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INFLUENCE OF DIFFERENT STORAGE TEMPERATURES ON
THE FLOUR QUALITY PARAMETERS DURING SHORT TERM
STORAGE
BY
SUJITHA JESURAJAN
FACULTY OF AGRICULTURE
EASTERN UNIVERSITY
SRILANKA
2016

INFLUENCE OF DIFFERENT STORAGE TEMPERATURES ON
THE WHEAT FLOUR QUALITY PARAMETERS DURING
SHORT TERM STORAGE
BY
SUJITHA JESURAJAN
A research report
Submitted in partial fulfillment of the requirements for
The advance course in
FOOD SCIENCE AND TECHNOLOGY
For the degree of
BACHELOR OF SCIENCE IN AGRICULTURE
Faculty of Agriculture
Eastern University, Sri Lanka
2015
APPROVED BY
…………………………….. …………………………
Mr. M.R Shiraj Muneer Prof.(Mrs).T.Mahendran
Assistant chemist, Professor and Supervisor,
RND department Department of Agric.Chemistry
Prima Ceylon PVT Ltd Faculty of Agriculture,
China bay Eastern University
Trincomalee Sri Lanka
Date:……………………. Date:…………………
………………………………….
Dr.(Mrs).P. Premanandarajah
Head, Dpartment of Agric.Chemistry,
Faculty of agriculture,
Eastern University,
Sri Lanka,
Date: ………………………

Dedicated to my mom and dad
Without you, I am nothing

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ABSTRACT
Wheat flour which is the milled product of wheat grain, can be produced to fulfill
different purposes (Cakes, bread, string hoppers, muffins, rotti.etc) by altering their
chemical compositions. Three differently produced wheat flour samples with different
chemical compositions, freshly milled in Prima Ceylon Private Ltd, Chinabay,
Trincomalee were stored for three months (In the period from November to January)
under different storage temperature conditions of room temperature storage and air
conditioned storage.
It was assumed that the ambient temperatures of air conditioned storage and room
temperature storage was remained constant and the relative humidity varied during
the storage in the dependence on the year season. But the changing relative humidity
was not considered as a factor influencing the quality parameters except the moisture
content.
Certain analytical characteristics (moisture, wet gluten, gluten index, ash content,
protein content, color value and falling number) and biological, microbiological
characteristics weevils count, bacterial count, yeast and mold count and E.Coli and
coliforms counts of three different flour samples were determined at regular intervals
under two different storage temperatures.
Flour moisture, protein content, ash content, color value and falling number changed
with the time of storage under two different storage temperatures but no explicit
influence of the storehouse conditions and the initial flour properties was proved.
Viscoelastic properties (Wet gluten and gluten index) of weaker flour samples
changed during storage more markedly than those of stronger flours in the sense of a
significant improvement of their quality. Biological and microbiological

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characteristics were influenced by storage temperature since the water activity was the
main factor influencing their survival.

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ACKNOWLEDGEMENTS
First of all, I wish to thank God Almighty for his blessings and guidance in every
effort of this project.
I offer my profoundest gratitude to my university supervisor Prof. (Mrs). T.
Mahendran, Professor in Food Science and Technology, Department of Agric.
Chemistry, Faculty of Agriculture, Eastern University, Sri Lanka and Prima company
supervisor Mr. M. R. Shiraj Muneer for providing me the opportunity to carry out the
research study and for giving me the dedicated help, guidance and support in each
and every steps of my project.
My grateful thanks go to Dr. P. Sivarajah, Dean, Faculty of Agriculture and Dr.
(Mrs). P. Premanandarajah, Head, Department of Agric.Chemistry, Faculty of
Agriculture, Eastern University, Sri Lanka, for their encouragement and support to
my project.
I express my heartfelt thanks to Mr.Careem, Deputy manager, Research and
development department, Prima Ceylon PVT Ltd and executive staffs, technicians and
helpers of RND Department, Prima for their valuable support and cooperation during
this project.
My special thanks go to Mr. T. Geretharan, Senior Lecturer in Department of Crop
science, Faculty of Agriculture, Eastern University, Sri Lanka for helping me with the
completion of statistical analysis.
Finally my everlasting love and sincere gratitude go to my parents and friends who
helped me in numerous ways to complete this project.

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TABLE OF CONTENTS
ABSTRACT……………………………………………………………………… I
ACKNOWLEDGEMENTS…………………………………………………….. III
TABLE OF CONTENTS………………………………………………………… IV
LIST OF TABLES…………………………………………………………………IX
LIST OF FIGURES…………………………………………………………….....X
LIST OF PLATES………………………………………………………………..XII
1.0 INTRODUCTION ......................................................................................................1
2.0 LITERATURE REVIEW..........................................................................................7
2.1 Wheat .............................................................................................................. 7
2.2 Components of the wheat kernel and their compositions ............................... 7
2.3 Chemistry of wheat grain................................................................................ 8
2.3.1 Wheat carbohydrates................................................................................9
2.3.2 Wheat proteins .......................................................................................10
2.3.3 Wheat lipids ...........................................................................................12
2.3.4 Wheat fiber.............................................................................................13
2.3.5 Minerals in Wheat..................................................................................14
2.3.6 Vitamins in Wheat .................................................................................15
2.3.7 Enzymes in wheat ..................................................................................15
2.4 Wheat flour Milling....................................................................................... 16

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2.4.1 Effect of milling on wheat flour quality.................................................18
2.5 Wheat flour quality........................................................................................ 18
2.5.1 Wheat flour quality components and the tests applied to them .............19
2.5.1.1 Moisture .............................................................................................20
2.5.1.2 Mineral content ..................................................................................21
2.5.1.3 Protein content....................................................................................22
2.5.1.3.1 Wet gluten content........................................................................22
2.5.1.4 Falling number ...................................................................................23
2.5.1.5 Color...................................................................................................24
2.5.1.6 Weevil Count Test..............................................................................24
2.5.1.7 Microbiology......................................................................................25
2.6 Storage of wheat flour................................................................................... 27
2.7 Environmental factors affecting wheat flour quality during storage............. 28
2.7.1 Water activity.........................................................................................28
2.7.2 Temperature ...........................................................................................29
2.7.3 Relative humidity...................................................................................30
2.8 Aging of wheat flour ..................................................................................... 31
2.9 Physical and chemical changes during flour storage .................................... 32
2.9.1 Effect of storage and aging on baking properties of wheat flour...........34
2.9.2 Effect of storage and aging on wheat starch ..........................................34
2.9.3 Effect of storage and aging on wheat protein and gluten.......................35

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2.9.4 Effect of storage and aging on wheat lipids...........................................35
2.10 Packaging materials used to pack wheat flours............................................. 36
2.11 Dry storage of wheat flour ............................................................................ 37
2.12 Shelf life of wheat flour ................................................................................ 37
3.0 MATERIALS AND METHODS.............................................................................39
3.1 Experiment Material...................................................................................... 39
3.2 Experiment design......................................................................................... 39
3.3 Experiment timeline ...................................................................................... 40
3.4 Experiment parameters.................................................................................. 40
3.4.1 Physiochemical analysis ........................................................................40
3.4.1.1 Long moisture test (Air oven method) ...............................................41
3.4.1.2 Moisture analyzer test ........................................................................42
3.4.1.3 Wet Gluten test (Glutomatic method) ................................................42
3.4.1.4 Gluten index.......................................................................................43
3.4.1.5 Falling number (Hagberg Perten Method) .........................................44
3.4.1.6 Protein test..........................................................................................46
3.4.1.7 Ash content.........................................................................................47
3.4.2 Biological test ........................................................................................48
3.4.2.1 Weevil test..........................................................................................48
3.4.3 Microbiological test ...............................................................................48
3.4.3.1 E.coli and Coliforms counting test.....................................................49

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3.4.3.2 Aerobial Plate Count Test ..................................................................55
3.4.3.3 Yeast and Mold Count........................................................................56
4.0 RESULTS AND DISCUSSIONS.............................................................................58
4.1 The Impact of Storage Temperature and Storage Periods on the Physical,
Chemical, Biological and Microbiological properties of different wheat flour
samples.......................................................................................................... 58
4.1.1 Effect of storage temperature and storage period on flour moisture
content …………………………………………………………………………58
4.1.2 Effect of Storage Temperature and Storage Time on Wet Gluten.........62
4.1.3 Effect of storage temperature and storage period on flour gluten index65
4.1.4 Effect of storage temperature and storage period on flour protein content
of wheat flour........................................................................................................68
4.1.5 Effect of storage temperature and storage period on flour ash content
and color value......................................................................................................72
4.1.6 Effect of Storage Temperature and Storage Period on Falling Number of
flour …………………………………………………………………………77
4.1.7 Effect of different storage temperatures on the Weevil Counts of three
different wheat flour samples ...............................................................................80
4.1.8 Effect of storage temperature on the Bacterial Count of different wheat
flour samples.........................................................................................................80
4.1.9 Effect of storage temperature on the yeast and mold count of different
wheat flour samples ..............................................................................................83

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4.1.10 The effect of storage temperatures on the E.coli and coliform population
…………………………………………………………………………85
5.0 CONCLUSION .........................................................................................................86
SUGGESTIONS FOR FUTURE RESEARCH.......................................................90
REFERENCE……………………………………………………………………….91
APPENDIX………………………………………………………………………...10

ix
LIST OF TABLES
Table 2-1 The typical nutritional value per 100 g.........................................................9
Table 2-2 Protein distribution in Osbourne fraction ...................................................11
Table 2-3Essential Amino Acid content in hard red wheat (% by dry weight) and
recoveries in flour(% of wheat grain) ..........................................................................11
Table 2-4 Fatty acid composition of total lipids from wheat and wheat milled
fractions........................................................................................................................13
Table 2-5 Mineral composition of wheat ....................................................................14
Table 3-1 Table for the weight of sample to be taken for falling number according to
moisture content...........................................................................................................45
Table 3-2 Experimental setup for 10 g sample which includes 1 empty tube, 2
dilution tubes and 9 gas tubes. .....................................................................................50
Table 3-3 Population calculation method for coliforms and E.coli ............................53
Table 3-4 Most probable number of population per 1 g .............................................53
Table 3-5 IMViC Result Interpretion Method ............................................................55
Table 3-6 Dilution volumes for Total Plate Count method.........................................56
Table 3-7 Dilution volumes Yeast and Mold Count Method ......................................57
Table 4-1 The MPN population of E.coli and Coliforms present in samples A,B and C
......................................................................................................................................85

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LIST OF FIGURES
Figure 4-1 The effect of different storage temperatures (27.5oC and 37.5oC) on
moisture content of sample A over time. .....................................................................59
moisture content of sample B over time ......................................................................60
moisture content of sample C over time ......................................................................60
Figure 4-4The effect of different storage temperatures (27.5oC and 37.5oC) on wet
gluten content of sample A over time ..........................................................................62
Figure 4-5The effect of different storage temperatures (27.5oC and 37.5oC) on wet
gluten content of sample B over time ..........................................................................63
Figure 4-6 The effect of different storage temperatures (27.5oC and 37.5oC) on wet
gluten content of sample Cover time ...........................................................................64
Figure 4-7 The effect of different storage temperatures (27.5oC and 37.5oC) on gluten
index of sample A over time........................................................................................66
index of sample B over time ........................................................................................67
index percentage of sample C over time. .....................................................................67
protein content of sample A over time.........................................................................69
protein content of sample B over time. ........................................................................70
protein content of sample C over time. ........................................................................71

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Figure 4-13 The effect of different storage temperatures (27.5oC and 37.5oC) on ash
content and color of sample A over time. ....................................................................73
content and color of sample A over time. ....................................................................73
Figure 4-15 The effect of different storage temperatures (27.5oC and 37.5oC) on.....74
Figure 4-16 The effect of different storage temperatures (27.5oC and 37.5oC) on color
value of sample B over time ........................................................................................74
content of sample C over time .....................................................................................75
Figure 4-18 The effect of different storage temperatures (27.5oC and 37.5oC) on color
value of sample C over time ........................................................................................75
Falling number of sample A over time. .......................................................................77
Falling number of sample B over time. .......................................................................78
Falling number of sample C over time. .......................................................................79
bacterial count of sample A over time .........................................................................80
bacterial count of sample B over time .........................................................................81
bacterial count of sample C over time .........................................................................82
Figure 4-25 The effect of different storage temperatures (27.5oC and 37.5oC) on yeast
and mold population of sample A over time................................................................83

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and mold population of sample B over time ................................................................84
and mold population of sample C over time ................................................................84
LIST OF PLATES
Plates 3-1 Transfering of buffer solution into the gas tubes .......................................51
Plates 3-2 Detailed design of the confirmation test.....................................................52

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CHAPTER 1
1.0 INTRODUCTION
Wheat (Triticum aestivum) is grown in more land than any other food crop in the
world (Delcour and Hoseney, 2010). Wheat is so prevalent because of the hardiness
and adaptability of the plant and the demand for its end-products. Wheat is counted
among the ‘big three’ cereal crops, with over 600 million tons being harvested
annually. For example, in 2015, the total world harvest was about 2,531 million tons
compared with 652 million tons of rice and 785 million tons of maize
(http://faostat.fao.org/). Wheat was a key factor enabling the emergence of city-based
societies at the start of civilization because it was one of the first crops that could be
easily cultivated on a large scale, and had the additional advantage of yielding a
harvest that provides long-term storage of food. Wheat contributed to the emergence
of city-states in the Asian Fertile Crescent, including the Babylonian and Assyrian
empires.
The ﬁrst cultivation of wheat occurred about 10, 000 years ago, as part of the
‘Neolithic Revolution’, which saw a transition from hunting and gathering of food to
settled agriculture. These earliest cultivated forms were diploid (genome AA)
(einkorn) and tetraploid (genome AABB) (emmer) wheats and their genetic
relationships indicate that they originated from the south-eastern part of Turkey (Heun
et al., 1997; Nesbitt, 1998; Dubcovsky et al., 2007). Cultivation spread to the Near
East by about 9000 years ago when hexaploid bread wheat made its ﬁrst appearance
(Feldman et al., 2001).

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The grain (kernel) itself is the fruit of the grass, also known as the caryopsis. The
caryopsis consists of 3 major anatomical parts: germ, endosperm, and an outer bran
layer (Hoseney et al., 1986). The endosperm is primarily starch and contains the
gluten forming proteins. It is the majority of material in white flour. The germ is rich
in oils and minerals, and bran is a cellulose-rich fibrous structure.
There are six wheat classifications: hard red winter, hard red spring, soft red winter,
durum (hard), hard white, and soft white wheat. The hard wheat has the highest gluten
content and are used for making bread, rolls and all-purpose flour. The soft wheat is
used for making flat bread, cakes, pastries, crackers, muffins, and biscuits. Wheat is
one of the first cereals known to have been domesticated, and wheat's ability to self-
pollinate greatly facilitated the selection of many distinct domesticated varieties.
Milling is the process separating germ from bran and grinding the germ to produce
flour. Milling aims to separate the anatomical parts of the kernel to produce flour with
minimal inclusion of bran particles (Hoseney et al., 1986; Stone and Morell, 2009).
During the milling process, different parts of the wheat grain are used to make
different types of flour. White flour is made from the endosperm only. Wholemeal
flour uses all parts of the grain: the endosperm, the wheat germ and the bran layer.
Brown flour contains about 85% of the original grain, but some bran and germ have
been removed.
Bran and germ are rich in nutrients. However, the oil-rich germ can become rancid
fairly quickly, which can cause functional and chemical changes in flour. Flour
composition and functionality determine end product quality. Variation in wheat flour
composition is economically and functionally important for manufacturing processes
and the resulting end-products (Duyvejonc et al., 2011). Different types of wheat are

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differentially suitable for any particular end product. For example, flour for bread
production generally requires high water absorption, high gluten strength and
moderately high damaged starch.
Flour quality parameters are rarely optimal for bakery and pastry manufacturing,
which leads to an increased tendency to improve flour quality, leading to the
production of bakery and pastry products of consistent quality. The important quality
parameters which are analyzed for wheat flour performance and its purpose of use are
moisture content, protein content, wet gluten content, gluten index, mineral content,
flour color, falling number, weevils count and flour microbiology. These parameters
are the indicators of milling performance and flour quality. Gluten-forming proteins
are the storage proteins of wheat kernels (Hoseney et al., 1986). They are found in the
endosperm, where they form a continuous matrix around starch granules (Goesaert, et
al., 2005).
The gluten has the property of ‘viscoelasticity’, which is particularly important in
making leavened bread, as it will allow the entrapment of carbon dioxide released
during leavening. However, Gluten also underpin a range of other uses including
making unleavened breads, cakes, and biscuits, pasta (from durum wheat), and
noodles (from bread wheat). Gluten content is also exploited in the food industry
where gluten proteins may be used as a binder in processed foods. Hagberg falling
Number is required in bread dough to measure the activity of alpha amylase enzyme
which provides sugars for gas production. Too high activity of this enzyme leads to
excessive starch breakdown sticky crumb and collapsed loaves.
Moisture content is very important since it is the main factor determines the milling
performance of the grain and storage capacity or keeping quality of wheat flour. Low

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moisture levels of wheat flour increases the oxidative rancidity during storage and
high moisture levels favor the growth of bacterial growth http://www.campden.co.uk.
Since color values are directly related to ash content of the flour it is important
measure both parameters. High color values and high ash content indicate the bran
contamination of the wheat flour which means low quality.
Weevils are the biological components present in the wheat flour which deteriorate
the quality of the wheat flour by laying eggs and leading to the growth of fungal
growth. Molds are the fungi responsible for the growth of ropy fungal growth in
bakery goods and reduce the keeping quality of end products.
The storage time and storage conditions have an influence on the technological
quality of wheat and result in modifications of the flour parameters. Lukow and White
(1997). Wheat flours continue to be living biological stuffs also during the ensuing
storage. All processes connected with maturation work on for several days after
milling and their influence on the flour quality depends on the ambient storage
conditions. Flour aging is thought to be a natural occurring maturation in wheat flour.
The underlying mechanism of aging is thought to relate to oxidation of flour
components including fatty acids and proteins (Cenkowski et al., 2000). Optimal
maturation time depends on both the flour characteristics and storage conditions
(Hrušková and Machová, 2002).
The time of maturation is important for the achievement of the optimal flour bread-
making quality although this period is affected by many factors. The time required for
the optimal maturation depends both on the flour characteristics and on the storage
ambient conditions. Weaker flours need a longer time but flours with higher ash
contents reach the optimal characteristics sooner. The changes of the rheological

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dough properties are connected with the gluten quality and its alterations during
maturation. Gluten extensibility mostly decreases and its elasticity increases
(Cenkowski et al., 2000). As a rule, dough becomes less sticky and its ability to hold
up fermentation gases increases. During the flour maturation water absorption also
increases, the amylolytic activity becomes lower and the temperature of starch
gelatinization increases (Linfeng Fang and Flotes, 1999). An improved dough
handling and a better quality of products are caused by the changes mentioned above
(Hampl and Prihoda, 1985).Storage time and conditions have an influence on the
technological qualities of wheat, so modification of flour parameters may occur
(Hrušková and Machová, 2002). Such modifications may include increases in water
binding capacity and batter viscosity. Starch gelatinization temperature and viscosity
may also be altered.
Storage temperatures affect the keeping quality of wheat flours. Increasing the storage
temperatures will build up the heat in wheat flours, accelerating the enzymes activity,
exhausting the substrates and/or thermal inactivation of essential enzymes (Pomeranz
et al., 1982). Heat can damage the gluten proteins and discolor the flour (Halverson
and Zeleny, 1988). Protein quality of the wheat decreases at faster rates with high
temperatures over time (Jones and Gersdorff, 1941). It was proved that wheat flour
stored at 30oC deteriorated much faster rate than stored at 20oC (Glass et al., 1959).
Flour changes become less pronounced during storage at low temperatures. During
two-year storage at 0°C, flour characteristics did not change significantly (Yoneyama
et al., 1970).
Flour is a very hygroscopic material and its moisture changes with the changes in
temperature and humidity of the store environments. Flour moisture changes can
support the acidity alterations caused by the enzymatic breaking of fytin by fytase,

6
lipolytic fat hydrolysis and proteolysis (Hansen and Rose 1996). Changes in the
protein–protease complex of wheat flour as reflected in elasticity and extensibility of
gluten can exert a positive or a negative influence on the dough bread-making
characteristics. During a longer storage time, flour properties change by the effect of
nonsaturated fatty acids which can reduce gluten swelling and water absorption, and
increase starch resistance against gelatinization (Chen and Schofield 1996). This
results in a lower amylolytic activity and a lower gas production ability of flour
(Srivastava and Haridas Rao 1992).
In this present study, efforts have been made to study the changes occur in the
parameters determining the quality of wheat under different storage temperatures. The
keeping quality of the wheat flour was evaluated. The study was conducted for the
three differently produced wheat flour samples which varies in their chemical
compositions.
Therefore the research was conducted with following objectives;
1. To analyze the changes wheat flour quality parameters during short term
storage (8 weeks) under two different temperature storage conditions(room
temperature and a/c condition).
2. To study the rate of wheat flour maturation process occurring under two
different temperature storage conditions (room temperature and a/c condition).
3. To evaluate the shelf life capacity of the wheat flour produced in Prima
Ceylon PVT Ltd.

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CHAPTER 02
2.0 LITERATURE REVIEW
2.1 Wheat
Common wheat is the member of wild grasses (Gramineae family) native to parts of
western Asia. It belongs to the group of genus Triticum known as Triticum aestivum.
Remarkably it has been cultivated for about 10,000 years. ( Cauvain et al., 2003)
Kingdom: Plantae
Division: Magnoliophyta
Class: Liliopsida
Order: Poales
Family: Poaceae
Subfamily: Pooideae
Tribe: Triticeae
Genus: Triticum
Species: Triticum aestivum
2.2 Components of the wheat kernel and their compositions
The structure of the wheat is quite complex, consisting of many readily discernible
entities. The caryopsis as it is known botanically is ovoid in shape with a longitudinal
crease. The outer layer epidermis is the sheath for other layers of cells, which
constitute a pericarp about 50 µm thick. Beneath the layer of nucellar tissue is the
aleurone layer, before we encounter the starch rich endosperm from which the wheat
flour is made. The endosperm containing the stored food for the plant represents more
than 80% of the weight of the kernel. The endosperm contains the albumins, globulins

8
and major proteins of gluten complex; glutenins and gliadins apart from
carbohydrates. The germ situated toward the lower end of the kernel. It consists of
plumule, to which is attached the scutellum, an absorbing organ for food. The wheat
germ represents only 2-3% by the weight of kernel. But it is rich in protein (25%) and
lipid (8-13%). Bran acts as the barrier to protect the grain and makes up over 8% of
the weight of the kernel. It comprises water insoluble fibre. (Cauvain et al., 2003).
There are large differences between the levels of aminoacids in the aleurone layer and
those in wheat flour. Glutamine and proline levels are only about one half, while
arginine is treble and alanine, asparagine, glycine, histidine and lysine are double
those in wheat flour. (Fulcher et al., 1997).
2.3 Chemistry of wheat grain
The chemical composition of wheat grain (moisture 11–14%) is characterized by the
high content of carbohydrates. The available carbohydrates, mainly starch deposited
in the endosperm, amount to 56–74% and fiber, mainly located in the bran, to 2–13%.
The second important group of constituents is the proteins which fall within an
average range of about 8–11%. lipids belong to the minor constituents (2–4%) along
with minerals (1–3%). The relatively high content of B-vitamins is, in particular, of
nutritional relevance. (Koehler and Wieser et al., 2013). The chemical components of
wheat are not uniformly distributed in the grain. Hulls and bran are high in cellulose,
pentosans and ash. The aleurone layer of wheat contains 25 times more minerals than
the endosperm; whereas the lipids are generally concentrated in the aleurone and
germ. The endosperm, which contains mostly starch, has lower protein content than
the germ and the bran, and is low in fat and ash. The typical nutrional value of what
grain per 100 g is indicated in Table 2.1.

9
Table 2-1 The typical nutritional value per 100 g
Nutrition Amount(Value/100 g)
Energy 1,418 kJ (339 kcal)
Protein 13.70 g
Fat 1.87 g
Fiber 12.2 g
Calcium 34 mg
Phosphorus 346 mg
Iron 3.88 mg
Thaiamin 0.447 mg
Riboflavin 0.215 mg
Niacin 6.365 mg
Pantothenic acid 1.008 mg
Folate 44 μg
Source: https://en.wikipedia.org/wiki/Wheat_flour
2.3.1 Wheat carbohydrates
Chief wheat carbohydrate starch is a mixture of two polymers, amylose and
amylopectin. Both are classifies as D-Glycanase. Wheat starch is obtained by wet
milling of white flour, preferably that from softer grade wheats (Cornell and
Hoveling, 1998). Wheat starch is present at about 63-65% of the weight of the wheat
kernel, figures being higher for the soft wheat than for the hard wheat (Toepfer et al.,
1972). Starch is basically a polymer of glucose. Chemically, at least two types of
polymers are distinguishable: amylose and amylopectin. Amylose is a mostly linear α-

10
(1,4)- linked glucose polymer with a degree of polymerization (DP) of 1,000–5,000
glucose units. Amylopectin is a much larger glucose polymer (DP 105–106) in which
α-(1,4)- linked glucose polymers are connected by 5–6% α-(1,6)-linkages. Normal
wheat starch typically contains 20–30% amylose and 70–80% amylopectin (Rose et
al., 2007).
2.3.2 Wheat proteins
Wheat grains protein may vary from less than 6% to more than 20%. The content
depends on the genotype (cereal, species, variety) and the growing conditions (soil,
climate, fertilization); amount and time of nitrogen fertilization are of particular
importance. Proteins are distributed over the whole grain, their concentration within
each compartment, however, is remarkably different. The germ and aleurone layer of
wheat grains, for instance, contain more than 30% proteins, the starchy endosperm
~13%, and the bran ~7%. (Belitz et al., 2009; Grosch et al., 2009). In 1907 Osbourne
separated wheat protein on the basis of their solubility into four fractions; Water
soluble albumin, salt soluble globulins, 70% ethanol soluble prolamines and the
glutelins which remained in the flour residue. Among the Osborne fractions in cereals,
the prolamin fraction has been the most studied (Eliasson and Larsson 1993). This
fraction is called gliadin in wheat.
The high molecular weight subunits of prolamins constitute a higher percentage of the
total in wheat than in other cereals (Shewry and Mifflin 1985). The baking quality of
wheat flour from different varieties is influenced by the glutelin content (Eliasson and
Larsson 1993). The protein distribution in osbourne fraction is given in Table 2.2
below. The essential amino acids present in wheat flour is given in Table 2.3 below

11
Table 2-2 Protein distribution in Osbourne fraction
These four proteins represent the storage protein of the wheat and usually make up
10-14% of the kernel. (Cauvain et al., 2003).
Table 2-3Essential Amino Acid content in hard red wheat (% by dry
weight) and recoveries in flour(% of wheat grain)
Adapted from Teopfer et al., (1992)
Wheat is unique among cereals in its ability to form cohesive, viscoelastic dough,
when flour is mixed with water. Wheat dough retains the gas produced during
fermentation and this result in a leavened loaf of bread after baking. It is commonly
Fraction Percentage (%)
Albumin 14.7
Globulin 7.0
Prolamine 32.6
Gluteline 45.7
Amino Acid Content(%) Recovery in flour(%)
Lysine 0.43 69
Histidine 0.36 85
Arginine 0.76 72
Valine 0.74 90
Methionine 0.25 13
Isoleucine 0.62 98
Leucine 1.07 97
Phenylalanine 0.77 10
Tryptophan 0.27 83

12
accepted that gluten proteins (gliadins and glutenins) decisively account for the
physical properties of wheat dough. ( Köhler and Wieser, 2007). All glutenin proteins
being classified as prolamines, The distinction between gliadin and glutenin being
based on their different functional properties caused by glutenins being polymeric and
gliadin being monomeric. (Shewry et al., 1986).
Gliadins and glutenins are mainly located in the in the mealy endosperm and are not
found in the seed coat layers nor in the germ. Storage proteins in wheat are unique
because they are technologically active. They have no enzyme activity, but they have
a function in the formation of dough as they retain gas, producing spongy baked
products (Belderok et al., 2000).
2.3.3 Wheat lipids
Lipids are present only in a small extent in cereals but they have a significant effect
on the quality and the texture of foods because of their ability to associate with
proteins due their amphipatic nature and with starch, forming inclusion complexes. In
wheat, the maturing seed synthesizes fatty acids at different rates ( Šramková et al.,
2009). The germ has the highest amount of lipids (11%), but significant amounts are
also associated with the bran and the starch and proteins of the endosperm. Although
some attempts have been realized to manipulate the lipid composition in order to
improve the nutritional quality of the crops, there is a lack of such research in wheat.
(Anai et al., 2003; Murphy 2006). The fatty acid compostion of wheat flour is given
in Table 2.4.

13
Table 2-4 Fatty acid composition of total lipids from wheat and wheat
milled fractions.
Source: Koehler and Wieser 2013.
2.3.4 Wheat fiber
Numerous studies (McKee and Latner 2000; Philippe et al., 2006; Rave et al., 2008;
Weickert and Pfeiffer 2007) have demonstrated the beneficial effects of fiber
consumption in protection against heart disease and cancer, normalization of blood
lipids, regulation of glucose absorption and insulin secretion and prevention of
constipation and diverticular disease. These components are typically divided into two
categories. Soluble dietary fiber is those components that are soluble in water and
includes pectic substances and hydrocolloids.
Insoluble dietary fiber is those components that are insoluble in water and includes
cellulose, hemicellulose and lignin. (Bermink et al., 1994). Dietary fiber component
Fatty acid Wheat grain Endosperm Bran Germ
Myristic 0.1 Tr Tr Tr
Palmitic 24.5 18.0 18.3 18.5
Palmitoleic 0.8 1.0 0.9 0.7
Stearic 1.0 1.2 1.1 0.4
Oleic 11.5 19.4 20.9 17.3
Linoleic 56.3 56.2 57.7 57.0
Linolenic 3.7 3.1 1.3 5.2
Arachidic 0.8 Tr Tr Tr
Other 1.1 1.1 Tr 0.8

14
in straight run flour is not more than 2% and whereas in whole meal flour, it is about
11-13 %. (Cornell and Hoveling 1998).
2.3.5 Minerals in Wheat
Due to the high consumption of wheat in a variety of food products all over the world,
wheat is considered an important source of minerals (Galan et al., 1997). The
concentration of minerals in wheat flour is genetically determined by the choice of
cultivar and environmentally determined by soil, climate and management practices
(Pomeranz et al., 1982). The mineral composition of wheat is given in Table 2.5
below.
Table 2-5 Mineral composition of wheat
Minerals Amount(g/mg) DV%
Calcium 40.8mg 4%
Iron 4.7mg 26%
Phosphorus 415mg 42%
Magnesium 166mg 41%
Potassium 486mg 14%
Sodium 6.0mg 0%
Zinc 3.5mg 23%
Copper 0.5mg 23%
Manganese 4.6mg 228%
Selenium 84.8mcg 121%
Fluoride ~
Source: Koehler and Wieser 2013

15
2.3.6 Vitamins in Wheat
Wheat is a good source of vitamins from the B-group, and, in industrial countries, It
fulfill about 50–60% of the daily requirement of B-vitamins. The most important fat-
soluble vitamins are the tocopherols, which are present in concentrations exceeding
20 mg/kg. Like the minerals, vitamins are concentrated in the outer layers of the
grains, in particular in the aleurone layer as well as in the germ.( Koehler and Wieser,
2007)
2.3.7 Enzymes in wheat
The chief function of the starch enzymes of wheat and flour is changing starch to
sugars. The many carbohydrate-degrading enzymes include α -amylases, β -amylases,
debranching enzymes, cellulases, β -glucanases, and glucosidases. Amylases are
enzymes that hydrolyze the polysaccharides in starch granules. The most important
enzyme of the endohydrolase type is α -amylase. The enzyme hydrolyzes α -1,4-
glucosidic bonds of amylose and amylopectin and produces a mixture of dextrins
together with smaller amounts of maltose and oligosaccharides; the pH-optimum is
about 5. The other major amylase type is β -amylase, an exohydrolase, which
hydrolyzes α-1,4-glucosidic bonds near the non-reducing ends of amylose and
amylopectin to produce maltose. The maximum activity of beta-amylase occurs at pH
4.5 to 5.1, whereas that of alpha-amylase is at pH 5.6 to 5.8. The pH optimum is
similar to that of α -amylase.( Koehler and Wieser et al., 2007).
The α-Amylase enzyme is relatively thermostable up to 70°C, whereas β-amylase
loses about half of its activity at this temperature. Fungal amylase is the least
temperature stable, followed by cereal amylase, while bacterial amylase is stable at
higher temperatures. Wheat α –amylases is of considerable interest in wheat

16
chemistry as it is involved directly in the absorbing properties and gassing power of
dough. Addition of small amount of α -amylases to a sound flour may improve the
baking properties of bread such as increased loaf volume, improved crumb color,
increased moistness of crumb and keeping quality (Freeman and Ford, 1941).
The Lipoxygenase enzymes are non-heme iron containing dioxygenase that catalyse
the oxidation of polyunsaturated fatty acids containing a cis,cis-1,4-pentadiene
system, producing conjugate cis,trans-diene hydroperoxides (Siedow et al., 1991).
Lipoxygenase is present in high levels in the germ. It catalyzes the peroxidation of
certain polyunsaturated fatty acids by molecular oxygen. Its typical substrate is
linoleic acid containing a methylene-interrupted, doubly unsaturated carbon chain
with double bonds in the cis -configuration. (Koehler and Wieser et al., 2007).
Proteases are hydrolytic enzymes that hydrolyze protein by adding water across
peptide bonds and break them in smaller peptides in organic solvents (Shehri et al.,
2004).
2.4 Wheat flour Milling
Milling is the process by which wheat is ground into flour, separating the wheat grain
into its constituents. Essentially this is the separation of the bran and germ from the
endosperm and the reduction of the endosperm to a uniform particle size. This is done
by a sequence of breaking, grinding and separating operations (Pomeranz et al.,
1988).
Wheat flour milling has for many years been well automated and recent years
computer technology has played a large role in distribution and blending of different

17
wheat and flours and also with other mill operations so that all these processes can be
made more cost effective (Ward et al., 1993).
Most wheat is milled into flour using the conditioning roller milling process (Bass et
al., 1988). Milling process starts when wheat is thoroughly cleaned to remove metals,
chaff, stones, and other foreign materials. This is done by magnets, air aspirator,
milling separator and disc separator. Cleaned grain is conditioned before milling, this
process is called as tempering, which is done to separate bran more efficiently and to
improve the sieving efficiency (Posner and Hibbs, 2005). Wheat is tempered with
water sprayed on the grain being conducted in a conveyor. Hard wheat is conditioned
for 24-48 hours and 16.5% moisture content. For soft wheat, conditioning should be
done for 12-24 hours and 15%-15.5% moisture (Serna-Saldivar et al., 2008). After
tempering, kernels first passed through an abrasive machine, equipped with an air
aspirator system with aim of eliminating impurities located on the pericarp, as well as
break damaged kernels (Posner and Hibbs, 2005).
Milling is accomplished in two types of roller mills, break and reduction roller mills
(Bass et al., 1988) The.sifters are used to remove the bran from the kernels. The
purifiers separate the particles of same size, through an air current.
After milling, lab tests are run to ensure that the flour meets specifications. Millers
also conduct routine monitoring of indicator natural organisms. Although dry flour
does not provide an environment that is conducive to microbial growth, it is important
to understand that flour is a minimally processed agricultural ingredient and is not a
ready-to-eat product. Flour is not intended to be consumed raw.

18
2.4.1 Effect of milling on wheat flour quality
The insect fragments in flour are reduced by passing the grains through cylindrical
metal sieve. Purifies in mills are used to produce refined flours with lower ash content
and better color results (Posner and Hibbs, 2005).
Extraction rate commonly used by millers, refers to the amount of flour produced
from a given amounts of wheat. Generally the extraction rate varies from 72% – 78
%. Lower quality flours are obtained (higher ash, Lower color score, etc) when the
mill is set to obtain higher extraction rates (Saldivar et al., 2008).
Air classification in milling allows the production of at least two contrasting daughter
flours with different granulation and chemical composition. Air classified soft flours
yield a low protein and finer flour suited to cake mixes. Air classified hard wheat
flours yield a fraction with increased protein and stronger gluten compared to parent
flour (Posner and Hibbs, 2005).
2.5 Wheat flour quality
Flour quality means different things to the ultimate users of the product. It usually
represents conformance to several measurable characteristics, which experience has
indicated to be significant in terms of the end use. The ultimate definition of the flour
quality is the ability of the flour to produce uniform and attractive end products. Flour
strength has been synonymous with flour quality. The presence or absence of strength
factors governs the suitability of the flour for specific end use (NIIR board of
engineers, 2007). The quality of the wheat flour attributes may be divided into two

19
groups: those under genetic control and therefore inherent in particular variety and
those influenced by environmental factors (Simmonds et al., 1989).
2.5.1 Wheat flour quality components and the tests applied to them
Flour quality may be defined as the ability of the flour to produce an attractive end
product at competitive cost, under conditions imposed by the end product
manufacturing unit. The concept of quality differs from producer and consumer point
of view. However, in general, the term quality may refer to fitness of a raw material
or a product for a particular process or consumer (Khatkar, 2013).
The quality of the wheat flour can be adequately assessed by determination of
moisture, ash, acid insoluble ash, acidity, nitrogen, gluten and filth. The main sources
of methods of analysis for flours are the International Association for Cereal
Chemistry (ICC), The American Association of Cereal Chemistry (AACC), American
Association of Cereal Chemists, Kent-Jones and Amor (1967) and Pearson (Food and
Agricultural Organization of the United Nations, 1980). The quality of cereals and
various cereal products are determined by a variety of characteristics that may be
assigned different significant levels depending upon the desired end products. These
characteristics may be divided into chemical, enzymatic and physical (Vladimir and
Charles, 2000). There have been many reviews of the tests which have been suggested
and used for evaluating wheat and flour quality, particularly quality of flour for bread
making. Moisture, ash and protein tests are the chemical tests more widely applied to
the flour (Matz et al., 1991).

20
2.5.1.1 Moisture
Moisture is by far the most important factor determining the the rate of deterioration
of wheat (Pomeranz et al., 1982).Grain moisture is one of the most important factors
affecting the quality of flour. Higher lipolytic and proteolytic activities are known to
be related to higher moisture content, which leads to loss in nutrients (lipid and
protein) and production of more free fatty acids resulting in inferior sensory
characteristics (Kent and Evers, 1993).
Moisture determination is essential step in evaluating the quality of cereal grains and
their products. The behavior of the grains in both storage and milling depends to a
great extent on the moisture content. Moisture content also influences the keeping
quality of the flour and the bakery products. The knowledge of the moisture content is
required for comparing production data at a uniform level of dry solids (Vladimir and
Charles, 2000). Moisture content is an important variable affecting the actual amount
of sample taken for analysis, hence the final percentage expressed on a dry weight
basis. It is customary to express analytical results on whole grain on an 11% moisture
basis and for flour 13.5% moisture basis (Simmonds et al., 1989).
Moisture content of 14 percent is commonly used as a conversion factor for other
tests in which the results are affected by moisture content. Moisture is also an
indicator of grain storability. Wheat or flour with high moisture content (greater than
14.5 percent) attracts mold, bacteria, and insects, all of which cause deterioration
during storage.

21
Wheat or flour with low moisture content is more stable during storage. Moisture
content of flour is very important regarding its shelf life, lower the flour moisture, the
better its storage stability (Gooding and Davies, 1997). The deterioration of baking
quality is also less at lower moisture content which can be credited to retarded
respiration and activity of microorganisms (Staudt and Zeigler, 1973).
Moisture is an important factor in controlling grain infestation. Insects that live on
stored grains and their products depend upon the moisture supply. Generally, moisture
content of 9% or lower restricts infestation. Moisture is also of great importance for
the safe storage of cereals and their products regarding microorganisms, particularly
certain species of fungi. At lower moisture fungi will not grow but at about 14% or
slightly above, fungal growth takes place (Hoseney, 1994). When the moisture
content exceeds 16%, shelf life of the flour is greatly reduced. The moisture should be
maintained at the range of 14-15%, which when stored under appropriate conditions
(cool, dry, and aerated) will provide longer shelf life. Moisture content of the flour
could vary from 11% to 15%, depending on the storage conditions and hygroscopic
nature of the flour (Whiteley, 1970). The moisture content can be determined by using
the following different AACC and ICC standard methods; oven drying method
(AACC method 44-15A, 44-16, 44-18, 44-20, ICC standard 109/1, 110/1).
2.5.1.2 Mineral content
Mineral content which is most commonly known as ash content, changes with flour
extraction rate. It is determined by ashing a flour sample in a muffle furnace at 600oC
for upto 120 minutes. The mineral content of flour varies between a minimum and
maximum depending on the regulations of different countries. The ash content is

22
determined according to ICC standard 104/1. Other standardized procedures used are
AACC method 08-01, 08-02 and 08-03. A quick test is also possible using NIR
spectroscopy (Raquel and Paula, 2014) . As for protein, Ash is expressed on an 11%
moisture basis for wheat and a 13.5% moisture basis for flour. Ash content is a good
indicator of milling quality, since high ash content of flour indicates significant
contamination with aleurone cells and bran during milling operations. Flour yielding
high ash is of inferior milling quality (Simmonds et al., 1989).
2.5.1.3 Protein content
Protein is a fundamental quality test of wheat, because it forms the basis for payment
to farmers and is related to its end product processing potential. In wheat there are
several protein fractions that differ in solubility. It is the gluten forming protein
fraction of the endosperm that determine the baking properties of wheat flour. The
total protein content of the flour is determined by kjeldhal method (ICC standard
number. 105/2). The organic constituents are oxidized in the presence of catalyst. The
ammonia formed after another step is distilled and titrated. The calculated amount of
nitrogen by titration is multiplied by a conventional factor (Raquel and Paula, 2014).
Protein quality criteria are related primary factor to the gluten portion of the flour
(NIIR Board of Engineers, 2007).
2.5.1.3.1 Wet gluten content
The wet gluten content is the measure of the amount of swollen gluten in the wheat
flour, which can be determined by forming a paste from the flour sample and washing
it out. The amount of gluten in the flour is an index of protein content and the physical

23
properties of the washed out gluten provide an index of flour strength (Dill and
Alsberg, 1924). Mechanical determination of the wet gluten content of the wheat flour
(ICC standard 137/1) is carried out with glutomatic machine (Raquel and Paula,
2014). Wet gluten content is determined by washing the flour or ground wheat sample
with a salt water solution to remove starch from the sample. The result has to be
converted to correspond to flour moisture content of 14% (Carver et al., 2009). The
gluten index provides an indication of gluten strength (Carver et al., 2009). The gluten
index is determined by weighing the portion of gluten passing through the sieve. The
higher the proportion of gluten that has not passed through the sieve, the higher the
index and better the quality of the measured gluten. Flours with a gluten index of
more than 95 indicate strong gluten characteristics whereas low values (up to 30)
indicate weak gluten characteristics (Kulkarni et al., 1987).
2.5.1.4 Falling number
Falling number is inversely proportional to α-amylase activity. It has considerable
significance, since there is a direct relationship between enzyme activity and finished
product attributes (bread crumb quality, loaf volume etc.) (Kruger and Tipple, 1980).
The level of enzymatic activity can be measured by the falling number test. Yeast in
bread dough requires sugars to develop properly and therefore needs some level of
enzymatic activity in the dough. Too much enzymatic activity means that too much
sugar and too little starch are present. Since starch provides the supporting structure of
the bread, too much activity results in sticky dough during processing and poor
texture in the finished product. If the falling number is too high, enzymes can be
added to the flour. If falling number is too low, that makes the flour unusable. Falling
number instrument analyzes viscosity by measuring the resistance of a flour and water

24
paste to a falling stirrer and recorded as an index of the enzyme activity in flour
sample. It is expressed in time as seconds. High falling number (>300) indicates
minimal enzyme activity and sound quality of wheat flour. A low falling number
(<250) indicates substantial enzyme activity and sprout damaged starch. (Panguliri
and Kumar, 2013).
2.5.1.5 Color
Flour is tested for color for evaluating either its whiteness which primarily determines
the extent of the oxidation of carotenoid pigments by bleaching compounds, or the
presence of bran particles indicating milling performance. Testing flour for whiteness
may be based on measuring the light reflectance of the sample within the blue range
of the light spectrum. Since improvement in flour color results from the oxidation of
the pigments by the bleaching agents as well as natural oxidation during storage. The
measured values vary not only with the extent of bleaching, but the age of flour (Kulp
et al., 2000). Color measurement may be divided into two classes. The first is a
measurement of whiteness which primarily determines the extent of color removal
bleaching compounds. Second area of color measurement largely ignores whiteness
and concentrates on the influence of branny material present in the flour by measuring
the reflectance with a light source in the green band of light spectrum (NIIR Board of
Engineers, 2007).
2.5.1.6 Weevil Count Test
Species such as Sitophilus granaries is described as a primary pest since they attack
undamaged grain, leaving it susceptible to invasion by other insects, fungi and
bacteria. The generation time of weevils, i.e the time required to complete one cycle

25
from egg through larva to adult varies greatly and depends upon temperature and
humidity (Simmonds et al., 1989). Grain infesting insects are sensitive to temperature.
They multiply slowly, not at all below 15oC and they cannot survive in the
temperature of 42oC or above (Bailey 1982). They appear to thrive best at about 30oC
and at that temperature their life cycles may be short as 30 days (Cotton and Wilbur,
1982). If you want to eliminate the chance of having any weevils in your flour, you
can store it in 0oC for 2 weeks. This kills any weevils and their eggs (Diana et al.,
2009).
2.5.1.7 Microbiology
If flour quality is to conform to the definition, some brief mention of microbiology is
necessary even though consistently reliable and effective control is yet to be attained.
The more comprehensive sources indicate that fungal count in flour falls in the range
of 85-8100 per gram and in the parent wheat from 90-1400 per gram. Bacterial
population of flour ranges from less than 1000 to over 100,000 per gram and in wheat
from low as 1000 to 300,000 per gram.
Under generally acceptable product moisture level and storage conditions, the total
bacterial population decreases with time. The population level in wheat is related to
growth, harvest, transport and storage conditions (NIIR Board of Engineers, 2007).
Transport and storage can be controlled. Wheat which is dampened attracts fungi and
leading to mold infestations. They are unfit for consumption, and molds may produce
mycotoxins including aflatoxin and fumonisin (Warcing, 2002). The yeast and mold
counts in the flour samples ranged from 2.0 to 3.0 log CFU/g, being 2.0 log CFU/g
the most frequent count. In addition to fungi, a wide variety of spoilage bacteria are

26
also present in flour. Seiler reported that bacterial count in flour was, on average, 1.9
log CFU/g lower than the initial level present in the wheat (6.4 log CFU/g). Studies
have indicated that bacterial contaminants may survive in a latent state for extended
periods in wheat flour, despite the low moisture content, and emerge from dormancy
when flour is added to environments that are more receptive to growth, such as batter
or mixes (Eglezos, 2010).
Enteric pathogens, such as Salmonella spp. and Escherichia coli, may be among the
microflora of wheat grain creating a food safety risk in milled products. E.coli is the
most widely used indicator of the sanitary state of the fresh foods since it presents in
the fecal matter.
Bacterial contaminants cannot grow and multiply in dry flour because of the small
amount of available water (Casey and Condon, 2002). Nevertheless, these
microorganisms survive in a dormant state, retaining their viability and the potential
to multiply if flour is incorporated to a more receptive environment for microbial
growth, such as batter or mixes (Eglezos, 2010). However, some pathogenic bacteria
such as Salmonella spp. and E. coli do not need to grow to cause illness, since they
require only a small number of cells (infective dose) to begin an infection (Schmid
and Frank, 2007).
For determining the population of pathogenic coliforms and E.coli present in the flour
sample, Most Probable Number method is applied. In this method dilutions of food
samples are prepared, three serial aliquots or dilutions are then planted into 9 or 15
tubes of appropriate medium for 3/5 tubes method. Numbers of organisms in the
original sample are determined by using a standard Most Probable Number table.

27
Standard plate count method is applied for counting the population of bacteria and
fungi. In this method portions of food samples are blended or homogenized, serially
diluted in an appropriate diluent, planted in or onto a suitable agar medium and
incubated at an appropriate temperature for a given time, after which all visible
colonies are counted by use of an electronic counter. It is by far the most widely used
method for determining the numbers of viable cells or colony forming units in a food
product (Jay et al., 2005).
2.6 Storage of wheat flour
Wheat flours continue to be living biological stuffs also during the ensuing storage.
All processes connected with maturation work on for several days after milling and
their influence on the flour quality depends on the ambient storage conditions. flour
quality depends on the ambient storage conditions. Flour is a very hygroscopic
material and its moisture changes with the changes in temperature and humidity of the
store environments. The baking properties of freshly harvested wheat or freshly
milled wheat flour have been reported to improve during storage for a time depending
on the nature of the flour and conditions of storage. Subsequently a point is reached
where further storage no longer seems to be conducive for baking and the bread
making properties of flour deterioration. Temperature and relative humidity of the
place and the packaging material used are the important factors affecting flour quality
during storage (Wright et al., 1988).
Flour quality could be destroyed totally with prolonged storage i.e more than 4 years
for flour and more than 20 years for wheat grain. Flour components such as protein,
starch and lipid change and these changes directly affect dough rheological and

28
baking characteristics (Wang and Flore, 1999). High moisture content is by far the
most important factor influencing the deterioration of the stored grain or flour, but
other factors include condition of the grain or flour, in particular its temperature when
put into storage and physical condition (Jay et al., 2005).
2.7 Environmental factors affecting wheat flour quality during storage
2.7.1 Water activity
Water activity refers to the availability of free water which influences the several
undesirable changes occur in food (Rockland B and Beuchet R, 1987).
𝑤𝑎𝑡𝑒𝑟 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
Þ
Þo
Þ = Partial pressure of food moisture at temperature T
Þ o= Saturation vapor pressure of pure water at temperature T
According to Scott (1952), the storage quality of food does not depend on the water
content but on water activity. Storage of flour at low temperature is among the oldest
method for preservation of food with increased water content. So it is necessary to
know the effect of water on storage life before suitable conditions selected (Gustavo
et al., 2007).
Decreased water activity retards the growth of microorganism and slows enzyme
catalyzed reactions, and also it influences non enzymatic browning, lipid oxidation,
degradation of vitamins and other deteriorative reactions. Water activity affects the
stability, flow, caking and clumping of flour during storage (Peleg and Manaheim,

29
1977). Knowledge of water activity of flours as a function of moisture content and
temperature is essential during storage (Chuy and Labuza, 1994). Water activity is a
well-established parameter for controlling the growth of microorganisms in foods
(Chirife and Buera, 1995,1996). The temperature dependence of water activity is
ignored in determination of water activity for growth of microorganisms. Most
important critical values of water activity are 0.6 aw for the growth of microorganisms
and 0.86 aw for flours with pathogenic microorganisms (Chirife and Buera ,1996).
Note: aw is the unit of water activity.
2.7.2 Temperature
Temperature control has been found to be the most important factor in maintaining
product quality. Wheat flour is subjected to environmental temperature and is a poor
conductor of heat. Storage temperature affects the keeping quality of wheat flour.
Increase in temperature damage the gluten proteins and cause inactivation of several
enzymes (Pomeranz et al., 1982). The protein quality of wheat flour over time
decreased at faster rate at increased temperature (Jones and Gersdorff, 1941). Changes
in proteins were decreased solubility, protein breakdown shown by increased amino
nitrogen and decreased nitrogen precipitation.
Flour changes become less pronounced during storage at low temperatures. During
two-year storage at 0°C, flour characteristics did not change significantly (Yoneyama
et al., 1970). Wheat stored at 30oC deteriorated much faster than at 20oC. Free fatty
acids raised in ambient temperature twice the level found at 5oC. Free fatty acid level
is high at 24oC. Higher viscosity of the dough was found in flour stored at ambient
temperature (Glass et al., 1959).

30
There were Significant decrease in moisture content, amylose activity, pH and increase
in titratable acidity was observed during storage at 45oC (Rehman and Shah, 1999).
Storage temperature impacts the rate of microbial growth, insect growth and chemical
changes in flour. Storage fungi commonly grow at about 30-32oC and their growth
rate decreases as the temperature increases. Low temperature was as effective as
decreased moisture content in preventing damage by fungi. Wheat or wheat flour with
moisture content up to 16% may be stored without obvious deterioration for a year at
temperature of 10oC or below and moisture content up to 18% safely for as long as 19
months at temperature of -5oC (Papavizas and Christensen, 1958).
2.7.3 Relative humidity
Control of relative humidity is an important part of temperature management, as the
two combine to reduce water loss and protect produce quality. Storing wheat flour in
humidity above 20%, most of the rapid moisture absorption has occurred. Flour is
hygroscopic, and quickly scavenges any available humidity from its environment.
Relative humidity of the flour stored influences the moisture loss. Moisture loss
reduces with increased relative humidity of the flour stored. High humidity results in
smaller differences in partial pressure at the flour surface and in the air, thus reducing
the rate of moisture loss. Since increase in temperature increases the partial vapor
pressure above the flour surface and any difference between this and the partial vapor
pressure of the air being too small for significance. When the flour temperature does
not exceed the ambient flour temperature, The influence of relative humidity on the
intensity of moisture loss increases. Dry goods like flour should be stored in a well
ventilated and cool place with the relative humidity of 60-65% (Ballard et al., 1993).

31
An equilibrium relative humidity of 65% is accepted as a safe maximum level for
long storage of wheat or flours (Pomeranz et al., 1982).
2.8 Aging of wheat flour
Flour properties change during the process of maturing. The time of maturation is
important for the achievement of the optimal flour bread-making quality although this
period is affected by many factors. The time required for the optimal maturation
depends both on the flour characteristics and on the storage ambient conditions (Pyler
et al., 1973).The practice of aging wheat flour is a method that has been employed for
centuries to improve gluten structure and optimize physical properties of the finished
baked product. During maturation, oxidation occurs by way of atmospheric exposure
of oxygen to flour, which changes the molecular structure of gluten fractions and
results in the strengthening of gluten bonds. The outcome of the increase in
crosslinking of flour protein is stronger, more elastic dough with increased volume
potential and a lighter texture in the final product. (Mcgee et al., 2004).
Freshly milled flour is not good for bread making because the gluten is somewhat
weak and inelastic and the color may be yellowish. When the flour is aged for several
months, the oxygen in the air matures the proteins so they are strong and more elastic
and bleaches the color slightly (Wayne et al., 2005). Storage conditions are important
in wheat and flour aging. Storage temperature, moisture content, relative humidity,
atmospheric oxygen content, light and microbial activity all affect the aging process.
Flour aging could be accelerated or suppressed by altering some of these factors
(Wang and Flore, 1999). The performance of flour improves with age. To allow this
aging process to occur naturally requires considerable time, which may vary with

32
storage temperature and wheat type. The nature of the maturing process is one of the
oxidation as it relates to performance of the flour, specifically modification of the
protein (NIIR Board of Engineers, 2007).
Natural aging, a few weeks of storage following the production of flour tends to
whiten the flour by reducing the pigmentation; this is referred to as natural aging of
flour. In addition to the improvement in flour color, the aging process also has the
beneficial effects on the dough characteristics and the finished product quality. The
nature of the maturing process is one of the oxidation and as it relates to performance
of the flour, specifically modification of protein. In countries where flour is not
allowed to be treated chemically, it is kept in storage bins for about 1 week to 4
weeks. Flour can be aged or matured chemically. Flour milled from different types of
wheat has different requirements of maturing compounds. Until 1962, the principal
maturing agent employed was chlorine dioxide gas, other chemical agents used were
ozone, agene and persulfates (NIIR Board of Engineers, 2007). Flour may be
bleached with benzoyl peroxide to whiten. Chlorine would bleach but it also has other
undesirable influence (Chakraverty et al., 2003).
2.9 Physical and chemical changes during flour storage
When flour milling destroys the structure of the wheat kernels by breaking the cell
walls, the endosperm loses the protection of the outer layers and it is open to attacks
by enzymes, microbes, insects, environmental moisture and gases. Most enzymes are
located in the germ, thus flour with less germ contamination such as patent flour
deteriorates more slowly. Higher germ contamination in flour would increase the level

33
of enzymes such as glutamate dehydrogenase, malate dehydrogenase, alcohol
dehydrogenase etc. (Honold et al., 1967).
The effect of flour aging on microbe levels has been investigated. The bacterial counts
of different flours during storage varied enormously. The bacterial counts diminished
with flour storage. Some flours showed over a 50% decrease after storage of 26 days
(Jones and Amos, 1930). Bacterial numbers decreased as pH values decreases during
flour storage. These results indicated that the microbes were not the major cause of
flour quality improvement (Wright et al., 1938).
Storage fungi have specific moisture and temperature requirements. Increase in
moisture and temperature results in more rapid growth of storage fungi and as
temperature increases thermophile group species level increases. The importance of
storage temperature can be illustrated by the fact that the deterioration of the flour is
about 10 times faster at 25oC than 3oC (Sauer et al., 1988). The quality of the flour
stored for 27 years totally destroyed flour quality, which indicated that the prolonged
storage is the enemy of flour quality (Greer et al., 1954). The oxygen absorption of
flour occurred at low and high moisture contents, such as 5% and 18% respectively.
Oxygen absorption was lowest at about 12% moisture content. They explained that
insects and microbes were responsible for oxygen absorption at increased moisture
and auto oxidation occurred at low moisture levels (Halton and Fisher, 1937). When
flour was aged at 38oC for 3 months, the color of the flour did not change, this
indicated that the bleaching reaction in flour occurred at a very slow rate and was not
a major factor in short term flour aging (Watson and Shuey, 1977). Aged flour
showed some degree of acidity (Alsberg et al., 1924).

34
The pH of flour decreased with an increase in storage temperature and moisture. Flour
stored in warm conditions deteriorates faster than the flour stored in cool conditions
(Sharp et al., 1924).
2.9.1 Effect of storage and aging on baking properties of wheat flour
The rapid improvement of bread loaf volume was observed of UK bread wheat flour
aged at cool temperature 26oC. Loaf volume of bread made with freshly milled flour
was 1232±12 cc, after the flour was aged for 20days loaf volume increased to
1354±20 cc and decreased with storage (Chen and Schofield, 1996). Water absorption
of the flour increased with wheat aging (Bur et al., 1910).
The baking quality of the prolonged stored flour was seriously deteriorated by
prolonged storage and produce bread with low loaf volume ( Rao et al., 1978). Cake
baked with the flour stored at room temperature over a two months period showed
improvement in both crumb grain and volume (Johnson and Hoseney, 1980). At low
moisture content, oxidative rancidity of flour occurred and produced poor baking
quality (Cuendent et al., 1954).
2.9.2 Effect of storage and aging on wheat starch
The onset temperature of the starch gelatinization increased 2-3oC after wheat was
aged for 3 weeks, as a result water binding capacity of flour increased with flour
aging (Shelke et al., 1992). During long term wheat storage alpha and beta amylases
attack the starch and produce dextrins and maltose (Zeleny et al., 1954).

35
2.9.3 Effect of storage and aging on wheat protein and gluten
Wheat flour with 18% moisture stored for 4 months at 28oC, 30oC, and 37oC free fatty
acid increased and washed out gluten decreased with flour aging (Dafatory et al.,
1969). To soft flour gluten from aged flour had the same properties as the gluten from
fresh milled flour (Kozmin et al., 1935). In strong and weak gluten flour stored at
15oC, 30oC and 45oC for 3 months, gluten quality changed sharply when flour was
stored at higher temperature. But no changes occurred in flour stored at 15oC. After
flour was stored for 2 months at 45oC, the gluten washed from strong flour was very
brittle and more elastic (Kozmin et al., 1935).
The amino nitrogen increased with storage. The decreases were greater at increased
temperature. The changes were more noticeable in white flour stored at room
temperature in bags. These changes caused by proteolytic activity of flour proteinase
as well as oxidation (Jones and Gersdorff, 1941).
2.9.4 Effect of storage and aging on wheat lipids
During storage, wheat lipids may be either hydrolyzed or oxidized. Mold lipases
rather than wheat lipases were dominant in fat hydrolysis at increased temperature and
moisture levels (Dirks and Geddes, 1955). Fat acidity increased more in moist flour
samples with 18% moisture content than it did in dry sample with 12% moisture
content (Fisher et al., 1939). Elevated temperature and moisture accelerated fat
hydrolysis in wheat flour (Norris and Gesses, 1954). Wheat lipid hydrolysis occurred
during flour aging and the lipid changes in aged flour affected gluten characteristics
(Gracza et al., 1965). Fat acidity increased significantly in flour stored at 100oF. The
changes of fat acidity were not significant in flour stored at 40oF (Shellenberger et al.,

36
1958). The lipids deteriorates rapidly when flour was stored at higher moisture
content at normal condition. This deterioration of lipids was caused by prolific molds
(Dafatary and Pomeranz, 1965).
2.10 Packaging materials used to pack wheat flours
Packaging materials, due to their physical mechanical properties particularly barrier
characteristics, significantly affect the quality and sustainability of packaged food
products (Lazić et al.,1994). In order to achieve a better protective effect of packaging
material great advantages were realized by applying new materials with improved
properties, as well as introducing different conditions within the packaging unit like
modified atmosphere (MAP), vacuum, and aseptically intelligent package (Lazić, and
Novaković 2010). Packaging materials used for the storage of flour products include
plastic containers, polymeric and paper bags (Aryee et al., 2006; Opara and
Mditshwa, 2013).
The packaging type and storage conditions applied affect the quality, shelf-life and
safety of food products through their influences on moisture content, water activity
and nutrient compositions of the food product (Opara and Mditshwa, 2013). Previous
studies have shown that both moisture and package types contribute to influence the
microbial load, the shelf-life and other quality attributes of flour products (Butt et al.,
2004; Mridula et al., 2010; Robertson, 2012). When packaging films are used, the
permeability of the film to water vapour and gases is particularly important, especially
with regard to the shelf-life of dry products (Siracusa, 2012).

37
2.11 Dry storage of wheat flour
Dry storage is used for foods that do not require refrigeration or freezing. Flour can be
stored in dry storage area. Dry storage room temperature should be maintained
between 10°C and 20°C. Floors, walls, shelving, and light fixtures should be kept
clean and dry. All items should be stored 6 to 8 inches off the floor so that all areas of
the floor can be cleaned. All items should be dated when they are placed in storage.
Items removed from the original container should be placed in air-tight containers
made for food storage and labeled with the contents and date placed in storage.
National Food Service Management Institute (2002).
2.12 Shelf life of wheat flour
The shelf-life of food will depend upon the food itself, packaging, temperature, and
humidity. If the food is not sterilized, it will ultimately spoil due to the growth of
microorganisms.
Dry food staples such as flour should be stored in their original packages or tightly
closed airtight containers below 30°C (optimum 10°C to 20°C). Humidity levels
greater than 60% may cause dry foods to draw moisture, resulting in caked and staled
products. Wheat grains and flours may be stored at room temperature in tightly closed
containers to keep out moisture and insects. Whole wheat flour may be stored in the
refrigerator or freezer to retard rancidity of the natural oils. Stabilized flour, such as
stabilized whole grain wheat flour, exhibiting unexpectedly superior extended shelf
life and superior biscuit baking functionality, may be produced with or without
heating to inhibit lipase by subjecting whole grains or a bran and germ fraction or
component to treatment with a lipase inhibitor, such as an acid or green tea extract.

38
Treatment with the lipase inhibitor may be performed during tempering of the whole
grains or berries or during hydration of the bran and germ fraction or component.
Normally wheat flours including all purpose flour and white flour can be stored in
pantry up to 6-8 months, in refrigerators for one year and and in freezers they can be
stored for 1-2 years. Flour and other materials used in manufacturing food products
need to be packaged and stored properly prior to utilization to ensure the quality,
safety and storage stability. To realize the full potential of wheat flour in food
processing knowledge of the effects of package types and storage conditions on
quality and shelf-life stability of wheat flour is important.

39
CHAPTER 03
3.0 MATERIALS AND METHODS
This study was carried out in 2015 at the Prima Ceylon PVT Limited, Chinabay,
Trincomalee. This company is milling wheat grain into wheat flour, and producing
wheat flours used for different purposes by altering their chemical compositions,
mainly the gluten percentage. Thus the produced flour can be used for different
purposes such as cake, rotti, bread. etc. For my research study purpose, the company
has given me three different wheat flour samples with different chemical
compositions named as A, B and C. The company did not mention about the purpose
of the flour to be used.
3.1 Experiment Material
Fresh milled three different types of samples (sample A, Sample B, Sample C) were
packed in polypropylene bags.5 bags per sample, 15 sample bags for room
temperature storage and 15 sample bags for A/C storage, making the total of 30
sample bags. The weight of each sample bag was approximately 2 kg. The samples
were taken and packed in such a way that no chemicals or insect infestations were
allowed.
3.2 Experiment design
This experiment was designed to analyze how the quality parameters of wheat flour
change in two different type of storage conditions; Room temperature and A/C
condition in which the temperature of A/C condition kept constant and temperature of
normal room temperature storage was not constant due to fluctuations in climatic
conditions.

40
The tests for quality parameters of fresh milled samples were done to get the initial
readings. After the tests were done the three sample bags were discarded.
The rest of the 12 sample bags were stored in room temperature environment. And
other 12 bags were stored in an air conditioned room.
Degrees Storage temperature
1) Adjusting the stored air conditioned temperature to 27.5oCs ±0.5.
2) Adjusting the stored room temperature to 37oC±0.5.
3.3 Experiment timeline
The quality tests were done every two weeks intervalto analyze in what pattern of
quality parameters change, what quality changes this will bring to the wheat flour,
how will affect the storage capacity of the wheat flour and the shelf life capacity of
the wheat flour.
3.4 Experiment parameters
Physiochemical properties – Moisture content, Wet gluten, Gluten index, Falling
number, Protein content, Ash content.
Biological properties – Weevils count
Microbiological properties – Aerial plate count, Yeast and mold, Coliforms and
E.coli
3.4.1 Physiochemical analysis
The physiochemical analysis was done to three different types of wheat flour sample
for certain important quality parameters; Moisture, Gluten content, Protein, Ash, and
Falling number.

41
3.4.1.1 Long moisture test (Air oven method)
Principle - This test is done according to AACC method, 44 – 15. Moisture content is
to be the loss of weight due to water evaporation, expressed as percentage of the
weight of original sample.
Scope and objective - It is applicable to flour, farina, semolina, bread and wheat
grain. Flour moisture is influenced by weather and environmental or storage
conditions such as humidity and storage temperature. Such conditions affect the
keeping quality of a flour. Higher moisture may lead to spoilage and lump formation
during storage. Lower moisture content, on the other hand, cause loss to the baker in
terms of low dry matter. Several methods are available to determine moisture content
e.g. air oven method, direct distillation, chemical and electrical methods. In air over
method 5 gm sample is kept in a dish for one hour at 130°C. Electrical method could
also be used satisfactorily provided they are accurately calibrated.
Apparatus - Oven (either gravity-convection or mechanical convection). Capable of
being maintained at 130°C (+1°) and provided with good ventilation.
Method - The 3 different sealed sample bags were taken and the flour was mixed
thoroughly to get a uniform distribution of flour. The metal moisture cans were taken
(two for each sample) and weighed in analytical balance. Then the balance was zeroed
and uniformly mixed sample was added in the moisture cans to make 10 g. After that,
the moisture cans were placed in the oven capable of maintaining the heat at 130oC
for 1 hour. After 1 hour the moisture cans were taken out and kept in a desiccator for
15 minutes tills the cans are cooled. Then the can were weighed to check how much
moisture is lost.

42
Calculation
𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 % =
A − B
A − C
× 100
A = Weight of flour + moisture can before drying
B = Weight of flour + moisture can after drying
C = Weight of moisture can
3.4.1.2 Moisture analyzer test
The moisture analyzer equipment was calibrated to measure the moisture content of
flour samples. Then 5 g samples were taken from uniformly mixed sample bags and
placed in the analyzer and the moisture analyzer was allowed to be heated. After the
total moisture is removed, the results was displayed in percentage of total moisture
removed in the sample.
3.4.1.3 Wet Gluten test (Glutomatic method)
Principle - This test is done according to AACC method, 38 – 12. Wet gluten in
wheat flour is a plastic substance consisting of gliadin and glutenin proteins.
Objective – Viscoelasticity property of wheat flour which is particularly important in
making leavened bread is due to gluten present in wheat flour. As it allow the
entrapment of carbon dioxide released during leavening. However, they also underpin
a range of other uses including making unleavened breads, cakes, and biscuits, pasta
(from durum wheat), and noodles (from bread wheat).
Reagent - Needed for this test is salt solution prepared by dissolving 20 g sodium
chloride in 1 L water.
Apparatus - Gluten machine, Centrifuge.

43
Procedure ˗Test chambers were assembled with metal sieves between persplex tube
and perforated stainless steel bottom. The sieve was moistened thoroughly to achieve
a capillary water bridge which prevents the water loss. 10 g of each samples were
taken in test chambers, 5 ml sodium chloride salt solution was pipetted into the test
chamber, and gently shaken to spread the mixture evenly allowed to be washed in
gluten machine for 5 minutes. After the washing the test chamber was lowered and
the washed wet gluten was taken and it was allowed for centrifugation for 1 minute.
The centrifuged wet gluten was weighed and weight was converted to percentage
multiplying by ten.
Calculation
𝑊𝑒𝑡 𝑔𝑙𝑢𝑡𝑒𝑛 =
Weight of washed gluten
Initial weight of the sample
× 100
3.4.1.4 Gluten index
Principle - Gluten separated from whole wheat meal or wheat flour by the Glutomatic
equipment is centrifuged to force wet gluten through a specially constructed sieve
under standardized conditions.
Objectives - Wet gluten in wheat flour is a visco-elastic substance made of gliadin
and glutenin, which is obtained by means of the specified method contained in this
international standard. The Gluten Index is a measure of the gluten characteristics,
which indicates whether the gluten is weak, normal or strong.
Apparatus - gluten machine, Centrifuge.
Method – wheat flour samples of 10 g were taken in gluten test chambers, 15 ml
sodium chloride salt solution was added, and they were allowed to be washed in

44
glutomatic machine for 5 minutes. After that, the washed wet gluten was taken and it
was allowed for centrifugation. The wet gluten was taken from the centrifugation cups
in a way that the portion remained inside the cup separately and the gluten which
leaked outside the cup separately. Both portions were weighed. and the gluten index
was calculated.
Calculation
𝐺𝑙𝑢𝑡𝑒𝑛 𝑖𝑛𝑑𝑒𝑥 =
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝐺𝑙𝑢𝑡𝑒𝑛 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑙𝑒𝑎𝑘𝑒𝑑 𝑔𝑙𝑢𝑡𝑒𝑛
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑔𝑙𝑢𝑡𝑒𝑛
× 100
3.4.1.5 Falling number (Hagberg Perten Method)
Principle - This test was done according to AACC method 56 – 81B. Falling number
is measure of the Degree of Alpha-Amylase Activity in Grain and flour. Falling
number is defined as time in seconds required to stir and to allow viscometer stirrer to
fall a measured distance through a hot aqueous flour undergoing liquefaction.
Objectives - The method is applicable to meal and flour of wheat, rye, barley, as well
as to other grains and to starch containing and malted products. In this standard the
word "flour" also means meals and ground grains (whole meal).By converting the
Falling Number into the Liquefaction Number it is possible to calculate the
composition of flour mixtures of desired Falling Number.
Apparatus – Shakematic Machine, Perten falling number machine.
Method - According to the moisture content of the sample, the particular amount of
sample was taken inside the viscometer tube (i.e. if moisture content of the sample is
13.2% then 6.92 g sample should be taken inside the falling number tubes). Then 25

45
ml distilled water was added into each tubes, a rubber stopper was fitted on the
viscometer tube and shaken well by placing it in the shakematic machine for 30
seconds to get a homogenous suspension. After that rubber stopper was removed and
viscometer stirrer was placed inside the tube and the flour adhering on the side walls
were scraped down by the stirrer. Then the tubes with stirrer were fixed in the boiling
water bath of perten system and the lid was closed. Once the sample tubes were at
working position auto mixing begins. After the mixing is finished two readings can be
taken from the left and right sides of the machine. The amount of wheat flour needed
to be taken for particular moisture content of the sample is given in Table 3.1 below.
Table 3-1Table for the weight of sample to be taken for falling number
according to moisture content
Moisture content Weight of sample Moisture content Weight of sample
11.0 6.76 13.0 6.92
11.2 6.78 13.2 6.94
11.4 6.8 13.4 6.95
11.6 6.81 13.6 6.97
11.8 6.83 13.8 6.98
12.0 6.84 14.0 7.00
12.2 6.86 14.2 7.02
12.4 6.87 14.4 7.03
12.6 6.90 14.6 7.04
12.8 6.92 14.8 7.07

46
3.4.1.6 Protein test
Principle - This test is done according to AACC (46 – 12) method. Crude protein is a
conventional expression for the total content of the nitrogenous compounds of the
analyzed product, calculated by multiplying the total nitrogen content by the
conventional factor.
Objective - The organic matter of the sample is oxidized with concentrated sulfuric
acid in the presence of a catalyst: the product of the reaction (NH4)2SO4 is treated by
alkali; free ammonia is distilled and titrated.
Reagents - Nitrogen free sulphuric acid, Nitrogen free potassium disulphate,
concentric sodium hydroxide solution prepared by dissolving 450 g NaOH powder in
1 L distilled water, methyl red methyl blue mixed indicator prepared by dissolving 0.1
g methyl red in 50 ml(A) 0.05 g methyl blue in 25 ml ethanol (B) and mixing A and
B together, boric acid solution prepared by dissolving 90 g boric acid in 4.5 L water,
and Boric acid methyl red methyl blue receiver solution prepared by adding 12 ml
methyl red methyl blue solution and boric acid solution.
Apparatus – Protein digester, Kjeldhal Distillation Unit.
Method - Potassium disulphate powder 2.5g, 0.5 g selenium tablet, 1 g wheat flour
sample were weighed and taken inside each kjeldhal digestion flask and 15 ml 98%
sulphuric acid was added. Then 2.5 ml hydrogen peroxide solution was added and the
digestion flasks were fitted in the protein digester and allowed to run for 40
minutes.(the temperature of the protein digester should be 420 o C in order to run the
machine)
After 40 minutes when the clear yellow color was obtained, the flasks were taken out
and 75 ml cold distilled water was added to each flasks. And each flask was fixed in

47
the distillation unit, In this unit 60 ml NaOH solution was added by the machine itself,
in the receiver end conical flask containing 50 ml boric acid methyl red methyl blue
solution was placed and distillation occurred for accurate 5 minutes. After that the
distillate was taken and titrated with 0.1 N Sulphuric acid. Then the titrated volume
was multiplied by protein conventional factor to get the exact protein value. The
protein factor depends on the molarity of sulphuric acid used for titration.
Calculation
Protein content = Titrated volume of 0.1 H2SO4 ×conventional factor
3.4.1.7 Ash content
Principle - This test was done according to AACC method, 08 – 01. Total ash is the
inorganic residual remaining on incineration in a muffle furnace. This reflects the
quantity of mineral matter present in the flour. Acid insoluble ash reflects added
mineral matter in milled products such as dirt, sand, etc.
Objective - Ash, an index of the mineral content of the flour, gives an indication of
the grade or the extraction rate of the flour. This is because the mineral content of the
endosperm is very low, as compared to the outer bran layers. Thus, low-grade flours,
rich in powdered bran give higher ash contents as compared to more refined or patent
flours.
Apparatus – Electric Muffle Furnace.
Method - Clean ash crucible were taken and their weights were measured in digital
weight balance. Then the balance was zeroed and 5 g well mixed sample was weighed
in the ash crucible. After that, the ash crucibles were placed in the electric muffle

48
furnace and heated at 600 oC for 6 hours. Then the crucibles were cooled in a
desiccator after each crucible was weighed to get the true ash content of the sample.
Calculation
𝐴𝑠ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡% =
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑏𝑢𝑟𝑛𝑡 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑐𝑟𝑢𝑐𝑖𝑏𝑙𝑒
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
× 100
Duplicate determination should not vary more than 0.02 for 1% ash content, and for
more than 1% ash content should not vary more than 2. If there is a great variation the
procedure has to be repeated.
3.4.2 Biological test
This test was done to check whether any living macro organisms present in the flour
which is a clear indicator of the wheat flour quality. Main organism which is often
checked for biological test is weevil. The presence of weevil in a wheat flour sample
says that the flour quality is deteriorating.
3.4.2.1 Weevil test
After microbiological and physiochemical tests were done, the whole quantity of flour
sample was subjected to sieving by 250 micrometer pore sized sieve. After that
number of weevils present in the sieve was noted.
3.4.3 Microbiological test
Microbiological test was done to check for the presence of yeast and mold, Coliforms
and Escherichia coli, and other pathogenic bacteria. Doing the microbiological test
for the fresh milled sample gives you the initial microbiological load of the wheat
flour sample.

49
The samples should be separately taken in sterile bags under aseptic condition and
this should be done before doing any other physiochemical or biological tests.
3.4.3.1 E.coli and Coliforms counting test
This test was done to count the population of total coliforms and Escherichia coli
bacteria in the sample. These bacteria are gram negative and rod shaped.
Identification criteria used are production of gas from glucose and fermentation of
lactose within 48 hours at 35oC for coliforms and at 45.5oC for E.coli.
The bacteria are cultured in Lauryl sulfate tryptose broth media (LST). The LST
solution was prepared by dissolving 35.6 g LST powder in 1000 ml distilled water.
Then the solution was sterilized in the autoclave at 121oC temperature and 15 psi
pressure. And the solution was stored in the refrigerator.
For the stock buffer solution preparation, 34 g potassium dihydrogen phosphate was
dissolved in 500 ml distilled water and the pH was adjusted to 7.2 by adding Sodium
hydroxide and making the solution to 1 L by adding distilled water. Then preparation
of diluent buffer solution was done by dissolving 1.25 ml potassium hydro phosphate
(KH2PO4) in 1000 ml water.
To perform this test 10 ml Lauryl sulfate tryptose broth solution was pipette out into
the pre sterilized gas tubes with durham tubes. (durham tubes were added to capture
the gas released by bacteria and they should be placed in an upside down manner).
Then 9.5 ml KH2PO4 buffer solution was taken into test tubes. The experiment setup
is shown in the Table 3.2.

50
The inoculation procedure was done under the laminar flow hood. Before starting the
inoculation the laminar flow hood was thoroughly surface sterilized by 70% ethanol
solution and it was kept sterile by lighting a gas burner inside.
Table 3-2experimental setup for 10 g sample which includes 1 empty tube,
2 dilution tubes and 9 gas tubes.
For the inoculation procedure, sterile stomacher bag was taken and kept on the
electric balance and the balance was zeroed. Then 10 g sample was added by sterile
spoon into the stomacher bag and sterilized buffer solution was poured into the
stomacher bag to make the total weight as 100 g. (10-1 dilution). Then the bag was
thoroughly mixed by stomacher lab blender for 1 minute. After that the mixed flour
solution was poured into a sterilized empty test tube and inoculation procedure
started.
1 ml 10-1 flour solution was pipette out using the sterile serological micro pipette and
released into the dilution tube; making it as 10-2 solution and kept aside. 1ml solution
from the 10-1 flour + KH2PO4 solution was pipette out and released into 3 gas tubes.
Dilution tubes containing
buffer solution
Gas tubes containing LST medium
and fermentation tubes
For each 10 g
sample
10-1
10-2
10-3

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