EFFECT OF BEETROOT EXTRACT (BETA VULGARIS .

L) ON STORAGE

STABILITY OF BEEF BURGER PATTIES.

By

MAGAYA ROSEMARY NATSAI

C18134870D

A dissertation submitted in partial fulfilment of the requirements for the Bachelor of Science

Honours Degree in Food Science and Technology

at

Chinhoyi University of Technology

School of Agricultural Sciences and Technology

Department of Food Science and Technology

June 2022

Supervisor: Dr A. Mpofu
 DECLARATION

I, MAGAYA ROSEMARY N, declare that this thesis is the result of my own research and

investigation, other than where sources are explicitly acknowledged and referenced within the

body of the text. This project has not been submitted to any university or any other institute for

any other degree. Furthermore, I also authorise the University to lend this thesis to other

institutions or individuals solely for the purposes of scholarly research.

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

i
 APPROVAL

This dissertation entitled ‘Effects of Beetroot (Beta vulgaris L.) extracts on storage stability

beef burger patties’ meets the regulation governing the award of the Bachelor of Science degree

in Food Science and Technology at Chinhoyi University of Technology.

Signature……………………………… Date…………………………..

ii
 ACKNOWLEDGEMENT

A very popular African Proverb reads, “It takes a village to raise a child.” Although, I am no

longer classified as a “child” I believe the “village” of Chinhoyi University of Technology has

done an excellent job preparing me for my future.

Firstly, I am grateful to my academic supervisor, Dr A. Mpofu for his support and dedication

during the time of my project. Without his constant knowledge, l would not have been able to

complete this work.

I want to thank all Food Science Department lecturers, for helping me with some aspects of

this project because without their help this project would not have been possible. To my fellow

students thank you for the inspiration and companionship. Finally I am thankful to my parents

and siblings. Thank you for moulding me into the person I am today and encouraging me to

pursue my ams, thank you for always being a phone call and for the guidance. Without their

emotional, spiritual, and financial support these past four years would not have been possible.

iii
 DEDICATION

This project is dedicated to my incredible parents and siblings. Without their emotional,

spiritual, and financial support these past four years wouldn’t have been possible. ‘Have I not

commanded you? Be strong and courageous. Do not be afraid; do not be discouraged, for the

Lord your God will be with you wherever you go ‘Joshua 1:9’.

iv
 ABSTRACT

Beef burger patties are perishable beef products with a short shelf life when kept refrigerated

(4°C) due to lipid oxidation and microbiological development. To limit lipid oxidation the

Standards Association of Zimbabwe (SAZ) has specified some synthetic additives that are

authorized in fresh minced beef and meat products to prevent deterioration. Consumer concerns

about the need to eat healthier foods has sparked interest in antioxidants derived from natural

sources. The effects of beetroot extracts (BE) against butylated hydroxytoluene (BHT) on the

storage stability of beef burger patties were evaluated during refrigerated storage at 4°C for 9

days, with analysis performed every 3 days. The pH, lipid oxidation, total phenolic content

(TPC), antioxidant activity (AOA %) and microbial analysis were determined. 0.04%BHT,

0.05%BE, 0.1%BE, and a control sample with no additive (0%BE) were used to make beef

burger patties. The results obtained showed a significant difference (p<0.05) between samples

during storage. In samples with 0.1%BE, TPC increased from 18.82 mgGAE/g±0.07 to 20.3

mgGAE/g±0.02, in samples with 0.05%BE, TPC increased from 16.24mgGAE/g±0.02 to

16.83mgGAE/g±0.03, in 0.04%BHT, TPC remained constant, and in 0%BE, TPC decreased

from 12.90mgGAE/g±0.01 to 12.77mgGAE/g±0.01. In 0.01% BE and 0.05% BE, the AOA%

increased from 66.2 to 82.83% and from 40.7 to 57.2%, respectively, whereas in BHT, the

AOA% remained constant at 88.6% and in 0%BE, it decreased from 15.45 to 5.42% during

storage. Total plate count readings were recorded as 7.32LogCFU/ml at the conclusion of

storage. Total plate count values in samples containing 0.05% BE, 0.04 % BHT, % BE, and

0%BE were 7.32logCFU/ml, 7.17logCFU/ml, 6.6logCFU/ml, and 7.41logCFU/ml,

respectively, at the end of storage. The peroxide value was 1.39 meq/kg for 0.1% BE and 1.48

meq/kg for 0.04 % BHT at the end storage. The highest acceptability rating was 8.2 for 0.1 %

BE burger patties. The results of this study showed that BE can be utilised as natural antioxidant

so as to improve storage stability of beef burger patties as well as other meat products.

v
TABLE OF CONTENTS

DECLARATION.................................................................................................................................... i
APPROVAL .......................................................................................................................................... ii
ACKNOWLEDGEMENT ................................................................................................................... iii
DEDICATION...................................................................................................................................... iv
ABSTRACT ........................................................................................................................................... v
List of Figures..................................................................................................................................... viii
List of tables.......................................................................................................................................... ix
CHAPTER 1 .......................................................................................................................................... 1
1.0 INTRODUCTION........................................................................................................................... 1
1.1 Background of the study....................................................................................................... 1
1.2 Statement of the problem ..................................................................................................... 4
1.3 Problem justification............................................................................................................. 4
1.4 OBJECTIVES OF THE STUDY ......................................................................................... 5
1.4.1 Main objective ................................................................................................................... 5
1.4.2 Specific objectives ............................................................................................................. 5
1.5 Research hypothesis .................................................................................................................... 5
CHAPTER 2 .......................................................................................................................................... 6
2.0 LITERATURE REVIEW .............................................................................................................. 6
2.1 Ground beef ................................................................................................................................. 7
2.2 Mechanism of Lipid Oxidation in Meat and Meat Products .................................................. 8
2.3 Antioxidants............................................................................................................................... 10
2.3.0 Primary antioxidants ......................................................................................................... 10
2.3.1 Secondary antioxidants...................................................................................................... 10
2.4 Natural antioxidants ................................................................................................................. 11
2.5 Synthetic antioxidants .............................................................................................................. 11
2.6 Antioxidants, mode of action.................................................................................................... 12
2.7 Plants as natural sources of antioxidants. ............................................................................... 12
2.8 Mechanism of Action of Natural Antioxidants....................................................................... 13
2.9 Application of Plant Extracts in Fresh Meat and Meat Products ........................................ 13
2.9 Extraction of Bioactive Compounds ........................................................................................ 14
2.10 Beetroot .................................................................................................................................... 15
2.11 Betalains ................................................................................................................................... 16
2.12 Nitrites in beetroots................................................................................................................. 18
2.13 Phenolic Acids and Flavonoids .............................................................................................. 19

vi
 2.14 Application of beetroot in meat products ............................................................................. 19
CHAPTER 3 ........................................................................................................................................ 21
3.0 MATERIALS AND METHODS ................................................................................................. 21
3.1 Research Design ........................................................................................................................ 21
3.2 Experimental design ................................................................................................................. 22
3.3 Sample collection and sample preparation ............................................................................. 23
3.3.1 Extraction of beetroot extract ........................................................................................... 23
3.3.2 Preparation of the burger patties ..................................................................................... 23
3.4 Determination of total phenolic content.................................................................................. 24
3.4.1 Preparation of gallic acid .................................................................................................. 24
3.4.2 Preparation of sample for total phenolic content ............................................................ 24
3.5 Determination of antioxidant activity of beetroot extract ..................................................... 24
3.6 Determination of lipid oxidation in beef burger patties. ....................................................... 25
3.7 pH determination ...................................................................................................................... 26
3.8 Microbial analysis of burger patties ........................................................................................ 26
3.8.1 Total yeasts and molds analysis ........................................................................................ 26
3.8.2 Total Bacteria Count ......................................................................................................... 27
3.8.3 Total coliforms ................................................................................................................... 27
3.8 Sensory analysis ........................................................................................................................ 27
3.9 Statistical Analysis .................................................................................................................... 28
CHAPTER 4 ........................................................................................................................................ 29
4.0 RESULTS AND DISCUSSION ................................................................................................... 29
4.1 Total phenolic content .............................................................................................................. 29
4.2 DPPH assay: Antioxidant activity ........................................................................................... 32
4.3 pH ............................................................................................................................................... 34
4.4 Lipid oxidation .......................................................................................................................... 35
4.5 Microbiology.............................................................................................................................. 38
4.5.1 Total Plate Count (logcfu/ml)................................................................................................ 38
4.5.2 Coliforms (logcfu/ml) ............................................................................................................. 39
4.5.3 Yeast and moulds (logcfu/ml)................................................................................................ 40
4.8 Sensory evaluation. ................................................................................................................... 42
CHAPTER 5 ........................................................................................................................................ 44
5.0 CONCLUSION AND RECOMMENDATIONS ........................................................................ 44
5.1 Conclusion ................................................................................................................................. 44
5.2 Recommendations ..................................................................................................................... 44
REFERENCES .................................................................................................................................... 46

vii
Appendices ........................................................................................................................................... 50

List of Figures

Figure 1: Flowchart for ground meat preparation ..................................................................... 7

Figure 2: Flow chart diagram of experimental design. ............................................................ 22

Figure 3:Radical scavenging activity (%) during storage ........................................................ 32

Figure 4:pH values of treated and untreated beef burger patties during storage. .................... 34

Figure 5:Peroxide values of treated and untreated beef burger patties during storage. ........... 36

Figure 6:Sensory attributes of treated and untreated samples. ................................................ 42

viii
List of tables

Table 1:Total phenolic content during storage ....................................................................... 29

Table 2:Mean total plate count log (cfu/ml) during storage .................................................... 38

Table 3:Mean coliform counts log (cfu/ml) during storage. .................................................... 40

Table 4:Mean yeast and moulds log (cfu/ml) during storage. ................................................. 41

ix
 CHAPTER 1

1.0 INTRODUCTION

1.1 Background of the study

Meat is a highly nutritious and multi-use food, consisting of high quality proteins, B-complex

vitamins and minerals especially iron and zinc with a high level of bioavailability (Shah et al.,

2014). Fresh ground beef is widely used all over the world as a basic ingredient of various food

preparations including burger patty formulations. However fresh ground beef meat is highly

susceptible to quality deterioration because of high nutrient and moisture content. Meat quality

deterioration affects lipids, proteins, carbohydrates, vitamins, and pigments. Oxidative

deterioration cause mainly lipid oxidation and results in loss of nutritional quality, sensorial

attributes and reduce shelf-life of meat-based products (Liu et al., 2015).

Lipid oxidation, which is initiated in the unsaturated fatty acids fraction in subcellular

membranes, is a major cause of the deterioration and reduced shelf-life of meat products

causing changes in meat quality parameters such as colour, flavour, odour, texture and even

nutritional value (Kolakowska, 2002). Being susceptible to deterioration, food additives can be

added during the preparation of minced meat products.

A lot of meat preparations are preserved by synthetic antioxidants. The most popular synthetic

preservatives, butylated hydroxylanisole (BHA), butylated Hydroxyltoluene (BHT) and

tertiary butylhydroquinone (THBQ), are widely utilized as synthetic antioxidant to inhibit or

1
prevent the origination of free radicals, thereby reducing lipid oxidation and extending the

shelf-life of meat ( Lu and Pham-Mondala, 2018).

There is less use of natural based additives which are safe compared to the synthetic additives

(Munekata, 2021). The use of synthetic additives in the meat industry has some advantages

which include colour enhancement, maintaining nutritional value and most of the additives are

used to make shelf stable meat products. However data in literature have some evidence that

these synthetic additives can cause adverse effects on human health including allergy,

headache, asthma and dermatitis (Aliakbarlu et al., 2016). Due to these reasons the application

of natural pigments preservatives is a growing interest due to the safety concerns of synthetic

additives. The consumers have become very health conscious and as a result, these synthetic

additives are being closely assessed by consumers. Consumers are constantly requiring

nutritionally improved meat products of high quality, healthy and more natural which is

beneficial to human health. (Domínguez, 2020) stated that consumers are now requiring

products that are convenient, nutritional, and safe. Because of the increasing consumer demand

for “healthier” foods that are free of synthetic additives, the use of natural preservatives have

gained a lot of interests and suggestions have been made in order to use natural preservatives

which can potentially replace synthetic additives (Shawky, 2018). Natural preservatives have

shown a potential in providing effective preservation activities (antioxidants and antimicrobial)

while reducing negative health effects. Hence, meat manufacturers and researchers have begun

to consider the use of natural rather than synthetic preservatives.

There are many different types of vegetables that contain bioactive compounds, and additional

features of some these vegetables include uses as natural colorants, antimicrobial and

antioxidants. Natural antioxidants from medical plants such as clove, sage, rosemary, green

2
tea, oregano, and moringa olifera have been investigated (Ahn et al., 2007) on their effects on

microbial growth and lipid oxidation during storage of different types of meat and meat

products. These studies have shown some positive effects on preserving meat and meat

products (Canadanovic-Brunet, 2011).

Red beetroot (Beta vulgaris L) is considered the most significant source of betanin (Geogiever

et al., 2010). It is a root vegetable grown all over the world. Red beetroot is less consumed,

however it can be consumed raw or roasted, frequently served in soups or on salads. Red

beetroot is composed of 87.57% water, 9.56% carbohydrates (29.3% fibre and 70.7% sugar),

1.61% protein, and 0.17% lipids in addition to being a source of potassium, choline, vitamin

C, and niacin (Ceclu and Nistor, 2020; USDA, 2011).

Red beetroot is a popular vegetable grown in many parts of the world mostly in cold areas. Red

beetroot is popular in Germany, UK and Russia since the plant thrives in cold climates. For

this reason this is why the vegetable is seasonal in Zimbabwe. Red beetroot allows for the

extraction of a wide range of bioactive compounds, with a particular focus on meat products.

Colorants (betalains), antioxidants (betalains and phenolic compounds), and preservatives

(nitrates) are among the compounds that can be extracted from beetroot and added to food

products, reducing the usage of synthetic additives while also extending shelf life (Jasna, 2011).

Despite these advantages, the use of beetroot extracts in the meat industry is limited, and little

research has been done on employing beetroot extract as an antioxidant in beef burger patties.

The aim of this study is to determine the effects of red beetroot extract on the storage stability

of beef burger patties.

3
1.2 Statement of the problem

Beef burger patties are highly susceptible to quality deterioration because their main ingredient

ground beef meat can get spoiled due high nutrient content, moisture content and also it is

subjected to a lot of handling during preparation. Use of multiple synthetic antioxidants in the

meat industry has helped in limiting spoilage and quality deterioration. However, continuous

use of synthetic antioxidants in beef burger patties have harmful effects. Some of the

preservatives that are used in beef burgers patties include nitrites and nitrates and consumption

of high levels of nitrate may be a potential health risk as they result in the formation of

carcinogenic nitroso-compounds. However, consumers are now interested in consumption of

with a healthiness appeal including consuming foods with less or no artificial additives rather

they need natural additives since consumers tend to associate food with synthetic additives

being unhealthy. With this in mind, in order to achieve consumer friendly products of meat

which is shelf stable this study aims to study the effects beetroot extract on storage stability of

beef burger patties.

1.3 Problem justification

Red beetroot extracts can be used as preservatives due to their biological bioactive compounds

(antimicrobial and antioxidant properties) that can be very effective in slowing down the

deterioration of burger patties by preventing the lipid oxidation and spoilage and therefore

increasing the shelf life of patties (Ghazy, 2020). The use of red beetroot extracts as in beef

burger patties is of importance as it is a way of exploiting natural source of natural antioxidants

while limiting the use of synthetic additives. The use of extracts as an alternative natural source

of additives in burger patties could fulfil both consumers’ health demand and improve the

storage stability of other types of meat and meat products. Red beetroot can be a good source

4
of colorants in meat products including the beef burger patties since the antioxidants contain

the most dominant colouring compound betalains (Pavlović, 2013).

1.4 OBJECTIVES OF THE STUDY

1.4.1 Main objective

To evaluate the effects of beetroot extracts on the storage stability of beef burger patties.

1.4.2 Specific objectives

1.4.2.1 To determine the potential antioxidant capacity and total phenolic content of

Beetroot Extracts.

1.4.2.2 To determine lipid oxidation of beef burger patties incorporated with Beetroot

Extracts.

1.4.2.3 To determine the pH of beef burger patties incorporated with Beetroot Extracts.

1.4.2.4 To determine microbial stability of beef burger patties incorporated with

Beetroot Extracts.

1.4.2.5 To evaluate the sensory properties of beef burger patties incorporated with

Beetroot Extracts.

1.5 Research hypothesis

H0 - Red beetroot extract do not significantly affect the storage stability of beef burger patties

H1 - Red beetroot extract significantly affect the storage stability of beef burger patties.

5
 CHAPTER 2

2.0 LITERATURE REVIEW

Meat and poultry are considered an excellent proteins source, some fat soluble vitamins and

minerals especially for people living in the developed countries. These meats also comprises

of monounsaturated fatty acids and saturated fatty acids. Typical constituents of ground beef

is reported to be about 18 g lipids/100 g total mass and its fatty acids content is divided into

about 46 g/100 g saturated fatty acids, 51 g/100 g monounsaturated fatty acids, and 3 g/100 g

polyunsaturated fatty acids (Mensink, 2005). Fresh meat and meat products are susceptible to

quality deterioration and lipid oxidation because of the higher nutrient constitution, pH, water

activity and high fat content (Mokhtar and Youssef, 2014). Lipid oxidation and microbial

activity are two key factors that triggering deterioration of meat and meat products and these

are posing great challenges to the meat industry in preserving meat and meat products. Lipid

oxidation in food components is undesirable, since it causes rancidity, polymerization and off-

flavours, which eventually lead to reduction in shelf-life and nutritive value to food. Lipid

oxidation and the oxidation of myoglobin cause deterioration of the nutritional quality, colour,

flavour, texture and safety of meat and meat products (Domínguez et al., 2019) as well as

development of off flavours and odours.

Meat and meat products provide excellent growth media for a variety of microorganisms

(bacteria, yeasts and moulds) some of which are pathogenic (Datta, 2012). Ground beef meat

is considered a conducive environment that favours the growth of several spoilage and

pathogenic bacteria. The most common genera of bacteria found in meat before spoilage is

Staphylococcus, Bacillus, Campylobacter, Clostridium, Listeria, Salmonella which result in

quality deterioration which is responsible for shelf-life reduction (Osman and Faruk 2016).

6
2.1 Ground beef

The process of making ground beef include deboning and exsanguinating the carcass, dividing

meat into portions (arms, thighs and loin) and then each portion is cut into blocks and sliced.

In order to make ground beef obtained blocks of processed meat are grounded using a grinder

or a mincer (Shimizu and Iwamoto 2022).

Livestock Carcass portion Slice for meat Grinding

Ground
beef

Figure 1: Flowchart of the process for ground meat preparation.

Ground beef has always been one of the most popular beef items sold in retail and food service,

due to its affordable price and versatility. Recently there have been a substantial increase in the

production and consumption of minced beef worldwide. The reason for this increase is due to

rapid population growth, quick growth in the fast food market, and great nutritional value and

palatability of beef meat products (Tarig et al., 2018).

Due to changing life style beef burger is almost the most popular meat product consumed as a

fast meal by millions of people from all over the world. Beef burger patties are handled

extensively throughout the production process and their potential for contamination is high.

Grinding meat during the production of burger patties disturbs muscle membrane integrity,

exposes lipid membranes to metal ions, and enhances the interaction of pro-oxidants with

unsaturated fatty acids, resulting in the generation of free radicals and the progression of

oxidative reactions (Ibrahim et al., 2010; Yogesh and Ali, 2014). In fact, the grinding process

7
results in the leakage of tissue fluids, which serve as a rich source of nourishment for a variety

of bacteria, fostering rapid microbial growth (William 2011). Because of the larger surface area

that allows direct interaction between lipid and air, and the better accessibility of oxidation

promoters such as released heme and non-heme iron from meat pigments hemoglobin,

myoglobin, and phospholipids from disrupted cells, ground beef is more vulnerable to

oxidation than whole meat cuts.

When the animal dies, blood flow stops and metabolic processes are disturbed, causing the

onset of oxidation. Many chemical reactions form the steps that initiate the degradation process,

resulting in a variety of complex by products. Unsaturated fatty acids serve as the reaction's

substrate, and catalysis occurs as a result of the action of oxygen, light, heavy metals, and other

factors, making it one of the major causes of meat product deterioration.

2.2 Mechanism of Lipid Oxidation in Meat and Meat Products

Lipid oxidation can occur at three stages: prior to slaughter, during slaughter (when muscle is

converted to meat), and after slaughter (during processing and storage). The initiation,

propagation, and termination stages of lipid oxidation are defined as a chain reaction of free

radicals. During the reaction, a free radical combines with the fatty acid's hydrocarbon chain,

generating peroxides, which then react with other hydrocarbon chains, abstracting hydrogens

and forming hydroperoxides (Hiromi and Satoshi, 2022). Lipid oxidation is defined as the

degradation of saturated and unsaturated fatty acids in the presence of oxygen. This fatty acid

alteration is mostly accomplished through an autocatalytic mechanism of free radicals known

as auto-oxidation.

8
An alkyl radical is formed in the initiation stage when a hydrogen atom is abstracted from a

neighbouring carbon and attached to a double bond in an unsaturated fatty acid. The initiation

reaction can be catalyzed by heat, light, and transition metals (Hecke, 2017). The alkyl radical

from the initiation step can react with molecular oxygen in the propagation step to release

several radical species, including the peroxyl (ROO•), another free radical. These radicals then

take a hydrogen atom from another vulnerable molecule, resulting in the formation of lipid

hydroperoxides (Min and Ahn, 2005). This creates a new alkyl radical that is quickly

transformed into peroxyl radicals. The chain reaction continues until the chain-carrying peroxyl

radical combines with another radical to form stable lipid oxidation products in the termination

step (Dominguez, 2019). Although hydroperoxide is stable at physiological temperatures, it

decomposes when exposed to transitional metal ions or heated to high temperatures (Min and

Ahn, 2005). Hydroperoxides can degrade into a variety of volatile and non-volatile chemicals,

including carbonyls (e.g., ketones and aldehydes), alcohols, hydrocarbons (e.g., alkane and

alkene), and furans, all of which contribute to the flavor degradation of many foods.

In live animals, intrinsic factors such as enzymes (superoxide dismutase, catalase, etc.) and

specific proteins and their mechanisms (transport proteins), as well as oxidative reaction-

breaking antioxidants (vitamin E and C), are available to limit the oxidation reaction in muscle

tissues (Mokhtar and Youssef, 2014). Due to numerous post-slaughter conditions, such as

anaerobic environment, presence of pro-oxidants, and lack of enzymatic antioxidative

processes, these factors lose their antioxidative potential after slaughter making meat

susceptible to oxidation. This susceptibility of ground meat and its products to oxidation and

microbial spoilage has challenged the food technologists to come up with techniques to extend

its shelf life including the addition of food additives such as preservatives and antioxidants

(Ibrahim et al., 2010). These are allowed to be added in meat and meat product to slow down

9
or inhibit the oxidation and improve the storage stability. Improving the storage stability and

quality of meat products is critical for both economic and nutritional reasons.

2.3 Antioxidants

Antioxidants are substances that can prevent or slow damage to cells caused by free radicals.

These antioxidants increase the resistance of fats to oxidation and consequent deterioration or

rancidity. Antioxidants are substances that delay oxidation by inhibiting initial free radical

formation or by preventing them from producing more free radicals which can extend the

reaction (Santos-Sánchez et al., 2020).

Based on the source of origin, antioxidants can be divided into two types; natural antioxidants

and synthetic antioxidants. The synthetic antioxidants increase the risk of mortality in adult

due to rigorous toxicity and the increase risk of cancer that they may cause in comparison to

natural antioxidants.

2.3.0 Primary antioxidants: They are also known as chain-breaking antioxidative compounds.

Primary antioxidants react directly with lipid radicals to prevent or delay oxidation by

scavenging free radicals and converting them to more stable products by donating hydrogen

atoms or electrons.

2.3.1 Secondary antioxidants: They slow oxidation by binding metal ions (Fe2+, Fe3+, and

Cu2+), scavenging oxygen, converting hydroperoxides to non-radical species, absorbing UV

radiation or deactivating singlet oxygen, and inhibiting enzymes (Santos-Sánchez, et al 2020).

Some natural phenolic compounds have both primary and secondary antioxidant properties.

10
2.4 Natural antioxidants

Natural antioxidants are polyphenol compounds that have the potential to donate hydrogen and

are found primarily in natural plants such as herbs, spices, fruits, and vegetables (Reddy et al.,

2018). Due to the presence of phenolic acids, stilbenes, and flavonoids in their extracts, which

have a high antioxidant activity through three mechanisms: free-radical scavenging activity,

transition-metal chelating activity, and/or singlet-oxygen quenching capacity, these plants

show strong free radical scavenging ability and antibacterial activity (Georgios, 2020). As a

result, researchers have been trying to incorporate these natural antioxidants into meat products

in order to reduce lipid and protein oxidation.

2.5 Synthetic antioxidants

Meat and meat products can only contain antioxidants that have been approved by the FDA.

BHT, BHT, PG, and TBHQ are some of the most commonly used synthetic

antioxidants. These antioxidants can effectively reduce oxidative stress in meat products and

prevent damage to macromolecules such as lipid and protein fractions, and they are preferred

by food manufacturers due to their low cost. However, these antioxidants have the disadvantage

of being volatile and decomposing at high temperatures (Ergezer & Serdarolu, 2018; Jiao et

al., 2020). They are also reported as having carcinogenic properties (Martinez-Tome et al.

2001). BHT has also been shown to be harmful in at least one research, especially at large

doses. Synthetic antioxidants, on the other hand, have been found as antinutritional,

toxicological, and carcinogenic agents in several research (Forsh, 2013), and as a result,

consumer demand for natural antioxidants is increasing.

11
2.6 Antioxidants, mode of action

Antioxidants have a variety of modes of action. Antioxidants work by scavenging species that

induce peroxidation, chelating metal ions so that they cannot generate reactive species or

decompose lipid peroxides, quenching O2 to prevent peroxide formation, breaking the

autooxidative chain reaction, and/or lowering localized O2 concentrations (Dorman et al.,

2003; Shah et al., 2014).

Although a large number of compounds have been proposed to have antioxidant action, only a

few of them can be incorporated in food. The use of antioxidants in food is regulated by national

or international regulations (Xiu-Qin et al., 2009). Antioxidants can come from either synthetic

or natural sources. Concerns about the safety and toxicity of synthetic antioxidants, combined

with customer demand for natural and healthful products, have sparked interest in the use of

natural antioxidants in the food business, particularly those found in plants. Natural

preservatives are becoming more popular as a means of preventing oxidation and the growth

of microbes in food (Ghazy, 2020).

2.7 Plants as natural sources of antioxidants.

Plant-derived antioxidants can be found in fruits, vegetables, spices, herbs, cereals, grains, and

seeds. It is also worth noting that the co-products produced during the processing of these foods

can be a source of antioxidants, whose utilization would allow them to be revalued, avoiding

the significant environmental and economic costs connected with these wastes. The presence

of chemicals with substantial antioxidant activity, primarily polyphenols and terpenoids, in the

extracts derived from these plant components is mostly responsible for their effectiveness

(Geogios et al., 2020). It is also worth noting that the advantages attributed to plant extracts are

12
not due to a single class of compounds, but rather to the numerous contributions of several

bioactive components.

2.8 Mechanism of Action of Natural Antioxidants

The majority of natural antioxidants are phenolic compounds, with tocopherols, flavonoids,

and phenolic acids being the most important. Phenolic acids, phenolic diterpenes, flavonoids,

and volatile oils are the main antioxidative phenolics. Phenolics found in natural antioxidants

have a high radical-absorbance capacity or have a significant H• donating activity (Santos-

Sánchez et al 2019). These natural antioxidants work by decomposing peroxides, chain

breaking to prevent continued hydrogen abstraction by active radicals; decreasing localized

oxygen concentrations; preventing chain initiation by scavenging initiating radicals;

decomposing peroxides, chain breaking to prevent continued hydrogen abstraction by active

radicals. Some phenolics prevent the formation of free radicals and propagation of the reactive

oxygen species, whereas, other scavenge free radicals and chelate pro-oxidants (Dorman et al

2003). Phenolic acids trap free radicals; flavonoids scavenge free radicals and chelate metals

(Fe2+, Fe3+, and Cu2+) as well.

2.9 Application of Plant Extracts in Fresh Meat and Meat Products

Meat and meat products are prone to oxidation processes, particularly those involving the

breakdown of lipids, due to their composition (Kurmar et al., 2015). As a result, antioxidants

are needed to preserve the quality of these products. Natural antioxidants, particularly those

containing polyphenols, can be a potential strategy because of their ability to scavenge free

radicals. Several papers have focused on the use of natural antioxidants (extracted from herbs,

spices, fruits, and vegetables utilizing green solvents and/or developing technologies) in fresh

meat products to reduce lipid and protein oxidation (Aklyn et al., 2020). Numerous studies on

13
plant polyphenols, as natural preservatives in meat and meat products, have been reported.

These include rosemary (Hes et al., 2017), clove extracts (Zahid et al., 2020), green tea (Ozen

and Soyer, 2018), turmeric (Zhang et al., 2015), and Moringa oleifera leaf (Mukumbo et al.,

2019). In their review paper, (Das et al., 2019) and co-authors noted that although there are

many types of bioactive molecules of natural origin for meat preservation, such as those from

animal and microorganisms, the greatest interest for meat manufacturing is centered on using

plant extracts rich in bioactive molecules. In their ground beef patties, Mansour and Khalil

(2000) used potato peel, fenugreek seeds, and ginger rhizomes extracts. Aloe vera, fenugreek,

ginseng, mustard, rosemary, sage, and tea catechins extracts were used in pig patties by Mc

Carthy et al. (2001a). Extracts from the leaves and secondary branches of rosemary

(Rosmarinus officinalis) and hyssop (Hyssopus officinalis) were employed in hog flesh

(Frenandez-Lopez et al., 2003). In beef patties, extracts of myrtle (Myrtus communis myrtillus

L.), rosemary (Rosmarinus officinalis L.), nettle (Urtica dioica), and lemon balm (Melissa

officinalis L.) leaves were studied (Akarpat et al., 2008).

Tang et al., 2006; Garca et al., 2009; Velasco and Williams, 2011; Kumar et al., 2015; Nowak

et al., 2016, investigated the use of natural antioxidants for meat preservation through the

reduction of both microbial growth and lipid oxidation during storage; their involvement in

maintaining quality parameters and improving health benefits was also investigated.

2.9 Extraction of Bioactive Compounds

The goal of the extraction procedure is to isolate polyphenol molecules at a high yield of

molecules of the highest quality in terms of target chemical concentration and extract

antioxidant potential (Beya et al., 2021). Soxhlet extraction, maceration, and stirring (Machado

14
et al., 2017), supercritical fluid extraction (Wijngaard et al., 2012), subcritical water extraction

(Singh et al., 2011), and ultrasonic aided extraction are some of the strategies used to extract

and recover bioactive compounds from plants (Viklu et al., 2008). In most cases, the plant

material is washed, dried, and crushed into a fine powder before being extracted using a solvent.

For the extraction procedure, many solvents have been utilized, either separately or in

combination. Absolute ethanol, 90% ethanol, 80% ethanol, 70% ethanol, acetone, and other

solvent solutions have been used in extraction of different bioactive compounds.

2.10 Beetroot

The beetroot (Beta vulgaris L.) is an herbaceous plant that thrives in temperate climates and

belongs to the Chenopodiaceae family. Beetroot is the huge, fleshy root of the same-named

plant, which is consumed as a vegetable. Its thin, smooth skin comes in a variety of colors,

ranging from purple-pink to reddish-orange to a brownish tone. The pulp is usually a dark ruby

red color with purple tinges and has a sweet taste. It produces 20% of the world's sugar and can

be consumed raw or powdered, as a complement to juice, bread, and pickles, as well as pureed,

boiled, and in salads (Kumar, 2015). Micronutrients such as potassium, magnesium, folic acid,

iron, zinc, calcium phosphorous, and vitamin B6 are found in red beetroot extracts. They also

include a unique phytonutrient known as betalains, which are basically nitrogen-containing

pigments that are often used as a dye for commercial red food colorants (Ceclu and Nistor,

2020). Beetroot also contains antioxidants such as flavonoids and carotenoids, which suppress

cancer cell proliferation and improve low-density lipoprotein oxidation resistance (Kujala,

2001).

15
Beetroot ranks among the 10 most powerful vegetables with respect to its antioxidant capacity

ascribed to a total phenolic content of 50–60 mol/g dry weight. Beetroot is also one of the few

vegetables that contain a group of highly bioactive pigments known as betalains as well as other

bioactive compounds that include nitrate, phenolics, ascorbic acid and carotenoids (Kushwaha et

al., 2019). Red beet is a rich source of polyphenols together with the betalains, and these are

two families of compounds demonstrating a high antioxidant effect and radical scavenging

capacity (Pavlović, 2013). The food industry has been focused on using natural sources of

bioactive compounds with multiple functionalities to obtain healthy products. It is rich in

betalains and phenolic compounds. Beetroot is consisted of multiple biologically active

phytochemicals including betalains, flavonoids, polyphenols, saponins and inorganic (Diego et

al., 2017).

Due to the high content of betalains (pigments with a deep and powerful purplish-red color and

antioxidant activity), phenolic compounds (antioxidants), and inorganic nitrate, both juices and

beetroot powder offer an excellent opportunity to extend the shelf life of meat and meat

products while also limiting the use of synthetic additives and/or potentially replacing them

(Merredy and Sultanawa 2017; Aykn-Dinçer, 2020).

2.11 Betalains

Betalains are divided into two groups, the betacyanins, which have a red-violet coloration, and

betaxanthins, which have a yellow-orange coloration, are the most important compounds found

in red beetroot. Betalains are free radical scavengers that protect biological molecules from

active oxygen-induced and free radical-mediated oxidation (Georgiev et al., 2016). Some

researchers found that betacyanins accounted for 80–90% of total betalains, whereas others

16
discovered that betacyanin concentrations ranged from 50–70% of total betalains. Because

betalains are less harmful than synthetic colorants and do not induce allergic reactions, they

offer a great deal of promise for usage as natural colorants in meat products. A study by (Aykın-

Dinçer, 2020) beetroot extract was used as a natural colour and it had an acceptable colour.

The extracted betanin from betalains have been approved as a natural colouring by the Food

Additive Act.

In addition to the colouring properties many studies indicate that betalains have high

antioxidant power. Since their antioxidant activity have been defined in a wide range of assays

(Jasna 2011; Lawrence 2016; Edziri et al., 2020), and it was discovered that enriching human

low-density lipoproteins with betalains effectively enhanced resistance to oxidation, interest in

betalains has grown. The antioxidant and free radical scavenging activity of betalains in

beetroot is comparable to that of the synthetic antioxidant BHT. Betanin's antioxidant action is

related to the presence of hydroxyl and cyclic amine groups, which are good hydrogen and

electron donors and can stabilize reactive species.

Several research looked into using natural antioxidants to preserve meat by reducing microbial

growth and lipid oxidation during storage, as well as their role in maintaining quality attributes

and stability (Georgis, 2020; Shawky, 2018; Abandansarie, Ariaii and Langerodi, 2019; Kumar

et al., 2015).

Using the DPPH ASSAY, beetroot pulp showed a 70% inhibition (Chhikara et al., 2019).

Despite the fact that numerous studies indicate that betalains have significant antioxidant

activity, their usage as bioactive agents has not been widely studied, in addition to their coloring

properties and in contrast to other chemicals (Costa et al., 2014; Georgiev et al., 2010, and

17
Ozaki et al., 2021). Furthermore, betalains, which are comparable to the synthetic antioxidant

BHT, are well known for their significant antioxidant and free radical scavenging capabilities

in beetroot (Sheila, 2018). According to Costa et al., 2017, the antioxidant activities of beetroot

powder were enhanced by increasing the temperature from 60 to 70 °C. Furthermore, while

betalains have strong antioxidant properties, the high antioxidant capacity of beetroot can also

be attributed to the presence of other phytochemicals such as polyphenols, phenolic acids,

flavonoids, catechins (Costa et al., 2014), nitrites, and other compounds such as carotenoids

and ascorbic acid.

2.12 Nitrites in beetroots

Synthetic nitrate and nitrite ingredients are widely used in this industry because they ensure

colour stability and proper flavour development in meat products, as well as acting as powerful

antimicrobial agents against pathogenic organisms such as Clostridium botulinum, its spore

outgrowth, and the growth of other bacteria (Merredy et al., 2017). In general, nitrites offer the

typical cured red color, microbial contamination inhibition, antioxidant benefits, and meat

product shelf life extension (Honikiel, 2014).However, nitroso-compounds formed by nitrite

and nitrate are carcinogenic.

With this in mind beetroot is considered a good source of inorganic nitrite. The authors noted

that nitrate levels in beetroot ranged from 644 to 1800 mg/kg in a previous study (Lidder and

Webb, 2013). Beetroot, on the other hand, had higher nitrate levels in a more recent study.

Beetroot juice, for example, has 4965 mg/L of nitrate (Corleto et al., 2018), raw beetroot had

4420 mg/kg, but following dehydration, this number skyrocketed to 42,415 mg/kg (Sucu and

Turp, 2018). Similarly, beetroot powder contained 14,037 mg/kg of nitrate (Ozaki et al., 2020)

18
2.13 Phenolic Acids

The combination of phenolic content and betalains results in a vegetable with a high antioxidant

and radical scavenging activity (Ceclu and Nistor, 2020). According to prior research, 4-

hydroxybenzoic acid, benzoic acid, ferulic acid, and vanillic acid were some of the most

important phenolic chemicals discovered in beetroot extracts (phenolic acids). Isoferulic acid

and protocatechuic acid were also detected in abundance in beetroot products (fresh fermented

and juice), according to a recent study (Patosz et al., 2018). Several researchers discovered a

substantial link between the phenolic content of beets and antioxidant activity (Ceclu and

Nistor, 2020; De Oliveira, 2020; Kumar et al., 2020; Czyzosaka, 2018). However, as noted in

the section on betalains, betalains have a synergistic impact with phenolic acids and flavonoids,

enhancing antioxidant activities even further.

According to Tytti (2001), the total phenolic contents of red beetroot extracts prepared using

various extraction methods and solvents decreased in the following order: 80 percent aqueous

methanol extract (24.1 0.3 mg/g GAE), water extract (20.5 0.4 mg/g GAE), 80 percent aqueous

methanol extract prepared using a method other than the first extracts (18.8 0.3 mg/g GAE),

and water extract (17.4 0.4 mg/g GAE. This study shows that beetroot extracts contain some

phenolic compounds which can potentially inhibit lipid oxidation.

2.14 Application of beetroot in meat products

Since beetroot is known to contain a group of highly bioactive pigments known as betalains as well

as other bioactive compounds, these bioactive compounds consists of different properties which

include free radical scavenging and antioxidant activity from betalains and phenolic acids,

colouring from betalains, and stabilizing activity from nitrite/nitrate and these are of great

19
importance in the meat industry as they can be applied to meat and meat product (Dominguez,

2020).

Jin et al., (2014) examined the use of red beet as a natural colorant in pork sausages. It was

found that pH and moisture content of sausages was significantly increased by incorporating

red beet powder at 0.5% and 1.0% levels. Results revealed that all textural parameters, TBARs

values, and sensory properties, except color, showed an insignificant difference (p > 0.05) by

the addition of beet powder. The addition of beet powder affected color characteristics of

sausages. L* values of sausages were decreased and a* values increased significantly by the

addition of red beet powder. The authors concluded that red beet powder could be successfully

used as a functional ingredient in meat products with slight modification in beet processing for

better results.

Another study carried out by El-Gharably & Ashoush (2011) in which red beetroot powder,

pomegranate rind powder, and their mixtures were incorporated into beef sausages at different

levels. Antioxidant activity and total phenolic content of raw and cooked sausages were

increased by adding pomegranate rind and beet powder and their mixtures. Lipid oxidation was

reduced during the storage of sausages. It was found that the products added with 1% and 3%

red beet powder depicted highest sensory scores than other prepared products.

Another study by (Lages et al., 2021) on fresh beef sausage showed that when powdered

beetroot extract was mixed with a low concentration of thyme essential oil (0.0095 percent) for

28 days at 4 °C, it inhibited the formation of coagulase-positive Staphylococcus.

20
 CHAPTER 3

MATERIALS AND METHODS

3.1 Research Design

All experiments were conducted at Chinhoyi University of Technology in the Food Science

Laboratory and Chemistry Laboratory between February 2022 and March 2022. In this

research, the experimental unit used was beef burger patty. Beetroot was bought randomly in

different local super markets. Two control treatments were used in this study: the negative and

the positive treatments.

For the treatment under study, two different treatments were used Beetroot extract (BE) and

BHT

 Control treatment 1- raw ground beef without additives (0%BE)

 Treatment 2 - 0.05% beetroot extract (0.05%BE )

 Treatment3 - 0.1% beetroot extract (0.1%BE)

 Control treatment 4 - 0.04% BHT

All the experiments were done in triplicates. The samples were stored at 4± 1o C for 9 days

with 3 day interval examinations. Scientific methods were used to determine total phenolic

content and antioxidant activity, examine pH changes, microbial analysis [total bacteria count

(TBC), total coliform count (TCC) and yeast and moulds], lipid oxidation and sensory

evaluation of the ground beef burger patty during storage.

21
3.2 Experimental design

Ground beef burger patties were prepared by mixing ground beef, salt, water and black pepper

and the butter base was subjected to different forms of treatments and different analysis were

done as shown in figure 2.

Ground beef +salt + Beetroot

water+ black pepper

Beetroot
extract
Batter base

Positive 0.1%
0.05%
Negative control Beetroot Beetroot
control (no 0.04 % Extract Extract
additive) (BHT)

Analysis
Microbial
Chemical analysis
analysis
Total Plate
DPPH Sensory
Count
analysis
Peroxide value Yeasts and
Texture
pH Moulds
Aroma
Coliforms
Color
General
Appearance

Figure 2: Flow chart diagram of experimental design.

22
3.3 Sample collection and sample preparation

The sampling techniques used in this research are convenience sampling and simple random.

Simple random sampling was used when buying the beetroot. Convenience sampling was used

in this research to sample fresh minced beef meat at the nearest butcher. Purposive sampling

was used in determining the concentrations of the beetroot extracts (0.05% and 0.1%) and BHT

(0.04%). The sampling time and storage temperature (4±1oC) was determined taking into

consideration the studies that were done by other researchers.

3.3.1 Extraction of beetroot extract

The extraction of beetroot was obtained using the method described by Lee et al (2010) with

some modifications. 50 g of beetroot was washed and chopped into cubes (0.5*0.5*0.5 cm)

and then crushed using an electric blender. The crushed particles was macerated in 70% ethanol

for 24 hours and then filtered. After filtration the extract was evaporated to remove the

unwanted solvent using a rotatory evaporator at 50 °C to get a concentrated extract.

3.3.2 Preparation of the burger patties

Portions of uniform weight of the minced beef were mixed for 15 with salt (2%), water 7.7%

water and ground pepper 0.3%.The obtained batter was divided into 4 experimental units each

weighing 200g. The treatments were applied and assigned as follows: (1) negative control

without adding any extracts (0%BE); (2) positive control with 0.04% BHT; (3) 0.05% beetroot

extracts(0.05%BE) and ( 4) 0.1% beetroot extract After mixing, beef burgers of 50±1 g were

formed using a petri dish to obtain desired shape. The beef burgers were packed in polyethylene

bags and kept in refrigerator at 4±1 ºC for 9 days. pH ,lipid oxidation and microbial analysis

was examined at 3 day interval .

23
3.4 Determination of total phenolic content

3.4.1 Preparation of gallic acid

1 g of gallic acid was dissolved in 100 ml of methanol to get 1% solution of gallic acid (10

mg/ml) termed as standard 1 solution.

3.4.2 Preparation of sample for total phenolic content

Total phenolic content in the beetroot extracts was determined using Folin-Ciocalteu method

according to (Atoui et al 2005).1 mg of extract was mixed with 0.75 ml of Folin-Ciocalteu

reagent (1 ml of Folin-Ciocalteu in 10 ml of distilled water ) and left to stand for 5 min. After

which 0.75 ml of 7% aqueous sodium carbonate (100 mg/ml) was added and the volume of the

reaction mixture was made up to 10 ml by adding distilled water. The mixture was incubated

for 90 min. The standard curve was prepared using different dilutions of gallic acid (0.1, 0.5,

1, 2.5, and 5 mg/ml). The absorbance was measured at 760 nm using Shimadzu UV-VIS

Spectrophotometer. The total phenolic values were calculated using the linear equation based

on the calibration curve. The same procedure was used for samples that contained different

concentration of the extract.

Total phenol contents (TPC) were expressed as gallic acid equivalent (mg/GAE/g) and were

calculated using the following liner equation based on the calibration curve: y = 0.1856x -

0.2201 R² = 0.9751

Where: (y) is absorbance (x) is the concentration (mgGAE/g) sample (R²) is correlation

coefficient.

3.5 Determination of antioxidant activity of beetroot extract

The free radical scavenging activity was determined using DPPH assay according to a method

by (Lawrence, 2016). 3mL of the extracts were taken in various concentrations (20-120 μg/mL)

24
and mixed with 1 mL of 0.1 mM of DPPH solution in methanol. The setup was left at dark in

room temperature and the absorption was monitored after 30 minutes. Absorbance was

measured at 517nm using Shimadzu UV-VIS Spectrophotometer. The ability of the test sample

to scavenge DPPH radical (% inhibition) was calculated by the following formula:

[(Absorbance in control  Absorbance in sample) / absorbance in control]*100

Absorbance control was the absorbance of DPPH and methanol. Absorbance sample was the

absorbance of DPPH radical and the test sample.

3.6 Determination of lipid oxidation in beef burger patties.

Peroxide value (PV) was determined according to a method by (Hęś and Gramza-Michałowska,

2016). 3g of the burger patty was weighed into a 250mL conical flask. The sample was heated

for 3 minutes at 60oC in a water bath to melt fat. The flask was thoroughly agitated with 30ml

acetic acid-chloroform solution (3:2 v/v) to dissolve fat. The obtained filtrate was filtered to

remove beef particles using Whatman filter paper.0.5mL of saturated potassium iodide was

added to filtrate and 0.5 ml of starch indicator was added. Titration was done against standard

solution of sodium thiosulfate.

PV = (V2-V1) × N × 1000/M.

Where V1 and V2 are the titrated volume of sample and control in ml (respectively), N is the

normality of sodium thiosulfate solution, and M is the weight of oil sample (gr).

25
3.7 pH determination

The pH was determined according to the method recommended by (Korkeala et al., 2004). Raw

beef burger sample from each experimental 10 g was homogenized in 90 ml distilled water for

60s in a blender. The pH values were measured using a standardized electrode attached to a

digital pH meter. The pH was recorded by directly dipping the electrodes in the sample until

stable readings were obtained.

3.8 Microbial analysis of burger patties

Microbial analysis was assessed by the method reported by American Public Health

Association (APHA, 1992) with slight modifications. Total bacterial counts (TBC), Total

coliforms count (TCC) and yeast and mould counts (Y&M) in the samples were enumerated

as log cfu/ml of sample.

Peptone water was prepared by suspending 10 grams of peptone salt solution in 500ml of

distilled water and stirred till it completely dissolved. The peptone water was poured (9ml) into

serial dilution bottles then autoclaved at 15 lbs pressure (121°C) for 15 minutes. All apparatus

to be used in the microbial analysis were autoclaved including micro pipettes, stirring and

spreading rods. Serial dilution from 10-1 to 10-6 was done by adding 1ml of each sample (from

the homogenate) into 9ml of the sterilized peptone water.

3.8.1 Total yeasts and molds analysis

Potato dextrose agar (19.5 g) was suspended in 500ml distilled water. It was boiled with stirring

to dissolve the medium and prevent lump formation. After boiling the agar was sterilized in an

autoclave at 15 lbs pressure (121°C) for 15 minutes. After auto-claving the agar was cooled for

45 minutes. A suitable quantity of sterilized medium was poured aseptically into the petri-

plates containing 1 ml of the serial dilutions (10-3 and 10-6 ). Plates were allowed to cool and

26
incubated at room temperature in the dark for 5 days to allow for the growth of yeast and molds.

After 5 day colonies appearing on the plates were counted and expressed as colony-forming

units per gram of sample. This was then converted to log cfu/ml

3.8.2 Total Bacteria Count

For TBC, 17.5 grams of Plate Count Agar (PCA HIMEDIA) was added in 1000 ml distilled

water in a conical flask then heated up to boiling to dissolve the medium completely. The

solution was then autoclaved at 15 lbs pressure (121°C) for 15 minutes to sterilize the media.

The media was then cooled to 45-50°C before pouring into sterile Petri plates. 1 ml of 10-3 and

10-6 dilution was transferred in to the petri dishes aseptically and a suitable quantity of sterilized

medium was poured aseptically into the petri-plates. The plates were incubated at 37oC for 48

hrs.

3.8.3 Total coliforms

For coliforms, 41.53 grams of Violet Red Bile Agar (VRBA HIMEDIA) was added in 1000

ml distilled water then heated with stirring up to boiling to dissolve the medium completely.

The media was not autoclaved and pour-plate method was used. Medium was poured into petri

dishes containing 1ml of serial diluted solution (10-3 and 10-6) swirled gently to mix the medium

and sample well. Medium was incubated at 35°C for 18 – 24 hours. Purple-red colonies were

examined and counted.

3.9 Sensory analysis

Sensory evaluation was assessed by a five-member untrained panel (Altieri et al., 2005) in

terms of colour, odour, and texture and general appearance). The control samples were used as

standard. Panellists scored each sample with a 9-point scale, on which 9 meant the most liked

and 1 the least liked.

27
3.10 Statistical Analysis

All data was recorded using Kruskal Wallis test from IBM SPSS Statistics 20.0 software

program of variance. The significance difference was defined at P < 0.05.

28
 CHAPTER 4

RESULTS AND DISCUSSION

4.1 Total phenolic content

Table 1 shows the phenolic content in mgGAE/g obtained during storage. The total phenolic

content of red beetroot extracts obtained were 28.21±0.02 mgGAE/g. Total phenolic content

in beef burger patties with 0.1% BE and 0.04% BHT burger patties was significantly higher

(p<0.05) with respect to control burger patties and 0.05%BE

Sample TPC(mgGAE/g) TPC(mgGAE/g) TPC(mgGAE/g) TPC(mgGAE/g)

Day 0 Day 3 Day 6 Day 9

Beetrootextract(BE) 28.21±0.02 28.47±0.02 29.21±0.02 30.07±0.02

Burger + 0%BE 12.90±0.01 12.88±0.01 12.85±0.01 12.77±0.01

Burger+0.04%BHT 23.65±0.05 23.78±0.06 23.81±0.01 23.92±0.02

Burger+0.05% BE 16.24±0.02 16.31±0.1 16.51±0.01 16.83±0.03

Burger+0.1% BE 18.82±0.07 19.43±0.12 19.92±0.08 20.3±0.02

Table 1:Total phenolic content(mgGAE/g) of treated and untreated burger patties during

storage.

In burger patties, phenolic content increase was correlated to the amount of BE added that is

the reason why 0.1%BE had more phenolic content (18.82mgGAE/g±0.07) compared to the

one containing 0.05%BE and also burger patty containing 0.1% BE had high total phenolic to

the BHT burger patties. The control sample also showed a small quantity

(12.90mgGAE/g±0.01) of phenols present. Generally total phenolic content can either decrease

29
or increase during storage (Dolatabi et al., 2015). With storage time the total phenolic content

was increasing slightly in all samples.

The total phenolic content of the BE obtained could be attributed to the bioactive compounds

that have been reported to be present in beetroot extracts by (Pulo et al.,2021), where the study

reported that beetroot extracts contains quite a number of phenolic acids including benzoic

acid, ferulic acid and vanillic acid. Also the high phenolic content could be attributed to the

environmental and genetic parameters, and conditions of harvesting beetroot. The presence of

phenolic compounds in animal diets to boost the antioxidant activity of the produced meat

could explain the minimal amount of polyphenols found in control patties (Mashau, 2021). As

the storage time increase the total phenolic content in 0.1% BE and 0.05% BE was increasing

in the total phenolic content could be attributed to the condition in which the burger patties

were stored at, that is cold storage. The results are in agreement with a study by (Dolatabi et

al., 2015) which showed that almond hull and shell extracts had an increase on the total

phenolic content during storage. Also another study by (Amoo et al., 2012) showed an increase

in the total phenolic content of Artemisia afra Clausena anisata Cussonia spicata Leonotis

intermedia and Spirostachys africana over long term storage. This is in agreement with the

study of Jakopic et al., who reported an increase of TPC of rutabaga root after four months of

cold storage. So the increase in the total phenolic content could be the result of changes in

phytochemicals during storage of the burger patties.

Besides studies agreeing with the current study results, the obtained results, are disagreeing

quite a number of studies which have shown a decrease in the total phenolic content. A study

by (Deng et al., 2018) who showed a decrease in the total phenolic content during storage at

30
4oC. The total phenol content obtained were higher as compared to results from a study by

(Guldiken et al., 2010) who reported a mean value of total phenolic content in fresh beetroot

of 2.55 mgGAE/g and this could be attributed to the extracting method and kind of solvent

used. Guldiken et al (2010) used water extraction where as in this study ethanolic extraction

was used and therefore results in high amounts of total phenolic content being obtained.

On the other hand, some studies found higher mean values for total phenols compared to the

obtained results in this study regarding total phenolic content of raw beetroot. A study by

(Edziri et al 2015) have proved to have high total phenolic content exceeding to as far as to 53-

56 mgGAE/g. A study by (Lameck et al., 2015) the total phenolic substance content in beetroot

extract was 27.72 mgGAE/mL.

31
4.2 DPPH assay: Antioxidant activity

The DPPH is a stable free radical that accepts an electron or hydrogen radical to form a stable

molecule in nature. A decrease in DPPH absorbance at 517 nm caused by antioxidants

contained in the extracts was used to determine its reducing nature. The results obtained from

beetroot extract were compared to BHT, which was used as a standard. Figure 3 shows the

obtained percentage inhibition in beetroot extract and negative control as well as other treated

samples.

Antioxidant Activity
100
90
80
Radical scavenging (%)

70
60
50
40
30
20
10
0
0 3 6 9
Storage (Days)

0.04%BHT 0.1% BE 0.05% BE 0% BE

Figure 3:Radical scavenging activity (%) during storage

The percentage inhibition (%inhibition) of the beetroot extract was found to be 88.05% ±0.03

at the end of storage. The obtained results were higher than that reported by (Vodnar et al.,

2016) who reported 45% in one gram of dried beetroot waste. Other studies have also shown

different % inhibition of different parts of beetroot, beetroot pulp 70% inhibition (Chhikara et

al., 2019), beetroot powder 65% (Ozaki et al., 2021). The high amount of % inhibition of

32
beetroot could be attributed to the type of extraction used which was ethanolic extract.

Geourgiv et al (2010) reported that beetroot extracts obtained from ethanolic extraction ranges

from 14.2-90% inhibition. Also the results showed that the radical scavenging activity

increased as the sample concentration increased.

Higher radical scavenging activity can be attributed to higher amounts of total phenolic

compounds that was present and also the presence of some biologically active phytochemicals

including betalains, flavonoids, polyphenols, saponins and inorganic nitrate (Diego et al.,

2017). Besides betalains, other beetroot compounds are reported as bioactive, including nitrate,

betaine, ascorbic acid, carotenoids, polyphenols, flavonoids, glycine, and folate (Chhikara et

al., 2019) and rutin, epicatechin, and caffeic acid, which also exhibit antioxidant activity

(Georgiev et al.,2010) and all these bioactive compounds could be attributed to high %

inhibition that was observed. The radical scavenging activity may reflect its antioxidant

potential.

It is also noted that as storage time progressed the % inhibition in BE treated samples was

increasing. Therefore, the DPPH scavenging abilities beetroot increased with storage time.

Burger patty samples containing BHT showed a significant increase (p<0.05) of the antioxidant

capacity depending on the storage time. The increase in the % inhibition could be attributed to

the increase in the total phenolic content. From the study beetroot extracts exhibited a high

antioxidant power, which encourages the meat industry to consider preserving meat products

with beetroots extract. From the results it was observed that concentration also had an effect

on the antioxidant as 0.1% BE showed a significant difference (p<0.05) with respect to

0.05%BE, 0.1%BE had a high antioxidant activity.

33
In this regard, previous researchers discovered a strong relationship between beetroot phenolic

content and antioxidant activity, and the study concluded that beetroot extract can be employed

as a natural antioxidant. The presence of phenolic compounds in beetroot extracts contributes

significantly to their antioxidant capacity, and several authors agree that there is a direct

relationship between total phenolic compounds determined by the Folin-Cioclateau method

and antioxidant power in many cases (Atoui et al., 2005).

4.3 pH

The effect of different treatments on pH values of beef burgers stored at 4±1 ºC is presented in

Figure 4.

pH
7.5
7
6.5
6
5.5
pH

5
4.5
4
3.5
3
0 3 6 9
Storage (Days)

0% 0.04%BHT 0.05%BE 0.1%BE

Figure 4:pH values of treated and untreated beef burger patties during storage.

The pH levels of all treatments were identical (p > 0.05) on day zero, indicating that there was

no significant difference between samples as the pH ranged between 5.5 and 5.6 showing that

pH was not affected by the antioxidants addition. These findings matched those of (Lee et al.,

34
2012), who found that red beet extract applied to pork patties had no effect on the pH value.

These findings echoed those of (Wójciak et al., 2011), who said that “the addition of plant

extracts to meat samples did not influence considerably the acidity of the samples during

chilling storage.” The pH values of all beef burger samples decreased during the first 3 days of

storage with the control (0%) having lowest pH with a value of 4.52 while other treatments

0.04%BHT, 0.05% BE and 0.1%BE ranged at 4.80, 4.83 and 4.81 respectively compared to

other treatment. The pH decrease is could be consequence of organic acids’(lactic acid

bacteria) production by Gram-positive bacteria in particular lactic acid bacteria generated

during anaerobic glycolysis and also the decrease was possibly due to the inorganic phosphate

generated during ATP degradation and lactic acid as proposed by (Hwang et al., 2017) in lows

salt frankfurters. After day 3 there was a gradual increase in all the treatments with control

having the highest pH value of 6.19. The increase in pH could be attributed to the build-up of

metabolites by microbial action in burger patties, where amino acids are released during protein

breakdown and ammonia and other alkaline compounds accumulate as a result of amino acid

degradation. Fatty acids and their oxidation products, on the other hand, would raise the pH

value and decrease the storage quality of beef patties (Hajlaoui et al., 2019). The pH of the beef

patties with 0.1%BE, 6.1 was significantly lower (p<0.05) than the control samples which had

7.2. The lower pH in BE-containing burger patties could be attributable to antibacterial

characteristics (nitrites) in the extract (Pulo et al., 2021) that have inhibited microbial action.

Various studies depicted that the final increase in the pH might be due formation of N non-

protein compounds and basic ammonium ions coupled with buffering actions of meat protein.

4.4 Lipid oxidation

Peroxide value is one of the most tests used for the measurement of primary oxidation in food

and it seems reasonable to determine the concentration of peroxide in order to estimate the

35
extent of initial oxidation in meat samples (Wang et al., 2015). Lipid oxidation for fresh beef

patties was assessed by measuring peroxide value at 4 °C, and the findings are shown in Figure

5.

Peroxide value
3.5

3
Peroxide value (meq/kg)

2.5

1.5

0.5

0
0 3 6 9
Storage (Days)

0%BE 0.04%BHT 0.05% BE 0.1%BE

Figure 5:Peroxide values of treated and untreated beef burger patties during storage.

The peroxide value of BHT and 0.1% BE had a significantly (p<0.05) lower peroxide value

those of the 0.05%BE and control due to the free radical scavenging activity of BHT as well

as BE at high concentration. Lowest peroxide values were observed in 0.1% BE which was

recorded as 1.37meqO2/kg whereas the control recorded 1.95meqO2/kg at the end of storage.

Initial peroxide values of beef burger samples were determined as 1.35, 1.29, 1.34, 1.24

meqO2/kg in the control, 0.04% BHT, 0.05%BE and 0.1% BE respectively.

The peroxide value of beef burger increased with storage, peaking after 6 days of chilling

storage. Furthermore, the peroxide value of all beef burger tended to rise with the progression

36
of time before decreasing at the end of the storage period. The conversion of hydroperoxide, a

primary oxidation product, to secondary lipid oxidation products could be attributed to a

decrease in peroxide value. These findings revealed that during the first 6 days of refrigerated

storage, the control samples experienced considerable lipid oxidation and attained their

maximal peroxide value. At day 6 all beef burger patties had the highest levels of peroxide

value, with 0.1%BE having lower values showing that indeed BE delayed lipid oxidation

during storage. A sharp decrease on the peroxide value was observed on day 9. This was in the

same trend with (Maqsood and Benjakul, 2010), who investigated the effects of tannic acid as

a phenolic antioxidant on the peroxide value of ground beef after chilling storage. They

discovered a progressive increase in peroxide value in all samples over the period of 15 days

of chilling storage (p≤ 0.05), with the exception of the samples without tannic acid treatment,

where the peroxide value reduced significantly after 15 days of chilling storage (p< 0.05).

Peroxide values were affected by the presence or absence of total phenolic. There was no

significant difference (p ≥0.05) observed between the samples on the first day of storage. The

inhibition effect of BE on lipid oxidation may be due to its ability to prevent the formation of

free radicals. Thus, it works to reduce the development of the oxidation process and protect the

meat patties from oxidation. Furthermore, during storage, oxidation occurs for lipid and thus

peroxides are formed, but it is the treatment by extracts that reduced the formation of peroxides

because they contain bioactive compounds that have inhibitory activity against the formation

of peroxides.

37
4.5 Microbiology

The results obtained show that the microbial population in all the samples increased

significantly (p<0.05) during the storage time on total bacteria, coliform, yeast and moulds

values.

4.5.1 Total Bacterial Count (logcfu/ml)

As shown in Table 2: The total bacterial count evaluated on PCA at day 0 of storage, all samples

remained below 4 log10 cfu/ ml having 3.55 log cfu/ml, 1.59 log cfu/ml, 1.78 log cfu/ml and

1.42 log cfu/ml in the control, 0.04% BHT, 0.05% BE and 0.1%BE respectively but these

values increased throughout the storage period.

Sample Day 0 Day 3 Day 6 Day 9

logcfu/ml logcfu/ml logcfu/ml logcfu/ml

0%BE 3.55±0.004 4.99±0.01 5.95±0.01 6.63±0.03

0.04%BHT 1.59±0.001 3.34±0.01 4.81±0.01 5.19±0.02

0.05%BE 1.78±0.03 4.57±0.02 5.65±0.01 5.71±0.01

0.1%BE 1.42±0.001 3.20±0.01 4.10±0.01 4.30±0.01

Table 2:Mean values of total bacterial count log (cfu/ml) of treated and untreated samples

during storage

An increase on the bacterial count may be attributed to the way the burger patties were

prepared. The ground meat, during its preparation, experience a lot of handling thereby making

the burger patty susceptible to microbial action. The variation in microbial counts on day 0

could be due to microorganisms' cell walls or outer membrane structure, which prevents the

plant extract from penetrating (Shawkey 2018). Control samples had the greatest total bacterial

count of 6.63 log cfu/ml on day 9, whereas treated samples with 0.1%BE had the lowest total

38
bacterial count of 4.30 logcfu/ml. This reveals that adding BE lowered overall bacterial count

in patties significantly (p<0.05). This is due to the fact that BE contains a variety of bioactive

compounds and nitrites with antibacterial properties (Fahey, 2005). As a result, the decreased

bacterial count in treated beef burger patties could be linked to these components.

The findings are agreeing with the ones obtained (Oji et al., 2018), who investigated the effect

of sage extract on the microbiological stability of sausage during storage for 8 days and found

that sage reduced total bacterial count during storage, with the control reaching 7.66 logcfu/g

and the treatment reaching 6.50 log cfu/g on day 8 of storage. Phenolic compounds may have

played a major role in the antibacterial properties of beetroot extract. The partial hydrophobic

nature of phenolic compounds present in the extract may destroy the cell wall, interact with

and disrupt the cytoplasmic membrane, harm membrane proteins, and interfere with

membrane-integrated enzymes, all of which can lead to cell death (Williams 2011).

4.5.2 Coliforms (logcfu/ml)

The number of coliforms counted are shown in table 3. In comparison to treated samples, the

control sample had the highest coliform count at day 0 (3.77 log cfu/ml), which increased to

5.20 logcfu/ml by day 9. During the 9-day treatment period, there was a significant increase

(p<0.05) in the number of coliforms in all treated samples

Sample Day 0 Day 3 Day 6 Day 9

0% BE 3.77±0.03 4.33±0.01 4.86±0.01 5.20±0.01

0.04% BHT 1.61±0.01 2.31±0.01 2.88±0.01 3.06±0.01

0.05%BE 1.66±0.01 2.67±0.01 3.29±0.01 3.88±0.01

0.1% BE 1.36±0.03 1.78±0.01 2.56±0.1 2.96±0.2

39
Table 3:Mean coliform counts log cfu/ml of treated and untreated samples during storage.

The presence of coliforms in both treated and untreated samples, however, could be attributed

to contaminated equipment and utensils, as well as inadequate sanitary handling and cross

contamination during preparation (Mashau, 2021). Moreover even though the coliform count

increased the total coliform count in 0.1% BE was low as compared to other treatments after

storage. This shows that beetroot extract slowed down the growth of coliforms due to the

present of some antimicrobial properties present in the extract. The obtained results were in

agreement with the ones that were observed by (Muthukumar, 2019) when moringa oleifera

leaves extracts were used and the coliform count was low in treated samples during cold storage

of ground patties, showing that plant extracts may negatively influence growth of coliforms.

4.5.3 Yeast and moulds (logcfu/ml)

The total number of yeast and moulds recorded are shown in table 4. At the end of storage the

values for the treated samples were 6.11 logcfu/ml (0.05 % BE), 4.83 logcfu/ml (0.1 % BE)

and 4.69logcfu/ml (0.04% BHT), with the control samples having the highest value of 6.25

cfu/ml

Sample Day 0 Day 3 Day 6 Day 9

(logCFU/ml) (logCFU/ml) (logCFU/ml) (logCFU/ml)

0%BE 5.78 ±0.02 5.93 ±0.03 5.98±0.08 6.25±0.01

0.04% BHT 2.04 ±0.05 3.30±0.01 3.74 ±0.01 4.69±0.01

0.05%BE 4.44±0.05 5.40 ±0.01 5.97 ±0.01 6.11 ±0.02

0.1%BE 2.58±0.02 3.27 ±0.05 4.09 ±0.01 4.83 ±0.03

40
Table 4:Mean yeast and moulds log (cfu/ml) of treated and untreated samples during storage.

The number of yeast and moulds counted over time in the control and treated samples differed

significantly (p<0.05). During storage days, however, all the burger patties showed a

significant increase. The growth of yeast and moulds could be attributed to the environment in

which the burger patties where prepared in which a settle plate showed that the environment

had yeast and moulds even though aseptic techniques were done during plating. However even

though there was an increase in the number of yeasts and moulds it was noted that 0.1%BE and

0.04%BHT recorded lower counts of yeast and moulds. (Sheila, 2017) demonstrated the

potential of beetroot extract to act as a preservative against pathogenic microbes such as

Staphylococcus aureus and Enterobacter aerogenes, as well as growing spoilage

microorganisms such as Escherichia coli and yeast and mould.

Overall results shows that samples containing 0.04%BHT and 0.1% BE showed slower

microbial growth than the control and 0.05% BE. Data pointed out the significant influence of

beetroot concentration on microbial growth the higher concentration used of the plant extract,

the lower the microbial counts as the microbial counts were higher in samples containing

0.05%BE than that of 0.1%BE. Even though there was an increase on the microbial load, it

was noted that the growth of microorganisms in treated samples both with BHT and the one

with BE was low as compared to the control samples. The antimicrobial action of beetroot

extracts due to the presence of nitrites, which are antimicrobial agents, and also the presence

of phenolic acids could be attributed to the lower count of total bacteria, total coliform, yeast,

and mould in treated beef patties compared to the control during storage.

41
4.8 Sensory evaluation.

Figure 6 shows the result of the sensory evaluation. Five untrained student panellists, noted

variations in color, odour, and overall appearance between burgers with various treatments and

control burgers

Sensory evaluation
Colour
10
8
6
4
Overal Acceptability Odour
2
0

General Appearance Tenderness

0% BE 0.05% BE 0.1% BE 0.04% BHT

Figure 6:Sensory attributes of treated and untreated samples.

Indeed, they appreciated the beneficial effect of beetroot on these parameters, as the vegetable

appeared to prevent discoloration of the beef burger patties and produce a good red colour in

beef burgers treated with 0.1 percent BE, owing to beetroot extract's high betanin content

(Geogiever et al., 2010). Nonetheless, high concentration of the extract could make beef

burgers unpleasant for the panellists. Similarly, the extract had an odour that panellists seemed

to enjoy when it was incorporated at 0.05%BE as compared to 0.1%BE and this could be

attributed to the fact that beetroot contains odorous compounds such as geosmin which could

be unpleasant to the panellist (Lu et al., 2003). Among the patties treated with beetroot,

panellists rated samples with 0.1% BE superior and they had high overall acceptability even

though the odour of this sample was least liked. The nice red colour is attributed to the pigments

42
of betalain and betanine (Aykın-Dinçer et al, 2020) present in BE. Addition of 0.05% BE

resulted in a pink to light red colour, while 0.1% BE added generated a darker colour. Beetroot

is known as a red pigment source that may affect the colour of food developed from it. The

majority of substances that contribute to the red colour is known as betalain. Controls strongly

evidenced a lower trend on colour probably due to microbial spoilage and the consequent

increased of pH values (Rowida, 2018) and the colour increased with the growing amount

vegetable extracts addition, as demonstrated by several authors like a study of beetroot powder

on Turkish fermented beef sausage (Sucu et al., 2018) and fresh pork sausage (Martínez et al.,

2006)

43
 CHAPTER 5

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

From the obtained results, it can be concluded that the use of natural and synthetic antioxidants

can reduce lipid oxidation, enhance colour stability and improve the sensory characteristics of

beef burgers if added in rightful concentrations. 0.1 % BE and 0.04% BHT showed positive

results as the peroxide value in 0.1 % BE was significantly (p<0.05) lower compared to 0.04%

BHT, 0.05% BE and control samples and microbial spoilage was reduced the least treatment

was 0.05% at the end of storage. Also 0.1% BE had high sensory acceptability (8.2) even

though the odour was affected negatively. The study showed positive results, although the

concentrations used were not strong enough to prevent spoilage to acceptable levels. Thus,

addition of natural antioxidants is one of the ways to extend the durability of meat and meat

products. The beetroot extract provided antioxidant protection against beef burgers

deterioration during 9 days storage period. Moreover, the beetroot extract showed a greater

potential which is almost same as that of to BHT in delaying the lipid oxidation and improving

the sensory attributes of beef burgers during refrigerated storage. The beetroot extracts proved

to be effective antioxidants in beef burgers. Therefore, utilization of beetroot extracts as natural

antioxidants in beef burger patties as well as other meat products could improve the quality and

therefore can serve the meat industry in manufacturing stable, functional and healthier meat

products which could meet the consumer demands.

5.2 Recommendations

For future research it is recommended that the identification of phenolic compounds or

processes responsible for the increase in total phenolic content of stored plant extracts be

studied extensively. It is also recommended that the effects of the addition of beetroot (or

44
beetroot extracts) in the reformulation of different types of meats and meat products should be

extensively studied. Also further studies should be done on extraction conditions of the

bioactive compounds from the beetroot as they must be optimized according to the meat

product application.

45
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49
Appendices

Standard Curve for Gallic acid.

1.4 Standard Curve for Gallic Acid

1.2 y = 0.1856x + 0.2201
Mean Absorbance (760nm)

R² = 0.9751
1

0.8

0.6

0.4

0.2

0
0 1 2 3 4 5 6
Contcentration (mg/ml)

Hypothesis test summary for pH determination during storage.

Descriptive Statistics
N Mean Std. Minimu Maximu
Deviation m m
Day 0 12 5.5750 .07065 5.40 5.63
Day 3 12 4.9467 .22576 4.80 5.32
Day6 12 5.8875 .18376 5.62 6.10
Day 9 12 6.5342 .44211 6.00 7.20

50
Hypothesis test summary for peroxide value during storage.

Descriptive Statistics
N Minimu Maximu Mean Std.
m m Deviation
Day 0 12 1.23 1.35 1.3008 .04660
Day 3 12 1.3400 2.0200 1.617500 .2958847
Day 6 12 1.5300 2.5300 1.864167 .4174916
Day 9 12 1.38 1.95 1.5792 .22641
Valid N
12
(listwise)

51
Hypothesis test summary for total bacteria count

Descriptive Statistics
N Minimu Maximu Mean Std.
m m Deviation
Day 0 12 4.6000 4.8000 4.725000 .0866025
Day 3 12 4.4000 4.7000 4.550000 .1167748
Day 6 12 5.5 7.7 6.450 .8785
Day 9 12 6.66 8.41 7.6400 .78101
Valid N
12
(listwise)

52
Hypothesis test summary for total coliforms

Descriptive Statistics
N Minimu Maximu Mean Std.
m m Deviation
Day 0 12 1.35 3.77 2.1017 1.01348
Day 3 12 1.7700 4.3300 2.773333 .9961137
Day 6 12 2.5500 4.8600 3.395000 .9238506
Day 9 12 2.95 5.20 3.7750 .93602
Valid N
12
(listwise)

53
Hypothesis test summary for yeast and moulds

Descriptive Statistics
N Minimu Maximu Mean Std.
m m Deviation
Day 0 12 2.0400 5.7800 3.710000 1.5565346
Day 3 12 3.27 5.93 4.4750 1.25828
Day 6 12 3.74 5.98 4.9450 1.08354
Day 9 12 4.6900 6.2500 5.470000 .7451662
Valid N
12
(listwise)

54
55