Development of Probiotic Cultured Milk Drink

Lin

Weihene Epage Dhammika Upul Shantha

Thesis submitted to the University of Sri Jayewardenepura, as the partial fulfillment

requirement for the award of the degree of

Masters of Science in Food Science and Technology

ii:

Department of Food Science and Technology

Faculty of Applied Sciences

University of Sri Jayewardenepura

Nugegoda

Sri Lanka

Declaration

The work described in this thesis was carried out by me under the supervision of Dr. K K D S

Ranaweera, Head, Department of Food Science and Technology, University of Sri

Jayewardenepura, Nugegoda, Sri Lanka and Mr. J N Ruwan Kumara, Quality Assurance

Manager, Fonterra Brands Lanka (Pvt) Ltd., Delgoda Road, Biyagama, Sri Lanka. I declare

that this report or any part of the report has not been submitted, presented or accepted in any

previous application for another degree.

W E D Upul Shantha

We hereby certify that the above statement made by the candidate is true to the best of our

knowledge and that this thesis is suitable for the submission to the University for the purpose

of evaluation

4 ;i •

Head

Department of Food Science and Technology

University of Sri Jayewardenepura

Sri Lanka.

Mr. J N Ruwan Kumara

Quality Assurance Manager

Fonterra Brands Lanka (Pvt) Ltd

100, Delgoda Road, Biyagama

Sri Lanka

Date Date.......................

Affectionately dedicated

To

My Wife; Niluka, My Parents, and Teachers.

Development of Probiotic Cultured Milk Drink

By

W. E. D. Upul Shantha

ABSTRACT

Cows milk is a good source of vital nutrients for humans. Fermentation further enhances the

nutritional value of milk. The functional food market is growing at a high speed every year.

Prohiotics, or live beneficial microorganisms in food, which is a very popular aspect in

functional food concept can he easily coupled with Fermented dairy products.

A Cultured l)airy Drink with Prohiotics will be developed under the research project.

A number of health benefits have been claimed for probiotic bacteria such as Laciobacillus

acidoplulus, Bifidobaclerium spp., Lactobacillus casci. Because of the potential health

benefits, these organisms are increasingly incorporated in to dairy foods. However to claim as

a probiotic product having health benefits it required to maintain a certain amount of viable

bacteria count of above I ().

A study was carried out to develop a probiotic cultured milk drink and to investigate its

physical, chemical and microbiological properties.

In the production of Probiotic Cultured Milk Drink, Milk fat and milk solid non fat levels were

standardized by adding required amounts of raw milk and skimmed milk powder. Then the

raw milk and skimmed milk powder mixture was homogenized at 200 psi using a laboratory

homogenizer and pasteurized by keeping in a water bath at 95 °C for 3 minutes. After that the

mixture was cooled to 42 °C and inoculated with the starter culture (ABT 3) and keep in

incubation room at 42 °C for 7 hours. Parallel to above, a mixture of sugar, stabilizer,

presrvatives, colour and flavour was dissolved by adding water. Then the dissolved mixture

was pasteurized at 200 psi and cool to 4 °C. Subsequently the pre-dissolved and pasteurized

ingredient mixture was added to the incubated product and mixed well. Then the final product

was filled in to bottles and transferred to the cool room at 4±1 °C.

Final product samples were evaluated for organoleptic properties by 30 panelists using a 9

point hedonic scale. The sample with 0.05% stabilizer, 10 mg/kg colour and 0.03% flavour

was selected as the best. Initial Lactobacillus acidophilus count was tested with increase of

amount of innoculum in the formulation and fount Out that the initial count of 108 when used

0.025%. 1)cveloped product was analyzed for crude protein, fat, ash and total solids and the

results were 3.5% protein, 2.4% of fat, 0.7% of ash, and 20.3 of total solids.

The product was analyzed for pH, acidity and the Lactobacillus acidophilus count over a

period of 30 days. pH was decreased from 4.1 to 3.92. The acidity was increased from 0.62 to

0.82. Although a significant decrease in pH with concomitant increase in acidity was observed

during the shelf life at cold storage, the values were with in the acceptable levels.

i)uring the cold storage of 30 days at 4±1 °C, a decrease of Lactobacillus acidophilus count

was observed. It has been decreased from 24 x 10 7 of initial count to 2 x 107 at the end of the

shelf life. The product can he claimed as prohiotic in terms of Lactobacillus acidophilus given

that the count has remained above the 106 level at end of the shelf life.

LII

ACKNOWLEDGEMENT

I wish to express my first and foremest appreciation to my external supervisor, Mr. J. N.

Ruwan Kumara, Quality Assurance Manager, Fonterra Brands Lanka (Pvt.) Ltd., for the

expert guidance and invaluable assistance given to me to make this research possible

I extend my sincere gratitude to my internal supervisor, Dr. K. K. D. S. Ranaweera, Head,

Department of Food Science and Technology, Faculty of Applied Sciences, for the kind

assistance and guidance given throughout the period of this research project.

I am also grateful to Mr. Sachith Kalana Muthukumarana and Ms. Nayana Perera, for the

assistance given to me in numerous ways throughout this period.

Finally I owe a special word of thanks to my wife Niluka for sharing the stress and tension

ff

Content

Abstract................................................................................................. 1

Acknowledgement.................................................................................... iii

Content..................................................................................................iv

Listof Tables ............................................................................................ Viii

Listof Figures........................................................................................ix

1.0 Introduction ......................................................................................... 01

1.1 Objectives of the Study...........................................................................03

2.0 Literature Review..................................................................................04

2.1. Prohiotics .......................................................................................... 04

2.1.1 Probiotic Microorganism..............................................................08

2.1.2 Probiotic Activity Mechanism of Lactic Acid Bacteria and Bifid..............11

2.1.3 Health Benefits of Probiotics..........................................................13

2.1.3.1 Intestinal Tract Function....................................................15

2.1.3.2 Cancer.........................................................................16

2.1.3.3 Immune Function............................................................17

2.l.3.4Allergy ............................................................... 18

2.1.3.5 Urogenital Health..................................................19

2.1.3.6 Cholesterol..........................................................20

2.1.3.7 Hypertension........................................................21

2.1.3.8 Other Health Effects...............................................21

2.2 Milk.................................................................................................................................22

2.2.1 The Composition of Milk.............................................................23

2.2.1.1 Milk protein..................................................................23

2.2.1.2MilkFat ...................................................................... 24

2.2.1.3 Lactose........................................................................25

2.2.1.4 Minerals......................................................................26

2.2.1.4 Minor Components and Micronutrients..................................26

lv

2.3 Cultured Dairy Products..........................................................................27

2.3.1 Classification of Fermented Milk....................................................28

2.3.2 Lactic Acid Bacteria (LAB)..........................................................29

2.3.3 Yoghurt..................................................................................29

2.4 Pectin ................................................................................................31

3.0 Materials and Research Method..........................................................................32

3.1 Location.............................................................................................32

3.2 Material Used.......................................................................................32

3.3 Special Equipments................................................................................32

3.4 Special Facilities Used............................................................................32

3.5 Production of Milk Drink.........................................................................32

3.6 Enumeration of Lactohacillus acidophilus.....................................................32

3.6.1 Plate Count Technique.................................................................32

3.7 Preliminary Studies................................................................................34

3.7.1 Preliminary Study I ....................................................................34

3.7.2 Preliminary Study 2....................................................................34

3.8 Selection of Level of Stabilizer to Be Used In the Product..................................34

3.8.1 Sensory Evaluation Test I ...........................................................34

3.8.2 Testing Criteria.........................................................................35

3.8.3 Serving of Samples......................................................................35

3.9 Shelflife Studies of the Developed Product .................................................... .35

3.9.1 Microbiological Analysis..............................................................35

3.9.2 Physico-Chemical Analysis...........................................................35

3.9.3 Sensory Evaluation Test 2.............................................................35

3.10 Proximate Analysis of the Developed Product ............................................... .36

3.10.1 Determination of Protein content...................................................36

3.10.2 Determination of Fat Content.......................................................36

3.10.3 Determination of Ash Content.......................................................36

V

4.0 Results .37

4.1 Result of Preliminary Study 1 .....................................................................37

4.2 Result of Preliminary Study 2....................................................................37

4.3 Results of the Sensory Evaluation I ............................................................37

4.3.1 The Effect of the Treatment on Appearance of the Milk Drink.................37

4.3.2 The Effect of the Treatment on Colour of the Milk Drink.......................38

4.3.3 The Effect of the Treatment on Flavour of the Milk Drink ............................. .38

4.3.4 The Effect of the Treatment on Mouthfeel of the Milk Drink ...................... .38

4.3.5 The Effect of the Treatment on Overall Acceptability of the Milk Drink......38

4.4 Results of Analysis of Pro biotic Cultured Milk Drink ...................................... ........ ... .39

4.4.1 Results of Microbiological Analysis ................................................................. .39

4.4.2 Results of Proximate Analysis of Probiotic Cultured Milk Drink....................39

4.5 Result of the Shelf life Evaluation Test ........................................................................... .39

4.5.1 Lactohacillus acidophilus Count Over the Shelf life of 30 days ...................... .39

4.5.2 Change of pH and Titratable Acidity over the shelf life of 30 days.............40

4.5.3. The Effect of the shelf life on the sensory attributes of the Milk Drink........42

5.0 I)iscussion ................................................................................................................. ..... 43

6.0 Conclusion and Suggestions ...... ... ... ......... ... .................... ......... .......... ........ ... ........ 46

References.................................................................................................................... ......... 47

ANEXTUREI .....................................................................................................................49

ANEXTURE2................................................................................................................ ..... 51

vi

List of Tables

Page

2.1 Potential and established effects of probiotic bacteria...............................13

3.1 Percentage of stabilizer used in different treatments.................................34

3.2 Sample Codes for sensory evaluation 2................................................ 35

4.1 Estimated Median of Sensory Evaluation Test I.....................................37

4.2 Estimated Median of Sensory Evaluation Test 2.....................................42

vii

List of Figures

Page

3.1 Probiotic Cultured Milk Drink Manufacturing Process .............................. ..3

4.1 Change of Lactobacillus acidophilus count over the shelf..........................40

4.2 Change of pH of the product over the shelf life.......................................41

4.3 Change of Titratable Acidity of the product over the shelf life.....................41

VIII

CHAPTER ONE

1.0 introduction

The functional food market is growing at an astonishing rate every year. Foods that

contain some health-promoting component(s) beyond traditional nutrients are generally

considered as functional foods. To become "functional", foods can be modified by various

ways. One very important aspect here is to improve food in a way that they will facilitate

the balance of gut microflora and increase the number of health beneficial microorganisms

in the gut.

One approach exists to increase the number of health-promoting organisms in the

gastrointestinal tract. The first is the oral administration of live beneficial microorganisms.

These microorganisms, called probiotics, have been selected mostly from lactic acid

bacteria and bifidohacteria that form a part of the normal intestinal microflora of humans,

since these organisms are indigenous to the colon, because of the numerous health benefits

claimed for them.

The main health benefits of probiotics are -

Increased digestion

Alleviation of digestive disorders

Enhanced assimilation and synthesis of nutrients (including vitamins, minerals and

protein)

Improved absorption of calcium

Protection against E.coli infections

Improved immune function

Improved lactose tolerance and digestibility of all milk products

Reduction of vaginal infections and yeast infections

Increased anti-carcinogenic (anti-cancer) activity

Reduction/prevention of peptic ulcers caused by H. pylori bacteria

Reduction in bad cholesterol

Reduction of acne

Ready- to Serve beverages (RTS) are popular among all ages but the nutritional quality

of most is questionable. Preparation of a RTS based on milk, which is fermented using a

selected culture would provide nutrition and promote the utilization of raw milk available

in Sri Lanka.

The origins of fermented or cultured dairy products probably date back to the dawn of

civilization. It is possible that serendipity, chance of contamination, favorable

environmental and climatic conditions together played a role in the development of many

of the cultured dairy products we know of today. From the current scientific understanding

of food preparation and preservation, two basic reasons for converting milk into cultured

dairy products can he ascribed. The first and the foremost reason is to preserve the high-

quality nutrients present in the readily perishable fluid milk in the form of relatively more

stable products. The second major reason is to provide variety in food by inducing changes

in body, texture, colour, flavour, and nutritive properties.

With the expansion of the functional food market, it clear that there is an important niche

for the pro-, pre and synhiotic approaches because of the strong commercial interests in

providing these supplements to both humans and animals. Therefore these concepts are

combined with almost every area in the food industry. This gave rise to the idea of using

them with fermented foods especially fermented dairy products. The production of

prohiotic cultured milk drink is a good example.

Cow's milk is a good source of calcium which is incomparable with the calcium fortified

beverages in which the calcium is hardly solublized, making it unavailable for the

consumer.

Fermentation of the beverage would further upgrade the product by flavor and nutrition.

Also, easily digestible to the consumer with lactose intolerance. It has also been found that

acidification induced by fermentation demineralize casein micelles which increase the

solublization of calcium collidal phohphates. Scientific research has proved that casein

phosphopetides improve the absorbance of Zinc and Calcium in the food.

2

1.1 Objectives of the Study

To develop suitable ingredient formulation for milk drink with probiotic culture.

To study the physico- chemical and microbial properties in relation to shelf life.

To study the change of selected probiotic bacteria count in the product during its shelf life.

CHAPTER TWO

2. LITERATURE REVIEW

2.1. Probiotics

There is a long history of health claims concerning living microorganisms in food,

particularly lactic acid bacteria in a Persian version of the Old Testament states that

'Abraham owed his longevity to the consumption of sour milk. in 76 BC the Roman

historian Plinius recommended the administration of fermented milk products for treating

gastroenteritis.

Since the advent of the microbiology era, some investigators (e.g., Carre, Tissier, and

Metchnikoff) attributed such health effects to shifts of the intestinal microbial balance.

The Russian Nobel Prize winner and father of modern immunology, Elie Metchnikoff,

working at the Pasteur Institute in Paris, first conceptualized Probiotics in 1908. He

believed that the flora in the lower gut was having an adverse effect on the host and that

consuming soured milk could ameliorate these adverse effects. In support of this he cited

the observation that Bulgarian peasants consumed large quantities of soured milk and also

lived to a great age. He isolated what he called the 'Bulgarian bacillus' from soured milk

and used this in subsequent trials. This organism was probably what became known as

Lactobacillus hulgaricus and is now called L. deibrueckii sub sp hulgaricus that is one of

the major organisms used to ferment milk and produce yoghurt. He hypothesized that the

growth of this Lactobacillus in the gastro-intestinal tract would displace other putrefactive

bacteria, reduce the concentrations of toxins in the gut and thus improve health.

In 1906 Henry Tissier, a French pediatrician, recommended the administration of

bifidohacteria to infants suffering from diarrhea, claiming that bifidobacteria supersede the

putrefactive bacteria that cause the disease. He showed that bifidobacteria were

predominant in the gut flora of breast-fed infants (Tissier, 1984).

Although the concept of prohiotics was introduced in the early 201h century, the term was

not coined until the 1960's. The definition of the term has evolved through the years

4