optimising the wagashie (a traditional cottage cheese

i

OPTIMISING THE WAGASHIE (A TRADITIONAL COTTAGE

CHEESE) PROCESS AND SENSORY QUALITY

BY

AKUA BOATEMAA ARTHUR

10443436

THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN

PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF

MASTER OF PHILOSOPHY IN RADIATION PROCESSING DEGREE

(FOOD SCIENCE AND POST HARVEST TECHNOLOGY)

DEPARTMENT OF NUCLEAR AGRICULTURE AND RADIATION PROCESSING,

SCHOOL OF NUCLEAR AND ALLIED SCIENCES

UNIVERSITY OF GHANA

July, 2016

University of Ghana http://ugspace.ug.edu.gh

ii

DECLARATION

This thesis is the result of the research work undertaken by Akua Boatemaa Arthur in the

department of nuclear agriculture and radiation proessing (N.A.R.P.) of the Graduate Shool of

Nuclear and Allied Sciences (GSNAS), University of Ghana, Legon under the supervision of

Prof. Wisdom kofi Amoa-Awua and Prof. Victoria Appiah. Except for the references of other

research works which have been duly cited, in this dissertation, this work has never been

presented either in whole or in part for any other degree in this University or elsewhere.

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

Akua Boatemaa Arthur

(Student)

Certified by:

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

Prof. Wisdom Amoa-Awua

(SUPERVISOR)

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

Prof. Victoria Appiah

(SUPERVISOR)


iii

DEDICATION

I dedicate this work to God almighty, my parents; Mr. and Mrs. Osei-Bonsu, my

children; Ebo and Nana, my super supportive sibblings (Serwah and Nketia) and my

husband; Ing Philip Bernard Arthur.


iv

ACKNOWLEDGEMENT

My unending gratitude goes to God almighty for his umlimited grace and favour throughout

my study. He is my source of inspiration. Also to my parents, Mr and Mrs Osei-Bonsu, my

husband; Ing Philip Bernard Arthur and my siblings; Afia Serwah Bonsu and Nketiah Osei-

Bonsu, you were very supportive and i am very grateful.

This work was carried out as part of the Danida funded project ‘Preserving African food

microorganisms for Green Growth. My profound gratitude therefore goes to the green growth

team for giving me the opportunity to carry out this project and for the experience acquired.

My sincere gratitude goes to my supervisors, Dr Wisdom Kofi Amoa-Awua and Prof.

Victoria Appiah for their encouragement, support and guidance throughout my study.

My gratitude also goes to Dr. Margaret Owusu, Mrs. Amy Atter and Mr. Theophilus Annan at

the Food Research Institute -CSIR for their assistance.

I am grateful to Prof. Saalia for his assistance in my experimental design and analysis. To

Mrs. Charlottte Oduro - Yeboah and Mr. Papa Toah, I am grateful for their enormous support

and assistance. I am also grateful to Dr. Abbey for his assistance during my instrumental

texture analysis.

My gratitude goes to all the laboratory technicians at the Food microbiology laboratory,

Nutrition test kitchen, Chemistry laboratory and the Food Processing laboratory at the Food

Research Institute –CSIR for their help and assistance especially, Alex, Yahayah, Auntie

Constance Boateng, Auntie Joyce, Auntie Alice, Edna, Solomon, Jemima, Mr Ameh, Frank

and all the service and attachment persons that were present at the time of the study.


v

My gratitude also goes to all the staff of Food Research Institute (CSIR) who took part in the

consumer and sensory evaluation for the project, thank you for your time and enthusiasm that

you exhibited throughout the activity.

Finally my gratitude goes to Mr Adu Gyamfi (BNARI) and the technicians at the Radiation

Technology Center (RTC-BNARI) for their help especially, Stanely and Jonathan.


vi

ABSTRACT

Wagashie is a traditional West African cottage cheese produced by the Fulani who are semi-

nomadic. It is a good protein source and can replace fish or meat in the diet of low income

families in Africa. However, it is a product with high moisture content (60%) which is

favourable for the growth of microorganisms and thus has a short shelf life of 3 days; it also

has a bland taste with limited patronage. This research was therefore carried out to reengineer

wagashie for a larger market with a focus on improving its sensory quality, safety and shelf

life. A brief survey was carried out to confirm the wagashie production procedure and

identify retailers and producers for collection of samples. The safety of market samples of

fresh and fried wagashie samples were determined by assaying for various indicator and

pathogenic microorganisms including aerobic mesophiles ,Yeast and moulds , coliform

bacteria, E. coli, Staphylococcus aureus Bacillus cereus, Salmonella spp ,Enterococcus,

Enterobacteriaceae. Studies were also carried out to replace the traditional coagulant of milk

which involves the use of plant extract of Sodom apple (Calotropis procera) with commercial

rennet used in industrial cheese production and ferment fresh milk used in the preparation.

The traditional method of preparation was also standardised to improve its sensory quality.

The process variables of wagashie, which are salt concentration, coagulant and fermentation

time, were thus optimised using the Box Behnken design which is a response surface

methodology and an affective testing was carried out to evaluate the consumer preference of

the product with a nine-point hedonic scale. The sensory profile of the ‘wagashie’ samples

were described by a Quantitative Descriptive Analysis by a trained 13 member panel. They

evaluated the wagashie samples for desirable and undesirable attributes of wagashie. The

rheology of ‘wagashie’ which involves Texture Profile Analysis (TPA) with a texture

analyser and colour determination using a chroma photometer were carried out on the

improved product which were the rennet coagulated fresh and smoked wagashie samples.


vii

Chemical analyses were carried out on the samples whereby the protein, ash, fat, free fatty

acids (FFA), moisture, pH and Titratable acididty (TTA) of wagashie were determined. The

safety of the laboratory prepared wagashie was assessed and shelf life studies were carried out

for 5 weeks.

The results of the microbiological tests carried out on the market wagashie showed that,

Salmonella and Staphylococcus aureus were not detected in the samples whiles Bacillus

cereus was detected in low counts in half of the samples. The rest of the microorganisms were

found in fairly high counts.

The optimum levels of the process variables which was used in standardizing the product

were 23 g of salt, 150 g of plant extract and 0 h fermentation for the traditional preparation

and 11 g of salt, 5.35ml of commercial rennet and 4 h fermentation for the improved

wagashie

The results of the consumer preference testing showed that the panelists preferred the non-

fermented product to the fermented wagashie for the traditional preparation (Sodom apple

extract as coagulant). The same panelist however preferred the fermented product to the non-

fermented product for the preparation with commercial rennet as coagulant. After a

confirmatory affective test where the wagashie samples were processed by frying and

smoking, the panelists rated the acceptability of the traditional non- fermented smoked and

the fermented rennet coagulated fried sample significantly higher at p<0.05, followed by the

fermented rennet coagulated smoked sample. However, all three samples were rated ‘like-

moderately’ on the 9-point hedonic scale. Thus the fresh and smoked samples were

considered for the rest of the study due to health concerns raised by consumers which

involved the reduction of fat in wagashie.


viii

In the Quantitative Descriptive Analysis, the panellists scored higher intensities of the

desirable attributes in the improved wagashie which were mainly milky aroma, milky taste,

cheesy aroma, cheesy taste, yoghurt aroma. The undesirable descriptors which were bitter

taste, bland taste, spoilt milk aroma, fermented cassava dough aroma were rated high in the

market samples. Generally, there were no significant differences between the plant extract

coagulated samples and the rennet coagulated samples prepared in the laboratory.

The Texture profile analysis showed that the wagashie samples were ‘hard’, ‘gummy’ and

‘chewable’. The instrumental colour determination showed that the fresh wagashie sample

had higher L* values which indicated a lighter colour while the smoked samples had lower L*

values which indicated a darker colour which corresponded to the results of the Quantitative

Descriptive analysis of the fresh and smoked wagashie samples.

The chemical analysis showed that the rennet coagulated smoked sample had the highest

protein content of 30.18 g/100g and the highest FFA value of 0.53. The fried samples had the

highest fat content of 25.32 g and the highest ash content of 2.0 g and the fermented fresh

samples had the highest moisture content of 56.10 g. Generally the FFA values for the

samples were low.

The safety of the improved wagashie samples was improved with fermentation and smoking,

this is because the count for microorganisms reduced when compared with the safety of the

market wagashie.The result of the shelf life study showed that preserving the wagashie

samples with vacuum packaging and irradiation extended the shelf life of wagashie from 3

days to 3 weeks of storage under ambient conditions.


ix

TABLE OF CONTENTS

DELARATION .......................................................................................................................... ii

DEDICATION .......................................................................................................................... iii

ACKNOWLEDGEMENT ........................................................................................................ iv

ABSTRACT .............................................................................................................................. vi

LIST OF TABLES ................................................................................................................... xii

LIST OF FIGURES ................................................................................................................. xiv

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

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

1.1.0 Justification .................................................................................................................... 4

1.2 Main Objecive .................................................................................................................. 4

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

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

2.1 Traditional food Processing .............................................................................................. 5

2.2.0 Traditional production of ‘wagashie’ a local cottage cheese ........................................ 6

2.2.1 Preservation of wagashie ` ............................................................................................ 7

2.2.2 Safety of wagashie......................................................................................................... 8

2.2.3 Spoilage and Pathogenic microorganisms in soft cheeses (‘wagashie’) ....................... 9

2.2.4 Calotropis procera ........................................................................................................ 10

2.3.0 Milk production in Ghana............................................................................................ 11

2.3.1 Milk consumption in Ghana ........................................................................................ 12

2.3.2 Traditional Fermented milk products in Ghana ........................................................... 12

2.3.3 Milk composition ......................................................................................................... 14

2.3.4 Milk Quality and Safety .............................................................................................. 17

2.3.6 Lactic acid fermentation of milk ................................................................................. 20

2.4.0 Cheese .......................................................................................................................... 21

2.5.0 Cheese Manufacture .................................................................................................... 29

2.5.7 pH of cheese ................................................................................................................ 35

2.6.0 Rheology of cheese ...................................................................................................... 36

2.7.0 Food Product Development ......................................................................................... 38

2.8.0 Packaging and Preservation of cheese ......................................................................... 39

2.8.1 Food Irradiation ........................................................................................................... 41

2.9.1 Descriptive Sensory Analysis ...................................................................................... 43

2.9.6 Principal Composite Analysis (PCA) .......................................................................... 47


x

2.10.0 Experimental Design ................................................................................................. 48

2.10.1 Response Surface Methodology and Box Behnken Design ...................................... 48

2.10.2 Box Behnken Design ................................................................................................. 49

CHAPTER THREE .................................................................................................................. 51

3.0 MATERIALS AND METHODS ....................................................................................... 51

3.1 Breif field study .............................................................................................................. 51

3.2 Sampling of wagashie ..................................................................................................... 51

3.3 Microbiological analysis of the market samples and laboratory prepared samples of

wagashie ............................................................................................................................... 51

3.4.3 Modification of the Wagashie process ........................................................................ 58

3.5.0 Fermentation of fresh cow milk using starter culture .................................................. 60

3.6.0 Design of Experiment for ‘wagashie’ preparation ...................................................... 62

3.7.0 Sensory Evaluation ...................................................................................................... 66

3.7.1 Hedonic Sensory Evaluation ....................................................................................... 66

3.7.2 Quantitative Descriptive Analysis ............................................................................... 67

3.8.0 Physicochemical Analyses .......................................................................................... 68

3.8.1 Determination of pH .................................................................................................... 69

3.8.2 Determination of Total Titratable acidity .................................................................... 69

3.8.3 Determination of protein ............................................................................................. 70

3.8.5 Determination of FFA ................................................................................................. 71

3.8.6 Moisture determination................................................................................................ 71

3.8.7 Determination of Ash content of the samples ............................................................. 72

3.8.8 Colour measurement of ‘wagashie’ ............................................................................. 72

3.8.9 Texture Profile Analysis (TPA) of ‘wagashie’ ............................................................ 72

3.9.0 Shelf life study of wagashie ............................................................................................ 73

3.9.1 Irradiation and Packaging of wagashie ........................................................................ 73

3.9.2 Storage of ‘wagashie’ .................................................................................................. 74

3.10 Statistical Analysis ....................................................................................................... 74

CHAPTER FOUR .................................................................................................................... 76

4.0 RESULTS ........................................................................................................................... 76

4.1.0 Market Survey ............................................................................................................. 76

4.1.1 Microrganisms present in market ‘wagashie’ samples ................................................ 76

4.2.2 pH of the market wagashie samples. ........................................................................... 78


xi

4.2.0 Fermentation trials for laboratory preparation of ‘wagashie’Rate of fermentation of

raw cow milk with cheese and yoghurt starter cultures........................................................ 78

4.3.0 Optimisation of the ‘wagashie’ process....................................................................... 81

4.3.1.0 Using the response surface methodology to optimise the ‘wagashie’ process

prepared with plant extract as coagulant. ............................................................................. 81

4.3.2.0 Using the Response Surface methodology to optimise the wagashie process using

commercial rennet as coagulant. .......................................................................................... 89

4.3.0 Sensory Evaluation .................................................................................................... 100

4.3.1 Affective Sensory Evaluation .................................................................................... 100

4.3.2 Quantitative Descriptive Sensory Evaluation ............................................................ 102

4.4.0 Pricipal composite analysis (PCA) ............................................................................ 108

4.5.0 Cluster Analysis ......................................................................................................... 111

4.5.1 Spider plot.................................................................................................................. 114

4.6.0 Proximate composition of ‘wagashie’ ....................................................................... 115

4.8.0 Rheology of Improved ‘wagashie’ ............................................................................ 116

4.9.0 Safety of improved ‘wagashie’ .................................................................................. 119

4.9.1 Shelf life of wagashie ................................................................................................ 120

4.9.4 pH of the wagashie samples during the 5 weeks storage period. .............................. 124

CHAPTER FIVE .................................................................................................................... 126

5.0 DISCUSSION .................................................................................................................. 126

5.1 Safety of wagashie ........................................................................................................ 126

5.2 Improving the quality of wagashie ............................................................................... 129

5.2.1 Affective testing ........................................................................................................ 129

5.2.2 Quantitative Descriptive Sensory Analysis ............................................................... 132

5.3 Chemical analysis of wagashie samples ....................................................................... 134

5.4 Rheology of wagashie .................................................................................................. 136

5.4.1 Colour Determination ................................................................................................ 136

5.4.2 Texture Profile Analysis (TPA) ................................................................................. 136

5.5 Shelf life of wagashie ................................................................................................... 137

5.5.1 pH of wagashie samples during the 5 weeks storage period .................................... 139

CHAPTER SIX ...................................................................................................................... 140

6.0 CONCLUSIONS AND RECOMMENDATIONS........................................................... 140

REFERENCES ....................................................................................................................... 142

APENDDIX ........................................................................................................................... 161


xii

LIST OF TABLES

Table 2.1: The most commonly used rennet and coagulants and their enzymes 28

Table 2.2: Classification of cheese 38

Table 2.3: The Box Behnken Experimental Design 50

Table 3.1: The Box Behnken Design matrix of variables (k=3) for the optimisation of

‘wagashie’ coagulated with Rennet 63

Table 3.2: Coded and actual levels of the factors for three levels Box Behnken design for

‘wagashie’ using plant extract as coagulant 63


‘wagashie’ coagulated with plant extract 64


‘wagashie’ coagulated with Rennet (ml) 65

Table 4.1: Mean microbial count on fresh and fried market wagashie samples in g/CFU 77

Table 4.2: Effect of deep frying and aseptic packaging on the microbial count of fresh

wagashie obtained from Nima in g/CFU. 77

Table 4.3: Mean scores for the confirmatory affective sensory evaluation 101

Table 4.4: Mean pH and TTA values of the optimised ‘wagashie’ samples after confirmatory

affective sensory evaluation 103

Table 4.5: Mean values for wagashie descriptors during the Quantitative Descriptive Analysis

108


xiii

Table 4.6: Mean values for the chemical composition of wagashie 116

Table 4.7: Mean values with standard deviations for the colour of improved wagashie 117

Table 4.8: Mean values for the textural characteristics of ‘wagashie’ 119

Table 4.9: Mean microbial counts in the improved ‘wagashie’ in CFU/g 121

Table 4.10: Changes in the mean microbial counts in the rennet coagulated fermented fresh

and smoked ‘wagashie’ samples as affected by packaging and irradiation for the 5 weeks

storage period in CFU/g (from day 0 to week 2) 123

Table 4.11: Mean pH values of the ‘wagashie’ samples during the 5 weeks storage period

125


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LIST OF FIGURES

Figure 3.1: Flow diagram for plant extract preparation (coagulant) 56

Figure 3.2 Flow diagram of wagashie production process (Traditional method) 57

Figure 3.3: Flow diagram of wagashie process with Plant extract and rennet for both non-

fermented and fermented preparations with cheese and yoghurt cultures 59

Figure 4.1: Mean pH of ‘wagashie’ sampled from the market 78

Figure 4.2: The rate of pH change after 24 hours fermentation in a water bath set at 45oC with

5mls and 10mls of cheese culture 79

Figure 4.3: The rate of pH change after 24 h fermentation of 2 L of pastuerized fresh cow

milk in a water bath set at 45oC with 10 ml and 10 ml of cheese culture. 80

Figure 4.4: Response Surface plot representing the effect of Fermentation time and Salt on the

score for Texture when the weight of extract is 150 g 82

Figure 4.5: Response Surface plot representing the effect of fermentation time and Salt on the

score for Colour when the Extract is 150 g 84

Figure 4.6: Response Surface plot representing the effect of Salt and Fermentation time on the

score for taste. 85

Figure 4.7: Response surface plot representing the effect of salt and fermentation time on the

score for Overall acceptability. 86

Figure 4.8: Contour plot for texture, taste, colour and overall acceptability of wagashie

overlaid on one axis of fermentation time (h) and salt (g) 88


xv

Figure 4.9: Mean pH values for wagashie samples coagulated with plant extract combined by

Box Behnken Design 89

Figure 4.10: The TTA of wagashie samples combined by Box Behnken Design using plant

extract as cogulant 90

Figure 4.11: Response Surface plots representing the effect of salt and Fermentation time on

the score for Texture when Rennet is 0.27%. 92


the score for colour when Rennet is 0.27%. 94


the score for Taste when Rennet is 0.27%. 95

Figure 4.14: Response Surface plot representing the effect of salt and Fermentation time on

the score for overall acceptability when Rennet is 0.27%. 97

Figure 4.15: Contour plot for texture, taste, colour and overall acceptability of wagashie

overlaid on one axis of fermentation time and salt at a constant rennet concentration of

5.35ml. 98

Figure 4.16: The mean pH of wagashie samples combined by Box Behnken Design using

Rennet as caogulant 99

Figure 4.17: The TTA of wagashie samples combined by Box Behnken Design using Rennet

as cogulant 100

Figure 4.18: PCA bi-plot of quantitative descriptive sensory used to describe the sensory

attributes of wagashie in their fresh, fried and roasted forms. 110


xvi

Figure 4.19: Dendogram from cluster analysis of wagashie samples considering sensory

attributes 113

Figure 4.20: Spider plot of the fresh and processed wagashie samples after the quantitative

descriptive sensory evaluation 114


1

CHAPTER ONE

1.0 INTRODUCTION

The slow progress in upgrading traditional food processing and preservation techniques in

Ghana contributes to food and nutrition insecurity. Simple, low-cost, traditional food

processing techniques are the bedrock of small-scale food processing enterprises that are

crucial to rural development. By generating employment opportunities in the rural areas,

small scale food industries reduce rural-urban migration and the associated social problems.

They are vital to reducing post-harvest food losses and increasing food availability. However,

rapid growth and development of small-scale food industries in Ghana are hampered by

adoption of inefficient and inappropriate technologies, poor management, inadequate working

capital and low profit margins. Some successes have however been achieved in upgrading

traditional food processing technologies in Ghana including the production of gari and

agbelema from cassava, the production of kenkey and banku from maize, the production of

dawadawa (a fermented condiment) , production of fura from millet and the traditional

cheese-making (wagashie) process from fresh cow milk. The scientific study of traditional

cheese making offers a growing understanding of the inherent nature, strength and limitations

of traditional food processing and preservation techniques. Cheese-making is one of the

oldest methods of preserving excess milk and is a major business worth billions of dollars in

many industrialized countries. Cheeses are now unique products in their own right and

cheese-making has advanced beyond being merely a food preservation technique.

‘Wagashie’ production thrives mainly in the peri-urban milk producing areas where it

provides employment mainly to women and increases the income of fresh cow milk sellers. A

higher demand for traditional soft cheese (wagashie) increased income of milk sellers by 54%

in Ghana (ILRI, 2006). The terminology with which this product is called has seen some


2

variations in West Africa; it is called woagachi (O‘Connor, 1993) and wara or warankashi

(Ogundiwin, 1978) by the people of Benin Republic and Nigeria respectively.

The process of making ‘wagashie’ was developed by the nomadic Fulani as a means of

preserving excess milk and is based on the milk coagulating properties of the juice from the

leaves and stems of the Sodom apple plant (Calotropis procera). The juice which is

obtained by crushing Sodom apple leaves or stems is mixed with raw cow milk gently

heated in a pot over fire wood or water. The leaves and stems of the Sodom apple plant

contain an organic acid called calotropin which has the ability to coagulate milk. Following

coagulation, the loose curd pieces are poured into small raffia baskets and allowed to drain

from the whey. Others produce ‘wagashie’ by storing milk in the abomasum of slaughtered

calves (Sanni et al., 1999). It is either sold in the fresh or fried forms.

Wagashie is a highly nutritious food with an excellent source of protein, fat, vitamins and

minerals such as calcium, iron and phosphorus. It is used to replace meat or fish, or in

combination with them in various food recipes especially for people with low income and can

contribute to solving problems related to protein deficiency in diets in Africa (Elkhider et al.,

2011). Its low lactose content makes it an acceptable food to many people who suffer from

lactose intolerance associated with milk consumption in Africa and Asia due to low levels of

intestinal β-galactosidase (lactase) (O‘Connor, 1993).The flower and other parts of the

Calotropis procera can be used to cure coughs, catarrh, asthma, stomach pains and

headaches.

The problem associated with the product is that;

Good hygienic practices during milking for wagashie production are not adhered to. Thus the

Fulani do not clean the udder of the cow and the equipments used in the milking process. The

environment in which the milking is done is also not clean; the milking is done mostly in the

kraal with the faeces and urine of the animals. Also, wagashie is not packaged after


3

preparation thus producers and retailers put the curds on metal trays and hand pick them into

flexible polyethylene films for consumers. This exposes the product to post production

contamination. The shelf life of the product may be affected and it also makes the product

unsafe for the consumer.

Traditional wagashie is known to have a bland taste as well as a bitter after taste. The bitter

taste results from the high non-specific proteolytic activity of Sodom apple which also affects

the yield of wagashie and the generation of excessive acid, bitter flavours and green

colouration in the product (Mahami et al., 2012). The product has a bland taste mainly

because the production process has not been standardised.

‘Wagashie' is referred to as a soft unripened cheese therefore it has a high moisture content of

about 50% which makes it highly perishable. Ashaye et al., (2006) observed that, the shelf

life does not exceed three days, after the second day of storage, ‘wagashie’ under ambient

temperature undergo considerable chemical changes. These changes which are moisture

change, proteolysis and Lipolysis are caused by increased activity of the resident lactic acid

bacteria and adventitious microbes. The moisture content reduces causing hardening.

Proteolysis sets in causing sourness and Lipolysis occurs imparting a rancid aroma to the

product. The change in the composition is accompanied by changes in the sensory quality of

the product.

Traditionally, ‘wagashie’ is preserved in its whey which extends the shelf life to two days or

boiled in water to make it tough which can increase the shelf life to 4 days when refrigerated.

It is sometimes fried, smoked or dried to enhance its keeping quality. However, all these

increase its shelf life by only a few days or a week at best. Soaking ‘wagashie’ in different

concentrations of brine ,preservation with biological plant extracts like Afromomum danielli

(Ashaye et al., 2006), ginger and garlic (Belewu et al., 2005) extended the shelf life up to

fifteen days under ambient conditions. However, the taste was affected. Application of


4

preservatives like propionic acid and sodium benzoate (Joseph and Akinyosoye, 1997) also

increased the shelf life up to 15 days but residues of the chemicals in the product altered the

taste.

1.1.0 Justification

The poor hygienic practices during milking and processing of wagashie by the Fulani’s raise

the need for its safety to be assessed.There is also the need to introduce a packaging material

for wagashie in order to minimise post production contamination and extend its keeping

quality.

Standardising the traditional process for wagashie and replacing the extract of sodom apple

plant with commercial rennet will eliminate the bitter after taste in the product.

Traditional and chemical processing and preservative methods affect the sensory quality of

‘wagashie’ thus there is the need to find an appropriate preservative method which will keep

the initial quality of the product and extend the shelf life.

1.2 Main Objecive

To improve the sensory quality, safety and preservation of wagashie

1.3 Specific Objectives

To assess the safety of wagashie

To improve the sensory quality of wagashie

To improve on the safety of wagashie

To extend the shelf life of wagashie


5

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Traditional food Processing

Traditional foods continue to play a central role in the eating habits of people in most part of

West Africa. High post-harvest food losses, arising largely from limited food preservation

capacity, are a major factor constraining food and nutrition security in the developing

countries of West Africa, where seasonal food shortages and nutritional deficiency diseases

are still a major concern. It is estimated that about 50% of perishable food commodities

including fruits, vegetables, roots and tubers and about 30% of food grains including maize,

sorghum,millet, rice and cowpeas are lost after harvest in West Africa. Ineffective or

inappropriate food processing technologies, careless harvesting and inefficient post-harvest

handling practices, bad roads, moribund rail systems, bad market practices and inadequate or

complete lack of storage facilities, packing houses and market infrastructures are some of the

factors responsible for high post-harvest food losses in West African countries (Aworh,

2008).The capacity to preserve food is directly related to the level of technological

development and the slow progress in upgrading traditional food processing and preservation

techniques in West Africa contributes to food and nutrition insecurity in the sub-region.

Traditional food processing provides a source of livelihood for a large number of traditional

food processors in the rural areas and currently in the urban areas (Lartey, 1975). Traditional

foods and traditional food processing techniques form part of the culture of the people and its

activities constitute a vital body of indigenous knowledge handed down from parent to child

over several generations. Some of these techniques include fermentation, drying such as

shallow layer sun drying, heat processing such as roasting, parboiling, cooking, milling such

as wet milling and dry milling and post harvest operations such as winnowing, threshing,


6

peeling, dehulling (Aworh, 2008). There is a vast array of traditional fermented foods

produced in West African Countries .These include foods staple such as gari, kenkey,

agbelima ,fufu, lafun and ogi ,fura; condiments such as dawadawa, ogiri (ogili) and ugba

(ukpaka); alcoholic beverages such as burukutu (pito or otika),shekete and agadagidi; and the

traditional fermented milks and cheese. Lactic acid bacteria and yeasts are responsible for

most of these fermentations (Cooke et al., 1987). The fermentation processes for these

products constitute a vital body of indigenous knowledge used for food preservation, acquired

by observations and experience, and passed on from generation to generation.

2.2.0 Traditional production of ‘wagashie’ a local cottage cheese

In West Africa, the Fulani pastoralists process surplus fresh milk into various stable products

like West African soft cheese (wagashie), Nono (fermented skimmed milk) ,nyarmie and

yoghurt. Wagashie is an unripened cheese consumed in several parts of West Africa mainly

Nigeria (wara or warankashi), Benin (woagachie) and Ghana (wagashie). Nomadic Fulani

who are pastoralists and are mainly involved in the rearing of cattle from one place to the

other are involved in milk production in Ghana. In herding families, only the children,

pregnant women and the elderly drink milk regularly while others get milk only on rare

occasions due to transportation problem or poor keeping quality of milk if not processed

(Belewu et al., 2005). The Fulani women process excess raw milk into a soft, unripened

cheese called “wagashie” as a way of preserving the excess milk for short term. Wagashie is

mainly consumed at home or sold on the market widely in the Northern and Volta regions of

Ghana. Wagashie is also produced in Ghana by small holder groups in the rural and peri-

urban communities under “indigenous” conditions using skills based on traditions (Mahami et

al., 2012). Wagashie processing involves the use of rudimentary equipment, and mostly


7

starter cultures are not used as processing conditions are not normally standardized or

optimized (Belewu et al., 2005).

Wagashie is traditionally prepared by heating fresh milk then coagulating it with an extract of

stems or leaves from the Sodom Apple plant (Calotropis procera). The formed cheese curd is

then poured into perforated calabashes to allow the whey to drain off (ILLR, 2006). The

leaves and stem extracts of pawpaw (Carica papaya) can also be used as a coagulant but the

extracts from Calotropis procera are preferred to the extracts of pawpaw because cheese

processed with Calotropis procera has a sweeter flavor compared to the cheese processed

with pawpaw leaf extracts (O’Connor, 1993). Wagashie is described as soft white unripened

cheese by (Ogundiwin, 1978); it has also been described as soft, wet, feta-like cottage cheese

made from whole milk (Jansen, 1990). It’s similarity with the cottage cheese is related to the

fact that they are both classified as soft cheese due to the high moisture content, they do not

undergo ripening and they have a curd like texture, however, cottage cheese involves the use

of starter culture whiles ‘wagashie’ does not (Ashaye et al., 2006).

Improvement of the product has been carried out by (Sanni et al., 1999) focusing on the

texture, aroma and the nutritional composition by the use of starter culture (lactococcus

lactis). It was discovered that consumers preferred the traditionally prepared ‘wara’ in terms

of appearance and texture but liked the aroma and palatability of the improved type.

Improving ‘wagashie’ with a different coagulant other than Calotropis procera has however

not been done.

2.2.1 Preservation of wagashie `

‘Wagashie’ provides a useful service in extending the shelf life of milk (Alalade and Adeneye

2006) by serving as an important preservation method for surplus milk in rural areas

especially during rainy season when milk is in abundance. (Aworh and Egounlety, 1985)


8

reported the processing technology of ‘wagashie’, its stabilization by heat treatment and the

use of chemical additives such as propionic acid and sorbates. (Kèkè et al., 2008) reported a

method of preservation of ‘wagashie’ using strains of Lactobacillus plantarum. The

preservation of ‘wagashie’ by chemical method has a negative effect on the sensory quality of

the product (Kèkè et al., 2008). Usually wagashie is stored in its whey at room temperature

(28oC), under this storage condition, wagashie is highly perishable and has a shelf life of 2 to

3 days ((Belewu et al., 2005, Adejunti, 2011). Therefore, ‘wagashie’ production must be

protected from spoilage microorganisms from production to the consumer. ‘Wagashie’ is

usually fried and used as a meat substitute in stews and soups, or smoke dried to enhance its

keeping qualities. Studies by (Appiah, 2000) showed that preserving ‘wagashie’ in different

concentrations of NaCl increased the shelf life up to twenty days during storage. However, all

these preservation methods increase its shelf life by only a few days or few weeks (Sessou et

al., 2013). Moreover the traditional practices for preservation of wagashie extends the shelf

life and preserves the sensory quality of the product for a relatively long time but the

exhaustive inventories of all these practices are not well documented (Sessou et al., 2013).

2.2.2 Safety of wagashie

Soft cheeses are good proteins source with high water content (60%) which is favorable for

the growth of microorganisms that affects its quality (Sessou et al., 2013). ‘Wagashie’ is a

type of fresh cheese and its production in Ghana has become a common sight without

supervision or quality due to increase in population and consumption. ‘Wagashie’ is delivered

to the market immediately after processing, under inadequate conditions, poor handling

techniques, inappropriate packaging materials and lack of adequate storage facilities,

however, dairy products including cheese must be safe, acceptable and meet consumer's

satisfaction. The United States Food and Drugs Administration (FDA, 2005) stated that, soft


9

raw milk cheeses can cause serious infectious diseases including Listeriosis, Brucelleosis,

Salmonellosis and Tuberculosis. Recently, the consumer desire for healthy microbiologically

safe foods has been increased; therefore the importance of production of cheese being

properly packed in convenience, smaller size packages, and longer product shelf life is

important.

‘Wagashie’ preparation does not involve the use of starter cultures and lack of adherence to

hygienic principles in production line leads to contamination in cheese with non-starter lactic

acid and Psychotrophic bacteria as investigated by Muehlenkamp-Ulate and Warthesen

(1999); and Sousa et al., (2001). Also according to (Ashaye et al., 2006), wagashie has a

neutral pH of 6.0 to 6.5 and a low salt content which makes it highly susceptible to the

growth of spoilage and pathogenic microbes.

2.2.3 Spoilage and Pathogenic microorganisms in soft cheeses (‘wagashie’)

High moisture content and high pH of soft fresh cheeses make them susceptible to pathogenic

and spoilage microorganisms like E. coli, L. monocytogenes, Salmonella spp. and

Staphylococcus aureus (Kousta et al., 2010). Manufacture of fresh soft cheeses with

pasteurized milk is necessary to reduce non essential microorganisms in milk prior to cheese

preparation. Addition of salt, good sanitation of the cheese plant, the use of good starters

noted by acid production and strict control of storage and processing temperatures are

important factors considered in cheese making (Farkye and Vedamuthu, 2002). Gram

negative psychrotrophic microorganisms such as pseudomonas spp, Coliform bacteria, yeast

and moulds are associated with the spoilage of fresh cheeses. These organisms can cause

sliminess, bitter tastes, off flavours and colour defects due to their high proteolytic and

lypolitic activities.


10

2.2.4 Calotropis procera

Calotropis procera (Sodom apple) is a member of the plant family Asclepiadaceae, a shrub

about 6m high and is widely distributed in West Africa and other parts of the tropics (Irvine,

1961). The plant is erect, tall, large, much branched and perennial with milky latex

throughout. In Nigeria traditional medicine, C.procera is either used alone or with other herbs

to treat common diseases such as fevers, rheumatism, indigestion, cold, eczema and diarrhea

also, preparations from latex with honey are also used as antirabies and also in the treatment

of toothache and cough (Kew, 1985). The secretion from the root bark is traditionally used for

the treatment of skin diseases, enlargement of abdominal viscera and intestinal worms in

India (Parrotta, 2001). In Senegal, the milky latex is locally applied in the treatment of

cutaneous diseases such as ringworm, syphilitic sores and leprosy (Kew, 1985). Leaf extracts,

chopped leaves and latex of C.procera have shown great promise as a nematicide in vitro and

in vivo (Khirstova and Tissot, 1995). The potentials of C.procera leaves in water treatment

and its ability to reduce total viable count have also been reported (Shittu, et al., 2004). In

Ghana, the latex from the leaves and stems of Calotropis procera is used to prepare

‘wagashie’ a local cheese; the extract however causes bitterness in the cheese due to its high

proteolytic activitiy (Mahami et al., 2012).The latex is used as an abortifacient, spasmogenic

and carminative properties, antidysentric, antisyphilitic, antirheumatic, antifungal,

mullusccide, diaphoretic and for the treatment of leprosy, bronchial asthma and skin

infection. Different parts of the plant have been reported to possess a number of biological

activities such as proteolytic, antimicrobial, larvicidal, nematocidal, anticancer, anti-

inflammatory (Basu and Chaudhury, 1991). The flowers have digestive and tonic properties.

On the contrary, the powdered root bark has been reported to give relief in diarrhoea and

dysentery. The root of the plant is used as a carminative in the treatment of dyspepsia. The

root bark and leaves of Calotropis procera are used by various tribes of central India as a


11

curative agent for jaundice, ulcer and leprosy (Shamar et al., 2009). C. Procera is drought-

resistant, salt-tolerant to a relatively high degree, and it disperses seeds through wind and

animals. It quickly becomes established as a weed along degraded roadsides, lagoon edges

and in overgrazed native pastures. It has a preference for and is often dominant in areas of

abandoned cultivation especially sandy soils in areas of low rainfall; assumed to be an

indicator of over-cultivation (Shamar et al., 2009).

2.3.0 Milk production in Ghana

Ghana has about 1.25 million cattle and the West African Shornhorn is the most popular

breed selected for high milk production (Otchere and Okantah, 2001). Currently local milk

production is conservatively estimated at 36.5 thousand tonnes which are mostly from

agropastorial producers. Milk is collected by small holder herdsmen for home consumption

and for sale. Milking is done often in the morning in the presence of the calf to induce milk

let down (Okantah et al., 1999). Okantah (1990), reported a mean daily partial milk yield of

0.9 kg and 0.7 kg for wet and dry season on the Accra plains, Karbo et al., (1998a) reported

similar observations also on the Accra plains. These observations together showed that cattle

kept by smallholders are low milk producers. However large quantities of milk are available

from several thousands of low yielding cattle in small holder system (Okantah, 1990). The

Ministry of Food and Agriculture has therefore initiated a pilot milk collection project on the

Accra plains, peri-urban Kumasi and Sekyedumasi in the Ashanti Region. In 1998, the pilot

collected 85,587 litres of milk from small holder farmers in peri-urban Accra. In 2001, the

annual milk production was estimated at 36.5 thousand tonnes with most of it coming from

small holder agropastorial producers. Also, according to the Animal Production Directorate of

MOFA, (2003), the Sanga was crossed with Friesian to increase daily milk production to


12

between 6 and 8 L as compared to the local breeds which produced less than 2 L of milk per

day. Since the milk production in Ghana is low, the per capital consumption of milk is low.

2.3.1 Milk consumption in Ghana

Milk production in 2006 was estimated at 34,000 metric tonnes and an average of 37,195

metric tonnes of liquid milk equivalent (LME) was imported yearly into the country

(Government of Ghana (GOG)/Food and Agricultural Organisation (FAO), 2002). In Ghana,

few people consume pasteurized raw milk whiles most people consume the condensed full

cream evaporated milk. This is because milk production in Ghana is low resulting in a low

per capital income milk consumption. There is therefore a shortfall between domestic milk

production and consumption. The deficit is made up through imports of milk and milk

products. From 1995 to 1999 the total volume of dairy products that was imported into the

country was 39,831.4 x 103 t (Otchere and Okantah, 2001).

However, in peri-urban Tamale milk producers obtained a fair daily income from the sale of

milk annually (Karbo et al., 1998). According to Nsiah-Ababio (1998) who conducted a study

revealed that the potential income of milk producers was the same as the income contribution

from crop producers. Also a study conducted in the Techiman District of Brong Ahafo

revealed that making ‘wagashie’ (cottage cheese) from fresh local milk added a value of 54%

to milk compared with sales of the fresh product (SFSP–GTZ–MOFA, 1998).

2.3.2 Traditional Fermented milk products in Ghana

Milk is the most abundant fermented animal product in Africa, although the extent to which

milk is used in the dairy diet varies to a great extent (Jespersen, 2003). Fermented milk

products are very important for people suffering from lactose intolerance, social value and as


13

a means of generating income (Beukes et al., 2001). The art of making these products is

handed down from one generation to generation (Caplice and Fitzgerald 1999). In Ghana,

apart from ‘wagashie’, various fermented milk products are produced; there is nunu, nyarmie,

and recently Burkina.

2.3.2.1 Nunu

Traditionally, nunu is prepared by inoculating freshly drawn cow milk with a little of the

leftover as a starter and then it is allowed to ferment for about 24 h at room temperature.

During fermentation, some of the lactose is converted to lactic acid. At the end of

fermentation period, the milk butter is removed by churning for further use, and the

remaining sour milk, nono, is a delicious and refreshing beverage (Olalokun, 1996).

Fermentation is said to be essentially brought about by various species of bacteria especially

members of the genus Lactobacillus and other Lactic Acid Bacteria (LAB), moulds and

yeasts and variations in milk composition, bacterial flora and ambient temperatures have been

noticed to be responsible for products of varying qualities (O’Connor and Tripachi,1995).

2.3.2.2 Burkina

Burkina is also prepared by adding cooked millet to partially fermented milk, sugar and

flavour may be added. Milk powder is mostly used for the preparation. The milk powder is

mixed with water and heated. It is allowed to cool to room temperature and inoculated with

prepared yoghurt or sometimes by natural fermentation and is allowed to occur overnight.

After fermentation sugar and flavour is added and sieved steamed millet is added to the milk

and served in plastic bottles (personal observation).


14

2.3.2.3 Nyarmie

Nyarmie is another fermented product consumed in Ghana by the Fulani tribe. It is produced

by pasteurising raw cow milk at 60 to 75 0 C for 30 – 45 mins. The milk is cooled and kept

overnight at 28 0C for natural fermentation to occur without the use of starter cultures A thick

product is formed; it is then whipped and served. There is however variability among

producers. It is preferred to both the fresh and the pasteurized milk and can be left and

consumed for 5 days unrefrigerated or refrigerated for several weeks at 4 oC. Freshly

produced nyarmie has a pleasant taste and a pH of about 4.2; however, the pH drops to below

4.0 after a day and becomes sour (Obodai and Dodd, 2006).

2.3.3 Milk composition

Since the middle of the 19th century, knowledge of the chemistry of milk has been developed

and today there is extensive literature on the chemistry of the major and minor constituents of

milk, especially cow milk. Variations exist in the composition of milk for various species.

The composition of cow milk varies for a number of reasons, e.g. the individuality of the

cow, the breed and age, stage of lactation, health of the cow, climatic conditions and herd

management which includes feeding and general care (O’Connor, 1993).Milk consists of

protein (caseins and whey proteins), lipid, lactose, minerals (soluble and insoluble), minor

components (enzymes, free amino acids, peptides) and water.The nitrogenous fraction of

cows’ milk typically consists of casein, whey protein and non protein nitrogen (urea,

proteose-peptones, peptides) at levels of 78, 18 and 4 g 100 g−1, respectively, of total

nitrogen (Law and Tamime, 2010).


15

2.3.3.1 Casein

Caseins make up over 80% of the total protein content and they can be further divided into

five groups; the alphas1, alphas2, beta, gamma, and kappa caseins. Caseins do not have an

organized structure, thus they cannot be denatured by heating. The casein fraction coexists

with the insoluble minerals as a calcium phosphate–casein complex. Casein in milk exists in

the form of spherical-shaped colloid particles (40–300 nm diameters) known as casein

micelles (Fox & Brodkorb, 2008; McMahon & Oommen, 2008). Casein, which is typically

present at a level of 2.5 g 100 g−1 in cows’ milk, is the main structural protein of both rennet-

and acid-induced milk gels (cheese) (Law and Tamime, 2010).

2.3.3.2 Whey protein

Whey is the liquid remaining after milk has been cudled and strained during the manufacture

of cheese. It is used to produce ricotta and brown cheeses and is an additive in many

processed foods, such as breads, crackers, pastries, and animal feed. A high level of casein to

whey protein interaction, induced by high heat treatment of the milk, is highly favoured in the

manufacture of yoghurt and smooth-textured cheeses with a high moisture to protein ratio,

such as cream cheese and ultra filtration produced Quark (Guinee et al., 1995). High heat

treatment of milk is generally undesirable for rennet curd cheeses as denatured protein at

levels of ≥25% impedes the ability of the milk to gel on rennet addition, causes deterioration

in melt properties of the cheese and reduces the recovery of fat from milk to cheese (Rynne et

al., 2004). However, a higher than normal heat treatment that gives a moderate degree of

whey protein denaturation may be desirable as a means of modulating the texture of reduced

fat cheese (Guinee, 2003; Rynne et al., 2004).


16

2.3.3.3 Lactose

Lactose or milk sugar is the major carbohydrate of milk occurring at 4.5 to 4.9% levels. It is a

disaccharide composed of glucose and galactose. Lactose is the most abundant of the milk

solid, its crystallization is important in the manufacture and utilization of several dairy

ingredients. Lactose is a useful source of energy and it promotes the absorption of calcium.

However, lack of the enzyme lactase results in the difficulty in digesting lactose, which

results in gastrointestinal distress. Some milk products are available that have lactase

introduced aseptically into the previously sterile product before packaging. The relative

sweetness of lactose is small (20%) when compared to sucrose which is about 100%,

(http://www.lactose.com/basic/physiological_properties.html).

2.3.3.4 Minerals

Cow milk contains minerals, which comprises Potassium, Calcium, Chlorine, Phosphorus,

Sodium, Magnesium, Sulphate and Citric acid (O’Brien et al., 1999c). These minerals are

partitioned into varying degrees between the serum (soluble) and the casein (colloidal or

insoluble) in native milk at room temperature at a pH of 6.6–6.7. Ash which is the white

residue after incineration of a given weight of milk is used as a measure of the mineral

content of milk. It is not identical to milk mineral level because of the decomposition and

volatilization of certain minerals due to heat (Law and Tamime, 2010).Their concentration is

less than 1% in milk, but they are involved in heat stability and alcohol coagulation of milk,

age thickening of sweetened condensed milk, feathering of coffee cream, rennin coagulation,

and clumping of fat globules upon homogenisation. The calcium level of milk influences the

firmness of curd during cheese making


http://www.lactose.com/basic/physiological_properties.html

17

2.3.3.5 Lipids

Lipids exist in the form of an oil-in-water type of emulsion, with fat globules varying from

0.1 to 22 μm in diameter.The lipid content of milk fat is 97–98%, triacylglycerols 2–1%

phospholipids, 0.2–0.4% sterols, and traces of fatty acids, and vitamins A, D, E, and K. The

cholesterol content of whole milk (3.3% fat) and skimmed milk is 14 mg/100 ml and 2

mg/100 ml, respectively. The fat in milk exists in the form of dispersed globules surrounded

by a lipoprotein membrane (milk fat globule membrane, MFGM) (Keenan & Maher, 2006).

Inadvertent damage of the membrane, by manhandling of the milk (e.g. excessive shearing,

turbulence), is highly undesirable in cheese manufacture. It leads to free fat in the cheese

milk, lower recovery of milk fat to cheese, lipolysis of the fat by lipases that survive

pasteurisation treatment, high levels of FFA and undesirable flavours (e.g. bitter, soapiness,

metallic), especially in some cheese types (e.g. Emmental, Gouda, Cheddar).

2.3.4 Milk Quality and Safety

Dairy product safety is an additional concern related to milk quality. Milk safety hazards are

associated with undesirable substances or organisms that contaminate milk and constitute a

risk to the health of the consumer (Anon., 2003).Milk quality may be defined under a broad

range of characteristics notably; microbial (Pathogenic and non Pathogenic bacteria),

chemical, compositional, physicochemical, enzymatic and issues of adulteration. Milk

produced under hygienic conditions from healthy cows should contain not more than 50, 000

bacteria per millilitre (O’Connor, 1993). Sources of microbial pathogens that can contaminate

milk are endogenous sources, such as the cow and exogenous sources, such as the

environment (soil, water, manure or human contact), collection and processing equipment,

milk handlers on the farm and in the factory (Tybor and Gilson, 2003).


18

Milk quality usually may also be defined by the somatic cell count and the bacterial count of

pre-pasteurized bulk tank milk. The largest factor that influences the somatic cell count of

milk is mastitis (Hamman, 2003). The somatic cell count of a cow that is not infected with

mastitis is usually less than 200,000 cells/ml and many cows maintain somatic cell count

values of less than 100,000 cells/ml. A somatic cell count greater than 200,000 cells/ml is

almost always caused by mastitis. High somatic cell count in milk reduces the shelf life of

dairy products and diminishes the quality and quantity of milk protein, thereby reducing

cheese yields (Barbano, et al., 1991). It is important that milk, whether it is for direct

consumption or for the manufacture of dairy products, is of good hygienic quality (O’Connor,

1993). Milk that is of poor hygienic quality will result in poor quality products with low

consumer acceptabilityContaminated milk may cause illnesses to humans including

tuberculosis, brucellosis, sore throats, diarrhoea and abdominal pains (O’Connor 1993).

2.3.5 Microbes and sources of microbes in milk

Milk is an excellent medium for growth of microorganisms; it provides rich nutrients i.e.

proteins, fats, lactose, vitamins and minerals for microbes because of the high moisture

content and neutral pH. Raw milk can potentially contain pathogenic bacteria such as

Salmonella spp., Staphyloccocus spp., E. coli, Bacillus spp., Enterobacteriacea, yeasts, molds

and coliform bacteria (Jayarao and Henning 2001). However, recent cheeses produced in

industries use pasteurised milk thus reducing the risk to public health. Consideration of

pathogenic bacteria is necessary where cheese is manufactured from raw unpasteurised milk

or the manufacture of speciality cheese types although pasteurised milk is used. Cheeses can

easily be contaminated with pathogenic bacteria through mishandling mostly in the

development of smear type cheeses where a complex and different microbial system evolves

on the surfaces of unpackaged cheeses with high pH during ripening in humidified


19

atmospheres . Common Salmonella species (spp.) which contain several strains that cause

illness in humans are the Enteriditis and Typhimurium and are found in the intestinal tracts of

all warm blooded animals including humans can be destroyed by pasteurization (Kongo,

2013). The rate of spoilage of many dairy foods is slowed by the application of one or more

of the following treatments: reducing the pH by fermenting the lactose to lactic acid; adding

acids or other approved preservatives; introducing a desirable microflora that restricts the

growth of undesirable microorganisms; adding sugar or salt to reduce the water activity (aw);

removing water, vacuum packaging and freezing (Ledenbach and Marshall, 2009).

In cheese production, slow lactic acid production by starter cultures favours the growth and

production of gas by coliform bacteria, with coliforms having short generation times under

such conditions. In soft, mold ripened cheeses, the pH increases during maturing which

increases the growth potential of coliform bacteria (Frank, 2001).

Bacterial contamination of raw milk can originate from different sources: air, milking

equipment, feed, soil, faeces and grass. The number and types of microorganisms in milk

immediately after milking are affected by factors such as animal and equipment cleanliness,

season, feed and animal health (Rogelj, 2003). It is hypothesized that differences in feeding

and housing strategies of cows may influence the microbial quality of milk. Rinsing water for

milking machine and milking equipment also influence the presence of a higher number of

micro-organisms including pathogens in raw milk (Bramley, 1990). After milking, milk is

cooled, which additionally influence the dynamic of microbial process (Rogelj, 2003). Entry

of food borne pathogens through contaminated raw milk into dairy food processing plants can

lead to the perseverance and establishment of pathogens in the form of bio films, subsequent

contamination of processed milk products and exposure of consumers to the pathogens.

Insufficient processing may result in the survival of definite pathogens and such contaminants

become a public-health threat (Oliver et al., 2005). The conditions during storage and


20

transport in refrigerated tanks cause the raw milk micro biota to change from predominantly

Gram-positive to predominantly Gram-negative organisms as they grow.

2.3.6 Lactic acid fermentation of milk

Lactic acid bacteria (LAB) are Gram-positive, usually non-motile, acid tolerant

microorganisms. They have complex nutritional requirements and a fermentative metabolism.

Phylogenetically the lactic acid bacteria belong to the clostridial branch of the Gram-positive

bacteria. They are catalase negative, non spore forming, cocci, cocobacilli or rods that have

less than 55 mol% G+C content in their DNA (Stiles and Holzapfel, 1997). Species of lactic

acid bacteria (LAB) belong to numerous genus under the family of Lactobacillaceae.

Fermentation, also known as bio preservation, is a cheap, widely accessible method of food

preservation. Bio preservation with lactic acid bacteria (LAB) is indeed one of the oldest and

highly efficient forms of non-thermal processing method. Fermentation is generally

considered as a safe and acceptable preservation technology of food and fermentation using

LAB can be categorized into two groups based on the raw material used, non-dairy and dairy

fermentation. Milk from different mammalian animals can be used in dairy fermentation to

produce several products. Milk of cow followed by milk of goat and sheep are the most

widely used raw materials to produce particular economic value fermented milk products

worldwide (Widyastuti et al., 2014). The presence of LAB in milk fermentation can be either

spontaneous or inoculated starter cultures. Milk is one of the natural habitats of LAB

(Delavenne, et al., 2013, Fox and Mcsweeny,2004) The most important properties of LAB

are their ability to reducing the pH of milk and to generate flavour and texture, by converting

milk protein due to their proteolytic activities (Griffiths and Tellez,2013). LAB has the ability

to ferment sugars, especially glucose and galactose, to produce lactic acid and aroma

substances that give characteristic flavors and tastes to fermented products. LAB also release


21

antimicrobial metabolites called bacteriocins, which are considered safe and natural

preservatives, with great potential to be used on their own or synergistically with other

methods in food preservation (Kongo, 2013). ). Among the bacteriocins produced by LAB,

nisin produced by Lactococcus lactis spp., is the only bacteriocin that has been officially

employed in the food industry and its use has been approved worldwide (Zacharof and Lovitt,

2012). Recently LAB isolates from traditional Portuguese raw-milk cheeses, revealed several

lactobacilli having antibacterial activity against pathogens such as Listeria monocytogenes,

Staphyloccus aureus, Salmonella Newport and E.coli (Kongo, 2013). LAB have a long and

safe history of application and consumption in cheese processing (Wood, 1997; Wood &

Holzapfel,1995, Caplice & Fitzgerald, 1999 Giraffa et al., 2010) thus being generally

regarded as safe (GRAS). Lactic acid bacteria represent the most extensively studied

microorganisms for milk fermentation (Olson, 1990 and Maragkoudakis et al., 2006) and

imparts the mild acid taste and pleasant fresh characteristics to fermented milk products such

as yoghurt and cheese.

2.4.0 Cheese

Cheese is a concentrated protein gel, which absorb fat and moisture (Law and Tamime,

2010). According to CODEX STAN 283(1978) Cheese is the matured or not fully matured

soft, semi-hard, hard, or extra-hard dairy product, which may be coated, and in which the

whey protein to casein ratio does not exceed that of milk by coagulating wholly or partly the

protein of milk, skimmed milk, partly skimmed milk, cream, whey cream or buttermilk, or

any combination of these materials, through the action of rennet or other suitable coagulating

agents, and by partly draining the whey resulting from the coagulation, while respecting the

principle that cheese making results in a concentration of milk protein and that therefore, the

protein content of the cheese will be definitely higher than the protein level of the blend of the


22

milk supplies from which the cheese was made and by processing techniques involving

coagulation of the protein of milk and products obtained from milk which give an end-

product with the same physical, chemical and organoleptic characteristics.Cheese is one of

the commonest dairy products in the world (Belewu et al.,2012). It is today, a major business

worth billions of dollars in many industrialized countries (Aworh, 2008). Cheeses are now

unique products in their own right and cheese-making has advanced beyond being merely a

food preservation technique (Aworh, 2008).

2.4.1 Ingredients for cheese making

2.4.2 Milk

2.4.2.1 Selection of milk for cheese making

Cheese manufacture commences with the selection of milk of high microbiological and

chemical quality, cheese milk must be free of antibiotics. In commercial practice, milk for

cheese is normally cooled to 4°C immediately after milking and may be held for several days

(Fox, 1993). Although raw milk is still used in cheese making, most cheese milk is

pasteurized before use. Pasteurization alters the indigenous micro flora and facilitates the

manufacture of cheese of more uniform quality, but unless due care is exercised, it may

damage the ability of the rennet to coagulate and the curd forming properties of the milk

even when properly pasteurized. Pasteurization of cheese milk minimizes the risk of

cheese serving as a carrier of food poisoning or pathogenic microorganisms, thus high

quality raw milk may be unacceptable for cheese manufacture (Fox, 1993). Milk for cheese

production should be free of any visible impurities, must not have any abnormal taste or

odour, have a pH of 6.6 or slightly higher at the milking time, must not be contaminated by

pathogenic microorganisms which may prove undesirable for the production of cheese. The


23

milk must also contain no foreign substances such as antibiotics, antiseptics and cleaning

products.

2.4.2.2 Starter culture

Certain cheese varieties require pure cultures of lactic acid bacteria containing organisms

with specific functions while for traditional cheeses, a natural fermentation is allowed using

whey from the previous lot (Kongo et al., 2013). The recipe will indicate the type and

quantity of starter to be used and temperature conditions.

2.4.2.3 Lactic acid bacteria as starter culture in cheese making

Cheese making is based on the use of lactic acid bacteria ( LAB) in the form of defined or

undefined starter cultures that are recognized to cause a rapid acidification of milk through

the production of lactic acid, with a resultant decrease in pH, thus affecting a number of

aspects of the cheese making process and in the end cheese composition and quality (Kongo,

2013).The initial productions of cheeses were based on the natural fermentation resulting

from the growth of the microflora naturally present in the raw milk and its environment. It is

known that while unprocessed milk can be stored for few hours at room temperatures, cheeses

may reach a shelf life up to five years depending on the variety (Kongo, 2013). The quality of

the cheese is as a result of the microbial load and range of the raw materials. Natural

fermentation was later optimized through back slopping, which is inoculating of the raw

material with the whey from a previously performed successful fermentation, the end product

characteristics depended on the best adapted strains dominan. Fresh cheeses with unlimited

shelf life have the primary proteolysis which is performed by the coagulating agents and to a

lesser extent plasmin residual coagulants and enzymes from the starter organisms (Sousa et

al., 2001). Starter cultures of LAB used in cheese making can be either mesophilic from the


24

genera of Lactococcus and Leuconostoc or thermophilic from the genera of Streptococcus and

Lactobacillus (Fox and Mcsweeny, 2004). Among species, Lactococcus lactis (Dias and

Weimer 1998, Hannon et al., 2007), Streptococcus thermophilus (Helinck et al., 2004) and

Lactobacillus helveticus (Dias and Weimer, 1998) are intensively studied. L. helveticus is

specialized in milk species and belong to the member of dairy niche species (Slaterry et al.,

2010). Several cheese products are based on L. helveticus as starter culture. It is also known

that L. helveticus have significant role in production of specific flavour compounds in Italian

cheese types and removing the bitterness in cheese (Gatti et al., 2003, Rossetti et al., 2008).

The starter cultures added during the production of fresh cheese are mesophilic group

including L. Latis sub sp. lactis and L. lactis sub sp. cremoris with different capacity of

producing citrate. Diacetyl is a major product of citrate metabolism of lactococci and is

desired in many fresh cheese varieties such as cottage cheese (Fernández et al., 1994).

2.4.2.4 Chemicals

Chemicals such as calcium chloride and sodium nitrate are recommended for some varieties

of cheese to improve curd quality and prevent the growth of organisms which may cause

problems during the ripening or maturing of the cheese (Cooker et al., 2005).

2.4.2.5 Coagulants

Coagulation, or clotting of the milk, is the basis of cheese production. Coagulation is brought

about by physical and chemical modifications to the constituents of milk and leads to the

separation of the solid part of milk, the curd from the liquid part, the whey. Milk coagulation

being one of the most important steps in the cheese manufacturing process determines the

final cheese properties (García, et al., 2012). The difference in protein matrix degradation as a

result of the agents used in the clotting process affects the changes that take place in the yield,


25

the cheese texture (elasticity, fragility, adhesiveness, hardness, gumminess and chewiness)

and the development of flavours (especially a bitter taste), through the production of

hydrophobic peptides and the hydrolysis of caseins (Macedo et al., 1993).The two types of

coagulants used in cheese making are, rennet (Animal and Microbial rennets) and plant or

vegetable extracts. In the olden days, many cheeses were made with vegetable or plant

coagulant but some are now made with animal or microbial coagulants. Animal and microbial

coagulants give more consistent products and are also cheaper and easier to use, thus avoiding

the labour intensive and expensive collection of plants (Roseiro et al., 2003)

2.4.2.5.1 Rennet coagulants

Rennet is the most common coagulant. The most commonly used rennet contains the

enzymes chymosin and pepsin, either as an extract from the abomasums of calf or as the

recombinant products (microbial source). The development of products with new sensory and

textural features is one of the main areas of innovation in cheese making (García, et al.,

2012). Calf rennet, has until lately been the reference product against which alternative

products are measured. Adult bovine rennet is the most widely used alternative to calf rennet

because it contains the same active enzymes as calf rennet. Bovine rennet has a high pepsin

content which gives the product a high sensitivity to pH, and a higher general proteolytic

activity. Also lamb ovine and kid-caprine or caprine rennet are very similar to calf or adult

bovine rennet, but they are best suited for clotting milk of their own species (Foltman, 1992).

Animal rennet is usually mixed with lipases, especially during the manufacture of South

Italian cheeses, in which they produce a characteristic flavour. Such products are called

rennet paste, and they are made by maceration and drying of stomachs from suckling calves,

lambs or kid-caprine, which have their stomachs filled with milk. Thus rennet paste contains


26

a mixture of rennet and lipase (pregastric and possibly gastric) enzymes in an un-standardised

ratio (Law and Tamime, 2010).

2.4.2.5.2 Microbial coagulants

The most popular microbial coagulants used for cheese making are of fungal origin. Most

bacterial proteases identified as milk clotting enzymes are unsuitable due to their high

proteolytic activity (Law and Tamime, 2010). The most regularly used microbial coagulants

are proteases derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria

parasitica, R. Miehei, have been used as a substitute of animal rennet for almost 40 years

(Jacob et al., 2011). C. parasitica proteases cleave the Ser104-Phe105 bond in κ-casein, while

R. miehei cleaves the Phe105-Met106 bond. Also, the higher heat stability of the derivatives

obtained from R. miehei may be due to excessive proteolysis, with a reduced ripening time

and bitter cheeses. Coagulants with greater heat stability than calf rennet should be avoided

and there should be differences in the coagulation temperature to limit excessive proteolysis

(Sousa et al., 2001).

2.4.2.5.3 Fermentation Produced Chymosin

FPC is chymosin produced by fermentation of a Genetically Modified Organism

(GMO).They contain chymosin similar to the chymosin from animal source, thus they have

the same amino acid sequence as chymosin from animal stomach. The main FPC, which

contains bovine chymosin B, is now considered to be the ideal milk clotting enzyme against

which all other milk-clotting enzymes are measured. The production and application of

bovine type FPC has been reviewed by Harboe, (1992a, 1993); Repelius, (1993) and recently,

a new production of FPC, equal to camel chymosin, has been manufactured. FPC (camelus)

has been found to be an effective coagulant for bovine milk than FPC (bovine), and is


27

characterised by its very high specificity against caseins, which leads to high cheese yields

without creating any bitterness (Law and Tamime, 2010).

2.4.2.5.4 Vegetable or Plant Coagulants

Cheeses made with vegetable coagulant can be found mainly in the Mediterranean, West

African and Southern European countries. Spain and Portugal have the largest variety and

production of cheeses using Cynara sp. as the vegetable coagulant. Rennet substitutes of plant

origin have been increasingly used to manufacture cheese. Juice extracts from fruits and

plants have long been used as milk coagulants (O’Connor, 1993). Application of plant

coagulants allows target cheese production, and hence contributes to improve the nutritional

input of those populations on whom restrictions are imposed by the use of animal rennets

(Gupta & Eskin, 1997). Several plant preparations have been shown to clot milk (Aworth and

Muller, 1987; Edwards and Kosikowski, 1983; Padmanabhan et al., 1993; Pozsar et al., 1969;

Tamer, 1993) to clot milk; however, the majority proved unsuitable for cheese production due

to their excessively proteolytic activity which reduces cheese yield and produce bitter

flavours in the final cheese (Lo Piero et al., 2002). Cynara sp. (cardoon) extract has been

widely used for centuries for making traditional Portuguese and Spanish ewe’s milk cheeses.

Similarly, Calotropis procera (Sodom apple) which grows abundantly in many parts of

Africa has been used for traditional cheese making in West African countries, such as

Nigeria, the Republic of Benin and Ghana.Other types of plant or vegetable extracts used as

cheese coagulants are lemon juice (Adetunji et al., 2007), Carica papaya leaves and sap, the

berries of Solanum elaeagnifolium (trompillo or silverleaf nightshade) (Martinez-Ruiz et al.,

2013) pineapple (bromelin), castor oil seeds (ricin) and latex of the fig tree and the plant. The

types of rennet and coagulants and their characteristics have been reviewed by several authors

(Harboe, 1992b; Guinee & Wilkinson, 1992; Garg & Johri, 1994). These extracts are suitable


28

for softer curd cheese which is consumed within a few days. The extracts are not suitable for

hard cheese with long maturing periods because of their excessive proteolytic activity which

results in bitter flavours in the ripened cheese (O’Connor, 1993).Rennet and coagulants are

most efficiently categorised according to their source.

Table 2.2 shows the predominant types of coagulant used for cheese making and their active

enzyme components.

Table 2.1: The most commonly used rennet and coagulants and their enzymes

Group Source Examples of rennet

and coagulants

Active enzyme

components

Animal Bovine stomachs

Ovine stomach

Caprine stomach

Calf rennet, adult

bovine rennet

Rennet paste

Lamb rennet, ovine

rennet

Kid-caprine rennet,

caprine rennet

Bovine chymosin A,

B and C,

pepsin A and

gastriscin

The same as above,

plus lipase

Ovine chymosin and

pepsin

Caprine chymosin

and pepsin

microbial Rhizomucor miehei

Cryphonectria

Parasitica

Miehei coagulant

type L, TL, XL

and XLG/XP

Parasitica coagulant

Rhizomucor miehei

aspartic

proteinase

Cryphonectria

parasitica aspartic

Proteinase

FPCa Aspergillus niger

Kluyveromyces

marxianus var. Lactis

CHY-MAXTM

CHY-MAXTM M

Maxiren R _

Bovine chymosin B

Camelus chymosin

Bovine chymosin B

Vegetable Cynara cardunculus Cardoon Cyprosin 1, 2 and 3

and/or cardosin

A and B

Source: Law and Tammine, 2010

2.4.2.3 Salt

Salt (sodium chloride) may be added to some varieties of cheese, the quantity and method of

addition depending on the recipe. Salt may be added directly to the milk or curd pieces; it


29

may be rubbed into the finished cheese or the cheese may be immersed in a brine solution

(O’Connor, 1993).

2.5.0 Cheese Manufacture

Cheese manufacture is one of the classical examples of food preservation, dating from 6000-

7000 BC (Fox, 1993). It preserves the most important constituents of milk (i.e. fat and

protein) which are determinant factors of cheese yield, (Banks et al., 1981), as cheese exploits

two of the classical principles of food preservation, i.e. lactic acid fermentation and reduction

of water activity through removal of water and addition of salt (NaCl) and the establishment

of a low redox potential, as a result of bacterial growth which contributes to the storage

stability of cheese (Coker et al., 2005). Cheese making remained an art rather than a science

until relatively recently. With the gradual acquisition of knowledge on the chemistry and

microbiology of milk and cheese, it became possible to direct the changes involved in cheese

making into a more controlled fashion (Fox, 1993). A number of developments have taken

place which helps the cheese maker to produce a better and more consistent quality cheese.

These developments include ; Pasteurisation, Acidification, Coagulation, Synerises, Salting,

Ripening

2.5.1 Pasteurisation

Milk may be heat treated or pasteurized at 73oC for 15 seconds (O’Connor, 1993).

Pasteurisation of the milk kills nearly all the microorganisms present, including the harmful

pathogenic bacteria that cause diseases, such as tuberculosis and leptospirosis, and other

undesirable microorganisms such as yeasts and coli forms that may alter the cheese

characteristics by producing carbon dioxide and undesirable proteolysis (Coker et al., 2005).


30

The milk may be standardised, thus the fat content may be increased or reduced or the casein

to fat ratio may be adjusted (O’Connor, 1993).

2.5.2 Acidification

One of the basic operations in the manufacture of most cheese varieties is a progressive

acidification throughout the manufacturing stage for some varieties during the early stages of

ripening, thus acidification commences before and transcends the other manufacturing

operations (Fox, 1993). Acidification of the milk is important for the proper release of whey

from the cheese curd and to control the growth of many undesirable bacteria (Coker et al.,

2005). Acidification controls the growth of many species of non-starter bacteria in cheese,

especially pathogenic food poisoning and gas producing microorganisms. Properly made

cheese is a very safe product from the public health viewpoint. In addition to producing

acid, many starter bacteria produce probiotics that also restrict or inhibit the growth of

non starter microorganisms (Fox, 1993). It is usually accomplished by the addition of lactic

acid bacteria that convert lactose to lactic acid. Most varieties of cheese cannot be made

without the addition of a "starter" which is a culture of carefully selected lactic acid-

producing bacteria. The large volumes of starter required for cheese making are made in

special bulk starter fermentation pots in which the milk is heat treated to destroy unwanted

bacteria, spores and phages and cooled to about 22°C, a temperature suitable for starter

growth. The frozen starter is mixed in and fermentation continues for about 6 to 16 hours.

The amount of starter required varies for the different cheese varieties (Coker et al., 2005).

Good quality starter is required, the type and quantity will be determined by the cheese

recipe. For some cheese varieties commercial starter preparations are not used; natural

fermentation or whey from the previous lot of cheese made may be used (O’Connor, 1993).


31

2.5.3 Coagulation

During coagulation, modifications of the milk protein complex occur under defined

conditions of temperature and by action of a coagulant agent, which changes the physical

aspect of milk from liquid to a jelly-like mass. Various coagulants are available, lemon juice,

plant rennet and proteolytic enzyme such as chymosin (rennin) and proteolytic enzymes from

the mould Rhizomucor miehei obtained by biotechnology. These enzymes have an acidic

nature, thus they have optimum activity in a slightly acidic environment. Therefore, the action

of lactic acid bacteria (LAB) in this phase is crucial as they are required to rapidly release

enough lactic acid, to lower the milk pH from 6.7 to about 6.2 which creates an appropriate

environment for optimum activity of rennin and to a pH of 4.5 as the processing proceeds,

creating an unsuitable environment for unwanted microbes, thus increasing the safety of the

end product (Kongo, et al., 2013). A rennet coagulum consists of a continuous matrix of

strands of casein micelles, which incorporate fat globules, water, minerals and lactose and in

which microorganisms are entrapped (Coker et al., 2005). The coagulants bring about, under

defined conditions of temperature, quantity and time to coagulate the milk into a firm jelly-

like mass (O’Connor, 1993).

2.5.4 Syneresis

Syneresis is the rearrangement of casein molecules, which results in a tightening of the casein

network. The end result is that moisture is squeezed out of the casein network. (Law and

Tamime, 2010). Syneresis, or shrinking, of the coagulum is largely the result of continuous

rennet action. It causes loss of whey, and is accelerated by cutting, stirring, cooking, salting

or pressing the curd, as well as the increasing amount of acid produced by the starter, and

gradually increases during cheese making. As a result, the cheese curd contracts and moisture

is continuously expelled during the cooking stages (Coker et al., 2005). The rate and extent


32

of syneresis are influenced by, milk composition, especially Calcium and casein, pH of

the whey, cooking temperature, rate of stirring of the curd-whey mixture and time. The

composition of the finished cheese is determined by the extent of syneresis (Fox, 1993).

2.5.5 Salting

Salt is added to cheese as a preservative and because it affects the texture and flavour of the

final cheese by controlling microbial growth and enzyme activity. The salt can be added

either directly to the curd after the whey is run off and before moulding or pressing into

shape, or by immersing the shaped cheese block in brine for several days following

manufacture (O’Connor, 1993).The level and method of salting have a major influence on pH

changes in cheese. The concentration of NaCI in cheese is between ( 0·7--4% and 2-10%)

thus salt in the moisture phase is sufficient to halt the growth of starter bacteria. Some

varieties, mostly of British origin, are salted by mixing dry salt with the curd towards

the end of manufacture and hence the pH of the curd for these varieties must be close

to the ultimate value (pH 5·1) at salting (Fox, 1993). Addition of salt to the cut curd

draws more whey from the cheese curd and some of the salt diffuses into the curd. The pH of

the curd, the contact time and the salt particle size and structure are all important in

determining how much salt is absorbed by the curd. Salt is also involved in physical changes

in cheese protein solubility and conformation, which influence cheese rheology and texture.

Another important function of salt in cheese is as a flavour or a flavour enhancer (Coker et

al., 2005). Salt also retards or prevents the growth of bacteria which may cause flavour and

other defects in the cheese (O’Connor, 1993).


33

2.5.6 Ripening or Maturation

Some cheeses are consumed fresh, however most cheese varieties are not ready for

consumption at the end of manufacture and undergo a period of ripening (curing,

maturation) which varies from about three weeks to more than two years. The ripening

process of cheese is very complex and involves microbiological, biochemical, structural,

physical and sensory changes during storage to the curd resulting in the flavour and texture

characteristic in the particular variety (Mcsweeny, 2004). Cheese texture softens during

ripening as a consequence of hydrolysis of the casein micelle by proteolysis and changes to

the water-binding ability of the curd and changes in pH which may cause other changes such

as the migration and precipitation of calcium phosphate (Mcsweeny, 2004). It has a major

effect on the quality of most cheese varieties with the exception of unripened cheeses

including fresh acid curd cheeses (Quark and Cream cheese) and some ingredient cheeses

(Law and Tamime, 2010). Cheese ripening involves the primary degradation of milk

constituents by glycolysis, lypolysis and proteolysis (Marilley and Casey, 2004).

Glycolysis occurs when lactose is metabolised completely to lactic acid and catabolised to

form acetic and propionic acids, carbon dioxide, esters and alcohol by the enzymes of the

starter cultures or secondary cultures in the milk (Fox et al., 2000). The presence of residual

lactose persisting in cheese during maturation is undesirable as it makes the cheese less

suitable for lactose intolerant consumers (Lomer et al., 2008), and also because it can be used

as a growth substrate by non starter lactic acid bacteria, which can affect the flavour and

quality when present in high numbers >108 cfu g−1 (Beresford &Williams, 2004). Fat is a

major component in most cheese varieties, with the exception of some low fat fresh acid

cheeses, such as Quark and Cottage cheese, and contributes directly and indirectly to

rheology, texture, cooking properties and flavour. During ripening lipids are broken down to

form free fatty acids, and catabolised to form ketones, lactones and esters by natural milk


34

enzymes (milk lipase) and lipids that are added to enhance flavour in particular cheese

varieties. Lipase action is high in raw milk compared to pasteurized milk cheeses. According

to (Vlaemynck 1992), pasteurization of milk partially inactivates milk lipase. The optimum

pH and temperature of milk lipase is 8.0 to 9.0 and at 35oC to 40oC respectively. A

combination of low pH and high salt concentration also inhibit the activity of milk lipase.

Though Lipolysis is needed for flavour enhancement, too much of it imparts a rancid flavour

which is undesirable in fresh cheeses (Ashaye et al., 2006).

Proteolysis also involves the gradual breakdown of proteins (caseins) to form peptides and

amino acids by the enzymes of the coagulant (residual chymosin), the natural milk enzymes

(peptidases) and the enzymes of the starter culture (Bylund, 1995). Proteolysis in cheese is

important for flavour and texture development due to the breakdown of the protein network,

decrease in water activity through water binding by carboxyl and amino acid groups and

increased pH (Sousa et al., 2001).The sequence of residues of the casein is strongly

hydrophobic and confers intact casein with strong self association and aggregation tendencies

in the cheese environment. Its cleavage is generally considered to be a major factor

contributing to the decrease in the rubberyness of young internal ripened hard cheeses, such

as Cheddar, Gouda, and Mozzarella, and their conversion to smooth bodied mature cheeses

(Law and Tamime, 2010). Proteolysis also has a major effect on the cooking properties of

cheese, increase in proteolysis generally coincide with increases in the levels of protein

hydration, free fat and of heat induced flow ability (Guinee, 2003). The degree of

stretchability of the melted cheese also increases progressively with proteolysis to a level,

which depends on the variety and decreases afterwards (Law and Tamime, 2010).


35

2.5.7 pH of cheese

The pH of cheese is a very important physicochemical parameter affecting the texture,

flavour and microbiological safety of cheese. The addition of starter cultures for milk

fermentation, the de-acidification of some varieties of cheese during maturation and the

ability of the curd formed to resist changes in pH determine the final pH of cheese. The main

components of cheese that affects the pH of cheese are 0=-the caseins. The degradation of

casein produces inorganic phosphates and organic acids and their levels in cheese are

influenced by milk composition, curd treatment and its effect on syneresis (Lucey et al.,

1992). Lipolysis also increases the acidity of cheese by the production of free fatty acids

(Law and Tamime, 2009).Decrease in pH of cheeses during maturation is due to the

continued production of lactic acid lactic acid bacteria and the liberation of amino acids such

as aspartic and glutamic acids during proteolysis (Sallami et al., 2004). The pH of soft fresh

cheeses ranges from approximately 4.1 to 5.4 (ICMSF, 1996), however, the buffer maximum

which is around pH of 5.0 is very important in cheese making since the optimum pH for most

cheese ranges from 5.0 - 5.2. As the pH of cheese is reduced towards pH 5.0 by lactic acid

fermentation, the buffer capacity is also increasing. The effect is to give the cheese maker

substantial room for disparity in the rate and amount of acid production. Without milk's built

in buffers it would be difficult to produce cheese in the optimum pH range.

(http://www.uoguelph.ca/foodscience/cheesemaking-technology/section-b-analytical/process-

and-quality-control-proceudures/ph


http://www.uoguelph.ca/foodscience/cheesemaking-technology/section-b-analytical/process-and-quality-control-proceudures/ph


36

2.6.0 Rheology of cheese

2.6.1 Texture measurement

Cheese texture may be defined as a ‘composite sensory attribute’ resulting from a

combination of physical properties that are perceived by the senses of touch (including

kinaesthesis and mouth-feel), sight and hearing. It can be measured directly using a trained

sensory panel; however, owing to the difficulty and cost in assembling sensory panels, they

are not routinely used for gauging cheese texture. Instead, cheese texture is generally

measured indirectly using rheological techniques (O’Callaghan & Guinee, 2004). The

rheology of hard or semi-hard cheese is commonly assessed by compression of a cylindrical

or cubic cheese sample between two parallel plates of a texture analyser (Fenelon & Guinee,

2000; Everard et al., 2007c). The cheese sample is placed on a base plate, and is compressed

at a fixed rate (typically 20 mm min−1) to a predetermined (e.g. 75% of its original height) by

the mobile plate (cross head). The compression may be carried out in one or two cycles

(bites). Analysis of the force – displacement or stress – strain curves, often referred to as

texture profile analysis, enables the determination of a number of rheological parameters e.g.

fracture stress, fracture strain, firmness and springiness, which are related to sensory textural

characteristics, such as brittleness, shredability, hardness and chewiness (O’Callaghan &

Guinee, 2004; Dimitrelia & Thomareis, 2007).

2.6.2 Colorimetry

Colour is an important measure of quality in the food industry because it is considered by

consumers to be related to product freshness, ripeness, desirability and food safety (McCraig,

2002; Jeli´nski et al., 2007). Colour measurement instruments, in accordance with the

standards developed by the Commission Internationale de l’ ´ Eclairage, transform or filter

reflected spectra to produce reproducible colour space coordinates, namely, L* (index of


37

whiteness), a* (index of redness), and b* (index of yellowness) (Commission Internationale

de l’ ´ Eclairage, 1986; MacDougall, 2001). Colour measurements are normally carried out in

a laboratory based instrument (HunterLab meter or Minolta Chroma meter) but they can also

be acquired by online instruments. Owing to ageing effects of light sources and detector

systems, regular calibration of colorimetric equipment against colour standards is essential.

Colorimetry is used routinely in quality control and product development to assess the colour

of curd and cheese. Colour is related to diet of cow, addition of colouring and cheese variety.

Recent studies also showed the potential role of colorimetry in assessing ripening of smear-

ripened cheese (Dufoss´e et al., 2005; Olson et al., 2006) and for measuring defects, such as

browning, during cheese maturation (Carreira et al., 2002).

2.6.3 Classification of cheese

There are hundreds of varieties of cheese, but each relies on similar principles of coagulating

the proteins in milk to form curds and then separating them from the liquid whey. The

percentage of water present in cheese, the microorganism used in ripening, and the length of

the maturing period of the cheese differentiates the many types of cheese present today

(Coker et al., 2005). Cheeses may be broadly classified into ‘soft’, ‘semi-hard’ and ‘hard’

cheeses (Fellows, 2014).Table 2.1 shows the classification of cheese based on their moisture

conten


38

Table 2.2: Classification of cheese

Type of

cheese

Moisture

content (%)

Fat content

(%)

Texture Shelf-life

Soft cheeses 45–75 <40 Soft, white,

spreadable

A few days

or weeks

Semi-hard

cheeses

35–45 <35 Firm,

crumbly, can

be sliced

Several

weeks

Hard cheeses 30–40 <30 Very firm,

dense

Several

months

CTA 2014 http://knowledge.cta.int/ Peter Fellows

2.7.0 Food Product Development

Product Development is a systematic, commercially oriented research to develop products

and processes satisfying a known or suspected consumer need. Product development is a

method of industrial research in its own right. It is a combination and application of natural

sciences with the social sciences of food science and processing with marketing and

consumer science into one type of integrated research whose aim is the development of new

products. The most widely referenced normative product development models are those of

Booz, Allen and Hamilton Inc. (1982) and Cooper and Kleinschmidt (1986). There are

essentially four basic stages in these models for every product development process. These

are: product strategy development; product design and development; product

commercialization; product launch and post-launch. The food industry appears to be

populated with companies that prefer to re-develop existing products (incremental change),

rather than create new products (radical change). Because food product development is


39

considered a highly risky venture, the incremental change strategy may be an attempt to

increase success rates. Ironically, this apparently ‘safe’ approach perpetuates the problem of

high food product failure, since truly innovative products are often more successful for a

company (Stewart-Knox & Mitchell, 2003). However, there are some indications that certain

factors may improve the number of the success rate in product development. Three important

factors that contribute to new product success were cited by Ilori et al., (2000). They were:

marketing and managerial synergy, strength of marketing communications and launch effort,

and market need, growth and size. These factors emphasize the role of marketing in the

product development process. Tetra Pak (2004) found one or more of the following features

are typical of new products that succeed in the marketplace. Therefore, these could be used as

criteria while screening ideas in the product development process: noticeable advantages for

the consumer; the more the better; distinctive details that are important to the consumer;

satisfy the consumers’ need for convenience, youth, better diet, less stress, perfect taste and

variation; reliable brand; advertising breakthrough. Stewart-Knox & Mitchell (2003) found

that understanding consumer needs and expectations and retailer involvement in product

development were associated with product success.

2.8.0 Packaging and Preservation of cheese

Increasing the shelf life of food using different preservative methods has always been a major

concern. Several preservation techniques are available to extend the shelf storage of food

products, among which packaging is the most capable. The packaging process undertakes

several basic roles such as preventing microbial and chemical quality deterioration and

enhancing the handling and marketing for packaged products. Dairy products are an

important food group highly suggested by nutritionists and it is one of the most perishable

category foods thus extending their shelf life and keeping quality for a long time is important.


40

Consumers are aware of the possible hazards of preservatives therefore technologists and

researchers have attempted to introduce new non preservative methods (Coker et al., 2003).

One technique is the modified atmosphere packaging (MAP), which changes the natural gas

surrounding the product in the package to slow down spoilage. Vacuum packaging is a type

of packaging whereby the air within the pack is removed and the pack sealed creating a

vacuum around the product. The gaseous atmosphere of the vacuum package is likely to

change during storage due to metabolism of the product or microorganisms thus the

atmosphere becomes indirectly modified. Vacuum packaging has so far been the most widely

used packaging technique for cooked products (Borch et al., 1996, Korkeala et al., 1985 and

Samelis et al., 2000). Perishable products have exhibited superior quality under vacuum or

MAP storage than under ambient conditions. Recent cheese packaging protects the food from

microorganisms and prevents moisture loss. Unripened cheeses are packaged immediately

after the curd is collected and must be immediately refrigerated. Ripened cheeses go through

various procedures during packaging for preservative reasons. Some ripened cheeses are

coated in wax to protect them from mould contamination and to reduce the rate of moisture

loss. Cheeses that naturally develop a thick, tightly woven rind, such as Swiss, do not require

waxing. A second method of ripened cheese packaging involves applying laminated

cellophane films to unwaxed cheese surfaces. The most common packaging film consists of

two laminated cellophane sheets and a brown paper overlay or a metal foil wrap.

Cheeses that are made and matured in large blocks are usually cut into appropriate size and

shrink wrapped in an atmosphere of carbon dioxide, which dissolves into the body of the

cheese, or vacuum sealed in a special "top-and-bottom" "webbed" package. The subsequent

anaerobic environment prevents mould growth on the cheese surface. Many cheeses, such as

the Brie and Camembert are packaged in special aerating wraps and stored in porous boxes

Coker et al., 2003).


41

2.8.1 Food Irradiation

Food safety is a subject of growing importance to consumers. One reason is the emergence of

new types of harmful bacteria or evolving forms of older ones that can cause serious illness.

Scientists, regulators and lawmakers, working to determine how best to combat food borne

illness, are encouraging the use of technologies that can enhance food safety worldwide,

(FDA, 1997). Irradiation can be an effective way to help reduce food-borne hazards and

ensure that harmful organisms are not in the foods we buy. During irradiation, foods are

exposed briefly to a radiant energy source such as gamma rays or electron beams within a

shielded facility. Irradiation is not a substitute for proper food manufacturing and handling

procedures.

Cheese is an important integral part of diet around the world especially in Europe, America

and it is consumed almost three times in a day. Soft fresh cheeses like cottage cheese and

‘wagashie’ have a short shelf life due to the high moisture content which creates a suitable

environment for spoilage and pathogenic microorganisms. Food irradiation however is a

preservation method that improves the safety and shelf life of food products. It could be used

to replace chemical preservatives as well as thermal treatment. It is considered as cold

pasteurization of food and does not leave any residue or render the food radioactive. It is

permitted in 35 countries worldwide for 40 different food products (Robert, 1998; Loaharanu,

2005 and Thayer, 2005). The use of gamma radiation from a Cobalt 60 source in dairy

product is considered as one of the most important peaceful application of nuclear energy

(FDA, 1997 and WHO, 2005). There was no hazard caused by irradiation up to 10 kGy which

could not cause cancer, genetic mutation or tumours (Mason, 1993; Sofos, 2002; Mehran et

al., 2005). Therefore, hospitals use irradiated food for patients with severely impaired

immune system (Lee, 1994; FAO, 1998; Leuschner & Boughtflower, 2002; Bernnand, 2006

and Konteles et al., 2009).


42

Bongirwar and Kumta (1967) reported that Cheddar cheese developed off-flavors when

irradiated at 0.5 kGy; however, none was detected when the dose was reduced to 0.2 kGy. A

dose greater than 1.5 kGy, when applied to Turkish Kashar cheese, not only resulted in off-

flavor development but also contributed to colour deterioration (Jones and Jelen, 1988).By

decreasing the dose to 1.2 kGy the sensory problems were eliminated and the mold-free shelf

life was extended from 12 to 15 days when stored at room temperature. In contrast, non-

irradiated cheese became moldy within 3 to 5 days. When combined with refrigeration

storage, irradiation increased the shelf-life period of the cheese fivefold. With Gouda cheese,

however, no taste difference was reported between irradiated (3.3kGy) and non-irradiated

samples (Rosenthal et al., 1983). Among the preservation methods to ensure safety of whey

cheeses are irradiation combined with vacuum packaging (Tsiotsias et al., 2002) and using of

antimicrobial compounds (Samelis et al., 2000) both of which had been applied to typical

Greek whey cheese.

2.9.0 Sensory Evaluation

Sensory evaluation has been defined as a scientific discipline used to evoke measure, analyze

and interprete reactions to those characteristics of food and materials as they are perceived by

the senses of sight, smell, taste, touch and hearing. Sensory characteristics are an important

determinant in the choice of the food products by the consumer. Therefore, the measurement

of sensory characteristics is an important point for the producer. Sensory analysis is the most

direct and thus the most valid way of measuring the organoleptic characteristics of food

(Piggott, 1995).

Instrumental measurements could replace the sensory analysis only if they have been

validated by experiments showing strong correlations between both sets of data and if the

predictive value of the instrumental measurements have been demonstrated. Sensory


43

measurements are often described as 'subjective' measurements; this is true as the measuring

instrument is a panel of subjects, but does not mean that the subjects are not objective.

Subjects in a panel are requested to disregard their personal liking and concentrate on the

description of their perceptions. To perform such rneasurements, trained subjects are used,

whereas consumers (naive subjects) are used for hedonic measurements (Issanchou and

Schlich, 1997).

Cheese is assessed and graded by cheese panellists and quality control personnel in industries

to ensure that its texture and flavour conform to a generally agreed consensus for a particular

variety (van Hekken et al., 2006; Sameen et al., 2008). Grading of cheeses for attributes like

appearance, flavour, body and texture or for precise defects like bitterness, mottled

appearance on an agreed scale is carried out to determine the grade acceptability of cheese for

specific markets. Quality scoring remains the most commonly used type of sensory evaluation

in the cheese industry to determine acceptability or rejection on the basis of scores obtained,

(Law and Tamime, 2010).

2.9.1 Descriptive Sensory Analysis

According to Delahunty & Drake (2004), Descriptive Sensory Evaluation refers to a

collection of techniques that discriminate between the sensory attributes of an product

including a range of cheeses and to determine quantitative and qualitative description of all

the sensory differences that can be identified. The sensory characteristics are defined in terms

of a lexicon of agreed attributes assigned by trained consumer panellists. Each attribute is

scored on a linear scale and the resultant data are typically presented in the form of spider

web diagrams or principal components loading plots, for the purpose of discriminating

between cheeses, (Law and Tamime, 2010).


44

Descriptive Data Analysis is mainly used as a tool for development of new cheeses and as a

quality control tool, provided that a standard cheese of acceptable quality is available for

comparison with other samples. Thus, the degree of excellence of cheeses may be

differentiated as determined by consumer acceptance, market research, and ‘difference from a

standard determined by consumer panels (Law and Tamime, 2010).

The Descriptive Data Analysis is simply a set of attributes or descriptors that a panel has

agreed upon that enables them to fully describe the sensory properties of the products being

evaluated. Descriptive sensory analysis addresses some of the problems of language use,

interpretation and scaling difficulties. To achieve this, a sensory quality program is organized

where time and effort is taken to recruit and train panelists. This procedure also helps to

obtain reliable data on the product being evaluated. Methods for generating descriptors are

classified according to whether the results are qualitative or quantitative even though one

could be transformed to another. An example of a qualitative method is Aroma profile.

Examples of the quantitative type are Texture Profile, Quantitative Descriptive Analysis, Free

Choice Profiling, Spectrum Analysis, Diagnostic Descriptive Analysis (Stone and Sidel,

1993) and Repertory Grid (Gains 1990). After the generation of descriptors, it is necessary to

determine which of the descriptors sufficiently describe the product. A descriptive sensory

analysis is useful when details of products need to be characterized in both academic and

industrial research.

2.9.2 The selection of a descriptive analysis Panel

The most important point in sensory measurements is the panel. Thus, care is required in

setting up the panel. The first step is to recruit the subjects within or outside the company. In

both cases, the first criteria to be taken into account are motivation and availability (Issanchou


45

et al., 1997). All descriptive methods require a panel with some degree of training or

orientation. This is achieved by screening and selection of panelist per the project demand.

An important issue is the number of assessors required to carry out profiling. In literature, the

descriptive panel size varies from eight, the recommended minimum number to 24 (Muir and

Hunter, 1991-1992).The most important thing that ensures success in training is the

commitment and motivation of the panelist to the project, panelist should attend all training

and evaluation sessions. Individual interview can be used to determine the commitment and

motivation, and availability can be determined by filling out a time table of available hours

per week (Murray et al., 2001). Personality of the panelist is very important in determining

the success or failure of sensory panelists. Studies by Piggot and Hunter (1999) showed that

elaborative screening procedures did not determine the ability of a panelist to perform well

but a concentration and personality test may be the best predictor of a good panelist together

with verbal creativity and test of discrimination ability (Murray et al., 2001).

2.9.3 Training of the panel

The panel is trained to use a common frame of reference to define the product attributes and

their intensity in the product under test. This is done by exposing the panel to the range of

products under test. The panel mentally refers to the background information and reference

points which serve as a frame of comparison when evaluating products (Munoz and Civille,

1998). Before the training, the panel is allowed to use their own frame of reference to

describe the product qualitatively with their own words to describe perceptions and

quantitatively by using previous experiences to rate intensities ability (Murray et al., 2001).

Trained panelists acquire a common qualitative and quantitative frame of reference that uses a

standard language to describe sensory concepts and a common scale. The Panel is advised to

rate products based on the term generation and concept formation sessions and not on their


46

own personal experiences (Murray et al., 2001). (ASTM, 1996; ISO 8586-1, 1993; ISO 8586-

2, 1994) recommends the use of a trained or expert panel in performing sensory profiling.

This is necessary because training the panel enables the panelists to take on an analytical

frame of mind. Conversely, untrained consumers tend to act non-analytically when scoring

attributes (Lawless and Heymann, 1998). Free choice profiling however, does not require

trained panelists and has been used for cheeses (R´etiveau et al., 2005; Drake et al., 2001),

dairy desserts (Gonz´asalez and Costell, 2006), and fresh products. Training procedures to

facilitate concept alignment in descriptive analysis should be very extensive, but will depend

on the approach or method chosen, the time available and the products under test.

2.9.4 Descriptive Attribute Generation

Selecting and defining descriptors for the products being tested is the critical step in a

descriptive sensory analysis. A well developed lexicon would help sensory researchers to

conduct a precise sensory analysis and compare the results from different sites. The training

phase of descriptive analysis starts with the formation of a common language which describes

the product attribute very well. Usually using a new panel will help in generating a new

sensory language but an experienced panel leader may be included to assist the process

(Murray et al., 2001). The panel is usually exposed to a wide range of products in the

category under test. Then products are assessed together and the descriptive profile of one

product is compared to and in the contest of the other products. This is the most important

stage as the product under assessment is very well defined (Murray et al., 2001).

Drake et al., (2001, 2002) have developed the descriptive language for Cheddar cheese

flavour by using a large representative sample set and validated the lexicon at three different

locations. The expanded lexicon for the flavour attributes of French cheeses has been

developed by Rétiveau et al., (2005).


47

2.9.5 Quantitative Descriptive Analysis (QDA)

Quantitative descriptive analysis was developed in 1970 to correct some perceived problems

associated with flavour profile method (FMP) (Stone and Sidel, 1998). Quantitative

descriptive analysis is a non technical common language to avoid biasing response behaviour

that may occur by giving out a language. It involves correct and non- correct answers

(Murray et al., 2001). QDA uses reference standards when a problem with a particular term is

noticed and a reference is needed for the subjects (Stone and Sidel, 1993). The panel leader is

not allowed to be a participant of the process to prevent bias, unstructured line scale is used to

score the intensity of the rated attributes. The panel is trained for approximately 10 to 15

hours to enable them understand the meaning of the attributes. QDA uses relative differences

among the product for evaluation and not the absolute differences, thus results of QDA will

show that the panellists are calibrated based on the relative differences among the samples.

Analysis of Variance is usually used to analyze QDA results and the cobweb or spider

diagram is used to graphically represent the data (Murray et al., 2001).

2.9.6 Principal Composite Analysis (PCA)

Drake, (2007) defined PCA as a multivariate data compression technique that allows multiple

treatments to be graphically displayed as they are differentiated by multiple variables.

Principal component analysis forms the basis for multivariate data analysis. PCA has been

described by Pearson as finding lines and planes of closest fit to systems of points in

space.The most important use of PCA is indeed to represent a multivariate data table as a

low-dimensional plane, consisting of 2 to 5 dimensions, such that an overview of the data is

obtained. This overview may reveal groups of observations, trends, and outliers. This

overview also uncovers the relationships between observations and variables, and among the


48

variables themselves. Statistically, PCA finds lines, planes and hyper planes in a dimensional

space that approximate the data as well as possible in the least squares sense (Drake, 2007). It

is easy to see that a line or a plane that is the least squares approximation of a set of data

points makes the variance of the co-ordinates on the line or plane as large as possible. The

technique is mostly used to assess how several products are differentiated by several sensory

descriptors. Attributes that are highly positively correlated will lie close to each other

(Lawless and Heyman, 1998).

2.10.0 Experimental Design

2.10.1 Response Surface Methodology and Box Behnken Design

Response surface methodology is a statistical mathematical method which uses quantitative

data in an experimental design to determine and simultaneously solve multivariate equations

(Giovanni, 1983). RSM is a collection of mathematical and statistical techniques useful for

the modeling and analysis of problems in which a response of interest is influenced by several

variables (Montgomery, 2005; Kiran et al., 2007). It aims at building a regression model that

is closest to the true regression model based on observation data and the model is empirical,

thus the true regression model is usually never known (Montgomery, 2005 and Kiran et al.,

2007). The empirical model technique is devoted to the evaluation of the relationship between

a set of controlled experimental factors and the observed results (Annadurai and Sheeja,

1998). The main aim of response surface methodology is to optimize the factors that produce

the maximum and minimum value of the response. Since the quality characteristics of a

production may not be linear with the input variables, a second degree polynomial is used if

there is a curvature in the system. Empirical evidence has shown that the higher order

polynomial or the quadratic model is enough for the optimum region (Meyers and

Montgomery, 2002). The response surface can be used to graphically make judgements about


49

the relationship between explanatory and response variables (Tong, et al., 2011). This helps

to know the treatment combinations that give the optimum response (Hinkelmann and

Kempthorne, 2007). Applications of the response surface methodology are the development

and formulation of new products and the improvement of an existing product. To analyze a

process mutually with a response, Y which depends on the input factors X1, X2, …, Xn, the

correlation between the response and the input process parameters are described as Y = f (X1,

X2, …, Xn) + ε (1)

Where f is the real response function, its format being unknown and ε is the residual error

which describes the differentiation that can be incorporated by the function f. Because the

correlation between the response and the input variables can be described as a surface of the

X1, X2…, Xn coordinates in the graphical sense, so the investigation of these relationships is

named as the response surface study. The most common designs, i.e. central composite design

(CCD) and Box-Behnkendesign (BBD), are the principal response surface methodology that

has been widely used in various experiments (Box et al., 1978).

2.10.2 Box Behnken Design

The Box Behnken design which is a type of Response Surface Methodology is an

independent quadratic design in that it does not contain an embedded factorial or fractional

factorial design. It was developed by Box and Behnken in 1980 and it is used to develop

second – order response surface models. Some three-level designs which have been proposed

by Box and Behnken are formed by combining 2k factorials with incomplete block designs. .

The level of one factor is fixed at the center level while combinations of all levels of the other

factors are applied (Montgomery, 2005). Box-Behnken which is a spherical and revolving

design has been applied in optimisation of chemical and physical processes (Oscar et al.,

1999; Muthukumar et al., 2003) because of its reasoning design and excellent outcomes. Box-


50

Behnken design does not contain any points at the vertices of the cubic region created by the

upper and lower limits for each variable and results in the reduced number of required

experimental runs. This could be advantageous when the points on the corners of the cube

represent factor-level combinations that are prohibitively expensive or impossible to test

because of physical process constraints (Box and Behnken, 1960; Montgomery, 2005).

Table 2.3 shows a three level experiment using the Box Behnken method with the 15

combinations generated by the design.

Table 2.3: The Box Behnken Experimental Design

Rank

A B C

1 -1 -1 0

2 1 -1 0

3 -1 1 0

4 1 1 0

5 -1 0 -1

6 -1 0 1

7 -1 0 1

8 1 0 1

9 0 -1 -1

10 0 1 -1

11 0 -1 1

12 0 1 1

14 0 0 0

15 0 0 0


51

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Breif field study

A brief field study was conducted to identify wagashie production sites and to obtain samples

for analysis.

3.2 Sampling of wagashie

‘Wagashie’ samples were obtained from Nima and Ashaiman markets. Six samples were

purchased from both areas. Samples were packaged in flexible polyethylene bags, kept in an

ice chest containing ice packs and transported to the Food Research Institute (CSIR) for

micobiological analyses.

3.3 Microbiological analysis of the market samples and laboratory prepared samples of

wagashie

To assess the safety of the wagashie samples,enumeration or detection of the following

indicator and enteric pathogens were carried out; Aerobic mesophiles, Feacal coliforms,

E.coli, Staphylococcus aureus, Salmonella spp., Bacillus cereus, Enterobacteriaceae,

Enterococcus, yeast and moulds.

3.3.1 Serial Dilution


52

Ten (10) grams of each sample was added to 90 ml of sterile Saline Peptone Solution (SPS)

containing 0.1% peptone and 0.8% NaCl with pH adjusted to 7.0. The sample was

homogenized in the stomacher (Lab Blender, Model 4001, Seward Medical, London,

England) for 90 s at normal speed to obtain 1:10 dilution. Further dilutions were made to

obtain a 10 fold dilution after which 1ml of each dilution was transfered into sterile petri

dishes and the appropriate media added. All analyses were done in triplicate.

3.3.2 Enumeration of Aerobic Mesophiles

Aerobic mesophiles were enumerated by the pour plate method using Plate Count Agar

(Oxoid CM ; Oxoid Ltd Basingstoke,Hamshire,UK). About 10 to 15 ml of the media was

poured on the plates, swirled gently and allowed to solidify for 10 min. The plates were

incubated at 30 °C for 72 h in accordance with the Nordic Committee on Foods Analysis

Method ( NMKL. No. 86, 2006). Plates with 25 to 250 colonies were selected and counted

using a colony counter.

3.3.3 Enumeration of Yeast and Moulds

Yeast and moulds were enumerated by the pour plate method using Oxytetracycline Glucose

Yeast Extract agar (Oxoid CM, ; Oxoid Ltd Basingstoke Hamshire,UK) supplemented with

Oxytetracycline to inhibit the growth of bacteria,the pH was adjusted to 7.0 About 10 to 15

ml of the media was poured on the plates, swirled gently and allowed to solidify for 10 min.

The plates were incubated at 30 °C for 120 h in accordance to ISO 7954,1987(E) . Colonies

were counted after 3,4 and 5 days of incubation and a microscopic examination was done to

distinguish colonies of yeast and moulds.


53

3.3.4 Enumeration of Total Coliform

Feacal coliforms were enumerated by pour plate method on Trypton Soy Agar (Oxoid

CM0131; Oxoid Ltd Basingstoke Hamshire,UK a non-selective agar) at pH 7.3 and overlaid

with Violet Red Bile Agar (Oxoid CM0107 ; Oxoid Ltd Basingstoke Hamshire,UK a

selective agar,) at pH 7.4 and allowed to cool for 10 min. The plates were incubated in

inverted positions at 44 °C for 24 h . Plates with 10 to 100 colonies were selected for

counting, colonies with purplish red colonies with 0.5 mm diameter or greater were counted.

Colonies were confirmed by selecting 5 suspected colonies and inoculating into Brilliant

Green Bile Broth containing Durham tubes (Oxoid CM0329; Oxoid Ltd Basingstoke

Hamshire,UK) at a of pH 7.4. The tubes were incubated at 37 °C for 24 h ( in accordance

with NMKL No. 44 2004) . Gas production at the bent portion of the Durham tubes indicated

a positive reaction.

3.3.5 Enumeration of E. coli

E. coli were enumerated by the pour plate method using a non-selective agar; Trypton Soy

Agar (Oxoid CM0131 ). About 5 ml of the TSA was transfered into the petri dish at a pH of

7.3 , overlaid with a selective agar; Violet Red Bile Agar (Oxoid CM0107) at a pH of 7.4 to a

ratio of 2:1.The inocula and the substrates were mixed thoroughly and the plates were

incubated in inverted positions at 44 °C for 24 h. Plates with 10 to 100 suspicious and typical

colonies which were dark red with 0.5mm diameter were selected. Colonies were confirmed

using EC Broth (Oxoid CM 0469) at a pH of 6.9, followed by Trypton Water (Oxoid

CM087) at a pH of 7.5.


54

The tubes were incubated at 44 °C for 24 h (NMKL. No. 125, 2005) and tested for indole

reaction. A red colour change was recorded as a positive reaction.

3.3.6 Enumeration of Staphylococcus aureus

Enumeration of Staphylococcus aureus was by the spread plate technique on Baird-Parker

Agar (BP, Oxoid CM 0275, Hampshire, England.). 0.1ml of the aliquot was innoculated on

the surface of the media and spread on the surface with a sterile glass rod. Egg Yolk Tellurite

Emulsion (SR54) was added and the plates were incubated at 37oC for 48 h, (NMKL No. 66

4th Ed 2009) . Colonies were confirmed using Rabbit Plasma Serum for coagulate positive

test

3.3.7 Enumeration of Bacillus cereus

Bacillus cereus was enumerated by the spread plate method on Bacillus cereus agar (Oxoid

CM 617 and SR99) supplemented with polymycin B and egg yolk emulsion. 0.1 ml of the

aloquot was innoculated on the surface of the media and a sterile rod was used to spread the

innoculum on the surface of the media. The plates were incubated at 30 0C for 24 h (NMKL

No 67, 2010). Suspected colonies were confirmed on Blood Agar Base (Oxoid CM 617) and

microscopy done.

3.3.8 Detection of Salmonella spp.


55

Approximately 25g of the food sample was weighed aseptically into sterile Stomacher bag,

225 ml of Buffered Peptone Water was added and homogenized in a stomacher at medium

speed. The sample solution was incubated at 37 °C for 16 h. After incubation, 0.1ml of the

sample solution was inoculated into 10 ml of Rappaport-Vasilliadis (RV) broth and incubated

in a water bath at 42 °C for 24 h. Following enrichment, in the Rappaport-Vasilliadis (RVS)

broth, the culture was streaked on XLD (Xylose Lysine Deoxycholate) in Petri -dishes, and

the plates were incubated at 37°C for 24 h. Biochemical and seriological test were done to

confirm the presence of Salmonella

3.3.9 Enumeration of Enterococcus

Enumeration of Enterococcus was done by the Pour Plate Method on a Tryptone soy agar

(TSA) overlaid with Slanetz and Bartley (S&B). About 1 ml of the aliquot was pippeted into

the petri dish to cover the base and the medium was added. The plates were incubated at

440C for 48hrs (NMKL No 68. 5th Ed.2011).

3.3.10 Enumeration of Enterobacteriaceae

Enterobacteriaceae were enumerated by pour plate method on Voilet Red Bile Glucose

Agar (VRBGA) (Oxoid CM0107 ; Oxoid Ltd Basingstoke Hamshire,UK a selective

agar,) at pH 7.4 . The plates were incubated in inverted positions at 37 °C for 24 h .

Plates with 10 to 100 colonies were selected for counting, colonies with purplish red

colonies with 0.5 mm diameter or greater were counted. Colonies were confirmed by

selecting 5 suspected colonies and inoculating into Brilliant Green Bile Broth at a pH of

7.4 containing Durham tubes (Oxoid CM0329; Oxoid Ltd Basingstoke Hamshire,UK).


56

The tubes were incubated at 37 °C for 24 h ( in accordance with NMKL No. 144 2004) .

gas production at the bent portion of the Durham tubes indicated a positive reaction.

3.4.0 Laboratory preparation of traditional ‘wagashie’

3.4.1 Preparation of Coagulant

Fresh stems of Calotropis procera obtained from the premises of Food Research Institute,

Accra were washed thoroughly and cut on a chopping board .The stems were crushed in a

mortar with a pestle. The crushed stems were collected in a bowl and weighed. About 500ml

of water was added to the stems and a weighed amount of salt was added. The mixture was

left to stand for a maximum of 10 minutes and filtered. Figure 3.1 shows the flow diagram

of the plant extract preparation.

Fresh stems of Calotropis procera (Sodom apple)

Wash thoroughly

Cut stems into pieces

Crush weighed stems in a mortar

Add 500mls of water

Add salt (NaCl)

Leave mixture to stand for 10 minutes

Filter with a colander

Plant Extract

Figure 3.1: Flow diagram for plant extract preparation (coagulant).

Collect in a bowl and weigh


57

3.4.2 Preparation of ‘wagashie’ (Traditional method)

Two (2) litres of raw cow milk (obtained from Animal Research Institute, Council for

Scientific and Industrial Research), was pasteurized at 80oC for 30 min and cooled to a

temperature of 40 o C. The coagulant was filtered and added to the milk. The mixture was

stirred briefly and cooked under low temperature to enhance coagulation. Curd formation

began after 10 min. when there was a clear separation between the curd and the whey, the

curd was boiled for 20 min to enhance curd firmness. The curd was poured into a colander to

expel the whey and the curds collected were transferred into a muslin cloth where it was tied

and pressed with a 6 kg load for further whey drainage for about 10 min. The matted curd was

cut into equal shape and size with a mould and packaged prior to sensory evaluation. Figure

3.2 shows the flow diagram for ‘wagashie’ preparation (Traditional method).

Raw cow milk

Pasteurize milk at 80 ͦ C for 30 min

Add plant extract

Curd formation

Boil curd for 20 min

Drain Whey

Press curd in a muslin cloth

Remove curd and cut

Package

Figure 3.2 Flow diagram of ‘wagashie’ production process (Traditional method)


58

3.4.3 Modification of the Wagashie process

In order to improve on the product quality of the traditional wagashie,various modifications

of the production method were carried out and the product evaluated by sensory evaluation

and physicochemical analysis.

The modifications were as follows;

1. The use of commercial rennet used in the cheese industry tocoagulate the fresh milk

instead of the extract of the Sodom apple.

2. Fermentation of fresh cow milk with a starter culture (Chr Hensen 10-12 Boege Alle

DK- 2970 Hoersholm-Denmark) prior to coagulation to modify the taste and improve

on the safety of wagashie.

3. Optimisation of the quantities of ingredients and processing parameters with respect

to the sensory quality of wagashie.

3.4.4 Preparation of ‘wagashie’ using commercial rennet

The process for making the improved ‘wagashie’ was similar to the traditional ‘wagashie’

process but the Calotropis procera was replaced with commercial rennet. In the rennet

prepation, the milk was pasteurised to 80 ͦ C for 30 min and allowed to cool to about 40 ͦ C

before the rennet was added with a stirile pippet and stirred. It was left to stand for 10 mins to

enable curd formation and boiled for 20 min to make the curds firm before boilling and

draining the curds.

Figure 3.3 is the flow diagram showing the process of making the improved ‘wagashie’ with

rennet and plant extract as coagulant.


59

Preparation with Rennet Preparation with plant extract

Pasteurisation of milk Raw milk Pasteurisation of milk (80 ͦ C,30min)

cool to 40 ͦ C cool to 40 ͦ C

Fermentation (starter culture) Fermentation (starter culture)

Addition coagulant (Rennet) Heat milk to 60 ͦ C

Curd formation Addition of coagulant (plant extract)

Curd boiling

Whey drainage curd formation

Curd boiling

Curd pressing whey drainage

Cutting

Curd pressing & cutting

Packaging Packaging

Figure 3.3: Flow diagram of ‘wagashie’ process with Plant extract and rennet for both

non-fermented and fermented preparations with cheese and yoghurt cultures.

Non

fermented

wagashie

Non-

fermented

wagashie


60

3.5.0 Fermentation of fresh cow milk using starter culture

As part of the modification process, raw cow milk was fermented and coagulated with

Calotropis procera or rennet. The fermentation was done by inoculating the fresh milk with

the starter culture at a temperature of 42 ͦ C in a water bath.

The procedure for traditional wagashie preparation was then applied. Figure 3.3 shows the

flow diagram for wagashie prepared with fermented and non-fermented raw cow milk using

plant extract and rennet as the coagulating agents.

3.5.1 Starter culture for the fermentation of raw cow milk

3.5.1.1 Fermentation using Yoghurt culture

One (1) litre of fresh cow milk was inoculated with 10 ml of yoghurt culture (obtained from

Animal Research Institute, CSIR-Accra) containing Lactobacillus thermophilus and

Lactobacillus thremoduric.

3.5.1.2 Fermentation using freeze dried cheese culture.

Freeze dried cheese culture obtained from Chr Hensen lab 10-12 Boege Alle DK- 2970

Hoersholm-Denmark was activated by two successive transfers in 9 ml of MRS broth for 24 h

and 16 h respectively, incubated at 30oC. The mixture was transferred into sterile centrifuge

tubes and centrifuged at 4500 g per minute for 15 minutes. The cultures were washed twice

with sterile distilled water. It was then diluted with 9mls of sterile distilled water and

homogenised with a votex mixer. Ten or 5 mls of the culture was inoculated into 2 L of milk

and allowed to ferment in reciprocal shelves at 42 oC.


61

3.5.1.3 Enumeration of LAB in yoghurt and cheese cultures

Enumeration of Lactic acid bacteria was done by the pour plate method using deMan, Rogosa

and Sharpe (MRS, Oxoid CM361) agar with pH 6.2. 0.1% cycloheximide supplement was

added to suppress yeast growth and Cystein HCL to achieve anaerobic conditions during

incubation without having to use Anaerocult A. The plates were incubated anaerobically in an

anaerobic jar at 30°C for 120 h.

3.5.2 Fermentation of milk with cheese culture and yoghurt culture

The rate of acidification of the yoghurt and cheese cultures were determined by 24 h

fermentation of pasteurised raw cow milk with both cultures in a water bath (M 25 LAUDA)

set at 420C. Ten (10) ml of the yoghurt culture and 10 ml and 5 ml of the cheese culture was

inoculated into 2 L of pasteurized raw cow milk. The initial pH of the milk was recorded and

the change in pH of the milk after every hour was recorded for 24 h.

3.5.3 Selection of appropriate pH after 24 h of fermentation

After the 24h fermentation, 2 h, 4 h and 6 h were selected for milk fermentation with 10 ml

cheese culture. The durations were selected based on their corresponding pH values attained

after the 24 h fermentation. A preliminary work was done where wagashie was prepared with

milk fermented for the selected periods. An informal sensory evaluation was done by an

untrained seven member panel where the taste, texture and colour of the samples were

assessed. Wagashie prepared with milk fermented for 4 h was accepted to be the maximum

period of fermentation and the minimum was zero h fermentation which was used in the box

Behnken design to optimise the wagashie process. The samples assessed were fresh, fried and

smoked.


62

3.6.0 Design of Experiment for ‘wagashie’ preparation

A three variable Box Behnken Design for response surface methodology was used to study

the combined effect of the coagulants (Extract and Rennet), salt (NaCl) and Fermentation

time on the responses; texture, colour, taste and overall acceptability of ‘wagashie’ over three

levels. The range and levels of the variables optimised are shown in Table 3.1 and Table 3.2

for plant extract used as coagulant and rennet used as coagulant for ‘wagashie’ preparation

respectively. The Box-Behnken design is suitable for the exploration of quadratic response

surfaces and generates a second degree polynomial model which is then used to optimise a

process using a small number of experimental runs. The design requires an experimental

number of runs according to:

N=K2+K+CP

Where k is the factor number which is 3, Cp is the number of replications at the centre point

which is 3. The design which was developed using Minitab14 resulted in 15 experimental

runs shown in Table 2. The 15 experimental runs were randomized to maximise the effects of

the independent variables based on preliminary experiments. In determining the design matrix

of the Box Behnken design, the level of one of the factors was fixed at the centre point while

combinations of all levels of the other factors were applied (Meyers and Montgomery, 2002).

Tables 3.1 and 3.2 show the coded and actual values for the level of the process variables for

the plant extract and rennet coagulated wagashie’.


63

Table 3.1: Coded and actual levels of the factors for three levels Box Behnken design for

‘wagashie’ using plant extract as coagulant.

Independent Variables Symbols coded and actual values

-1 0 +1

Fermentation Time(hr) X1 0 2 4

NaCl (g) X2 14 19 24

Extract weight (g) X3 100 150 200

Table 3.2: Coded and actual levels of the factors for three levels Box Behnken

design for ‘wagashie’ using Rennet as coagulant

Independent Variables Symbols coded and actual values

-1 0 +1

Fermentation Time(hr) X1 0 1 3

NaCl (g) X2 7 10.25 14

Rennet conc. (ml) X3 0.7 5.35 10


64

3.6.1 Optimisation of the ‘wagashie’ process using Box-Behnken Design (plant extract

preparation)

The Box Behnken design which is a type of the response surface methodology was used to

generate 15 combinations for the process variables fermentation time h, salt (g) and coagulant

(plant extract (g) and rennet (ml)). Tables 3.3 and 3.6 shows the 15 combinations for both

plant extract and rennet preparations combined by the Box Behnken De sign


‘wagashie’ coagulated with plant extract

Run

number

X1 X2 X3

Fermentation

time(hr)

Extract(wt/g)

Salt

(NaCl)(g)

1 -1 1 0 0 200 19

2 0 -1 1 2 100 24

3 -1 -1 0 0 100 19

4 0 -1 -1 2 100 14

5 -1 0 1 0 150 24

6 1 0 -1 4 150 14

7 0 0 0 2 150 19

8 0 1 -1 2 200 14

9 0 1 1 2 200 24

10 -1 0 -1 0 150 14

11 0 0 0 2 150 19

12 1 0 1 4 150 24

13 1 -1 0 4 100 19

14 0 0 0 2 150 19

15 1 1 0 4 200 19

Table 4.3 shows the 15 combinations of the process variables, fermentation time (h), salt (g)

and extract weight 9(g) generated by the Box Behnken design which provided the

optimisation quality attributes (taste, texture, colour and overall acceptability) for the sensory

evaluation of ‘wagashie’. Table 3.3 shows the coded and the actual values of the process


65

variables that were used for the optimisation process. X1 represents the minimum value or

level, X2 represents the middle value or level and X3 represents the maximum value or levels

of the process variables which were fermentation time (h), extract weight (g) and weight of

salt (g) for the product. The main effect of the sensory characteristics of ‘wagashie’ as a

function of fermentation time, extract weight and weight of salt which include colour, taste,

texture and overall acceptability is discussed in the results.

3.6.2 Optimisation of the ‘wagashie’ process using Box Behnken Design (commercial

rennet preparation)


‘wagashie’ coagulated with Rennet (ml)

Table 4.6 shows the 15 combinations of the process variables, fermentation time (h), salt (g)

and rennet (ml) generated by the Box Behnken design which provided the optimised quality

attributes for sensory evaluation of ‘wagashie’. Table 3.4 shows the coded and the actual

values of the process variables that were used for the optimisation process. X1 represents the

Run

order

X1 X2 X3 Fermentation

time(hr)

Salt (g) Rennet

(ml)

1 1 1 0 3 14.00 5.35

2 -1 1 0 1 14.00 5.35

3 1 -1 0 3 7.00 5.35

4 0 -1 -1 2 7.00 0.70

5 0 0 0 2 10.50 5.35

6 -1 -1 0 1 7.00 5.35

7 0 1 -1 2 14.00 0.70

8 0 0 0 2 10.50 5.35

9 1 0 -1 3 10.50 0.70

10 0 -1 1 2 7.00 10.00

11 0 1 1 2 14.00 10.00

12 0 0 0 2 10.50 5.35

13 -1 0 1 1 10.50 10.00

14 1 0 1 3 10.50 10.00

15 -1 0 -1 1 10.50 0.70


66

minimum value or level, X2 represents the middle value or level and X3 represents the

maximum value or levels of the process variables which were fermentation time (h), rennet

concentration (ml) and weight of salt (g). The main effect of the sensory characteristics of

‘wagashie’ as a function of fermentation time, rennet concentration and salt which include

colour, taste, texture and overall acceptability is discussed in the results.


Hedonic sensory evaluation and the quantitative descriptive sensory evaluation was used to

evaluate the ‘wagashie’ samples. A 9- point hedonic scale was used to rate the acceptability

and an unstructured 10cm line scale was used to measure the intensity of the sensory

attributes; taste, texture, colour and aroma of the ‘wagashie’ samples.

3.7.1 Hedonic Sensory Evaluation

Two hedonic sensory evaluations were carried out. The first hedonic sensory was used to

discriminate among the fifteen wagashie samples generated by the box behnken design. The

second hedonic sensory was carried out for confirmatory affective testing (consumer

preference) of the optimised ‘wagashie’ samples. Fifteen voluntary panellists were selected

for the first sensory and twenty voluntary panellists were selected for the second sensory.The

selection was done based on their familiarity with cheese or wagashie and were regular

consumers of the product. The panellists were to rate the acceptability of the ‘wagashie’

samples. Ten (10) grams of ‘wagashie’ was served to each panellist. The samples served were

coded with three digit random numbers and evaluated at room temperature with uniform

lighting conditions in a well structured sensory evaluation laboratory at the CSIR-Food

Research Institute. Each panel was seated in individual cubicle, water and tissues were served


67

for mouth rinsing and hand cleaning after every evaluation. Evaluation of the wagashie

samples was done based on the sensory attributes; colour, taste, texture and overall

acceptability using a 9-point hedonic scale of 1 to 9 ;(1 = ‘dislike extremely’, 2 = ‘dislike

very much’, 3 = ‘dislike moderately’, 4 = ‘dislike slightly’, 5 = ‘neither like nor dislike’, 6 =

‘like slightly’, 7 = ‘like moderately’, 8 = ‘like very much’, 9 = ‘like extremely’). Fifteen

fresh samples were assessed for the first hedonic sensory whiles seven samples (fresh, fried

and smoked) were assessed during the affective sensory.

3.7.2 Quantitative Descriptive Analysis

3.7.2.1 Selection of Panellists

After optimising the product, a twelve member panel from CSIR-Food Reaserch Institute

were selected and trained for a Quantitative Descriptive Sensory Evaluation.

3.7.2.2 Training of Panellists

The training for the Quantitative descriptive sensory evalution was done for three days, two

hours per day. A breif introduction about the origin, process, consumption of ‘wagashie’ and

the purpose for the gathering was given. The panellists developed words (descriptors) which

described the sensory attributes; aroma, texure, taste and colour of laboratory- prepared

‘wagashie’ (fermented and non-fermented) and ‘wagashie’ sold on the market (control).The

laboratory- prepared ‘wagashie’ samples were fresh, fried and smoked whiles the market

‘wagashie’ samples were fresh and fried.


68

3.7.2.3 Generation of Descriptors for ‘wagashie’

An agreed consensus was reached for the descriptors and were defined by the panellists.The

selection of the descriptors were confirmed using reference samples whose sensory attributes

(aroma or taste or texture or colour) are closely related or the same as the descriptors

developed for ‘wagashie’. The panellists were trained on how to quantify the descriptors on

an unstructured 10cm line scale.

3.7.2.4 Assessment of wagashie using QDA

The XLSTAT V. 14 was used to develop a Balanced Block Design where the pattern for

seving the samples were completely randomised to eliminate any form of bias during the

evaluation. The sensory evaluation was done in a well structured sensory evaluation

laboratory with individual booths or cubicles and uniform lighting conditions. Eleven of

samples served were fresh, fried and smoked samples were served. The fried ‘wagashie’

samples were fried for 2 minutes in sunflower oil at 110 0C, the smoked samples were

smoked on a coalpot for 20 minutes under low heat. 10g of each sample was served to each

panellist for assessment. The panellists were served with water for mouth rinsing after

assessing each sample and tissues for cleaning of the hand to minimise all forms of error.

The evaluation was done for three days; six samples were assessed on the first day,two

samples on the second day and five samples on the third day. Panelists quantified the

attributes ; Taste, Texture, Colour and Aroma on an ustructured 10cm line scale.

3.8.0 Physicochemical Analyses

The following physical and chemical properties were determined in the ‘wagashie’ samples;


69

3.8.1 Determination of pH

Ten (10) g of each of the ‘wagashie’ sample was weighed into a stomacher bag, 100 mls of

distilled water was added. The content was homogenised in a stomacher for 20s and the pH

reading was determined by dipping the probe of a calibrated Mettle Toledo pH meter into the

sample.

3.8.2 Determination of Total Titratable acidity

To 10 g of test portion, water was added to a volume of 105 ml, the mixture was agitated and

filtered. 25 ml of the aliquot filtrate which represented 2.5 g of the test portion was measured

into a conical flask and titrated against 0.1M NaOH using phenolphthalein as indicator. The

results were expressed as lactic acid.

1 ml 0.1 M NaOH = 0.0090 g lactic acid or ml 0.1 M NaOH/100 g. (AOAC, 2006).

% acid (wt/wt) = NxV1x Eq wt

V2 x 10

Where,

N= Normality of titrant, NaOH (meq/ml) = 0.1

V1= Titre (ml)

Eq wt = Equivalent weight of Lactic acid (mg/meq) = 90.08

V2 = Volume of Sample (ml) = 25ml


70

3.8.3 Determination of protein

Kjeldahl method was used to determine the crude protein content of the samples. About 0.2 g

of the sample was weighed and ground on a filter paper. The filter paper was folded and

dropped into a digestion tube and concentrated sulphuric acid (15 ml) and a catalyst (Kjeltab)

tablet were added. The digester was set at 400 oC to digest the sample. When there was a

colour change of the sample from black to green; the digestion process was put to a stop. The

sample was then distilled with 80 ml of water and 80 ml of 40% sodium hydroxide in a

distillation unit. During distillation the steam generated by a heating apparatus distilled the

ammonia and water into a receiving flask containing 25 ml of boric acid. The boric acid and

NH3 + H2O formed was titrated with standardised 0.1M HCl which resulted in a change from

colourless solution to a pink colour formation at the end point. A blank was run under the

same condition as with the sample. Total nitrogen content was then calculated according to

the formula:

(Titre (of sample) – blank) x concentration of standardised HCl x 14.007

10 x weight of sample

The total nitrogen was converted to crude protein by multiplying it with a factor 6.25.

3.8.4 Determination of Fat

About 2g of the macerated sample was dried in an oven as described for moisture

determination below. The dried sample was then placed in an extraction thimble and stopped

with grease-free cotton. Prior to extraction, the round bottom flask was dried, cooled and

weighed. The thimble was placed in the extraction chamber and 240 ml of petroleum ether

was added to extract the fat. The extraction was done for 15 h at a condensation rate of 5 - 6


71

drops per second. The fat extracted was then dried in an oven at 103 ± 2 oC for 1 h. The dried

fat was then cooled and weighed. A blank was run with the same procedure but without the

sample.

3.8.5 Determination of FFA

About three drops of phenolphthalein was added to 40 ml of ethanol and neutralised with 0.1

N Sodium hydroxide. About 5 g of the well-mixed sample was added; the mixture was boiled

on a hot plate and titrated with 0.1N NaOH. The titre was recorded and the free fatty acid

(FFA) was determined according to the calculation below:

FFA = Titre x Normality of NaOH (0.1) x Factor of dominant FFA (Oleic)

10 x weight of sample

The FFA determined was expressed as oleic acid.

3.8.6 Moisture determination

Five grams of well-mixed portion of the sample was weighed on an analytical balance with an

error of 0.0001g. This was preceded by heating the metal can made of alumina for 20 minutes

at 103 ± 2 oC and cooling in a desiccator to constant weight. The can with the sample was

then dried in an oven (Genlab Ltd, England) at 103 ± 2 oC for 4 hours. The drying started at

the time the oven attained a temperature of 103± 2 oC. The % moisture was determined at

room temperature according to the calculation below.

Moisture (%w/w)=[(wt of dish + fresh sample) – (wt of dish + dried sample)]x 100%

[(wt of dish + fresh sample) – (wt of dish)]


72

3.8.7 Determination of Ash content of the samples

Ash is the inorganic residue obtained by burning off the organic matter of feedstuff at 400-

600 OC in muffle furnace for 4hours. 2g of the sample was weighed into a pre-heated crucible.

The crucible was placed into muffle furnace at 400-600 OC for 4 hours or until whitish-grey

ash was obtained. The crucible containing the ash was then placed in the desiccator prior to

weighing.

%Ash = wt. of crucible+ash – wt. of crucible

Wt. of sample

3.8.8 Colour measurement of ‘wagashie’

The colour of ‘wagashie’ was determined by using the L a b colour notation system. The

equipment used was the Minolta Chroma Meter (Model Cr- 200 Minolta Camera, Japan). The

‘wagashie’ samples were sliced and arranged in a Petri dish and covered. The colour

measurement for the processed sample (smoked) was done on the inside and outside of the

‘wagashie’ samples. The colour was measured by placing the equipment on the surface of the

petri dish containing the sample. The readings were taken randomly from three spots and the

mean reading was calculated. Before measuring, the chroma meter was calibrated with a

white tile and checked for recalibration in between measurements, although no modifications

were required. Colour values were recorded as L* = darkness/lightness (0= black, 100 =

white), a* (–a* = greenness and +a* = redness), and b*(–b* = blueness, +b* = yellowness).

3.8.9 Texture Profile Analysis (TPA) of ‘wagashie’

Texture properties of the ‘wagashie’ samples were determined by a Texture Analyzer TA-

XT2 (Stable Micro Systems Ltd., Surrey, UK). The samples were shaped uniformly with a


73

cylindrical cork borer (10 mm in diameter). Fifteen measurements were taken on each

‘wagashie’ sample. TPA parameters measured were hardness, adhesiveness which is the

degree of stickiness by mouth feel when chewed five times, springiness (the force with which

the sample returns to its original shape or size after partial compression it is also known as

elasticity of cheese), gumminess (which is the density during chewing time required to break

up a semi solid food until it is suitable for chewing) and chewability (which is the number of

chews needed to masticate the sample to a consistency suitable for swallowing). These

parameters were measured by the software with a P175 75m compression platern and a

cylindrical probe with pre-test speed of 2 mm/sec, test speed 1.5 mm/sec, post-test speed 5

mm/sec, distance 10mm, time 3sec and a contact force of 5.0g. The samples were uniformly

shaped with a weight of 1g, width of 10mm and a height of 10mm. Samples were penetrated

to 75% of their original height with a constant speed penetration of 5 mm/ sec.

3.9.0 Shelf life study of wagashie

3.9.1 Irradiation and Packaging of wagashie

In order to select an appropriate dose for the irradiation of ‘wagashie’, a preliminary study

was carried out whereby the improved ‘wagashie’ samples were subjected to four different

doses; 1kGy, 2kGy, 3kGy and 4kGy from Cobalt-60 source. These doses were selected based

on the purpose of the radiation process which was to extend the shelf life of ‘wagashie’ by

decontaminating the product of all microorganisms that may shorten the shelf life of the

product. The dose absorbed by the samples per hour was 1.61kGy thus about 2 h and 30 min

was used to deliver all four doses. For purpose of preliminary trial, unpasteurised cow milk

was used to prepare the ‘wagashie’ samples. Microbiological analysis was carried out after

the preparation of the samples. The samples were vacuum packaged with a vacuum


74

packaging machine in high density polyethylene bags obtained from Kasoa in the Central

region of Ghana. The gauge of the polyethylene was 150mm by 30mm in length and width

respectively before irradiation. After irradiation, the samples were stored under ambient

conditions (28 ͦ C for 14 days) and analysed by an informal sensory evaluation. The colour by

appearance, the texture by hand feel and the aroma were assessed. After the evaluation, all the

samples except the sample irradiated with a dose of 4kGy had developed green and yellow

discolouration, off flavour and the very soft texture with whey exudates in the packaging

material. The 4kGy was therefore chosen for the continuation of the study. The actual

samples for the shelf life study were prepared from pasteurized raw cow milk. The samples

were divided into two sets, the irradiated and non irradiated samples, the non irradiated

samples served as a control for the irradiated samples

3.9.2 Storage of ‘wagashie’

The vacuum and normal packaged irradiated and non irradiated samples were stored under

ambient conditions. Microbiological tests were carried out daily for the fresh samples (shorter

shelf life) and weekly for the roasted samples until spoilage was observed in the samples. The

pH of the samples were measured and recorded.

3.10 Statistical Analysis

Analysis of variance (one way ANOVA) was used to determine the significant differences

among sensory parameters, physicochemical properties, the safety assessment of

‘wagashie’,shelf life study and the rheological analysis of ‘wagashie’.

Minitab V 17 was used to generate model equations for each sensory parameter which was

used to plot three dimensional response surface plots.


75

Microsoft Excel 2007 and XLStat 2014 was used generate the spider plot and the principal

composite analysis respectively for the Quantitative Descriptive

Analysis.

Fresh wagashie

Smoked wagashie

Soddom apple plant

Fried wagashie


76

CHAPTER FOUR

4.0 RESULTS

4.1.0 Market Survey

4.1.1 Microrganisms present in market ‘wagashie’ samples

Table 4.1 Shows the mean population of microorganisms detected in market wagashie

samples. Significant differences occurred between the count of microorganisms detected in

the fried and fresh ‘wagashie’ samples at p<0.05. The highest count of microorganisms was

1.38x109 CFU/g (aerobic mesophiles) which was detected in the fresh sample obtained from

Nima. Bacillus cereus was not detected in the fried samples but was detected in low counts in

the fresh samples. Yeast and moulds were detected in all samples except the fresh sample

obtained from Ashaiman. There was no detection or counts for Salmonella and

Staphylococcus aureus respectively.

Table 4.1 also shows the effect of deep frying and immediate packaging on the safety of the

market wagashie samples. Superscript letters ‘a’ and ‘b’ show the significant differences

between the fresh and the fried wagashie samples at p<0.05. Aerobic mesophiles were

detected in high count in the fresh sample. Moulds and Enterococcus were detected in low

counts whiles Salmonella, Bacillus cereus, Enterobacteriaceae, Stahpyloccocus, yeast and

mould, E. coli and coliforms were not enumerated or detected in the fried samples . The fresh

samples however contained almost all the pathogens except Salmonella and Staphylococcus.

The highest count of microorganisms was 2.4x1010 (aerobic plate count) and was recorded in

the fresh sample and the lowest count of microorganism recorded was 1.0x101 (yeast) which

was recorded in the fried sample.


77

Table 4.1: Mean microbial count in fresh and fried market ‘wagashie’ samples in g/CFU.

Means in the same column with the same letters are not significantly different (p<0.05)

Table 4.2: Effect of deep frying and aseptic packaging on the microbial count of fresh ‘wagashie’ obtained from Nima in g/CFU.

Means in the same column with the same letters are not significantly different (p<0.05)

Sample

Aerobic

Plate count

Yeast

Moulds

Coliform

Bacteria

E. coli

Entero

bacteriaceae

Entero

coccus

B. cereus

Salmonell

a spp.

Staphy

lococc

us

aureus

Fresh

Nima

1.4x109b 1.1x107a 1x15a 5.6x105b 3.6x104b 3.9x106b 8.1x104a 7.0 x102b Not

detected

Not

detecte

d

Fresh

Ashiama

n

3.0x109a 4.7x106b Not

detected

2.3x107a 4.1x104a 5.7x106a 1.0x104b 1.1x103a Not

detected

Not

detecte

d

Fried

Nima

8.3x104c 1.3x102c 5.0x10b 2.6x103d 1.0 x10c 1.9x106c 6.0x102c Not

detected

Not

detected

Not

detecte

d

Fried

Ashiama

n

2.9x106c 2.0x10c 1.0x10c 3.7x105c Not

detected

5.8x105d 3.3x102c Not

detected

Not

detected

Not

detecte

d

Sample

Aerobic

Plate

count

Yeast

Mould

Coliform

Bacteria

E. coli

Entero

bacteriaceae

Entero

coccus

B.

cereus

Salmone

lla spp.

Staphylo

coccus

aureus

Fresh 2.4x1010b 1.68x107 2.0x105b 1.8x107 1.4x106 2.1x108 7.4x106b 9.0x102 0 0

Fried 5.6x107a 0.0 1.0x10a 0.0 0.0 0 2.0x10a 0 0 0


78

4.1.2 pH of the market wagashie samples.

From fig.4.1, Significant differences occurred in the pH values of the market samples.

The highest pH recorded in the samples was 5.56 which was for a fried sample and the

lowest pH was 5.09 which was a fresh sample. the fresh samples had low pH values with

high count of enteric pathogens and the fried samples recorded high pH values with low

counts of enteric pathogens.

Figure 4.1: Mean pH of ‘wagashie’ sampled from the market

4.2.0 Fermentation trials for laboratory preparation of ‘wagashie’; Rate of

fermentation of raw cow milk with cheese and yoghurt starter cultures

4.2.1 Fermentation with different concentrations of cheese culture

To choose the best concentration of the starter culture for the fermentation of milk, this

experiment was carried out. Figure 4.2 shows the rate of fermentation of 1 L of

pasteurized raw cow milk with two different concentrations of cheese culture 5 ml and 10

ml. From the graph there was not much difference in the rate of fermentation between the

4.8

4.9

5

5.1

5.2

5.3

5.4

5.5

5.6

5.7

FreshAshiaman

Fresh Nima FriedAshiaman

Fried Nima Aseptic Friedwagashie

Freshwagashie

pH

Sample


79

two cultures at the end of the 24 h fermentation period, but the rate of pH change for the

cheese culture with a concentration of 10 ml was faster after two hours of fermentation

than the cheese culture with a concentration of 5 ml. The cheese culture with

concentration 10 ml, reduced from a pH of 6.12 to a pH of 3.92 at the end of the 24 h

fermentation whiles the pH of the cheese culture with concentration 5 ml, reduced from

6.12 to 4.14 at the end of the fermentation period. Since the rate of fermentation of the

cheese culture with concentration of 10 ml was faster than the 5 ml concentration, the 10

ml was selected for the fermentation of raw cow milk for the study.

Figure 4.2: The rate of pH change after 24 hours fermentation in a water bath set at

45oC with 5mls and 10mls of cheese culture

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

pH

of

Raw

co

w m

ilk p

er

1 h

r fe

rme

nta

tio

n

Fermentation time (hr)

pH 1

pH 25 mls of cheese culture

10 mls of cheese culture


80

4.2.2 Fermentation with 10 ml of both cheese and yoghurt cultures

Figure 4.3 shows the rate of acidification for the cheese and yoghurt cultures at a

concentration of 10 ml in 1 L of pasteurized raw cow milk for both cultures after 24 h

fermentation. From figure 4.3, pH 1 represents 10 ml of yoghurt culture whiles pH 2

represent 10 ml of cheese culture. With an initial pH of 6.04 ,the pH of the yoghurt

culture reduced after 1 h of fermentation whiles the pH of the cheese culture started

reducing after 2 h of fermentation. However, there was not much difference in the pH of

the milk at the end of the 24 h fermentation for both cultures. The fermentation was

carried out under the same temperature condition in a water bath set at 42 oC. This

experiment was carried out to know how effective the yoghurt culture ferment milk as

compared with the cheese culture so that in case of unavailability of the cheese

culture,the yoghurt culture could be substituted.

Figure 4.3: The rate of pH change after 24 h fermentation of 2 L of pastuerized

fresh cow milk in a water bath set at 45oC with 10 ml of yoghurt culture and 10 ml

of cheese culture.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

pH

Fermentation time (h)

pH 1

pH 2 10 mls of cheese culture

10mls of yoghurt culture


81

4.3.0 Optimisation of the ‘wagashie’ process

4.3.1.0 Using the response surface methodology to optimise the ‘wagashie’

process prepared with plant extract as coagulant.

The response surface methodology was used to determine the effects of salt (g),

fermentation time (h) and plant extract (g) on the sensory attributes: colour, taste,

texture and overall acceptability of ‘wagashie’. The Box Behnken design was used to

study the effects of variation in the levels of milk fermentation time (0h-4h), quantity

of salt (14g – 24g) and extract weight (100g – 200g) for ‘wagashie’ production on

the various responses. The relationships between the various responses are

represented by a three dimensional response surface. The percentage variability (R2)

obtained were high (close to 100%) for the various responses which indicates a good

model which can best be used to describe the effects of the variations on the

responses. Results imputed were obtained using a nine point hedonic sensory

evaluation. The discussions are based on the trends of the visualised effects and the

significant terms in the fitted model.

4.3.1 Texture

The analysis of the coefficients for the regression models showed that the

independent variables had significant effects except plant extract (g) which had

marginal significance on the texture of ‘wagashie’. The fitted regression model (R2)

for texture was 99.8% which denotes a good model and can be used to best describe

the texture of ‘wagashie’. Figure 4.4 shows the response plots for texture of wagashie

showing the effect of fermentation time (h) and salt (g) at a constant extract weight

of 150g (7.5%). The response plots shows a significant linear effect on fermentation

time and salt, a significant quadratic effect on fermentation time (h) , salt (g) and


82

extract (g) and a significant interaction effect of extract (g) and salt (g) on taste.The

linear effects showed that texture decreased or increased continiously in the

experimental field, the quadratic effect showed the possibility of an optimal region

for salt weight (14g - 24g) and fermentation time (0h-4h) and the interraction showed

that the texture of wagashie is affected by fermentation time, exract (g) and salt (g).

From figure 4.4, the score for texture increased with decreasing fermentation time (h)

and increasing quantity of salt (g) but decreased with increasing fermentation time

and increase in the quantity of salt (g).

Figure 4.4: Response Surface plot representing the effect of Fermentation time

and Salt on the score for Texture when the weight of extract is 150 g.

Fitted regression model equation for texture = 8.93544- 0.785552x1+0.01397x2-

0.23233x3 + 0.00239x1x2 + 0.00239x2x3 + 0.017019x12 + 0.00005x2

2 +0.01653x32

T exture

6.0

6.5

0.0

Fermentation time (hr)Fermentation time (hr)

0.0 1.53.0

T exture

7.0

7.5

4.54.5

22.520.0

17.515.0

22.520.0

Salt(g)

Hold Values

Extract(wt/g) 150


83

4.3.2 Colour

Colour gives the first impression of food quality thus it is one of the most important

attributes affecting the consumer acceptance of food. The analysis of the coefficient

estimates for the regression model showed that the independent variables had no

significant effect on the colour of ‘wagashie’. The R2 was 91.7%. There was no

significance (>0.05) in the linear and quadratic effects and the interaction of the

independent variables on the score for colour. Figure 4.5 shows the response surface

plot of the effect of fermentation time (h) and salt (g) on the score for colour when

the extract (g) is constant at 150g (7.5%), the response surface showed linear,

interactive and quadratic effects of the factors. The linear effect showed that colour

decreased or increased continuously in the experimental field. Colour score increased

with increase in the quantity of salt (g) and decrease in fermentation time (h).It

decreased with increasing fermentation time (h) and increase in the quantity of salt

(g). The quadratic effect showed the existence of an optimal level for colour in the

experimental field.


84

Figure 4.5: Response Surface plot representing the effect of fermentation time

and Salt on the score for Colour when the Extract is 150 g.

The fitted regression model for colour = 9.81519- 0.06669x1+0.00519x2-0.17858x3 -

0.00479x1x2 – 0.00100 x2x3 - 0.000099x12 - 0.00007x2

2 +0.00864x32

4.3.3 Taste

Taste is one of the most important attributes of ‘wagashie’ aside texture and it mostly

determines the acceptability of the product. Figure 4.6 shows the response plot for

taste which showed a linear, quadratic and interaction effects of fermentation time

(h) and quantity of salt (g) at a constant extract weight of 150 g representing 7.5%.

From the surface plots, the score for taste increased with increase in the quantity of

salt (g) and decrease in fermentation time (h). It decreased with increasing

fermentation time and decreasing quantity of salt (g). The analysis of the coefficient

estimates for the regression model showed that there was a significant linear effect of

Colour

6.8

7.0

7.2

0.0 1.53.0


Colour7.2

7.4

4.5

22.520.0

17.515.0

22.520.0

Salt(g)

Hold Values

Extract(wt/g) 150


85

the quantity of salt (g), a marginal significant effect of fermentation time (h) and a

non significant linear effect of plant extract (g) on the score for taste.There was a

significant linear effect of salt (g), a significant quadratic effect of fermentation time

and salt (g) and a significant interaction effect of salt (g) and extract (g) on taste. The

fitted regression model had an R2 of 96.7% which depicts a good model and can best

describe the taste of ‘wagashie’ prepared with extract as coagulant.

Figure 4.6: Response Surface plot representing the effect of Salt and

Fermentation time on the score for taste when extract is 150 g.

The fitted regression model for colour = 14.1206- 0.4577x1 + 0.0103x2 - 0.8404x3 -

0.0022x1x2 – 0.0011 x2x3 - 0.0769x12 - 0.0000x2

2 +0.270x32

4.3.4 Overall Acceptability

The analysis of the coefficient estimates for the regression model showed that the

independent variables had no linear significant effect on the score for overall

T aste

6.5

7.0

0.0


0.0 1.53.0

T aste7.0

7.5

4.54.5

22.520.0

17.515.0

22.520.0

Salt(g)

Hold Values

Extract(wt/g) 150


86

acceptability of ‘wagashie’ but showed the quadratic effect of fermentation time (h)

as the significant term. From the response plot (fig 4.7),the score for overall

acceptability increased with increasing weight of salt (g) and decreasing fermentation

time (h) and decreased with increasing fermentation time (h) and increasing quantity

of salt (g). In fig 4.7, the linear effect showed that overall acceptability increased or

decreased continuously in the experimental field. The quadratic effect showed that

there is an optimal level within the fermentation time (h) range and the interactive

effect showed that overall acceptability was not affected by the combined effect of

the variables.

Figure 4.7: Response surface plots representing the effect of salt and

fermentation time on the score for Overall acceptability when plant extract

weight is 150 g.

The fitted regression model for Overall acceptability = 11.0888- 0.530x1 - 0.0065x2 -

0.3693x3 - 0.0099x1x2 – 0.0015 x2x3 - 0.1379x12 - 0.0001x2

2 +0.0166x32

Overall acceptability

6.5

7.0

0.0


0.0 1.53.0

Overall acceptability 7.0

7.5

4.5

22.520.0

17.515.0

22.520.0

Salt(g)

Hold Values

Extract(wt/g) 150


87

4.3.5 Optimisation process

To optimise the process variables (salt, fermentation time and extract) for ‘wagashie’

production with respect to the responses, the contour plots were overlaid. Fig 4.8

shows an overlay of contour plots for the responses, texture, taste, colour and overall

acceptability of ‘wagashie’. From fig 4.8, the optimum levels for the preparation of

‘wagashie’ with maximum texture, taste, colour and overall acceptability were 0 h

fermentation time, 23g to 24g of salt and 150g of the plant extract. Corresponding to

the optimised values for the process variables the predicted values for taste, texture,

colour and overall acceptability were 7.38, 7.18, 7.47 and 7.36 respectively at 23g of

salt and 0 h fermentation time. For the purpose of the study the optimum conditions

were chosen and prepared which was then compared with ‘wagashie’ prepared with

milk fermented for 4h with extract weight of 150g and 23g of salt. Using a 9- point

hedonic scale, a sensory evaluation was carried out to confirm the predicted values

obtained after the optimisation process. The products assessed were in the fresh, fried

and smoked for

Figure 4.8: Contour plot for texture, taste, colour and overall acceptability of

‘wagashie’ overlaid on one axis of fermentation time (h) and salt (g)


Salt

(g)

43210

24

22

20

18

16

14

Hold Values

Extract(wt/g) 150


88

Figure 4.9: Mean pH values for wagashie samples coagulated with plant extract

combined by Box Behnken Design

Figure 4.9 shows the mean pH values of ‘wagashie’ recorded after preparation.

Overlapping error bars indicates a non significant difference at p<0.05 whiles error

bars that do not overlap represents a significant difference between the samples.

From figure 4.6 the highest pH value was 7.02 and the lowest pH value recorded was

6.1. These samples were non-fermented and fermented samples respectively.

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7

7.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

pH

Run Number


89

Figure 4.10: The TTA of wagashie samples combined by Box Behnken Design

using plant extract as cogulant

Figure 4.10 shows the Titratable acidity of the ‘wagashie’ samples after preparation.

Overlapping error bars indicate a non significant difference at p<0.05 whiles error

bars that do not overlap represents a significant difference between the samples. The

TTA recorded showed the amount of lactic acid present in the ‘wagashie’ as a result

of the lactic acid fermentation of the milk. The TTA values recorded were low

ranging from 0.001 to 0.89.

4.4.0 Using the Response Surface methodology to optimise the wagashie process

using commercial rennet as coagulant.

The response surface methodology was used to determine the effects of salt (g),

fermentation time (h) and rennet (ml) on the sensory attributes, colour, taste, texture

and overall acceptability of ‘wagashie’. The Box Behnken design was used to study

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TTA

Run Number


90

the effects of variation in the levels of milk fermentation time (0h-4h), weight of salt

(7g – 14g) and rennet concentration (0.7ml – 10ml) for ‘wagashie’ production on the

various responses. The relationships between the various responses are represented

by a three dimensional response surface and an overlaid contour plots. The

percentage variability (R2) obtained for the various responses; texture, colour, taste

and overall acceptability were 97.06%, 91.49%, 89.53% and 74.67% which indicates

a fairly good model may be used to describe the effects of the variations on the

responses. Results imputed were obtained from a sensory evaluation using fifteen

untrained panellists. The discussions are based on the trends of the visualised effects

and the significant terms in the fitted regression model

4.4.1 Texture

The analysis of the coefficients for the regression model showed that the independent

variable rennet (ml) had no significant effects on the score for texture.However the

quantity of salt (g) had a significant effect and fermentation time (h) had a marginal

significant effect on the score for the texture of ‘wagashie’. The fitted regression

model (R2) for texture was 97.06% which denotes a good model and can be used to

describe the texture of ‘wagashie’. Figure 4.11 shows the effect of fermentation time

(h) and salt (g) at a constant rennet concentration of 5.35ml (0.27%) on the sensory

score for texture of wagashie. From the results of the analysis of variance, there was

a significant linear and quadratic effect of the variables on the score for texture and a

non significant interaction effect of the variables on the texure score. There was a

significant linear and quadratic effect of salt and a marginal significant interaction

effect of fermentation time (h) and rennet (ml) on texture .From the response plot


91

(figure 4.11),the score for texture increased with increasing fermentation time (h) and

increasing quantity of salt (g) but decreased with increase fermentation time (h) and

decrease in the quantity of salt. The linear effects showed that the sensory score for

texture decreased or increased continously in the experimental field, the quadratic

effect showed possibility of an optimal region for the quantity of salt (11g - 14g) and

fermentation time (0h-4h) and the interraction showed that the score for the texture

of wagashie was marginally affected by fermentation time (h) and rennet

concentration (ml). Generally, the texture of cheese depends on the initial process

during which milk acidify (fermentation), duration of ripening and the changing

miosture content of the cheese.

Figure 4.11: Response Surface plot representing the effect of salt and

Fermentation time on the score for Texture when Rennet is 0.27%.

The fitted regression model for texture = 5.00 - 0.258 x1 + 0.510 x2 - 0.1844 x3

+ 0.0160 x12 - 0.02986 x2

2 + 0.01168 x32 + 0.0160 x1x2 + 0.02151 x1x3

T exture

5.5

6.0

0.01.5 3.0

Fermentation time(hr)

T exture6.5

7.0

1412

10 Salt (g)8

4.5

Hold Values

Rennet (ml) 5.35


92

4.4.2 Colour

The analysis of the coefficient estimates for the regression model showed that there

was a significant linear effect of the variables on taste, however there was no

significant effect of fermentation time on the colour of ‘wagashie’ but there was a

significant effect of salt and rennet concentration on the score for taste . The fitted

regression model R2 was 91.49%.There was no significance in the quadratic and

interaction effect of the independent variables on the score for the colour of

‘wagashie’. Figure 4.12 shows the response surface plots of the effect of

fermentation time (h) and salt (g) on the score for colour when the rennet

concentration is constant at 5.35 ml (0.27%). The response surface showed linear,

interactive and quadratic effects on the colour of wagashie. The linear effect showed

that colour increased or decreased continuously in the experimental field. From the

response surface, colour increased with increasing quantity of salt (g) and

fermentation time (h) and decreased with increasing fermentation time (h) and

decreasing quantity of salt (g). The quadratic effect showed the existence of an

optimal region for colour in the experimental field and the interactive effect showed

that colour was not significantly affected by the levels of fermentation time and

weight of salt. A significant lack of fit showed that a higher model is needed to

explain the effect of the factors on the sensory score for the colour of ‘wagashie’.


93


Fermentation time on the score for colour when Rennet is 0.27%.

The fitted regression model for Colour = 7.079 - 0.184 x1+ 0.151 x2 - 0.0660 x3 -

0.0028 x12 - 0.01255 x2

2 + 0.00214 x32 + 0.01429 x1x2 + 0.00753 x2x3

4.4.3 Taste

Fig. 4.13 shows the effects of fermentation time and salt on taste at a constant rennet

concentration of 5.35ml representing 0.27%. From the surface plots, the score for

taste increased with increase in the quantity of salt and increase in the fermentation

time (h). It however, decreased with decrease in fermentation time (h) and increase in

the quantity of salt (g). The analysis of the coefficient estimates for the regression

model showed that there was no significant linear effect of fermentation time (h) and

rennet concentration (ml) on taste but there was a significant linear effect of salt (g)

on the score for taste. The quadratic effect and the interaction of the variables

Colour

6.8

7.0

8 10 12Salt (g)

Colour

7.2

7.4

14

3.0

1.5 Fermentation time(hr)0.0

4.5

3.0


Hold Values

Rennet (ml) 5.35


94

showed no significant effect on the taste. The fitted regression model, R2 was

89.93%. A significant lack of fit shows that a higher model is needed to better

explain the effect of the factors on the taste of ‘wagashie’ prepared with rennet as

coagulant. Cheese flavour is regarded as a component of taste and aroma and taste

refers to the water soluble fraction which includes peptides, amino acids, organic

acids, salts and amines.


Fermentation time on the score for Taste when Rennet is 0.27%.

The fitted regression model for Taste = -3.11 - 0.211x1 + 1.795x2 -

0.472x3+ 0.0449x12 - 0.0806x2

2 + 0.0109x32 + 0.0185x1 x2+ 0.0019x1 x3 + 0.0310x2

x3

T aste

6.5

7.0

0.0

Fermentation time(hr)Fermentation time(hr)

0.01.5 3.0

T aste

7.5

4.5

1210

8

1412

Salt (g)

Hold Values

Rennet (ml) 5.35


95

4.4.4 Overall acceptability

The analysis of the coefficient estimates for the regression model showed that the

independent variables had no significant effect on the score for overall acceptability

of ‘wagashie’. The linear and quadratic effects and the interaction had no significant

effect on the score for overall acceptability of ‘wagashie’. From the response plot

(fig 4.14) overall acceptability increased or decreased continuously within the

experimental field thus overall acceptability increased with increase in the quantity

of salt (g) with an increase in the fermentation time (h) and decreased with an

increase fermentation time (h) and a decrease in the quantity of salt (g). The fitted

regression model R2 for overall acceptability was 74.67% and the adjusted R2 was

29.08%, which shows that the model cannot be used to explain the sensory score for

the overall acceptability of ‘wagashie’and that a higher model is needed to best

explain.


96


Fermentation time on the score for overall acceptability when Rennet is 0.27%.

The fitted regression model for Overall acceptability = 7.27 - 0.339 x1 + 0.093 x2 -

0.085 x3 + 0.0378 x12 - 0.0107 x2

2 - 0.0208 x32 - 0.0114 x1x2 + 0.0427 x1x3)

+ 0.0237 x2x3

4.4.5 Optimisation process

To optimise the process variables (salt (g), fermentation time (h) and rennet

concentration (ml)) for ‘wagashie’ production with respect to the responses, the

contour plots were overlaid on one axis of fermentation time (h) and salt (g) at a

constant rennet concentatration of 5.35 ml. Fig 4.15 shows an overlay of contour

plots for the responses, texture, taste, colour and overall acceptability after the

consumer assessment of ‘wagashie’. From Fig 4.15, the optimum levels for the

Overall Acceptability

6.5

7.0

0.0

Fermentation time(hr)Fermentation time(hr)

0.01.5 3.0

Overall Acceptability 7.0

7.5

4.5

1210

8

1412

Salt (g)

Hold Values

Rennet (ml) 5.35


97

preparation of ‘wagashie’ with maximum texture, taste, colour and overall

acceptability were 4 h fermentation time, 11 to 14 g of salt and 5.35ml of rennet. The

corresponding optimised values of the process variables and the predicted values for

taste, texture, colour and overall acceptability were 7.37, 6.68, 7.37 and 7.22

respectively at 11 g of salt and 4 h fermentation time. For the purpose of the study,

the optimum conditions were chosen and prepared and compared with ‘wagashie’

prepared with non fermented milk with rennet concentration of 5.35 ml and 11g of

salt. Using a 9- point hedonic scale, a sensory evaluation was carried out in order to

confirm the predicted values obtained after the optimisation process. The products

assessed were in the fresh, freid and smoked forms.

Figure 4.15: Contour plot for texture, taste, colour and overall acceptability of

‘wagashie’ overlaid on one axis of fermentation timeand salt at a constant

rennet concentration of 5.35ml.


Sa

lt (

g)

43210

14

13

12

11

10

9

8

7

Hold Values

Rennet (ml) 5.35


98

Figure 4.16: The mean pH of wagashie samples combined by Box Behnken

Design using Rennet as coagulant

Figure 4.16 shows the mean values for the pH of the wagashie samples. High pH

values were recorded in the samples. Overlapping error bars indicates a non

significant difference at p<0.05 whiles error bars that do not overlap represents a

significant difference between the samples. The highest pH recorded was as 6.83

and the lowest was recorded as 6.15. Significantly, not much differences were

observed in the samples.

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

pH

Run Number


99

Figure 4.17: The TTA of wagashie samples combined by Box Behnken Design

using Rennet as cogulant

Table 4.9 shows the TTA of the ‘wagashie’ samples determined after preparation.

Overlapping error bars indicates a non significant difference at p<0.05 whiles error

bars that do not overlap represents a significant difference between the samples. The

TTA values recorded ranged from 0.25 as the minimum and 0.79 as the maximum

values recorded in the samples.

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TTA

Run Number


100


4.5.1 Affective Sensory Evaluation

The optimised ‘wagashie’ samples were prepared and processed and an affective

sensory test was carried out to confirm the predicted ratings. The results obtained

after the consumer preference testing carried out on the wagashie samples are shown

in table 4.3.

Table 4.3: Mean scores for the confirmatory affective sensory evaluation

Sample code Taste Colour Texture Overall acceptability

980 5.95±1.91bc 7.45±0.89a 6.80±1.28a 6.35±1.87ab

591 6.70±1.69ab 7.15±1.31a 6.80±1.39a 6.65±1.42ab

115 7.45±1.23ab 7.15±1.31a 7.55±1.23a 7.65±1.09a

744 7.00±1.84ab 7.00±1.84a 7.20±1.85a 7.00±1.78ab

410 7.81±0.85a 6.77±1.65a 7.10±1.39a 7.73±0.83a

483 6.64±1.81abc 7.73±1.03a 7.05±1.17a 6.86±1.61ab

276 5.13±2.08c 6.96±1.66a 6.81±1.22a 5.73±2.07b

Means with the same alphabet are not significantly different from each other at

p<0.05.

980: Fermented- Rennet coagulated- fresh

591: Fermented- extract coagulated- fresh

115: Fermented- rennet coagulated- fried

744: Rennet coagulated- fermented smoked

410: Non fermented- extract coagulated - smoked

483: Non fermented -Rennet coagulated- fresh

276: Non fermented -rennet coagulated -smoked

Table 4.3 shows the scores for the confirmatory affective test. Scores for taste ranged

between 5.1 and 7.8, with Extract coagulated non- fermented smoked ‘wagashie’

being rated significantly higher. The rating for colour and texture however was not

significantly different among the various treatments. This observation suggests that,


101

regardless of the treatment, this attribute was equally liked by the panellists. The

most liked ‘wagashie’ was the “Extract coagulated non-fermented smoked”. This

sample obtained a score of 7.7, which may be interpreted as “like very much” on the

9-point hedonic scale, whereas the least preferred was “Rennet coagulated non-

fermented -smoked”. Significantly, there was no difference in the acceptability of

the non fermented extract coagulated smoked sample and the fermented rennet

coagulated fried sample at p< 0.05.The rennet coagulated smoked sample had a score

of 7.00 which was rated as liked very much on the 9-point hedonic scale.

The pH and TTA of the ‘wagashie’ samples obtained after preparation are recorded

in table 4.4. Significant differences were observed in the samples for pH and TTA

values at p<0.05. Low pH values with corresponding high TTA values were recorded

in the fermented samples as observed in the fermented- rennet coagulated- fresh and

smoked samples; 980 and 744 respectively and the fermented–extract coagulated-

fresh sample; 591 ,except the fermented rennet coagulated fried sample; 115. The

non fermented samples also recorded high pH values with low TTA values as

observed in the non fermented extract coagulated smoked sample; 410, the non

fermented – rennet coagulated- fresh sample;483 and the non fermented- rennet

coagulated –smoked sample;276. The pH and TTA values recorded for the samples

that were used for confirmatory affective testing are shown in Table 4.4.


102

Table 4.4: Mean pH and TTA values of the optimised ‘wagashie’ samples after

the confirmatory affective sensory evaluation.

Sample Ph TTA

980: Fermented- Rennet coagulated - fresh

5.81±0.01cd 0.95±0.43a

591: Fermented- extract coagulated - fresh 5.73±0.01d 0.88±0.08a

115: Fermented- rennet coagulated - fried

5.87±0.01c 0.34±0.08ab

744: Rennet coagulated- fermented- smoked

5.33± 0.06e 0.79±0.00a

410: Non fermented- extract coagulated - smoked

6.89±0.01b 0.36±0.00ab

483: Non fermented -Rennet coagulated - fresh

7.09± 0.01a 0.11±0.05b

276: Non fermented -rennet coagulated - smoked

7.08± 0.01a 0.34±0.03ab

Means with the same alphabet are not significantly different from each other at

p<0.05

4.5.2 Quantitative Descriptive Sensory Evaluation

Twenty-two descriptive terms were generated for the ‘wagashie’ after which a

formal evaluation was carried out. After the formal evaluation, complete data sets

were obtained for the 13 judges. Some of the descriptors were considered as

desirable and undesirable. The results of the ANOVA for the attribute ratings across

the 14 samples for the 13 judges are summarized in Table 4.5.Significant differences

occurred between the samples with regard to the texture, taste, colour and aroma.

4.5.2.1 Taste

Significant differences occurred between the samples for taste. However no

significant differences were observed in the fermented- rennet coagulated- fresh

sample; 614, the fermented - extract coagulated –fresh sample; 616, the fermented

rennet coagulated - fried sample;421 and the fermented -extract coagulated- fried


103

sample; 706 for sour taste at p<0.05. No significant differences were observed in

the samples for bland and cheesy taste. There were significant differences between

the Fermented- rennet coagulated- fried sample; 421 and the Non -fermented-

rennet coagulated- fresh sample; 110 for milky taste. The fresh market sample; 997

was scored highest for bitter and bland tastes while the fried market sample;246 was

scored highest for sour taste. The fermented- rennet coagulated- fried sample; 421

was scored highest for fried egg taste and salty taste.

4.5.2.2 Colour

The colour of ‘wagashie’ was assessed for white colour, for the interior part of the

product and brown colour, for the exterior part of the product. Significant

differences occurred in the scores for colour for the samples.However, there were

no significant differences between the whitish colour of the non fermented -rennet

coagulated- smoked sample; 203, the fermented- rennet coagulated- fried sample;

961, the fermented- extract coagulated- fried 706 and the non fermented extract

coagulated fried; 707. There were also no significant difference in the brownish

colour of the fermented rennet coagulated smoked; 417; Non fermented extract

coagulated fried sample; 707, Non fermented extract coagulated smoked

sample;961 at p<0.05. There were no significant differences in the whitish colour of

the fermented and non-fermented rennet and extract coagulated fresh samples, a

simmilar trend was observed in the fried samples at p<0.05. For the smoked

samples, there were significant differences between the fermented –rennet

coagulated smoked sample; 417and the non- fermented rennet and extract-

coagulated –smoked samples 203 and 961 respectively for the interior white colour


104

of the product. Colour and flavour attributes are very important cheese acceptance

criteria for consumers.

4.5.2.3 Aroma

From table 4.5, significant differences occurred in the aroma of the samples

however there were no significant differences for cheesy aroma, beefy aroma and

fermented cassava dough aroma in the samples. There was a significant difference

between the fried market sample 246 and the improved fried samples (Fermented-

rennet coagulated- fried sample; 421, non fermented -rennet coagulated- fried

sample; 101, fermented -extract coagulated- fried sample; 706, non fermented -

extract coagulated –fried sample; 707) for spoilt milk aroma. Significant differences

occurred in the samples for milky aroma, yoghurt aroma, fried sweet potato aroma,

doughnut aroma, fried ripe plantain aroma and smoked chevon aroma. Aroma is an

important component of the sensory property of cheese and is one of the first

stimuli to be perceived before consumption.

4.5.2.4 Texture

Generally, significant differences were observed in the texture of the samples.

Considering the various descriptors for texture, there were no significant differences

in the scores for soft texture between the fermented extract coagulated fried sample;

706, the fried market sample; 246 and the non fermented- rennet coagulated- fresh

sample; 110. Significant differences were observed in the fermented-rennet

coagulated- smoked sample; 417, the fermented-extract coagulated fresh sample;

616, the non fermented extract coagulated fresh sample; 355 and the fresh market

sample; 997 for spongy texture. No significant differences were observed in the


105

fermented rennet coagulated smoked sample; 417, the non- fermented – extract

coagulated smoked sample; 961, the non- fermented –rennet coagulated-freid

sample; 101, the non fermented-extract coagulated- fried sample; 707 and the non

fermented- extract coagulated-fresh sample; 355 for smooth texture. The fresh

market sample; 997 had the highest score for soft texture,the non fermented-extract

coagulated- fried sample;707 had the highest score for spongy texture, the

fermented-extract coagulated fresh sample; 616 had the highest score for smooth

texture and the non fermented- extract coagulated- fresh sample 355 had the highest

score for crumbly texture.


106

Table 4.5: Mean values for wagashie descriptors during the Quantitative Descriptive Analysis

Sample

Sensory attributes

Soft

texture

Spongy

texture

Smooth

texture

Crumbly

texture

Milky

aroma

Yoghurt

aroma

Cheesy

aroma

Beefy

aroma

Fried sweet

potato

aroma

Fried ripe

plantain

aroma

Smoked

chevon

aroma

spoilt

milk

aroma

F 110 5.33±2.3abc 6.63±1.7a 4.58±2.8abcd 5.48±2.4a 5.98±2.2a 4.18±3.0abcd 5.34±2.7a 4.25±2.7a 0.93±1.5bcde 0.89±1.6cdef 1.20±1.8bcd 1.26±1.6b

355 3.74±1.9bcde 6.28±1.4a 3.72±3.0cd 6.35±2.0a 6.01±2.2a 4.61±2.7abcd 4.32±2.7a 3.84±3.0a 0.77±1.5cde 0.57±1.0def 1.98±2.7abcd 1.97±2.0ab

101 3.53±2.3cde 6.67±1.6a 3.53±2.7cd 5.56±2.8a 4.40±2.4ab 2.99±2.6cd 3.69±2.5a 2.76±2.6a 3.82±2.5a 3.31±2.9b 1.26±1.5bcd 1.14±1.5b

838 6.52±1.5ab 4.86±2.3ab 7.01±1.5ab 4.46±2.5ab 6.03±1.9ab 5.76±2.2adc 5.09±1.7a 2.77±2.0a 0.20±0.3de 0.18±0.3abcde 1.25±1.7bcd 2.42±1.8ab

616 7.63±1.2a 2.31±2.0b 7.54±1.7a 2.03±2.3b 6.21±1.8a 7.54±0.9a 5.61±2.1a 2.43±2.4a 0.27±0.4de 0.19±0.3ef 0.75±1.4bcd 1.64±1.9ab

614 5.19±2.0abcd 3.88±2.2ab 5.42±2.1abc 5.54±2.2ab 5.03±1.8ab 5.83±1.5abc 4.49±2.3a 1.93±2.5a 0.16±0.2de 0.18±0.2ef 0.15±0.2cd 3.32±3.5ab

997 7.07±1.3a 5.00±1.5ab 6.98±1.6ab 5.84±2.1a 5.56±2.3ab 6.43±2.3ab 3.82±2.7a 1.61±2.4a 0.21±0.4e 0.14±0.2f 0.19±0.3d 4.45±3.2a

f 246 5.40±1.9abc 5.98±1.5a 4.80±1.4abcd 5.39±1.8a 4.03±2.4ab 2.40±2.5cd 3.17±2.2a 2.00±2.4a 3.11±2.5abcde 3.61±2.8abc 1.12±1.7bcd 2.54±2.4ab

421 4.03±2.0bcde 5.53±2.5a 3.86±1.6bcd 4.83±1.9ab 3.07±1.5ab 3.09±2.2bcd 2.59±1.9a 1.98±1.7a 4.24±2.2a 4.16±3.0ab 0.75±1.1bcd 0.72±0.9b

706 5.39±1.2abc 6.25±1.5a 5.58±1.9abc 3.63±2.2ab 2.52±1.3b 2.61±2.2cd 2.02±1.8a 1.34±1.3a 3.73±2.6ab 3.73±2.6abcd 0.77±1.5bcd 0.74±0.8b

707 3.32±2.6cde 6.76±2.1a 3.05±2.3cd 5.23±2.5ab 4.56±2.8ab 2.86±2.6cd 4.14±2.7a 2.26±3.2a 3.68±3.0ab 4.37±3.2a 1.42±2.1bcd 1.03±1.1b

S 203 2.15±1.9e 6.17±2.0a 2.02±2.0d 5.90±2.1a 4.86±2.3ab 2.99±2.7cd 3.94±2.3a 3.85±3.3a 3.16±3.4abcd 2.42±2.1abcdef 3.05±3.2abc 1.96±2.1ab

961 1.71±1.8e 6.07±2.7a 2.67±2.6cd 5.64±2.3a 5.59±2.3ab 3.23±3.0cd 4.44±1.9a 3.71±3.2a 1.93±2.3abcde 1.63±2.5abcdef 3.58±3.1ab 2.16±2.5ab

417 2.47±1.8de 4.90±2.3ab 3.04±2.2cd 4.43±2.9ab 3.35±2.7ab 1.70±1.9d 2.55±2.2a 2.85±2.7a 1.54±2.4abcde 1.22±1.9bcdef 4.79±3.0a 0.85±1.4b

Means in the same column with the same letters are significantly different (p<0.05), F= Fresh, f= fried, S= Smoked

Fresh samples: Fermented rennet coagulated; 614, market sample; 997, Non fermented- rennet coagulated; 110, Non fermented- extract

coagulated; 355, Fermented- extract coagulated with extra fat of 63g; 838, Fermented- extract coagulated; 616.

Fried samples: Fermented -rennet coagulated; 421, fermented -extract coagulated; 706, market sample; 246, Non fermented extract coagulated;

707, non fermented -rennet coagulated; 101

Smoked samples: Fermented rennet coagulated; 417, Non -fermented extract coagulated; 961, Non fermented rennet coagulated; 203


107

Table 4.5 contd: Table 4.5: Mean values for wagashie descriptors during the Quantitative Descriptive Analysis

Means in the same column with the same letters are significantly different (p<0.05), F= Fresh, f= freid, S= smoked

Fresh samples: Market wagashie; 997, Fermented rennet coagulated; 614, Fermented- extract coagulated; 616, Fermented- extract coagulated+

63g of fat; 838, Non- fermented- rennet coagulated; 110, Non- fermented- extract coagulated; 355.

Fried samples: Fermented -rennet coagulated; 421, Fermented -extract coagulated; 706, Market wagashie; 246, Non- fermented - extract

coagulated; 707, Non- fermented - rennet coagulated; 101

Smoked samples: Fermented- rennet coagulated; 417, Non - fermented extract coagulated; 961, Non- fermented rennet coagulated; 203

Sample Sensory Attributes

Fermented

cassava

dough

aroma

Doughnut

aroma

Sour taste Bland

Cheesy

taste Milky taste Bitter taste

Fried egg

taste Salty taste

Whitish

colour

Brownish

colour

F 110 0.40±0.8a 0.54±0.8ab 1.10±0.1.0b 1.16±1.7b 5.16±2.3a 6.30±1.5a 0.16±0.2c 2.27±2.5abc 1.12±1.4bc 6.56±1.4a 1.58±2.5d

355 0.50±1.1a 0.45±0.8ab 1.62±2.2b 1.27±1.8b 3.84±2.4a 5.88±2.7ab 0.33±0.5c 3.09±2.9abc 1.25±1.6abc 5.97±1.3ab 0.65±1.9d

101 0.45±0.9a 1.87±1.9ab 1.30±1.6b 0.59±0.8b 4.74±2.1a 5.14±2.2ab 0.38±0.7c 4.15±2.3a 1.97±2.2abc 6.05±1.4ab 5.14±2.4bc

838 1.14±1.5a 0.22±0.3ab 3.06±2.5ab 1.57±2.1ab 4.20±2.4a 4.79±1.9ab 0.28±0.3c 0.71±1.0bc 2.29±2.4abc 5.89±2.0ab 0.29±0.7d

614 1.04±1.3a 0.19±0.2ab 3.13±2.9ab 0.62±0.6b 4.56±2.3a 4.54±1.5ab 0.59±1.2c 0.37±0.3c 4.10±2.8ab 5.20±1.8ab 0.26±0.5d

616 1.44±1.7a 0.28±0.4ab 3.01±2.4ab 1.80±1.8ab 4.71±2.6a 5.02±2.5ab 0.41±0.41c 0.74±1.1bc 1.72±2.2abc 6.09±2.1ab 0.16±0.4d

997 3.57±3.0a 0.26±0.5b 2.13±1.9b 2.85±2.8a 3.70±2.8a 3.38±2.9ab 2.07±2.0a 0.48±0.9c 1.03±1.6bc 6.78±1.5a 0.15±0.2d

246 0.60±0.9a 1.19±1.5ab 5.48±2.5a 1.61±2.0b 4.0±2.0a 3.73±2.43ab 2.33±2.1b 2.24±2.2abc 1.99±1.8abc 5.78±1.4ab 7.92±1.9d

f 707 0.39±0.9a 2.12±2.0a 1.13±1.7b 1.03±1.4b 4.26±2.2a 5.08±2.5ab 0.17±0.3c 4.60±2.7a 1.29±1.7abc 5.53±1.5ab 4.84±1.8bc

706 0.79±1.9a 1.73±1.ab 4.05±2.3ab 1.16±1.4b 3.11±2.1a 3.52±2.0ab 1.07±1.5c 2.47±2.3abc 3.26±3.0abc 5.76±1.4ab 6.84±2.0abc

421 0.32±0.4a 2.38±1.6a 3.49±1.9ab 0.72±0.8b 2.81±1.7a 2.70±1.2b 0.83±1.1c 2.92±2.3abc 4.21±3.0a 5.38±1.4ab 7.23±1.5abs

S 203 0.61±1.3a 1.00±1.7ab 1.57±2.0b 0.89±1.3b 3.67±2.5a 4.75±2.8ab 0.42±0.6c 3.98±2.2ab 1.15±1.2bc1 5.50±1.7ab 4.48±2.3c

961 0.67±1.3a 0.85±1.74ab 1.92±2.3b 0.89±1.1b 3.67±2.1a 5.06±2.1ab 0.28±0.31c 4.08±2.6a 1.80±2.1abc 4.98±1.6ab 5.49±1.4bc

417 0.66±1.8a 0.60±1.0ab 1.39±1.7b 2.69±2.6b 2.86±2.3a 3.35±3.1ab 0.24±0.3c 2.38±2.5abc 0.55±0.9c 3.94±1.9b 5.51±2.2bc


108

4.6.0 Principal composite analysis (PCA)

In the PCA, all attributes were considered, and it was applied on the mean values of

each individual attribute per ‘wagashie’ sample as obtained from quantitative

descriptive sensory analysis of aroma, texture, taste and colour of wagashie. Figure

4.8 graphically represents a PCA bi-plot of the attributes and the position of the

different ‘wagashie’ samples relative to the attributes that were rated.

Figure 4.18 is a graphical representation of the relationship between the sensory

attributes and the ‘wagashie’ samples. The total variation of the data for F1 and F2

was 65.97%. Factor F1 accounted for 41.3% of the total variation which shows the

highest percentage variation among the samples. It was defined mainly by the

attributes, milky taste, milky aroma, cheesy aroma, cheesy taste and yoghurt aroma

on the positive side and beefy aroma, smoked chevon aroma, fried egg aroma,

crumbly, and spongy on the negative side. F2 accounted for 24.66% which shows the

second largest percentage variation among the samples. The non fermented- rennet

coagulated- fresh sample; 110, the Non fermented extract coagulated fresh; 355, the

fermented extract coagulated sample with extra fat of 63g; 838 and the Fermented

extract coagulated; 616 had high intensities for milky taste, milky aroma, cheesy

aroma, cheesy taste and yoghurt aroma which denotes a positive correlation, this was

represented in the second quadrant. However, they had low intensities for fried

sweet potato aroma, doughnut aroma, fried ripe plantain aroma, salty taste and

brownish colour because they in opposite quadrants and thus are negatively

correlated.


109

Figure 4.18: PCA bi-plot of quantitative descriptive sensory used to describe the sensory attributes of ‘wagashie’ in their fresh, fried and

roasted forms.

997246

203

110

355961

707 101838

614

616

706421

417

Soft

Spongy

Smooth

Crumbly

Milky aroma

Yoghurt aroma

Cheesy aroma

Beefy aroma

Fried sweet potato aroma

Fried ripe plantain aroma

Smoked chevon aroma

spoilt milk aroma

Fermented cassva dough aromaDoughnut aroma

Sour taste

Bland

Cheesy taste

Milky taste

Bitter taste

Fried egg taste

Salty taste

Whitish colour

Brownish colour

-10

-8

-6

-4

-2

0

2

4

6

8

10

-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

F2 (

24

.66

%)

F1 (41.31 %)

Biplot (axes F1 and F2: 65.97 %)

Non-fermented samples

Fermented samples

Market samples

Fresh samplesFried samples

Smoked samplesNB:101 and 707 are fried Fresh samples


110

Also, they have similar attributes with the Fermented rennet coagulated; 614 and the

market sample; 997 because they were all fresh ‘wagashie’ samples and were on the

same side (positive) on the F1 axis. The Fermented rennet coagulated fresh sample;

614 and the fresh market sample; 997 had positive correlation with whitish colour,

spoilt milk aroma, fermented cassava dough aroma, soft, smooth, bitter taste, sour

taste and bland taste .They however had low intensities for beefy aroma, smoked

chevon aroma, fried egg aroma, crumbly, and spongy. The fermented rennet

coagulated -smoked sample; 417, the Non -fermented extract coagulated –smoked

sample; 961, Non fermented rennet coagulated- smoked sample; 203, Non fermented

extract coagulated- fried sample; 707 and the non fermented -rennet coagulated fried

sample; 101 had high intensities for beefy aroma, smoked chevon aroma, fried egg

aroma, crumbly texture, and spongy texture and have positive correlation. However,

they had low intensities for whitish colour, spoilt milk aroma, fermented cassava

dough aroma, soft, smooth, bitter taste, sour taste and bland taste because they were on

opposite quadrants and were negatively correlated. The fresh market sample; 997, had

positive correlation with almost all the undesirable descriptors which were bitter taste,

fermented cassava dough aroma, spoilt milk aroma and bland taste. The fermented

rennet - coagulated fresh sample; 614 was slightly associated with the undesirable

descriptors which were bitter taste, fermented cassava dough aroma, spoilt milk aroma

and bland taste because they were in the same quadrant.


111

4.6.1 Cluster Analysis

The results achieved by the Hierarchial Agglomerate Cluster Analysis using

XLSTAT 2014 for the thirteen ‘wagashie’ samples considering all the sensory

variables are presented as a dendrogram in Figure 4.19. Five clusters were formed.

Group A (997,838,614 and 616) was made up of fresh fermented samples, both

rennet and extract coagulated except sample 997(market sample). Group B was

formed by 246,706,421, which were all fried fermented and non- fermented both

rennet and extract coagulated samples. Group C was made up of 4 samples (two

smoked non fermented rennet and extract coagulated samples; 203, 961 and two fried

non -fermented both rennet and extract coagulated samples; 101 and 707). Group D

was formed by two samples; which were non fermented rennet coagulated fresh

‘wagashie’ sample, 110 and a non fermented extract coagulated fresh ‘wagashie’

sample, 355. Group E was formed by one sample; 417 representing fermented -

rennet coagulated- smoked sample. These results showed that there were differences

among the ‘wagashie’ samples in different groups and the sensory data may contain

enough information to attain differences in the ‘wagashie’ samples based on the

classes established. However, samples that belong to the same class had similar

attributes and were not different from each other. The cluster analysis was used to

ascertain the similarities and the disimilarities among the samples.


112

Group A Group B Group C Group D Group E

Figure 4.19: Dendogram from cluster analysis of ‘wagashie’ samples considering sensory attributes

997

616

838

614

246

706

421

110

355

417

707

101

203

961

0

50

100

150

200

250

300

Dis

sim

ilari

ty

Dendrogram


113

Figure 4.20: Spider plot of the fresh and processed ‘wagashie’ samples after the quantitative descriptive sensory evaluation

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

SoftSpongy

Smooth

Crumbly

Milky aroma

Yoghurt aroma

Cheesy aroma

Beefy aroma

Fried sweet potato aroma

Fried ripe plantain aroma

Smoked chevon aromaspoilt milk aroma

Fermented cassva dougharoma

Doughnut aroma

Sour taste

Bland

Cheesy taste

Milky taste

Bitter taste

Fried egg taste

Salty taste

Whitish colour

Brownish colour

997

246

203

110

355

961

707

101

838

614

616

706

421

417


114

4.6.2 Spider plot

Figure 4.20 shows the spider plot of the ‘wagashie’ samples assessed during the

quantitative descriptive sensory evaluation which shows the descriptive sensory

analysis profile more vividly. Each spoke symbolizes one attribute, and the relative

intensity corresponds to that point in which the product line crosses, with the lowest

and highest intensities toward the centre point and farthest from the centre,

respectively. From the spider plot, the non fermented- extract coagulated- fried

sample; 707 had the highest intensity, whiles the rennet coagulated fresh sample; 614

had the lowest mean for fried egg taste. The market fresh sample; 997 had the

highest intensity with a mean of 4.45 for spoilt milk aroma. The market sample 997

had the highest intensity with a mean of 6.78 for whitish colour. The fried market

sample; 246 had the highest intensity with a mean of 7.92 for brownish colour. The

fried market sample; 246 with a mean of 2.33 and the fresh market sample with

mean; 2.07 had the highest intensity for bitter taste whiles the non- fermented-rennet

coagulated fresh sample; 110 had the lowest intensity for bitter taste with a mean of

0.16. The non- fermented-rennet coagulated fresh sample; 110 had the highest

intensity with a mean of 6.30, 5.16 for milky and cheesy tastes, whiles the

fermented-rennet coagulated fried sample; 421 with a mean of 2.70, 2.81 had the

lowest intensity for milky and cheesy tastes respectively. The fresh market sample;

997 with a mean of 2.85, had the highest intensity for bland taste. The fermented-

rennet coagulated fried sample; 421 with a mean of 2.38 had the highest intensity for

sour taste. The fermented –rennet coagulated -smoked sample; 417 with a mean of

4.79 had the highest intensity followed by the non-fermented rennet coagulated-

smoked sample; 203 with mean 3.05 for smoked chevon aroma. The non-fermented

extract coagulated fresh sample; 355 with a mean of 6.35 had the highest intensity


115

for crumbly texture whiles the fermented-extract coagulated fresh sample; 616 with a

mean of 2.03 had the lowest intensity for crumbly texture. The fermented-extract

coagulated fresh sample; 616 with a mean of 7.54, had the highest intensity smooth

texture.The non-fermented rennet coagulated –fresh sample; 110, with a mean of

5.16 and 6.30 had the highest intensities for cheesy and milky tastes respectively.

The fermented extract –coagulated fresh sample; 616 with a mean of 5.61 and 7.54

had the highest intensities for cheesy and yoghurt aroma. Significant differences

occurred among the samples at p<0.05 in relation to attribute intensities and this is

shown by the different superscript alphabet in table 4.5, except fermented cassava

dough aroma, cheesy taste, bland taste, beefy aroma and cheesy aroma.

4.7.0 Proximate composition of ‘wagashie’

The chemical analysis of the ‘wagashie’ samples was carried out to determine the

nutritive value of the fermented rennet coagulated wagashie and the results can be

observed in table 4.15.

Table 4.6: Mean values for the chemical composition of ‘wagashie’

Sample

Ash g/100g FFAg/100g Protein Fat/100g Moistureg/100g Fermented Smoked

wagashie

1.21±0.014c 0.53±0.01a 30.18 ± 0.71a 21.40±0.69b 41.99 ± 0.09c

Non-Fermented fresh wagashie 1.95±0.007b 0.21±00b 23.20 ± 0.24c 14.49±1.33c 45.71±0.12b

Fermented fresh wagashie 0.95±0.056d 0.16±0.07b 19.28 ± 0.18d 15.48±0.06c 56.10±0.21a

Non –fermented

Fried wagashie

2.09±0.311a 0.16±0.08b 24.97±0.12b 25.32±0.31a 37.69±0.11d

Means with the same superscript alphabets in the same column are not significantly

different at p<0.05

From Table 4.6, significant differences were observed in the mean value for ash at

p< 0.05, the mean values for ash ranged from 0.95 g to 2.09 g with the non-


116

fermented fried ‘wagashie’ sample rated significantly higher. The mean values for

FFA ranged from 0.16g to 0.53g. The mean values for fat ranged from and 14.49 g

to 25.32 g . Also the mean values of protein ranged from 19.28 to 30.18 with the

fermented smoked wagashie sample significantly rated higher . The mean values for

moistureranged from 37.69 to 56.10 with the fermented fresh ‘wagashie’ sample

significantly rated higher. No significant differences were observed between the

mean value of the fat and FFA in the fermented and non –fermented - fresh

‘wagashie’ sample at p<0.05. The non fermented- fried ‘wagashie’ sample recorded

the highest mean value for ash and fat whiles the fermented smoked ‘wagashie’

sample recorded the highest mean value for protein. The fermented fresh sample

recorded the highest value for moisture.

4.8.0 Rheology of Improved ‘wagashie’

4.8.1 Colour

The average values of colour parameters for ‘wagashie’ obtained from instrumental

measures are shown in Table 4.7.

Table 4.7: Mean values with standard deviations for the colour of improved

‘wagashie’

Sample

L* a* b*

Fermented smoked extract (interior) 79.25±1.5c -7.15±0.3a +15.42±0.1ab

Fermented smoked extract (exterior) 57.00±2.8d +2.26±0.5d +17.50±3.2b

Fermented smoked rennet (interior) 79.84±0.4bc -6.42±0.2abc +18.42±0.4ab

Fermented smoked rennet (exterior) 59.83±1.1d +0.69±0.6e +15.69±0.3b

Fermented extract fresh 83.36±0.9ab -5.58±0.2ab +17.50±0.4ab

Fermented rennet fresh 84.11±1.2a -5.58±0.2c +19.43±0.7a

Non fermented rennet fresh 84.34±0.5a -6.13±0.1bc +19.29±0.6a

Means with the same alphabets in the same column are not significantly different

from each other at p<0.05


117

Table 4.7 shows the mean values with standard deviations for the colour of the fresh,

fried and smoked ‘wagashie’ samples at p<0.05. Significant differences occurred in

the L* value of the samples however, no significant difference was observed

between the L* mean value of the fermented and non-fermented rennet coagulated

fresh samples at p<0.05. Significant differences were not observed in the exterior

colours of the rennet coagulated and extract coagulated smoked samples.The

lightness index of the ‘wagashie’ ranged from 84.34 to 57. The higher the L* value,

the lighter the colour and the lower the L* value the darker the colour. The mean

value for a* colour ranged from -0.69 to +7.15. The interior colour of the smoked

samples had positive mean values for a* whiles the interior colour of the fresh

samples had negative mean values of a*. Significant differences, thus occurred

between the samples for a* value at p< 0.05. Significant differences occurred

between the samples for the mean values of b*, however, no significant difference

were observed between the interior colours of the extract coagulated - smoked

sample, the rennet coagulated -smoked sample and the extract coagulated fresh

sample. Also significant differences were not observed in the mean values of b* for

the exterior colour of both extract and rennet coagulated smoked samples and

between the rennet coagulated fermented and non fermented fresh samples. The

mean values for b* ranged from +19.43 to +15.42.

4.8.2 Texture

Evaluation of texture by instrumental and sensory analysis is important to new

product development.The results for Texture Profile Analysis (TPA) for ‘wagashie’

are compiled in Table 4. 8. Five parameters were measured; hardness, adhesiveness,


118

springiness, gumminess and chewiness. The samples analysed were rennet

coagulated fermented and non –fernemented smoked, fried and fresh samples

Table 4.8: Mean values for the textural characteristics of ‘wagashie’

Texture

Characteristics

Sample Springiness

(mm)

Adhesiveness

g/sec

Chewiness

(N)

Gumminess

(N)

Hardness Hardness (g)

Non fermented

fresh

0.915 ± 0.02a -1.425 ± 1.97a 2086 ± 106c 1984 ± 352cd 4862 ± 890bc

Non fermented

fried

0.912 ± 0.03a -1.014 ± 0.35a 6397 ± 896a 6982 ± 1152a 12144 ± 3078a

Non fermented

smoked

0.909 ± 0.03a -0.790 ± 0.35a 2822 ± 106bc 2729 ± 602bc 4578 ± 1300bc

Fermented fried 0.866 ± 0.08a -1.274 ± 0.17a 658 ± 104d 1222 ± 223de 6372 ± 951b

Fermented

smoked

0.713 ± 0.08b -0.872 ± 0.66a 2930 ± 345b 3475 ± 473b 3931 ± 590c

Fermented fresh 0.635 ± 0.06c -0.993 ± 0.64a 516 ± 124d 695 ± 200cd 4164 ± 1218c

Mean with the same superscript alphabets in the same column are not sisgnificantly

different at p<0.05

From table 4.8, Textural differences were observed in the samples though there was

no significant difference between the values for adhesive texture at p>0.05. The

mean values for adhesive texture ranged from –0.790 to -1.425. Significant

differences were observed between the fermented smoked and fresh samples for

springiness at p<0.05. However, no significant difference were observed between the

non fermented fresh, fried and smoked samples and the fermented fried sample for


119

springiness at p<0.05, the values for springiness ranged from 0.653 to 0. 915.

Significant differences were observed for chewiness at p<0.05, however, no

significant differences were observed between the fermented fresh sample and the

fermented fried sample at p<0.05. The mean value for chewiness ranged from 516 to

6397. Also significant differences were observed in the samples for gumminess at

p<0.05. The mean values for gumminess ranged from 694 to 6982. There was no

significant difference between the hard texture of the fermented fresh sample and the

fermented smoked sample. The non fermented fried sample recorded the highest

value for hardness while the fermented smoked sample recorded the lowest mean

value for hard texture.

4.9.0 Safety of improved ‘wagashie’

To ascertain the safety of the improved ‘wagashie’, microbiological tests were

carried out on the samples. The tests carried out were similar to the tests carried out

on the market ‘wagashie’ samples. Table 4.9 shows the results of the microbiological

tests done on the improved ‘wagashie’ samples. Significant differences were

observed between the two samples for the count for coliform, E. coli and yeast with

the fermented fresh sample rated significantly higher than the fermented smoked

sample at p<0.05. Generally, it was observed that the count for microorganisms in

the fermented fresh sample was significantly higher than the fermented smoked

sample. There were no counts for Salmonella, Staphylococcus aureus, Bacillus

cereus and mould in the two samples.


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Table 4.9: Mean microbial counts in the improved ‘wagashie’ in CFU/g to

ascertain the safety of the lobratory prepared samples


different at p<0.05

4.10.0 Shelf life of wagashie

Table 4.10 shows the mean count for aerobic mesophiles, yeast and mould during the

ambient storage of the ‘wagashie’ samples for 5 weeks.

Twenty grams samples were analysed immediately after preparation for day 0 and

the rest were packaged for irradiation. The microbial count in the irradiated samples

reduced after irradiation (Day 3) whiles that for the non-irradiated samples increased.

Sample Coli

form

E. coli Yeast Moul

d

Aerobic

mesophiles

Salmonel

la

Staphyloc

occus

Bacillus

cereus

Fermented

fresh

3x10a 3x10a 9 x 103a 0 2.9x105 a 0 0 0

Fermented

smoked

0b 0b 8x102b 0 0b 0 0 0


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From Table 4.10, after the third day, there was a slight increase in the count for aerobic

mesophiles and yeast and no count for mould. During the first week, there were counts for

mould mostly in both the irradiated and non-irradiated control samples (normal packaged).

The non-irradiated vacuum packaged samples however recorded a slight growth for mould

whiles the irradiated vacuum packaged samples did not have counts for mould. The counts for

aerobc mesophiles and yeast also increased. During the second week of storage growth of

aerobic mesophiles, yeast and mould increased in the normal/non- vacuum packaged

irradiated and non-irradiated samples (nNS, nNF, nIF, nIS). From the third week to the fifth

week of storage, there were no mould growth in the irradiated vacuum packaged samples.The

growth of aerobic mesophiles and yeast in the irradiated and non-irradiated vacuum packaged

samples increased in the 3rd and 4th weeks. A slight increase was recorded in week 5 with the

non-irradiated vacuum packaged fresh sample recording the highest count for aerobic

mesophiles, yeast and mould.


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Table 4.10: Changes in the mean microbial counts in the rennet coagulated fermented fresh and smoked ‘wagashie’ samples as affected

by packaging and irradiation for the 5 weeks storage period in CFU/g (from day 0 to week 2)

PCA Yeast Mould

Sample Day 0 Day 3 Week 1 Week 2 Day 0 Day 3 Week 1 Week 2 Day

0

Day

3

Week 1 Week 2

VIF - 1.0 x 10 1c 1.0 x 102d 2.3 x 103b - 5.0 x 10c 1.0 x 102c 6.0 x 10 2d - - - -

nIS - 4.0 x 10 1c 2.5 x 104cd 1.6 x 106ab - 1.5 x 10c 5.0 x 102c 1.1 x 105cd - - 2.5 x 10 2d 1.5 x 10 2b

VIS - 1.0 x 10 1c 4.0 x 101d 3.0 x 102b - 0 0 2.5 x 10d - - 0 -

nIF - 3.0 x 10 1c 3.7 x 104c 2.8 x 106ab - 2.0 x 10c 2.5 x 102c 3.0 x 106b - - 2.0 x 103c 3.5 x 104b

nNF 2.9x102 3.0 x 103a 2.8 x 105a 3.7 x 107a 9.0x103a 8.0 x102a 9.0 x 103a 1.9 x 107a - - 1.0 x 104a 5.0 x 105a

nNS - 2.0 x 103b 2.5 x 104b 1.5 x 106ab 8x102b 5.0 x102b 5.0 x 103b 1.9 x 106b - - 1.3 x 103b 5.0 x 104b

VNF - 1.0 x 103c 2.0 x 103d 4.3 x 103b - 1.2 x 10c 8.0 x 102c 1.1 x 103d - - 2.0 x 102d -

VNS - 2.0 x 103c 1.7 x 103d 1.5 x 103b - 6.0 x 10c 1.3 x 10c 2.4 x 102d - - 1.0 x 102d -

Means with the same superscript alphabets in the same column are not significantly different at p<0.05, ‘ –‘ = none

Legend: V- Vacuum packaged, n- non- vacuum packaged I- Irradiated, N- Non-irradiated, F- Fresh, S-Smoked


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Table 4.10 contd.: Changes in the mean microbial count in the rennet coagulated fermented fresh and smoked ‘wagashie’ samples as

affected by the packaging material and irradiation for the 5 weeks storage period in CFU/g (week 3 to week 5).

PCA

Yeast

Mould

Sample Week 3 Week 4 Week 5 Week 3 Week 4 Week 5 Week 3 Week 4 Week 5

VIF 1.1 x 10 4c 2.3 x 10 5b 9.0 x 10 6b 6.0 x 10 3c 2.0 x 10 4b 1.7 x 10 4c - - -

VIS 1.3 x 10 3d 3.0 x10 5b 8.0 x 10 5c 1.2 x 10 3c 4.0 x 10 4b 1.3 x 10 4c - - -

VNF 2.5 x 10 6a 2.0 x10 7a 5.0 x 10 7a 8.0 x 10 5a 2.0 x 10 5a 4.0 x 10 6a 1.6 x 10 3a 8.0 x 10 4a 7.0 x 10 4a

VNS 1.9 x 10 5b 1.2 x10 5b 4.0 x 10 6b 1.1 x 10 4b 8.0 x 10 4b 2.0 x 10 5b 4.0 x 10 3b 8.0 x 10 3b 1.0 x 10 4b

Means with the same alphabets are in the same column are not significantly different at p<0.05, ‘–‘= none

Legend: V- Vacuum packaged, I- Irradiated, N- Non-irradiated, F- Fresh, S-Smoked


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4.10.1 pH of the wagashie samples during the 5 weeks storage period.

The pH values recorded for the product in the fresh and smoked forms for the

radiated and non-irradiated samples during the 5 weeks storage period are

summerised in Table 4.11.

Table 4.11: Mean pH values of the ‘wagashie’ samples during the 5 weeks

storage period

Sample

Day 0

Day 3

(after

irradiation) Week 1 Week 2 Week 3 Week 4 Week 5

VIF 4.99 bc 4.96e 4.93f 4.94b 4.96c 5.30c

nIS 4.93 d 4.94f 4.92f 5.15a 5.15b 5.20d

VIS 5.00 b 4.99d 5.03e 4.89b 5.22a 5.63a

nIF 4.88 e 4.95ef 4.88 g 4.89b 5.25a 5.58b

nNF 4.99 a 4.97c 5.22a 6.44a - - -

nNS 4.90b 5.05a 5.15b 5.54c - - -

VNF 4.92d 4.92g 6.09c - - -

VNS 4.88 e 5.07c 6.28b - - -


different at p<0.05

Legend: V- Vacuum packaged, n- normal packaged/non vacuum packaged I-

Irradiated, N- Non-irradiated, F- Fresh, S-Smoked, ‘–‘= none

From Table 4.11, significant differences occurred in the pH values of the samples at

p<0.05. There was a slight increase in pH on the Day 3 (after irradiation). Significant

differences were not observed in the pH values recorded for the non-irradiated

vacuum packaged smoked sample and the irradiated normal packaged fresh sample


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and like wise between the non-irradiated- vacuum packaged fresh sample and the

normal- packaged –irradiated smoked sample on the 3rd day after storage. On week 1,

there was a slight increase in pH for the irradiated vacuum packaged samples and the

non-irradiated vacuum packaged samples (IVS, IVF, NVS and NVF), however there

was a wide increase in the pH values for the non- vacuum packaged samples

(nNF,nNS,nIS, nIF). On week 2, there was an increase in pH values for the non-

irradiated and irradiated non vacuum packaged samples. On week 3 there was a

slight increase in pH for the irradiated vacuum packaged fresh samples; IVF and the

non –irradiated vacuum packed smoked sample; NVS. There was an increase in pH

for the irradiated vacumm packaged smoked sample; IVS (from 4.92 in weeks 2 to

5.15 in week 3) and a slight decrease in the non-irradiated vacuum packaged fresh

sample; NVF. On week 4, an increase in pH was observed in the non-irradiated

vacuum packaged samples; NVS, NVF and slight increase in the irradiated vacuum

packaged fresh sample;IVF, however,no increase was observed in the irradiated

vacuum packed sample;IVS. On week 5, a general increase in pH was observed in all

thesamples.


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CHAPTER FIVE

5.0 DISCUSSION

‘Wagashie’ a traditional cheese in West Africa usually coagulated with coagulant

from plant origin (Calotropis procera) is considered as a type of soft cheese due to

its high moisture content of about 50% (Ashaye et al., 2006). Extracts from the

succulent leaves and stems of Calotropis procera is used for the coagulation and has

been reported to contain rennet enzymes called calotropin that coagulates milk

(Belewu and Aina, 2000). Although coagulants from plant sources like Calotropis

procera are available for milk coagulation, their excessive proteolytic nature reduces

cheese yield and increases the perception of bitter tastes, making its use more

difficult for cheese making (Lo Piero et al., 2002 and Roseiro et al., 2003). Also

traditional ‘wagashie’ due to its low salt content has a bland taste and a short shelf

life of three days after preparation (Ashaye et al., 2006). Thus this study was carried

out to develop the existing product by improving the safety, sensory quality, and

shelf life.

5.1 Safety of wagashie

The safety of market ‘wagashie’ samples was assessed in the laboratory to ascertain

its safety on the market. It was then compared to the safety of the improved

‘wagashie’ prepared under laboratory conditions. Various indicator and pathogenic

microorganisms including aerobic mesophiles, Yeast and moulds, coliform bacteria,

E. coli, Staphylococcus aureus Bacillus cereus, Salmonella spp, Enterococcus,

Enterobacteriaceae were assayed. The results of the microbiological analysis of the


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market wagashie samples indicated that fresh wagashie had high counts of enteric

pathogens than fried wagshie samples. However, the level of contamination in both

fresh and fried samples as shown in the results of the microbilogical analysis, were

above the acceptable limits in accordance with GSB Standards, (1998) and the

Standard method for the examination of dairy products, 2001 (Ledenbach and

Marshal, 2009). To determine the means of reducing levels of contamination, fresh

market ‘wagashie’ samples were fried, aseptically packaged and tested

microbiologically in the laboratory. Frying reduced the counts for indicator and

pathogenic organisms but could not reduce them to meet acceptable standards per

GSB standard 1998 because of the high counts in aerobic mesophiles (>106 CFU/g).

The high microbial load found in the wagashie samples investigated in the present

study is in agreement with the work by Elkhider et al., (2011) who reported that

cheese samples collected from different producers in rural areas of eastern Sudan

indicated that the level of hygiene and production methods, source of raw milk and

its handling could be the main factors responsible for high microbial loads which

affect the quality of cheese.

It was however observed during the market survey that the high microbial counts

recorded in the market wagashie samples was because the curds were not packaged

but rather exposed on metal trays for retailing. Retailers also hand picked ‘wagashie’

curds into flexible polyethylene bags which served as a point of exposure to post

production contamination.Tohibu,et al., (2013), also reported that, ’wagashie’ is not

packaged after preparation thus producers hand pick the curds into flexible

polyethylene films exposing the product to contamination, reducing the shelf life and

making the product unsafe for the consumer. The improved ‘wagashie’ samples

produced in this study were fermented to improve the safety of the product due to the


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antimicrobial activity of lactic acid bacteria. According to Adams and Moss, (1999),

lactic acid bacteria especially lactobacillus spp. has been found to produce

bacteriocin in addition to lactic acid and hydrogen peroxide during their lactic

fermentation. Bacteriocin have been found to inhibit a wide range of bacteria

including Gram positive and Gram negative food spoilage and pathogenic bacteria

such as E. coli (Ogumbanwo et al., 2003). The milk was fermented for 4 h to a pH of

5.06 with10 ml and 20 ml of cheese and yoghurt cultures. The concentration of cells

responsible for the fermentation of milk was 24x1010 CFU/ml and 4x1010 CFU/ml for

cheese and yoghurt cultures respectively after plating out. The yoghurt culture was

used at a point to replace the cheese culture because of the inactivity of the cheese

culture which resulted from the inconsistencies in storage conditions.

Fermentation reduced the counts for indicator and pathogenic organisms in the

improved wagashie compared to the results of the market ‘wagashie’ samples.

Smoking eliminated aerobic mesophiles and reduced the yeast count to 103 CFU/g

which was within the acceptable limits for microorganisms in food products in

accordance with GSB Standard (1998) and the Standard method for the examination

of dairy products, (Ledenbach and Marshal, 2009). The improved ‘wagashie’

therefore had lower counts for pathogenic and indicator organisms than market

‘wagashie’ samples.

Packaging the wagashie samples after preparation reduced the risk of post-

contamination of the product. According to Poças and Pintado, (2010), packaging is

increasingly recognized as an important factor in protecting and controlling the

quality and safety of cheese, as well as addressing consumer issues.

The pH values recorded for the maket samples were generally high, although pH of

the fresh samples was relatively lower than the fried samples. This observation


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accounted for the high microbial load in the market samples. However the pH of the

laboratory prepared samples were low and thus the low microbial counts.

5.2 Improving the quality of wagashie

5.2.1 Affective testing

To improve on the sensory quality of ‘wagashie’, the process variables involved in

the preparation of traditional ‘wagashie’ and the improved ‘wagashie’ were

optimised by a three variable Box Behnken design to compare their acceptability,

taste, colour and texture with a 9-point hedonic sensory evaluation. Though milk

used for the traditional ‘wagashie’ preparation was not fermented, fermentation was

included mainly to improve on the taste and safety of the product. The results of the

response surface generated by the Box Behnken design as a result of sensory

evaluation showed that the panellists preferred the non fermented wagashie better

than the fermented wagashie. This is because, the texture , colour, taste and overall

acceptability of the traditional ‘wagashie’ was rated higher when fermentation time

was 0 h ,the quantity of salt was 23 g and 150g of plant extract was used in the

preparation. The predicted scores for texture, colour, taste and overall acceptability at

these levels were 7.18, 7.47, 7.38 and 7.36 respectively which was rated as ‘like

moderately’on the hedonic scale. The results of the overlaid contour plots for all the

attributes; texture, colour, taste and overall acceptability which were overlaid on one

axis of fermentation time and salt also confirmed this finding. The panellists

generally, rated the fermented sample lower because the bitter aftertaste imparted by

the plant extract combined with the sourness imparted by the fermented milk made

the product very bitter.

Commercial rennet was used to replace the plant extract to eliminate the bitter taste

imparted into the product. From the results of the response surface generated by the


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Box Behnken design for the improved ‘wagashie’ as a result of sensory evaluation,

the texture , colour, taste and overall acceptability was rated higher when the milk

was fermented for 4 h, the quantity of salt used was 11g and the concentration of

commercial rennet used was 5.35ml. the overlaid contour plots generated for the four

attributes overlaid on one axis of fermentation time and salt with a constant rennet

concentration of 5.35 ml also confirmed the finding stated earlier .The predicted

scores for the attributes texture, colour, taste and overall acceptability for the

wagashie samples were 6.67, 7.37, 7.37 and 7.22 respectively. The score can be

interpreted as ‘like moderately’ on the 9- point hedonic scale except texture which

was rated as ‘like slightly’. These results showed that when commercial rennet was

used as the coagulant, only a sour taste was perceived. Since rennet does not

influence the taste of the product, this was considered as desirable by the panellists as

with the case of European cheese.

The results from the two preparations showed that, when wagashie was prepared

using the extract of Sodom apple, the panel preferred that the product was not

fermented at all. However when wagashie was prepared with commercial rennet, the

panel preferred that the milk was fermented for 4 h. Also the plant extract coagulated

wagashie and the fermented rennet coagulated wagashie were both rated as ‘like

moderately’ by the sensory panel.

The results of the pH and TTA values recorded for the ‘wagashie’ samples were

relatively high as compared to other fresh cheeses. This was because most of the

acidity was lost into the whey during draining and pressing of the curd. This is

because a sour taste was observed upon tasting the whey. The loss of acidity and

increase in pH during draining and pressing also resulted from the insufficient lactic

acid produced by the starter culture in the milk during fermentation. This was


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because of the low concentration of the starter culture introduced for the milk

fermentation. The concentration of starter culture was therefore increased to achieve

the required pH at draining. This was in line with the finding by Lucey and Fox,

(1992) who stated that, curd is cooked to expel moisture at a temperature which

normally adversely affects the starter bacteria. The cheese maker must therefore

exert judgement to ensure that the desired acid development in the curd is reached at

about the same time as the required moisture content. To compensate for seasonal

changes in milk composition it is necessary to vary the percentage of innocula of the

starter culture to achieve the required acidity at draining.

The second hedonic sensory evaluation was carried out to confirm the consumer

preference of the optimised ‘wagashie’ prepared with the extract from the Sodom

apple plant and commercial rennet. In this evaluation, the non-fermented plant

extract coagulated sample was rated highest for taste followed by the rennet

coagulated fermented fried sample. However, significant differences were not

observed in their acceptability.The non-fermented rennet coagulated-smoked sample

was rated significantly low among the samples for taste; this observation confirmed

the results of the discriminatory hedonic sensory evaluation used for the product

optimisation where the panellists rated the non-fermented rennet coagulated fresh

sample low. The optimisation procedure was able to standardize the traditional

production method which eliminated the bitter aftertaste imparted by the plant

extract. The non fermented extract coagulated sample was rated significantly lower

than the other samples for colour. This can be as a result of the green pigmentation

imparted in the wagashie by the extract from the sodom apple plant as in agreement

with Chikpah et al., (2014) who stated that, the use of the Calotropis plant as

coagulant of soya milk resulted in a green colouration and bitter flavours. The


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fermented rennet coagulated smoked sample scored highest for texture whiles the

fermented rennet and extract coagulated fresh samples scored least.It therefore

showed that, smoking improved the texture of the fermented product.

The pH values of the non-fermented samples used for the confirmatory affective test

were significantly higher than the fermented samples. This was because the inoculum

was increased (20 ml per L instead of 20ml per 2 L of yoghurt culture) to improve on

the safety and the sensory quality of the product.

5.2.2 Quantitative Descriptive Sensory Analysis

During the results of the Quantitative Descriptive Sensory evaluation, 22 lexicons

were generated for ‘wagashie’ by the 13 panellists used. They described the sensory

characteristics of the fresh, fried and smoked forms of the fermented and non-

fermented- rennet coagulated wagashie, laboratory prepared fermented and non

fermented plant extract coagulated wagashie, and the fresh and fried market

‘wagashie’. PCA was performed to illustrate graphically the correlation ratings given

to the different descriptors and to visualise how the ‘wagashie’ samples were related

to each other and their relationship with the generated attributes. It was observed

that, all the fermented samples were grouped together whiles the non-fermented

samples were also grouped together. Also all fresh samples were grouped together in

the same quadrant because they had similar attributes. The smoked samples were

grouped in one quadrant and the fried samples were also in the same quadrant except

the non- fermented- rennet coagulated and the non-fermented –extract coagulated

fried samples which were in the same quadrant with the smoked samples This is

because, the rating was influenced mostly by fermentation and the processing

methods applied . From the groups formed by the cluster analysis, it was observed


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that, there were not much difference between the samples prepared with commercial

rennet and the samples prepared with the extract from Sodom apple. This shows that

the coagulant used had little or no influence on the acceptability of the product. The

rennet coagulated preparation can therefore replace the plant extract prepation

without any diificulty in consumer acceptance. This will save producers from going

through the stress involved in the preparation of the plant extract. It will also reduce

the bitter effect it imparts in ‘wagashie’ observed in the market samples which is as

a result of the excessive proteolytic nature of the Calotropis plant which reduces

cheese yield and increases the perception of bitter tastes, making its use more

difficult for cheese making (Lo Piero et al., 2002, Roseiro et al., 2003). The mean

intensity rating for each attribute was used to create a sensory descriptive analysis

profile and the spider plot showed the descriptive sensory analysis profile more

vividly (Stone, 1992). The non –fermented extract and rennet coagulated samples

were mainly described to have milky aroma, milky taste, cheesy taste, cheesy aroma

and yoghurt aroma. The fermented fresh samples (both plant extract and rennet

coagulated) were described to have, yoghurt aroma, spilt milk aroma, fermented

cassava dough aroma and whitish colour. The rennet coagulated sample was rated

more whitish than the extract coagulated sample, was also described to have a sour

taste, soft texture and a smooth texture. This is similar to the findings by Winwood,

(1983); Koth and Richter, (1989), who reported that the organoleptic properties

which typify soft unripened cheeses include, milky, white colour, soft body, smooth

texture, a good spreadability, no signs of syneresis on the cheese surface, no dryness

or grittiness and a mild to acidic flavour.

The fresh market wagashie samples were described to have a bland taste, bitter

aftertaste, sour taste, soft texture and smooth texture.They had the highest intensities


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for bland and bitterafter taste which were considered as undesirable. The smooth

texture of the fresh market sample was because it was being stored in its whey at the

retailing point. The sourness resulted from the activity of lactic acid bacteria present

in the natural microbial flora in the milk. These organisms can induce fermentation

in the product when conditions are favourable causing the sourness. It is possible

because fresh milk used for the preparation of traditional wagashie is not pasteurized.

The smoked samples were described to have smoked chevon aroma because of the

smoke that was imparted into the product as done with goat meat.They were

described as beefy aroma which resulted from the milk from the cow origin. They

were also described as crumbly texture because smoking reduced the moisture

content of the product.

The fried samples were described to have spongy texture because the samples soaked

some of the oil that was used during frying. The fried samples were described to

have fried egg taste, fried ripe plantain aroma, doughnut aroma, fried sweet potato

aroma. This is because the panel likened the taste and aroma of the fried ‘wagashie’

to be the taste sensations observed after eating egg that is fried and the aroma

sensations observed as a result of frying sweet potato, egg, ripe plantain and

doughnut respectively. They were described as brownish in colour because of the

processing methods applied (frying and smoking). They were also described to have

salty taste because of the salt which is in the form of sodium chloride added.

5.3 Chemical analysis of wagashie samples

Cheese is a nutrient dense food which provides fat, high quality proteins,

oligopeptides, amino acids, vitamins and minerals. The results of the proximate

analysis carried out on the samples showed that, the fermented rennet coagulated


135

smoked sample had the highest FFA and protein content. It also had high fat content

with reduced moisture.This is because smoking reduced the moisture content by

evaporation which caused, the protein and fat to become more concentrated in the

cheese. This showed that the fat content in the milk used in making the product was

high, thus the current conserns about the intake of high fat and it relation to coronary

heart diseases has caused consumers to be concerned about their fat intake. The

smoked sample was therefore considered for the rest of the study.The low moisture

content recorded in the smoked ‘wagashie’ may prolong its shelf life whereas the

high FFA value can cause spoilage to set in by the action of lipolysis.

The fried ‘wagashie’ sample had the highest fat content because the oil used in

frying was absorbed into the product. It also recorded the lowest moisture content

due to moisture loss during frying. The fermented sample had the lowest ash content

because of the breakdown of lactose, calcium phosphate and other minerals in the

milk by the starter culture introduced for fermentation, excessive breakdown of

calcium phosphate affects the texture of the final product. Lucey and Fox,(1992)

stated that, the relationship between the rate of moisture (and lactose) removal versus

rate of lactic acid production by the lactic acid bacteria to lower the curd pH has

profound effects on the characteristics of the final cheese.This was observed in the

fermented fresh sample which was brittle in texture. This effect has been reported by

Lucey and Fox, (1992), that the rapid and extensive acid production will remove

more calcium and phosphate, to produce a brittle cheese with a lower mineral

content.The non-fermented sample however had the highest moisture and ash

contents. The wagashie samples had high moisture content which is in agreement

with a report by Barabano and Rasmussen (1992) and Dave et al., (2003) who found


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that, cheese made with fermentation produced chymosin contained higher moisture

than cheese made from other coagulants.

5.4 Rheology of wagashie

5.4.1 Colour Determination

The results for the rheology of the improved wagashie showed that the colour of the

wagashie sample were lighter in colour because of their high L* values. This is

because milk which is the major ingredient in ‘wagashie’ is light in colour and

therefore the product basically takes the colour of the milk. The lower L* values

for the outer colour of the smoked and fried samples is mainly due to browning

reactions, which are influenced by the distribution of water and the reaction of

reducing sugars and amino acids (Kent and Evers 1994). The samples had positive

b* values, which was an indication of slightly yellowish colour. The samples also

had positive a* value for exterior part of the product which showed a slightly green

colour and negative a* values for the interior part of the product which showed a

slightly red colouration.

5.4.2 Texture Profile Analysis (TPA)

The results for the TPA analysis also showed that texture of the samples were hard

and chewy because of the high values recorded. The hardness resulted from the high

protein content in the ‘wagashie’ samples. This has been confirmed by Simoes et

al.,( 2013) who stated that lower concentration of protein reduced hardness in cheese

and and higher concentrations increase hardness. The protein matrix also produces

elasticity and it is the main factor responsible for flexibility and recovery after


137

tension.The values for chewiness in the sample were considered high when

compared to those reported for cheese of fete type, which is a type of cottage cheese

made from buffalo milk (Kumar et al., 2011). Simoes et al., (2013) stated that

chewiness value decreased with decrease in the concentration of added cow milk.

5.5 Shelf life of wagashie

In order to extend the shelf life of wagashie, the samples were vacuum packaged and

irradiated. Tsiotsias et al., (2002) also confirmed that among the preservation

methods to ensure safety of whey cheeses are irradiation combined with vacuum

packaging. The shelf life of the wagashie samples was carried out for 5 weeks. It was

observed after the study that all the control samples which were the non-irradiated

and non- vaccum/ normal packaged samples had undergone spoilage at the end of the

second week. The spoilage was characterised mainly by profulous mould growth, off

odour, moisture exudation, discolouration and bloated packaging as a result of gases

produced by yeast and other spoilage organisms in the samples, and rancidity. Poças

and Pintado, (2010) reported that, chemical failures associated with excessive water

loss or rind decolourization due to excessive exudation, off-flavours, and defatted

tastes originate from oxidation (rancidity) or through microbial metabolism

(bitterness, rancidity, acidity), and openings or irregular holes formed by

uncontrolled microbial fermentation. According to GSB Standard 1998, the

minimum count for yeast and mould should be <104 CFU/g, and 1.0 x 106 CFU/g for

total plate count. Also the Standard method for the examination of dairy product

2001 (Lebenach and Marshall, 2009), which is the regulatory standard for indicator

organisms for European Union and the United States stated that, the maximum

acceptable limits for total plate count should be <106, thus, the samples had exceeded


138

the maximum limits and were discarded. The samples that remained for rest of the

storage period were the irradiated vacuum packaged freshand smoked samples and

the non-irradiated vacuum packaged fresh and smoked samples (IVS, IVF.NVS and

NVF).

The vacuum packaged non-irradiated samples were able to stay on the shelf for 2

weeks. According to Kreft, (2008), vacuum packaging increased the shelf life of

Gouda cheese to 10 weeks. It also increased the shelf life of Parmigiano-Reggiano

cheese to 6 months (Severini et al., 1998). Their spoilage was however characterised

by bloated packaging as, result of gas production by yeast and other microorganisms

and few mould growth. The irradiated vacuum packaged fresh sample also

deteriorated on the 3rd week and was characterised by bloated packaging. The

bloating of the packaging material was as a result of the anaerobic condition and the

activity of yeast created in the packaging material. This condition must therefore be

controlled. Poças and Pintado (2010), stated that anaerobic condition inside packaged

cheese must be controlled because Clostridium spp. may grow and produce gas due

to anaerobic fermentation of lactate. They also stated that, active packaging systems

with antimicrobials, as well as packages with adequate oxygen permeability, may

solve some of these frequent microbial failures.

Though the smoked sample had microbial count which did not exceed the maximum

acceptable limits of spoilage or indicator organisms in food with regard to GSA

Standard, (1998) and the Standards for the examination of dairy products as

mentioned earlier,the product had developed off- odour but no visual defects were

observed. This was also observed by Bongirwar and Kumta (1967) who reported that

Cheddar cheese developed off-flavours when irradiated at 0.5 kGy. However,

according to Chincholle, (1991) Camembert cheese suffered no off-flavour


139

development up to a dose of 3 kGy. No mould growth was observed in the irradiated

vacuum packaged samples during the 5 weeks storage period. Jones and Jelen,

(1988) observed also that when Turkish Kashar cheese was exposed to 1.2 kGy, a

mould free shelf life was obtained for 12 to 15 days of storage. Though ‘wagashie’ is

known to be a perishable product with a shelf life 2 to 3 days (Ashaye et al., 2006),

its shelf life was extended to a maximum of 3 weeks when preserved by vacuum

packaging and 4 kGy of gamma radiation from a Cobalt 60 source.

5.5.1 pH of wagashie samples during the 5 weeks storage period

Fresh cheeses have high pH when acid formed during milk fermentation is lost

during whey drainage and pressing; the high pH creates a suitable condition in the

cheese for the growth of spoilage organisms like Bacillus cereus, yeast and moulds

etc. The pH obtained after milk fermentation for ‘wagashie’ preparation was 5.06

and decreased to 4.9 after preparation.

The results of the pH of the samples during the 5 weeks storage, showed that the pH

values of the samples increased as the microbial count increased. This observation is

contrary to the findings by Nobile et al., (2009) who stated that the pH of Ricotta

cheese packaged under modified atmosphere increased as the microbial count

reduced upon storage. This may be influenced by the type of organisms responsible

for the spoilage and the chemical changes that occur in the product.The high pH

recorded was as a result of proteolysis which is the gradual breakdown of proteins to

form amino acids and peptides which increase alkalinity. This is influenced by the

enzymes in the coagulant and the activity of the microorganisms present in the

cheese (Bylund, 1995).


140

CHAPTER SIX

6.0 CONCLUSIONS AND RECOMMENDATIONS

6.1 CONCLUSIONS

Wagashie sold on the market pose a health hazard to consumers since they generally

contain indicator and pathogenic microorganisms which exceed the limits prescribed

by GSB and other regulatory bodies. However, the hazards are reduced when the

wagashie sold is in a fried form.

The traditional process of wagashie production can be improved or industrialised by

replacing the use of the plant extract with rennet and other commercial cheese

coagulants. This eliminates the bitterness imparted to wagashie and makes it easier to

standardise the process.

By fermenting the pasteurised milk used to produce wagashie, both the sensory

quality and the safety of the product were improved due to the antimicrobial

properties of the lactic acid bacteria used for the fermentation and a more

pronounced taste of the acidified product.

The combined effect of fermentation, smoking and vacuum packaging extended the

shelf life of wagashie to 2 week without refrigeration. However when combined with

irradiation, the shelf life extended to 3 weeks.

.


141

6.2 RECOMMENDATIONS

The selection of the optimum packaging system must be considered which should

include the fact that cheese is a complex dynamic matrix in which several microbial,

physical, and biochemical changes occur during storage. This can be done by

incorporating Modified Atmosphere packaging whereby CO2 can be introduced into

the vacuum packaging material and the application of low doses of radiation (1kGy

to 3 kGy) to further extend the shelf life of ‘wagashie’ whiles maintaining its

quality.

The chemical analysis as well as sensory evaluation of ‘wagashie’ during storage

should be carried out along with microbiological tests to observe their influence on

each other.

The texture of the fermented ‘wagashie’ sample should be improved by maturing it

under controlled conditions to further improve on the product.

Studies to improve on the yield of the rennet coagulated ‘wagashie’ should be carried

out.


142

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161

APENDDIX

Apendix I

Principal Component Analysis:

Correlation of the discriptors of wagashie

Factor loadings:

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

Soft 0.6801 0.5407 0.1734 -0.3253 0.2713 0.0913 -0.1363 0.0176 -0.0122 -0.0073 0.0643 0.0623 -0.0149

Spongy

-

0.6882

-

0.1278 0.4562 0.2634 0.3818 -0.1692 0.0812 -0.0061 0.0602 -0.0703 0.1903 -0.0811 0.0152

Smooth 0.7059 0.5022 0.0229 -0.2626 0.2977 0.2236 -0.0649 -0.0513 0.0572 -0.1642 0.0295 -0.0330 0.0484

Crumbly

-

0.4493

-

0.1451 0.4723 0.5535 0.0722 -0.3389 0.2922 0.1006 -0.1098 0.0364 -0.0483 0.1230 0.0367

Milky aroma 0.5378

-

0.5508 0.4614 0.2078 -0.1576 0.1899 -0.1225 -0.0987 0.0501 0.1936 0.1440 0.0600 -0.0372

Yoghurt aroma 0.9343

-

0.0302

-

0.0374 -0.2207 0.0131 -0.0686 0.2170 -0.0384 -0.1179 0.0675 0.0572 0.0070 -0.0288

Cheesy aroma 0.5487

-

0.5290

-

0.0498 -0.3360 -0.4147 -0.0081 -0.1177 0.2890 0.1524 0.0438 0.0800 0.0060 0.0529

Beefy aroma 0.0941

-

0.5143 0.1413 -0.1703 0.8014 0.0340 0.0140 0.0498 0.1379 0.0960 -0.0316 0.0330 -0.0366

Fried sweet

potato aroma

-

0.7147 0.2519 0.4368 -0.2390 -0.1226 0.2208 -0.1101 0.2892 -0.1222 -0.0091 -0.0213 -0.0337 -0.0416


162

Fried ripe

plantain aroma

-

0.6786 0.3585 0.3195 -0.3359 -0.0778 0.3276 0.1178 -0.1287 -0.0892 -0.0303 0.1364 0.1534 0.0349

Smoked chevon

aroma

-

0.4045

-

0.2054

-

0.6330 0.5266 0.1914 0.1588 0.0072 0.1342 -0.0066 0.0876 0.1519 -0.0690 0.0269

spoilt milk

aroma 0.5370 0.3776 0.3755 0.5538 -0.1809 0.0052 0.2739 0.0032 -0.0053 -0.0403 0.0549 -0.0924 -0.0192

Fermented

cassva dough

aroma 0.6868 0.4027 0.0791 0.4596 -0.1541 0.2523 0.1892 0.1217 0.1023 0.0093 0.0043 -0.0024 -0.0112

Doughnut aroma

-

0.7379 0.3846 0.3148 -0.3145 0.0293 0.2395 0.0807 0.1430 -0.1024 0.1046 0.0036 -0.0546 -0.0042

Sour taste 0.1144 0.9063 0.2328 0.2045 -0.0054 -0.1330 -0.1493 -0.0767 0.1054 0.0915 0.0001 0.0019 0.0649

Bland 0.1294 0.1498

-

0.4397 0.6650 0.1753 0.5154 0.0224 -0.0411 -0.0782 0.0832 -0.1132 0.0215 0.0198

Cheesy taste 0.1700

-

0.4409 0.7765 0.2321 0.0348 0.1936 0.0323 0.1311 0.1931 -0.1003 -0.1108 0.0552 0.0160

Milky taste 0.2336

-

0.7972 0.4173 -0.2023 0.0054 0.0670 -0.0935 -0.1346 -0.1587 0.1513 -0.0618 -0.0830 0.0683

Bitter taste 0.1205 0.7466 0.3080 0.3783 -0.0621 -0.2247 -0.3433 -0.0355 0.0061 0.1237 -0.0017 0.0215 -0.0288

Fried egg taste

-

0.6271

-

0.4913 0.3244 0.1722 -0.2768 0.2169 -0.0320 -0.2864 0.1081 -0.0936 -0.0030 -0.0507 -0.0277

Salty taste

-

0.3602 0.4788

-

0.0528 -0.5641 -0.0770 -0.0060 0.4637 -0.0831 0.2300 0.1868 -0.0497 -0.0424 0.0029

Whitish colour 0.6105 0.1866 0.7321 -0.1026 0.1367 -0.0009 -0.0044 0.0033 -0.1183 0.0005 -0.0390 -0.1079 0.0071

Brownish colour

-

0.8845 0.3043 0.0642 0.0896 0.0223 0.0216 -0.2943 -0.0008 0.1385 0.0641 -0.0384 -0.0243 0.0019

Factor scores:


163

Observation F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

997 4.8591 3.2630 2.1102 3.5438 -0.3949 0.4483 1.0498 -0.0260 0.0402 -0.1213 -0.0295 -0.1492 0.0290

246

-

1.0383 3.1858 0.9035 0.9275 -0.0486 -1.1839 -1.8244 0.1382 0.2130 0.6393 0.2202 0.4383 -0.0868

203

-

1.4521

-

2.4828 0.6833 1.0232 -0.5336 -0.4594 -0.1020 -0.8118 0.0448 -0.0092 -0.5321 -0.2823 -0.4018

110 1.7131

-

2.0619 1.7021 -0.7035 3.5628 -0.0254 0.2077 0.0871 0.3692 0.0193 -0.2774 0.2463 0.0361

355 0.8085

-

2.1088 0.1521 -0.0877 0.0394 -1.4332 0.5261 -0.4307 -1.3273 0.5417 0.3572 -0.1299 0.1475

961

-

1.7646

-

2.9796

-

0.0350 1.4325 -0.9221 -0.3502 -0.4040 -0.2188 1.1376 0.0668 0.0998 -0.2674 0.3175

707

-

2.4387

-

1.4209 1.7824 0.0336 -0.8061 1.7571 0.0508 -0.6234 -0.3339 -0.4893 0.4124 0.6038 0.0096

101

-

1.9925

-

1.1097 2.2418 -0.6150 -0.6949 0.5296 -0.1646 1.8570 -0.3676 0.0347 -0.1422 -0.2514 -0.0280

838 3.0930

-

0.3293

-

1.0721 -1.6923 0.1862 -0.3681 -0.1853 0.2033 0.4082 -0.5458 0.9600 -0.2708 -0.1928

614 1.7820

-

0.3546

-

1.8955 -1.2591 -1.5304 -1.3479 1.1615 0.4320 0.2531 -0.2846 -0.4458 0.5784 0.0092

616 4.7089

-

0.1553

-

1.6046 -1.9928 -0.7164 1.7101 -1.2057 -0.3084 -0.1426 0.5016 -0.3849 -0.0945 0.0599

706

-

2.0749 3.1787

-

0.1832 -1.0078 0.5295 -0.6218 -0.8564 -0.3751 -0.4469 -1.0309 -0.3826 -0.2661 0.1573

421

-

3.8324 3.3316 0.0597 -2.4658 0.1672 0.5226 1.5713 -0.3698 0.4218 0.6404 0.0878 -0.1949 -0.0152

417

-

2.3712 0.0438

-

4.8447 2.8633 1.1621 0.8222 0.1752 0.4463 -0.2696 0.0374 0.0571 0.0398 -0.0416


164

Appendix II

Generated Attributed by the 13 member panel during the Quantitative

Descriptive sensory evaluation of ‘wagashie’ on ‘taste’, ‘texture’, ‘colour’ and

‘aroma’.

During the training of the panel members for the descriptive analysis 23 descriptors

were generated in a focus group discussion. These descriptors were documented and

used to evaluate the 14 samples. Table 4.10 shows the attributes and the generated

descriptors by the 13 member panel for the quantitative descriptive sensory

evaluation

Table 4.5: Generated attributes and descriptors for ‘wagashie’

Attributes Descriptors

Taste Sour

Bland

Cheesy

Milky

Bitterness

Fried egg

Salty

Texture Soft

Smooth

Crumbly

Spongy

Aroma Milky

Yoghurt

Cheesy

Beefy

Spoilt milk

Fermented cassava dough

Doughnut

Fried ripe plantain

Smoked chevon

Fried sweet potato

Colour Whitish

Brownish


165

Definition of generated attributes

Some of the descriptors generated were considered as desirable and undesirable,

table 4.6 shows the desirable and undesirable descriptors and their definitions.

Table 4.6: The desirable and undesirable descriptors generated by the 14

member panel during the training

Attribute Desirable Definition Undesirable Definition

Taste Sour Taste

sensation

associated

with fermented

milk

Bland Tasteless

sensation in a

food product

Cheesy Taste

sensation

associated

with cheese

Bitter taste Taste

sensation of

quinine or

caffeine

Milky Taste

associated

with fresh milk

Fried egg Taste

associated

with fried egg

Salty Taste

produced by a

solution of

sodium

chloride

Texture Soft

The degree of

stickiness in

the mouth

Smooth The

spreadability

of the cheese

Spongy The texture


166

characteristics

of a bouncy

cheese

Crumbly The extent to

which the

cheese breaks

in the mouth

Aroma Milky Aromatics of

milk from

dairy origin

Spoilt milk Aromatics of

spoilt milk

Yoghurt Aromatics of

plain yoghurt

Fermented

cassava dough

Aromatics of

fermented

cassava dough

Cheesy The aromatic

sensation of

cheese

Beefy

Smell

associated

with cow meat

Smoked

chevon

The aromatics

associated

with grilled

goat meat

Fried sweet

potato

The smell

associated

with fried

sweet potato

Fried ripe

plantain

The smell

associated

with fried ripe

plantain

Doughnut The smell

associated

with fried


167

doughnut

Colour Whitish Lighter colour

Brownish Darker colour

Sample Questionnaire for the Acceptability of Wagashie

Name:…………………………………………………………………………

Date:………….

Please rate the texture, colour, taste and overall acceptability of the samples

provided. Indicate your choice with a tick. Please rinse your mouth in-between

sample tasting.

Texture:

Sample code 1st 2nd 3rd 4th 5th

9. Like extremely

8. Like very much

7. Like moderately

6. Like slightly

5. Neither like nor dislike

4. Dislike slightly

3. Dislike moderately

2. Dislike very much

1. Dislike extremely

Apenddix III

Response Surface Regression: Texture, versus Ferment time, ...


168

Response Surface Regression: Texture versus Ferment time, Extract(wt/g, Salt(g)

The analysis was done using uncoded units.

Estimated Regression Coefficients for Texture

Term Coef SE Coef T P

Constant 11.2921 3.15705 3.577 0.016

Ferment time -1.1441 0.38526 -2.970 0.031

Extract(wt/g) 0.0300 0.02148 1.395 0.222

Salt(g) -0.5506 0.24774 -2.222 0.077

Ferment time*Ferment time 0.1143 0.03771 3.031 0.029

Extract(wt/g)*Extract(wt/g) -0.0000 0.00006 -0.749 0.488

Salt(g)*Salt(g) 0.0249 0.00603 4.125 0.009

Ferment time*Extract(wt/g) 0.0043 0.00145 2.985 0.031

Ferment time*Salt(g) -0.0032 0.01449 -0.224 0.831

Extract(wt/g)*Salt(g) -0.0024 0.00058 -4.123 0.009

S = 0.2898 R-Sq = 96.9% R-Sq(adj) = 91.3%

Analysis of Variance for Texture

Source DF Seq SS Adj SS Adj MS F P

Regression 9 13.1486 13.14861 1.460956 17.39 0.003

Linear 3 8.7824 1.45320 0.484400 5.77 0.044

Square 3 2.1857 2.18568 0.728561 8.67 0.020

Interaction 3 2.1805 2.18047 0.726825 8.65 0.020

Residual Error 5 0.4200 0.41997 0.083993

Lack-of-Fit 3 0.4069 0.40690 0.135633 20.76 0.046

Pure Error 2 0.0131 0.01307 0.006533

Total 14 13.5686

Unusual Observations for Texture

Obs StdOrder Texture Fit SE Fit Residual St Resid

1 1 4.600 4.918 0.251 -0.318 -2.19 R

13 13 6.870 6.553 0.251 0.317 2.19 R

R denotes an observation with a large standardized residual.

Response Surface Regression: Colour versus Ferment time, Extract(wt/g), Salt(g)


Estimated Regression Coefficients for Colour


Constant 10.2946 2.82625 3.642 0.015

Ferment time -0.1657 0.34489 -0.480 0.651

Extract(wt/g) 0.0045 0.01923 0.237 0.822

Salt(g) -0.2766 0.22178 -1.247 0.268


169

Ferment time*Ferment time -0.0383 0.03376 -1.136 0.308

Extract(wt/g)*Extract(wt/g) 0.0000 0.00005 0.235 0.824

Salt(g)*Salt(g) 0.0113 0.00540 2.086 0.091




S = 0.2594 R-Sq = 86.5% R-Sq(adj) = 62.2%

Analysis of Variance for Colour


Regression 9 2.1541 2.1541 0.23934 3.56 0.088

Linear 3 1.2937 0.1353 0.04511 0.67 0.606

Square 3 0.4086 0.4086 0.13619 2.02 0.229

Interaction 3 0.4518 0.4518 0.15060 2.24 0.202

Residual Error 5 0.3366 0.3366 0.06731

Lack-of-Fit 3 0.2299 0.2299 0.07663 1.44 0.435

Pure Error 2 0.1067 0.1067 0.05333

Total 14 2.4906

Response Surface Regression: Taste versus Ferment time, Extract(wt/g), Salt(g)


Estimated Regression Coefficients for Taste


Constant 15.5101 4.91673 3.155 0.025

Ferment time -0.5930 0.59999 -0.988 0.368

Extract(wt/g) 0.0192 0.03346 0.573 0.591

Salt(g) -1.0134 0.38583 -2.627 0.047



Salt(g)*Salt(g) 0.0316 0.00940 3.358 0.020




S = 0.4514 R-Sq = 81.0% R-Sq(adj) = 46.8%

Analysis of Variance for Taste


Regression 9 4.33817 4.33817 0.48202 2.37 0.178

Linear 3 1.33535 1.84830 0.61610 3.02 0.132

Square 3 2.42915 2.42915 0.80972 3.97 0.086

Interaction 3 0.57367 0.57368 0.19123 0.94 0.488

Residual Error 5 1.01860 1.01860 0.20372


170

Lack-of-Fit 3 0.97000 0.97000 0.32333 13.31 0.071

Pure Error 2 0.04860 0.04860 0.02430

Total 14 5.35677

Unusual Observations for Taste

Obs StdOrder Taste Fit SE Fit Residual St Resid

1 1 5.400 5.875 0.391 -0.475 -2.10 R

13 13 6.800 6.325 0.391 0.475 2.10 R

R denotes an observation with a large standardized residual.

Response Surface Regression: Overall acce versus Ferment time, ...


Estimated Regression Coefficients for Overall acceptability


Constant 13.4324 5.04147 2.664 0.045

Ferment time -0.9158 0.61521 -1.489 0.197

Extract(wt/g) 0.0152 0.03430 0.443 0.676

Salt(g) -0.7157 0.39562 -1.809 0.130



Salt(g)*Salt(g) 0.0257 0.00963 2.666 0.045




S = 0.4628 R-Sq = 83.0% R-Sq(adj) = 52.5%

Analysis of Variance for Overall acceptability


Regression 9 5.2432 5.2432 0.58258 2.72 0.141

Linear 3 1.7995 1.2774 0.42581 1.99 0.234

Square 3 1.7186 1.7186 0.57288 2.67 0.158

Interaction 3 1.7251 1.7251 0.57504 2.68 0.157

Residual Error 5 1.0709 1.0709 0.21419

Lack-of-Fit 3 0.9237 0.9237 0.30789 4.18 0.199

Pure Error 2 0.1473 0.1473 0.07363

Total 14 6.3142

Response Surface Regression: Texture versus Fermentation time, salt(g), Rennet(ml)

The following terms cannot be estimated and were removed:


171

salt(g)*Rennet(ml)

Method

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Model 8 1.72837 0.216046 12.38 0.031

Linear 3 0.70253 0.234178 13.42 0.030

Fermentation time 1 0.14981 0.149813 8.59 0.061

salt(g) 1 0.24000 0.240000 13.76 0.034

Rennet(ml) 1 0.02821 0.028213 1.62 0.293

Square 3 0.55729 0.185763 10.65 0.042

Fermentation time*Fermentation time 1 0.00988 0.009882 0.57 0.506

salt(g)*salt(g) 1 0.32120 0.321202 18.41 0.023

Rennet(ml)*Rennet(ml) 1 0.15301 0.153015 8.77 0.059

2-Way Interaction 2 0.18138 0.090688 5.20 0.106

Fermentation time*salt(g) 1 0.02138 0.021376 1.23 0.349

Fermentation time*Rennet(ml) 1 0.16000 0.160000 9.17 0.056

Error 3 0.05233 0.017444

Lack-of-Fit 1 0.01307 0.013067 0.67 0.500

Pure Error 2 0.03927 0.019633

Total 11 1.78070

Model Summary

S R-sq R-sq(adj)

0.132077 97.06% 89.22%

Coded Coefficients

Term Effect Coef SE Coef T-Value P-Value VIF

Constant 6.5133 0.0763 85.42 0.000

Fermentation time 0.3533 0.1767 0.0603 2.93 0.061 1.44

salt(g) -0.6000 -0.3000 0.0809 -3.71 0.034 1.84

Rennet(ml) -0.1533 -0.0767 0.0603 -1.27 0.293 1.25

Fermentation time*Fermentation time 0.1283 0.0642 0.0853 0.75 0.506 1.22

salt(g)*salt(g) -0.7317 -0.3658 0.0853 -4.29 0.023 1.22

Rennet(ml)*Rennet(ml) 0.5050 0.2525 0.0853 2.96 0.059 1.25

Fermentation time*salt(g) 0.223 0.112 0.101 1.11 0.349 1.70

Fermentation time*Rennet(ml) 0.4000 0.2000 0.0660 3.03 0.056 1.00

Regression Equation in Uncoded Units

Texture = 5.00 - 0.258 Fermentation time + 0.510 salt(g) - 0.1844 Rennet(ml)

+ 0.0160 Fermentation time*Fermentation time - 0.02986 salt(g)*salt(g)

+ 0.01168 Rennet(ml)*Rennet(ml) + 0.0160 Fermentation time*salt(g)

+ 0.02151 Fermentation time*Rennet(ml)

Response Surface Regression: Colour versus Fermentation time, salt(g), Rennet(ml)


172

The following terms cannot be estimated and were removed:

Fermentation time*Rennet(ml)

Method



Model 8 0.522550 0.065319 5.37 0.061

Linear 3 0.344981 0.114994 9.46 0.027


salt(g) 1 0.189113 0.189113 15.56 0.017

Rennet(ml) 1 0.148519 0.148519 12.22 0.025

Square 3 0.077544 0.025848 2.13 0.240


salt(g)*salt(g) 1 0.070917 0.070917 5.83 0.073


2-Way Interaction 2 0.100025 0.050012 4.11 0.107


salt(g)*Rennet(ml) 1 0.060025 0.060025 4.94 0.090

Error 4 0.048619 0.012155

Lack-of-Fit 2 0.048619 0.024309

Pure Error 2 0.000000 0.000000

Total 12 0.571169

Model Summary

S R-sq R-sq(adj) R-sq(pred)

0.110248 91.49% 74.46% 0.00%

Coded Coefficients


Constant 7.3300 0.0637 115.16 0.000

Fermentation time -0.1813 -0.0906 0.0477 -1.90 0.130 1.13

salt(g) -0.3075 -0.1537 0.0390 -3.94 0.017 1.00

Rennet(ml) 0.3338 0.1669 0.0477 3.50 0.025 1.13

Fermentation time*Fermentation time -0.0225 -0.0113 0.0637 -0.18 0.868 1.08

salt(g)*salt(g) -0.3075 -0.1538 0.0637 -2.42 0.073 1.03



salt(g)*Rennet(ml) 0.2450 0.1225 0.0551 2.22 0.090 1.00


Colour = 7.079 - 0.184 Fermentation time + 0.151 salt(g) - 0.0660 Rennet(ml)

- 0.0028 Fermentation time*Fermentation time - 0.01255 salt(g)*salt(g)


+ 0.00753 salt(g)*Rennet(ml)


173

Response Surface Regression: Taste versus Fermentation time, salt(g), Rennet(ml)



Model 9 6.48237 0.72026 2.85 0.210

Linear 3 3.37098 1.12366 4.45 0.126


salt(g) 1 3.03882 3.03882 12.03 0.040

Rennet(ml) 1 0.07008 0.07008 0.28 0.635

Square 3 2.29669 0.76556 3.03 0.193


salt(g)*salt(g) 1 1.87072 1.87072 7.40 0.072


2-Way Interaction 3 0.57631 0.19210 0.76 0.586



salt(g)*Rennet(ml) 1 0.43574 0.43574 1.72 0.280

Error 3 0.75793 0.25264

Lack-of-Fit 1 0.09127 0.09127 0.27 0.653

Pure Error 2 0.66667 0.33333

Total 12 7.24031

Model Summary


0.502637 89.53% 58.13% *

Coded Coefficients


Constant 6.537 0.290 22.52 0.000

Fermentation time 0.688 0.344 0.229 1.50 0.231 1.44

salt(g) 2.135 1.068 0.308 3.47 0.040 2.13

Rennet(ml) -0.242 -0.121 0.229 -0.53 0.635 1.44


salt(g)*salt(g) -1.974 -0.987 0.363 -2.72 0.072 1.68




salt(g)*Rennet(ml) 1.008 0.504 0.384 1.31 0.280 1.71


Taste = -3.11 - 0.211 Fermentation time + 1.795 salt(g) - 0.472 Rennet(ml)



+ 0.0019 Fermentation time*Rennet(ml) + 0.0310 salt(g)*Rennet(ml)


174

Response Surface Regression: Overall Acceptab versus Fermentation tim, salt(g),

Rennet(ml)



Model 9 2.57655 0.28628 1.64 0.305

Linear 3 0.40912 0.13637 0.78 0.554


salt(g) 1 0.08000 0.08000 0.46 0.529

Rennet(ml) 1 0.12751 0.12751 0.73 0.432

Square 3 0.91690 0.30563 1.75 0.273


salt(g)*salt(g) 1 0.06361 0.06361 0.36 0.573


2-Way Interaction 3 1.25053 0.41684 2.38 0.185



salt(g)*Rennet(ml) 1 0.59290 0.59290 3.39 0.125

Error 5 0.87402 0.17480

Lack-of-Fit 3 0.70222 0.23407 2.72 0.280

Pure Error 2 0.17180 0.08590

Total 14 3.45057

Model Summary


0.418097 74.67% 29.08% 0.00%

Coded Coefficients


Constant 7.030 0.241 29.12 0.000

Fermentation time -0.317 -0.159 0.148 -1.07 0.332 1.00

salt(g) -0.200 -0.100 0.148 -0.68 0.529 1.00

Rennet(ml) 0.252 0.126 0.148 0.85 0.432 1.00


salt(g)*salt(g) -0.263 -0.131 0.218 -0.60 0.573 1.01

Rennet(ml)*Rennet(ml) -0.898 -0.449 0.218 -2.06 0.094 1.01

Fermentation time*salt(g) -0.160 -0.080 0.209 -0.38 0.718 1.00


salt(g)*Rennet(ml) 0.770 0.385 0.209 1.84 0.125 1.00


Overall Acceptability = 7.27 - 0.339 Fermentation time + 0.093 salt(g) - 0.085 Rennet(ml)


- 0.0208 Rennet(ml)*Rennet(ml) - 0.0114 Fermentation time*salt(g)

+ 0.0427 Fermentation time*Rennet(ml) + 0.0237 salt(g)*Rennet(ml)


175


optimising the wagashie (a traditional cottage cheese

Documents