FORMULATION OF FUNCTIONAL FOODS OF KODO MILLET(Paspalum scrobiculatum) ENRICHED WITHPROBIOTICS AND TO EVALUATE THEIR

HEALTH POTENTIAL

Thesisby

SHAKSHI SHARMA(F-2013-24-M)

Submitted to

Dr Yashwant Singh Parmar Universityof Horticulture & Forestry, Solan (Nauni)

HP-173 230 INDIA

in

Partial fulfilment of the requirements for the degree

of

MASTER OF SCIENCEMICROBIOLOGY

(DEPARTMENT OF BASIC SCIENCES)

2015

Dr. Nivedita SharmaProfessor

Department of Basic Sciences(Microbiology Section)College of ForestryDr. Y S Parmar University of Horticultureand Forestry, Nauni, Solan – 173 230 (HP)

CERTIFICATE - I

This is to certify that the thesis entitled, “Formulation of functional foods of

kodo millet (Paspalum scrobiculatum) enriched with probiotics and to evaluate

their health potential ”, submitted in partial fulfilment of the requirements for the

award of degree of MASTER OF SCIENCE MICROBIOLOGY to Dr. Yashwant

Singh Parmar University of Horticulture and Forestry, Nauni, Solan (HP) is a

bonafide record of research work carried out by Ms Shakshi Sharma (F-2013-24-M)

daughter of Sh. Tirlok Sharma under my guidance and supervision. No part of this

thesis has been submitted for any other degree or diploma.

The assistance and help received during the course of investigations have been

fully acknowledged.

Place: Nauni, Solan

Dated: , 2015

(Dr. Nivedita Sharma)Chairperson

Advisory Committee

CERTIFICATE - II

This is to certify that the thesis entitled “Formulation of functional foods of

kodo millet (Paspalum scrobiculatum) enriched with probiotics and to evaluate

their health potential”, submitted by Ms Shakshi Sharma (F-2013-24-M) daughter

of Sh. Tirlok Sharma to Dr. Yashwant Singh Parmar University of Horticulture and

Forestry, Nauni, Solan (HP) in partial fulfilment of the requirements for the award of

degree of MASTER OF SCIENCE MICROBIOLOGY has been approved by the

Student’s Advisory Committee after an oral examination of the same in collaboration

with the external examiner.

Dr. Nivedita Sharma External ExaminerChairperson

Advisory Committee

Members of Advisory Committee

Dr. R. K. Gupta Dr. Anjali Chauhan(Professor) (Asstt. Professor)

Department of Basic Science Department of Basic Science

Dr. Vipin Sharma(Asstt. Professor)

Department of Vegetable Science

Dean’s Nominee

Professor and HeadDepartment of Basic Sciences

DeanCollege of Forestry

CERTIFICATE - III

This is to certify that all the mistakes and errors pointed out by the external

examiner have been incorporated in the thesis entitled “Formulation of functional

foods of kodo millet (Paspalum scrobiculatum) enriched with probiotics and to

evaluate their health potential”, submitted by Ms Shakshi Sharma (F-2013-24-M)

daughter of Sh. Tirlok Sharma to Dr. Yashwant Singh Parmar University of

Horticulture and Forestry, Nauni, Solan (HP) in partial fulfilment of the requirements

for the award of degree of MASTER OF SCIENCE MICROBIOLOGY.

Dr. Nivedita SharmaChairperson

Advisory Committee

Professor and HeadDepartment of Basic Sciences

Dr. Y S Parmar UHF, Nauni, Solan (HP)

ACKNOWLEDGEMENTSPraise the LORD. Give thanks to the LORD, for he is good; his love endures forever

Parents are the beauty of our present life and dream of our future. I wish to thank my parents fortheir sincere encouragement and inspiration throughout my life, whatever I am today or will bein future, I owe to them. I would also like to thank my family members and my brother Varun,who’s always there for supporting and motivating me.

I would like to express my very great appreciation to my supervisor Dr. (Mrs.) Nivedita Sharma(Professor), Department of Basic Science, for her valuable and constructive suggestions duringthe research work. Her willingness to give her time so generously has been very muchappreciated.

I express my sincere thanks to Dr. P K Mahajan (Professor and Head of Department of BasicSciences) for providing all necessary facilities. Also I express my vulnerable thanks to themembers of my advisory committee Dr. R K Gupta, Dr. (Mrs.) Vipin Sharma and Dr. (Mrs.)Anjali Chauhan for their guidance and cooperation in the course of investigation. My sincerethanks and obligations to Dr. (Mrs.) Sunita Devi, Dr. (Mrs.) M. Kaur for their advice andassistance and providing a homely environment for stepping ahead.

I sincerely acknowledge the help received from my lab seniors Dr. Shweta Handa and Dr. Shrutipathania the most for their unconditional help and support. Thanks are due to Jasveen di,Poonam di, Ranjana di, Dr. Geetanjal, Dr. Anupama, and Dr. Pankaj. Also I would like to thankmy sweet juniors Kanika and Pushpi for their fun and entertainment in the lab.

I make mistakes, I know I’m not perfect, that’s why I am thankful for the true friends who stick byme knowing how I am, without them my Journey is not so much joyful thanks to Mtko, Anilkapoor, Bhuvnesh, Akshay walia. Also my heartfelt gratitude to Ambika di, Pankaj Sir, Abhishek,Gillu, Vishal, Nini, Malu, Nehu, Shilpa, Jeenu, Sanju, Neha Sayal, , abhishek pht and Babu. Aspecial call out to my lovely roommates Rosie, Neha, Phiba and Nidhi thank you for toleratingmy frustration and anger. Thanks are due to my hostel friends Diksha, Deepanjali, Manju di,Aasu, Mandy. The co-opertaion and help received by office, Joshi sir, Store incharge sir, Stenomam and Prakash bhaiya is also duly acknowledged.

Thanks to one and all and to those whose names could not appear but who at one stage or theother has helped me.

Needless to say, errors and omissions are solely mine.

Nauni, Solan

Date: (Shakshi Sharma)

CONTENTS

Chapter Title Pages

1. INTRODUCTION 1-3

2. REVIEW OF LITERATURE 4-23

3. MATERIALS AND METHODS 24-43

4. RESULTS AND DISCUSSION 44-74

5. SUMMARY AND CONCLUSIONS 75-77

LITERATURE CITED 78-87

ABSTRACT 88

APPENDICES I-IV

LIST OF TABLES

Table Title Page(s)

Chapter 2 : Review of Literature

1 Millet area and production across the globe 10

2 Top ten producers of millet in the world 10

Chapter 3 : Materials and Methods

1 Mineral estimation 33

Chapter 4 : Results and Discussion

1 Isolation of bacteria from raw kodo millet showing theirmorphological characteristics

45

2 Isolation of bacteria from malted kodo millet showing theirmorphological characteristics

46

3 Biochemical characteristics of isolated bacteria from kodomillet

49

6. Biochemical characteristics of isolated bacteria from maltedmillet

50

5 Preliminary screening of isolated bacteria from raw kodo millet onthe basis of their antagonistic pattern against tested bacterialindicators by bit/disk method

53

6 Preliminary screening of isolated bacteria from malted kodo milleton the basis of their antagonistic pattern against tested bacterialindicators by bit/disk method

54

7 Identification of finally screened bacterial isolates 55-56

8 Nutritional evaluation of kodo millet grains 59

9 TLC Rf values of polyphenols extracted from kodo millet 60

10 HPLC values of polyphenols extracted from kodo millet 61

11 Antagonistic spectrum of polyphenols extracted from kodomillet by spot method

64

12 Inhouse Probiotic microorganisms used for preparation of foodproducts

65

13 Nutritional chart of malt beverage 67

14 Sensorial evaluation of malt beverage 68

15 A profile of microbial count of malt beverage 69

16 Nutritional chart of RTE porridge 71

17 Sensorial evaluation of RTE porridge 71

18 Standardization of different ratio of wheat and kodo milletbased on physical attributes

72

19 Nutritional chart of multigrain bread 73

20 Sensorial evaluation of bread 74

LIST OF FIGURESFigure Title Between

PagesChapter 2 : Review of Literature

1 Different types of millet 9

2 Millet production rate of world 11

Chapter 4 : Results and Discussion

1 Morphology of isolated microorganisms from raw kodomillet

45-46

2 Morphology of isolated microorganisms from malted kodomillet

45-46

3 Biochemical characteristics of bacterial isolates from rawkodo millet

47-48

4 Biochemical characteristics of bacterial isolates frommalted kodo millet

47-48

5 Antagonistic potential of isolated microorganisms formkodo millet against test indicators

53-54

6 HPLC chromatogram of kodo millet (a) standard solutionof ferulic caid in acetone, (b) standard solution of cinnamicacid, (c) acetone extracted of sample of kodo millet

61-62

7 HPLC chromatogram of kodo millet (a) standard solutionof ferulic caid in methanol, (b) standard solution ofcinnamic acid, (c) methanol extracted of sample of kodomillet

61-62

8 Sensorial evaluation of malt beverage 67-68

9 Total viable count of malt beverage 69-70

10 Sensorial evaluation of RTE porridge 71-72

11 Sensorial evaluation of multigrain bread 73-74

LIST OF PLATESPlates Title Between

Pages1. Germination of kodo millet grains 45-46

2. Inhibitory spectrum of potential microorganisms againsttest indicators by bit/disk diffusion method

53-54

3. Colony morphology of screened isolates isolated frommalted kodo millet

55-56

4. Identification of best screened isolate KR5 by 16S rRNAgene technique

55-56

5. Thin layer chromatography of polyphenols extracted fromkodo millet, (a.) Polyphenols extracted from acetone (b.)Polyphenols extracted from methanol ; Rf – 0.684(Cinnamic acid), Rf – 0.52 (Ferulic acid), Rf – 0.15(caffeic acid), Rf - 0.07-0.10 (Flavonoids-glycosides)

61-62

6. Inhibitory spectrum of polyphenols extracted from kodomillet using acetone, methanol and water as solventsagainst tested indicators by spot method; AC: Acetonecontrol, AS: Acetone Sample; MC: Methanol control, MS:Methanol Sample

63-64

7. Inter compatibility of probiotic microorganisms 65-66

8 Probiotic enriched malt beverage 67-68

9 RTE porridge 71-72

10 Multigrain bread [Wheat : Kodo millet (50 : 50)] 73-74

List of ABBREVIATIONS

ᵒC - Degree centigrade% - Per cent& - Andµg - MicrogramBp - Base pairCfu - colony forming unitcm - CentimeterC - ControlDNA - Deoxyribonucleic aciddNTPs - deoxyribonucleotide triphosphateFAO - Food and Agriculture OrganizationFig. - Figureg/l - Gram per litreh - HourHPLC - High Performance Liquid Chromatographyi.e - That isLAB - Lactic acid bacterial - LitreM - Molarmg - Milligrammin - Minuteml - Millilitremm - MillimeterMRS - De Man Rogosa Sharpe agarNA - Nutrient Agarnm - Nano meterOD - Optical densityppm - Parts Per Millionpsi - Per square inchPCR - Polymerase Chain ReactionRNA - Ribonucleic AcidrDNA - Ribosomal DNArRNA - Ribosomal RNARTE - Ready To Eatrpm - Rotations per minuteSDS - Sodium Dodecyl Sulphatesp. - SpeciesTLC - Thin Layer ChromatographyUV - Ultra violetv/v - Volume/Volumeviz. - Visuallyw/v - Weight/VolumeWHO - World Health Organization

Chapter-1

INTRODUCTION

Cereals and cereal products are significant and important human food resources and

livestock feeds worldwide. Cereals form a major portion of human diet and are a vital source of

starch and other dietary carbohydrates, which play an important role in energy requirement and

nutrient uptake in humans The main cereal grains used for foods include corn (maize), wheat,

barley, rice, oats, rye, millet, and sorghum. By any nutritional parameter, millets are far ahead of

other cereals in terms of their mineral content, compared to rice and wheat. Millets play very

specific role in human nutrition because of their multiple qualities. Millets are rich in vitamins,

minerals, sulphur containing amino acids and phytochemicals, and hence are termed as “nutri-

cereals”. They are nutritionally comparable or even superior to staple cereals such as rice and

wheat (Gopalan et al. 2004). Each one of the millets has more fibre than rice and wheat. Finger

millet has thirty times more Calcium than rice while every other millet has at least twice the

amount of Calcium compared to rice. In their Iron content, foxtail and little millet are so rich that

rice is nowhere in the race. While most of us seek a micronutrient such as Beta Carotene in

pharmaceutical pills and capsules, millets offer it in abundant quantities. The much privileged

rice, ironically, has zero quantity of this precious micronutrient. In this fashion, nutrient to

nutrient, every single millet is extraordinarily superior to rice and wheat and therefore is the

solution for the malnutrition that affects a vast majority of the Indian population.

Millets are one of the important cereals which occupy highest area under cultivation.

Millets were used as staple food for thousands of years by people in Asian and African countries

before rice become a common commodity to man. Millets are important crops in the semiarid

tropics of Asia and Africa (especially in India, Nigeria, and Niger), with 97% of millet

production in developing countries. Millets are of great importance as they have a short growing

season and yield higher productivity. The millets have high fiber content, and proteins

composition contributes significantly to nutritional security of large section of population (Desai

et al. 2010). The term “millets” is used for any of several small seeded annual grasses that are of

importance mainly in Asia and Africa. The most important characteristic of millet is their

2

unique ability to tolerate and survive under adverse condition of continuous or intermittent

drought as compared to most other cereals like maize and sorghum (LCRI, 1997). Millets are

principally food sources in arid and semi-arid regions of the world.

India is the leading producer of small millets namely, finger millet (ragi), kodo millet

(kodo), foxtail millet (kangni), barnyard millet (sawan), proso millet (cheema) and little millet

(kutki) (Majumdar et al. 2006). Small millets form an important component of the traditional

cropping systems and contribute significantly to the regional food and nutritional security and

diversity in the national food basket. The millets include five genera of the Panaceae family (

Panicum, Setaria, Echinochola, Pennesetum and Eleusine). The most important cultivated

species are: Proso millet (Panicum miliaceum), Foxtail millet (Setaria italica), Japanese

barnyard millet (Echinochloa frumentacea), Finger millet (Eleusine coracana) and Kodo millet

(Paspalum scrobiculatum). Among them kodo millet (Paspalum scrobiculatum) is grown

primarily in India, and also in the Philippines, Indonesia, Vietnam, Thailand, and in West Africa

where it originated. It is a very hardy crop that is drought tolerant and can survive on marginal

soils where other crops may not survive, and can supply 450–900 kg of grain per hectare. Kodo

millet has large potential to provide nourishing food to subsistence farmers. The grain varies in

color from light red to dark grey and is enclosed in a tough husk that is difficult to remove.

Traditional fermented foods are receiving extensive scientific attention globally and

many traditional preparations have been analyzed for their microbiological, enzymologial and

biochemical changes (Omemu et al, 2007). Various millet based traditional preparations are also

available throughout the world and fermentation of millet is a common practice. Fermentation

has a positive influence on grains. Millet is the major source of energy and protein and has many

nutritious and medical functions (Obilana and Manyasa, 2002). Millet grains are easily digested,

have a longer shelf life and do not contain gluten, hence are advisable for celiac patients

(Chandrasekhar and Shahidi, 2010). Millets are recognized nutritionally for being a good source

of minerals magnesium, manganese and phosphorus. Millets are also rich in phytochemicals,

including phytic acid (Shashi et al. 2007), which is believed to lower cholestrol, and phytate,

which is associated with reduced cancer risk. Thus millets have great potential for being utilized

in different food systems by virtue of their nutritional quality and economic importance

3

Therefore there is an enormous scope growing in this crop to explore the technological

possibilities of its utilization in food industry for the preparation of various food products.

Additionally a major development in functional foods also pertains to foods containing

probiotics and prebiotics which impart immense health benefits to human system. Functional

foods with probiotics are establishing worldwide at rapid scale and these have become extremely

popular among consumers recently (Saarela et al. 2000). Therefore, health foods containing

probiotics/synbiotics constitute current and future waves in the evolution of the food

development cycle.

Keeping in view the high potential to process underutilized millet grains into value-added

food and beverages, the different novel, nutraceutical food products enriched with probiotics

have been formulated in the present study entitled “formulation of functional foods of kodo

millet enriched with probiotics and to evaluate their health potential” with the following

objectives:

To prepare innovative health foods of kodo millet.

To evaluate nutritional potential of prepared products.

To assess the storage stability and exceptional healthcare properties of these products.

Chapter-2

REVIEW OF LITERATURE

Millet is a general category for several species of small grained cereal crops and is a

food staple in parts of India, Africa, China and elsewhere. The term millet is employed for

several related genera, some used to produce grain, or forage or both. Millet has been

cultivated since prehistoric times in regions of North Africa and Central Asia, though its

origin is ambiguous. Mostly millet is produced in Asia and Africa. In Europe and the United

States, millet is grown mainly as forage for poultry and as bird feed. Millets are a group of

highly variable grasses which are called as “little giant”. Millets are mainly classified into

two types i.e. major millets and minor millets. Major millets are maize (Zea mays), great

millet or pearl millet (Pennisetum typhoideum). Minor millets includes grain crops like little

millet (Samai or Panicum sumatrense), proso millet (Panivaragu or Panicum milliaceum),

foxtail millet (Varagu or Paspalum serobiculatu), finger millet (Ragi in Tamil vernacular or

Eleusine coracana), kodo millet (kodra or Paspalum scrobiculatum). Millet contains an

average of 10 - 12% protein. While its protein is superior to that of wheat or corn in terms of

content of essential amino acids, it nonetheless contains less than half the amount of the

essential amino acid lysine that is found in high quality protein sources such as meat. Millet

lacks gluten, the wheat protein that makes dough prepared from wheat flour elastic; hence

millet flour generally is used in making flat cakes and breads. The whole grain is used in

soups, stews or as a cooked cereal. Millet is also popped; roasted or sprouted (Robert Ronzio,

2004).

Millets are cereal species growing in an equally broad range of environments. The

most widely cultivated millets are finger millet (Eleusine coracona), foxtail millet (Setaria

itallica), pearl millet (Pennisetum typhoideum), proso millet (Panicum miliaceum), kodo

millet (Paspalum scrobiculatum), barnyard millet (Echinochooa colona), etc. Millets are

considered the least important of cereals, with annual production less than 2% of the world’s

grain. However they are of great local importance as staples and as reserve crops in marginal

areas. The use of millets not only provides farmers with a market for their products but also

saves foreign exchange, which would otherwise be required to import cereals. Particularly in

the developed countries, there is a growing demand for gluten-free foods and beverages from

5

people with celiac disease and other disease and other intolerances to wheat that cannot eat

products from wheat, barley or rye. Since literature exclusively on kodo millet is scarce due

to limited work done on it, therefore, this review has included related studies on whole Millet

Family.

2.1 Types of millet and their Chemical Composition:

a. Finger millet: Finger millet also known as ‘ragi’ in India is an important staple food for

people belonging to the low socio-economic group. It is also known as African millet, and is

an important staple food in Africa and India. Finger millet, a chief dry land crop has the

ability to withstand adverse weather conditions when grown in soils having poor water

holding capacity. It is grown in arid regions of Eastern and Southern Africa, India and Nepal.

The small millet seeds can be stored safely for many years without insect damage, which is

invaluable in farmers risk avoidance strategies in drought prone areas. Finger millet is the

third most important millet in India, next to sorghum and pearl millet, covering an area of 2

million hectares with annual production of 2.15 million tones. In Karnataka, it is grown in an

area of 0.8 Mha with an annual production of 1.34 mt. Finger millet is generally grown in

higher rainfall areas (600-1200 mm) and is one of the better crops for acid soils. It matures

within 100 to 130 days. Finger millet is an important staple food in East and Central Africa

and in India (Hulse et al. 1980). In Uganda, finger millet is the second most important cereal

after maize (Esele, 1989). Uganda is regarded as the centre of its origin, and was probably

taken to India some 3000 years back (Hulse, 1980).

Finger millet grown on marginal land provides a valuable resource in times of famine.

Its grain tastes good and is nutritionally rich (compared to cassava, plantain, polished rice and

maize meal) as it contains high levels of calcium, iron and manganese. It has a carbohydrate

content of 81.5%, protein 7.3%, crude fiber 4.3% and mineral 2.7% that is comparable to

other cereals and millets. Its crude fiber and mineral content is markedly higher than wheat

(1.2% fiber, 1.5% minerals) and rice (0.2% fiber, 0.6% minerals); its protein is relatively

better balanced; it contains more lysine, threonine and valine than other millets. The millet

straw is also an important livestock feed, building material and fuel. Finger millet contains

methionine, an essential amino acid lacking in the diets of hundreds of millions of the poor

who rely mostly on starchy staples (Ravindran, 1991).

6

b. Sorghum: Sorghum is the king of millet cereals and is one of the important food crops in

dry lands of tropical Africa, India and China (Shobha et al. 2008). India ranks second in the

world for sorghum production and first with respect to many regionally important crops like

millets and pseudo-cereals. Sorghum is the principal staple food of Maharashtra, and is also

an important food of Karnataka, Madhya Pradesh, Tamil Nadu and Andhra Pradesh.

Sorghum can be milled to produce starch or grits (semolina) from which many ethnic and

traditional dishes can be made. The most common products are leavened and unleavened

breads, porridges, boiled grains and steam cooked products such as couscous.

Sorghum contains 10.4 % protein, 1.9% fat and around 8.3% total dietary fiber. Most

of the fiber is present in the pericarp and cell walls. Sorghum contains 6.5 – 7.9% insoluble

fiber and 1.1 – 1.2% soluble fiber. Insoluble dietary fiber increased during food processing

due to increased levels of bound protein mainly kafirins, and enzymes-resistant starch.

Kafirins (the sorghum prolamin proteins) and glutelins comprise the major protein fractions

in sorghum. These fractions are primarily located within the protein bodies and protein matrix

of the endosperm, respectively. The germ and aleurone are rich in fat-soluble and B-vitamin.

Sorghum contains 0.3 – 0.8 μg/g of α – tocopherols and 9 – 11.5 μg/g of τ– tocopherols.

Precursors of vitamin A (carotenes) are found in yellow and heteroyellow endosperm

sorghums. Sorghum is an important source of minerals that are located in the pericarp,

aleurone and germ. Phosphorus is the mineral found in greatest amounts, its availability is

negatively related to the amount bound by phytates. Phytase activity during malting and

fermentation significantly increases availability of phosphorus and other minerals as well.

The sorghum aleurone layer is not a major source of endosperm-degrading enzymes. The

scutellum of sorghum is where α-amylase is formed and diffuses into the endosperm.

Sorghum does not respond to gibberellins to enhance production of

amylases during malting. α-amylase activity in sorghum starts 24–36 h after germination.

Sorghum malt has high levels of α-amylase activities but it has reduced β – amylase

activities.

Condensed tannins (proanthocyanidins) are not present in all sorghums; however, all

sorghums contain phenolic acids, and most contain flavonoids. Kernels that contain

condensed tannins have a thick, highly pigmented testa. These sorghums were referred to as

7

brown sorghums but are not classified as tannin sorghums. Tannins protect the kernel against

pre-harvest germination and attack by insects, birds and molds.

The tannin sorghums are potent sources of antioxidants. Bran fractions and extracts

from them have significantly higher oxygen radical absorbance capacity (ORAC) levels, a

measure of antioxidant strength, than most fruits and vegetables. Bakery products containing

this bran have increased fiber content, higher antioxidant potential, and attractive natural

brown or chocolate color. Tannin sorghums can also be transformed into excellent whole

grain snacks by extrusion. The extrusion process significantly reduced the degree of

polymerization of tannins, which may be beneficial in human foods (Waniska et al. 2004).

Sorghum and millet have considerable further potential to be used as a human food and

beverage source. In developing countries the commercial processing of these locally grown

grains into value-added food and beverage products is an important driver for economic

development (Taylor, 2004).

c. Foxtail Millet: Foxtail millet is commonly known as Italian millet, German millet,

Chinese millet, Hungarian millet, dwarf setaria, giant setaria, liberty millet, and Siberian

millet. The seeds are small and measure around 2mm in diameter. They are encased in a thin,

papery hull which is easily removed upon threshing. Seed color can vary greatly between

varieties grown and range from a pale yellow, through to orange, red, brown and black. A

thousand of these seeds weighs approx. 2 grams. The protein in Foxtail millet is known to be

deficient in lysine, and its amino acid scores are comparable to that of maize. In different

grain varieties, higher the protein content, lower is the lysine content in the protein. It is

relatively high in leucine and methionine. The starch in some foxtail millet varieties contain

100% amylopectin, and the starches contained in foxtail, proso and barnyard millets are more

digestible than maize starch. The total ash content of foxtail millet is good and is much higher

than the more commonly consumed cereal grains including sorghum, however de-hulling of

the grain, like in other millets, causes considerable nutrient losses.

d. Barley: Barley is one of the major millet crops of the world, characterized for its small

seeds. It is of major importance in the west but a stable in diets of African and Asian people.

Barley is important millet used for malting and brewing because of its high diastatic power

(Pawar et al. 2006)

8

e. Little Millet: Little millet is a relative of proso millet and is grown throughout India but is

of little importance elsewhere and has received very little attention from plant breeders as a

crop source. The plant varies in size between 30-90 cm and its oblong panicles ranged from

14 to 40 cm long. The seeds of little millet are much smaller than proso millet. It has

reasonably good levels of protein, but very poor amino acid values. It also has the highest fat

content of all the millets.

f. Browntop Millet: Browntop millet is another native of India but was introduced to the

U.S.A. in 1915. It is grown in the south eastern states mainly for hay and pasture, and often

for bird and quail feed plantings on game preserves. It has a short growing season and finer

stems that allow for easier curing for hay production. Seed and forage yields of this plant are

low in tests and it has been found that it doesn't compete well with weeds.

g. Barnyard Millet: Barnyard or Japanese millet is a domesticated relative of barnyard grass

and there exists several varieties. It is the fastest growing of all the millets and produces a

crop in six weeks. In India, Japan and China it is often used as a substitute for rice when the

paddy crop fails. In the U.S.A. it is grown primarily for forage and can produce up to eight

harvests a year. It is comparable to proso millet in protein and fat content, but the actual

quality of the protein, like that of little millet have the poorest amino acid values of all the

millets. It is very high in fiber.

h. Kodo Millet: Kodo millet is a minor grain crop in India but is of much greater importance

in the Deccan Plateau. It is an annual grass species that grows to around 90 cm high. Some

varieties of kodo millet are prone to attacks from mycotoxins. The grain varies in color from

light red to dark grey and is enclosed in a tough husk that is difficult to remove. It has high

protein content, being around 11% and the nutritional value of the protein is regarded as

being slightly better than that of foxtail millet, but comparable to the other millets. It is

however deficient in the amino acid tryptophan. It is also reasonably low in fat with high

fiber content. Due to high antioxidant content, it is beneficial in protecting against oxidative

stress and maintaining glucose levels in type-2 diabetes (Taylor, 2004).

9

RagiKodo millet

Foxtail millet Pearl millet

Proso millet Barnyard millet

Little millet Sourgham

Fig 1. Different types of Millets

10

2.2 Geographical Distribution and Production of Millets

Table 1 represents the millet area and production across the globe in 2002 and 2009.

According to FAO statistics (2009), the world production of millets was 26702000 metric

tons from an area of 33692000 Hectare. Nearly a decade earlier (2002), the world production

of millets was down to 23338000 metric tons from an area of 33396000 Hectare. Africa was

the largest producer of millet in 2009 (20626000 metric tons), followed by Asia (12492000

metric tons) and in particular India (10500000 metric tons). Table 2 showed the top ten

producers of millet in world and millet production rate is represented in Fig. 1. Relative to

wheat, rice, maize and barley, sorghum ranks fifth in importance, in terms of both production

and area planted, accounting for 5% of the world cereal production (Obilana, 2004).

Table 1: Millet area and production across the globe [(FAO, (2002);http://www.fao.org)]

Region Production(in tons)

2002 2009

Area harvested(in ha)

2002 2009

WorldAfricaAsiaIndia

23338 2670213633 1490810078 88106150 8810

33396* 33692*20626* 20631*11359* 12492*12527 10500**

* May include official, semi official or estimate data

** Unofficial data

No symbol – Official data

Table 2: Top ten Producers of Millet in the world [(FAO (2002); http://www.fao.org)]

CountryProduction ( in tons)

2002 2009India 10078 8810Nigeria 6105 4885China 2126 1226Burkina Faso 726 971Mali 759 1390Sudan 496 630Uganda 534 841Chad 259 709Ethiopia 320 560No symbol – Official data

In – FAO data based on imputation methodology

11

Fig1. Millet production rate of world (http://www.fao.org).

2.3 Distribution of Millets in India

India is the world's largest producer of millets. In 1970’s all of the millet crops

harvested in India were used as staple food. By 2000’s the annual millets production had

increased in India, yet per capita consumption of millets had dropped by between 50% to

75% in different regions of the country. As in 2005, the majority of millets produced in India

were being used for alternative applications such as livestock fodder and alcohol production.

Consumption of millets also dropped, as India experienced rapid economic growth and

witnessed a significant increase in per capita consumption of other cereals. Further, in each

of the millet growing areas at least 4 to 5 species are cultivated either as primary or allied

crop in combination with the pulses, oilseeds, spices and condiments.

India is the top most producers of millets followed by Nigeria for the year 2000 and

2009 (Table 1.2). In India, eight millets species (sorghum, finger millet, pearl millet, foxtail

millet, barnyard millet, proso millet, kodo millet and little millet) are commonly cultivated

under rain fed conditions. Further, in each of the millet growing areas at least 4 to 5 species

are cultivated either as primary or allied crop in combination with pulses, oilseeds, spices and

12

condiments. For instance, while pearl millet and sorghum are primary crop and allied crops

respectively in the desert regions of Rajasthan, in the eastern parts of Rajasthan and Gujarat.

However, in spite of a rich inter/intra-species diversity and wider climatic adaptability

cultivation of diverse millet species/varieties is gradually narrowing in the recent past. In a

way, lack of institutional support for millet crops in contrast to the institutional promotion of

rice and wheat continue to shrink the millet-growing region. Over the last 50 years, the share

of ‘coarse grains’, which include pearl millet, sorghum, maize, finger millet, barley and 5

other millet species known as ‘small millets’, in terms of total area has registered 25.3%

decline from 38.83 Mha. (1949-50) to 29.03 Mha. (2004-05). In spite of this, several

communities in the dry/rain fed regions having known the food-qualities of millets over

generations continue to include a range of millets in the traditional cropping patterns, which

recognize millets as an essential part of the local diet.

2.4 Economic and social impact of millets

Millets, in most cases, have been grown in difficult conditions, and it is scarcely

surprising that they involve high production risks (Dogget, 1989). They have always been

crops for situation where there is a risk of famine, as well as offering a low but more reliable

harvest relative to other crops. Although it is found in other countries, finger millet has

gained little importance outside Africa and India. Equally important to note is that, common

millet has received little attention from plant breeders (Hulse et al. 1980).

In most parts of the world, millet is grown as a subsistence crop for local

consumption. Commercial millet production is risky, especially in Africa because the absence

of large market outlets means that fluctuations in output cause significant price fluctuations,

in areas where millet is the main food crop (FAO and ICRISAT, 1996). Apart from grain

production, millet is also cultivated for grazing, green fodder or silage.

2.5 Neutraceutical and functional properties of millet:

Like other cereals, major and minor millets are predominantly starchy. The protein

content is nearly equal among these grains and is comparable to that of wheat and maize.

Pearl and little millet are higher in fat, while finger millet contains the lowest fat. Barnyard

millet has the lowest carbohydrate content and energy value. One of the characteristic

features of the grain composition of millets is their high ash content. They are also relatively

13

rich in iron and phosphorus. Finger millet has the highest calcium content among all the food

grains. High fiber content and slow digestibility of carbohydrates are other characteristic

features of sorghum and millet grains.

Millets are rich in vitamins, minerals, sulphur containing amino acids and

phytochemicals, and hence are termed as “nutri-cereals”. They have higher proportions of

non starchy polysaccharides and dietary fibre. Millets release sugars slowly and thus have a

low Glycemic index. Millets have great potential for being utilized in different food systems

by virtue of their nutritional quality and economic importance. Millets are rich in B vitamins

(especially niacin, B6 and folic acid), calcium, iron, potassium, magnesium and zinc.

Generally the whole grains are important sources of B-complex vitamins, which are mainly

concentrated in the outer bran layers of the grain.

Millets do not contain gluten, which makes them appropriate foods for those with

celiac disease or other forms of allergies/intolerance of wheat. However, millets are also a

mild thyroid peroxidase inhibitor and probably should not be consumed in great quantities by

those with thyroid disease. Nutritional potential of millets in terms of protein, carbohydrate

and energy values are comparable to the popular cereals like rice, wheat, barley or bajra.

Finger millet contains about 5–8% protein, 1–2% ether extractives, 65–75% carbohydrates,

15–20% dietary fiber and 2.5–3.5% minerals (Chethan and Malleshi 2007). It has the highest

calcium content among all cereals (344 mg/100 g).

However, the millet also contains phytates (0.48%), polyphenols, tannins (0.61%),

trypsin inhibitory factors, and dietary fiber, which were once considered as “anti nutrients”

due to their metal chelating and enzyme inhibition activities (Thompson, 1993) but nowadays

they are termed as neutraceuticals. The seed coat of the millet is an edible component of the

kernel and is a rich source of phytochemicals, such as dietary fiber and polyphenols (0.2–

3.0%) (Hadimani and Malleshi, 1993; Ramachandra et al. 1977). It is now established that

phytates, polyphenols and tannins can contribute to antioxidant activity of the millet foods,

which is an important factor in health, aging and metabolic diseases (Bravo, 1998).

Cereal grains, including soft wheat flour, are low in protein (7 to 14%) and are

deficient in some amino acids such as lysine and certain other amino acids (Claughton and

Pearce, 1989).

14

Legumes on the other hand, are higher in proteins (18 to 24%) than cereal grains and

can be used to support certain amino acids such as lysine, tryptophan, or methionine

(Rababah et al. 2006).

Soy protein is preferred because of its low cost, accessibility, widely varying

functional properties and high content of good quality protein. While soy protein is rich in

lysine, cereals are rich in sulphur containing amino acids, especially methionine and hence

blending of these two in appropriate quantities will make up the individual deficiencies

(Prasad et al. 2007).

Value addition through processing of nutritious cereals should also be explored and

popularized to make them popular among consumers. Some of the broad steps in making

them popular are a large scale awareness campaign about it and moreover the barrier of low

social status attached to these nutritious cereals should be removed by terming them as health

foods (Seetharama and Rao, 2004).

2.5.1 Polyphenols

Nowadays, there has been a renewed interest in polyphenols as “life span essentials”

due to their role in maintaining body functions and health throughout the adult and later

phases of life (Chandrasekara and Shahidi, 2010). Polyphenols are a large and diverse class

of compounds, many of which occur naturally in a range of food plants. Phenolics

(hydroxybenzenes) especially polyphenols (containing two or more phenolic groups) are

ubiquitous in plant foods consumed by human and animals and one of the widest groups of a

dietary supplements marketed worldwide (Ferguson, 2001). The main polyphenols in cereals

are phenolic acids and tannins, whilst flavonoids are present in small quantities (Rao and

Muralikrishna, 2002).

Although, these compounds play no known direct role in nutrition (non-nutrients),

many of them have properties, including antioxidant (Sripriya et al. 1996), anti-mutagenic,

anti-oestrogenic, anti-carcinogenic and anti-inflammatory, antiviral effects and platelet

aggregation inhibitory activity that might potentially be beneficial in preventing or

minimising the incidence of diseases (Ferguson 2001). The tiny finger millet grain has a dark

brown seed coat, rich in polyphenols compared to many other continental cereals such as

barley, rice, maize and wheat (Viswanath et al.2009).

15

Chethan et al. (2008) suggested that phenolics in finger millet grain are detrimental to

its malt quality, as they inhibited malt amylases. Siwela et al. (2010) determined type of

phenolics type, fungal load, germinative energy (GE) and the malt quality of finger millet

grains differing in colour and phenolic contents and reported that phenolics influenced malt

quality positively by contributing to attenuation of the fungal load on the germinating grain.

Finger millet types with higher level of phenolics had superior malt quality than the low-

phenol varieties, with respect to diastatic power (DP), and α- and β-amylase activities.

According to them, GE, DP and α-amylase activity positively correlated with total phenolics

and the phenolics content (p<0.05) and negatively correlated with total fungal count (p<0.01).

2.5.2 Antimicrobial properties

Plant phenolics have been implicated for minimising the intensity of several diseases

and also to inhibit the in vitro growth of an assortment of fungal genera (Baranowski et al.

1980; Bravo 1998). Seetharam and Ravikumar, (1994) indicated that finger millet grain

phenolics including tannins may be involved in resistance of the grain to bacterial/fungal

attack. Phenolic compounds, particularly tannins in the outer layers of the grain serve as a

physical barrier to the fungal invasion. The acidic methanol extracts from the seed coat

showed high antibacterial and antifungal activity compared to whole flour extract due to high

polyphenols content in seed coat (Viswanath et al. 2009). Siwela et al. (2009) reported that

the fungal load (total fungal load and infection levels) of the unmalted millet grain and its

malt, were negatively correlated (p<0.05) with total phenolics and phenolic type (condensed

tannins, anthocyanins and flavan-4-ols).

Oxidation of microbial membranes and cell components by the free radicals formed,

irreversible complexation with nucleophilic amino acids leading to inactivation of enzymes

are major biochemical benefits of polyphenols towards the antifungal activity. Besides, loss

of their functionality and also the interaction of phenolic compounds, especially tannins with

biopolymers such as proteins and polysaccharides and complexing with metal ions making

them unavailable to micro-organisms are some of the mechanisms involved in the inhibitory

effect of phenolic compounds on microorganisms (Cowan, 1999; Scalbert, 1991). The

extremely good storage property of finger millet and its processed foods could be attributed

to its polyphenol content. The seed coat extract of millets showed higher antimicrobial

activity against Bacillus cereus and Aspergillus flavus compared to whole flour extract

(Mathangi and Sudha, 2012).

16

2.5.3 Antioxidant properties

Antioxidant compounds are gaining importance due to their main roles as lipid

stabilizers and as suppressors of excessive oxidation that causes cancer and ageing. Their

stable radical intermediates prevent the oxidation of various food ingredients, particularly

fatty acids and oils. Phenolic acids and their derivatives, flavonoids and tannins present in

millet seed coat are of multifunctional and can act as reducing agents (free radical

terminators), metal chelators, and singlet oxygen quenchers (Sripriya et al. 1996). The

potency of phenolic compounds to act as antioxidants arise from their ability to donate

hydrogen atoms via hydroxyl groups on benzene rings to electron- deficient free radicals and

in turn form a resonance stabilized and less reactive phenoxyl radical. Studies were carried

out on the natural antioxidants in edible flours of small millets.

Total antioxidant capacity of finger, little, foxtail and proso millets were found to be

higher and their total carotenoids content varied from 78–366 mg/100 g in the millet

varieties. Total tocopherol content in finger and proso millet varieties were higher (3.6–4.0

mg/100 g) than in foxtail and little millet varieties (~1.3 mg/100 g). HPLC analysis of

carotenoids for the presence of β-carotene showed its absence in the millets, and vitamin E

indicated a higher proportion of ᵧ-and α-tocopherols; however, it showed lower levels of

tocotrienols in the millets. Edible flours of small millets are good source of endogenous

antioxidants (Ashrani et al. 2010).

The antioxidant activity of millet phenols and their health benefits have also been

reported. For instance, in Japanese barnyard millet, the antioxidant activity of luteolin was

nearly equal to that of quercetin; however, the activity of tricin was lower than luteolin.

Finger millet is a potent source of antioxidants and has potent radical-scavenging activity that

is higher than that of wheat, rice, and other millets; these results corresponded to their

phenolic content. The brown or red variety of finger millet, which is commonly available,

had higher activity (94%) than the white variety (4%) using the DPPH method (Sripriya et al.

1996). Kodo millet quenched DPPH by nearly 70% higher than other millets (15–53%);

white millet varieties had lower activity (Hedge and Chandra, 2005).

2.6 Effect of Processing on the Nutrient Composition of Millets

Cereals and millets are the primary sources of minerals in most vegetarian diets,

secondary sources being legumes. Besides inherent factors such as phytate, tannin, and fiber

17

negatively influencing the bioavailability of zinc and iron from these food grains, the same

may also be influenced by processing, such as cooking, boiling, roasting or germination

which these food grains undergo. Food processing by heat generally alters the bioavailability

of nutrients – both macro and micro. The digestibility and consequently absorption of

micronutrients such as iron is believed to be improved upon heat processing by softening the

food matrix, releasing of protein bound iron and thus facilitating its absorption. In addition,

heat processing of food is also likely to alter the inherent factors that inhibit mineral

absorption, such as phytate and dietary fiber, especially the insoluble fraction (Amparo et al.

2003; Abdalla et al. 1998).

Soaking, germination and boiling resulted in a significant reduction of phytate

phosphorus. The concentrations of calcium, magnesium, iron and zinc increased upon

soaking and germination, while boiling decreased calcium, magnesium and iron

concentration. Solubility of minerals was higher in soaking and germination than in boiling

(Sushma et al. 2008). Major biochemical changes occurred during fermentation (48 h) of

finger millet compared to its germination (24 h). The processing decreased the pH from 5.8 to

3.8 and increased the total sugars, reducing sugars and free amino acids. The phytate content

decreased by 60% while the phytate Ca/Zn molar ratio decreased from 163 to 66.2, indicative

of an increased Zn bioavailability. The study revealed that a combination of germination and

fermentation is a potential process for decreasing the antinutrient levels and enhancing

mineral availability (Sripriya et al. 1997).

2.7 Fermentation

Fermentation is one of the oldest transformation and preservation techniques for food.

This biological process allows not only the preservation of food but also improves its

nutritional and organoleptic qualities (relating to the senses; taste, sight, smell, touch). A well

conducted fermentation will favour useful flora, to the detriment of undesirable flora in order

to prevent spoilage and promote taste and texture. Fermentation occurs when microorganisms

grow in food and cause desirable changes. It can occur in both animal food (e.g. sausage,

cheese) and plant food (pickles, bread). Fermentation is necessary to decompose and return

natural material to soil and air. Although it is often desirable to slow or prevent the growth of

microorganisms to prevent spoilage or food borne illness, many different foods are actually

produced by microorganisms.

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Lactic acid fermentation comprises of the chemical changes in foods accelerated by

enzymes of lactic acid bacteria resulting in a variety of fermented foods (Bladino et al.

2003).Lactic acid fermentation processes are the oldest and most important economical forms

of production and preservation of food for human consumption. It is reported that fermented

foods globally contribute 20 to 40% of the food supply and usually, a third of the food

consumed by man is fermented (Sanni AI, 1993). This renders fermented foods and beverages

a significant component of people’s diets globally. Fermentation, by certain LAB and yeasts,

removes or reduces the levels of antinutritional factors (Frontele et al., 2008). During

fermentation, optimal pH conditions prevail for enzymatic degradation of the antinutritional

factors. This results in better bioavailability of minerals such as iron, zinc and calcium.

Strains of Lb. plantarum degraded phytic acid in the cereals after incubation at 37 °C for 120

hours (Holzapfel WH, 2002). This degradation can be ascribed to the hydrolysis of the

phosphate group by phytases from the raw cereal substrate and produced by the fermenting

microorganisms (Chelule et al. 2010). Fermentation alone reduced the phytate content by

39%. The combined effect of fermentation plus the addition of exogenous phytase, resulted in

a reduction of 88% of the phytates in tannin sorghum gruel (Towo, 2006).

2.8 Food Preparations of Millets

Millets have considerable potential in foods and beverages. As they are gluten free

they are suitable for celiacs. The major categories of traditional foods where millets can be

effectively used are fermented and unfermented flat breads, fermented and unfermented thin

and thick porridges, steamed and boiled products, snack foods, alcoholic and nonalcoholic

beverages. As millets are less expensive compared to cereals and is a staple for the poorer

sections of population, studies have been carried out to explore the possibility of the millet as

a vehicle for fortification. Millets have been successfully utilized in food products, beverages,

convalescent and weaning foods.

2.9 Functional Foods

The term “functional food” was originated in Japan in the 1980s and this functional

food concept obtained the legal status in 1991 by setting up “Foods for Specified Health Use”

(FOSHU) regulatory system (Staton et al. 2001; Prado et al. 2008). The demand for

functional foods was increased in recent years broadening the market for functional foods

(Staton et al. 2001). Functional foods are designed foods with some modifications to be

“functional” (Shah, 2007). There are numerous definitions for functional foods. The Institute

19

of Food Technologists (IFT) expert report defines it as “foods and food components that

provide a health benefit beyond basic nutrition” (IFT, 2005). This may include conventional

foods, fortified, enriched or enhanced food and dietary supplements. The American Dietetic

Association (ADA) defines functional foods as “Food, that includes whole foods and

fortified, enriched or enhanced foods, have a potentially beneficial effect on health when

consumed as part of a varied diet on a regular basis, at active levels.” According to ADA

there are four different functional food categories: conventional foods, modified foods,

medical foods, and foods for special dietary use. Fruits and vegetables, which are rich in

phytochemicals and yogurt that is rich in probiotics are some examples for conventional

foods. Modified foods are functional foods that have enriched, fortified, or enhanced with

bioactive components such as calcium-fortified milk and orange juice. Medical foods are

formulas administered only under physician supervision for specific health problems. Foods

for special dietary use such as gluten free products target specific health issues, but, a

physician recommendation is not required (Furguson, 2009).

In many Asian and African countries millet is the staple food of the people and are

used to prepare various traditional foods and beverages (Chandrasekara and Shahidi, 2011).

Most of the millets produced in India are used as staple food and less in ready-to-use and

convenient food products due to non-availability of proper milling technology. The major

constraints for widespread utilization of millet are its coarse fibrous seed coat, coloured

pigments, astringent flavour and poor keeping quality of the processed products (Desikachar,

1975).

Although millets are nutritionally superior to other cereals, yet their utilization is not

wide spread. One possible way of extending their utilization could be by blending them with

wheat flour after suitable processing. Kodo millet is an important food crop for vast sections

of the tribal community in Central India. The people in Himalayan foothills use millet as a

cereal, in soups, and for making dense, whole grain bread called Chapatti. In Maharashtra

state flat thin cakes called Roti are often made from sorghum/millet flour and used as the

basis for meals. It is possible to incorporate 50–75% barnyard millet flour in preparation of

rotis, idlies, dosa, chakli idli, pakora, vedai, adai and sweet halwa, kolukattai from finger

millet; Navane sampali, huggi, burfi or kabab from foxtail millet; and Samai dosa, porridge,

paddu and paysam from little millet as traditional recipes in different millet growing states in

India (Veena et al. 2004).

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‘Kodo ko jaanr’ is the most common fermented alcoholic beverage prepared from dry

seeds of finger millet in the Eastern Himalayan regions of the Darjeeling hills and Sikkim in

India. Chhang is also a fermented finger millet beverage popular in Ladakh region in India.

Koozh is another fermented beverage made with pearl or finger millet flour and rice, and

consumed by ethnic communities in Tamil Nadu (Ilango and Antony, 2014). Mahewu is a

non-alcoholic beverage prepared in Zimbabwe from finger millet (1/3) and sorghum (2/3)

malt by traditional fermentation (Gadaga et al. 1999).

Kamaraddi and Shanthakumar, (2003) incorporated the small millet flours to

commercial wheat flour and studied the effect of incorporation of refined millet flours on

chemical, rheological and baking characteristics. It was found that substitution of wheat flour

with millet flours was possible from 10 to 20% level. Barnyard millet and proso millet can be

added 20 and 15% respectively. The optimum level of addition of finger millet, foxtail millet

and little millet was 10%. The increase in level of millets in blends increased the ash content

and decreased the gluten and sedimentation value; loaf volume of dough; per cent damaged

starch and protein whereas crust colour and shape of bread remained unaffected but colour of

crumb changed from creamish white to dull brown.

Singh et al. (2005) prepared composite flours of foxtail, barnyard and finger millet

with wheat flour by adding 10-30% millet flour and observed that addition of milled millet

flour to wheat flour increased the concentration of protein, fat and ash but decreased the

carbohydrates. Addition of milled barnyard millet flour increased significantly the level of

protein, crude fat and total ash contents but whole barnyard flour decreased significantly the

level of protein. With the increase in the level of finger millet flour in the blend, protein

content decreased from 11.59 to 10.99% whereas fat and ash contents increased from 1.06 to

1.37 and 0.55 to 1.37% respectively with non significant variation in carbohydrate content.

Bakery products are popular all over the world and the production has risen by many

folds due to their low cost, varied taste and textured profiles with attractive package and

longer shelf-life to suit easy marketing (Patel and Rao, 1996). The use of millets in bakery

products will not only be superior in terms of fibre content, micronutrients but also create a

good potential for millets to enter in the bakery world for series of value added products

(Verma and Patel, 2013). These are mostly prepared from the wheat flour but efforts are

being made to replace few portion of it with millets in order to provide an alternative and

reduce over dependence on wheat and make gluten free bread. Finger millet and foxtail millet

21

flour can be incorporated in bakery items like biscuits, nan-khatai, chocolate, cheese, cakes,

muffins, etc.

Research findings have revealed that substitution of 40% wheat flour with finger

millet flour in baked products like cake and biscuits is possible. Sehgal and Kawatra, (2007)

prepared sweet, salty and cheese biscuits using pearl millet flour (40- 80%), refined wheat

flour (10-50%) and green gram flour (10%) and found highly acceptable with nonsignificant

difference. Biscuits prepared from maida finger millet flour blend (80:20) can have self life

period of 120 days at 65% RH at 27˚C. Saha et al. (2010) prepared biscuits from flour

composites containing 60:40 and 70:30 (w/w) finger millet : wheat flour and found that

hardness of biscuit dough was more in 60:40 combination than in 70:30 level. The

adhesiveness and resistance of biscuit dough increased with the increasing levels of wheat

flour but expansion of biscuit and breaking strength after baking was more in 70:30

composite than in 60:40. Wheat composite flour (40 g/100 g) had higher water absorption

capacity than in 30 g/100 g composite.

Biscuits prepared by substituting 50% of refined wheat flour with barnyard millet

flour had lower glycemic index, GI (50.17) compared to the GI of wheat biscuits (73.58)

without much difference in the nutrient composition (Srivastava and Singh, 2003). The burfi

was prepared by substituting Bengal gram flour with foxtail millet flour upto 57% and a

control. It was found that both types of burfi had similar sensory score (8.2) but millet burfi

had less GI (51) than control (68). It was also observed that there was significant reduction in

serum glucose and serum cholesterol due to foxtail millet biscuits and burfi.

Vidyavati et al. (2004) prepared millet papad (rolled, circular and thin sheets) by

substituting 50% of mixture of black gram dhal flour and sago flour with finger millet flour

and compared with black gram (Phaseolus mungo) dhal papad. The finger millet flour papad

had higher sensory score of 4.7 on a five point hedonic scale and were rich in Ca (102 mg%

in roasted and 109 mg% in fried) compared to black gram dhal papad (82 mg% in roasted and

99.6 mg% in fried).

Asma et al. (2006) prepared weaning blends composed of 42% sorghum

supplemented with 20% legumes, 10% oil seeds, and 28% additives (sugar, oil, skim milk

powder, and vanillin) as per FAO/WHO/UNU recommendations and processed in a twin-

roller drum dryer. The blends were found to contain good proportion of protein 16.6% to

22

19.3%, fair fiber content of 0.9% to 1.3%, satisfactory energy level 405.8 to 413.2 kcal per

100 g and a healthy iron content of 5.3 to 9.1 mg/100 g. The calcium content ranged from

150 to 220 mg/100 g and lysine content improved considerably for all blends. Thakkar and

Kapoor, (2007) found roti, upma and idli (Indian breakfast recipes) prepared from gum acacia

and finger millet showed lowest glycemic index (41–48%). Similarly Arora et al. (2003)

found that finger and barnyard millet preparations with legumes and fenugreek seeds (Sharma

and Raghuram, 1990) reduce the GI with non-significant difference between them.

Fermented foods like Dosa and Idli are popular and common breakfast foods and even

as the evening meals in many parts of India. Millets are good source of protein but the protein

quality in terms of lysine and tryptophan content is low, hence there is growing emphasis on

the improvement of protein quality. Fermentation not only improves the taste but at the same

time enriches the food value in terms of protein, calcium and fibre, B vitamins, in vitro

protein digestibility and decreases the levels of anti-nutrients in food grain (Chavan and

Kadam, 1989; Maha et al. 2003; Verma and Patel, 2013). Fermentation of the ground

germinated pearl millet grains gives higher protein digestibility. Khetarpaul, (2003)

fermented the pearl millet by inoculating the micro flora namely, Saccharomyces diastaticus,

Saccharomyces cerevisiae and Lactobacillus brevis and incubated at 30 ˚C for 72 h in single

culture, mixed culture and sequential culture fermentation. The samples were oven dried and

ground to fine flour and found that controlled pure culture fermentation did not change the

protein and ash content of pearl millet (sprouted and flour) and increased the starch

digestibility of flour significantly.

Fermentation is one of the most economic and effective measure for reducing

polyphenols and phytic acid significantly and improves HCL-extractability of zinc (Sripriya

et al., 1996; Murali and Kapoor, 2003), iron, copper, calcium and manganese but maximum

reduction is brought out by sequential fermentation. Dry heating and acid treatment of pearl

millet also increases the mineral availability significantly (Arora et al. 2003).

Probiotics are beneficial bacteria. They favorably alter the intestinal microflora

balance, inhibit the growth of harmful bacteria, promote good digestion, boost immune

function and increase resistance to infection (Soccol et al., 2010). Other physiological

benefits of probiotics include removal of carcinogens, lowering of cholesterol, enhancing the

bioavailability of nutrients, alleviation of lactose intolerance and immunostimulation (Modi,

2014). Thus probiotics have a great potential in medicine, prevention and treatment of

23

gastrointestinal infections, inflammations and allergic reactions or as carrier and adjuvant in

vaccination (Shigwedha et al., 2014). There are varied sources of probiotic microorganisms

and among them fermented food are their rich habitats.

The cereal fermented foods and the predominant LAB are generally regarded as safe

(GRAS). Some of the LAB in the fermented food beverages are of human origin and have

been used for centuries knowingly or unknowingly. The dominant microorganisms involved

in the fermentation of cereal-based beverages have no reported health risk to human life.

Germination and probiotic fermentation cause significant improvement in the contents of

thiamine, niacin, total lysine, protein fractions, sugars, soluble dietary fibre and in vitro

availability of Ca, Fe and Zn of food blends (Arora et al. 2011).

Chapter-3

MATERIALS AND METHODS

3.1 Collection of samples

Kodo millet grains were collected from different districts of Himachal Pradesh i.e.

Kangra, Mandi and Hamirpur and brought to the laboratory. All samples were segregated,

cleaned and stored in air tight containers till further use.

3.1.1 Malting

Malting was done by germination of kodo millet seeds by keeping them in dark place

having 25-30˚C temperature and relative humidity. After germination the seedlings were

dried and stored.

3.1.2 Grinding

Native and malted millet grains were separately grinded in a mixer to get whole flour

of 1.0 mm sieve size and stored in air tight containers till further use.

3.2 Evaluation of microbial profile

3.2.1 Isolation of microorganisms

The pooled kodo millet samples of each district were crushed separately in clean

sterilized pestle and mortar by adding distilled water and slurry so prepared was

homogenized for 15 min on vortex mixture. From each of these samples, stock was made by

adding 0.1 ml of sample in 9.9 ml of sterilized distilled water. All samples were diluted by

serial dilution in the dilution range of 10-2 to 10-12. The samples (0.1 ml each) from each

dilution were mounted by spread plate method on sterilized petriplates containing solidified

selected media i.e. de Man, Rogosa, Sharpe (MRS) agar for lactic acid bacteria and nutrient

agar for other bacteria. Lactic acid bacteria containing plates were kept in anaerobic jar and

incubated at 37°C for 48 h in the incubator while nutrient agra plates were kept inverted in

the incubator at 37°C for 24h. After incubation, individual colonies were selected and

purified using streak plate technique on respective selected medium. Pure cultures so

obtained were further preserved on slants and 40% glycerol in deep freezer (-20°C).

25

Composition of de Man, Rogosa, Sharpe (MRS) Agar (Aneja, 2003)

i) Peptone : 10 g

ii) Beef extract : 10 g

iii) Yeast extract : 5 g

iv) Dextrose : 20 g

v) Ammonium citrate : 2 g

vi) Agar : 20g

vii) Distilled water : 1000 ml

viii) pH : 6.5

Composition of Nutrient Agar (Aneja, 2003)

i) Peptone : 5g

ii) Beef extract : 3g

iii) NaCl : 5g

iv) Agar : 20g

v) Distilled water : 1000 ml

vi) pH : 7.0

The isolates were primarily examined according to their colony morphology, catalase

reaction and gram reaction

3.2.2 Gram staining (Gram, 1984)

Cultures were grown in appropriate mediums at 37°C for 24 h under anaerobic

conditions. Cells from fresh cultures were used for gram staining. After incubation, cultures

were transferred aseptically into 1.5 ml eppendrof tubes and centrifuged for 5 min at 6000

rpm. Then, supernatant was removed and cells were resuspended in sterile water. Gram

staining procedure was followed (Gram, 1984). Afterwards, gram reaction of purified isolates

was observed under light microscopy.

3.2.3 Catalase test (Aneja, 2003)

Catalase test was performed for isolates in order to observe their catalase reaction.

Overnight cultures of isolates were grown on selective medium at suitable conditions. After

24 h 3% hydrogen peroxide solution was dropped onto randomly chosen colony. Also fresh

liquid cultures were used for catalase test by dropping 3% hydrogen peroxide solution onto 1

26

ml of overnight cultures. Therefore, isolates which did not give gas bubbles (catalase

negative) were chosen for further study.

3.2.4 Long term glycerol preservation of isolates

Gram positive and catalase negative isolates were preserved in selective broth

medium containing 20% (v/v) glycerol as frozen stocks at -80˚C. The glycerol stocks of

sample were prepared by mixing 0.5 ml of active culture and 0.5 ml selective medium

including 40% sterile glycerol.

3.2.5. Biochemical tests

Following biochemical tests were performed with selected isolates viz. Cellulase test,

MRVP test, Amylase, Pectinase and Casein hydrolysis.

3.2.5.1 Methyl-Red and Voges-Proskauer (MRVP) test (Aneja, 2003)

Tubes of MRVP broth (pH 6.9) were inoculated with the isolated strains separately

followed by the incubation at 35°C for 48 h. Then tubes were examined for change in the

color of methyl red for MR test and crimson-to ruby pink for VP test.

Composition of MRVP broth (Aneja, 2003)

i) Peptone : 7.0 g

ii) Dextrose/Glucose : 5.0 g

iii) Potassium phosphate : 5.0 g

iv) Distilled water : 1000 ml

v) pH : 6.9

3.2.5.2 Casein hydrolysis (Aneja, 2003)

Skimmed milk agar medium was autoclaved at 15 lb pressure for 15 min. The

medium was poured into sterile petridish and isolates were allowed to solidify. The plates

were streaked with each of the isolated strains separately followed by incubation at 37°C for

24 h in an inverted position. Presence or absence of clearance around the line of growth was

examined.

27

3.2.5.3 Cellulase production test (Aneja, 2003)

Czapek-mineral salt agar medium was autoclaved at 15 lb pressure for 15 min. The

medium was poured into sterile petridish and allowed to solidify. The plates were then

inoculated with each of the isolated strains separately followed by incubation at 25°C for 2-5

days in an inverted position. The plates were flooded with 1% hexadecyltrimethyl ammonium

bromide. Presence or absence of formation of zone around the growth was examined.

Composition of Czapek-mineral salt agar medium

i) Sodium nitrate : 2.0g

ii) Potassium phosphate : 1.0g

iii) Magnesium sulphate : 0.5g

iv) Potassium chloride : 0.5g

v) Carboxymethyl cellulose : 5.0g

vi) Peptone : 2.0g

vii) Agar : 20.0g

viii) Distilled water : 1000 ml

ix) pH : 6.5

3.2.5.4 Amylase production test (Aneja, 2003)

Starch agar medium was autoclaved at 15 lb pressure for 15 min. The medium was

then poured into sterile petriplates and allowed to solidify. The plates were streaked with

each of the isolated strain separately followed by incubation at 37°C for 48 h. The plates were

then flooded with iodine solution with a dropper for 30 seconds. Presence or absence of

clearance around the line of growth was examined.

3.2.5.5 Pectolytic production test (Aneja, 2003)

Hankin’s medium was autoclaved at 15 lb pressure for 15 min. The medium was then

poured in sterilized petriplates and allowed to solidify. The plates were then inoculated with

each of the isolated strains separately followed by incubation at 25°C for 2-5 days in an

inverted position. The plates were flooded with 1% hexadecyltrimethyl ammonium bromide.

Presence or absence of formation of zone around the growth was examined.

28

Composition of Hankin’s medium (Aneja, 2003)

i) Pectin : 5.0 g

ii) Monopotassium phosphate : 4.0 g

iii) Disodium phosphate : 6.0 g

iv) Ammonium sulphate : 2.0 g

v) Yeast extract : 1.0 g

vi) Ferrous sulphate : 0.2 g

vii) Magnesium sulphate : 10 mg

viii) Calcium chloride : 1 mg

ix) Boric acid : 10 mg

x) Manganese sulphate : 10 mg

xi) Zinc sulphate : 70 mg

xii) Copper sulphate : 50 mg

xiii) Molybdenum trioxide : 10 mg

xiv) Agar : 15.0 g

xv) Distilled water : 1000 ml

xvi) pH : 5.5±0.5

3.3 SCREENING OF POTENTIAL ISOLATED MICROORGANISMS BYBIT/DISC METHOD

3.3.1 Procurement of indicator microorganisms

Different bacterial indicators viz. Staphylococcus aureus IGMC, Enterococcus

faecalis MTCC 2729, Listeria monocytogens MTCC 839, Clostridium perfringens MTCC

1739, Bacillus cereus CRI, Escherichia coli IGMC, Pseudomonas syringae IGMC,

Leuconostoc mesenteroids MTCC 107, Lactobacillus plantarum CRI and Pectobacterium

carotovorum MTCC 1428 were used to check antagonistic activity of the isolates. All the

indicators used to check the antagonistic activity of given isolates were maintained on

nutrient agar slants at 4˚C. All indicators were sub cultured periodically at 35˚C.

3.3.2 Growth of indicator microorganisms

3.3.2a Growth of isolates and indicator microorganisms

A loopful of each of the selected isolates as well as the indicator bacteria was added

into different test tubes containing 10 ml of respective nutrient broth. The cultures were

incubated at 35˚C until they reached 1.0 OD which was checked periodically after every 24 h.

29

Composition of nutrient broth (Aneja, 2003):

i) Peptone : 5g

ii) Beef extract : 3g

iii) NaCl : 5g

iv) Distilled water : 1000ml

v) pH : 7.0

3.3.3 Antimicrobial activity

The antimicrobial activity of the selected isolates against the bacterial indicator was

checked by bit/disc method.

3.3.3a Bit/Disk preparation of potential isolates (Barefoot and Klaenhammer, 1983).

3.3.3b Lawn preparation of indicators

1 ml inoculum of each indicator microorganisms (1.0 OD) was swabbed properly on

pre-poured sterilized petriplates using sterilized cotton bud. The swabbing was done in such a

way that indicator culture covered the whole surface of nutrient agar plate.

3.3.3c Bit/Disk preparation

The isolated strains (1.0 OD) were grown on respective plate containing selective

medium for their growth for 24 h at 37°C. Then with the help of sharp, sterilized borer bit of

10 mm diameter of isolates was cut. The bit of isolated strains was kept on lawn of indicator

microorganisms with the help of sterilized inoculating needle in such a way that surface on

which isolates grew faced the lawn of indicator microorganism and the activity was noted in

terms of zone of inhibition formed around the bit. The diameter of zone formed was

measured as its zone size.

3.4 MOLECULAR CHARACTERIZATION OF SELECTED BACTERIALISOLATES USING 16S rRNA GENE TECHNIQUE

The best screened bacterial isolates were identified at genomic level by using 16S

rRNA gene technique as given below:

3.4a Isolation of bacterial genomic DNA

Genomic DNA of bacterial isolates were isolated by using DNA prep kit of Banglore

genei, India make, following their protocols as given below:

30

Reagents

i) Lysis buffer I

ii) Lysis buffer II

iii) Wash buffer I

iv) Wash Buffer II

v) Absolute ethanol

vi) Elution buffer

vii) RNase A

viii) Proteinase k

ix) Lysozyme

Procedure

18 h old bacterial culture was centrifuged at 10,000 rpm for 10 min. Supernatant

obtained after centrifugation was discarded and pellet was suspended in 100μl of bacterial

lysis buffer containing lysozyme at a final concentration of 20 μg/ml and incubated at 37°C

for 30 min. Then 180 μl of lysis buffer I and 20μl of Proteinase k was added followed by

incubation at 55°C for 1-3 h. To the solution 4 μl RNase A (100 mg/ml) was added followed

by vortexing. This mixture was incubated at room temperature for 5 min. 200 μl of lysis

buffer II was added followed by slow vortexing. Then incubation of 20 min was given at

70°C. 200 μl of absolute ethanol was added and mixed properly by vortexing. Genei column

was kept in a 2 ml of collection tube and mixture prepared above was added in it which was

centrifuged at 10,000 rpm for 5 min. Collection tube with a flow through was discarded.

Genei column was kept in a fresh 2 ml collection tube in which 500 μl of wash buffer I

(diluted with 3 volumes of ethanol) was added. Column containing wash buffer was spinned

at 10,000 rpm for 1 min. Collection tube with wash sample was discarded. Then the column

was again kept on fresh collection tube, 500 μl of wash buffer II (diluted with 3 volumes of

ethanol) was added in column followed by centrifugation at 10,000 rpm for 3 min. Wash

fraction collected after centrifugation was discarded and collection tube was retained for next

step. Spin the empty column for 2 min at 10,000 rpm. After spinning collection tube was

discarded and Genei column was placed in a new 1.5 ml vial and incubated for 2 min at 70°C

at dry bath. 200 μl of elution buffer was added in a column which was incubated for 5 min. at

room temperature. Finally DNA was eluted by spinning column at 10,000 rpm for 1-2 min.

Eluted DNA was stored at -20°C.

31

3.4b PCR amplification of 16S rRNA region

PCR amplification was done to confirm the identity of the bacterial strain and the

small sub unit 16S rRNA genes were amplified from the genomic DNA with 16SU

(5’AGAGTTTGATCMTGGCTCAG3’) and 16SD (5’ACCTTGTTACGACTT3’) universal

primers to get an amplicon size of 1500 bp. Amplification were carried out in 50 μl reaction

volume consisting of 10 x buffer, 5.0 μl; 2mM dNTPs, 5.0 μl; 3 U/μl Taq DNA polymerase,

0.33 μl; 100ng/μl of each primer, 2 μl; 50 – 100 ng template DNA, 1μl and H2O 34.67 μl in a

Astech thermocycler (Japan make) using the PCR conditions 95°C for 2 min (denaturation),

58°C for 1 min (annealing) and 72°C for 1 min (extention). The total numbers of cycles were

40, with the final extension of 72°C for 10 min. The amplified products (50 μl) were size

separated on 0.8% agarose gel prepared in 1% TAE buffer containing 0.5 μg ml-1 ethidium

bromide and photographed with the gel documentation system (Alpha Imager 2200). A 100

bp ladder was used as molecular weight size markers.

3.4c Purification of the PCR product

The PCR product was purified from contaminating products by electro elution of the

gel slice containing the excised desired fragment with Qiaquick gel extraction kit (Sigma).

The elution was carried out in 30 μl of nuclease free water.

3.4d Nucleotide sequencing

Sequencing preparation- The PCR amplicons obtained by amplifying PCR products

was diluted in Tris buffer (10 mM, pH 8.5), dilutions used was 1:1000. In order to obtain the

DNA concentration required for sequencing (30 ng/μl), the sequencing reaction required 8 μl

DNA. The primer used in all sequencing reactions was 16 SU at a concentration of 3 μM.

Sequencing was then performed using an automated sequencer (ABI PRISM 310, Applied

Biosystem, USA) by Europhins, India Pvt. Ltd.

3.4e BLAST analysis

Translated nucleotide sequence was then analyzed for similarities by using BLASTN

tool (www.ncbi.nlm.nih.gov:80/BLAST/).

3.4f Inference

Isolate KR5 was identified as Paenibacillus jamilae.

32

3.5 EVALUATION OF NUTRITIONAL POTENTIAL OF MILLET GRAINS

3.5.1 Proteins (Ranganna, 1997)

Sample of 0.5 – 1 g weight along with 0.5 g digestion mixture (2.5 g SeO2 + 20 g

CuSO4.5H2O + 100 g K2SO4) was digested in 25 ml concentrated H2SO4 for 5h or till it

became colourless. Digestion flasks were allowed to cool overnight at room temperature. The

digest was transferred to 100 ml capacity volumetric flask and made up to volume with glass

distilled water. The nitrogen was estimated by modified Kjeldhal method.

Nitrogen (%) =(Sample titre - blank titre) x Normality of HCL x 14 x 100

Weight of sample x 1000

Crude protein (%) in the sample was then calculated by multiplying percent nitrogen

with the factor 6.25.

3.5.2 Carbohydrates (Sadasivam and Manickam, 1992)

The phenol-sulphuric acid method was used to estimate carbohydrates as described by

Sadasivam and Manickam, (1992). To the diluted sample, 1ml of phenol solution [5 % (v/v)]

was added and mixed properly in a test tube. Then, 5 ml of 96 % (v/v) sulphuric acid was

added and shaken well. The tubes were kept in a water bath at 25-30°C for 20 min, the

absorbance was recorded at 490nm and compared with standard curve prepared with glucose.

Standard of glucose was prepared. The standard curve was prepared using different

concentrations i.e. 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml of glucose.

3.5.3 Starch content (Hedge and Hofreiter, 1962)

0.5 g of the sample was homogenized in hot 80% (v/v) ethanol and centrifuged to

retain the residue and was dried. The residue was added with 5.0 ml of water and 6.5 ml of

52% (v/v) perchloric acid and extracted at 0°C for 20 min. The sample was centrifuged at

5000 rpm for 20 min and the supernatant was collected. 0.1 ml of the supernatant was pipette

out and make up the volume to 1 ml. The standard curve was prepared using different

concentrations i.e. 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml of glucose.

3.5.4 Antioxidant activity (Free Radical Scavenging Activity, FRSA) (Brand et al.1995)

DPPH (2, 2-diphenyl-1-picrylhydrazyl) was used as a source of free radical. A

quantity of 3.9 ml of 6×10-5 mol/L DPPH in methanol was put into a cuvette with 0.1 ml of

33

sample extract and decrease in absorbance was measured at 515 nm for 30 min or until the

absorbance become steady. The remaining DPPH concentration was calculated using the

following equation:

Free radical scavenging activity (%) = Ab(b)- Ab(s)

Where

Ab(b) = Absorbance of blank

Ab(s) = Absorbance of sample

3.5.5 Phenols (Bray and Thorpe, 1954)

The amount of total phenols in the sample was determined with the Folin-Ciocalteau

reagent according to method of Bray and Thorpe, (1954) using catechol as a standard. 1 gm

of sample was taken and mixed with 10 ml of 80 % ethanol in pestle and mortar followed by

centrifugation for 20 min at 1000 rpm and then filteration was done. Filterate was evaporated

in an oven up to dryness and residue was dissolved in 5 ml distilled water. 0.2 to 2 ml aliquot

was taken in separate test tubes and volume was made upto 3 ml with water. Then 0.5 ml

Folin-ciocalteau reagent was added. After 3 min, 2ml of sodium carbonate [20% (w/v)] was

added and mixed. Test tubes were placed in boiling water bath for 1 min and then cooled.

Optical density of sample was recorded at 650 nm with the help of UV/Vis

spectrophotometer. The concentration was determined as per the standard procedure from

standard curve. The standard curve was prepared using different concentrations i.e. 0.2, 0.4,

0.6, 0.8 and 1.0 mg/ml of catechol and results were expressed as mg/100g on fresh weight

basis.

3.5.6 Minerals (Ranganna, 1997)

Minerals viz. iron, phosphorus and magnesium were estimated using wet digestion

method. The estimation was performed in accordance with instrument setting, standardization

and reading with reference to manufacturer’s specification as follow in Table 3.

Table 1: Mineral estimation

1. PhosphorusVando-molybdate phosphoric yellowcolor method

2. IronAtomic absorption spectrophotometer,AA-175 series, Australia

3. MagnesiumAtomic absorption spectrophotometer,AA-175 series, Australia

34

3.5.7 Crude fiber (AOAC, 2007)

Distilled water (200 ml) was added to the sample (100 g) and the contents were

brought nearly to a boil. After adding 25 ml of 50% (w/v) sodium hydroxide solution, the

contents were boiled for five minutes. The material was transferred to previously weighed

screen and washed thoroughly with water until whole of the sodium hydroxide had been

removed. The presence of sodium hydroxide was checked by using phenolphthalein

indicator. The contents were dried at 100oC for 2 h in hot air oven and fibre content was

expressed in percentage.

Fiber content (%) =( )( ) × 100

3.5.8 Crude fat content (Folch, 1957)

Dried sample of 5 g was extracted with petroleum ether in Soxhlet extraction

apparatus for 6 hr. The ether extract was filtered in pre-weighed beakers. Petroleum ether was

evaporated completely from the beakers and the increase in weight of beaker represented the

fat content. The fat (%) obtained was estimated as (g fat/ g dry biomass) × 100

3.5.9 Flavonoids (Madaan et al. 2012)

The aluminum chloride method was used for the determination of the total flavonoid

content of the sample.

Materials

i. Methanolii. Sodium potassium tartarate

iii. Aluminium chloride

Procedure

Aliquots of extract solutions were taken and made upto the volume of 3ml with

methanol. Then 0.1ml aluminium chloride (10% w/v), 0.1ml sodium potassium tartarate and

2.8 ml distilled water were added sequentially. The test solution was vigorously shaken.

Absorbance at 415 nm was recorded after 30 min of incubation. A standard calibration plot

was generated at 415 nm using known concentrations of quercetin.

35

Calculations

The concentrations of flavonoid in the test samples was calculated from the

calibration plot and expressed as mg quercetin equivalent /g of sample.

3.6 Extraction of polyphenols (Banerjee et al. 2012)

The defatted 1g of sample was suspended in 100ml of different polar solvents

(methanol, acetone and water) to extract the polyphenols. The polar solvents were acidified

with 1% HCl and the extraction was carried out by refluxing each of the extract for about 60

min using a water bath. The individual extracts were centrifuged at 5000 rpm for 20 min and

clear supernatant was collected. The residue was re-extracted with 50ml of fresh solvent and

the process was repeated till the residue tested negative (with Folin-Ciocalteu’s phenol

reagent) for polyphenols. The extracts were pooled, freeze dried and used for further studies.

3.6.1 Antagonistic activity of fractioned polyphenolics

3.6.1a Procurement of indicator microorganisms

Different bacterial indicators viz. Staphylococcus aureus IGMC, Enterococcus

faecalis MTCC 2729, Clostridium perfringens MTCC 1739 and Bacillus cereus CRI were

used to check antagonistic activity of the isolates. All the indicators used to check the

antagonistic activity of given isolates were maintained on nutrient agar slants at 4˚C. All

indicators were subcultured periodically at 35˚C.

3.6.1b Growth of indicator microorganisms

A loopful of each of the selected isolates of indicator bacteria was added into a test

tube containing 10 ml of nutrient broth. The cultures were incubated at 35˚C until they

reached 1.0 OD which was checked periodically after every 24 h.

Composition of nutrient broth:

i) Peptone : 5g

ii) Beef extract : 3g

iii) NaCl : 5g

iv) Distilled water : 1000ml

v) pH : 7.0

36

3.6.2 Antimicrobial activity

The antimicrobial activity of the selected isolates against the indicator bacterial strains

was checked by spot method.

3.6.2 Spot method

3.6.2a Lawn preparation of indicators

As mentioned in section 3.3.3b

3.6.2b Spot on lawn preparation (Schillinger and Lucke, 1989)

In spot method the sample was applied as a spot on the respective plate containing

selective medium for their growth. The sample was taken with the help of micropipette and

was spotted on the plate. The plates were then incubated at 37˚C for 24 h. the diameter of

zone formed was measured as its zone size.

3.7 Thin layer chromatography for identification of polyphenols (Sadasivam andManickam, 1992)

TLC was used to characterize, separate and identify polyphenols

Materials

i. Glass plate (20×20 cm or 20×10 cm)

ii. Glass tank with lid

iii. Spreader

iv. Developing solvents

v. Adsorbent silica gel G

vi. Sample

vii. Standards

viii. Spraying agent

3.7.1 Procedure

3.7.1a Preparation of plates

Slurry of adsorbent was prepared in water in the ratio 1:2. The slurry was stirred

thoroughly for 1-2 min and poured into the glass plate. The slurry over the glass plate was

coated uniformly from one end to other. The plate was kept for drying at room temperature

for 15-30 min. The plate was then heated in an oven at 100-120˚C for 1-2 h to remove

moisture and to activate the adsorbent on the plate.

37

3.7.1b Sample application

The sample i.e. polyphenols extracted in methanol and acetone, was applied 2.5cm up

from the edge of the plate by means of micropipette as small spot. All the spots were placed

at equal distance from one end of the plate. The samples were then dried.

3.7.1c Development chromatogram

The developing solvent i.e. ethylacetate: formic acid: water (90 : 6 : 6) was poured

into the tank to a depth of 1.5 cm. It is allowed to stand for at least an hour with a cover plate

over the top of the tank to ensure that the atmosphere within the tank becomes saturated with

the solvent vapour. After that the cover plate was removed and the thin layer plate was placed

vertically in the tank with the spotted end dipped in the solvent. The cover plate was again

placed. The separation of compound was observed as the solvent moves upward. When the

solvent reached at the top of the plate, it was removed from the tank.

3.7.2 Identification

Spraying agent i.e. iodine was used for identification and the analysis was done on the

basis of Rf value.

Calculation

Distance travelled by sample (cm)

Distance travelled by mobile phase (cm)

3.8 High Performance Liquid Chromatography (HPLC) of polyphenols forquantification of polyphenolic compounds (Banerjee et al. 2013)

The polyphenols extract was membrane (0.45 μ) filtered and an aliquot (20 μl) of the

filtrate was fractionated in a reverse phase HPLC system [Shimadzu (Kyoto, Japan)], LC-8A

integrated system controller, a Spherisorb C-18 reverse-phase column (250 × 4.6 mm; ODS

2; 5 μm particle size), Waters corp., Massachusetts, USA and a Helwlett Packard 1040 UV

diode array detector with an attached HP analysis computer and data storage system. The

gradient elution schedule was standardized based on the resolution of the sample, with 50

min run of 15% methanol and 1% of acetic acid in water followed by a linear gradient to 40%

methanol over 40 min at a flow rate of 1 ml / min. Elutes were detected by a Waters 2487

dual wavelength detector at 295 nm and the peaks were recorded. Scanning was performed

Rf =

38

from 200 to 600 nm. The constituent phenolic compounds were identified by comparing the

retention times and also the UV–visible spectra of the pure standards to indicate the

preparations of standards and the range of calibration curves. The analysis were replicated (n

= 3) and the contents were given as mean values, plus or minus the standard deviation. The

results were expressed as milligrams of each compound per 100 g of dry weight.

3.9 Inter Compatibility test of bacterial probiotic strains

Compatibility of different isolates was checked by using cross streak method.

3.9.1 Cross streak method (Barefoot and Klaenhammer, 1983)

In this method, on the prepoured properly sterilized selective medium i.e. MRS, two

bacterial isolates were cross streaked against each other on each petriplate i.e Pediococcus

acidilactici L1 and Lactobacillus plantarum L2, Pediococcus acidilactici L1 and

Lactobacillus fermentum F3 and Lactobacillus plantarum L2 and Lactobacillus fermentum

F3 Then these plates were incubated at 37˚C for 24 h and their growth patterns were noticed.

The strains showing best compatibility were chosen for probiotic consortia formulations.

3.10 Formulation of functional foods

3.10.1 Malting (Verma and Patel, 2013)

Kodo Millet seeds were soaked in water for germination. During soaking the soaked

water was required to be changed once or twice to prevent excessive growth of

microorganisms. After soaking, millet seeds were germinated and their germination time was

standardized i.e. 12, 24, 36, 48 and 72 h. After germination, the seeds were dried at a

moderate temperature not exceeding 75˚C in an oven. The sprouted grains were dried to final

moisture of nearly 10-12%. These grains were then roasted uniformly at 70-80˚C by using

conventional toasting pan and grinded. The malt so obtained was then pulverized to convert it

into RTE form.

3.10.2 Fortification of malt with probiotics

In house potential probiotic strains, Pediococcus acidilactici L1, Lactobacillus

plantarum L2 and Lactobacillus fermentum F3 with accession number KM251713,

KM251714 and KC242235 were added into the malt extract. The schematic representation of

beverage preparation has been given below:

39

3.10.3 Malt beverage

3.10.3.1 Ingredients

Kodo millet grains 250 g

Autoclaved distilled water 1500 ml

Sugar 3 %

P. acidilactici L1 108 cfu/ml

L. plantarum L2 108 cfu/ml

L. fermentum F3 108 cfu/ml

3.10.3.2 Recipe

Kodo millet grains

Washing

Soaking (12h)

Germination (48-72 h)

Drying (75˚C, 6h)

Grinding (3 min)

Slurry

(1500 ml sterilized water)

Boiling (100˚C, 20 min,)

Filteration

Sugar

Heating (100˚C, 15 min,)

Cooling (25˚C)

Inoculation (Probiotic culture)

40

Set I Set II Set III Set IV

P. acidilactici L1 L. plantarum L2 L. fermentum F3 P. acidilactici L1

(1.5 ml) (1.5 ml) (1.5 ml) +

L. plantarum L2

+

L. fermentum F3

(0.5 ml each)

Transfer of inoculated malted beverage (100 ml each) to sterilized containers

Fermentation (37°C, 4 h)

Refrigeration (4°C)

Evaluation of quality attributes

Cold storage

Preparation of malt beverage

3.10.3.3 Sensorial Evaluation

Nine point hedonic scale method as given by Amerine et al. (1965) was followed for

conducting the sensory evaluation of probiotic food products. The panel of 10 judges were

selected to evaluate malt beverage.

3.10.3.4 Microbial evaluation during storage

The colony count was observed during storage period by standard spread plate

method. MRS agar was used to enumerate lactic acid bacteria while nutrient agar, yeast

extract agar and PDA were used to enumerate total aerobic mesophilic bacteria including

yeast and mold, respectively.

41

3.10.3.5 Nutritional evaluation of malt beverage:

3.10.3.5a Proteins: as mentioned in section 3.5.1

3.10.3.5b Carbohydrates: as mentioned in section 3.5.2

3.10.3.5c Antioxidant: as mentioned in section 3.5.4

3.10.3.5d Crude fibers: as mentioned in section 3.5.7

3.10.3.5e Total fats: as mentioned in section 3.5.8

3.10.3.5f Statistical analysis

Data pertaining to the physicochemical attributes of probiotic product was analyzed

by Completely Randomized Design (CRD). Data on sensorial evaluation of probiotic

products were analyzed by using Randomized Block Design (RBD) as described by Mahony

(1985).

3.10.4 Ready to Eat (RTE) Porridge

RTE porridge was prepared as given below:

3.10.4.1 Ingredients

i. Kodo millet seeds 10 g

ii. Barley seeds 10 g

iii. P. acidilactici L1 108 cfu/ml

iv. L. plantarum L2 108 cfu/ml

v. L. fermentum F3 108 cfu/ml

3.10.4.2 Recipe

Kodo Millet seeds: Barley seed(50 : 50)

Soaking (6h)

Consortium of probiotics[P.acidilactici L1+ L.plantarum L2+ L.fermentum F3 (0.5 ml each)]

Drying (75̊ C, 6h)

Roasting (80˚C, 5 min)

Grinding (5 min)

RTE Porridge

42

3.10.4.3 Sensorial evaluation: same as in section 3.10.3.3

3.10.4.4 Microbial evaluation during storage

Bioavailability of this product was carried out after 30 days of storage as mentioned in

section 3.10.3.4.

3.10.4.5 Nutritional evaluation of RTE porridge

3.10.4.5a Proteins: same as in section 3.5.1

3.10.4.5b Carbohydrates: same as in section 3.5.2

3.10.4.5c Antioxidant: same as in section 3.5.4

3.10.4.5d Crude fibers: same as in section 3.5.7

3.10.4.5e Total fats: same as in section 3.5.8

3.10.4.5f Statistical analysis: same as in section 3.10.3.5f

3.10.5 Multigrain bread

3.10.5.1 Formulation of composite flour

Multigrain flour was prepared by combining wheat and kodo millet in different ratios

i.e. 3:7, 4:6, 5:5, 6:4 and 7:3 (wheat: kodo millet). Among these, 5:5 ratio of wheat and kodo

millet was standardized for further studies.

3.10.5.2 Preparation of dough and fermentation

3.10.5.3 Ingredients

i. Millet flour : 50 g

ii. Wheat flour : 50 g

iii. Yeast : 1 mg

iv. Sugar : 10 g

v. Salt : 5 g

vi. Oil : 10 ml

43

3.10.5.4 Recipe:

Multigrain flour

[50:50 (wheat: kodo millet)]

Salt (5 g) Sugar (10 g)

Yeast (108 cfu/ml) Oil (10 ml)

Knead (water, 100 ml)

Fermentation (37̊ C, 2 h)

Baking

(450˚F for 30 min)

Flow chart of steps followed in preparation of bread

3.10.5.5 Nutritional evaluation of bread

3.10.5.5a Proteins: same as in section 3.5.1

3.10.5.5b Carbohydrates: same as in section 3.5.2

3.10.5.5c Total fat: same as in section 3.5.8

3.10.5.5d Crude fiber: same as in section 3.5.7

3.10.5.5e Antioxidants: same as in section 3.5.4

3.10.5.5f Sensory evaluation: same as in section 3.10.3.3

3.10.3.4 Microbial evaluation during storage

Bioavailability of multigrain bread in dough after 6 h of fermentation of dough on

YEMA.

3.10.5.5f Statistical Analysis: same as in section 3.10.3.5f

Chapter-4

RESULTS AND DISCUSSION

4.1 Collection of samples

Kodo millet seeds were collected from different sites of 3 districts of Himachal

Pradesh viz. Mandi, Kangra and Hamirpur. The selected kodo millet grains were cleaned,

pooled area wise and then stored in sterilized air tight glass jars.

4.1.1 Grinding

A part of native millet seeds was grinded in a mixer and kodo flour was stored in air

tight containers for further use.

4.1.2 Malting

Another part of the selected kodo millet grains was malted. Malting induces important

beneficial biochemical changes and microflora. The germination of millet seeds was done by

keeping them in dark place having 25-30˚C temperature and relative humidity (Plate 1). After

germination the seedlings were dried and then grinded in a mixer to a sieve size 1.0 mm and

stored till further used.

4.2 Isolation of microorganisms

4.2.1 Isolation from native (raw) sample

Since, unique natural microflora are associated with kodo millet grains and flour, so

an attempt has been made to isolate them and to study their different characteristics. In total,

13 isolates from native grinded millet were isolated aerobically and anaerobically on selective

medium of respective pH. The morphological characters i.e. color, form, elevation and

margins of potential isolates were noted down and presented in Table 1 and Fig. 1 (a, b and

c). The color of colonies varied from white, cream and yellowish cream. Majority of isolates

were cream and white in color i.e. 46% of each and remaining 8% were yellowish cream. All

isolates exhibited mainly two types of forms i.e., circular and irregular. Out of 13 isolates,

isolate KR3, KR6, KR9, KR7, KR8, SR1, SR4, SR6 (62%) were circular, whereas, KR5, SR,

SR2, SR3, SR8 (38%) were irregular. All isolates had two different margins i.e. entire and

45

undulated and had different elevations (flat, raised and convex). Out of 13 isolates, 10

isolates had entire margin i.e. KR3, KR6, KR9, KR7, KR8, SR1, SR3, SR4, SR6 and SR8

(77%) and 3 had undulated margin i.e. KR5, SR and SR2 (23%).

Majeed et al. (2015) showed the morphological characteristics of bacterial isolates

from wheat. In total 5 strains were isolated, out of which maximum were round and wavy in

shape. The bacteria showed white to milky white colonies with variable sizes and margins.

The cells were mostly motile, rod shaped showing Gram-negative reaction.

Table 1: Isolation of bacteria from raw kodo millet showing their morphologicalcharacteristics

Sr. no. Name ofisolate

Food source Color Form Margin Elevation Texture

1 KR3 Raw kodo millet Cream Circular Entire Flat Smooth

2 KR5 Raw kodo millet Yellowish cream Irregular Undulate Raised Smooth

3 KR6 Raw kodo millet Cream Circular Entire Convex Smooth

4 KR9 Raw kodo millet White Circular Entire Flat Smooth

5 KR7 Raw kodo millet Cream Circular Entire Flat Smooth

6 KR8 Raw kodo millet White Circular Entire convex Smooth

7 SR Raw kodo millet Cream Irregular Undulate Flat Smooth

8 SR1 Raw kodo millet Cream Circular Entire Flat Smooth

9 SR2 Raw kodo millet White Irregular Undulate Raised Mucoid

10 SR3 Raw kodo millet White Irregular Entire Raised Smooth

11 SR4 Raw kodo millet White Circular Entire Flat Mucoid

12 SR6 Raw kodo millet White Circular Entire Flat Smooth

13 SR8 Raw kodo millet Cream Irregular Entire Raised Smooth

4.2.2 Isolation from malted sample:

In total, 16 microbial isolates were isolated from malted powdered sample aerobically

and anaerobically on selective medium of respective pH. The morphological characters i.e.

color, form, elevation and margins of potential isolates were noted down and presented in

Table 2, Fig. 2 (a, b and c). The color of colonies varied from white, cream, yellowish cream

and transparent. Majority of isolates were cream in colour i.e. 56%, 19% were yellowish

cream, 12% were white and 13% were transparent in colour. All isolates exhibited form

Plate 1: Germination of kodo millet grains

a. Color

Fig. 1. Morphology of microorganisms isolated from raw kodo millet

Cream (46%)

White (46%)

Yellowish cream(8%)

b. Form

circular (62%)

irregular (38%)

c. Margin

entire (77%)

undulate (23%)

Fig. 2. Morphology of microorganisms isolated from malted kodo millet

a. Color

cream (56%)yellowish cream (19%)white (12%)transparent (13%)

b. Form

circular (62%)

irregular (19%)

punctiform (19%)

c. Margin

entire (69%)

undulate (31%)

46

mainly in three types i.e. circular, irregular and punctiform. Out of 16 isolates, 10 isolates

(62%) KM4, KM8, SM1, SM5, SM6, SM9, SM10, SM7, KM9 and KM5 were circular, 3

isolates i.e. SM4, SM3 and KM3 (19%) were irregular and the remaining 3(19%) i.e. KM7,

SM8 and KM1 were punctiform. All isolates had two different margins i.e entire and

undulated margin and had different elevations (flat, raised, convex and umbonate). Out of

total, 11 isolates had entire margin i.e. KM7, SM1, SM4, SM6, SM5, SM8, SM9, SM10,

KM1, KM3 and KM5 while 5 had undulated margin i.e. KM4, KM8, SM3, SM7 and KM9.

Table 2: Isolation of bacteria from malted kodo millet showing their morphologicalcharacteristics

Sr. no. Name ofisolates

Food source Color Form Margin Elevation Texture

1 KM4 Malted kodomillet

Yellowishcream

Circular Undulate Umbonate Smooth

2 KM7 Malted kodomillet

Cream Punctiform Entire Flat Smooth

3 KM8 Malted kodomillet

Cream Circular Undulate Raised Smooth

4 SM1 Malted kodomillet

Cream Circular Entire Raised Smooth

5 SM4 Malted kodomillet

Cream Irregular Entire Raised Smooth

6 SM6 Malted kodomillet

Yellowishcream

Circular Entire Flat Mucoid

7 SM5 Malted kodomillet

Cream Circular Entire Flat Mucoid

8 SM8 Malted kodomillet

Cream Punctiform Entire Convex Smooth

9 SM9 Malted kodomillet

Yellowishcream

Circular Entire Umbonate Smooth

10 SM3 Malted kodomillet

Cream Irregular Undulate Flat Mucoid

11 SM10 Malted kodomillet

Cream Circular Entire Flat Smooth

12 SM7 Malted kodomillet

White Circular Undulate Flat Smooth

13 KM1 Malted kodomillet

Transparent Punctiform Entire Flat Mucoid

14 KM3 Malted kodomillet

White Irregular Entire Flat Smooth

15 KM9 Malted kodomillet

Cream Circular Undulate Flat Smooth

16 KM5 Malted kodomillet

Transparent Circular Entire Raised Smooth

Malt is partially germinated barley. Malting process, which involves soaking,

germination and drying, aims to change grains into malt with high enzymes and vitamins

content. Malted finger millet (sprouted seeds) is a nutritious food which is easily digested and

recommended particularly for infants and elder people. In terms of malting qualities, finger

47

millet could be the key to provide cheap and nutritious foods for solving the malnutrition that

kills millions of infants throughout the tropics. Malting is the process of germinating finger

millet to activate enzymes that break down the complex structures of starch into sugars and

other simple carbohydrates that are easy to digest. Because of its nutritive properties, the crop

has medicinal value and it is used in treatment of measles, anemia and diabetes (Taylor,

2004). Malting of finger millet improves its digestibility, sensory and nutritional quality.

Malting characteristics of finger millet are superior to other millets (Pawar et al. 2007).

Similar studies have been cited in literature, where Kwarteng et al. (2010) isolated a

total of 70 isolates of lactic acid bacteria from the traditional processing of millet into fura, a

popular millet based dumpling. The selected isolates were gram positive and catalse negative.

Among these rods accounted for 36 isolates, whereas cocci accounted for 34 isolates. The

LAB were grouped into five genera belonging to Lactobacillus (51.42%), Pediococcus

(21.4%), Streptococcus (14.3%), Leuconostoc (8.5%) and Enterococcus (4.3%).

Geetha and Kalaichelvan, (2010) reported that during the finger millet fermentation,

22 lactic acid bacteria isolates were taken at different stages of fermentation and subjected to

characterization study. Among them 36% were cocci and tetrad shaped, hence identified as

Pediococcus sp; 27% were heterofermentative Lactobacillus. Others were homofermentative

Lactobacillus sp (18%) and Leuconostoc sp. (18%). About 20 lactic acid bacteria isolates

were isolated from pearl millet fermentation. The results of characterization are as follows:

45% of isolates belongs to Leuconostoc sp; 35% were of homofermentative Lactobacillus

group and rest belong to Pediococcus (5%) and heterofermentative Lactobacillus (10%)

group.

4.3 Screening and identification of potential microorganisms

4.3.1 Physiological and biochemical characterization

Physiological and biochemical characterization of isolated potential microorganisms

from raw and malted kodo millet samples had been done and their characteristics were noted

down as given in Table 3 and 4 and Fig. 3( a, b, c and d) and 4(a, b, c and d). Gram staining

was done to check Gram’s reaction and the shape of bacteria. Gram positive microorganisms

appeared blue-purple colored, while gram negative appear pink by gram staining. The results

showed that 54% (7) and 46% (6) of bacterial cultures of raw millet were rods and coccus

Fig. 3. Biochemical characteristics of bacterial isolates from raw kodo millet

a. Shape

rods (54%)

coccus(46%)

b. Gram's reaction

gram +ve(92%)

gram -ve(8%)

c. Catalase test

catalase+ve (31%)

catalase -ve (69%)

Aerobes(46%)

Obligateanaerobes(31%)

Facultativeanaerobes(23%)d. Mode of growth

Fig. 4. Biochemical characteristics of bacterial isolates from malted kodo millet

a. Shape

coccus(56%)rods(44%)

b. Gram's reaction

gram +ve(87%)

gram -ve(13%)

c. Catalase test

catalase+ve(25%)

catalase -ve (75%)

d. Mode of growth

aerobes(44%)

facultativeanaerobes(31%)obligateanaerobes(25%)

48

(Fig. 3a), while in malted millet sample 56% (6) of bacterial cultures were coccus and 44%

(7) were rods respectively (Fig. 4a).

Further catalase test of the selected isolates was performed and 9 isolates out of 13 in

raw millet were found to be catalse negative, while 4 were catalase positive as shown in Fig

3(c), whereas in case of malted millet 12 were catalase negative and 4 were catalase positive

(Fig. 4 c). Catalase is an enzyme produced by many microorganisms that breaks down the

hydrogen peroxide into water and oxygen and causes gas bubbles. The formation of gas

bubbles indicates the presence of catalse enzyme i.e. 2H2O2 2H2O + O2

Various biochemical tests have been performed viz. indole, amylase, casein, pectin,

cellulase and MRVP test with isolated potential bacterial isolates as shown in Table 3 and 4.

In raw millet, only 4 isolates out of 13 were noticed to hydrolyze casein while 9 were unable

to do so. Methyl Red and Voges-Proskauer (MRVP) test was performed for high acid

production during carbohydrate fermentation. In raw millet KR3, KR5, KR6, SR1, SR8 and

SR6 showed poor acid production while, rest of them i.e. KR7, KR8, KR9, SR, SR2, SR3,

SR4, KR9 and KR8 were found to be high acid producers, while only KM4 and SM1 in

malted millet sample showed poor acid production, the remaining 14 were high acid

producer.

After physiological and biochemical characterization, bacterial isolates of raw and

malted kodo millet, were tentatively identified as lactic acid bacteria (LAB) (Lactococcus and

Lactobacillus), Bacillus and Coccus. Mode of growth of isolated potential microorganisms

was ascertained on the basis of sensitivity to oxygen. The growth conditions revealed that

46% isolates were aerobic, 31% were obligatory anaerobic and 23% were facultative

anaerobic and in case of raw kodo millet, while in malted kodo millet 44% were aerobic,

31% were facultative anaerobic and 25% were obligatory anaerobic. Lactic acid bacteria is an

important group of bacteria being placed in group 19 with important biochemical characters

that is gram’s reaction , catalase negative, casein hydrolysis as authenticated in Bergey’s

Manual of Determinative Bacteriology (7th Edn.).

Lactic acid bacteria (LAB) form a phylogenetically diverse group, widely distributed

in nature and defined as gram-positive, non-sporulating and catalase negative, which are

devoid of cytochromes, fastidious, acid tolerant and strictly fermentative bacteria that secrete

lactic acid as their major end product of sugar fermentation (Pelinescu et al. 2009).

49

Table 3: Biochemical characteristics of bacterial isolates from raw kodo millet

Sr.no.

Isolates Gram staining Catalasetest

Indole Amylase Casein Pectin Cellulase MRVP Mode of growth Tentative identification

Shape Gram’sreaction

1 KR3 Rods +ve +ve -ve +ve +ve -ve -ve MR-VP+ Aerobes Bacillus

2 KR5 Rods +ve +ve -ve +ve +ve -ve -ve MR-VP+ Aerobes Bacillus

3 KR6 Rods -ve +ve -ve +ve -ve -ve -ve MR-VP+ Aerobes Bacillus

4 KR9 Coccus +ve -ve -ve +ve -ve -ve -ve MR+VP+ Aerobes Coccus

5 KR7 Coccus +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Aerobes Coccus

6 KR8 Coccus +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactococcus

7 SR1 Coccus +ve -ve -ve -ve -ve -ve -ve MR-VP+ Aerobes Coccus

8 SR2 Coccus +ve -ve -ve +ve -ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactococcus

9 SR3 Rods +ve -ve -ve +ve -ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactobacillus

10 SR Coccus +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Obligateanaerobes

Lactococcus

11 SR4 Rods +ve -ve -ve -ve +ve -ve -ve MR+VP+ Obligateanaerobes

Lactobacillus

12 SR8 Rods +ve -ve -ve -ve +ve -ve -ve MR - VP+ Obligateanaerobes

Lactobacillus

13 SR6 Rods +ve +ve -ve +ve -ve -ve -ve MR- VP+ Obligateanaerobes

Lactobacillus

50

Table 4: Biochemical characteristics of bacterial isolates from malted kodo millet

Sr.no.

Isolates Gram staining Catalasetest

Indole Amylase Casein Pectin Cellulase MRVP Mode of growth Tentative identification

Shape Gram’sreaction

1 KM7 Rods +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Aerobes Bacillus

2 KM4 Rods +ve +ve -ve -ve -ve -ve -ve MR -VP+ Aerobes Bacillus

3 KM8 Rods +ve -ve -ve +ve -ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactobacillus

4 SM1 Rods -ve +ve -ve +ve +ve -ve -ve MR-VP+ Aerobes Bacillus

5 SM4 Coccus +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Aerobes Coccus

6 SM6 Coccus -ve +ve -ve +ve +ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactococcus

7 SM5 Rods +ve -ve -ve -ve -ve -ve -ve MR +VP+ Facultativeanaerobes

Lactobacillus

8 SM8 Coccus +ve -ve -ve -ve -ve -ve -ve MR+ VP+ Facultativeanaerobes

Lactococcus

9 SM9 Coccus +ve -ve -ve +ve -ve -ve -ve MR+ VP+ Aerobes Coccus

10 SM3 Rods +ve -ve -ve -ve +ve -ve -ve MR+ VP+ Aerobes Bacillus

11 SM10 Rods +ve +ve -ve +ve -ve -ve -ve MR+ VP+ Aerobes Bacillus

12 SM7 Rods +ve -ve -ve -ve -ve -ve -ve MR+VP+ Facultativeanaerobes

Lactobacillus

13 KM1 Coccus +ve -ve -ve -ve -ve -ve -ve MR+VP+ Obligateanaerobes

Lactococcus

14 KM3 Coccus +ve -ve -ve +ve -ve -ve -ve MR +VP+ Obligateanaerobes

Lactococcus

15 KM9 Coccus +ve -ve -ve +ve -ve -ve -ve MR+ VP+ Obligateanaerobes

Lactococcus

16 KM5 Coccus +ve -ve -ve -ve -ve -ve -ve MR +VP+ Obligateanaerobes

Lactococcus

51

Lactobacilli were the first genus of bacteria proved to have beneficial health effects.

They have been shown to be present in gastrointestinal tract of most animals and birds. It is

one of many friendly species of intestinal microflora considered as beneficial bacteria in its

ability to aid in breakdown of protein, carbohydrates and fats in food and help absorption of

necessary elements and nutrients such as minerals, amino acids and vitamins by the host.

They are also referred as “live enzyme factory” as they produce wide range of enzymes,

which can breakdown even complex carbohydrates, hence beneficial to the host

(Anonymous, 2002).

Badau, (2006) reported that gram positive, non spore forming, rods encountered at the

various malting stages were Lactobacillus delbruekii and Lactobacillus plantarum. The green

malt had the highest microbial count which could be due the exposure of the germinating

grains to various sources of contamination. During germination, grains could be exposed.

Green malt had the highest microbial count, followed by dry malt and polished malt. Malt

flour and unmalted grain had the least. Steeped grain did not show any growth. Total

bacterial count, mold count, staphylococcal count and coliform count, ranged from 4.08 to

5.28 log10 CFU/g, 2.50 to 3.71, 1.78 to 4.20 and 2.65 to 3.65 respectively.

4.3.2 Preliminary screening of microorganisms isolated from kodo millet

Preliminary screening of 13 isolates of raw kodo millet and 16 of malted kodo millet

was done on the basis of their antagonistic pattern to select best isolates out of them for

further studies.

4.3.2.1 Antagonistic spectrum of microorganisms by Bit/Disk method

Tentatively identified microflora isolated from raw and malted kodo millet were

further tested for their antagonistic activity against selected food borne/spoilage causing

bacteria viz. Staphylococcus aureus IGMC, Enterococcus faecalis MTCC 2729, Listeria

monocytogens MTCC 839, Clostridium perfringens MTCC 1739, Leuconostoc mesenteroids

MTCC 107, Bacillus cereus CRI, Escherichia coli IGMC, Pseudomonas syringae IGMC,

Pectobacterium carotovorum MTCC 1428 and Lactobacillus plantarum CRI. The data on

inhibitory spectrum of isolated bacteria by bit/disk method is shown in Table 5, 6, Plate 2 and

Fig. 5. Those isolates having clear zones less than 9 mm diameter against their respective test

strain indicated poor activity, while the other strains which made appreciable halos greater

than 12 mm shown to have good and strong antimicrobial activity against their corresponding

52

bacterial indicators. Antagonistic pattern of different bacteria varied against test pathogens

i.e. some showed antagonism against maximum number of test indicators viz. isolate KR5

found to inhibit maximum 8 bacterial test indicators, whereas SM1 and SM3 inhibit 5

bacterial test indicators. Isolate KR3, KR9 and KM1 inhibited 4 bacterial test indicators,

while KM8, KR8, SM6 and SR8 inhibited 3, while KM5, SM5, SM10, SM7, SR2, SM8,

SM9 and KM7 inhibited 2 bacterial test indicators, whereas KM4, SR, KR7 inhibited only 1

bacterial test indicator. Isolate SR6, KM9, KM3, SR4, SR3, SM4, KR6 and SR1 inhibit none

of the tested bacterial indicators.

The inhibitory action of LAB is mainly due to accumulation of main primary

metabolites such as lactic and acetic acids, ethanol, carbon dioxide; or antimicrobial

compounds such as formic, benzoic acids, hydrogen peroxide, diacetyl and acetoin

(Yukesdag and Aslim, 2010). In addition, LAB has shown to possess inhibitory activities due

to bactericidal effect of protease sensitive bacteriocins (Jack et al. 1995). By producing these

antimicrobial compounds, probiotic microorganisms gain an edge over other microorganisms

to survive in the adverse conditions of gastrointestinal tract (El-Nagger, 2004).

The studies pertaining to antimicrobial activity of different microorganisms against

food borne pathogens by bit/disk method have been well documented in literature. The

inhibitory substances produced by potential strains act differently on different indicator

strains. Pundir et al. (2013) isolated a total of 26 lactic acid bacteria, purified and screened

them for their antimicrobial activity against seven human pathogenic MTCC strains counting

three test fungal strains such as Aspergillus fumigatus, Aspergillus sp. and Candida albicans,

and four test bacterial strains (two gram-negative namely Escherichia coli, Salmonella

enterica ser. Typhi and two gram-positive Staphylococcus epidermidis and Bacillus

amyloliquifaciens).

Wakil and Osamvonyi, (2012) isolated a total of 26 lactic acid bacteria on MRS agar.

LAB isolates were screened for antimicrobial activity against selected indicator organisms.

The zones of inhibition ranged from 5mm - 18mm in diameter. The highest inhibitions

(18mm) were from isolate FL9 against Pseudomonas aeruginosa and Staphylococcus aureus,

isolate FL19 against P. aeruginosa and S. aureus, isolate FL14 against S. aureus and isolate

FL 20 against Pseudomonas flourescens while lowest inhibition (5mm) was by isolate FL18

against Salmonella species. P. flourescens shows the highest susceptibility to LAB isolates

while Salmonella species showed the least susceptibility. 22 of the 26 LAB isolates showed

53

Table 5: Preliminary screening of isolated bacteria from raw kodo millet on the basis of their antagonistic pattern against testedbacterial indicators by bit/disk method

Sr.No. Nameofisolates

Source E. coli(mm)

B.cereus(mm)

C. perfringens(mm)

L. monocytogenes(mm)

S.aureus(mm)

E.faecalis(mm)

L.plantarum(mm)

P.carotovorum(mm)

P.syringae(mm)

L.mesenteroids(mm)

MeanPercentage

inhibition(%)

1 KR3 Raw kodomillet

- - - 10.3 - 9.3 - 9.0 9.7 - 3.8 40

2 *KR5 Raw kodomillet

10.3 12.7 11.3 10.3 11.7 - 14.7 14.3 12.3 - 9.8 80

3 KR6 Raw kodomillet

- - - - - - - - - - 0.0 0

4 KR9 Raw kodomillet

14.7 8.3 - - 15.3 9.0 - - - - 4.7 40

5 KR7 Raw kodomillet

12.3 - - - - - - - - - 1.2 10

6 KR8 Raw kodomillet

- 9.0 13.0 - - 10.0 - - - - 3.2 30

7 SR1 Raw kodomillet

- - - - - - - - - - 0.0 0

8 SR2 Raw kodomillet

12.3 - - - - 9.0 - - - - 2.13 20

9 SR3 Raw kodomillet

- - - - - - - - - - 0.0 0

10 SR Raw kodomillet

- - 10.3 - - - - - - - 1.0 10

11 SR4 Raw kodomillet

- - - - - - - - - - 0.0 0

12 SR8 Raw kodomillet

11.7 - 9.0 - 15.3 - - - - - 3.6 30

13 SR6 Raw kodomillet

- - - - - - - - - - 0.0 0

Zone size >20 mm = strong activityZone size >12 mm = good activityZone size <9 mm = poor activity*Showing broadest/strongest antagonism

Indicator: Staphylococcus aureus Indicator: Escherichia coli

Indicator: Bacillus cereus Indicator:Clostridium perfringens

Indicator: Enterococcus faecalis Indicator: Pseudomonas syringae

Plate 2: Inhibitory spectrum of potential microorganisms against test indicators bybit/disc diffusion method

SM5

SM8 SR2

KR8

SR2

SM4

SM1

KM1SM3

KR9

SM1

SR2

SR1 KR9

KR5

R5

SM1

KR8

SRKR5

KR7

KR6

SM8

KR9KR3

KR6

KR9KR55

KR3

SM3

Fig 5: Antagonistic potential of isolated microorganisms from kodo millet against testindicator

0

10

20

30

40

50

60

70

80

KR3

KR5

KR6

KR9

KR7

KR8

Inhi

bitio

n (%

)

Fig 5: Antagonistic potential of isolated microorganisms from kodo millet against testindicator

KR8

SR1

SR2

SR3 SR SR4

SR8

SR6

KM4

KM7

KM8

SM1

SM4

SM6

SM5

SM8

Isolates

Fig 5: Antagonistic potential of isolated microorganisms from kodo millet against testindicator

SM8

SM9

SM3

SM10

SM7

KM1

KM3

KM9

KM5

54

Table 6: Preliminary screening of isolated bacteria from malted kodo millet on the basis of their antagonistic pattern against testedbacterial indicators by bit/disc method

Sr.No. Name ofisolates

Source E. coli(mm)

B.cereus(mm)

C. perfringens(mm)

L.monocytogenes

(mm)

S.aureus(mm)

E.faecalis(mm)

l.. plantarum(mm)

P. carotovorum(mm)

P.syringae(mm)

L.mesenteroids(mm)

Mean Percentageinhibition

(%)

1 KM4 Malted kodo millet - - - - 13.0 - - - - - 1.3 10

2 KM7 Malted kodo millet - - - - - - - - 9.7 9.0 1.8 20

3 KM8 Malted kodo millet - 9.0 11.0 - 12.3 - - - - - 3.2 30

4 *SM1 Malted kodo millet 10.3 11.0 9.3 - 13.3 - - - 9.0 - 5.3 50

5 SM4 Malted kodo millet - - - - - - - - - - 0.0 0

6 SM6 Malted kodo millet - - - - 10.3 9.0 - 9.0 - - 2.8 30

7 SM5 Malted kodo millet 14.3 - - - - 9.3 - - - - 2.3 20

8 SM8 Malted kodo millet 13.0 - - - - 19.7 - - - - 3.2 20

9 SM9 Malted kodo millet 11.7 - - - 12.7 - - - - - 2.4 20

10 *SM3 Malted kodo millet - 10.7 10.3 11.7 12.3 - - - 19.3 - 6.4 50

11 SM10 Malted kodo millet 13.3 - - 10.3 - - - - - - 2.3 20

12 SM7 Malted kodo millet 12.7 - - - - 14.7 - - - - 2.7 20

13 KM1 Malted kodo millet - - 9 - 9.7 13.5 - 15.3 - - 4.7 40

14 KM3 Malted kodo millet - - - - - - - - - - 0.0 0

15 KM9 Malted kodo millet - - - - - - - - - - 0.0 0

16 KM5 Malted kodo millet - 8.3 - 19.3 - - - - - - 2.7 20

Zone size >20 mm = strong activityZone size >12 mm = good activityZone size <9 mm = poor activity*Showing broadest/strongest antagonism

55

antimicrobial activity; isolates FL1, FL3, FL5 and FL22 did not show antimicrobial activity

against any of the indicator organisms. Lactic acid bacteria that showed antimicrobial activity

were further characterized using various physiological and biochemical tests including

growth at different pH, gram reaction, catalase test, growth in different salt concentrations

etc. Based on these results and the results of the biochemical and physiological

characterization, the antimicrobial producing LAB were identified as L. plantarum, L.

fermentum, L. jensenii, L. sp., L. mesenteriodes, L. brevis and P. acidilactici. The result

showed that L. plantarum was dominant in occurrence with 45% occurrence, L. fermentum

showed 18.2% occurrence, L. jensenii, L. sp. and L. mesenteriodes all had 9.1% occurrence,

L. brevis and P. acidilactici showed 4.5% occurrence.

Thus, antagonistic pattern on the basis of percent inhibition and the mean of zone of

inhibition of isolated microorganisms was one of the important factors for preliminary

screening. Out of total of 29 isolates, three isolates viz. KR5, SM1 and SM3 emerged as best

strains on the basis of broadest and strongest antagonism ranging between 50-80% of overall

inhibition of tested indicators and thus were further selected for their identification studies.

4.3.2.2 Genotypic Characterization

The best selected three bacterial isolates were identified at genomic level by using

16S rRNA gene technique. Genomic DNA of three best selected bacterial isolates was

isolated using DNA purification kit (Bangalore Genei, make). The isolated DNA was used in

PCR to amplify small subunit of 16S rRNA using universal primers having expected product

size of 1500 bp. The PCR product so obtained after amplification was visualized using

ethidium bromide on 2% agarose gel. Amplified PCR products were purified and got

sequenced by the services provided by Europhins, India Pvt. Ltd. to confirm the results.

Nucleotide Sequencing

Following sequences of best screened one isolate was obtained after sequence

analysis and shown in Table 7

Table 7: Identification of finally screened bacterial isolates

a) On the basis of Biochemical test

Name of isolates Source Tentative identificationSM3 Malted kodo millet Bacillus sp.SM1 Malted kodo millet Bacillus sp.

a.) SM1

b.) SM3

Plate 3: Colony morphology of screened isolates isolated from malted kodo millet

a.) KR5

b.) Genomic DNA c.) PCR product

Plate 4: Identification of best screened isolate KR5 by 16S rRNA gene technique

KR5

100 bp

900 bp

700 bp

500 bp

300 bp

1300 bp

bpbp

1500 bp

56

b) On the basis of 16S rRNA

Name ofisolates

Source Closesthomologue(organism)

Identity(%)

16S rRNAIdentification

Accession no.

KR5 Rawkodomillet

Paenibacillusjamilae

94% Paenibacillusjamilae

KT831773

Sequence of isolate KR5

ACCGGAAACGGTAGCTAATACCCGATACATCCTTTTCCTGCATGGGAGAAGGAG

GAAAGGCGGAGCAATCTGTCACTTGTGGATGGGCCTGCGGCGCATTAGCTAGTT

GGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGAT

CGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG

GAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAA

GGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGTCTTGTAGAGTAACTG

CTACAAGAGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAG

CCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGC

GCGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGCGTCGC

ACTGGAAAACTGGGAGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGT

AGCGGTGAAATGCGTAGAGATGTGGAGGAACCACCAGGTGGCGAAGCGACTCTC

TGGGCTGTAACTGACGCTGCATGCTGATCCGCGATTACTAGCAATTCCGACTTCA

TGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGACCGGCTTTTCTAGGATTGGC

TCCACATCGCTGCTTCGCTTCCCGTTGTACCGGCCATTGTAGTACGTGTGTAGCCC

AGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA

CCGGCAGTCTGCTTAGAGTGCCCAGCTTGACCTGCTGGCAACTAAGCATAAGGGT

TGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACACCAT

GCACCACCTGTCTCCTCTGTCCGAAGGAAAGGTCTATCTCAGACCGGTCAAGGGA

GTCAGACCTGGTAGGTTCTTCGCGTTGTTCGAATTAACCACATACTCCACTGCTTG

TGCGGGTCCCGTCATTCCTTGATTTCATCTGCGACCGTCTCCCCGGCGGATGCTTA

TGTGTTACTTCGGCCCAGGGTATCAAACCCTAACACTAGCATTCATCGTTACGGC

GTGGACACCAGGTATCTATCTGTTGCTCCCACGCTTCCGCTCACGTCATTACGCC

AGAGATCGCTTCCCACTGTGTTCTCACATCTTACGCATTCACGCTACAGTGGATTC

CCTCTCTCTCTGCCTCAGCTCCCATTTCCGTGCACCGAGTGACCTCGGATAACACA

GACTAAGAGCGCCGCCGGCTTAGCCCATATTCCGACACGCTGCCCTACTATACGC

57

GCTGCGGCCTATTACCGGGCTTCTCCAGTACGCACCTGTACAGTATCTACAGCGT

CTCCTGCACGACTTACATCGAAACTCACATCAGCGCGTGTCGTAGCTTCCCATGC

GAAATCCTATGTGCTCCTAGATCGGGCGG

Colony morphology of SM1 and SM2 were shown in Plate 3.Sequence similarity

search for the KR5 (BLAST, NCBI) showed 94% homology with the available sequence of

Paenibacillus jamilae having accession no. KT831773 has been represented in Plate 4.

4.4 Evaluation of nutrient profile of kodo millet grains

The nutrient profile of kodo millet grains collected from different districts of

Himachal Pradesh was evaluated and compared and the following results as mentioned below

were obtained:

The protein content of kodo millet grains from different districts of Himachal Pradesh

was evaluated and it was found that kodo millet grains from district Mandi contain highest

3.8% protein, followed by 3.5% from kangra and 3.2% from Hamirpur district. Similarly, the

carbohydrates content of millet grains was found to be 55.6 mg/g, 55 mg/g and 52 mg/g in

the respective districts. Starch is the main constituent of carbohydrates, the starch content in

kodo millet grains was found to be 42.5 mg/g from district Hamirpur, 42 mg/g and 41 mg/g

from district Mandi and Kangra, respectively. The phenol content of kodo millet grains was

found to be 5.65 mg/g in Hamirpur, 5.6 mg/g in Kangra and 5.45 mg/g in Mandi district.

Whereas crude fibers was found to be maximum i.e. 6.9% in district kangra, followed by

6.8% in Mandi, while 6.5 % in Hamirpur district.

Among the nutrients of kodo millet grains collected from different districts, protein

content of Mandi district kodo grains was statistically significantly different from the Kangra

and Hamirpur district, whereas crude fibers of Mandi and Kangra were significantly different

from Hamirpur district. In case of Carbohydrates the values found in Kangra district were

significantly at par with Mandi and Kangra while, starch and total phenols in district

Hamirpur were significantly at par with Mandi and Kangra. This variation in nutrient

contents proves the areawise variability in different varities of kodo millet.

Davis et al. (1981) reported, protein and carbohyrate contents in wheat that ranges

from 8.3 to 19.3% for protein and 65.4% to 78% for carbohydrates. Azim and Ali, (1989)

58

observed that the protein content was 30%, whereas crude fibers content ranges from 18.35 to

42.33 % in maize.

Verma and Patel, (2013) evaluted the proteins and carbohydrates content in kodo and

ragi millets and reported that it contains 66.6 g of carbohydrates, 9.8 g of proteins and 9.0 g

of crude fiber. The protein content of pearl millet is comparable to wheat (11.6 vs 11.8 g/100

g), is higher than rice (6.8 g/100g), sorghum (10.4 g/100g) and maize (4.7 g/ 100g) as per the

Nutritive value of Indian foods (NIN, 2003). Pearl millet has lower starch and higher protein

and oil content as compared to sorghum. The nitrogen intake and absorption were higher for

pearl millet as compared to corn and the digestibility of nitrogen was similar for pearl millet

and corn. Net protein utilization was lower (p<0.05) in pearl millet when compared to corn

(Adeola and Orban, 1994).

Saldivar, (2003) reported starch content in different types of millet and found that

kodo millet contains maximum 72% followed by pearl millet which contains 60.5% of starch,

whereas 59.1 and 59.0% starch content in foxtail and finger millet, respectively. Pearl millet

has high fiber (1.2 g/100g). Finger millet contains about 5-8% protein, 65-75% carbohydrates

and 15-20% dietary fibers (Chetahan and Malleshi, 2007). The total dietary fibers (22.0%) of

finger millet grain were reported relatively higher than that of many other cereal grains (e.g.

12.6%, 4.6% and 12.8% respectively for wheat, rice, maize and sourgham) (Siwela et al.

2010). However, the dietary fiber content in pearl millet ranges between 8 to 9% (Taylor,

2004). Kamath and Belavady, (1980) found 18.6% dietary fibers and 3.6% crude fiber in

finger millet.

The crude fat content in kodo millet sample was evaluated and it was found that it

contains 0.10 to 0.11 g of crude fat in sample from Mandi and 0.10 from Hamirpur and

Kangra district respectively. Also the antioxidant activity was found to be 45%, 43% and

42% from Mandi, Kangra and Hamirpur district of Himachal Pradesh. The following

minerals i.e. phosphorus, iron and magnesium content in kodo millet was evaluated. It was

found that sample from district Kangra contains, 0.29%, 0.15% and 8.0% of phosphorus,

magnesium and iron, respectively, while 0.32% phosphorus, 0.13% magnesium and 7.0%

iron in district Mandi. Whereas, 0.35% phosphorus, 0.14% and 7.45% magnesium and iron in

district Hamirpur. The flavonoids content was ranged from 1.24 -1.29µg/ml in above

mentioned districts respectively as shown in Table 8.

59

The iron content of finger millet ranged from 3.3 to 14.8 mg (Babu et al. 1987). Singh

and Srivastava, (2006) reported the iron content of 16 finger millet varieties ranged from 3.61

mg/100 g to 5.42 mg/100 g with a mean value of 4.40 mg/100g. According to Vijayakumari

et al. (2003) finger millet is the richest source of calcium and iron.

The phosphorus content ranged from 130 to 295 mg% with a mean value of 180.42

mg% (Singh and Srivastava, 2006). Millet also contains iron, potassium, magnesium and zinc

(Vachanth et al. 2010). Overall mineral content of pearl millet was found to be 2.3 mg/100g

(NIN, 2003). Barnyard millet showed highest concentration of iron (40.2 ppm), followed by

finger millet with 34.15 ppm, little millet with 32.71 ppm, kodo millet with 32.28 ppm and

foxtail millet with 27.19 ppm.

Rao, (1994) reported that total iron decreased in finger millet from 4.4 to 1.8 mg/100

g and in white finger millet from 12.0 to 2.8 mg/g. Similarly, Hemanalini et al. (1980) have

reported that malted finger millet flour resulted in 32, 26 and 33% losses in calcium,

phosphorus and iron respectively. Sprouted finger millet contained 230 mg phosphorus and 5

mg iron. Deosathale, (2002) reported ionisable iron content to be 88.3% in malted finger

millet as compared to 7.4% in raw finger millet.

Table 8: Nutritional evaluation of kodo millet grains

Sr.No.

Component Kodo millet Mean CDMandi Kangra Hamirpur

1 Proteins (%) 3.7 3.5 3.2 3.80 0.192 Carbohydrates

(mg/g)54 55.6 54 55.0 1.64

3 Starch (mg/g) 41 41 42.5 42.0 1.644 Total phenols

(mg/g)5.5 5.6 5.65 5.50 0.16

5 Crude fibers (g) 6.7 6.8 6.45 6.80 0.166 Antioxidant

activity (%)44 42 44 45.0 1.99

7 Phosphorus (%) 0.33 0.28 0.35 3.20 0.018 Magnesium (%) 0.13 0.15 0.14 1.30 0.029 Iron (%) 6.9 7.9 7.45 7.00 0.1610 Crude fat (g) 0.12 0.11 0.11 1.20 0.0211 Flavonoids

(µg/ml)1.28 1.24 1.28 1.28 0.02

60

4.5 Analysis of polyphenols using Thin Layer Chromatography

Analysis of polyphenols was done after their extraction using polar solvents i.e.

methanol, acetone and water using Thin Layer Chromatography (TLC). TLC analysis of

polyphenols extracted with acetone and methanol showed the presence of total 6 spots by

different samples as shown in Table 9 and Plate 5. When the computed Rf (Retention factor)

value of the spots were compared with the literature Rf value, the compounds were identified

as ferulic acid (Rf = 0.52), cinnamic acid (Rf = 0.68) and caffeic acid (Rf = 0.15), whereas

rest of the spots whose Rf value lies between 0.07-0.10 were identified as flavonoids-

glycosides.

Table 9: TLC Rf values of polyphenols extracted from kodo millet

Source Spots Rf

(Retention factor) of

polyphenols extractedfrom kodo millet

Rf

in

literature

Compoundidentified

Reference

Polyphenolsextracted usingAcetone

1 0.09 0.09 Flavonoids-glycosides

Vladmir etal., 2011

2 0.52 0.52 Ferulic acid

Polyphenolsextracted usingMethanol

1 0.07 0.07 Flavonoids-glycosides

2 0.100.10

Flavonoids-glycosides

3 0.150.15 Caffeic acid

4 0.68 0.68 Cinnamic acid

Rf = Distance travelled by solute/ Distance travelled by solvent

Sitarski and Bojanowska, (1993) evaluated phenolic acids from rye and wheat grain

using Thin Layer Chromatography (TLC) on silica gel plate. The dominant form of phenolic

acid in both rye and wheat grain was ferulic acid, although isoferulic, coumaric, syringic, and

caffeic acids were detected in minor amounts by thin-layer chromatography. In addition, p-

hydroxybenzoic acid was present in rye grain. In the water-soluble fraction of rye grain, the

spots corresponding to caffeic and syringic acids were relatively more intense than those in

the whole grain.

4.6 Analysis of polyphenols using High Performance Liquid Chromatography

As TLC results had shown the presence of ferulic and cinnamic acid in kodo millet,

further quantication of these compounds was done by using High Performance Liquid

61

Chromatography (HPLC). The polyphenols extracted from kodo millet seed coat using

different polar solvents i.e. 1 % HCL methanol, acetone and water was fractioned by High

Performance Liquid Chromatography (HPLC). The phenolic compounds identified were

ferulic acid and cinnamic acid as shown in Table 10. HPLC analysis depicted that kodo millet

contains 109.45 mg/ 100 g and 111.45 mg/ 100 g ferulic and cinnamic acid respectively in

polyphenols extracted in acetone as in Fig 6. Similarly, polyphenols extracted in methanol

from kodo millet contains 359.2 mg/ 100 g of ferulic acid and 79.01 mg/100 g of cinnamic

acid as shown in Fig. 7. Phenolic compounds in millets exist as free, soluble conjugates and

insoluble bound forms. HPLC results showed that methanol extracted polyphenols contain

ferulic acid as the major free form phenolic acid and cinnamic acid as major bound form.

While, in acetone extracted polyphenols both the polyphenols were found as major free form

phenolic acid. According to the results acetone has been found to be very effective solvent for

the extraction of kodo millet polyphenols. The presence of phenolic acids in cereal grains has

been confirmed in several studies (Busch and Fulcher, 1999). The polyphenolic content in

cereals is usually less than 1% of dry matter, except for some sorghum cultivars. The main

polyphenols in cereals are phenolic acids and tannins, while flavonoids are present in small

quantities (Subba Rao and Muralikrishna, 2002).

The results obtained in the present study when compaired with other cereals it has

been noticed that polyphenols present in kodo millet i.e. cinnamic acid (79.01 mg/ 100 g in

methanol and 111.45 mg/ 100 g in acetone) and ferulic acid (359.2 mg/g in methanol and

109.45 mg/g in acetone) were found to be much higher i.e. maize having ferulic acid 0.405

mg/ 100g, wheat having 47 mg/ 100g, barley 30 mg/ 100g, oat flour 36 mg/ 100g and rice

having 30 mg/ 100g of ferulic acid, whereas finger millet had 0.405 mg/ 100g of ferulic acid

and 0.035 mg/ 100g of cinnamic acid (Phenol-explorer, database). The values of constituent

phenolics extracted with different solvents varied significantly. Variations in the yields and

Table 10: HPLC analysis of polyphenols extracted from kodo millet

Compound Retention time(min)

Quantification(mg/100 g)

Methanol Acetone

Ferulic acid 3.98 359.2 109.45

Cinnamic acid 2.71 79.01 111.45

Plate 5: Thin layer chromatography of polyphenols extracted from kodo millet, (a.)Polyphenols extracted from acetone (b.) Polyphenols extracted from methanol; Rf – 0.684 (Cinnamic acid), Rf – 0.52 (Ferulic acid), Rf – 0.15 (caffeic acid), Rf

- 0.07-0.10 (Flavonoids-glycosides)

3

1 12

2

4

a. b.

Plate 5: Thin layer chromatography of polyphenols extracted from kodo millet, (a.)Polyphenols extracted from acetone (b.) Polyphenols extracted from methanol; Rf – 0.684 (Cinnamic acid), Rf – 0.52 (Ferulic acid), Rf – 0.15 (caffeic acid), Rf

- 0.07-0.10 (Flavonoids-glycosides)

3

1 12

2

4

a. b.

Plate 5: Thin layer chromatography of polyphenols extracted from kodo millet, (a.)Polyphenols extracted from acetone (b.) Polyphenols extracted from methanol; Rf – 0.684 (Cinnamic acid), Rf – 0.52 (Ferulic acid), Rf – 0.15 (caffeic acid), Rf

- 0.07-0.10 (Flavonoids-glycosides)

3

1 12

2

4

a. b.

Fig 6: HPLC chromatogram of kodo millet (a) Standard solution of ferulic acid inacetone, (b) Standard solution of cinnamic acid in acetone (c) Acetoneextracted sample of kodo millet

c.

b.

a.

Fig 7: HPLC chromatogram of kodo millet (a) Standard solution of ferulic acid inmethanol, (b) Standard solution of cinnamic acid in methanol (c) Methanolextracted sample of kodo millet

(a)

(b)

(c)

62

their phenolic contents of various extracts are attributed to the polarities of the phenolics

present in the seeds. Extraction of phenolics from any of the natural material depends on the

solubility of their polyphenols (Naczk and Shahidi, 2006). Such differences have been

reported for other cereals also (Ragaee et al. 2006).

Polyphenols are secondary metabolites of plants and are generally involved in defense

against ultraviolet radiation or aggression by pathogens. Phenolic acids are found abundantly

in foods and divided into two classes: derivatives of benzoic acid and derivatives of cinnamic

acid. The hydroxybenzoic acid content of edible plants is generally low, whereas the

hydroxycinnamic acids are more common than hydroxybenzoic acids and consist chiefly

of p-coumaric, caffeic, ferulic and sinapic acids. In food, polyphenols may contribute to the

bitterness, astringency, color, flavor, odor and oxidative stability. Polyphenols may be

protective against cardiovascular diseases and have antioxidant, anti-platelet, anti-

inflammatory effects as well as increasing HDL. Effect of polyphenols on human cancer cell

lines, is most often protective and induce a reduction of the number of tumors or of their

growth (Yang et al. 2001).

In a recent study, over 50 phenolic compounds has been identified in several whole

millet grains like kodo, finger, foxtail, proso, little and pearl using HPLC and also their

antioxidant and antiradical activity was estimated (Chandrasekara and Shahidi, 2010).

Millets extract from the seed coat were reported to have shown high antibacterial and

antifungal activity compared to whole flour extract due to high polyphenols content in seed

content (Vishwanath et al. 2009; Xu et al. 2011).

According to Hilu et al. (1978), majority of phenolic compounds present in millet

exit in the form of glycosides, whereas Rao and Muralikrishna, (2002) reported ferulic acid

as the major bound phenolic acid (18.60 mg/ 100 g) and protocatechuic acid as the major

free phenolic acid (45.0 mg/ 100 g) of the millet. The major bound phenolics present in

finger millets were ferulic acid and p-coumaric acid, and the bound phenolic fraction

accounts for 64-96 % and 50-99% of total ferulic acid and p-coumaric acid content of millet

grains respectively. The main polyphenols in cereals are phenolic acids and tannins, while

flavonoids were present in small quantities. Acidic methanol (1% HCL in methanol) has

been shown to be very effective for extraction of polyphenols (Ramachandra et al. 1977).

63

Sripriya et al. (1996) reported the phenolic of 51.4 and 43.1 mg/100 g in pearl millet

and sourgham, respectively. Sharma and Kapoor, (1996) had reported the phenols in pearl

millet grains as 608.1 mg/ 100 g and that in pearl millet flour as 761 mg/100 g.

Chandrasekara and Shahidi, (2010) revealed that the phenolic extract of kodo millet exhibited

higher inhibition activities against oxidation of LDL cholesterol and liposome than that of

pearl millet. Hydroxycinnamic acids, mainly ferulic and p-coumaric acids contributed to the

observed action of millet phenolics in addition to hydroxybenzoic acids and flavonoids

identified in pearl millet. The main phenolic constituent in finger millet was gallic acid, p-

coumaric, vanillic, syringic, ferulic, trans-cinnamic acids and quericitn (Mathangi and Sudha,

2012).

According to McDonough and Rooney, (2000) ferulic, p-coumaric and cinnamic acids

are the major phenolics in finger millet. Nearly 70% of finger millet phenolic acids were free

and 30% in bound form and ferulic acid (18.60 mg/ 100 g) is the major bound phenolic acid,

whereas protocatechuic acid (45.0 mg/ 100 g) is the major free phenolic acids. Studies on the

changes in free and bound phenolic acids and their antioxidant properties during malting of

ragi were also reported (Rao and Muralikrishna, 2002). Chandrasekara and Shahidi, (2011)

reported that hydroxycinnamic acids and their derivatives constitute the insoluble bound

fraction whereas flavonoids are present in free phenolics. Kodo millet had the highest total

phenolic content, whereas proso millet contains the least. Ferulic and p-coumaric acids

present in higher amount in the bound fractions compare to the soluble phenolics

(Chandrasekara and Shahidi, 2010).

4.6.1 Antagonistic spectrum of polyphenols

Polyphenol extracts obtained from the seed coat to whole flour were used to

determine the antimicrobial activity as they were found to be good sources of polyphenols

amongst the various fractions studied. Since the crude extract of the finger millet polyphenols

was used for the antimicrobial activity, the degree of inhibition could not be attributed to the

constituent phenolics. The inhibitory action of polyphenols extracted using different solvents

i.e. acetone and methanol was tested against 4 bacterial test indicators viz. Staphylococcus

aureus, Leuconostoc mesenteroides, Bacillus cereus and Escherichia coli. The methanol and

acetone extract (sample and control) of kodo millet showed inhibition against all the 4 tested

indicators as shown in Table 12 and Plate 6.

Indicator: Staphylococcus aureus Indicator: Escherichia coli

Indicator: Leuconostoc mesenteroids Indicator: Bacillus cereus

Plate 6: Inhibitory spectrum of polyphenols extracted from kodo millet using acetone,methanol and water as solvents against tested indicators by spot method; AC:Acetone control, AS: Acetone Sample; MC: Methanol control, MS: MethanolSample

MC

MSAS

AC

MS

MC

AC

AS

MS

MCAC

AS

ASAC

MC

MS

64

The results showed that zone of inhibition for polyphenols extracted with methanol

was maximum against Staphylococcus aureus i.e. 24.3 mm for sample, whereas when

polyphenols were extracted with acetone showed less inhibition as compared to methanol i.e.

the values ranged upto 15.0 mm. However, no inhibition was observed in water extract

indicating the possibility that none of polyphenols could be solublised in water.

Table 11: Antagonistic spectrum of polyphenols extracted from kodo millet by spotmethod

Sr.

no.

Solvents Bacterial indicators

Zone size (mm)

S. aureus L. mesenteroids B. cereus E. coli

Control Sample Control Sample Control Sample Control Sample

1 Methanol 18.0 24.3 11.7 13.7 12.0 14.0 10.7 11.7

2 Acetone 10.0 10.7 10.0 11.7 1.0 1.0 13.0 15.0

3 Water - - - - - - - -

It is cited in the literature, that polyphenols have the property of inhibiting the

proliferation of microorganisms. Banerjee et al. (2012) evaluated that finger millet

polyphenols showed proliferation inhibitory activities on Staphylococcus aureus, Bacillus

cereus, Escherichia coli, Listeria monocytogenes, Streptococcus pyogenes, Klebsiella

pneumonia and Pseudomonas aeroginosa. Cinnamic acid and its derivatives provide natural

protection against infections by pathogenic microorganisms. Cinnamic acid affects plasma

membrane ATPase activity of Saccharomyces cerevisae.

Mathangi and Sudha, (2012) evaluated that the acidic methanol extract from the seed

coat showed higher antibacterial activity as compared to whole flour extract due to high

polyphenols content in seed coat. The extremely good storage property of finger millet could

be attributed to its polyphenol content.

Viswanath et al. (2009) examined the crude extract of finger millet from the seed coat

to whole flour for their antimicrobial properties. The minimum inhibition concentration of the

polyphenols was found to be 30% for the seed coat and 50% for the inhibition of Bacillus

cereus in the anti-bacterial experiment. The zone of inhibition for the seed coat and whole

flour were 15 and 13 mm, respectively, for the same experiment. Since the seed coat in whole

contained the highest polyphenol concentrations it had been inferred that the polyphenols

65

were responsible for the microbial inhibition. The seed coat polyphenols exhibited a higher

inhibitory response than the whole flour polyphenols due to its higher polyphenol content.

4.7 Inter compatibility of isolates for probiotic formulations

Probiotic microorganisms used for the preparation of functional foods of kodo millet

were inhouse Pediococcus acidilactici L1, Lactobacillus plantarum L2 and Lactobacillus

fermentum F3 as shown in Table 12. In order to formulate probiotic consortia, compatibility

of these three probiotic potential isolates was determined by cross streak method. In this

method all the three screened probiotic isolates were cross streaked against each other on

prepoured selective medium plate i.e. MRS for three plates followed by incubation at 35 ˚C

for 48 h. The intercompatibility of probiotic strains is shown in Plate 7 a, b and c.

Table 12: Inhouse probiotic microorganisms used for preparation of food products

Sr. No. Probiotic microorganism Accession number

1 Pediococcus acidilactici L1 KM251713

2 Lactobacillus plantarum L2 KM251714

3 Lactobacillus fermentum F3 KC251713

Probiotics can be bacteria, mould or yeast. But most probiotics are bacteria. Among

bacteria, lactic acid bacteria group is more popular. Lactobacillus acidophilus, L. casei, L.

lactis, L. salivarius, L. plantarum, L. fermentun, L. delbrueckii, L. johnsonii, L. reuteri, L.

rhamnosus, Streptococcus thermophilus, Enterococcus faecium, E. faecalis, Bifidobacterium

bifidum, B. breve, B. longum, Bacillus subtilis and Saccharomyces boulardii are commonly

used probiotics. A probiotic used may single microbial strain or preferably a consortium as

well (Gilliland and Speck, 1977). The most commonly utilized probiotic preparations include

specific strains of either alone or in combination – Lactobacilli, Streptococci and

Bifidobacteria as these three genera are important gastrointestinal flora, considered to be

harmless, and capable of preventing the overgrowth of pathogenic organsisms (Wadher et al.

2010).

Regular intake of probiotics (i.e. a fermented milk drink containing a mixture of L.

rhamnosus GG, Bifidobacterium, L. acidophilus and S. thermophilus) has been demonstrated

to reduce potentially pathogenic bacteria in the gastrointestinal tract of humans (Wang et al.

2004). Some in vitro and experimental animal studies, have proved that probiotic were

a.) Pediococcus acidilactici L1 & Lactobacillus fermentum F3

b.) Pediococcus acidilactici L1& Lactobacillus plantarum L2

c.) Lactobacillus plantarum L2& Lactobacillus fermentum F3

Plate 7: Inter compatibility of probiotic microorganisms

a.) Pediococcus acidilactici L1 & Lactobacillus fermentum F3

b.) Pediococcus acidilactici L1& Lactobacillus plantarum L2

c.) Lactobacillus plantarum L2& Lactobacillus fermentum F3

Plate 7: Inter compatibility of probiotic microorganisms

a.) Pediococcus acidilactici L1 & Lactobacillus fermentum F3

b.) Pediococcus acidilactici L1& Lactobacillus plantarum L2

c.) Lactobacillus plantarum L2& Lactobacillus fermentum F3

Plate 7: Inter compatibility of probiotic microorganisms

66

potential to reduce colon cancer risk in experimental animals. Intake of beverage and specific

probiotic culture had been shown to reduce the development of precancerous lesions

(aberrant crypts) and chemically induced tumors, although the findings appeared to be both

species- and strain-dependent (Wollowski et al. 2001).

4.8 Functional food

Different health promoting foods with additional health benefits have been formulated

by adding potential probiotic strains in different combinations as given below:

4.8.1 Malt beverage

Fermented kodo malt beverage can be used as health drink or energy drink. In the

present investigation, an attempt has been made to prepare probiotic enriched malt beverage

by adding inhouse potential probiotic strains i.e. Pediococcus acidilactici L1 KM251713,

Lactobacillus plantarum L2 KM251714 and Lactobacillus fermentum F3 KC242235 as

single cultures/cocultures. The malt beverage was prepared in four different sets. Set-A was

fermented only with Pediococcus acidilactici L1, while set-B was inoculated with

Lactobacillus plantarum L2, set-C with Lactobacillus fermentum F3. In set-D, consortia of

probiotic isolates i.e. Pediococcus acidilactici L1 + Lactobacillus plantarum L2 +

Lactobacillus fermentum F3 (@ 108cfu/ml) were added (Plate 8). After inoculation

fermentation of each set was carried out at 37˚C. The fermentation was terminated by

keeping these sets at 4˚C and all these sets were subjected for further nutritional evaluation,

microbiological and physicochemical analysis of each prepared set of malt beverage was

performed at regular interval during storage.

Nutritional facts of fresh malt beverage had been presented in Table 13. Nutritional

facts of RTE beverage indicated that this product was rich in proteins and maximum protein,

carbohydrates and crude fibers was in set D i.e. 24.2 g per 100 ml of protein, 14.17 g per 100

ml carbohydrates and 8.3 g per 100 ml of dietary fibers. Statistically, it was observed that set

D significantly contains the highest amount of nutritional contents compared to set B, C and

D. As is globally known being malt beverage a healthy drink as it contains good amount of

carbohydrates, fats and measurable amount of vitamin A, E and K, thiamine, riboflavin,

vitamin B12, Ca, Fe, Mg, P, Zn, Co (Ershidat et al. 2010).

67

Table 13: Nutritional chart of malt beverage

Nutritional factsper 100 ml

ControlSamples

CD

Set A Set B Set C Set D

Antioxidants(%)

48.0 51.28 50.54 52.34 53.11 0.81

Protein (g) 19.5 23.5 20 23 24.2 1.16

Total Fat (g) 5.3 5.3 4.8 4.6 5.5 0.16

Carbohydrate(g)

13.9 12.11 12.15 14.12 14.15 0.08

Crude fibers (g) 7.5 7.7 8.2 7.9 8.3 0.18

CD0.05 0.94 0.43 0.89 0.87 0.24

Control: without inoculamSet A: P. acidilactici L1Set B: L. plantarum L2Set C: L. fermentum F3Set D: P. acidilactici L1 + L. plantarum L2+ L. fermentum F3

Ingestion of LAB has been suggested to confer a range of health benefits including

immune system modulation (Bielecka et al. 2002; Tannock, 2001), increased resistance to

malignancy and infectious illness (Krasaekoopt et al. 2003). Clinical studies hence suggested

the efficacy of the administration of probiotics in maintaining the remission of the pouchitis,

ulcerative colitis, and crohn’s disease (Gupta and Garg, 2009). Recent studies have also

suggested that probiotics could have beneficial effects for some metabolic disorders such as

hypertension. A probiotic may also be a functional food. The Lactic acid fermentation of

cereals simultaneously also led to enhance the nutritional content of that product. Therefore,

an aim to prepare important cereal based functional food to deliver the probiotic health

effects for mankind has been successfully delivered.

Beverages are food that are distinguished by its principal characteristics from other

foods, first they are liquid that are consumed in liquid state and secondly, they are either

consumed for their thirst quenching properties or for their stimulating effect. Llango and

Antony, (2014) studied microbial quality of “koozh” a fermented beverage made from millet

flour and rice. In all koozh samples, LAB were found to be dominant and yeast-mould counts

were comparatively lower. LAB counts on MRS showed significant differences (p ≤ 0.05)

with TBC and counts on M17 and yeast counts. The LAB counts on MRS showed a very

strong correlation with counts on M17 (r = 0.9396) as both are selective media used for LAB

enumeration.

Plate 8: Probiotic enriched malt beverage

Fig. 8: Sensorial evaluation of malt beverage

0123456789Control

Set A

Set BSet C

Set DAppearance/color

Flavor

Texture

Taste

68

Malted finger millet is used to produce alcoholic beverage. Traditionally opaque beer

was produced by malting sorghum, converting cooked sorghum and maize grits into

fermentable sugars, souring the mash and finally fermenting the sugars into alcohol (Waniska

et al. 1999).

4.8.1.1 Sensorial evaluation

Freshly prepared malt beverage samples were assessed by 10 panelist using a 9 point

sensory hedonic scale for some sensory parameters (viz. appearance/colour, flavour, texture,

taste and overall acceptability), as described by Amerine et al. (1965). In a sensory evaluation

malt beverage set A was least accepted whereas malt beverage set D had a maximum

acceptability as it scored 7.97 out of 10 (Table 14 and Fig. 8 ). Statistically sensorial

evaluation was carried out by Randomized Block Design (RBD). The result showed

significantly higher acceptable effect of set D based on different treatments on sensory

attributes of malt beverage. The results of above experiment also indicated that the types of

bacterial strain contributed a significant influence on the overall acceptability of the product.

Table 14: Sensorial evaluation of malt beverage

Parameter Appearance / Color Flavor Texture Taste Overallacceptibility

Control 7 6 7 7 6.75

Set A 7 6.5 6.3 6.8 6.65

Set B 7.3 6.5 7.1 6.5 6.85

Set C 6.5 7.8 6.5 6.8 6.9

Set D 8.4 7.9 8.3 7.3 7.97

CD0.05 1.15 0.82 0.82 0.82 0.36

4.8.2.2 Microbiological evaluation

The microbiolocal evaluation of malt beverage was carried out to validate predicted

growth values during storage period. Table 15 and Fig. 9 revealed the data regarding viable

colonies in terms of log cfu/ ml. The viable colonies each treatment were enumerated on 0th

day, 3rd, 5th, 7th and 15th day of fermentation. It was observed that there was an increase in

number of bacterial cells during storage conditions in each treatement. At 0 h maximum

cfu/ml were in set D (10.20) and nil in control. Viable count become maximum for set A, B

and D on 5th day with log cfu/ml 14.80 for set A and 16.50 for set B and D. In addition to

69

Table 15: A profile of microbial count of malt beverage

Sr.No. Name ofTreatment

Storage Intervals (in days) CD

0 3 5

LAB OtherBacteria

Yeast Mold LAB OtherBacteria

Yeast Mold LAB OtherBacteria

Yeast Mold

1 Control 0.0 0.00 0.00 0.00 2.60 6.50 2.50 0.00 4.50 10.19 4.50 2.50 2.6

2 Set A 10.20 0.00 0.00 0.00 12.50 2.50 0.00 0.00 14.80 4.50 2.50 0.00 0.01

3 Set B 10.05 0.00 0.00 0.00 13.50 3.05 0.00 0.00 16.50 2.50 3.05 0.00 0.01

4 Set C 10.18 0.00 0.00 0.00 11.50 0.00 0.00 0.00 12.85 0.00 0.00 0.00 0.008

5 Set D 10.20 0.00 0.00 0.00 13.80 0.00 0.00 0.00 16.50 0.00 0.00 0.00 0.008

CD 0.01 0.02 0.01 0.008 2.8 0.02 0.01 0.008

Treatment (0.24) Days(0.52) (T×D= 0.12)

Fig. 9: Total viable count of malt beverage

0

2

4

6

8

10

12

14

16

18

LAB

Oth

er B

acte

ria

Yeas

t

Mol

d

LAB

Oth

er B

acte

ria

Yeas

t

Mol

d

LAB

Oth

er B

acte

ria

Yeas

t

Mol

d0 3 5

Via

ble

coun

ts(L

og C

FU

/ml)

Storage Time (Days)

Control Set A Set B Set C Set D

70

lactic acid bacteria, total aerobic mesophilic bacteria, yeast and mold were also enumerated

and it was observed that total aerobic mesophilic bacteria, yeast and mold were below the

detection limit as shown in Fig. 9. Data obtained from analysis of the samples were evaluated

by variance of analysis and the difference among means were calculated. Statistically, it had

been confirmed that non significant change occurred in viability of microbial cells during

strorage and set D contains the highest number of beneficial LAB as compared to control, set

A, B and C.

Beverages are food that are distinguished by its principal characteristics from other

foods, first they are liquid that are consumed in liquid state and secondly, they are either

consumed for their thirst quenching properties or for their stimulating effect. Llango and

Antony, (2014) studied microbial quality of “koozh” a fermented beverage made from millet

flour and rice. In all koozh samples, LAB were found to be dominant and yeast-mould counts

were comparatively lower. LAB counts on MRS showed significant differences (p ≤ 0.05)

with TBC and counts on M17 and yeast counts. The LAB counts on MRS showed a very

strong correlation with counts on M17 (r = 0.9396) as both are selective media used for LAB

enumeration.

Malted finger millet is used to produce alcoholic beverage. Traditionally opaque beer

was produced by malting sorghum, converting cooked sorghum and maize grits into

fermentable sugars, souring the mash and finally fermenting the sugars into alcohol (Waniska

et al. 1999).

4.7.2 Ready To Eat (RTE) porridge

Porridge, a widely consumed nutritious product is usually prepared by grinding or

chopping grains. Grains used for porridge include rice, wheat, barley, corn and buckwheat. In

the present study, an absolutely novel health product i.e. RTE porridge was made by mixing

kodo millet and barley seeds in equal propotion (50 : 50) followed by their soaking for 6 h in

consortium of inhouse probiotics i.e. Pediococcus acidilactici L1, Lactobacillus plantarum

L2 and Lactobacillus fermentum F3. The grains were then dried, roasted and grinded to

coarse powdered form. This RTE porridge was stored upto a period of one month without any

apparent change (Plate 9).

Nutritional chart of RTE porridge had been presented in Table 17. This product was

found rich in antioxidants, crude fibers, carbohydrates and proteins. The RTE porridge was

71

found to have 32.1 g proteins, 58% antioxidants, 3.2 g of total fats, 24 g carbohydrates and

11.2 g of crude fibers.

Table 16: Nutritional chart of RTE porridge

Sr. no. Nutritional facts per 100 ml

1 Antioxidants (%) 58

2 Protein (g) 32.1

3 Total Fat (g) 3.2

4 Carbohydrate (g) 24

5 Crude fibers (g) 11.2

Porridge produced from various cereals and coarse cereals like wheat, oats, maize,

sorghum etc. are widely consumed owing to their ease of making and acceptability among all

age groups. Porridges are used as breakfast foods for adults as well as complimentary foods

for infant and are also dietary adjuncts for convalescents (Michaelsen, 1998). Andah and

Muller, (1973) evaluated the nutrient content of koko, a Ghanian fermented maize porridge.

The analysis was as follows: crude protein 96%, fat 4.3%, crude fibre 17%, ash 149 %.

4.7.2.1 Sensorial evaluation:

RTE porridge prepared was divided into three sets. In set I, RTE porridge was mixed

with water, in set II with milk, while in set III with curd. The freshly prepared RTE porridge

slurry samples were assessed by 10 panelist using a 9 point sensory hedonic scale for some

sensory parameters (viz. appearance/color, flavor, texture and overall acceptability), as

Table 17: Sensorial evaluation of RTE porridge

Samples Parameters

Appearance /Color

Flavor Texture Taste Overallacceptibility

Set I* 7.7 6.5 7.6 7.3 7.28

Set II** 8.7 7.7 7.6 8.6 8.15

Set III*** 7.3 7.0 7.5 7.5 7.33

CD0.05

0.19 0.19 0.24 1.22 0.02

Set I*: RTE porridge in waterSet II**: RTE porridge in milkSet III***: RTE porridge in curd

Plate 9: RTE porridge

Fig 10: Sensorial evaluation of RTE porridge

0

2

4

6

8

10

Appearance /Color

Flavor

Texture

Taste

Set I*

Set II**

Set III***

72

described by Amerine et al. (1965). In a sensory evaluation set I was least accepted whereas

set II had a maximum acceptability as it scored 8.15 out of 10 as shown in (Table 17 and Fig

10). Statistically sensorial evaluation was carried out by Randomized Block Design (RBD)

and significantly set B was accepted more as compared to other sets. The results showed a

significant effect of different treatments on sensory attributes of RTE porridge. The results of

above experiment also indicated that the type of bacterial strain contributed a significant

influence on the overall acceptability of the product.

4.7.2.2 Microbiological evaluation:

Bioavailability of probiotics in RTE porridge was carried out to validate predicted

growth value during storage period of one month. The viable count of lactic acid bacteria was

found to be 9.60 log cfu/ml after one month of storage. This is as per specification of WHO

showing efficacy of probiotics i.e., 108 cfu/ml (FAO/WHO, 2002) and meets out the criteria

of good probiotic food which should contain specific probiotic strains at a specified level

during storage time.

4.7.3 Multigrain bread

Multigrain bread of kodo millet was prepared by mixing wheat and kodo millet in

different ratios. Standardization of different ratios of wheat:kodo millet i.e. 30:70, 40:60,

50:50, 60:40, 70:30 was done. Baker’s yeast i.e. Saccharomyces cerevisae was added at the

rate 108 cfu/ml. The best ratio was selected on the basis of its physical attributes and 50:50

was finally selected shown in Plate 10 (a, b and c) and Table 18.

Table 18: Standardization of different ratio of wheat and kodo millet based on physicalattributes

Sr.no.

Ratios(wheat:kodo millet)

Colour Texture Taste Appearance Softness Mean

1 30:70 2 1 2 1 1 1.4

2 40:60 2 1 2 1 2 1.6

3 50:50 3 2 3 2 3 2.6

4 60:40 1 2 3 2 2 2.0

5 70:30 1 2 2 2 2 1.8

C D0.05

0.39 0.58 0.18 0.18 0.18

1 : poor2 : fair3 : good

73

Table 19 represents the comparison of multigrain bread (kodo millet : wheat) with the

commercial, wheat bread and the multigrain bread had been found to be rich in vital nutrients

i.e. proteins, fats and overall contents as compared to the wheat bread.

Table 19: Nutritional chart of multigrain bread (kodo millet : wheat) and wheat bread

ParametersNutritional facts per 100 g t-test

Multigrain bread Wheat or wholebread

(commercial)

Proteins (%) 9.7 3.0 165.72

Carbohydrates (mg/g) 80 144 9.89

Crude fibers (g) 21 - 75.7

Fats (g) 1.5 1.0 15.3

Total phenols (mg/ g) 6.13 - 22.07

Flavonoids (µg/ ml) 2.2 - 39.66*: calorieking.com

When nutrients facts were compared with commercial wheat bread, it has been

observed that kodo millet multigrain bread had much higher proteins fiber and antioxidants as

compared to commercial wheat bread. Thus, proving it to be a better product for consumers.

Clopicka et al. 2012 examined the phenolic contents of different kinds of flour and breads,

and were expressed as mg gallic acid per gram of dry weight. Buckwheat flour had the

highest phenolic content (7.25-0.23 mg/ g) and the next one was wheat (6.96-0.11 mg/g dw).

Amaranth and quinoa flour had the lowest phenolic content (2.71-0.1 mg/ g and 2.8- 0.1

mg/g, respectively) and the differences between them and the former two were statistically

significant.

Karwe et al. (2006) observed lower content of total phenolics in buckwheat white,

raw flour, but higher content of total phenolics of buckwheat dark, raw flour in comparison

with phenolic content in our buckwheat flour. Consistently with the above results, the content

of phenols in breads was highest in breads baked with 30 g/100 g addition of buckwheat flour

(2.65-0.10 mg/g ).

4.7.2.1 Sensorial evaluation:

Multigrain bread prepared was divided into two sets i.e. Set A and Control. The two

sets were assessed by 10 panelist using a 9 point sensory hedonic scale for some sensory

a.) b.)

c.)

Plate 10: Multigrain bread [Wheat : Kodo millet (50 : 50)]

Fig 11: Sensorial evaluation of multi grain bread

6.26.46.66.8

77.27.47.67.8

88.2

Color

Aroma

TasteTexture

Overallacceptibility

50:50:00

30:70

74

parameters (viz. appearance/color, flavor, texture and overall acceptability), as described by

Amerine et al., (1965). In a sensory evaluation control was least accepted whereas control

had a maximum acceptability as it scored 8.1 out of 10 as shown in (Table 20 and Fig 11).

Statistically sensorial evaluation was carried out by Randomized Block Design (RBD) and

significantly set A was accepted more as compared to other sets. The results showed a

significant effect of different treatments on sensory attributes of RTE porridge. The results of

above experiment also indicated that the type of bacterial strain contributed a significant

influence on the overall acceptability of the product.

Table 20: Sensorial evaluation of bread

50:50: Wheat : Kodo millet

30 :70: wheat: kodo millet

Kodo millet grains contain some natural microflora capable of suppressing broad

spectrum pathogens. In addition, high quality of polyphenols also contributes strongly for

antimicrobial activity thus imparting shelf stability to its products. The novel functional kodo

food items formulated in this research work have been assessed with high nutritional value

along with other exceptional health benefits, high antioxidants probiotic viability thus

fulfilling the main objectives of the present study.

S. no. Attributes Multigrain bread t-value

50:50 30:70

1 Color 7.4 6.9 12.75*

2 Aroma 8.1 7.4 17.85*

3 Taste 8.0 7.4 2.15

4 Texture 7.8 7.4 10.19*

Overall acceptibility 7.84 7.29 140.22*

Chapter-5

SUMMARY AND CONCLUSIONS

In the present investigation entitled “Formulation of functional foods of kodo millet

(Paspalum scrobiculatum) enriched with probiotics and to evaluate their health potential” an

attempt has been made to prepare different nutraceutical food products of kodo millet viz.

multigrain bread, malt beverage and RTE porridge. To evaluate their health potential,

schematically first of all, the natural microflora associated with kodo millet (raw and malted)

were isolated followed by their screening and characterization on biochemical and molecular

level. The major findings of the work include:

In total, 29 bacteria i.e. 13 from raw kodo millet and 16 from malted kodo millet were

isolated. The morphological and biochemical characteristics of all these isolates were

explored. 16 lactic acid bacteria, 7 bacilli and 6 cocci were isolated from raw and malted

kodo millet samples. Among them, 26 isolates were found to be gram +ve and only 3 were

gram –ve. Further, these 29 isolates were preliminary screened on the basis of their

antagonistic activity. The test indicator used in the present study were Staphylococcus aureus

IGMC, Enterococcus faecalis MTCC 2729, Listeria monocytogens MTCC 839, Clostridium

perfringens MTCC 1739, Leuconostoc mesenteroids MTCC 107, Bacillus cereus CRI,

Escherichia coli IGMC, Pseudomonas syringae IGMC, Pectobacterium carotovorum MTCC

1428 and Lactobacillus plantarum MTCC 1428. Out of 29 isolates, 6 bacterial isolates viz.

KR3, KR5, KR9, SM1, SM3 and KM1 exhibited broadest and strongest antagonism and on

its basis the best isolates were finally screened for further studies. Among all, three bacteria

KR5, SM1 and SM3 showed highest degree of antagonism and these isolates were identified

as Paenibacillus jamilae, Bacillus sp.SM1 and Bacillus sp. SM3 respectively.

In the next step of study, nutritional evaluation i.e. proteins, carbohydrates, starch,

minerals, dietary fibers, antioxidants, flavonoids, phenols and crude fat contents of kodo

millet grains collected from different sites of Mandi, Kangra and Hamirpur districts of

Himachal Pradesh was accomplished and it was found that grains collected from the Mandi

district comparatively had an edge in many nutrient contents over others. The result showed

that overall kodo millet grains contained 3.7 % proteins, 55.6 mg/ g carbohydrates, 5.6 mg/g

of total phenols, 6.7 g crude fibers and 44 % antioxidant activity.

76

The polyphenols present in kodo millet were extracted using three different solvents

i.e. acetone, methanol and water. TLC studies of the extracted polyphenols from kodo millet

showed the presence of predominantely ferulic acid (Rf - 0.52), cinnamic acid (Rf - 0.68) in

the millet. Further quantification of these polyphenols was done by using HPLC, analyzing

359.2 mg/ g of ferulic acid and cinnamic acid was 79.01 mg /g in methanol extract, whereas

in case of acetone extraction it ranged 109.45 mg /g of ferulic acid and 111.45 mg/ g of

cinnamic acid. Antagonistic spectrum of polyphenols extracted from kodo millet showed

inhibition against 4 bacterial test indicators viz. S.aureus, L. mesenteroids, B.cereus, E.coli

proving its antimicrobial action.

Formulation of probiotic enriched functional foods of kodo millet was done by using

inhouse potential probiotics i.e. Pediococcus acidlactici L1, Lactobacillus plantarum L2 and

Lactobacillus fermentum F3 as a single culture as well as consortia of them.

Intercompatibility of different microorganisms was analyzed for consortia formulation.

Probiotic enriched malt beverage was prepared in different sets by adding probiotic culture in

different permutations and combinations. Nutritional evaluation of different sets of malt

beverage i.e. proteins, carbohydrates, crude fibers and antioxidant activity was carried out

and the set in which consortia of probiotic culture was added, adjudged the best depending

upon the highest value of nutrients as compared to other sets and sensory evaluation as well

figuring out score of 7.97 on 9 point hedonic scale. Upon storage for 15 days, the malt

beverage was investigated for microbial count. The viable colonies of lactic acid bacteria and

aerobic mesophilic bacteria were counted in terms of log 10 cfu/ ml. It was found that viable

count of Lactobacilli was well maintained during storage period.

In the present study, an absolute novel health product i.e. probiotic enriched kodo

RTE porridge. This product was found to be rich in nutrient contents i.e. antioxidants, crude

fibers and carbohydrates. This RTE porridge was stored upto a period of one month without

any apparent physical change. Bioavailability of LAB added in the product remained

consistent throughout the storage period.

Another interesting innovation of the present study is multigrain bread prepared by

adding wheat and kodo millet in different ratios (kodo millet : wheat) 30:70, 40:60, 50:50,

60:40, 70:30. Out of these, the bread having ratio 50:50 of kodo and wheat flour was

accepted the most depending on its physical attributes. Further nutritional evaluation of

multigrain bread was done and the results showed that multigrain bread had the highest value

77

of protein and crude fibers as compared to white bread this finding was supported by

sensorial evaluation also for its qualitative traits proving its market potential.

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Dr. Y.S Parmar University of Horticulture & Forestry, Nauni, Solan-173230, H.P.Department of Basic Sciences

Title of thesis : “Formulation of functional foods of kodo milletenriched with probiotics and to evaluate their

health potential”Name of student : Shakshi SharmaAdmission No. : F-2013-24-MName of Major Advisor : Dr. (Mrs.) Nivedita SharmaMajor field : MicrobiologyMinor field(s) : i) BiochemistryDegree awarded : M.Sc.Year of award of degree : 2015No. of pages in thesis : 88No. of words in abstract : 374

ABSTRACT

The present investigation was carried out to prepare different nutraceutical, functional food products of kodo millet viz.

multigrain bread, malt beverage and RTE porridge. To evaluate their health potential schematically the natural microflora

associated with kodo millet (raw and malted) were isolated followed by their screening, characterization on biochemical and

molecular level. In total, 29 bacteria i.e. 13 from raw kodo millet and 16 from malted kodo millet were isolated. The

morphological and biochemical characteristics of all these isolates were explored. In total, 16 lactic acid bacteria, 7 bacilli and 6

cocci were isolated from raw and malted kodo millet samples. Among them, 26 isolates were found to be gram +ve and only 3

were gram –ve. Further, these 29 isolates were preliminary screened on the basis of their antagonistic activity. Among all, KR5,

SM1 and SM3 showed highest degree of antagonism, and were identified using 16S rRNA gene technique, KR5 was identified

as Paenibacillus jamilae and SM1, SM3 were tentatively identified as bacilli on the basis of biochemical characterization.

Nutritional evaluation i.e. proteins, carbohydrates, starch, minerals (Fe, P, Mg), dietary fibers, antioxidants, flavonoids, phenols

and crude fat content of kodo millet grains collected from different sites of Mandi, Kangra and Hamirpur districts of Himachal

Pradesh was accomplished and it was found that overall grains collected from the Mandi district comparatively had an edge in

many nutrients. TLC studies of the extracted polyphenols from kodo millet showed the presence of predominantely ferulic acid

(Rf - 0.52) and cinnamic acid (Rf - 0.68) in the millet. Further quantification of these polyphenols was done by using HPLC,

analyzing ferulic acid and cinnamic acid. Antagonistic spectrum of polyphenols extracted showed inhibition against 4 bacterial

test indicators viz. S.aureus, L. mesenteroids, B.cereus, E.coli proving its antimicrobial action. Various Functional foods of kodo

millet i.e. malt beverage, RTE porridge and multigrain bread was prepared by using different inhouse potential probiotic i.e.

Pediococcus acidlactici L1, Lactobacillus plantarum L2 and Lactobacillus fermentum F3.These food products were adjudged the

best depending upon nutritional as well as sensorial evaluation. The probiotic microorganisms used to prepare new

pharmaceutical functional foods of kodo millet to impart to betterment of health of public, in the present study have been proved

safe as well as highly effective.

Signature of Major Advisor Signature of Student

Countersigned

Professor and Head,Department of Basic Sciences,

Dr. Y.S. Parmar University of Horticulture and Forestry,Nauni, Solan - 173230 (H.P.)

i

APPENDIX I

Anova for Table 14

Source df Mean sum of square (MSS)Antioxidant Proteins Total fat Carbohydrate Crude fibers

Treatment 4 11.67 13.38 0.48 3.29 3.36Error 10 0.20 0.40 0.008 0.002 0.01

Analysis of Variance for Functional foods

Anova for sensorial evaluation of malt beverage

Source df Mean sum of square (MSS)Appearance Falvor Texture Taste Overall

acceptibilityTreatment 4 3.20 2.19 1.72 0.26 12.66Error 10 0.41 0.21 0.21 0.21 0.04

Anova for sensorial evaluation of RTE porridge

Source df Mean sum of square (MSS)Appearance Falvor Texture Taste Overall

acceptibilityTreatment 2 1.63 1.09 0.007 1.31 0.72Error 6 0.01 0.01 0.014 0.38 0.001

ii

Anova for Table 8

Source df Mean sum of square (MSS)Proteins Carbohydrates Starch Total

phenolsCrudefibers

Antioxidants Phosphorus Magnesium Iron Crude fat Flavonoids

Treatment 2 0.130 2.56 1.96 0.01 0.09 4.00 0.0004 0.0003 0.77 0.0001 0.001Error 6 0.01 0.67 0.67 0.006 0.007 1.00 0.0001 0.0001 0.006 0.0001 0.0001

iii

APPENDIX II

Sensory Evaluation Sheet

EVALUATION FOR SENSORIAL QUALITY OF FOOD PRODUCTS

Name of Penalist: Date:

Product Name: Malt beverage

Kindly evaluate the given samples on Hedonic scale (1 to 9) according to attributesmentioned below:

Sr. No. Samples Sensory parametersAppearance/

ColorFlavor Texture Taste Overall

Acceptibility1 Set A

2 Set B

3 Set C

4 Set D

Scores:1. Disliked extremely2. Disliked very much3. Disliked moderately4. Disliked slightly5. Neither liked nor disliked6. Liked slightly7. Liked moderately8. Liked very much9. Liked extremely

iv

APPENDIX III

Sensory Evaluation Sheet

EVALUATION FOR SENSORIAL QUALITY OF FOOD PRODUCTS

Name of Penalist: Date:

Product Name: RTE Porridge

Kindly evaluate the given samples on Hedonic scale (1 to 9) according to attributesmentioned below:

Sr. No. Samples Sensory parametersAppearance/

ColorFlavor Texture Taste Overall

Acceptibility1 Set I

2 Set II

3 Set III

Scores:1. Disliked extremely2. Disliked very much3. Disliked moderately4. Disliked slightly5. Neither liked nor disliked6. Liked slightly7. Liked moderately8. Liked very much9. Liked extremely

v

APPENDIX IV

Sensory Evaluation Sheet

EVALUATION FOR SENSORIAL QUALITY OF FOOD PRODUCTS

Name of Penalist: Date:

Product Name: Multigrain bread

Kindly evaluate the given samples on Hedonic scale (1 to 9) according to attributesmentioned below:

Sr. No. Samples Sensory parametersAppearance/

ColorAroma Texture Taste Overall

Acceptibility1 50:50

2 30:70

Scores:1. Disliked extremely2. Disliked very much3. Disliked moderately4. Disliked slightly5. Neither liked nor disliked6. Liked slightly7. Liked moderately8. Liked very much9. Liked extremely

Brief resume of Student

Name : Shakshi Sharma

Father’s Name : Shri. Tirlok Sharma

Date of Birth : 02.01.1993

Sex : Female

Marital Status : Unmarried

Nationality : Indian

Educational Qualifications :

Certificate/Degree Class/Degree Board/University Year

Matriculation First HP Board Dharamshala 2008

10+2 First HP Board Dharamshala 2010

B.Sc.(H) Biotechnology First HPU, Shimla 2013

Whether sponsored by some : No

State /Central Govt./ Univ./SAARC

Scholarship /Stipend/ Fellowship, : Yes

any other financial assistance received

during these study period

(Shakshi Sharma)