The Use of Natural Plant Stabilizers Extracts in the Production of Yoghurt

THE USE OF NATURAL PLANT STABILIZERS EXTRACTS IN THE PRODUCTION OF YOGHURT

By Maha Elsadig Abuzeid khalifa B.Sc. Honors (2005) University of Ahfad for Women

A dissertation submitted to the University of Khartoum in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Technology

Supervisor Prof. Elgasim Ali Elgasim

Department of Food Science and Technology Faculty of Agriculture University of Khartoum

August 2008

DEDICATION

To my Family Fiancée, Friends and to All Whom I Love

MAHA

I Acknowledgements

I would like to thanks all the people who devoted their time and efforts to let this work come true. A work of long hard weeks, months and non-stop night staying, this would not have happened without the help of those people. My admiration and appreciation goes to my supervisor Prof. Elgasim Ali Elgasim who with his knowledge, motivation and fatherly supervision helped me to accomplish the research project. I extend my thanks to the following: ¾ Dr. Abd El- Wahab Hassan, who provided me the psyllium seeds (Plantago ovata) and some valuable information. ¾ Dr. Ahmed Zaghloul, for providing me the chicory roots (Cichorium intybus) and for helping me in the processing and the analysis of the product. ¾ Staff of National Research Center, El-Doki, Cairo. ¾ Staff of Environmental Research Institute, especially, Mr. Magdi Hashim. And finally, to every one who worked behind the curtains, supporting me and pushing me to succeed in accomplish this project.

II Table of Contents

Title Page Dedication I Acknowledgements II Table of Contents III List of Tables VII English Abstract VIII Arabic Abstract IV

Chapter One 1 Introduction 1

Chapter Two 4 Literature Review 4 2.1 Milk and Milk Composition 4 2.1.1 Forms of Milk Products 4 2.2 Milk Fermentation 5 2.2.1 Types of Fermentation 5 2.2.2 Fermented Milk Products 5 2.3 Yoghurt 6 2.3.1 Composition of Yoghurt 7 2.3.2 Nutritional Value of Yoghurt 7 2.3.3 Types of Yoghurt 10 2.3.4 Manufacture of Yoghurt 11

III 2.3.4.1 Preparation of Yoghurt mix 11 2.3.4.2 Fermentation 13 2.3.4.3 Breaking and Cooling 13 2.3.4.4 Addition of Fruits and Filling 14 2.3.5 Nutritional Benefits of Yoghurt 14 2.3.6 Therapeutic Properties of Yoghurt 15 2.4 Stabilizers 16 2.4.1 Plantago ovata 16 2.4.1.1 Classification 17 2.4.1.2 Botany and Distribution 19 2.4.1.3 Plantago ovata Forsk 19 2.4.1.4 Phenotypic Description of Seed 19

2.4.1.5 Uses of Plantago ovata 20 2.4.2 Cichorium intybus 20 2.4.2.1 Classification 21 2.4.2.2 Botany Descriptions 22 2.4.2.3 Uses of Cichorium intybus 22 Chapter Three 24 Materials and Methods 24 3.1 Materials 24 3.1.1 Preparation of the Mucilage 24 3.1.2 Preparation of the Inulin 24 3.1.3 Yoghurt Manufacturing 25

IV 3.2 Methods of Analysis 25 3.2.1 pH Value Measurement 25 3.2.2 Titratable Acidity Determination 25 3.2.3 Wheying off Determination 25 3.2.4 Ash Content Determination 25 3.2.5 Total Nitrogen Determination 26 3.2.6 Total Carbohydrate Determination 26 3.2.7 Lactose Content Determination 27 3.2.8 Total Solid Determination 27 3.2.9 Acetaldehyde Concentration Determination 28 3.2.10 Diacetyl Concentration Determination 28 3.2.11 Statistical Analysis 29 Chapter Four 30 Results and Discussions 30 4.1 pH Values 30 4.2 Titratable Acidity 30 4.3 Wheying off 31 4.4 Ash Content 35 4.5 Total Protein Content 35 4.6 Total Carbohydrate 35 4.7 Lactose Content 39 4.8 Total Solid Contents 39 4.9 Acetaldehyde Concentration 42

V 4.10 Diacetyl Concentration 42 Chapter Five 45 Conclusion and Recommendations 45 5.1 Conclusions 45 5.2 Recommendations 45 Chapter Six 46 References 46 Appendix 50

VI List of Tables

Title Page

Table 2.1: The chemical composition of natural and flavored yoghurt 8

Table 2.2: Some typical values of the major constituents of milk and 8 yoghurt (all units per 100g)

Table 2.3: Some typical vitamin contents of milk and yoghurt (all 9 units per100g)

Table 2.4: Stabilizers commonly used in yoghurt and their properties 18

Table 4.5: Effect of stabilizers at different levels of treatment and the 32 storage period on the pH of the yoghurt

Table 4.6: Changes in titratable acidity of yoghurt treated with 33 different stabilizer stored for up to 10 days

Table 4.7: Effect of stabilizers treatment and storage period on 34 wheying-off of yoghurt

Table 4.8: Effect of stabilizers treatment and storage period on ash 36 content

Table 4.9: Protein content (%) of yoghurt treated with different 37 stabilizers and stored for up to 10 days

Table 4.10: Effect of stabilizers and storage period on yoghurt 38 carbohydrates content (mg/ml) stored for up to 10 days

Table 4.11: Changes in lactose percent of yoghurt treated with 40 different levels of stabilizers and stored for up to 10 days

Table 4.12: Effects of stabilizers and storage periods on the total solid 41 content of yoghurt stored for up to 10 days

Table 4.13: Acetaldehyde concentration (µ mol/100ml) in yoghurt 43 treated with different stabilizers and stored for up to 10 days Table 4.14: Diacetyl concentration (µ mol/100ml) of yoghurt treated with different stabilizers stored for up to 10 days 44

VII THE USE OF NATURAL PLANT STABILIZERS EXTRACTS IN THE PRODUCTION OF YOGHURT (M.Sc. Thesis) Maha Elsadig Abuzeid Khalifa

Abstract: The objective of this study was to make use of two natural plant stabilizers namely mucilage and inulin extracted from Psyllium seeds “Plantago ovata” and Chicory roots “Cichorium intybus” in yoghurt manufacture. Inulin was used at levels of 4% and 6% and mucilage at the level of 0.2% to manufacture yoghurt from skim milk powder. Immediately after processing, yoghurt was kept refrigerated at 5±1 ºC for up to 10 days. Parameters measured include: pH value, titratable acidity, wheying off, ash, protein, lactose, total carbohydrate, total solid, acetaldehyde and diacetyl. Addition of inulin at 4%, 6% and mucilage at 0.2% resulted in 30%, 58% and 50% reduction in the wheying-off of yoghurt, respectively compared to control. Different treatments did not show any effect on the ash content of yoghurt (p≥0.05). None of the stabilizers used in the study had any effect on the lactose content of yoghurt. However, a decrease in lactose content was observed with the increase in the storage period. There was a substantial decrease in lactose content from 2% to 0.78% on the 10th day of storage. The protein content of yoghurt treated with inulin 6%, mucilage 0.2% and control were significantly different (p≤0.05) and gave value of 4.56%, 4.37% and 4.19% respectively. Based on the findings of this study, mucilage and inulin are potential stabilizers to be used in yoghurt processing.

VIII ﺇﺴﺘﺨﺩﺍﻡ ﺍﻟﻤﺜﺒﺘﺎﺕ ﺍﻟﻤﺴﺘﺨﻠﺼﺔ ﻤﻥ ﺍﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﻁﺒﻴﻌﻴﺔ ﻓﻰ ﺇﻨﺘﺎﺝ ﺍﻟﺯﺒﺎﺩﻯ

( ﺃﻁﺭﻭﺤﺔ ﻤﺎﺠﺴﺘﻴﺭ)

ﻤﻬﺎ ﺍﻟﺼﺎﺩﻕ ﺃﺒﻭﺯﻴﺩ ﺨﻠﻴﻔﺔ

ﺍﻟﻤﺴﺘﺨﻠﺹ: ﺘ ﻬﺩﻑ ﻫﺫ ﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻼﺴﺘﻔﺎﺩﺓ ﻤﻥ ﺍﺜ ﻨﻴﻥ ﻤﻥ ﻤﺜﺒﺘـﺎ ﺕ ﺍﻟﻨﺒﺎﺘﻴـﺔ ﺍﻟﻁﺒﻴﻌﻴـﺔ ، ﻫﻤـﺎ

ﺍﻟﻤﻴﻭﺴﻼﺝ ﻭ ﺍﻻﻨﻴﻭﻟﻴﻥ ﺒﺎﻤﺴﺘﺨﻼﺼﻬﻤﺎ ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﺴﻴﻠﻴﻡ ”Plantago ovata“ ﻭ ﻤﻥ ﺠﺫﻭﺭ ﺍﻟﺸﻜﻭﺭﻴﺎ

”Cichorium intybus“ﻓﻰ ﺍﻨﺘﺎﺝ ﺍﻟﺯﺒﺎﺩﻯ.

ﺃﺴﺘﺨﺩﻡ ﻤﺴﺘﺨﻠﺹ ﺍﻻﻨﻴﻭﻟﻴﻥ ﺒﻨﺴﺏ 4% ﻭ 6% ﻭ ﺍﻟﻤﻴﻭﺴﻼﺝ ﺒﻨـﺴﺒﺔ 0.2% ﻓـﻰ ﺘـﺼﻨ ﻴﻊ

ﺍﻟﺯﺒﺎﺩﻯ ﻤﻥ ﺒﺩﺭﺓ ﺍﻟﺤﻠﻴﺏ ﻤﻨﺯﻭﻉ ﺍﻟﺩﺴﻡ . ﺒﻌﺩ ﺍﻟﺘﺼﻨﻴﻊ ﻤﺒﺎﺸﺭﺓ ، ﺘﻡ ﺤﻔﻅ ﺍﻟﺯﺒﺎﺩﻯ ﻓﻰ ﺍﻟﺜﻼﺠﺔ ﻋﻠﻰ ﺩﺭﺠﺔ

ﺤﺭﺍﺭﺓ º 5±1 س ﻭ ﺍﺴﺘﻤﺭﺕ ﻤﺤﻔﻭﻅﺔ ﻋﻠﻰ ﻫﺫﻩ ﺍﻟﺤﺎﻟﺔ ﻟﻤﺩﺓ 10 ﺃﻴﺎﻡ.

ﺍﺸﺘﻤﻠﺕ ﺨﺼﺎﺌﺹ ﺍﻟﺯﺒﺎﺩﻯ ﺍﻟﻤﺩﺭﻭﺴﺔ ﻋﻠﻰ ﺍﻻﺱ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻰ ، ﻨﺴﺒﺔ ﺍﻟﺤﻤﻭﻀـﺔ ، ﺍﻟـﺸﺭﺵ

ﺍﻟﻤﻨﻔﺼل، ﺍﻟﺭﻤﺎﺩ، ﺍﻟﺒﺭﻭﺘﻴﻥ، ﺍﻟﻜﺎﺭﺒﻭﻫﻴﺩﺭﺕ، ﺍﻟﻤﻭﺍﺩ ﺍﻟﺼﻠﺒﺔ، ﺍﻻﺴﻴﺩﺍﻟﺩﻫﺎﻴﺩ ﻭ ﺜﻨﺎﺌﻰ ﺍﻻﻴﺜﺎﻴل.

ﺃﺩﺕ ﺇﻀﺎﻓﺔ ﺍﻻﻨﻴﻭﻟﻴﻥ ﺒﻨﺴﺒ ﺔ 4%، 6% ﻭ ﺍﻟﻤﻴﻭﺴﻼﺝ ﺒﻨﺴﺒ ﺔ 0.2% ﺍﻟﻰ ﺇﻨﺨﻔﺎﺽ ﻨﺴﺒﺔ ﺍﻟﺸﺭﺵ

ﺍﻟﻤﻨﻔﺼل ﺒ ـ 30%، 58% ﻭ50% ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻰ ﻤﻘﺎﺭﻨﺔ ﺒ ﺎﻟﻌﻴﻨﺔ ﺍﻟﻀﺎﺒﻁﺔ . ﻟﻡ ﺘﺅﺜﺭ ﺍﻟﻤﻌﺎﻤﻼﺕ ﺍﻟﻤﺨﺘﻠﻔ ﻪ

ﻟﻠﺯﺒﺎﺩﻯ ﻋﻠﻰ ﻨﺴﺒﺔ ﺍﻟﺭﻤﺎﺩ ﻤﻌﻨﻭﻴﺎ (p≥0.05). ﻜﺫﻟﻙ، ﻓ ﺄﻥ ﻫﺫﻩ ﺍﻟﻤﻌﺎﻤﻼ ﺕ ﻟﻡ ﺘﺅ ﺜﺭ ﻋﻠﻰ ﻨﺴﺒﻪ ﺍ ﻟﻼﻜﺘﻭﺯ،

ﻟﻜﻥ ﺘﻼﺤﻅ ﺍﻥ ﻨﺴﺒﺔ ﺍﻟﻼﻜﺘﻭﺯ ﻗﺩ ﺍﻨﺨﻔﻀﺕ ﻤﻊ ﺍﺯﺩﻴﺎﺩ ﻓﺘﺭﺓ ﺍﻟﺘﺨﺯﻴﻥ ﻓﻘﺩ ﻜﺎﻥ ﻫﻨﺎﻟﻙ ﺍﻨﺨﻔﺎﺽ ﻭﺍﻀﺢ ﻓﻰ

ﻨﺴﺒﺔ ﺍﻟﻼﻜﺘﻭﺯ ﻤﻥ 2% ﺍﻟﻰ 0.78% ﻓﻰ ﺍﻟﻴﻭﻡ ﺍﻟﻌﺎﺸﺭ ﻟﻠﺘﺨﺯﻴﻥ.

IX ﻓﻰ ﻨﺴﺒﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﺯﺒـﺎﺩﻯ ﺍﻟﻤﻌﺎﻤـل ﺒـ ـ p≤0.05) %6) ﻜﺎﻨﺕ ﻫﻨﺎﻟﻙ ﻓﺭﻭﻗﺎﺕ ﻤﻌﻨﻭﻴﺔ

ﺍﻨﻴﻭﻟﻴﻥ، 0.2% ﻤﻴﻭﺴﻴﻼﺝ ﻭ ﺍﻟﻌﻴﻨﺔ ﺍﻟﻀﺎﺒﻁﺔ ، ﺤﻴﺙ ﻜﺎﻨﺕ ﺍﻟﻘﻴﻡ ﺍﻟﻤﺘﺤﺼل ﻋﻠﻴﻬﺎ 4.56%، 4.37% ﻭ

4.19% ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻰ.

ﺍﻋﺘﻤﺎﺩﺍ ﻋﻠﻰ ﻨﺘﺎﺌﺞ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ، ﻓﻘﺩ ﻭﻀ ﺢ ﺍﻥ ﺍﻟﻤﻴﻭﺴﻴﻼﺝ ﻭﺍﻹﻨﻴـﻭﻟﻴﻥ ﻴﻤﻜـﻥ ﺇﺴـﺘﺨﺩﺍﻤﻬﻤﺎ

ﻜﻤﺜﺒﺘﺎﺕ ﻓﻰ ﺼﻨﺎﻋﺔ ﺍﻟﺯﺒﺎﺩﻯ.

X Chapter One INTRODUCTION

The growing awareness of the relationship between diet and health has led to an increased demand for food products that support health above and beyond providing basic nutrition. One of these products, yoghurt is made from milk. Yoghurt essentially has all the nutritive components of milk (Vedamuthu, 1993). The word yoghurt is derived from the Turkish word “jugurt” for milk fermented by a lactic culture. The product has many local names, it is known in Armenia as matzoon, in Lebanon and some Arab countries as leben, in Bulgaria as naja, in Italy as gioddu, in Iraq as roba, in Egypt and Sudan as zabady, in India as dahlia and busa in Turkistan (Tamime and Deeth, 1980). In Sudan the common name is zabadi (Dirrar, 1993). Yoghurt is a popular fermented milk product consumed in many parts of the world. It is produced in different forms such as whole milk yoghurt, skim milk yoghurt, cream milk yoghurt, fruit yoghurt and liquid yoghurt. No one knows exactly where or how yoghurt originated, but apparently when the goat was first domesticated in the very old days about 5000 B.C., her milk was stored on the ground in hot weather, upon staying for some time it naturally forms a curd. Southwest Asia still represents a key area for yoghurt production and consumption (Kosikowski, 1982). Yoghurt (zabadi) is not a truly indigenous food of the Sudan. The product is only known to urban populations. Until 1950s, zabadi was only known to the inhabitants of Khartoum and a few relatively large towns. It is believed that the art of zabadi making came to the Sudan from Egypt through

1 the Egyptians, Greeks, Syrians and Turks. Its introduction most likely took place with Anglo-Egyptian invasion and subsequent colonization (1898-1956), (Dirrar, 1993). Yoghurt is used by all family members as a daily meal or supplements particularly for children, old people and pregnant women. It is also highly used by people suffering from most of the gastrointestinal problems. In parallel the dairy industry is coping with these requirements in their forms to satisfy different consumer needs. People who don’t drink milk because they can not digest lactose; consume yoghurt which contains less lactose. Yoghurt is considered a healthy food because it contains viable bacteria. Yoghurt is one of the most, unique yet universal dairy products. The uniqueness of yoghurt is attributable to the symbiotic fermentation involved in its manufacture. Yoghurt may be defined as the solids, custard-like fermented milk product made from fortified high-solids milk using a symbiotic mixture of Streptococcus salaivarius subsp. Thermophilus and Lactobacillus delbrueckii subsp. bulgaricus as starters (Vedamuthu, 1993). The regulation specify that yoghurt before addition of bulky flavors contains not less than 3.25% milk fat and not less than 8.25% milk solids-non- fat (MSNF) and has a titratable acidity not less than 0.9%, expressed as lactic acid. Three categories of the product recognized are yoghurt, low fat yoghurt and non-fat yoghurt. A product to be labeled yoghurt should meet all the aforementioned criteria. However, it is difficult to provide a universal make-procedure for yoghurt to suit every variation in the final product marketed by the industry. And what ever procedure is used, it is essential that the final product conforms

2 to the requirements of the code of federal and state regulation, is safe for public consumption, has a satisfactory shelf-life and meets consumer demands with respect to body, texture and flavor characteristics. A major concern of the yoghurt industry is the production and maintenance of a product with optimum consistency and stability. The factors known to improve consistency are increasing total solids, manipulation of processing variables, and characteristics of starter culture, (Omer, 2003) Stabilizers are used to produce a thick, cohesive body, smooth texture and to prevent wheying-off. Use of stabilizers also insures a uniform product with respect to body and texture from batch to batch according to Vedamuthu (1993). This study investigates some stabilizers of plant origin. These stabilizers include ‘mucilage’ from psyllium ovata (Plantago ovata Forsk) and ‘inulin’ from chicory roots (Cichorium intybus L.). The objectives of this study are to test some natural products as stabilizers for yoghurt include inulin with a different ratio and mucilage. Furthermore, the study will assess the properties of the yoghurt after using these stabilizers. The assumption is that if the stabilizers under investigation are effective, it will hopefully add a relatively new yoghurt stabilizer to the already existing ones.

3 Chapter Two LITREATURE REVEIW

2.1 Milk and Milk Composition

Milk is a white creamy suspension secreted by all species of mammals to supply nutrition and immunological protection to their infants. In its processed form may be whole full fat, semi skimmed and low fat milk (Jenness, 1988). Milk is composed of water 87.3%, fat 3.9% and solid non-fat (snf) 8.8%. The snf contains: protein 3.23%, lactose 4.6%, minerals 0.05%, acids 0.18% and little amount of enzyme, gases and vitamins (Pyke, 1999). 2.1.1 Forms of Milk Products Milk products are manufactured from fluid milk by various methods. Fluid milks include all of the plain products, with fat contents varying from those of whole to skim milk as well as flavored and fermented milks (Bassette and Acosta, 1988). a. Whole milk: Most fluid milk is consumed in the form of pasteurized, homogenized and vitamin D fortified whole milk or just boiled milk. b. Low fat milk: Consumption of this type has increased substantially over the past decade to avoided milk fats that cause obesity and increased cholesterol levels that are related to heart diseases. c. Skimmed milk: Obtained after all or most of the milk fat is removed from the whole milk. d. Fermented and Acidified milk: Fermented milk is cultured dairy product manufactured from whole, partially skimmed or slightly concentrated milk.

4 2.2 Milk Fermentation

Fermentation defined by Kosikowski (1982), is a process leading to the anaerobic breakdown of carbohydrates, such as organic acids, proteins and fats are fermentable in the broader view that fermentation is an energy yielding oxidation-reduction process. Fellows (2000) reported, a large number of cultured milk products produced throughout the world, (for example: yoghurt, cheese, Kefir, Koumiss, butter milk, sour cream and Leben). Differences in flavour are due to differences in the concentration of lactic acids and acetyl methyl carbinol (diacetyl). The last is produced by fermentation of citrate in milk and gives the characteristic ‘buttery’ aroma to dairy products. Changes in texture are due to lactic acid, which causes a reduction in electrical charge on the casein micelles. They coagulate at the iso-electric point to form characteristic flocs. Modifications to the starter culture, incubation conditions and subsequent processing conditions are used to control the size and texture of the coagulate protein flocs and hence produce the many different textures encountered. Preservation is achieved by chilling and increased acidity (yoghurt and cultured milks). 2.2.1 Types of Fermentation The major sugar and citric acid reactions in milk include lactic acid fermentation propionic acid fermentation, citric acid fermentation, alcoholic fermentation and butyric acid fermentation (Kosikowski, 1982). 2.2.2 Fermented Milk Products Milk can be fermented by bacteria, yeast and mould to produce a variety of products such as cream and yoghurt, cheese, sour butter milk. Modification of milk by micro-organisms affects both the physical and chemical properties and the economic value of milk. The physical and chemical changes are tested

5 in such properties as flovour, texture and nutritive value. The economic value of milk is enhanced by the increased shelf life of the product (Kilara and Shahani, 1987). In some countries of central Europe, the Mediterranean basin area, Asia and Africa fermented milks are more important than fresh milk. They form the staple food of numerous meals and their popularity is increasing. Fermented milks originated in the Near East and spread central and Eastern Europe a characteristic common to all fermented milks is the presence of lactic acid (Kosikowski, 1982). 2.3 Yoghurt Yoghurt is a fermented milk product, which is produced by fermenting milk with lactic acid bacteria, which are responsible for development of typical yoghurt flavour (Dalanc ,2004). In the United State, the definition and regulations governing yoghurt are set by the Food and Drug Administration (FDA). According to FDA, yoghurt is the food produced by culturing the following, namely, cream, milk, partially skimmed milk or skim milk either alone or in combination with characterization bacterial culture that contains the lactic acid-producing bacteria, L. bulgaricus and S. thermophilus. Yoghurt is highly nutritious and easily digestible due to the predigested nutrients by bacterial starter, it is less perishable in view of its unused lactose content, which can be utilized for the growth of indescribable micro-organisms responsible for spoilage (Durgo, 1986). The cultures used in yoghurt processing are S.thermophillus and L. bulgaricus, the first one grows rapidly to produce diacetyl and lactic, acetic and formic acids. L. bulgaricus possesses weak protease activity which release peptides from the milk proteins.

6 These stimulate the growth of S. thermophillus. The increased acidity then slows the growth of S. thermophillus and promotes L. bulgaricus, which is stimulated by formate produced in initial stage. L. bulgaricus produces most of the lactic acid and also acetaldehyde which together with diacetyl gives the characteristic flavour and aroma in yoghurt (Fellows, 2000). Yoghurt can be tolerated by some people who are unable to digest dairy products due to loss of the enzyme lactase during adulthood; this enzyme converts lactose to lactic acid. In its absence or low quantity milk products will stay undigested in the intestine, causing bloat, abdominal cramps and diarrhea. As lactose is already converted to lactic acid it is more easily digested by people with lactose intolerance. 2.3.1 Composition of Yoghurt Yoghurt composition is similar to milk. However, there are many aspects in which the composition of yoghurt and milk differ. These differences are either from deliberate addition of solids to milk or yoghurt, or from changes brought about by bacterial fermentation. The percentage of protein is increased, thus yoghurt will almost invariably have higher protein content than milk. The major differences result from fermentation by the production of lactic acid from lactose. However, several other important changes also occur as shown on Table [2.1]. 2.3.2 Nutritional Value of Yoghurt: In many developing countries, yoghurt is produced as naturally soured milk, and makes an important contribution to the diet as source of protein and calories [Table 2.2] and some vitamins as shown in [Table 2.3].

8 Table 2.1: The chemical composition of natural and flavored yoghurt

Component Natural Flavored/Fruit/Nut Moisture % 86 73-79 Fat % 1.0 0.9-2.6 Protein % 5.0 4.8-5.2 Lactose % 4.6 3.2-4.8 Total Carbohydrate 6.2 14-18 % (Egan et al, 1981)

Table 2.2: Some typical values of the major constituents of milk and yoghurt (all units per 100g)

Milk Yoghurt YOGURT Low Low fat + Whole Skim Full fat fat fruit Water (g) 87.8 91.1 81.9 84.9 77.0 Energy value 66 33 79 56 90 (kcal) Protein (g) 3.2 3.3 5.7 5.1 4.1 Fat (g) 3.9 0.1 3.0 0.8 0.7 Carbohydrate(g) 4.8 5.0 7.8 7.5 17.9 Calcium (mg) 115 120 200 190 150 Phosphorus (mg) 92 95 170 160 120 Sodium (mg) 55 55 80 83 64 Potassium (mg) 140 150 280 250 210 Zinc (mg) 0.4 0.4 0.7 0.6 0.5 (Tammime and Robenson, 1999)

9 Table 2.3: Some typical vitamin contents of milk and yoghurt (all units per100g)

Milk Yoghurt YOGURT Full Low fat + Whole Skim Low fat fat fruit Retinol(µg) 52 1 28 8 10 Carotene(µg) 21 Tr 21 5 4 Thiamin (B1)(µg) 30 40 60 50 50 Riboflavin (B2)(µg) 170 170 270 250 210 Pyridoxine (B4)(µg) 60 60 100 90 80 Cyanocobalamine 0.4 0.4 0.2 0.2 0.2 (B12)(µg) Vitamin C (mg) 1 1 1 1 1 Vitamin D(µg) 0.03 TR 0.04 0.01 0.01 Vitamin E (µg) 90 TR 50 10 10 Folic acid (µg) 6 5 18 17 16 Nicotinic acid (µg) 100 100 200 100 100 Pantothenic acid 350 320 500 450 330 (µg) Biotin (µg) 1.9 1.9 2.6 2.9 2.3 Choline (mg) 12.1 4.8 - 0.6 - TR = trace. (Tammime and Robenson, 1999).

10 2.3.3 Types of Yoghurt The differentiation of the various types of yoghurt is made according to their chemical composition, method of production, flavor and nature of post- incubation processing. However, based on the method of production and physical structure of the coagulum, there are many types of yoghurt according to Tamime and Deeth (1980). a. Set Yoghurt: This type of yogurt is incubated and cooled in the final package and is characterized by a firm like jelly in texture. b. Stirred Yoghurt: This type of yoghurt is incubated in a tank and the final coagulum is broken by stirring prior to cooling and packaging. The texture of stirred yoghurt will be less firm than a set yoghurt what some like a very thick cream. c. Drinking Yoghurt: This type of yoghurt is very similar to stirred yoghurt but the coagulum is severely broken. Little if any reformation of the coagulum appearing after packing is known. d. Frozen Yoghurt: is inoculated in the same manner as stirred yoghurt. However cooling is achieved by pumping through a whippier/ chiller/ freezer and the size and distribution of the ice crystals produced. e. Concentrated Yoghurt: this type of yoghurt is inoculated and fermented in the same manner as stirred yoghurt. Following the break of the coagulum the yoghurt is concentrated by boiling off some of the water, this is often done under vacuum to reduce the temperature required. Heating of low pH yoghurt can often lead to protein being denatured and producing rough and gritty textures. This is often called strained yoghurt due to the fact that the liquid that is released from the coagulum upon heating used to be strained off in a manner similar to making soft cheese.

11 f. Flavored Yoghurt: yoghurt with various flavours and aromas has become very popular. The flavours are usually added at or just prior to filling into pots. Common additives are fruits usually as a puree or as whole fruit in syrup. These additives often have as much as 50% sugar in them; many manufactures offer a low sugar and low fat version of their products. Low or no sugar yoghurts are often sweetened with saccharin or more commonly aspartame. The use of fruit sugar in the form of concentrated apple juice is sometimes found as away of avoiding added sugar to the ingredients. The declaration of this tends to be marketing policy and has no real added benefit. There are other types like acidophilus yoghurt, liquid yoghurt and yoghurt cheese (Kosikowki ,1982). 2.3.4 Manufacture of Yoghurt Manufacturing consists of five major steps according to (Vedamuthu, 1993), namely: 2.3.4.1 Preparation of yoghurt mix: a. Ingredients: quality of the ingredients used decides the quality of the final product. All the ingredients used should be of the highest quality. Yoghurt is generally made from whole, partially skimmed milk, condensed skim milk, cream and nonfat dry milk. Alternatively, milk may be partly concentrated by removal of 15-20% water in a vacuum pan. Supplementation of milk solids-not-fat with nonfat dry milk is the preferred industrial procedure. The most commonly used sweetener in yoghurt is table sugar or sucrose. Sucrose may be used in crystalline form or as concentrated syrup. Corn sugar (dextrose) and, in some cases, honey and high fructose corn syrup are also used. For dietary yoghurt, non-nutritive sweeteners like saccharin, aspartame etc., may be used.

12 b. Mixing and Homogenization: Depending upon the category of yoghurt manufactured like regular yoghurt, low fat yoghurt or non-fat yoghurt- fresh milk is standardized with respect to milk fat. The fortified milk is further warmed up to 63º C and the mix is homogenized at 3000 psi single state. Homogenization is a very important step. Homogenization breaks fat globules of varying sizes normally occurring in milk into small, uniformly-sized and evenly distributed particles. It prevents fat separation. Additionally, homogenization may aid in the dissolution and uniform distribution of powdered ingredients by breaking up any residual granules or grains. The end result is a smooth, creamy, rich product. c. Heat Treatment: After homogenization, the mix is transferred to a double- walled stainless steel vat designed for pressurized heating. The mix is further heated to 82º C and held at that temperature for 30 min. the high heat-treatment of yoghurt mix accomplishes the following: i. Destroys unwanted pathogenic and spoilage microorganisms, natural enzymes and heat labile inhibitors in the raw ingredient mix. ii. Heat-treatment liberates certain stimulatory compounds which promote the growth of yoghurt starter bacteria. iii. Heat-treatment at temperatures ranging from 82º-87º C for 30 min. that is usually used for yoghurt mixes results in the interaction of whey protein with k-casein to form a complex. When casein precipitates (forms a coagulum) as the pH falls down to 4.6 (isoelectric point of casein), the complexed whey protein also coagulates. This result in a thicker, heavier body in the yoghurt. d. Setting: Is the term used for procedures involved in preparing the mix for the starter followed by the addition and uniform mixing of the starter into

13 the mix and adjustment of condition for the actual fermentation of mix, setting involves the following: e. Adjustment of the mix temperature to the desired point at which the starter can be added. Usually the set temperature will range from as low as 35º C as high as 115º C. f. After adjusting the temperature to the desired point, the starter is added. Bulk starter or concentrated direct-vat-set starter may be used and should be mixed uniformly. Over-agitation should be avoided to prevent excessive air incorporation. g. Once the starter is mixed in, agitation should be stopped and temperature control is set to the desired point and the fermentation should be allowed to proceed in quiescent state. 2.3.4.2 Fermentation: Fermentation as applied to yoghurt, is that phase in its manufacture during which the microorganisms added in the form of starter to the prepared mix, convert part of the lactose or other sugars in the mix to lactic acid and trace metabolites (acetaldehyde, acetic acid, propionic acid, formic acid, diacetyl, etc.) and transform the mix to an acid coagulum which has a tart, typical green acetaldehyde flavor. Fermentation then, is the heart of yoghurt manufacture. 2.3.4.3 Breaking and cooling: Once the desired pH is reached usually between (4.3-4.5), the fermentation should be arrested to prevent excessive acid accumulation. This achieved by processes that would allow rapid cooling of the curd mass. The pH at breaking would depend upon the size of the fermentation vat and the rate at which the product could be cooled to <7º C. If a long time is needed to cool down the product, the pH at breaking should be higher. The same would apply

14 if the product has to be held for any length of time before filling. According to some European processors, the curd structure appears to undergo minimum damage when handled between 21º-24º C, and firmness is regained when the filled cartons are chilled in the cooler to <7º C. Flavor may be added, if needed during cooling. This is done for flavored yoghurt without fruit like vanilla and also for Swiss style yoghurt. 2.3.4.4 Addition of fruits and filling: Fruit may be distributed in the body of the yoghurt through an in-line fruit-feeder or may be mixed in, in a blending tank. For fruit-in-the bottom or fruit-on-the top Sundae style yoghurt, metered portions of the fruit are added at filling ports situated before or after the main fillers. Pneumatic or gravity fillers are recommended for yoghurt. The filling area should be protected from air drafts and unnecessary traffic. After filling, the containers should be transferred to a walk-in cooler with good air circulation. The product should be held in the cooler for at least a day for proper “knitting” and gelling to develop the desired body, texture and mouth-feel. 2.3.5 Nutritional Benefits of Yoghurt a. According to Vedamuth (1993), Yoghurt has all the nutritive components of milk, namely: casein, lactoalbumin, lactoglobulin and other minor nitrogenous components which are nutritionally high quality proteins. b. Cow’s milk, buffalo’s milk and whole or partially skim milk are used for conversion to yoghurt, this fermented product is a good source of vitamin A. c. Yoghurt is a suitable dairy product for persons who suffer from slight or moderate lactose intolerance.

15 d. According to Dirrar (1993), yoghurt (zabadi) is useful for the treatment of stomach disturbances and the individual with such complains are advised to use zabadi. 2.3.6 Therapeutic Properties of Yoghurt: The action of lactic acid in inhibiting the growth/metabolism of the putrefactive bacteria could be one such process. Whether acid in yoghurt can survive the neutralizing effect of the bile components is open to debate, but the fact remains that yoghurt could change slightly, the pH gradient within the intestine. If this change does occur, then there could be a basis of truth in the traditional belief of Bulgaria, (Tamime and Robinson, 1999). A hypocholestrolaemic action has been attributed to yoghurt, the exact reason for this effect is not clear, but the fact that yoghurt is more active in this respect than unfermented milk implies that some enzyme system or biochemical compound of bacterial origin may well be involved. Hydroxymethyl glutarate has been proposed as one metabolic of starter cultures that could limit cholesterol synthesis. The study with rats and mice found the consumption of yoghurt, live or pasteurized, inhibited the growth of certain types of tumor. Whether such results can be interpreted as applicable to humans is another matter, but it is a possible benefit of yoghurt consumption that cannot be dismissed. Stimulation of the normal microflora of the gut has been attributed to the regular consumption of yoghurt and is proposed that the lysing cells of the starter bacteria release vitamins or other growth factors that encourage the development of L.acidophilus for the example in the small intestine (Tamime and Robinson, 1999).

16 2.4 Stabilizers Stabilizers are added substances to yoghurt so as to keep the physical structure of yoghurt and give it shape. Stabilizers are used during the manufacture of some dairy products, but in yoghurt making only stabilizers are added to the milk base. The stabilizers are hydrophilic colloids that bind water and consequently increase the viscosity of yoghurt; they also help prevent the separation of whey from the yoghurt, a problem known as syneresis. The most beneficial quantity of a stabilizer to add to yoghurt mix has to be determined experimentally by each manufacturer. Problems related to stabilizer usage are usually attributable to under- stabilization or over-stabilization and improper use of stabilizers. Under- stabilization results in a weak body and separation of whey. Over-stabilization on the other hand, produces a slick, rubbery or “jello-like” spring body. Improper usage consists of application of wrong combination of stabilizers and lack of care in dissolving stabilizers. Too fast addition of stabilizer or adding stabilizer at improper temperature may cause lumping. Adding stabilizer to too hot a mix will cause “case-hardening” or the formation of a thick, leathery pellicle over stabilizer granules, which do not dissolve or hydrate. This results in a grainy texture in the finished product. There are many types of stabilizers which can be added to the yoghurt like Gelatin, Alginates, Carbo gum, Guar gum, Starch and Carboxy methyl cellulose, shows in [Table 2.4], according to Vedamuthu (1993). 2.4.1 Plantago ovata Plantago ovata is known in commerce as Blond psyllium or Indian Plantago. Seeds have been used in Europe as a domestic remedy since the 16th Century, but only since 1930 have they been extensively used in the USA as a popular remedy for constipation (Rizk, 1986).

17 Commercially, the most important plant product is the husk of the seed of P.ovata, which is produced in north-western India and further purified and processed in the USA. The seeds contain a holoside (identified as planteose, consisting of galactose, glucose and fructose; “6-(∝-D-galatopyranosyl)-B-D-fructofuranosyl-∝-D- glucopyranoside”, aucubin, sterols and mucilage’s). The mucilage’s obtained from the seeds remained in a gel form a not as a sol, a matter which made them useful as a laxative (Rizk, 1986 and Osman, 2000). The mucilage (extracted by cold water) has a higher uronic acid content and lower pentosan content than the polysaccharide isolated by extracting the residue with hot water. The sugar constituents of the mucilage are: D-xylose, L-arabinose, L-rhamnose and D-galacturonic acid (Rizk, 1986). The seed coat shows mainly linoleic, oleic and palmitic acids in decreasing concentrations, while the seed itself doesn’t contain any appreciable amounts of fatty acids (Rizk, 1986). 2.4.1.1 Classification: Plantago ovata Forsk classified according to (Psyllium/wiki, 2008). Kingdom: Plantae Subkingdom: Tracheobionta Super division: Spermatophyta Division: Magnoliophyta Class: Magnoliopsida Subclass: Asteridae Order: Plantaginales Family: Plantaginaceae Genus: Plantago L.

18 Table 2.4: Stabilizers commonly used in yoghurt and their properties:

Stabilizer Source Advantages(a)/Disadvantages(d) (a) Good stabilizer for yoghurt and frozen yoghurt. Gives a Hydrolysis of meat Gelatin smooth product. Dissolves well protein collagen between 55º C and 65º C. (d) Degrades at high temperatures. (a) Gives a smooth product. Heat stable. Complex with calcium and Alginates Sea weeds casein to give a good gel. Dissolves at room temperature. (a)Similar gelling mechanism as Carrageenan Sea weeds alginates. Dissolves between 50º C and 80º C. Carbo gum Seeds and a legume (a) Effective at low pH. (a) Good stabilizer. Stable at high Guar gum Seeds temperature. Soluble in the cold water. Starch Cereals (a) Good in combinations. Carboxymethyl (a) Effective for high temperature Cellulose cellulose processing. (Vedamuthu ,1993).

19 2.4.1.2 Botany and Distribution: This family is widely distributed in temperate regions and mountain in the tropics and possessing the following diagnostic features: plants are herbaceous, but some have persistent woody stems. The entire or variously divided leaves are alternate, in base rosettes or rarely opposite with basal sheath. Flowers are small usually bisexual and wind-pollinated, few or many in axillaries spike. Stamens are four and alternating with the corolla lobes. Anthers are versatile, dehiscing inwards. Ovary is superior of two fused carpils and two locules with one to several ovules an axile placenta in each locule. Fruits are a membranous circumscissile capsule or one seeded bony nut. Seeds, often sticky when wet, has a fleshy endosperm, in the middle of which is straight (Osman, 2000). 2.4.1.3 Plantago ovata Forsk: An annual, acquiescent herb, the stem of which is much ramified and bears linear or lanceolate leaves, dentate, and pubescent. The flowers are white and grouped into cylindrical spikes. The sepals are characterized by a distinct midrib extending from the base to the summit; the petal lobes are oval with mucronate summit. The seeds are oval and clearly carinate, grey-pink with brown line running along their convex surface (Osman, 2000). 2.4.1.4 Phenotypic Description of the Seed: The British Pharmaceutical Codex (BPC) described the seed as follows: it is about 1-3.5 mm long, and 1-1.75 mm wide. The Ovate in outline and boat- shaped, hard and pinkish-buff, with a reddish-brown oval spot and about one- quarter of the length of the seed in the center of the convex surface. Occasional seeds are uniformly reddish-brown in color. Hilum appearing as a brown spot in the center of the concave surface, which is more or less, covered with a whitish membrane having two perforations. Endosperm is hard because the embryo straight almost as long as the seed lying near the convex surface and

20 having two cotyledons with their contiguous flattened upper surface. In the median plane; epidermis mucilaginous and swelling in water to form a translucent colorless envelope of mucilage and it is free from taste and odor (Osman, 2000). 2.4.1.5 Uses of Plantago ovata: The seeds of p.ovata (Ispaghula) are useful in the treatment of chronic constipation (Rizk, 1986 and Osman, 2000). And due to their large content of mucilage can be used in treatment of bacillary dysentery and chronic diarrhea (Rizk, 1986). The fixed oil has been used in the form of sodium psylliate injection as a sclerosing agent in the treatment of varicose veins and internal hemorrhoids that are not prolapsed or for treatment of thrombosis. The branched-chain pentose containing polysaccharide is a good dietary fiber for use in diabetes (Rizk, 1986). The husks have the characteristics to act as an ice cream stabilizer and that they can be developed as a suitable economical substitute for sodium alginate (Rizk, 1986). The work of Sahay (2004) suggests that, the husk can be used as an alternative gelling agent for microbial culture. 2.4.2 Cichorium intybus (Chicory) Chicory (Cichorium intybus) is a root that has been known for its curative benefits since the first century A.D. Chicory, itself is a member of Asteracea family, which also includes artichokes, calendula, dandelions, burdock and Jerusalem artichokes. It belongs to the genus Cichorium. There are a large number of plants found in this genus. They include Belgian endive, culinary endive, escarole, and radicchio. Each of these plants differs considerably in appearance, color and to some extent, flavor, although they all

21 share a slightly bitter taste. Most of the members of this family produce a root that can be roasted, ground and combined with other roots to make a coffee substitute (Chicory-root, 2008). Inulin is an actually a carbohydrate found in large quantities in the chicory root. It is made up of many fructose chains (3 to 60 units) and terminates in one glucose molecule. Inulin can be extracted from the root in a pure form and utilized as a food ingredient or the roots can be sliced and dried and mixed with other ingredients and utilized in foods. Inulin belongs to a group of naturally-occurring carbohydrates containing non-digestible fructooligosaccharides. Inulin and fructooligosaccharides (FOS) are widely distributed throughout the plant kingdom. They have been increasingly used in various foods due to their beneficial nutritional attributes. The nutrition industry refers to them as FOS. Inulin is a plant starch (Inulin, 2008). Inulin occurs naturally in large quantities in some of the most famous herbs, such as burdock root, dandelion root, elecampane root and of course, chicory root. It is soluble only in hot water, which is why it has traditionally been consumed in hot teas, from (Chicory-root, 2008). 2.4.2.1 Classification: Chicorium intybus L. classified according to (chiory/wiki, 2008). Kingdom: Plantae. Division: Magnoliophyta. Class: Magnoliopsida. Order: Asterales. Family: Asteraceae. Tribe: Cichorieae. Genus: Chicorium.

22 23 2.4.2.2 Botany Description: Chicory is a perennial plant indigenous to Europe, India and Egypt. It was introduced to the US in the late 19th century. It grows as a weed in temperate climates and is widely cultivated in northern Europe. There are two principal types: the Brunswick variety has deeply cut leaves and generally spreads horizontally; the Magdeburg variety has undivided leaves and grows erect. Chicory has bright blue flowers that bloom from July to September. The dried root is the primary part of the plant used. 2.4.2.3 Uses of Chicorium intybus: The roots are used as a tonic, laxative and diuretic (Grieve, 1998) and to treat skin eruptions and fevers (Foster and Duke, 1990). Root decoctions were once taken to mitigate jaundice, gout, rheumatic complaints (Le Strange, 1977). Chicory leaves and roots are used as a vegetable. Roasted roots are ground and brewed. Chicory is a sedative with potential cardioactive properties. Chicory’s oligosaccharides are probiotic and are beneficial in maintaining healthy gastro intestinal flora. Inulin type fractions of the plant may help certain conditions including constipation, diarrhea, cancer and cardiovascular disease. Chicory has also been noted as an appetite stimulant and for dyspepsia (Chicory, 2008). The ancient Egyptians ate large amounts of chicory because it was believed that the plant could purify the blood and liver. This “purifying effect” is probably why it has been used beneficially with people who have arthritis, rheumatism and gout. Chicory supports the body's ability to absorb calcium, a nutrient that helps build and maintain strong teeth and bones. Raftilin inulin and raftilose oligofructose are fibers extracted from chicory root inulin that cannot be digested by the small intestine. Instead, they are fermented by “friendly”

24 bacteria in the large intestine, leading to the increased absorption of calcium and other minerals. The ingestion of moderate amounts of inulin actually acts as a prebiotic that nourishes the beneficial bacteria in both human and animal digestive systems which, in turn, promotes a healthy digestive tract and improves overall health. The major friendly floras that are fed by inulin are bifidobacterium. The increased growth of “good” healthy bifidobacterium inhibits the growth of pathogenic bacteria, from (Chicory-root, 2008). In addition to its beneficial effects on health, as a dietetic fibre and as a prebiotic ingredient, inulin shows interesting technological properties, as a low-calorie sweetener, as a fat substitute, or it can be used to modify texture. Its properties as a fat substitute are attributed to its capacity to form microcrystals that interact with each other forming small aggregates, which occlude a great amount of water, creating a fine and creamy texture that provides a mouth sensation similar to that of fat. The combined consideration of its nutritional and technological characteristics makes inulin a very attractive ingredient. In most cases, its addition to different foods has been done in order to increase fiber ingestion, in amounts that ranges from 3 to 6 g per portion, or to assure its bifidogenic nature, by adding 3–8 g per portion. Despite the great number of commercial products including it in their formulation, there is still little information available referring to its physical properties and to the effects produced by its incorporation on the physical characteristics of different types of foods. Some authors have analysed the effect of adding inulin on the rheological and sensorial characteristics of several dairy products, like ice-creams, yoghurts, fresh cheese and dairy desserts (Villegas and Costell, 2007).

25 26 Chapter Three MATERIALS AND METHODS

3.1 Materials Skim milk powder, chicory roots and yoghurt culture were obtained from the National Research Center, Cairo, Egypt. Psyllium ovata seeds obtained from University of Khartoum, Faculty of Agriculture, Shambat, Sudan. 3.1.1 Preparation of the Mucilage: Mucilage content of the seed was determined following the procedure of Khanna et al (1987). Fifty grams of seeds were suspended in 200 ml distilled water resulting in the formation of viscous mucilaginous suspension which was treated with 25 ml of 10% NaOH (aqueous) at room temperature for 5 minutes. The liquefied mucilage was filtrated through muslin cloth and filtrate was acidified with 5N HCl to pH 2.0. At this stage original high viscosity of mucilage returned with simultaneous precipitation of total mucilage. The precipitated mucilage was washed four times with distilled water using one liter each time, two times with 50ml methanol and one time with acetone. The washed mucilage was dried at 80º C in oven and percentage was calculated with relevance to the air-dried sample. 3.1.2 Preparation of the Inulin: Ten grams of chicory roots were washed with distilled water and heated in 200ml distilled water for up to 70 ºC and left to cool overnight in refrigerator at 5º C to for maximum extraction. The mixture was filtrated by muslin cloth and the filtrate is known as inulin.

27 28 3.1.3 Yoghurt Manufacturing: Yoghurt samples were made by adding 15 grams of skim milk powder per 100 ml de-ionized water. The yoghurt manufacturing was carried out using the method described by Tamime and Robinson (1999). The reconstituted milk was pasteurized in a water bath for an average of 15 min at 85 ºC. It is then cooled to 45 ºC. After cooling the inulin (4% and 6%) and the mucilage (0.2%) were added as stabilizers. The strains culture of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were inoculated at 3% and incubated at 45 ºC for 4-6 hrs. At the end of the incubation period, yoghurt was kept in a refrigerator at 5 ºC along with yoghurt manufactured without using stabilizers (control). 3.2 Methods of Analysis 3.2.1 pH Measurement: Hydrogen ion concentration (pH) was measured using laboratory pH meter with a glass electrode according to AOAC (2000). 3.2.2 Titratable Acidity Determination: Titratable acidity was estimated according to AOAC (2000). Ten milliliters of the yoghurt were placed in a conical flask and titrated with 0.1 N NaOH solution using phenolphthalein as indicator. Results were expressed as percent of lactic acid. 3.2.3 Wheying off Determination: Wheying off is made by a measuring cylinder taking the water separated from the set yoghurt. It was measured by sucking the water on the surface of the curd and pouring it in the cylinder according to AOAC (2000). 3.2.4 Ash Content Determination: Exactly 2.5 g of the sample (yoghurt) weighted in a silica crucible of constant weight. Ashing was first carried out on a weak flame furnace and then

29 completed in a muffle furnace at 650 ºC for 6 hr. the residue was weighted and calculated as the ash content of the sample according to AOAC (2000). 3.2.5 Total Nitrogen Determination: The total nitrogen was determined by the macro Kjeldahl method according to

AOAC (2000) except that NH3 in the steam distillate was received in 40 ml of 2% boric acid solution containing five drops of mixed indicator (10 ml of 0.1 % bromocresol green in 95% alcohol mixed with 2 ml of 0.1 methyl red in 95% alcohol) and then titrated with 0.1 N HCl solution. 3.2.6 Total Carbohydrate Determination: The method of Taylor (1995) was used as follows: Reagents:

1. Sulphuric Acid: 27 ml of concentrate H2SO4 (sp.gr 1.84) was diluted to 1 liter with distilled water.

2. 5% phenol solution: phenol solution was distilled and 5 ml of the distilled phenol were diluted with distilled water and made up to 100 ml.

3. Standard glucose solution: 0.05g of glucose (A.R) was dissolved and made up to 1 L with distilled water. The solution 0.5 mg glucose /1 ml. Procedure: Twenty milligrams of yoghurt were mixed with 10 ml of diluted sulphuric acid in a stoppered test tube and the tube was placed in an oven at 100º C for 4 hrs. The tube contents were transferred to 100 volumetric flasks and made up to volume with distilled water. One milliliter was mixed with 1 ml of 5 % phenol solution and 5 ml of concentrated sulphuric acid were added and left to cool for 10-15 min at room temperature and the optical density was measured at 490 nm wave length. The concentration of carbohydrate was calculated and expressed as glucose percentage from a standard curve of glucose ranging from 0.1 mg- 1 mg glucose.

30 3.2.7 Lactose Content Determination: Lactose content was determined according to Michel et al (1956) as follows:- One gram of the yoghurt sample was accurately weighed in a 500 ml volumetric flask. Twenty five ml of distilled water were added, followed by 2ml 1 N NaOH and mixed well until the sample was completely dissolved. The flask contents were made up to volume with distilled water and mixed thoroughly. One ml of the sample solution was pipetted into a stoppered test tube, followed by 1 ml of 5% phenol and mixed well. Five millitres, of concentrated sulphuric acid were added to the tube contents with care, mixed well and left to cool at room temperature. The optical density was measured at 490 nm wave length using a spectrophotmeter (UV - 240 Shimadzeau, Japan) and 1 cm glass cell. The lactose content of the sample was determined from a standard curve of lactose ranging from 10 - 100 µg / ml. 3.2.8 Total Solids Determination: The determination of the total solids content of yoghurt was made by following procedure according to AOAC (2000): Clean dried aluminum dish was weighed initially. Five ml of the yoghurt mix was added to the dish. The dish was placed in a drying oven for 2 hrs. at 105 ºC. The dish was then put in a disscator for half an hour and weighed. The dish was brought back to the oven for one hour. The sample was put again in the disscator and weighed; the process was repeated until a constant weight was obtained. Total solids content was calculated as follows: Total solids content = M2 – M0 ×100 M1 – M0

31 Where: M0 ≡ weight of empty dish in gram before drying. M1 ≡ empty dish weight + sample. M2 ≡ empty dish weight + sample after drying. (Round the value obtained to the nearest decimal). 3.2.9 Acetaldehyde Concentration Determination: Acetaldehyde was estimated as described by Lees and Jago (1969) using Conway micro diffusion semicarbazide method. Acetaldehyde reacts with semicarbazide to form semicarbazone which has maximum absorption at 224 nm. Procedure: One ml of 1 micromole semicarbazide solution was pipette in the inner wall of Conway micro diffusion cell. Three millimeters of yoghurt were rapidly pipetted in the outer compartment and the cell was covered and placed in an incubator at 30 ºc for 90 min. the solution in the inner wall was transferred to 10 ml volumetric flask and made up to volume. The absorption was measured at 224 nm using Schimadzu model 240 spectrophotometer. The concentration of acetaldehyde was calculated from standard curve of acetaldehyde solution ranging from 1µ mol to 20 µ mol/100 ml mixed with yoghurt and treated as samples 1µ mol. 3.2.10 Diacetyl Concentration Determination: The previous procedure was also utilized to determine diacetyle content with the exception of measuring the absorption at 270nm. using the same spectrophotometer, according to the method described by Lees and Jago (1970).

32 3.2.11 Statistical Analysis: The data collected from the different treatments were subjected to analysis of variance and whenever appropriate the mean separation procedure of Duncan was employed (Steel and Torrie, 1980). The SAS programme (SAS, 1988) was used to perform the general linear model (GLM) analysis.

33 Chapter Four RESULTS AND DISCUSSIONS

4.1 pH Values: The effect of the different stabilizers and storage periods on the pH value of set yoghurt is shown in Table [4.5]. Regardless of the treatments the pH of yoghurt showed significant changes (p≤0.05) with the increase in the storage period. At any one treatment, the pH at day zero was significantly (p≤0.05) higher when compared to the pH of the samples stored for 3, 7 and 10 days. On the other hand the pH value of samples stored for 3, 7 and 10 days were similar (p≤0.05). The changes in the pH value differ according to the different stabilizers used. Regardless of the storage period, the lowest pH value (p≤0.05) was reported for the control (without stabilizer) while that of treated got the highest pH value. Within inulin treatments, yoghurt treated with inulin 4% had similar pH value (p≤0.05) to that treated with inulin 6%. Within the stabilizers treatments i.e inulin 4%, inulin 6% and mucilage 0.2%, all the treatments showed similar pH values (p≥0.05). 4.2 Titratable Acidity: Titratable acidity (TA) values expressed as percentage of lactic acid is listed in Table [4.6]. Titratable acidity was significantly higher (p≤0.05) for samples stored for 10 days than that at the beginning of storage period (day zero). Generally within each stabilizer treatment TA increased significantly with the increases in storage period. A progressive increase in titratable acidity

34 with storage period could be noticed. The rate of increase varies from one stabilizer to the other, such results agree with the findings of Jogdand et al (1991). When the storage period was disregarded, the control yoghurt (no stabilizer added) had significantly lower TA values (p≤0.05). Yoghurt stabilized with 0.27% mucilage had higher TA values than the control yoghurt. When the effect of adding 4% or 6% inulin was compared with mucilage, the difference in titratable acidity value decreased with the inulin treatment. Titratable acidity of yoghurt treated with inulin 6% was significantly higher than that produced with inulin 4% however still less than that of mucilage 0.2%. When stabilizers treatments were disregarded, the TA increased by 11%, 18%, 26% at 3, 7 and 10 days respectively when compared with that at day zero. 4.3 Wheying off: Wheying off of the set yoghurt was expressed as volume per ml of whey water separated from yoghurt during the storage period is shown in Table [4.7]. Wheying off was significantly higher for the control samples and lower for inulin 6% (p≤0.05). All stabilizers investigated in this study, namely inulin and mucilage had marked effects on the wheying off of the set yoghurt. Apparently mucilage was much effective in reducing wheying off than inulin 4%, however both of them resulted in substantial reduction i.e. 50% and 33% respectively. Within inulin treatments, inulin 6% was more effective than inulin 4% in reducing the wheying off of set yoghurt. On the other hand inulin 6% was more effective than mucilage 0.27% in reducing wheying off of set yoghurt, a reduction of 58% and 50% could be observed respectively for the two treatments. Regardless of the treatments, the highest whey separated is found to be at day 10(p≤0.05), while no difference was observed for storage periods 0, 3 and 7.

35 36

Table 4.5: Effect of stabilizers at different levels of treatment and the storage period on the pH of the yoghurt Storage Period/ Day Treatment YOGURT 0 3 7 10 mean a b c d B Control 4.27 ±0.05 4.23 ±0.02 4.21 ±0.03 4.14 ±0.03 4.21 ±0.06 a b c d A Inulin 4% 4.43 ±0.03 4.37 ±0.07 4.23 ±0.04 4.20 ±0.04 4.31 ±0.11 a b c d A Inulin 6% 4.38 ±0.03 4.30 ±0.02 4.32 ±0.05 4.24 ±0.04 4.31 ±0.06 a b c d A Mucilage 0.2% 4.48 ±0.05 4.32 ±0.06 4.24 ±0.04 4.17 ±0.02 4.30 ±0.12 Storage Time 4.39A±0.09 4.31B±0.06 4.25C±0.05 4.19D±0.05 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Table 4.6: Changes in titratable acidity of yoghurt treated with different stabilizers stored for up to 10 days

Storage Period/ Day Treatment YOGURT 0 3 7 10 mean Control 0.97d±0.07 1.04c±0.02 1.12b±0.02 1.21a±0.02 1.09D±0.10 Inulin 4% 0.98d±0.02 1.43a±0.01 1.21c±0.00 1.27b±0.01 1.15B±0.11 Inulin 6% 1.07d±0.02 1.09c±0.01 1.13b±0.01 1.24a±0.01 1.13C±0.07 Mucilage 0.2% 0.99d±0.02 1.15c±0.01 1.25b±0.01 1.31a±0.01 1.17A±0.13 Storage Time 1.00D±0.05 1.11C±0.05 1.18B±0.06 1.26A±0.04 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Table 4.7: Effect of stabilizers treatment and storage period on the wheying-off of yoghurt

Storage Period/Day Treatment YOGURT 0 3 7 10 mean Control 0.17d±0.06 0.27b±0.11 0.23c±0.06 0.37a±0.06 0.26A±0.09 Inulin 4% 0.13c±0.06 0.17b±0.06 0.17b±0.06 0.23a±0.06 0.18B±0.06 Inulin 6% 0.13a±0.06 0.10b±0.00 0.10b±0.00 0.10b±0.00 0.11D±0.03 Mucilage 0.2% 0.17a±0.06 0.13b±0.06 0.10c±0.00 0.10c±0.00 0.13C±0.05 Storage Time 0.15B±0.05 0.17AB±0.09 0.15B±0.07 0.20A±0.12 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

39 No literature was sited to approve or disapprove these results. However, on the consumer point of view when ever the less wheying off the better yoghurt. 4.4 Ash Content: Table [4.8] shows the results of the ash analysis. Irrespective of the storage periods the ash content of all yoghurt samples were similar, showing no differences (p≤0.05). These results agree well with the findings of Tamime and Robinson (1999). Irrespective of the treatment, the ash content of yoghurt showed slight (p≥0.05) changes with the increase in storage period. 4.5 Total Protein Content: The protein content of yoghurt with or without stabilizer is shown in Table [4.9]. Irrespective of the treatments, the protein content was found to be similar (p≥0.05) through out storage periods tested. Regardless of the storage period, the different treatments showed significant differences (p≤0.05), the highest protein content was found for inulin 6% followed by mucilage and the lowest protein content was reported for inulin 4% and the control. Generally the results obtained agree well with the finding of Tamime and Robinson (1999). When the treatments were disregarded, the protein content of yoghurt was not effected (p≥0.05) by the storage period. 4.6 Total Carbohydrates Content: The effect of stabilizers and storage period on the carbohydrate content of yoghurt is shown in Table [4.10]. The content of carbohydrate at different storage periods showed significant differences, the highest (p≤0.05) content was reported for day 10 and the lowest value decreased for day 0. Irrespective of the storage periods, the highest (p≤0.05) carbohydrate content was observed

40 for mucilage 0.2%. It had about 4, 2.5 and 2 higher carbohydrate compared to the control, inulin 4% and inulin 6% respectively.

Table 4.8: Effect of stabilizers treatment and storage period on ash content

Storage Period/Day Treatment YOGURT 0 3 7 10 mean Control 0.98 a±0.00 0.97b±0.01 0.98a±0.02 0.98a±0.01 0.98A±0.01 Inulin 4% 0.98b±0.01 0.98b±0.01 0.99a±0.02 0.97c±0.01 0.98A±0.01 Inulin 6% 0.98b±0.01 0.97c±0.02 0.99a±0.02 0.98b±0.01 0.98A±0.01 Mucilage 0.2% 0.98a±0.01 0.97b±0.01 0.98a±0.02 0.98 a±0.01 0.98A±0.01 Storage Time 0.98A±0.01 0.97B±0.01 0.98A±0.02 0.98A±0.01 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Table 4.9: Protein content (%) of yoghurt treated with different stabilizers and stored for up to 10 days

Storage Period/ Day Treatment YOGURT Zero 3 7 10 mean Control 4.26a±0.04 4.26a±0.01 4.26a±0.02 3.97b±0.61 4.19C±0.29 Inulin 4% 4.09c±0.03 4.13b±0.02 4.16a±0.01 4.17a±0.01 4.14C±0.04 Inulin 6% 4.55c±0.02 4.57a±0.02 4.56b±0.02 4.57a±0.02 4.56A±0.02 Mucilage 4.28d±0.02 4.35c±0.03 4.41b±0.03 4.43a±0.02 4.37B±0.06 0.2% Storage 4.30A±0.17 4.33A±0.17 4.35A±0.16 4.29A±0.36 period mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Table 4.10: Effect of stabilizers and storage period on yoghurt carbohydrate content (mg/ml) stored for up to 10 days Storage Period/ Day Treatment YOGURT 0 3 7 10 mean control 0.05c±0.00 0.05c±0.00 0.07b±0.00 0.08a±0.00 0.06D±0.02 Inulin 4% 0.06d±0.00 0.23c±0.00 0.29b±0.00 0.35aa±0.00 0.23C±0.12 Inulin 6% 0.05d±0.00 0.34c±0.00 0.37b±0.00 0.42a±0.00 0.30B±0.15 Mucilage 0.2% 0.36d±0.00 0.48c±0.02 0.67b±0.00 0.81a±0.00 0.58A±0.18 Storage period 0.13D±0.14 0.28C±0.16 0.35B±0.22 0.42A±0.27 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

44 It seems that the mucilage had a better performance in keeping higher carbohydrate content and that might be explained by the fact that mucilage has higher carbohydrate precursors according to (Rizk, 1986). Within the inulin treatments, inulin 6% resulted in significantly higher carbohydrate contents (p≤0.05) than inulin 4% 4.7 Lactose Content: The total lactose content is shown in Table [4.11]. The results obtained for storage period showed decreased lactose content with the increase in storage period. The highest (p≤0.05) content was observed in day 0 and the lowest in day 10, which shows the effect of the starter culture. The addition of the different ratio of stabilizers showed similar lactose content (p≥0.05), these findings agree with the concept of reduced lactose and the ratio obtained was less than the results according to (Fellows, 2000). 4.8 Total Solids Content: The effects of stabilizers and storage period on the total solids content of yoghurt are shown in Table [4.12]. The results obtained for the different treatments indicated significant differences in total solids content (p≤0.05). A range of 13.56-14.26% was found for the different treatment the highest total solids were expressed by mucilage treatment followed by inulin 6%, inulin 4% and the control. These results agree well with the findings of Tamime and Robinson (1999) where they used skimmed milk for yoghurt manufacture. With respect to the storage period, the results showed a significant increase in total solids with the increase in storage periods (p≤0.05). This increase in total solids can be explained partially by the increase in titratable acidity and the increase in total carbohydrates.

45 46

Table 11: Changes in lactose (%) of yoghurt treated with different levels of stabilizers and stored for up to 10 days

Storage Period/Day Treatment YOGURT 0 3 7 10 mean Control 2.08a±0.08 1.56b±0.03 1.00c±0.03 0.82d±0.08 1.37A±0.52 Inulin 4% 1.91a±0.02 1.79b±0.02 1.20c±0.30 0.62d±0.03 1.38A±0.55 Inulin 6 % 2.06a±0.06 1.69b±0.02 1.13c±0.03 0.79d±0.01 1.42A±0.52 Mucilage 0.2% 1.96a±0.03 1.64b±0.05 1.00c±0.00 0.90d±0.01 1.37A±0.46 Storage period 2.00A±0.09 1.67B±0.09 1.08C±0.16 0.78D±0.11 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Table 4.12: Effect of stabilizers and storage periods on the total solid content of yoghurt stored for up to 10 days

Storage Period/Day Treatment YOGURT Zero 3 7 10 mean Control 13.09d±0.02 13.49c±0.05 13.67b±0.07 13.97a±0.03 13.56D±0.33 Inulin 4% 13.40c±0.04 13.88b±0.01 14.38d±0.02 14.74a±0.05 14.10B±0.33 Inulin 6% 13.28d±0.04 13.58b±0.03 14.37c±0.03 14.85a±0.04 14.02C±0.65 Mucilage 0.2% 13.62c±0.08 13.98a±0.03 14.53d±0.03 14.91b±0.03 14.26A±0.52 Storage period 13.35D±0.2 13.74C±0.21 14.24B±0.35 14.62A±0.40 mean n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

48 4.9 Acetaldehyde Concentration: The concentration of acetaldehyde in yoghurt samples is presented in Table [4.13]. With respect to storage period the results showed an increase in acetaldehyde concentration with the increase in storage periods. This increase with storage period is significantly different from each other (p≤0.05). The highest concentration is found in the day 10 followed by day 7 and day 3. The present results are in accordance with that reported by Lee and Jago (1976) who showed that Lactobacillus bulgaricus was able to produce acetaldehyde. In relation to the different treatments the results showed significant differences between the control, inulin 4% and inulin 6% (p≤0.05). Highest concentrations were reported for the control, inulin 4% and inulin 6% respectively. However, the concentration of the mucilage was found similar to the inulin 4%, inulin 6% and significantly different from the control. 4.10 Diacetyl concentration: The diacetyl concentration in yoghurt is shown in Table [4.14]. At any one treatment, the diacetyl concentration in yoghurt increased significantly (p≤0.05) with the increase in the storage period. The highest is reported for day 10, day 7, day 3 and day zero. The results of the treatments on the diacetyl concentration revealed a similar performance with respect to inulin 4% and inulin 6% (p≥0.05). Significant differences were observed for the control inulins and mucilage (p≤0.05).

Table 4.13: Acetaldehyde concentration (µ mol/100ml) in yoghurt treated

Storage Period/Day Treatment YOGURT 0 3 7 10 mean Control 0.14d±0.02 0.22c±0.02 0.26b±0.01 0.36a±0.02 0.25A±0.08 Inulin 4% 0.14d±0.01 0.20c±0.01 0.23b±0.02 0.33a±0.01 0.22B±0.07 Inulin 6% 0.12d±0.00 0.17c±0.02 0.23b±0.01 0.33a±0.01 0.21C±0.08 Mucilage 0.2% 0.12d±0.01 0.16c±0.01 0.24b±0.02 0.34a±0.02 0.22BC±0.09 Storage period 0.13D±0.01 0.19C±0.02 0.24B±0.02 0.34A±0.02 mean with different stabilizers and stored for up to 10 days n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Storage Period/Day Treatment YOGURT 0 3 7 10 mean Control 0.03d±0.00 0.04c±0.00 0.06b±0.00 0.07a±0.00 0.05C±0.02 Inulin 4% 0.04d±0.00 0.05c±0.01 0.07b±0.00 0.08a±0.00 0.06B±0.02 Inulin 6% 0.03d±0.00 0.05c±0.00 0.07b±0.00 0.08a±0.00 0.06B±0.02 Mucilage 0.2% 0.09b±0.00 0.09 b±0.00 0.09b±0.00 0.10a±0.00 0.10A±0.01 Storage period 0.05D±0.03 0.06C±0.02 0.07B±0.01 0.08A±0.01 mean

Table 4.14: Diacetyl concentration (µ mol/100ml) of yoghurt treated with different stabilizers and stored for up to 10 days n=3. a-d = means in the same row bearing different superscript small letters are significantly different (p≤0.05). A-D = means in the last row or last column bearing different superscript capital letters are significantly different (p≤0.05).

Chapter Five Conclusion and Recommendations

5.1 Conclusion Based on the results obtained from physical and chemical analysis of yoghurt made using different stabilizers; inulin 4%, inulin 6% and mucilage 0.2%, it was found that these stabilizers contain nutrients and flavor compounds suitable for yoghurt manufacture. Fiber content, acceptability evaluation, viscosity, digestibility and metabolism of this product not included since they are outside the scope of the present study. However, these parameters need further research and investigation. 5.2 Recommendations 1. There is a potential for the use of inulin and mucilage as stabilizers in the manufacture of yoghurt. 2. Inulin and mucilage could be good substitutes or partial replaces to some of the well known stabilizers in yoghurt industry. 3. Further research is recommended to study some stabilizer aspects of inulin and mucilage to address the chemical composition of these stabilizers.

Chapter Six References

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57 Appendix

Cups of Control Yoghurt

Cups of Mucilage 0.2% Yoghurt

Cups of Inulin 4% Yoghurt

Cups of Inulin 6% Yoghurt