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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
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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).
20
‘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.
LITERATURE CITED
Abdalla AA, El Tinay AH, Mohamed BE and Abdalla AH. 1998. Effect of processing onphytate and mineral content of pearl millet. Food Chemistry 63 (1): 79 – 84
Adekunle AA. 2012. Agricultural innovation in sub-saharan Africa: experiences frommultiple stake holder approaches. Forum for Agricultural Research in Africa, Ghana.ISBN 978-998
Adeola O and Orban JI. 1994. Chemical composition and nutrient digestibility of pearl milletfed to growing pigs. Journal of Cereal Science 22: 177-184
Amerine MA, Pangborn RM and Rossler EB. 1965. Principles of sensory evaluation of food.Academic Press, New York, 254 p.
Amparo S, Reyes B, and Rosaura F. 2003. Bioaccessibility of calcium, iron and zinc fromthree legume samples. Food 47 (6): 438-441
Andah A and Muller HG. 1973. Koko, a Ghanian fermented maize porridge. Journal ofAgriculture Sciences 6: 103-108
Aneja KR. 2003. Experiments in Microbiology, Plant pathology and Biotechnology.Biochemical activities of microorganisms, 4theds., New age International Publishers, NewDelhi, pp. 245-275
Anonymous. 2002. Agriculture: A vision for the future-probiotics ‘What’s the Hype’. Bio-Age News Letter. http://www.bioag.com/info/newsletters/enews2.html. Accessed during22nd January 2008
AOAC. 2007. Official methods of analysis of association of official analytical chemists,16theds., Association of Official Analytical Chemists. Arlington, Virginia, USA
Arora P, Sehgal S and Kawatra A. 2003. Content and HCl-extractability of minerals asaffected by acid treatment of pearl millet. Food Chemistry 80: 141-144
Arora S, Jood S and Khetarpaul N. 2011. Effect of germination and probiotic fermentation onnutrient profile of pearl millet based food blends. British Food Journal 113(4): 470-481
Asharani VT, Jayadeep A, Malleshi NG. 2010. Natural antioxidants in edible flours ofselected small millets. International Journal of Food Property 13(1): 41–50
Asma MA, El Fadil EB and El Tinay AH. 2006. Development of weaning food fromsorghum supplemented with legumes and oil seeds. Food Nutrition Bulletin 27(1): 26-34
Azim A, Naseer Z and Ali A. 1989. Nutritional evaluation of maize fodder at two differentvegetative stages. Journal of Animal Sciences 2(1): 27-34
Babu BV, Ramana T and Radhakrishna TM. 1987. Chemical composition and protein inhybrid varieties of finger millet. Indian Journal of Agriculture Sciences 57(7): 520-522
Badau MH. 2006. Microorganisms associated with pearl millet cultivars at various maltingstages. International Journal of Food Safety 8: 66-72
79
Banerjee S, Sanjay KR, Chethan S and Malleshi N G. 2012. Finger millet polyphenols:Investigation of their antioxidant capacity and antimicrobial activity. Academic Journals6(13): 362-374
Baranowski JD, Davidson PM, Nagel CW and Brannen RL. 1980. Inhibition ofSaccharomyces cerevisiae by naturally occurring hydroxyl cinnamates. Journal of FoodScience 45: 592–594
Barefoot SF and Klaenhammer TR. 1983. Detection and activity of Lactacin B, a bacteriocinproduced by Lactobacillus acidophilus. Applied and Environmental Microbiology 45(6):1808-1815
Bhumika T, Kalpana P. 2010. Finger millet (Eleucinecoracana) flour as a vehicle forfortification with zinc. Journal of Trace Elements in Medicine and Biology 24: 46 – 51
Bielecka M, Biedrzycka E and Majkowska A. 2002. Selection of probiotics and prebiotics forsynbiotics and confirmation of their in vivo effectiveness. Food Research International35: 125-131
Blandino A, Al-Aseeri M E, Pandiella SS, Cantero D and Webb C. 2003. Cereal basedfermented foods and beverages. Food Research International 36: 527-534
Brand WW, Cuvelier ME and Berset C. 1995. Use of free radical method to evaluateantioxidant activity. Food Science and Technology 28: 25-30
Bravo L. 1998. Polyphenols: chemistry, dietary sources, metabolismand nutritionalsignificance. Nutritional Review 56: 317–333
Bray HG and Thorpe WV. 1954. Analysis of phenolic compounds of interest in metabolism.Methods of Biochemical Analysis 52: 1-27
Busch RH, Fulcher RG. 1999. Evaluation of wheat phenolic acids during grain developmentand their contribution to fusarium resistance 47(4): 1476-1482
Chandrasekara A, Shahidi F. 2010. Content of insoluble boundphenolics in millets and theircontribution to antioxidant capacity. Journal of Agriculture Food Chemistry 58:6706–6714
Chavan JK and Kadam SS. 1989. Nutritional improvement of cereals by fermentation.Critical Reviews in Food Science and Nutrition 28(5): 349-400
Chetan S and Malleshi N G. 2007. Finger millet polyphenols: characterization andneutarceutical potential. American Journal of Food Technology 2: 582-59
Chethan S, Dharmesh SM, Malleshi NG. 2008. Inhibition of aldosereductase from cataractedeye lenses by finger millet (Eleusine coracana) polyphenols. Bioorganic MedicalChemistry 16:10085–10090
Claughton SM and Pearce RJ. 1989. “Protein enrichmentof sugar-snap cookies withsunflower protein isolates”. Journal of Food Science 54: 354
Clydesdale FA. 1997. Proposal for establishment of scientific criteria for health claims forfunctional foods. Nutrition Review 55: 413-22
80
Cowan MM. 1999. Plant products as antimicrobial agents. Clinical Microbiological Review12:564–582
Dave S, Yadav BK, and Tarafdar JC. 2008. Phytate phosphorus and mineral changes duringsoaking, boiling and germination of legumes and pearl millet. Journal of Food Science andTechnology 45 (4):344 – 348
Davis MJ. 1981. Isolation and culture of the bacteria associated with phony peach disease andplum leaf scald. Phytopathology 71: 869-870.
De MJ, Rogosa M and Sharpe M. 1960. A medium for the cultivation of lactobacilli. Journalof Applied Bacteriology 23: 130-135
Deosthale YG. 2002. The nutritive value of foods and the significance of some householdprocesses. http://www.unu.edu. p. 6
Desai AD, Kulkarni SS, Sahu AK, Ranveer RC and Dandge PB. 2010. Effect ofsupplementation of malted ragiflour on thenutritional and sensorial quality characteristicsof cake. Advanced Journal of Food Science and Technology 2(1): 67-71
Dogget H. 1989. Sorghum longmans. Journal of Food Science and Technology 19: 70-80
El-Nagger MYM. 2004. Comparitive study of probiotic culture to control the growth ofEscherichia coli and Salmonella typhimurium. Biotechnology 32: 173-180
Esele JP. 1989. Cropping systems, production technology, pests and diseases of finger milletin Uganda. In: Seetharam A, Riley K W and Harinarayana G (eds.). small millet in globalagriculture. Oxford and IBH, Delhi, India
Ferguson LR. 2001. Role of plant polyphenols in genomic stability. Mutation Research 475:89–111
Folch J, Lees M and Sloane-Stanley G. 1957. A simple method for isolation and purificationof total lipids from animal tissues. Journal of Biological Chemistry 226: 497-509
FAO/WHO. 2002. Food and Agricultural Organization of the United Nations and WorldHealth Organization: Guidelines for the evaluation of probiotics in food.WWW.fao.org/es/esn/food/foddanfood_probio.
FAO/WHO. 2006. Probiotic in foods. Health and nutritional properties and guidelines forevaluation. In: FAO Food and Nutrition pp 85
Fuller R. 1989. Probiotics in man and animals: A review. Journal of Applied Bacteriology 66:365-378
Gadaga TH, Mutukumiraa AN, Narvhusb JA and Feresu SB. 1999. A review of traditionalfermented foodsand beverages of Zimbabwe. International Journal of Food Microbiology53: 1-11
Gaggia F, Gioia DD, Baffoni L and Biavati B. 2011 The role of protective and probioticcultures in food and feed and their impact in food safety. Food Science and Technology22:58-66
81
Geetha T and Kalaichelvan G. 2010. Microbial succession and biochemical changes occurring infermentation of koozhu prepared from ragi and cumbu. International Journal of ChemicalSciences 8(5): 585-594
Gilliland SE and Speck ML. 1977. Deconjugation of bile acids by intestinal Lactobacilli. Appliedand Environmental Microbiology 33: 15-18
Gopalan C, Ramashastri BV and Balasubramanium SC. 2004. Nutritive Value of IndianFoods. ICMR, New Delhi
Gram HC. 1984. Uber die isoliertefarbung der Schizomuceten in Schnitt- undTrockenpraparaten (In German). Fortschritte der Medizin 2: 185-189
Gupta V and Garg R. 2009. Use of probiotic microorganisms in food. Indian Journal ofMedical Microbiology 27: 202-209
Hadimani NA, Malleshi NG. 1993. Studies on milling, physicochemical properties, nutrientcomposition and dietary fiber content of millets. Journal of Food Science and Technology30: 17–20
Harju S, Fedosyuk H and Peterson KR. 2004. Rapid isolation of yeast genomic DNA: bust n’grab. Biomedical Central Biotechnology 4: 8
Hazel T and Johnson IT. 1989. Influence of food processing on iron availability in-vitro fromextruded maize-based snack foods. Journal of Science of Food and Agriculture 46: 365 –375
Hedge JE and Hofreiter BT. 1962. In: Methods in carbohydrate chemistry, Academic press,New York, pp. 10-12
Hegde PS and Chandra TS. 2005. ESR spectroscopic study reveals higher free radicalquenching potential in kodo millet (Paspalumscrobiculatum) compared to other millets.Food Chemistry 92:177–182
Hemanalini G, Umapathy KP, Rao JR and Saraswathi G. 1980. Nutritional evaluation ofsprouted ragi. Nutrition Report International 22(2): 271-277
Hilu KW, De JMJ and Seigler D. 1978. Flavonoids patterns and systematic in Eleusine.Biochemistry System Ecology 6: 247-249
Hulse JH, Laing EM, Peason OE. 1980. Sorghum and millets: Their composition andnutritive Value. Academic Press, London
http://www.ICRISAT/FAO, 1996.ICRISAT/FAO
Ilango S and Antony U. 2014. Assessment of themicrobiological quality of koozh, afermented milletbeverage. African Journal of Microbiological Research 8(3): 308-312
Ikwelle MC, Lube DA and Nwasike CC. 1993. Millet production in Nigeria: constraints andprospects. In: Proceedings of the regional pearl millet improv. W/shop (O. Youm, and K.A. Kumar, ISC, Niger
Jack RW, Tagg JR and Ray B. 1995. Bacteriocins of gram positive bacteria. MicrobiologyReview 59: 171-200
82
Jackson M L. 1978. Kaolinite intercalation for all sizes and types with X-ray diffractionspacing distinctive from other clays. Trans. 11th Int. Congr. Soil Science 1:228-229
Khetarpaul N. 2003. Improvement of nutritional value ofpearl millet by fermentation andutilization of thefermented products. In: Recent Trends in MilletProcessing andUtilization, CCS Hisar Agril. Univ. Hisar, India, pp. 67-73
Kimura H, Sashihara T, Matsusaki H, Sonomoto K and Ishizaki A. 1998. Novel bacteriocinof Pediococcus sp. ISK-1 isolated from well – aged bed of fermented rice bran. Annals ofNew York Academy of Science 864: 345-348
Kligler B, Hanaway P and Cohrssen A. 2007. Probiotics in children. Pediatric Clinics ofNorth America 54: 949-967
Krasaekoopt W, Bhandari B and Deeth H. 2003. Evaluation of encapsulation techniques ofprobiotics for yoghurt. International Dairy Journal 13(1): 3-13
Kwarteng JO, Debrah KT, Glover RLK and Akabanda F. 2010. Process characteristics andmicrobiology of fura produced in Ghana. Nature and Science 8(8): 41-51
Lilly DM and Stillwell RH. 1965. Probiotics: Growth-promoting factorsproduced bymicroorganisms. Science 147: 747–748
Llango s and Antony U. 2013. Assessment of microbiological quality of koozh, a fermentedmilk beverage. African Journal of Microbiology Research 8(3): 308-312
MacDonough CM, Rooney LW and Saldivar SO . 2000. The millets. Food science andtechnology: Handbook of cereal science and technology .CRC Press. 2nd ed. 177-210
Madaan R, Bansal G, Kumar S and Sharma A. 2012. Estimation of total phenols andflavonoids in extract of Actaeaspicata roots and antioxidant studies. Indian Journal ofPharmaceutical Sciences 73(6): 666-669
Mahony MO. 1985. Sensory evaluation of food: statistical methods and procedures. MarcelDekker Inc., New York, 132 p.
Majeed A, Abbasi MK, Hameed S, Imran A and Rahim N. 2015. Isolation andcharacterization of plant growth promoting rhizobacteria from wheat rhizosphere andtheir effect on plant growth promotion. Fronteirs in Microbiology 6: 198
Majumdar TK, Premavalli KS and Bawa AS. 2006. Effect of puffing on calcium andironcontents of ragivarieties and their utilization. Journal of Food Science andTechnology 42(5): 542-545
Mathangi SK and Sudha K. 2012. Functional and phytochemical properties of finger milletfor health. International Journal of Pharmaceutical, Chemical and Biological Sciences2(4): 431-438
Metchnikoff II and Mitchell PC. 1910. Nature of man or studies in optimisticphilosophy,Kessinger Publishing, Whitefish,MT, USA
Michaelsen KF. 1998. Complementary feeding: A global perspective. Nutrition 14:763-766
83
Modi N. 2014. Probiotics and necrotisingenterocolitis: The devil (as always) is in the detail.Neonatology 105: 71-73
Murali A and Kapoor R. 2003. Effect of natural and pureculture fermentation of finger milleton zincavailability as predicted from HCL-extractabilityand molar ratios. Journal of FoodScience and Technology 40(1): 112-114
Naczk M, Shahidi F. 2007. Phenolics in cereals, fruits and vegetables: occurrence, extractionand analysis. Journal of Pharmacy Biomedical Analysis 43(2): 798
NIN, Nutritive value of Indian foods, Ed Gopalan and Deosthale, National Institute ofNitrogen, Hyderabad, 2003
Nkama I. 1998. Traditional food preparations of pearl millet in Nigeria. In: Pearl millet inNigeria Agriculture: Production, utilization and research priorities. Proc. of the pre-seasonnational coordination and planning meeting of NCRP for pearl millet (A. M. Emechebe;M. C. Ikwelle; O. Ajayi; M. A. Kano and A. B. Anaso) (eds.) LCRI Maiduguri. pp. 179 –208
Obilana J and Manyasa K. 2002. Encyclopedia of Grain Science: Sorghum – Production,ICRISAT, ed- Colin Wrigley, Harold Corks, Charles E Walker, Elsevier Ltd.
Oliver MM and Reid V. 2009. Use of probiotics in child care. Journal of Pediatric HealthCare 23: 194-197
Omemu AM, Oyewole OB and Bankole MO. 2007. Significance of yeasts in the fermentationof maize for ogiproduction. Food Microbiology 24: 571–576.
Onyeneho SN and Hettiarachchy NS. 1992. Antioxidant activity of durum wheta bran.Journal of Agriculture Food Chemistry 40:1496-1500
Parvez S, Malik KA, Kang SAH and Kim HY. 2006. Probiotics and their fermented foodproducts are beneficial for health. Journal of Applied Microbiology 100: 1171-1185
Patel MM and Rao V. 1996. Influence of untreated, heattreated and germinated black flourson biscuitmaking quality of wheat flour. Journal of FoodScience and Technology, 33(1):53-56
Pawar VD, Machewad GM. 2006. Changes in availability of iron in barely during malting.Journal of Food Science and Technology 43 (1):28 – 30
Pawar PA and Dhanvijay VP. 2007. Weaning foods: An overview. Beverage Food World34(11): 27-33
Pelinescu D, Chifiriuc MC, Ditu LM, Sarbu I, Bleotu C, Vassu T, Stoica I, Lazar V,Corcionivoschi N and Sasarman E. 2011. Selection and characterization of the probioticpotential of some lactic acid bacteria isolated from infant feces. Romanian BiotechnologyLetters 16(3): 6179-6189
www.phenol-explorer.eu/reports/41.databases on polyphenols content in food
Pundir RK, Rana S, Kashyap N and Kaur A. 2013. Probiotic potential of lactic acid bacteriaisolated from food samples: an in vitro study. Journal of Applied Pharmaceutical Science3(3): 85-93
84
Rababah TM, Majdi A, Al-Mahasneh MA and Ereifej KI. 2006. “Effect of chickpea, broadbean, or isolated soyprotein additions on the physicochemical and sensory properties ofbiscuits. Journal of Food Science 71(6): 438-442.
Ragaee S, Abdel-Aal EM, and Noaman M. 2006. Antioxidant activity and nutrientcomposition of selected cereals for food use. Food Chemistry 98: 32–38
Ramachandra G, Virupaksha TK, Shadaksharaswamy M. 1977. Relationship between tanninlevels and in vitro protein digestibility in finger millet. Journal of Agricultural FoodChemistry 25: 1101–1104
Ranganna S.1997. Handbook of analysis and quality control for fruit and vegetableproducts, 2nd e., dn. Tata McGraw Hill Publishing Comapany Ltd., New Delhi, India, 1109p.
Rao PU. 1994. Evaluation of protein quality of brown and white ragi (Eleusinecoracana)before and after malting. Food Chemistry 51: 433- 436.
Rao SS , Muralikrishna G. 2002. Evaluation of the antioxidant properties of free and boundphenolic acids from native and malted finger millet (ragi, Eleusinecoracana Indaf-15).Journal of Agriculture Food Chemistry 50: 889–892
Ravindran G. 1991. Studies on millets: Proximate composition, mineralcomposition, and phytateand oxalate contents. Food Chemistry 39 (1): 99 – 107
Ravindran V, Ravindran G, Sivkanasam R and Rajaguru SB. 1995. Biochemical andnutritional assessment of tubers from 16 cultivairs of serrt potato (Ipomeabatatas). Journalof Agricultural and Food Chemistry 43: 2646-2651
Robert R. 2004. The encyclopedia of nutrition and good health, 2nd edition, viva books PvtLtd, New Delhi
Sadasivam S and Manickam A. 1992. Biochemical methods for agricultural science. Willeyeastern limited, New Delhi, pp. 10-11
Saha S, Gupta A, Singh SRK, Bharti N, Singh KP, Mahajan V and Gupta HS. 2010.Compositionaland varietal influence of finger millet flour onrheological properties ofdough and quality ofbiscuit. Food Science and Technology, 44:616 -621
Saarela M, Mogensen G, Fonden R, Matto and Mattila-Sandholm T. 2000. Probioticbacteria:safety, functional and technological properties. Journal of Biotechnology 84: 197-215
Saldivar S. 2003. Cereals: Dietary Importance. In: Caballero B, Trugo L, Finglas P (ed)Encyclopedia of Food Science and Nutrition, ReinoUnido: Academic Press, Agosto,London, pp. 1027-1033
Schillinger U and Lucke FK. 1989. Antibacterial activity of Lactobacillus sake isolated frommeat. Applied Environmental Microbiology 55: 1901-1906
Seetharama N and Rao DB. 2004. Sustaining nutritional security. The Hindu Survey ofIndian Agriculture. Pp. 37
85
Seetharam A, Ravikumar RL. 1994. Blast resistance in finger millet— its inheritance andbiochemical nature. In: Riley K W, Gupta S C, Seetharamn A, Mushonga J N (eds)Advances in small millets. International Science Publisher, New York, pp 449–465
Sehgal A and Kwatra A. 2007. Use of pearl millet andgreen gram flours in biscuits and theirsensory andnutritional quality. Journal of Food Science and Technology 44(5): 536-538
Sharma A, and Kapoor AC. 1996. Levels of antinutritional factors in pearl millet as affectedby processing treatments and various types of fermentation. Plant Foods Human Nutrition49(3): 241-52
Sharma R and Raghuram T. 1990. Hypoglycemic effectof fenugreek seeds in non-insulindependent diabetesmellitus. Nutrition Research 10: 731-739
Shashi BK, Sharan S, Hittalamani S, Shankar AG and Nagarathna TK. 2007. Micronutrientcomposition, antimicronutirent factors and bioaccessibility of iron in differentfinger millet(Eleucinecoracana) genotype. Karnataka. Journal of Agriculture Science 20(3):583-585
Shigwedha N, Zhang L, Sichel L, Jia L, Gong P, Liu W, Wang S, Zhang S, Han X and GaoW. 2014. More than a few LAB alleviate common allergies: Impact of paraprobiotics incomparison to probiotical live cells. Journal of Biosciences and Medicines 2: 56-64
Shobha V, Kasturiba B, Naik RK, Yenagi N. 2008. Nutritive Value and QualityCharacteristics of Sorghum Genotypes. Karnataka Journal of Agricultrure Sciences20:586-588
Shrivastva B, Jain KK, Kalra A and Kuhad RC. 2014. Bioprocessing of wheat straw intonutritionally rich and digested cattle. Scientific Reports 4: 6360
Singh P, Singh G, Srivastava S and Agarwal P. 2005.Physico-chemical characteristics ofwheat flour andmillet flour blends. Journal of Food Science andTechnology 42(4): 340-343
Singh P and Srivastava S. 2006. Nutritional composition of 16 new verities of finger millet.Journal of Community Mobilization Sustainable Development 1(2): 81-84
Singh P and Srivastava S. 2007. Development and quality evaluation of iron rich biscuitmixes using finger millet. Journal of Community Mobilization Sustainable Development1(2): 89-94
Sitarski KR and Bojanowska KR. 1993. Ferulic acid in rye and wheat grain and grain dietaryfibers. Cereal Chemistry 70: 55-59
Siwela M, Taylor JRN, De milliano WAJ and Duodu KG. 2010. Influence of phenolics infinger millet on grain and malt fungal load, and malt quality. Food Chemistry 121:443–449
Soccol CR, Vandenberghe LPS, Spier MR, Medeiros ABP, Yamaguishi CT, Lindner JDD,Pandey A and Soccol VT. 2010. The potential of probiotics: a review. Food Technologyand Biotechnology 48(4): 413–434
Sreeramaiah H, Patel K, Srinivasan K. 2007. Influence of heat processing on thebioaccessibility of zinc and iron from cereals and pulses consumed in India. Journal ofTrace Elements in Medicine and Biology 21:1 – 7
86
Sripriya G, Chandrasekharan K, Murty VS, Chandra TS. 1996. ESR spectroscopic studies onfree radical quenching action of finger millet (Eleusinecoracana). Food chemistry 57(4):537–540
Sripriya G, Antony U and Chandra TS. 1997. Changes in carbohydrate, free amino acids,organic acids, phytate and HCl extractability of minerals during germination andfermentation of finger millet (Eleusinecoracana). Food Chemistry 58 (4): 3455 – 3501
Sushma D, Yadav BK, and Tarafdar JC. 2008. Phytate phosphorus andmineral changesduring soaking, boiling and germination of legumes and pearlmillet. Journal of FoodScience and Technology. 45 (4): 344 – 348
Tannock GW, Crichton C, Welling GW, Koopman JP and Midtvedt T. 2001. Ecologicalbehaviour of Lactobacillus reuteriis affected by mutant of the luxS gene. AppliedEnvironmental Microbiology 71: 8419-8425
Taylor JRN. 2004. Grain production and consumption: Africa. In: Wrigley C, Corke H, Walker CE (eds.) Encyclopedia of Grain Science. Elsevier, London, pp. 70 – 78
Thakkar R and Kapoor R. 2007. Enrichment of rice and finger millet based preparations withgum acacia andtheir effects on glycemic response in non-insulin dependent diabeticsubjects. Journal of Food Science and Technology 44(2): 183-185
Thompson LU. 1993. Potential health benefits and problems associated with antinutrients infoods. Food Research International 26: 131–149
Vachanth MC, SubbuRathinam KM, Preethi R and Loganathan M. 2010. Controlledatmoshpheric storage techniques for safe storage of processed little millet.AcademicJournal of Entomology 3(1): 13- 16.
Valerie G, Polycarpe K, Muriel G, Isabelle R and Claire M R. 2011. Changes in iron, zincand chelating agents during traditional African processing of maize. Food Chemistry 10:10-16
Veena B, Chimmad BV, Naik RK and Malagi U. 2004.Development of barnyard millet basedtraditional foods. Karnataka Journal Agricultural Science 17(3): 522-527
Verma V and Patel S. 2013. Value added products from nutria-cereals: Finger millet. Journalof Food Agriculture 25(3): 169-176
Vidyavati H, Mustai Begum J, Vijayakumari J, Gokaki S and Shemshad Begum. 2004.Utilization of fingermillet in the preparation of papad. Journal of Food Science andTechnology 41(4): 379-382
Vijayakumari J, Mushtari Begum J, Begum S, Gokavi S (2003) Sensory attributes of ethinicfoods from finger millet (Eleusinecoracana). Recent Trends in Millet Processing andutilization. In: Proceeding of National Seminar on Processing and Utilization of Millet forNutrition Security held on October 7-8,2003 organized under RNPSI (NATP) atCCSHAU, Hisar. pp. 7-12.
Viswanath V, Urooj A, Malleshi NG. 2009. Evaluation of antioxidant and antimicrobialproperties of finger millet polyphenols (Eleusine coracana). Food Chemistry 114: 340–346
87
Wadher KJ, Mahore JG and Umekar MJ. 2010. Probiotics: living medicines in healthmaintenance and disease protection. International Journal of Pharma and Bio Sciences1(3): 1-9
Wakil SM, Osamwonyi UO. 2012. Isolation and screening of antimicrobial producing lacticacid bacteria from fermenting millet gruel. International Research Journal ofMicrobiology 3(2): 72-79
Wang YC, Yu RC and Chou CC. 2006. Antioxidative activities of soymilk fermented withlactic acid bacteria and Bifidobacterium. Food Microbiology 23: 128-135
Waniska RD, Rooney LW. 2002. Sorghum grain quality for increased utilization.In: Leslie, J.F.(ed.), Sorghum and Millets Diseases. Iowa State Press, USA, pp. 327 – 335
Wollowski I, Rechkemmer G and Pool-Zobel B L. 2001. Protective role of probiotics andprebiotics in colon cancer. American Journal of Clinical Nutrition 73: 451-455
Xu W, Wei L, Qu W, Liang Z, Wang J and Huanga K. 2011. A novel antifungal peptide fromfoxtail millet seeds. Journal of Science Food Agriculture 91: 1630-1637
Yang ZY, Tu YY, Xia HL, Jie GL, Chen XM and He PM. 2007. Suppression of free-radicalsand protection against H2O2-induced oxidative damage in HPF-1 cell by oxidized phenolcompounds present in black tea. Food Chemistry 105(4):1349–1356
Yuksekdag ZN and Aslim B. 2010. Assessment of potential probiotic and starter properties ofPediococcus spp. Isolated from Yurkisk-Type fermented sausages. Journal ofMicrobiology and Biotechnology 20(1): 161-168
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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)
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