Moringa seed powder (MSP) indicated a significantly lower pH and higher titratable acidity,

compared to the control yoghurt during the storage period (14days). The pH and titratable

acidity of yoghurt samples enriched with MSP showed a concentration-dependent pattern

(p<0.05). Treatment T3 (1%), had the least pH (3.9± 0.05), and treatment T0 (0%) (4.24 ±

0.03) had the highest pH at day 14 of storage. Treatment T3 (1%) had the highest titratable

acidity % (1.3± 0.05) and treatment T0 (0%) (1.09 ± 0.03) at day 14 of storage. These data

show that % increase in the incorporation of Moringa seed powder results in decrease in pH

and increase in titratable acidity. Changes in acidity and pH values correlates with the growth

of yoghurt starter culture (LAB) and their ability to break down lactose to form lactic acid.

Moringa encompasses phytochemicals like organic acids, phenolic acids, and flavonoids.

30
These components are considered to have a prebiotic role, they enhance the growth of LAB

and cause an accelerated drop in pH and a rise in titratable acidity (Hsieh & Ofori, 2007). In

previous studies, probiotic yoghurt supplemented with Moringa leaves extract also showed a

similar trend of pH changes during the storage period.

As shown in Figure 5, the data revealed that the values of acidity gradually increased

significantly while the values of pH gradually decreased significantly across all the samples

as cold storage progressed. Indicated that yoghurt culture is active even at low temperatures

and can ferment lactose into lactic acid, resulting in pH reduction and acidity formation

(Hsieh & Ofori, 2007). Streptococcus thermophilus being oxygen tolerant, converts lactose to

lactic acid thereby reducing pH to 5.2 while at pH 4.4 growth is dominated by Lactobacillus

thermophilus which acid tolerant and continues producing lactic acid at slow pace during

refrigerated storage. The optimum acidic conditions of commercial yoghurt should be in the

range of 7.0–9.0 mg/g of lactic acidity and pH 4.0–4.4 to avoid excessive acidic taste of the

yoghurt (Gadzirai et al.,2021).

31
4.5. Water holding capacity of yoghurt samples

WHC
100
90
80
70
60
WHC %

50
40
30
20
10
0
Day 0 Day 7 Day 14
Storage (Days)

T0(0%) T1(0.25%) T2(0.5%) T3(1%)

Figure 10. Water holding capacity of yoghurt samples

Water holding capacity is an essential physical property which can determine the

acceptability of yoghurt. It is linked to the capability of a protein to hold water in the yogurt

gel structure (Da-Hee Kim et al, 2019). In this study there was a significant difference

(p<0.05) between the different treatments at different storage days. Yoghurt samples enriched

with Moringa seed powder (MSP) indicated a significantly higher water holding capacity

(WHC %), compared to the control yoghurt during the storage period (14days). Treatment T3

(1%), had the highest WHC % (94± 0.05), and treatment T0 (0%) had the lowest WHC %

(86± 0.02) at day 14 of storage. The yoghurt samples enriched with MSP showed a

concentration-dependent pattern (p<0.05). This is due to some interactions between the

components i.e. polyphenols of MSP and the proteins in the yogurt. MSP is rich in

polyphenolic compounds, as shown by the total phenolic content assay (Fig. 6). These

polyphenols have a high affinity to proteins, they can form complexes with milk proteins

such as casein. This protein-polyphenol complex can contribute to the observed increase in

32
the WHC of MSP enriched yoghurt (Zhang et al, 2019). The polyphenol–protein complexes

formed during that period might have caused WHC values to increase until the end of the

storage period. Dietary fibers could retain water, increasing the WHC of yogurt. The

abundant dietary fiber in MSP may explain the high WHC of the yogurt (Da-Hee Kim et al,

2019).

33
Table 4. Microbial analysis of yoghurt produced with different concentration of Moringa
oleifera seed powder

Treatmen
Storage
Type of culture t (log
(day)
CFU/ml)
T1(0.25%
T0(0%) T2(0.5%) T3(1%)
)
0 9.3±0.01 9.4±0.05 9.5±0.01 9.5±0.01
Lactic acid bacterial 10.4±0.0
5 9.5±0.05 10.1±0.03 10.3±0.03
(LAB) 3
10 8.9±0.06 9.8±0.03 9.9±0.01 10±0.02
0 <1 <1 <1 <1
Yeast and Moulds 5 <1 <1 <1 <1
10 1.48±0.15 <1 <1 <1
0 <1 <1 <1 <1
Coliforms 5 <1 <1 <1 <1
10 <1 <1 <1 <1

The data obtained from this research shows no significant difference on the LAB count on

day 0. Yoghurt samples enriched with Moringa seed powder (MSP) indicated a significantly

higher LAB count, compared to the control yoghurt at day 5. In particular, treatment T3

(1%), the one which had the highest percentage of Moringa seed powder had the highest LAB

count, (10.4±0.03 logCFU/ml) compared to treatment T0 (0%) the control yoghurt (9.5±0.05

logCFU/ml) at day 5 of storage. These data show that MSP enhance fermentation activity. It

increases the growth of lactic acid bacteria (LAB) during yoghurt fermentation and this due

to the components of MSP such as polyphenols (Zhang et al., 2019). In addition, MSP is rich

in fiber, which can act as a prebiotic for the LAB thereby enhancing its growth (Izza et

al.,2012).

The total LAB count decreased but not significantly (p>0.05) by the 10th day of storage

across all samples. During the storage of the samples, pH values also decreased. The decline

in pH resulted in a highly acidic environment which is unsuitable for the growth of acid

34
intolerant Streptococcus thermophilus caused its death. This might also be due to low

refrigeration temperatures which limits the growth of LAB. LAB counts in all yoghurt

samples were over 7.0 Log CFU/g, which is the minimum requirement according to the

Codex Alimentarius (Da-Hee Kim et al, 2019)

Yeast and moulds were not detected in any of the yoghurt samples from day 0 to day 5. The

availability of lactic acid bacteria in yoghurt prevents the growth of fungi in yoghurt

(Osundahunsi et al.,2007). The low pH (<5) also limits the growth of yeast and moulds. Yeast

and moulds were not detected in the yoghurt enriched with MSP at day 10 whilst they were

present in the control yoghurt (1.48±0.15 log cfu/ml). Yeast and moulds count increased with

increase in storage time. The increase in the population of yeasts and moulds in T0(0%) is

due to reduction in pH or potential oxygen during fermentation process thereby creating

suitable environment for growth of yeasts and moulds. Levels above 10.0 cfu/g yeast and

moulds are unacceptable, as they can produce toxic metabolites i.e. mycotoxin leading to

food poison (Izza et al.,2012). Yeast and moulds were not detected in any of the yoghurt

samples enriched with MSP from day 10. Polyphenols in MSP prevented potential spoilage

by inhibiting the growth of spoilage microorganisms such as yeasts and molds during

fermentation. This shows that MSP has antimicrobial properties (Zhang et al.,2019).

Coliforms were not detected in any of the yoghurt samples from day 0 to day 10 of storage.

The absence of coliform bacteria (TCC) shows high level of hygiene and that the yoghurt

samples are free from fecal contamination. This corresponds with the statement of MacGraw

(1997) who stated that processed milk should contain no trace of coliform. Coliforms

generally must not be detectable in any 100 ml of yoghurt sample. The Coliforms were

supposed to be absent in yoghurt due to high temperature short time pasteurization and

effective cleaning and good hygienic procedures done (Adepoju & Selezneva, 2020).

35
4.6. Sensory evaluation of yoghurt samples

Sensory evaluation of yoghurt samples (9-point hedonic scale)

T0(0%) T1(0.25%) T2(0.5%) T3(1%)
Colour
10

5
overal aceptability Flavour

Consistency Taste

Figure 7.
11.Sensory
Sensoryevaluation
evaluationofofyoghurt
yoghurtsamples
samples

The scores for the sensory attributes (colour, consistency, taste, flavor, and overall

acceptability) of the yoghurt samples were illustrated in figure 6. by a radar chart. Generally,

the addition of MSP resulted in an increased consistency, but decreased overall acceptability

and flavor. The highest scores were shown for treatment T0(0%) on taste, colour, flavour and

overall acceptability. This is because most people are more familiar with the quality

properties of commercially available yoghurts enriched with fruits rather than herbs that have

a seemingly different taste (Zhang et al., 2019). The lowest score for all the sensory

properties except consistency was observed for T3(1%). The addition of MSP particularly

caused a notable decrease in the flavor score, compared to the control yogurt, T0. (P < 0.05).

This is due to the dominant herbal flavor of Moringa seed powder (El-Gammal et al., 2017).

However, the addition of 0.05% and 0.25% MSP to yoghurt did not significantly influence

the overall acceptability, compared to the control yoghurt. In relation to the sensory

evaluation data obtained in this research, yoghurt sample T2 (0.05%) could be used for the

36
production of MSP enriched yoghurt. At this concentration, the yoghurt sample showed

improved quality characteristics without negatively impacting the sensorial attributes.

CHAPTER 5

5.1. Conclusion

According to the findings obtained by the current study, it can be concluded that Moringa

oleifera seeds powder contributes to the increase in TPC and AOA without significantly

decreasing its sensory acceptability. The effects are concentration-dependent. MSP exhibited

prebiotic effects. The fiber and polyphenols present in the MSP can enhance the viability of

yoghurt culture (LAB). The physicochemical parameters of the Moringa enriched yoghurt

were slightly higher as compared to the control, they showed reasonable ranges for pH, TA

and WHC during storage. The best yoghurt sample was obtained at 0.5% of MSP, it showed

improved physicochemical properties without negatively affecting the sensorial properties. It

remained stable during storage. This work sheds light on the usefulness of Moringa seed to

produce novel functional yoghurt, which can provide a therapeutic strategy to mitigate non-

communicable diseases through its consumption.

5.2 Recommendations

 Further research into limiting or preventing post-fermentation acidification is

recommended by the researcher as the process reduces the shelf life of the product

while also increasing acidity, whey syneresis, and undesirable flavour.

 Further studies should be done on the in vivo tests to help in the justification of the

functional yoghurt claim.

 Further studies are should be done on Moringa oleifera seed powder to determine its

safety and toxicity for human consumption, sub-chronic and chronic toxic effects.

37
There should be guidelines as to the standard amount of MSP to be incorporated in

food products since over consumption may lead to adverse health effects.

38
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APPENDICES

Appendix 1: Questionnaire for sensory assessment of yoghurt samples using 9-point hedonic

scale.

Instruction: You are given different yoghurt samples coded, please evaluate them on the,

nine points hedonic scale shown below:

Remove the lid from the cups and evaluate the colour, taste, flavour, consistency and overall

acceptability. For consistency break down the yoghurt gel with spoon and gently mix the

samples. After placing product in your mouth evaluate the taste, flavour and overall

acceptability.

Then tick on the table below:

Overall Flavour Taste Colour Consistency

Acceptabi

lity

Like extremely

Like very much

Like moderately

Like slightly

Neither like or

dislike

Dislike slightly

Dislike moderately

45
 Dislike very much

Dislike extremely

Additional comments on the samples

…………………………………………………………………………………………………

…………………………………………………………………………………………………

………………………………………………………………………………………………....

Appendix 2: Standards

Standard curve of Gallic acid

Standard curve of Gallic acid (TPC)

1.4

1.2 f(x) = 0.219914304824012 x + 0.144489298553632

R² = 0.976872262485837

0.8
Absorbance (A)

0.6

0.4

0.2

0
0 1 2 3 4 5 6

Concentration (mg/ml)

46
Standard curve of Ascorbic acid

Graph between inhibition and Concentration (DPPH)

80

70 f(x) = 0.583775811209439 x + 13.3608652900689

R² = 0.991738943513485

60
Radical scavenging %

50

40

30

20

10

0
0 20 40 60 80 100 120

Concentration ug/ml

47
Appendix 3: Statistics
Descriptive

N Minimum Maximum Mean Std. Deviation

Day 0 12 4.3700 4.8000 4.436667 .1225734

Day 7 12 4.2700 4.4500 4.327500 .0687717

day 14 12 4.1200 4.2500 4.165000 .0466125

Valid N (listwise) 12

Anova

Sum of df Mean of F Sig.

squares squares

Day 0 Between Groups .000 5

.000
Within groups .000 12
.000
Total .000 7
Day 7 Between Groups 58.278 5
11.656 11.42 .000
Within Groups 12.667 12
1.056
Total 70.944 7
Day14 Between Groups 153.333 5
30.667 12.837 .000
Within Groups 28.667 12
2.389
Total 182.000 7

48
 Descriptive

95% Confidence Interval for Mean

N Mean Std. Std. Error Lower Bound Upper Bound
Deviation

T0 6 12.567 2.3544 1.3593 .1 18.415

T1 6 6.333 .5774 .3333 .0687717 7.768

T2 6 12.233 1.5948 .9209 .0466125 17.195

T3 6 4.233 .6807 .3930 2.542 5.924

Total 24 8.961 4.5556 1.0738 6.696 11.227

49