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natural polysaccharides
A THESIS SUBMITTED
TO THE
UNIVERSITY OF MUMBAI
FOR THE
SUBMITTED BY
SATISH DASHARATH SHEWALE
UNIVERSITY OF MUMBAI
INDIA
JUNE-2008
STATEMENT BY THE CANDIDATE
As required by the University Ordinance 770, I wish to state that the work
embodied in this thesis titled “Studies in the Enzymatic depolymerisation of
natural polysaccharides”, forms my own contribution to the research work
carried out under the guidance of Prof. A. B. Pandit at the Institute of Chemical
Technology, University of Mumbai, Matunga, Mumbai. This work has not been
submitted for any other degree of this or any other university. Whenever
references have been made to the previous work of others, it has been clearly
indicated as such and included in the Bibliography.
Satish D. Shewale
(Research Student)
Certified by,
The research work described in this thesis has been carried out by Mr. Satish D.
Shewale under my supervision. I certify that this is his bonafide work. The work
described is original and has not been submitted for any other degree of this or any
other university. Further that he was a regular student and has worked under my
guidance as a full time student at Institute of Chemical Technology, University of
Mumbai, Matunga, Mumbai – 400 019 until the submission of the thesis to the
University of Mumbai.
Date:
Place: Matunga, Mumbai-400 019.
List of publications
UICT is the place, where I have spent about 6 years of my life and these years
are mainly responsible for my way of being today. So firstly I thank UICT and
all the people associated with UICT. I thank all the teaching staff of UICT in
M. Chem. Engg. for taking my chemical engineering knowledge to new
heights.
I would like to thank Prof. A. M. Lali for providing CELBEADS for the
present work and allowing me to use lab facilities from his lab.
I would like to thank Prof. D. N. Bhowmick for allowing me to use facilities
from his lab.
My special thanks are due to Mr. Potdar from workshop, who always being
there in case of any instrument problems. Without his efforts, it would have
been really very difficult to complete this work.
I thank my very special friends during the entire time, i spent in UICT,
Yogesh Doshi, my “saala” – “Awaara” Balu and Ajit (my langoti yaar in
chemical engineering) for their timely help and constant encouragement. I am
really very thankful to god to give me such wonderful friends.
I would like to thank all my ABP labmates Parag, Virendra, Gopal, Rajesh,
Shashank (KS), Mohan, Amit (ask him any problem about computer,
amazingly he has solution), Pratap, Prashant (Birra), Ambu, Parag (kanthu),
Preeti, Shailesh (Sher), Shankar, Vishwa, Haresh, Naresh, Ajaykumar,
Pramod, Hemant, Sandip, Shravan, Bijal, Aditi, Apoorva, and all others who
have newly joined the ABP group.
My special thanks to all AML labmates, especially Kishor (for giving me the
taste of Trekking), Amol, Amit, Pratap, Pooja, Archana, Ann, Ganesh, Umesh,
Amrutraj, Abhijar, Monika, Rashmi, Sandip, and all others for making the
working in lab joyful.
I would like to thank my parents ‘aai and nana’ for providing all the support
needed; their support and faith in me always encouraged me. Sweet
memories of my aaji come to my mind. She would have been very happy to
me for completing the highest qualification. I also thank my younger brother
and my best friend ‘Chetan’ for everything he did for me. I also thank my
elder brother ‘Suresh’, Mugdhavahini, my darling neice ‘Lekha’, and my in
laws ‘mama, mami, Smita and all others’ for their love for me.
I would like to thank my dear wife ‘Supriya’ for her love, encouragement,
understanding, and awesome support throughout the Ph D work. Thank you
for bearing with my late hours, work weekends, bad moods (which being
consequences of not getting results), and going for treks with freinds. Now,
last but certainly not least my son, ‘Malhar’. He always is a source of joyful
moments for me. His presence around me always kept me in fresh mood to
do my work.
It is quite possible that I might have missed name of few people in spite of
their valuable assistance, both from a professional and personal perspective. I
thank all of them.
Satish D. Shewale
CONTENTS
1. Introduction 1
References 218
Appendix A. Analytical methods 233
Appendix B. Matlab code to find kinetic constants 243
Synopsis
Chapter 1: Introduction 1
1. Introduction
cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and
maize. Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons
(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina
(5.4%) (www.fas.usda.gov). Sorghum is a staple food crop for many of the world’s
poorest people, and constitutes a major source of energy and proteins for millions of
people in Africa and Asia. In India, sorghum is grown in the kharif (rainy season) and
rabi (postrainy season). Rabi crop is almost entirely used for human consumption,
whereas kharif crop is not very popular for human consumption and is largely used
for animal feed, starch, and by the alcohol industry. Maharashtra is the largest
sorghum producing state in India with production of 5.8 million Tonnes in 2001-2002.
with limited use of pesticides and it has a potential to adapt itself to the given natural
environment. Sorghum is valued because of its ability to grow in areas with marginal
rainfall and high temperatures (i.e. semi arid tropics and sub tropical regions of the
world), where it is difficult to grow any other cereal, and also because of its relatively
short growing season requirement, thus its suitability for double cropping and crop
decreasing with change in the way of living due to increased urbanization, increased
per capita income of the population, and easy availability of other preferred cereals in
of staple food for humans, it also serves as a source of feed for cattle and other
livestock in case of scarcity of maize, but at lower prices. Also, about 10-20 % of the
production gets wasted due to damage and inadequate transport and storage facilities.
Industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are
available in large quantity at Food Corporation of India (FCI) at 10 times lower rate
than the fresh grains (Suresh et al., 1999a). Damage includes chalky appearance,
cracked, broken, mold, infection etc. These damaged grains are not suitable for
that are harmful to health and productivity of human and animal (Bandyopadhyay et
al., 2000). Hence, damage caused by insect infection and attack of fungus (blackened
sorghum or grain mold) because of wet and humid weather makes sorghum grains
farmers, through value added products. There is very small amount of research done
1996; Aggarwal et al., 2001), production of ethanol (Wu et al., 2007; Suresh et al.,
1999a,b; Zhan et al., 2003 and Zhan et al., 2006) and isolation of starch (Yang and
Sieb, 1996; Xie and Seib, 2002; Higiro et al., 2003; Perez-Sira and Amaiz, 2004; Park
et al., 2006). The reason for the lower level of industrial exploitation can be
and Pflugfelder, 1986; Chandrashekar and Kirleis, 1988; Zhang and Hamaker, 1998;
Elkhalifa et al., 1999; Ezeogu et al., 2005) and reduced protein digestibility (Duodu et
al., 2003) after cooking i.e. heat-moisture treatment of sorghum flour. There is no
Research aim
glucose and maltose from different qualities of sorghum i.e. healthy, germinated and
blackened. In the present work, sorghum flour was used directly for liquefaction and
saccharification rather than isolating starch and using it for liquefaction and
around 50–60% i.e rest part (40–50%) gets wasted or does not fetch much price.
Such methodology of direct hydrolysis was first used by Kroyer in 1966 using corn
Thesis Outline
Starch structure and chemistry, action of different types of enzymes used for
different starch hydrolysis products are also discussed in Chapter 2. Attempt is made
yield of sorghum in the world and India are also briefly discussed in chapter 3.
Applications of sorghum viz. food use and industrial utilization, and problem areas
content) and factors affecting them in industrial utilization of sorghum are also briefly
reviewed in chapter 3. Then lastly literature on production of ethanol and starch from
operation of the system, easy separation of product from the enzyme etc.) that
immobilized enzyme have over the free enzyme, it was first decided to develop a
process for the production of glucose from sorghum flour using immobilized
sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of
before studying this, it was necessary to first immobilize BLA on beads and study its
rigid superporous (pore size ∼ 3 µm) cross-linked cellulose matrix (CELBEADS) and
Chapter 5 covers the work to add value to three different varieties of sorghum
viz. normal healthy, germinated, and blackened through production of glucose and to
processes were optimized with the use of normal sorghum flour as a starting material
for the production of glucose. Effect of ultrasound treatment on the sorghum slurry
optimized conditions.
Chapter 6 details the work to add value to three different varieties of sorghum
viz. normal healthy, germinated, and blackened through production of maltose and to
pullulanase) process was optimized with the use of normal sorghum flour as a starting
material for the production of maltose. Effect of ultrasound treatment on the sorghum
slurry prior to liquefaction and use of pullulanase during saccharification was studied
List of the references used in the present work and synopsis are provided at the
end of thesis. All analytical methods are provided in the appendix A and code in
Matlab used to find kinetics in hydrolysis of soluble starch using immobilized BLA is
2.1. Starch
initial plant growth. Man harvests this reserve and with little preparation may use it
directly or after separation and purification by relatively simple processes. Starch has
been important ingredient of human diet over centuries mainly as a high calorie food
source. (Zobel, 1992) Starch pastes and gels are used to control consistency and
textures of sauces, soups and spreads. In addition to its use in human food, it has
material. Sweetener and fermentation industries are two of the main consumers of the
namely maltodextrins, high maltose syrup, maltose, glucose syrup, dextrose, and
1998; Denyer et al., 2001; Emes et al., 2003; Smith et al., 1997; Tester and Karkalas,
2002 as cited in Tester et al., 2004a). Sucrose (derived from photosynthesis) is the
starting point for alpha-glucan deposition. In the cell cytosol the sucrose is converted
least, G-1-P may be (a) translocated directly into the amyloplast or (b) be converted
(also) converted to ADP-glucose and provides glucose residues for amylose and
amylopectin biosynthesis. Starch synthases (of which there are commonly considered
to be two major classes, ‘granule bound’ and ‘soluble’, with a number of isoforms of
each) add glucose units to the nonreducing ends of amylose and amylopectin
amylose and is considered to be responsible for the synthesis of this polymer. Soluble
linear chains (branches) to the growing amylopectin molecule. (Tester et al., 2004a)
Starch granules are synthesized in a broad array of plant tissues and within
(α-glucan, lipid, moisture, protein and mineral content) are mainly reflection of their
botanical origin (Table 2.1; Tester et al., 2004a). For industrial production of starch,
most important sources of starches are cereal grains, pulses and tubers, with maize
are mainly composed of α-glucan, lipids, protein and moisture. Starch granules
consist of two types of α-glucan viz. amylose and amylopectin, which represent
amylopectin varies according to the botanical origin of the starch. Starches are
defined as waxy when amylose content is less than 15%, normal when amylose
content is in the range of 15-35% and high-amylose (amylo-) when amylose content
2.1.1.1. Amylose
and 1% α(1→6) linkages and differs in size and structure depending on botanical
11 chains per molecule. Each chain contains approximately 200–700 glucose residues
of amylose is shown in the Fig. 2.1. Buleon et al. (1998) concluded that though few
branch points (i.e. α(1→6) linkages) are present in the amylose, they do not
significantly alter the solution behavior of amylose chains, which remains identical to
that of strictly linear chains. One end of the linear chain has a free C1 hydroxyl group
and is the reducing end. Another specific feature of interest concerning amylose is its
capacity to bind iodine. Amylose complex with iodine and produces deep blue color,
this property is generally used as qualitative method to identify the presence of starch.
Figure 2.1. Basic structure of amylose and amylopectin. Adapted from Tester et al.
(2004a)
2.1.1.2. Amylopectin
of 1 × 107 – 1 × 109 and a heavily branched structure built from about 95% α(1→4)
common with amylose, the molecular size, shape, structure and polydispersity of the
molecule varies with botanical origin. Unlike amylose, however, there is great
additional variation with respect to the unit chain lengths and branching patterns.
Amylopectin unit chains are relatively short compared to amylose molecules with a
(chain lengths, CL) and consequently position within starch granules. The A and B1
chains are the most external (exterior) and form double helices (and crystallites)
within the native granules. Their CL is typically, 12–24 depending on genetic origin
and starches with ‘A-type’ crystallinity, (most cereals) having shorter chain lengths on
average than ‘B-type’ starches (like potato). With the exterior chains of amylopectin
(A- and B1) comprising a range from CL 12–24 as previous mentioned, the A-type
chains are typically CL 12–16 and B1 CL 20–24. Amylopectin molecules from high
amylose starches contain relatively high proportions of very long chains. With respect
linked by B-chains which in turn can be linked to other B-chains or the ‘backbone’ of
the amylopectin molecule, the single C-chain (which contains a sole reducing group).
within the native granule, B chains are referred to as B1–B4 (one to four clusters).
Typical CLs for A, B1–B4 chains for different starches (after debranching with
isoamylase) are 12–16, 20–24, 42–48, 69–75 and 101–119, respectively. The ratio of
A- to B-chains depends on the starch source and is typically of the order of ,1:1 to .2:1
on a molar basis or ,0.5:1 to .1:1 on a weight basis. (As reviewed by Tester et al.,
2004a)
amino acids and nucleic acids. Mineral fractions are negligible in cereal starches in
contrast to tuber starches. Cereal starches contain phosphorus that is mainly in the
Lipids represent the most important fraction associated with the starch
small amounts of free fatty acids, tocopherols, and sterols. Cereal starch granules
contain internal (or intragranular) lipids, which are exclusively monoacyl lipids i.e.
free fatty acids (FFA) and lysophospholipids (LPL), and can complex with amylose.
Internal lipids are proportional to the amylose fraction in all normal cereal starches
and the LPL may account for up to 2% of starch weight in high amylose cereal
starches. (Morrison, 1988) Since the lipid fraction within starch granules is
insufficient to saturate entire quantity of amylose, amylose exists in two forms; free
usually composed of a typical left-handed amylose helix, in which the aliphatic part of
the lipid is included i.e. fatty acid chains occupy a hydrophobic core located within
the single amylose helix (Nebesny et al., 2002; Tester et al., 2004a).
phospholipids and free fatty acids derived from the amyloplast membrane and non-
starch sources. These differ from internal lipids, which are composed exclusively of
the FFA and LPL. (Tester et al., 2004a) Normal and high-amylose cereal starches
contain more internal, than granule surface lipids, whereas waxy cereal starches,
potato and bean starches contain small amounts of granule surface lipids and probably
gelatinization. They will restrict swelling, dispersion of the starch granules and
solubilization of amylose, thus generating opaque pastes with reduced viscosity and
to 18% depending on the source, with cereal starches at the lower end and tuber
starches at the Upper end. Purified starches contain low levels of protein (<0.5%)
which largely represent the residues of biosynthetic enzymes involved in the synthesis
There are few reviews (Oates 1997; Buleon et al., 1998; Tester et al., 2004a),
which have analyzed and discussed starch granule structure. Starch granules are
(1→6) branch regions of amylopectin and amylose (Fig. 2.3). Both amylose chains
and exterior chains of amylopectin molecule (A and B1) form double helices, which
in turn associate to form crystalline domain. From Fig. 2.3 it is apparent that the
(‘dark’) growth rings which comprise about 16 crystalline lamellae, which are of
approximately the same width as the interspersed amorphous (‘light’) growth rings
Three types of starches, designated as type A, type B, and type C, have been
identified based on X-ray diffraction patterns. These depend partly on the chain
lengths making up the amylopectin lattice, the density of packing within the granules,
and the presence of water. Although type A and type B are real crystalline
modifications, type C is a mixed form. (Sajilata et al., 2006) The important features of
units. The hydrogen bonding between the hydroxyl groups of the chains of
between these micelles, linear chains of amylose moieties are packed by forming
hydrogen bonds with outer linear chains of amylopectin. This pattern is very common
in cereals.
glucose molecules with water inter-spread. This is the usual pattern of starches in raw
glucose molecules, a combination of type A and type B, which is typical of peas and
beans.
Starch granule B A
Figure 2.3. (A) Structure of amylopectin. (B) Organization of the amorphous and
crystalline regions of the structure generating the concentric layers that contribute to
the “growth rings” those are visible by light microscopy. (C) Orientation of the
amylopectin molecules in a cross section of an idealized entire granule. (D) Shows the
likely double helix structure taken up by neighboring chains and giving rise to the
extensive degree of crystallinity in granule (www.lsbu.ac.uk; van der Maarel et al.,
2002)
amylopectin clusters and randomly interspersed among clusters in both crystalline and
and crystalline domains accordingly. The location of amylose with respect to the
crystalline and amorphous regions is dependent on the botanical source of the starch.
In wheat starch, amylose is mainly found in the amorphous region, but in potato
that are present in the granule core are able to participate in double helices with
amylopectin, whereas smaller amylose molecules present at the granule periphery are
Gelatinization of starch is crucial step for many starch based industrial and
gelatinization process is usually carried out with a 30–35% dry solids starch slurry.
Higher starch concentrations may yield higher volumetric efficiencies and lower
energy consumption (Baks et al., 2008), but are difficult to handle mechanically.
The starch granules are very organized (specific to their botanical origin) with
amylose having helical coil like structure and amylopectin having tree like structure.
gelatinization takes place. Starch granules absorb water, swell, lose crystallinity
(crystalline form gets transformed into amorphous form), and leach amylose during
thermal gelatinization. Gelatinization is used as a collective term for the changes that
starch granules faces when they are heated in the presence of water. These changes
and swelling, change in the shape and size of granule, and leaching of amylose.
Ultimately, granule structure is completely lost and a thin paste (<~4%) or gel (>~4%)
the starch-water ratio. When the moisture content of the starch-water mixture is low,
complete swelling and disruption of the starch granules is not possible and only part
of the crystallinity of the starch granules is lost. (Leach, 1965; Tester et al., 2004b;
Figure 2.4. Idealized diagram of the swelling and gelatinisation of a starch granule in
the presence of water. Adapted from Tester et al. (2004b).
disintegration of the granules is less (or the granules even remain intact), amylose
Sources for industrial production of starch are plant seeds, roots, and tubers.
Basic process steps include cleaning, steeping, grinding, separation, and isolation of
finished products. Products include starch, and co-products based on protein, fiber,
and oil components. In 2005 world starch production was 60 million tones, out of
which ~ 70% starch had origin as maize (www.starch.dk). Maize can be processed
through Dry or Wet milling operations for the production of starch. However in
industry wet milling process is more popular because products by Dry mill process
consists of protein adhering to starch and also due to this protein it is also not suitable
cold water. Since swelling occurs in cold water, these starches are used in instant food
starches can have lowere granule gelatinization temperature and gels that show less
firming with age, as well as freeze thaw stability and better clarity. Due to good film
forming property hydroxypropyl starch give smooth paper surfaces and ink holdout.
Cross linking is most commonly accomplished using adipic acid and phosphorous
granules to achieve needed functionality. Oxidized starch, thin boiling starch and
Starch has become a very important biopolymer and is used in many industries
as a feedstock material. Sweetener and fermentation industries are two of the main
consumers of the starch. Nutritive sweeteners are mainly starch hydrolysis products
namely maltodextrins, high maltose syrup, maltose, glucose syrup, dextrose, which
are used in food and pharmaceutical industry (Table 2.3). Two excellent textbooks
(Schenck and Hebeda, 1992; Kearsley and Dziedzic, 1995) on the basics concepts of
the production of starch hydrolysate were mainly referred to write this entire section
2.2. Three steps in which enzymes are used in starch hydrolysis and processing are as
follows:
gelatinized starch)
maltose
3. Isomerization of glucose
Starch is a polymer of glucose units, in which glucose units are linked together
In order to manufacture these products α(1→4) and α(1→6) glucosidic linkages must
be cleaved. This cleavage of the glucosidic linkages can be achieved by either acid
Previously heat and acid treatments were used for generating starch hydrolysis
products. Though effective, these methods were not specific and undesirable by-
products and off-flavors were also formed due to harsh reaction conditions. Enzymes
catalyze reaction under moderate conditions of pH and their use has led to better
Processes involving biocatalysts are potentially energy saving and are very well suited
industrially in an ecologically safe way and are renewable, degradable and non-toxic
(Uhlig, 1998).
starch into products for use in beverages, food, pharmaceuticals, and industrial
ethanol production (Teague and Brumm, 1992). They are present in all living cells,
where they perform vital functions by controlling the metabolic processes and causing
stability, and they lose their activity in due course of time. (Davidson, 1999) The
essential role of enzymes in almost all physiological processes stems from two key
features of enzymatic catalysis: (1) enzymes greatly accelerate the rates of chemical
produce specific reaction products. Together these properties of rate acceleration and
substrate specificity afford enzymes the ability to perform the chemical conversions
of metabolism with the efficiency and fidelity required for life. (Copeland, 2000) In
some cases, enzyme action is specific to certain bonds in the compounds with which,
they react (Roberts, 1989). Enzymes are classified based on the types of reactions
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
great value over the last two decades, was the starch industry. In 1950s, fungal
amylase was used in the manufacture of syrup, containing a range of sugars, which
could not be otherwise prepared using conventional acid hydrolysis. But it was in
enzyme which hydrolyses the O-glycosyl linkage of starch. The α-amylase family, is
a large enzyme family that constitutes about 20 enzymes having different reaction and
energy and carbon. Microorganisms mainly bacteria, fungi produce various starch
amylase, glucoamylase, and β-amylase, which can be derived from bacteria, fungi, or
plants and their classification, are summarized in the Table 2.4. Action pattern of all
enzymes is diagrammatically represented in the Fig. 2.5. Nigam and Singh (1995),
Guzman-Maldonado & Paredas-Lopez (1995) and Kuriki & Umanaka (1999) have
from starch. Van der Maarel et al. (2002) have reviewed properties and applications
comprise 30% of the world’s enzyme consumption (Van der Maarel et al., 2002). Use
Figure 2.5. Schematic representation of action pattern of starch hydrolyzing enzymes (Guzman-Maldonado & Paredas-Lopez, 1995;
and Kuriki & Umanaka, 1999 as cited in Nair, 2006)
Starch granules
bacterial α-amylase have reducing group in α configuration. It can bypass but cannot
further used for production of glucose and maltose syrup. During liquefaction α(1→4)
bonds are hydrolyzed in random manner and it results into reduction in the viscosity
hydrolysis.
amylase is around 40, but prolonged treatment leads to the formation of maltulose (4-
gelatinized starch to ease subsequent processing and also to avoid further possible
thermostability. The industrial needs for such specific thermostable enzyme and
improvements required to maximize their application in the future are also suggested.
predominantly produces maltose from starch with significant quantities of glucose and
maltotriose. It has ability to bypass α(1→6) glucosidic bonds i.e. it does not attack
α(1→6) bond, but overlook it and attack next α(1→4) bonds in the chain. It is used in
the production of high maltose syrup from liquefied starch. Prolonged incubation of
liquefied starch with fungal α-amylase results in the production of large amounts of
2.2.1.3. Glucoamylase
amylase, amyloglucosidase) cleaves first α(1→4) glucosidic bond from non reducing
end of starch and glycogen in exo-manner and then liberates β-glucose unit. Its
speciality exist in the ability to cleave α(1→6) glucosidic bond. Hydrolysis proceeds
in the stepwise manner. Maltotriose (G3) and particularly maltose are hydrolyzed at
lower rates than higher saccharides and α(1→6) linkages are broken more slowly than
α(1→4). It has activity towards α(1→2), α(1→3), α(1→4), and α(1→6) glucosidic
glucose and this fact has been used for development of starch assay. At high
normally used in the production of glucose syrup and dextrose. (Teague and Brumm,
2.2.1.4. β-amylase
enzyme. It cleaves second α(1→4) glucosidic bond from non reducing end of starch
or glycogen polymer and releases β-maltose unit. β-amylase can neither cleave
2.2.1.5. Pullulanase
α(1→4) and α(1→6) glucosidic bonds and produces maltose and maltotriose (van der
2.2.1.6. Isoamylase
is cleaves α(1→6) linkages. It is the only enzyme that debranch glycogen. These
enzymes have no effect on pullulan (this fact distinguishes it from pullulanase), while
it cleaves all the α(1→6) linkages in amylopectin and glycogen. (Olsen, 1995; van der
fructose. An excellent review on this enzyme and its industrial application has been
2.2.2. Maltodextrins
the market since the first commercial product Frodex 15 (later called Lo-Dex 15) was
introduced by the American Maize Products Company in 1959. The United States
Food and Drug Administration define maltodextrin as (21 CFR paragraph 184.1444):
primarily by α(1→4) bonds and that has a DE (dextrose equivalent) of less than 20.
and maltose syrups, and cyclodextrins are not considered here. Design of the desired
2.2.2.1. Production
Single stage and double stage processes are normally used for the production of
maltodextrins from starch. In single stage process, either acid or enzyme conversion is
performed at high temperature (~105 °C) using thermostable bacterial α-amylase. The
dual stage process involves first liquefaction at high temperature (105 °C) with acid or
enzyme to a low DE (usually < 3), followed by treatment at high temperature (~120-
conversion products of acid hydrolysis contain more glucose than their enzymatically
undesirable for this type of product. Furthermore, they have a low stability caused by
the presence of linear chains that have not been shortened sufficiently to avoid
carried out using acids, acid hydrolysis is being replaced by enzymatic hydrolysis for
the production of tailor-made maltodextrins. (Reeve, 1992; Teague and Brumm, 1992;
can even have different properties in various applications that reflect differences in
determines both its physical and biological functionality. Factors influencing the
saccharide composition are type and source of enzyme, source of starch, starch
processing and extraction of products during hydrolysis (Marchal et al., 1999) and
Enzymes
Downstream processing
Temperature
Extraction of products
Potential
Source of starch
Organic solvents
Concentration Immobilization
Pressure
Costs
Starch Maltodextrin a
Dextrose equivalent
0 5 10 15 20
Viscosity/ Bodying
agent
Browning reaction
Cohesiveness
Freezing point
depression
Hygroscopicity
Sweetness
Prevention of coarse
crystals
Solubility
Osmolality
Figure 2.8. Increase or decrease in functional properties of maltodextrins as a
function of DE (Alexander et al., 1992)
2.2.2.2. Application
the food processing and pharmaceutical industries. These applications are based on a
absorption by human. The dextrose equivalent (DE) value and saccharide composition
agent, texture provider, fat replacer, tablet expicient, film former, sport beverage,
parenteral and enteral nutrition products (Alexander, 1992; Marchal et al., 1999).
Carrier / Bulking agent. Used in dry mix products (includes puddings, soup, and
carbohydrate source in the nutritional fluids like energy bar, infant formula
(i.e. non milk or non lactose based fluids), enteral products, parental nutrition
excipients, isotonic solution that can be directly infused into veins of patient.
Fat replacer. It produces soft reversible gel and gives creamy fatlike mouthfeel. Gel
can be used to replace part of fat or oil in high fat products like ice cream and
Confectionery. It is perfect for candy coating and soft-centre candies, for frosting and
glazing, for nut and snack coating, in lozenges and for binding, plasticizing
characteristics.
saccharides obtained from starch. Glucose syrups are largely composed of glucose
maltose) and have DE values between 20 and 80. Major source of starch for
2.2.3.1. Production (Reeve, 1992; Teague and Brumm, 1992; Howling, 1992)
Acid conversion
water boiling conditions at atmospheric pressure), rapid hydrolysis takes place, with
concentrations of both acid and starch. Acid catalyzed starch hydrolysis is remarkably
limitation is that in the production of glucose syrup with DE above 55, acid promote
These undesirable products serve as impurities and also impart undesirable color to
syrup.
limited to the range of 30 - 55 DE. In this range of DE, products are reproducible in
terms of saccharide composition and high quality (i.e. color stability and clarity).
The range of glucose syrup i.e. 30-55 DE, produced with acid conversion, is
widened by use of enzyme in a dual conversion with acid used for liquefaction. Acid
55-60 °C. Second stage enzymatic hydrolysis can be done using bacterial α-amylase,
enzymes, depending upon the properties of required glucose syrup i.e. DE value and
were made by hydrolysis of 17-20 DE acid substrate or 40-42 DE acid substrate using
saccharide composition, enzyme and substrate used for a range of acid-enzyme syrups
(Howling, 1992).
DE of Acid catalyzed
17 20 40 40
hydrolysate, 1st stage
Enzyme used in second Bacterial β-amylase fungal α-amylase β-amylase
stage α-amylase + glucoamylase + glucoamylase
DE 26 42 63 63
Dextrose % on db i.e.
4 6 36 37
dry basis
Maltose % on db 4 45 30 32
Higher sugars % on db 92 49 34 31
Enzyme-Enzyme conversion
°C and hydrolysate of 12-15 DE was produced. Then this hydrolysate was further
saccharide composition.
only provided better control over saccharide composition but also reduced the
purified and deodorized by removing trace impurities that remained after separation
of bulk of the protein and lipid by filtration. Impurities that remain consist of protein
or protein hydrolysate, peptides and amino acids, color precursors, and flavor and
or calcium phosphate haze forms due to the presence of calcium, which is usually
added as a cofactor for bacterial α-amylase. Also, syrup might contain sulphate and
phosphate picked up from filer aid and / or tap water. Hence demineralization is also
required, which gave an improved product in terms of color removal and color
stability. This demineralization step will also remove color precursors, which are
basically ionic. This refining step will be required and will be mostly same for all
After refining, glucose syrups require concentration to about 80% solids for
store at temperatures that do not reduce shelf life by color formation. Evaporation is
sweetness, and viscosity, which are mainly dependent on DE. The increase or
decrease in these properties with an increase in DE is same as that shown in the Fig.
2.8 for maltodextrins. Glucose syrups are used in a variety of food and food
glucose syrups.
Confectionary. Glucose syrups are used in various confectionary products like hard
candies, toffees/ caramel/ fudge, Gums and jellies, fondants, marshmallows, and
Nondairy creamer. Product is made by mixing 50% glucose syrup solids (25-30 DE)
with 40% hardened vegetable oil, emulsifier, stabilizer, flavor etc at 82 °C and
spray dried.
concentration, they are used in products like jams, preserves, conserves, and
marmalades.
Candied fruit. 63 DE syrup used due to sweetness, preservative property & low
viscosity.
Frozen desserts.
Bakery goods. Used in preparation of bakery products like bread, rolls, doughnuts,
Breakfast cereals. 42 DE syrup used to coat cereal products due to good film forming
property and also imparts good shelf life and enhancement in flavor of the
product.
and fine chemicals, 2. Medicated confectionary and 3. as a carrier for liquid cough
It contains D-glucose in excess of 90% on dry basis (db) with a typical value between
sometimes also termed as high DE glucose syrup. The products produced from this
feedstock include crystalline dextrose, liquid glucose and high fructose syrup. Liquid
dehydration reactions. The acid-enzyme process will yield 93% dextrose, since acid
There are mainly two steps involved in the enzymatic production of Dextrose
pullulanase
Liquefaction
thermostable and hydrolyzes only internal α(1→4) linkages in the starch. Most
bacterial α-amylase are discussed earlier in the section 4.2.1. Hydrolysis of starch
using bacterial α-amylase is random in nature except that linkages near either end of
polymer chain and those close to a branch point are resistant. Hydrolysis of starch
α(1→6) linkages.
stability are disadvantages of this process. Added calcium salts must be removed at
enhanced by high pH (pH > 6), high temperature (>110 °C), long residence times in
the liquefaction, and is more pronounced at high DE values. Maltulose precursors are
produced due to chemical isomerization of the reducing end glucose units during
final dextrose level attainable. Liquefaction is usually performed with reaction time of
1-2 h.
amylase with ability to cleave both α(1→4) and α(1→6) linkages. Hence it seems that
(db) with very low concentration (<1% w/v) of starch as substrate solution. As starch
concentration increases maximum dextrose attainable decreases (at 30% w/v starch
for an economic process), the dextrose level rises to the maximum indicated and then
favored by high substrate concentration. These reversion reactions will proceed till
mixture. But increase in the dosage of amyloglucosidase will also enhance the rate of
Hydrolysates obtained from 30-35% w/v starch slurry are economically and
slurry (30-35% w/v), a 33-39% w/v solution is obtained with DE of 97-98 and
0.8% maltotriose and 0.6-1% higher saccharides. Normal refining (carbon, strong
occurs.
cleave α(1→6) linkages faster (which are cleaved, but relatively slowly by
reduction in the reversion rate will result into an increase in the dextrose level (>1%).
1992)
purity syrup of 71% ds and 99.5% db D-glucose has been the usual method of
Dextrose hydrolysate, crystalline dextrose and liquid dextrose are mainly used
Industrial Applications. Liquid glucose and solids are used in adhesives, building
leather.
syrup is classified into high maltose, extra high maltose, and high conversion syrup.
without pullulanase.
α(1→4) bonds yielding large quantities of maltose and maltotriose. β-amylase is exo-
amylase, which cleaves second α(1→4) bond from non-reducing end to produce
maltose. Since it does not have the ability to bypass α(1→6) bond, saccharification
For the production of very high maltose syrup with maltose content of 70-
amylase. Debranching enzyme cleaves α(1→6) linkages and produces linear dextrins,
maltose level attainable is the liquefact DE. As the liquefact DE increases, quantity of
or β-amylase has been done mainly on economic rather than technical basis. Ratio
maltose/dextrose ratio.
2.2.5.2. Applications
concern people. Maltose syrup invariably scores as a favorite alternative to sugar for
health conscious but sweet-loving people. It is often used in bread, cake and beer
brewage because of its well volatility. Meanwhile, Maltose Syrup is also widely used
in other fields such as candy, drinks, bakery goods, frozen foodstuff and seasoning
and so on. Its applications are mostly same as that of glucose syrup.
2.2.6.1. Production
column performance, while refining removes calcium ions detrimental to the activity
8.3. The magnesium maximizes and stabilizes the enzyme activity while
counteracting the inhibitory effect of residual calcium ions. The system will tolerate
in 20 fold excess over calcium. Sulfite or bisulfite salts are added to feed and is
usually aerated. These two adjustments lengthen enzyme half life. Isomerization is
function of column age, decrease in activity with time, operating conditions and
produce HFS-90 containing 90% fructose. HFS-42 and HFS-90 are blended to
produce syrup with desired fructose/dextrose ratio; mostly HFS-55 with 55% fructose.
Syrup is further filtered, refined and evaporated to 77-80% w/v. (Teague and Brumm
2.2.6.2. Applications
development, and colligative properties (e.g. freezing point depression and osmotic
Animal feed.
Baking industry and snack foods. Biscuits, breads, cakes, caramel color, cookies,
Canners and packers. Berries, candied fruits, fruit fillings, fruit pectin, soups, tomato
sauces, vegetables
Chemicals, drugs, pharmaceuticals. Acids, amino acids, antibiotics, food and drug
cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and
(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina
(5.4%) (www.fas.usda.gov). Sorghum ranks third in the major food grain crops in
India. In India, Maharashtra is a largest sorghum producing state with share of around
50%. Sorghum is a staple food for about 300 millions people worldwide. The seed or
caryopsis of sorghum provides a major source of calories and protein for millions of
people in Africa and Asia. In addition to being a major source of staple food for
humans, it also serves as an important source of cattle feed and fodder. It is grown by
United States, Australia and other developed countries for animal feed. Sorghum
grows comparatively quicker and gives not only good yields of grain but also very
variability in wild and cultivated species is still found today. It was probably
domesticated in Ethiopia between 5000 and 7000 years ago. From there, it was
distributed along trade and shipping routes around the African continent, and through
the Middle East to India at least 3000 years ago. It is believed that from India it was
carried to China along the silk route and through coastal shipping to South-East Asia.
Sorghum was first taken to America through the slave trade from West Africa. It was
introduced into the United States for commercial cultivation from North Africa, South
Africa and India at the end of the 19th century and subsequently spread to South
America and Australia. It is now widely cultivated in dry areas of Africa, Asia, the
and Russia and 40°S in Argentina. (Kimber et al., 2000; Balole & Legwaila, 2005)
arid regions of the world. Sorghum is also found in temperate regions and at altitudes
crops like maize under good environmental and management conditions. It is one of
the most widely grown dry land food grains in India. It does well even in low rainfall
areas. Sorghum is also termed as “Nature-cared crop” because it has strong resistance
other crops, it is usually grown as a low-level chemical treatment crop with limited
use of pesticides and it has a potential to adapt itself to the given natural environment.
Sorghum is valued because of its ability to produce in areas with marginal rainfall
(400 – 600 mm) and high temperatures (i.e. semi arid tropics and sub tropical regions
of the world), where it is difficult to grow any other cereal, and also, because of its
relatively short growing season requirement, thus its suitability for double cropping
In Africa, a major growing area runs across West Africa south of the Sahara,
through Sudan, Ethiopia and Somalia. It is grown in upper Egypt and Uganda,
Thailand, in central and northern China, Australia, in the dry areas of Argentina and
Brazil, Venezuela, USA, France and Italy. Sorghum is called by various names in
guinea-corn, dawa or sorgho in West Africa, durra in the Sudan, mshelia in Ethiopia
and Eritrea, mtama in East Africa, kaffircorn in South Africa and mabele or amabele
and milo in Spain. In the Indian subcontinent, it is known as jowar (Hindi), jwari
(Maharashtra), jonna (Andhra Pradesh), cholam (Tamil Nadu) and jola (Karnataka).
3.2. Taxonomy
Pliny (ca. 60 to 70 A. D.) was the first to give a written description of sorghum
and after that there was hardly a mention of it until the sixteenth century. Moench in
1794 established the genus Sorghum and brought the sorghums under the name
Sorghum bicolor. Harlan and de Wet (1972) developed a simplified classification that
has real practical utility for sorghum workers. Sorghum (L.) Moench comprises about
20-30 species. Sorghum Bicolor (L.) Moench is primarily cultivated specie. Other
Sorghum bicolor (L.) Moench subspecies bicolor i.e. grain sorghum contains
all of the cultivated sorghum and is sub classified into different races on the basis of
grain shape, glume shape, and panicle shape. Five basic races are Bicolor, Guinea,
kafir, Caudatum, and Durra. There are 10 intermediate races, which are caused by
inflorescences and long clasping glumes that enclose the usually small grain at
maturity. Cultivars are grown in Africa and Asia, some for their sweet stems to make
syrup or molasses, others for their bitter grains used to flavor sorghum beer, but they
often pendulous at maturity; the grain is typically flattened and twisted obliquely
between long gaping glumes at maturity. Guinea sorghum occurs primarily in West
Africa, but it is also grown along the East African rift from Malawi to Swaziland and
it has also spread to India and the coastal areas of South-East Asia. Many subgroups
can be distinguished, e.g. with cultivars especially adapted to high or low rainfall
regimes. In the past the grain was often used as ship’s provisions because it stored
well.
shape, elliptical sessile spikelets and tightly clasping glumes that are usually much
shorter than the grain. Kafir sorghum is an important staple across the eastern and
insensitive to photoperiod and most commercially important male sterile lines are
4. Caudatum: It is characterized by turtle-backed grains that are flat on one side and
curved on the other; the panicle shape is variable and the glumes are usually much
shorter than the grain. Cultivars are widely grown in north-eastern Nigeria, Chad,
Sudan and Uganda. The types used for dyeing also belong here and are known as
sessile spikelets, and creased lower glumes; the grain is often spherical. Cultivars are
widely grown along the fringes of the southern Sahara, western Asia and parts of
India. The durra type is predominant in Ethiopia and in the Nile valley in Sudan and
Egypt. It is the most specialized and highly evolved of all races and many useful
genes are found in this type. Durra cultivars range in maturity from long to short-
Nigeria and Sudan, and guinea-kafir is grown in East Africa and India. Kafir-
caudatum is widely grown in the United States and almost all of the modern North
American hybrid grain cultivars are of this type. Guinea-caudatum with yellow
endosperm and large seed size is used in breeding programmes in the United States.
The species Sorghum bicolor covers a wide range of varieties, from white and yellow
follows:
corn, common wild sorghum, Drummond broomcorn, durra, Egyptian millet, feterita,
forage sorghum , great millet, guinea corn, jowar, Kaffir-corn, Kaffircorn, milo,
shallu, shatter cane, shattercane, sorghum, Sudan Grass, sweet sorghum and wild
cane.
Synonyms for Sorghum bicolor ssp. Bicolor – Holcus bicolor, Holcus sorghum,
var. caffrorum, Sorghum vulgare var. durr, Sorghum vulgare var. roxburghii,
Sorghum is an important cereal crop which is grown globally for food and
feed purposes. It is most widely grown in the semi-arid tropics, where water
grow sorghum.
Sorghum cultivation is distributed throughout the world (Figs. 3.1 and 3.2). In
Asia, it is grown in China, India, Korea, Pakistan, Thailand and Yemen. Australia
and USA grow the crop too. Here one thing should be remembered that these
developed countries cultivate sorghum for animal feed, whereas, developing countries
in Asia and Africa cultivate it for use as human feed. In Southern and Eastern Africa,
and Zimbabwe. In West and Central Africa, the crop is grown in Benin, Burkina
Faso, Burundi, Cameroon, Central African Republic, Chad, Egypt, Gambia, Ghana,
Rwanda, Senegal, Sierra Leone, Sudan, Togo, Tunisia and Uganda. In Latin America,
Europe, it is grown in France, Italy, Spain, Albania and Romania. (Deb et al., 2004)
Figure 3.1. Distribution of sorghum area, 1999-2001. (Source: Deb et al., 2004)
4.2 4
5 19.9
5.4 United States Nigeria
India Mexico
Sudan Others
Argentina Ethiopia
17.9 15.5
Australia China
7 11.3
9.8
Nigeria, India, Mexico, Sudan, and Argentina with production in million metric tones
of 12.8 (19.9%), 10 (15.5%), 7.3 (11.3%), 6.3 (9.8%), 4.5 (7%) and 3.5 (5.4%),
Table 3.1 shows top fifteen countries in terms of sorghum cultivation area,
sorghum production and yield. India has the largest area under sorghum cultivation of
10.06 million ha (Table 3.1, Fig. 3.1). The second largest sorghum cultivating
country is Nigeria, followed by Sudan, USA and Niger. More than 90% of the
world’s sorghum area lies in the developing countries, mainly in Africa and Asia.
The area under sorghum in countries across the world has recorded a mixed trend over
the last three decades (Table 3.2). Area significantly declined in many major
sorghum-growing countries like Argentina, China, India and USA (Table 3.2).
However, sorghum-growing countries like Brazil, Mali, Mexico, Niger, Sudan and
Tanzania experienced notable increases in area at the end of the 20th century
compared to the early 1970s, and this increase has been consistent over the last three
decades. Though Nigeria experienced a decline in area under sorghum in the early
1980s, it increased in the early 1990s and, at the end of the 20th century, was 42%
higher than in the early 1970s. Niger is at 5th position in sorghum cultivation area, but
is at 18th position in production due to very poor yield i.e. 217 kg/ha. Countries like
Australia, Burkina and Egypt shows neither significant increase nor significant
Table 3.1. Area, production and yield of sorghum in different countries, 1999-2001.
Rank
Area Production Yield Area Production Yield
Country ('000 Ha) ('000 T) (kg/Ha) wise wise wise
India 10055.7 8231.7 818.6 1 2 72
Nigeria 6816 7647.3 1122 2 3 54
Sudan 4306.5 2441 566.8 3 7 89
USA 3352.7 13379.8 3990.7 4 1 12
Niger 2286.2 500.9 219.1 5 16 98
Mexico 1992.4 6092 3057.6 6 4 19
Burkina Faso 1301.9 1130.6 868.4 7 10 68
Ethiopia 1189.8 1377.6 1157.9 8 9 53
China 941.5 2947.7 3130.9 9 6 16
Chad 879.4 529.6 602.2 10 15 85
Mali 718.5 649.5 903.8 11 14 67
Argentina 690.5 3159.1 4575.3 12 5 8
Tanzania 638.9 653.6 1023 13 13 57
Australia 601.8 1810 3007.8 14 8 20
Brazil 452.8 742.9 1640.4 15 12 38
Egypt 162.7 945.1 5810.3 30 11 6
Colombia 66.2 212.2 3203 41 27 15
France 59.8 364.2 6094.3 42 21 5
Italy 33.5 216.6 6457.8 53 26 3
Uruguay 26.8 95 3539.1 54 39 13
Spain 8.5 44.1 5164.1 64 50 7
Yugoslavia, 2.2 9.1 4102.7 75 67 11
Israel 1.1 13.4 12663.5 79 60 1
Croatia 0.1 0.5 4115.4 89 86 10
Peru 0.1 0.3 3268.9 90 90 14
New Caledonia 0.1 0.1 1366.7 91 95 43
Algeria 0.1 0.4 6400 92 87 4
Kazakhstan 0.1 0.2 4392.6 93 91 9
Jordan Negligible 0.3 11710.5 98 89 2
Other countries 5273.4 5406.2 975.4 - - -
World 41859.3 58556.5 1398.9 - - -
Countries belonging to top 15 positions in Area, Production and Yield are mentioned.
Source of data: Deb et al. (2004)
Table 3.2. Trend in area, production and yield of sorghum in major sorghum
producing countries during 1971 – 2001. Source of data: Deb et al. (2004) and
www.fas.usda.gov
Country Average Area ('000 Ha)
1971-73 1981-83 1991-93 1999-2001
Argentina 2074 2411 721 690
Australia 629 671 460 602
Brazil 50 117 159 453
Burkina Faso 1038 1073 1417 1302
China 5072 2704 1368 941
Egypt 205 166 144 163
India 16335 16469 12574 10056
Mali 373 534 875 719
Mexico 1077 1520 1305 1992
Niger 531 1075 2315 2286
Nigeria 4792 2216 4535 6816
Sudan 1974 3682 5345 4307
Tanzania 338 500 642 639
USA 6077 5101 4160 3353
Average production ('000 Tones)
1971-73 1981-83 1991-93 1999-2001 2005-07 2007-08
Argentina 4140 7935 2626 3159 2800 3500
Australia 1181 1160 915 1810 1769 2700
Brazil 85 224 274 743 1698 1575
Burkina Faso 489 626 1280 1131 1679 1800
China 8680 7343 5151 2948 2324 2600
Egypt 846 623 740 945 900 900
India 7929 11578 10588 8232 7340 7300
Mali 284 452 716 649 n. a. n.a.
Mexico 2799 5286 4582 6092 5733 6300
Niger 200 345 424 501 683 800
Nigeria 3072 3589 4832 7647 10333 10000
Sudan 1527 2300 3323 2441 4058 4500
Tanzania 172 493 619 654 853 900
USA 21951 18614 16839 13380 9518 12827
Average yield (kg/Ha)
1971-73 1981-83 1991-93 1999-2001
Argentina 1953 3306 3635 4585
Australia 1912 1738 1931 3003
Brazil 2231 1953 1739 1639
Burkina Faso 471 583 903 867
China 1711 2716 3765 3124
Egypt 4120 3747 5149 5811
India 485 703 839 819
Mali 765 848 833 902
Mexico 2601 3485 3513 3056
Niger 370 322 185 217
Nigeria 637 1620 1064 1122
Sudan 775 619 616 568
Tanzania 509 1134 965 1027
USA 3625 3596 4001 3986
In terms of annual production during 1999-2001 (Table 3.1), USA tops the list
with 13.38 million T, followed by India (8.23 million T), Nigeria (7.65 million T),
Mexico (6.09 million T) and Argentina (3.16 million T). Here it should be noted that
India was second largest producer of sorghum in 1999-2001 with 8.23 million T. But
at present in the 2007-2008, Nigeria is the second largest producer of sorghum with
(Table 3.2). Though, USA is largest sorghum producing country, its annual
of that in 1971-72; whereas annual production sorghum in Nigeria is about three times
of that in 1971-72 (Table 3.2). In India annual sorghum production has been steadily
None of these major sorghum producing countries have highest global yields
e.g. India, Nigeria, Sudan, and Niger have yields of 819, 1120, 567 and 220 kg/ha,
respectively (Table 3.1). Whereas the largest sorghum producing country (USA) has
yield of 3990 kg/ha (Table 3.1). Highest sorghum yields during 1999-2001 (Table
3.1) were recorded by Israel (12664 kg/ha), followed by Jordan (11711 kg/ha), Italy
(6458 kg/ ha) and Algeria (6400 kg/ha). Maximum yield of 12664 of Israel is around
15 times the sorghum yield of India. Thus, while Asian and African countries like
India and Nigeria had the largest area devoted to sorghum cultivation, countries in
West Asia (like Israel and Jordan) and Europe (Italy and France) reaped the highest
yields. It should be noted that Israel and Jordan are not major sorghum-growing
countries. The average area under the crop during 1999-2001 was 1006 ha and
3.3.2. Trend in cultivation area, production and yield of sorghum in India and
In India sorghum is grown in the kharif (rainy season) and rabi (postrainy
season). The share of kharif is higher both in terms of area under cultivation and
production. The kharif sorghum crop accounts for 55% of the total area under
cultivation and 68% of the total production. It can be seen from the Figure 3.4 that
cultivation area under sorghum initially increased from 1950-51 to 1960-61. India
was having maximum land under sorghum cultivation during 1960 to 1970. But,
thereafter continuous decline in the sorghum cultivation area was observed. Reason
for this could be attributed to increase in the % coverage under irrigation (Fig. 3.4).
As the irrigation facility is improved, tendency of farmers to grow cash crops like
20
18
16
14
12
Sorghum cultivation area (million ha)
10
% coverage under irrigation
8
6
4
2
0
1
1950-51 11
1960-61 21
1970-71 31
1980-81 41
1990-91 51
2000-01
Year
Figure 3.4. Trend in sorghum cultivation area (million ha) and % coverage under
irrigation (Source of data: www.agricoop.nic.in)
Similarly, it can be seen from Fig. 3.5 that annual sorghum production
increased from 1950 to 1980. The decade 1980-90 has seen peak production of
sorghum in the country. But, after that annual sorghum production steadily decreased
from 11 to 7.3 million tones. This decrease is primarily due to decrease in the
sorghum cultivation area. It can be seen from the Fig. 3.6 that sorghum yield in the
14
12
Production (Million Tones)
10
0
1950-51
1 1960-61
11 1970-71
21 1980-81
31 1990-91
41 2000-01
51
Year
the early 1970s to 10 million ha in the late 1990s (Fig. 3.4). Sorghum production was
increasing until the early 1980s but declined after that Yield of sorghum has increased
over time (Figs. 3.5 and 3.6). Average sorghum yield in the late 1990s was 826 kg/ha
against 543 kg/ha in the early 1970s. Decrease in sorghum production was primarily
Table 3.3. Trend in Area, production and yield of sorghum in different states of India
1200
1000
600
400
200
0
1950-51
1 1960-61
11 1970-71
21 1980-81
31 1990-91
41 2000-01
51
Year
around 50% (Fig. 3.7). The trends in the area, production and yield of sorghum in
major sorghum-growing states in India are presented in Table 3.3. The area under
growing states (Andhra Pradesh, Gujarat, Madhya Pradesh, Rajasthan and Tamilnadu)
compared to the early 1970s, early 1980s and early 1990s. In fact, the niche of
(Table 3.3).
that of corn. The height of the plant varies from 0.5 m to 5 m. Sorghum produces one
or several tillers, which emerge initially from the base and later from the stem nodes.
The long, wide leaves grow from the stalk. The seed is small and round. A seed head
of about 25 cm to 36cm is seen on the top of the stalk of a mature sorghum plant. The
flower is a panicle, usually erect, but sometimes recurved to form a goose neck.
Grain sorghum has a large, erect stem terminating in a semi compact or compact head
or panicle.
Botanical parts of sorghum plant are shown in the Fig. 3.8. Sorghum plant
Root system
The roots of the sorghum plant can be divided into a primary and secondary
system. The primary roots are those which appear first from the germinating seed.
The primary roots provide the seedling with water and nutrients from the soil. Primary
roots have a limited growth and their functions are soon taken over by the secondary
roots. Secondary roots develop from nodes below the soil surface. The permanent root
system branches freely, both laterally and downwards into the soil. If no soil
m early in the life of the plant. The roots are finer and branch approximately twice as
Leaves
Sorghum leaves are typically green, glasslike and flat, and not as broad as
maize leaves. Sorghum plants have a leaf area smaller than that of maize. The leaf
blade is long, narrow and pointed. The leaf blades of young leaves are upright but the
blades tend to bend downwards as leaves mature. Stomata occur on both surfaces of
the leaf. A unique characteristic of sorghum leaves is the rows of motor cells along
the midrib on the upper surface of the leaf. These cells can roll up leaves rapidly
during moisture stress. Leaves are covered by a thin wax layer and develop opposite
one another on either side of the stem. Environmental conditions determine the
Stem
The stem of the plant is solid and dry, to succulent and sweet. Stalk is the main
stem of plant. Under favorable conditions more internodes develop, together with
leaves, producing a longer stem. The stem consists of internodes and nodes. A cross
section of the stem appears oval or round. The diameter of the stem varies between 5
and 30 mm. The internodes are covered by a thick waxy layer giving it a blue-white
color. The waxy layer reduces transpiration and increases the drought tolerance of the
plants. The root band of nodes below or just above the soil surface develops prop
roots. The growth bud develops lateral shoots. Sometimes the growth buds higher up
Inflorescence (panicle)
shape and color of the panicle varies between cultivars. Panicles are carried on a main
stem or peduncle with primary and secondary branches on which the florets are borne.
The peduncle is usually straight and its length varies from 75 to 500 mm. Each
panicle contains from 800 to 3000 kernels, which are usually partly enclosed by
glumes. The colour of the glumes may be black, red, brown or tan. The flowers of
sorghum open during the night or early morning. Those at the top of the panicle open
first and it takes approximately 6 to 9 days for the entire panicle to flower. Because of
the structure of the flower, mainly self-pollination takes place. A small percentage of
Seed
which are removed during threshing and/or harvesting. The shape of the seed is oval
to round and the colour may be red, white, yellow, brown or shades thereof. If only
the pericarp is coloured, the seed is usually yellow or red. Pigmentation in both the
Better drought tolerance of sorghum than most other grain crops (Plessis
3. The leaves fold up more efficiently during warm, dry conditions than that of
maize.
4. It has an effective transpiration ratio of 1:310, as the plant uses only 310 parts
of water to produce one part of dry matter, compared to a ratio of 1:400 for
maize.
5. The epidermis of the leaf is corky and covered with a waxy layer, which
6. The stomata close rapidly to limit water loss. During dry periods, sorghum has
the ability to remain in a virtually dormant stage and resume growth as soon as
conditions become favorable. Even though the main stem can die, side shoots
can develop and form seed when the water supply improves.
(Plessis, 2008):
Growth of sorghum plant is not very rapid up to the 8-inch height, while the
plant establishes a root system and starts to take up nutrients much more rapidly.
Shortly after reaching the 8-inch height, the growing point of the plant changes from
producing leaves to producing the head. For a medium-maturity sorghum, this occurs
the plant since the plant’s total number of leaves is determined at this stage. At this
point, when the plant has completed about 5 percent of its growth, it has taken up 10
to 15 percent of the nutrients it will use during the entire season. During the next 30 to
35 days, until flowering, the plant grows rapidly. It produces much of the leaf area,
which will be important during the grain-filling period. During this time, the head
develops and the stalk grows rapidly. First, the lower portion of the stalk grows,
pushing the head up into the flag leaf sheath into the boot stage. Later, the upper stalk
(the peduncle, which holds the head) grows rapidly, pushing the head out of the flag
leaf sheath, where flowering and pollination can occur. If something happens during
this stage of growth, the head may not fully emerge from the sheath, may not be fully
pollinated, or may cause problems at combining. This period (from when the head
first starts to form until lowering) is a time for rapid growth and rapid nutrient uptake.
At flowering, the plant will have produced about half of its total weight at maturity;
however, between 60 and 70 percent of the total nutrient uptake already will have
occurred. The final stage of growth, from flowering to physiological maturity, is the
important grain-filling period. During this time, total production of the plant is going
into the grain. Materials stored in the stalk are being moved into the grain, and the
plant is taking up approximately the final one-third of the nutrients. If drought occurs,
both uptake and growth may be limited. The end of this period occurs when the grain
percent, and it must dry considerably before it can be harvested and placed in
conventional storage. For high moisture grain or early harvest and artificial drying,
sorghum can be harvested at any time after physiological maturity. (Vanderlip, 1993,
1998)
inherit yield potential, are a deep well-drained fertile soil, a medium to good and
fairly stable rainfall pattern during the growing season, temperate to warm weather
1. Day length. Sorghum is a short-day plant, which means that the plant requires short
varieties initiate the reproductive stage, when the day lengths return to 12 hours.
The optimum photoperiod, which will induce flower formation, is between 10 and
The tropical varieties are usually more sensitive to photoperiod than the quick,
flower initiation.
2. Rain fall. Sorghum can grow in rain weather and also does well in semiarid areas. It
is more important in areas which are too dry for maize. In strongly seasonal
climates, when the rains stop, the plant often begins flowering. In natural flood
irrigation areas such as the decrue system regions of West Africa, the entire life
cycle is completed during the dry season. Sorghum is produced in areas with
3. Altitude. Sorghum can grow at altitudes from sea level to 3000 m. The high figure
for good germination and growth. Seeds germinate well at 10 to 35 °C. Optimum
temperatures. Breeding efforts have extended its range into cooler area. Base
flower primordia are delayed with increased day and night temperatures. Plants
with four to six mature leaves that are exposed to a cold treatment (temperatures
less than 18 °C) will form lateral shoots. However, for plants in or beyond the
eight-leaf stage, apical dominance will prevent the formation of lateral shoots.
Temperatures below freezing are detrimental to sorghum and may kill the plant.
below freezing point, but at 7 °C below zero, plants are killed. Plants older than 3
weeks are less tolerant to low temperatures and may die off at 0 °C.
mainly grown on low potential, shallow soils with high clay content, which
usually are not suitable for the production of maize. Sorghum usually grows
poorly on sandy soils, except where heavy textured subsoil is present. The
tolerable range of pH of soil varies from 5.0 to 8.5. Sorghum can better tolerate
short periods of water logging compared to maize. Soils with a clay percentage of
3.9): pericarp (outer layer), endosperm (storage tissue), and germ (embryo) with
percentage of total mass of 4.3-8.7, 8-11 and 81-86.5, respectively. Waniska and
Rooney (2000) have reviewed and given the grain morphology in detail.
individual mature caryopsis. The outer layer or pericarp originates from the ovary
wall and is comprised of three segments viz. epicarp, mesocarp, and endocarp. The
outermost layer (epicarp) is generally covered with a thin layer of wax. The epicarp is
two or three cell layers thick and consists of rectangular cells that often contain
pigmented material. The thickness of the mesocarp, the middle structure, varies from
the very thin cellular layer (containing small amount of starch granules) to 3 or 4
cellular layers containing a large amount of starch granules. Sorghum is the only food
grade crop that is reported to contain starch in this anatomical section. The innermost
endocarp is composed of cross cells and tube cells. The inner tube cells conduct
water during grain germination, whereas, the outer cross cells form a layer that
A stylar area is located on the tip of the caryopsis, opposite the germ. The
black layer, or hilum, is the colored placenta scar tissue that develops at the germ tip,
when the caryopsis reaches physiological maturity. Cell walls of sorghum pericarp,
esters of ferulic acid. The seed coat or testa is derived from the ovule integuments.
The thickness of the testa ranges from 8 µm to 40 µm and varies within individual
caryopses. The thickest area usually is observed below the style and the thinnest on
the side.
The endosperm tissue is triploid, resulting from the fusion of a male gamete
with two female polar cells. It is composed of the aleurone layer, peripheral, corneous
and floury areas. The aleurone is the outer cover and consists of a single layer of
rectangular cells adjacent to the testa or tube cells. The cells possess a thick cell wall,
large amounts of proteins (protein bodies, enzymes), ash (phytin bodies), and oil
containing more protein and smaller starch granules than the corneous area. Both the
peripheral and corneous areas appear translucent, or vitreous, and they affect
processing and nutrient digestibility. Waxy sorghums contain larger starch granules
The corneous and floury endosperm cells are composed of starch granules,
protein matrix, protein bodies, and cell walls rich in cellulose, β-glucans, and
hemicellulose. Starch granules and protein bodies are embedded in the continuous,
protein matrix in the peripheral and corneous areas. The protein bodies are largely
circular and 0.4–2.0 µm in diameter. High-lysine cultivars contain fewer and smaller
protein bodies than do regular sorghums, and thus contain significantly less alcohol
soluble kafirins. The starch granules are polygonal and often contain dents from the
protein bodies. Size of starch granules varies from 4 µm to 25 µm, the average being
15 µm. Granules present in the corneous endosperm are smaller and angular whereas
those in the floury endosperm are larger and spherical. The opaque, floury endosperm
(located near the center of the caryopsis) has a discontinuous protein phase, air voids,
and loosely packaged, round, starch granules. The presence of air voids diffracts
The germ is diploid due to the sexual union of one male and one female
gamete. It consists of two major parts: the embryonic axis and scutellum (Fig. 3.9).
The protein of the germ contains high levels of lysine and tryptophan that are
excellent in quality. The embryonic axis contains the new plant and is divided into a
radicle and plumule. Upon germination and development, the radicle forms primary
roots whereas the plumule forms leaves and stems. The scutellum is the single
cotyledon and contains reserve nutrients, i.e., moderate amounts of oil, protein,
enzymes, and minerals, and serves as the bridge or connection between the endosperm
and germ. The vitreous endosperm has a continuous protein matrix, which is attached
to the starch granules, protein bodies, and cell walls. The floury endosperm has a
discontinuous protein matrix with many small voids between the starch granules. Rate
of endosperm development is faster in sorghum with hard endosperm than that with
softer endosperm.
Figure 3.9: Diagrammatic representation of sorghum grain (Source: Chandrashekar and Mazhar, 1999)
Sorghum grain consists of starch, proteins, fibers, lipids etc. Physical (i.e.
Table 3.4. Composition of sorghum seed in % (Source: Waniska and Rooney, 2000)
(pentosans, cellulose, and hemicellulose). Starch is most abundant and others have
low content.
Starch
properties and uses similar to those of maize starch and the procedure for wet milling
of sorghum is similar to the one used for maize. Pigments in the sorghum pericarp
discolors the starch, yielding light pink color. Bleaching with NaClO2 or rinsing with
105 and 8 to 10 × 106 kD, respectively. Sorghum that has three recessive wx genes
produces caryopses that contain mostly amylopectin and are termed as waxy sorghum.
heterowaxy2 (Wxwxwx i.e. two wx genes) and waxy (wxwxwx) sorghum were 24,
23, 17.3 and 1, respectively. Starch isolated from corneous endosperm has lower
iodine binding capacity and higher gelatinization temperature than that isolated from
Protein
The second major component of sorghum and millet grains is protein. Protein
content and composition varies due to genotype, water availability, temperature, soil
fertility and environmental conditions during grain development. The protein content
of sorghum is usually 11-13% but sometimes higher values are reported (Dendy,
1995). Grain proteins are broadly classified into four fractions according to their
acid solutions).
al. (2006). In common with other cereals, the major storage proteins (kafirins) in the
prolamins. Belton et al 2006 have classified prolamins into four groups, called α, β, γ
-kafirins (based on their relationships to the zeins revealed by their amino acid
compositions and sequences, their molecular masses and their immunochemical cross-
reactions), and δ-kafirin (related to the d-zeins of maize, which has been identified
from the sequences of cloned DNAs but has not been characterised at the protein
level).
composition, and traditionally, the bread which cannot be baked from sorghum and
The crude fat content of sorghum is 3 percent, which is higher than that of
wheat and rice but lower than that of maize. The germ and aleurone layers are the
main contributors to the lipid fraction. The germ itself provides about 80 percent of
the total fat. As the kernel fat is mostly located in the germ, in sorghum mutants with
a large embryo fraction the fat content is higher (5.8 to 6.6 percent) than normal.
Neutral lipid fraction was 86.2 percent, glycolipid 3.1 percent, and phospholipid 10.7
percent in sorghum fat. Fatty acid was significantly higher in kafir, caudatum and
wild sorghum than in the bicolor, durra and guinea groups. On the other hand,
caudatum types had the lowest linoleic acid and bicolor, durra and guinea varieties
had more than wild and kafir sorghum. Oleic and linoleic acids were negatively
correlated with each other. The fatty acid composition of sorghum fat (linoleic acid 49
percent, oleic 31 percent, palmitic 14 percent, linolenic 2.7 percent, stearic 2.1
percent) was similar to that of corn fat but was more unsaturated.
categories viz. Human Food, and industrial use (includes Animal Feed, alcohol
industries etc.). It can be seen from Table 3.5 that in 1979-81, an estimated 39
percent of global production was used as human food and 54 percent for animal feed,
whereas in 1992–94, 42 percent of total utilization was for human food and 48 percent
for animal feed. The proportion of food utilization has gradually increased as a result
of a greater food use in Africa and the substitution of sorghum by other grains (mainly
each year during the 1992–94 period (Table 3.5), almost the entire amount in Africa
and Asia. It is a key staple cereal in many parts of the developing world, especially in
the drier and more marginal areas of the semi-arid tropics. Per capita annual food
considerably higher, than in urban centers. And within these rural areas, consumption
variety of forms that vary from region to region. In general, it is consumed as whole
grain or processed into flour, from which traditional meals are prepared. There are
• Flat bread, mostly unleavened and prepared from fermented or unfermented dough
Table 3.5. Sorghum utilization by type and region (Source: ICRISAT/FAO, 1996)
security crop is highest in Africa. For example, per capita annual consumption is 90–
100 kg in Burkina Faso and Sudan; sorghum provides over one-third of the total
calorie intake in these two countries. In Asia, sorghum continues to be a crucial food
security crop in some areas (e.g., rural Maharashtra in India, where per capita annual
However, both production and food utilization have fallen during the 1980s
consumers are shifting to wheat and rice which taste better and are easier and faster to
cook. This trend is accentuated by rapid urbanization and the growing availability of
a range of convenience foods based on wheat and rice. ICRISAT/FAO (1996) have
discussed sorghum economy in detail. Several previous reviews have addressed the
subject of traditional foods from sorghum in depth, for example Murty and Kumar
(1995), Rooney and Waniska (2000), and Rooney and Serna-Saldivar (2000).
The most common and simplest food prepared from sorghum and millets is
the whole seed before milling and sifting has been applied. The treatments procedures
are steeping, fermentation, malting, alkali or acid treatment, popping, roasting (dry or
wet), parboiling, and drying. One of the aims of seed treatment is to remove the
polyphenolic compounds from the seed. Others are to improve storage quality, or to
make many kinds of snacks and other popular foods. The traditional art of food
preparation is not standardized and routine procedures have been passed on to the
women through generations. The stiff porridge prepared from maize or cereal
mixture (maize, sorghum, pearl millet, finger millet, etc.) in Kenya, Uganda and
Tanzania is commonly called ugali. The most important fermented thin porridge that
steamed, granulated product called couscous, made from cereal flours (mostly wheat)
is highly popular. In West Africa, sorghum, pearl millet, maize, and fonio are used to
important food product in China. Sorghum is used for tortilla preparation either alone
Mexico. Roti is an unfermented dry roasted pancake made in India from wheat,
sorghum, pearl millet and maize flour. Sorghum grain is used in the production of
two types of beer: clear beer and opaque beer. The latter is a traditional, low-alcohol
African beer that contains fine suspended particles. Sorghum is traditionally a major
ingredient in home-brewed beer. Small quantities are used in the beer industries in
Mexico and USA. Sorghum is a good source of starch, cellulose, and glucose syrup.
Although domestication was primarily for food (and also for beer and sweet stems in
Africa, and for brooms in China), crop residues have been valued as animal fodder,
puffing, extrusion, micronizing) new sorghum and millet products of good quality and
Taylor et al. (2006) have reviewed role of sorghum in nutrition and health of
human, and novel food and non food uses of sorghum. In the developed countries,
nowadays there is a growing demand for gluten-free foods and beverages from the
people with coeliac disease and other intolerances to wheat, who cannot eat products
the mucosa of the small intestine caused by ingestion of certain wheat proteins and
related proteins in rye and barley. The gliadins and glutenins of wheat gluten have
been shown to contain protein sequences that are not tolerated by coeliacs. The
average worldwide prevalence has been estimated as high as 1: 266. Estimates place
the number of persons with coeliac disease in the USA at roughly 3 million. The
cornerstone treatment for coeliac disease is the total lifelong avoidance of gluten
ingestion. This means that wheat, rye, and barley have to be avoided, including durum
wheat, spelt wheat, kamut, einkorn, and triticale. Sorghum is often recommended as a
safe food for coeliac patients, because it is only distantly related to the Triticeae tribe
cereals wheat, rye and barley, being a member of the Panicoideae sub-family which
also includes maize and most millets. Sorghum therefore, provides a good basis for
gluten-free breads and other baked products like cakes and cookies (biscuits) and in
activity. Their use as nutraceuticals and in functional foods are reviewed in the paper
by Dykes and Rooney (2006). In addition to the potential health benefits of sorghum
phenolics, sorghum wax may also have unique health properties. Long-chain fatty
cholesterol. Policosanols (fatt alcohols in sorghum wax) are a promising resource for
the prevention and therapy of cardiovascular disease. Crude lipid extract from whole
kernel sorghum, which comprised a wide range of lipid substances including plant
Taylor et al. (2006) reviewed literature on novel and non traditional sorghum
foods like Gluten-free leavened breads (starch bread and additives, flour breads and
additives, effect of cultivar and Theoretical basis for sorghum functionality in gluten-
free bread making), Cakes and cookies, Tortillas, snack foods, parboiled sorghum,
and noodles.
The main industries using sorghum in India are the animal feed sector, alcohol
distilleries, and starch industries. Only rainy-season sorghum is used for industrial
purposes. Post rainy-sorghum is a highly valued food grain, and thus too expensive to
(human food use constitutes about 42 percent). In contrast to food utilization, which is
relatively stable, utilization for feed sorghum changes significantly in response to two
factors: rising incomes, which stimulate the consumption of livestock products, and
the price competitiveness of sorghum vis-à-vis other cereals, especially maize. While
income elasticities for livestock products (and hence the derived demand for feed) are
Demand for animal feed is concentrated in the developed countries and in middle-
income countries in Latin America and Asia, where demand for meat is high and the
contains higher carotene levels than sorghum, so meat from maize-fed animals tends
to be more yellow than meat from sorghum fed animals. In Japan for example,
ingredient in some compound feed rations (for poultry, pigs and some breeds of beef
there generally prefer poultry meat and egg yolks with a deeper yellow colour.
(ICRISAT/ FAO)
Table 3.6. Summary of industrial demand (‘000 t) for sorghum in India (Kleih et al.,
2007).
The limited inclusion of sorghum in poultry feed and its relatively low status
3.7.
Advantages Disadvantages
• Low cost • Lower energy content than maize
• Energy alternative to maize • Risk of aflatoxins (often associated with
• Easy availability blackened grain)
• Good pelletability • Risk of tannins
• Not always available
• Problems with grinding; mash becomes
powdery reducing feed intake by birds
• Low palatability and digestibility
• Varying quality; grain often infested with
weevils, fungi, etc.
• Sorghum lacks the carotenoid pigments
present in yellow maize, which are necessary
for egg yolk colour
• Feed including sorghum is more difficult to
sell
• Absence of standard varieties in the market
composition to maize, and, as with all cereals, its composition varies significantly due
to genetics and environment. Starch ranges of 60–77% and 64–78% have been
reported for sorghum and maize, respectively. As such, sorghum grain would be
appropriate for use in fermentation similar to the use of maize for the production of
bioethanol. Its use may be of particular benefit in countries where rainfall is limiting
and maize does not grow well. Taylor et al. (2006) concluded from the available data
that 1.2–2.3 million metric tons sorghum was used for ethanol production, 3.7–7.5%
of the grain used for ethanol production was sorghum, and 0.13–0.25 billion gallon
(0.49–0.95 billion litres) of ethanol originated from sorghum. (Taylor et al., 2006)
While discussing the potential for using sorghum in alcohol production, one
must keep in mind that in India, molasses (a byproduct of sugar manufacture using
sugarcane) constitutes the most important raw material in this industry. It is estimated
that about 95% of the alcohol manufactured in India is from molasses and the rest
comes from grains, and roots and tubers. Although, the quantity of sorghum grain
presently used by the alcohol sector in India is comparatively low (Table 3.6), it
seems to be the most "enthusiastic" user of the crop as an industrial raw material.
preference for varieties with a higher starch content and less protein. Distilleries had
no objection to using severely blackened grain as long as the starch content was
acceptable. In general, like most other industrial users, distilleries purchase rainy-
season sorghum through traders or brokers in main producing centers. Problem about
this system could be the misuse of the position by brokers to "control" the market. In
this context, contract farming may be an option providing better linkages between
material for alcohol production because of its lower market price. Maharashtra, the
main producer of rainy season sorghum, regularly faces the problem of finding
disadvantages of using sorghum in alcohol production are given in the Table 3.8.
Literature on use of sorghum for alcohol production is reviewed briefly in section 3.9.
review of early research on wet milling of sorghum can be found in Munck (1995).
the pericarp and/or glumes, which, stains the isolated starch. Due to this reason
sorghum is not much popular in starch producing industries. Sorghum is used for
starch production only when maize is not available. Literature on use of sorghum for
starch was produced from corn. Other raw materials being wheat, sweat potato,
of starch was produced in 1998, out of which 0.6 million t was produced from maize
Malting and brewing with sorghum to produce lager and stout, often referred
to as clear beer as opposed to traditional African opaque beer, has been conducted on
a large, commercial scale since the late 1980s, notably in Nigeria. Nigeria brews in
excess of 900 million litres of beer annually. Brewing with sorghum is now also
taking place in east Africa, southern Africa, and the USA. (Taylor et al., 2006)
In India, easily available barley malt is preferred as the principal raw material
for brewing. Sorghum is not currently used for beer production in India either as malt
There has been extensive research and development work and several
and brewing technology (Agu and Palmer, 1998; Hallgren, 1995; Owuama, 1997,
1999; Taylor and Dewar, 2000, 2001). Major outstanding problem areas (like use of
tannin sorghum in malting and brewing, starch gelatinization, the role of the
endosperm cell walls and beta-amylase activity in malt) in sorghum brewing that are
specific to characteristics of sorghum grain and sorghum malt are reviewed by Taylor
et al. (2006).
on the mechanisms of resistance to insect pests and fungal pathogens in sorghum and
millets. Teetes and Pendleton (2000), Frederiksen (2000) and Stahlman and Wicks
(2000) have discussed Insect pests of sorghum, Diseases and disease management in
sorghum
Sorghum has the distinct advantage (compared to other major cereals) of being
drought resistant and many subsistence farmers in these regions cultivate sorghum as
a staple food crop for consumption at home. Therefore sorghum acts as a principal
source of energy, protein, vitamins and minerals for millions of the poorest people
living in these regions. In this way, sorghum plays a crucial role in the world food
However there are few problem areas in the utilization of sorghum food as well as
Gelatinization of the starch is most important initial step, due to which starch
section 2.1.3.
°C and 71–81 °C for sorghums grown in southern Africa and in India, respectively
in the range of 75–80 °C (Palmer, 1992) and 60–80 °C (Wu et al., 2007).
quoted for barley starch of 51–60 °C. This factor becomes important in the malting
and brewing of sorghum. Due to this difference in the gelatinization temperature the
simultaneous gelatinisation and hydrolysis of starch that occurs when mashing barley
malt, is problematical with sorghum malt. Sorghum grain or sorghum malt is first
cooked to gelatinize the starch and then the starch is hydrolyzed using barley malt,
Taylor et al. (2006) have reviewed the literature on starch gelatinization with
context of its use in malting and brewing. In a study of 30 sorghum varieties, Dufour
et al. (1992) found a few with low gelatinisation temperatures, approaching that of
barley. More recently, Beta et al. (2000a–c) found that Barnard Red, a traditional
South African sorghum variety which was selected for its good malting and opaque
beer characteristics, had a low onset starch gelatinisation temperature of 59.4 °C and
gave high paste viscosity, even though the starch had a normal amylose-amylopectin
ratio. It is suggested that waxy sorghums gelatinize more rapidly, have a relatively
weak endosperm protein matrix and are more susceptible to hydrolysis by amylases
and proteases than normal endosperm sorghums and hence should be better for
brewing (Del Pozo-Insfran et al., 2004). Figueroa et al. (1995) investigated mashing
of 20 sorghum adjuncts of varying endosperm structure with barley malt. They found
that the waxy and heterowaxy types gave much shorter conversion times (time to
starch disappearance as indicated by iodine yellow colour) than normal types. They
attributed this to the lower starch gelatinisation temperatures, 69.6 °C for waxy type,
71.1 °C for the heterowaxy type and 71.1–73.3 °C for the normal types. Interestingly,
Ortega Villican and Serna-Saldivar (2004) found that when brewing with waxy
sorghum adjunct, highest beer ethanol content and lowest residual sugar content were
obtained if the adjunct was first heated at 80 °C, then pressure cooked.
gelatinisation (using the β-amylase and pullulanase method) was lower in hard
endosperm sorghum (with high kafirin protein content) than in soft endosperm types
(low kafirin content). The addition of the reducing agent 2-mercaptoethanol during
degree of starch gelatinization was more in harder high kafirin sorghum than that in
the softer low kafirin sorghum. They concluded that the endosperm protein matrix
which envelops the starch granules limits starch gelatinisation. They also reported
that after treatment of pepsin with uncooked sorghum flour (soft sorghum), flour
particles lose their structural integrity and only free starch granules with some
adhering protein bodies remain indicating that the integrity of sorghum particles is
maintained by protein. Their SEM work showed that flour particles from the hard
grains were most often covered with cell wall and when exposed the starch granules
cell walls appeared sloughed off the particle and far fewer protein bodies surrounding
the starch granule. Starch granules, protein bodies and cell wall appear to be linked
together by strands of protein. This gets supported by the fact that organized structure
in the both hard and soft grains was lost due to treatment with pepsin. They suggested
that the linking proteins that hold the particle together are strands of glutelin. Thus,
the protein matrix in the hard grain contains both protein bodies and matrix strands,
rural household food security (ICRISAT/ FAO). A nutritional constraint to the use of
because it would provide more amino acids for absorption on proteolysis. In vivo
studies using pepsin and in vitro studies show that the proteins of wet cooked
sorghum are significantly less digestible than the proteins of other similarly cooked
cereals like wheat and maize. (Duodu et al., 2003) In an excellent review on factors
affecting sorghum protein digestibility, Duodu et al. (2003) divided these factors into
Exogenous factors: These refer to factors that arise out of the interaction of sorghum
Endogenous factors: These refer to factors that arise out of changes within the
sorghum proteins themselves and do not involve interaction of the proteins with non-
protein components.
Duodu et al. (2003) have discussed all these factors in detail in the review.
Since protein digestibility is not topic of interest in the present work, this will not be
value of sorghum as well as production of ethanol from sorghum. Sorghum flour with
high starch digestibility will have good food value as well as will be a good candidate
of the waxy gene in sorghum. Due to pronase treatment of the sorghum flour, starch
digestibility increases in all four types of sorghum and slightly lesser than that of
isolated sorghum starch. The pronase treatment significantly increased the rate of
starch hydrolysis because it hydrolyzed the protein matrix (which surrounds starch
granules) and increased the surface area of the starch in contact with
Table 3.9. Effect of the waxy gene of kafir and pronase treatment on in vitro starch
hydrolysis (mg glucose/g starch) using amyloglucosidase. Lichtenwalner et al. (1978)
Genotype Amylose Ground grain Pronase pretreated Purified
content % ground grain starch
Normal (WxWxWx) 24 434 540 550
Heterowaxy (WxWxwx) 23.1 467 565 598
Heterowaxy (Wxwxwx) 17.3 545 703 745
Waxy (wxwxwx) 1 741 966 1035
digestibility with special emphasis on sorghum and corn. The digestibility of starch is
affected by the composition and physical form of the starch, protein ~ starch
and the physical form of the feed or food material. Starch exists inside the endosperm
3.10). Starch digestibility is affected by the plant species, the extent of starch-protein
interaction, physical form of the granule, inhibitors such as tannins, and the type of
starch. Among the cereals, sorghum generally has the lowest raw starch digestibility
cooked forms, while waxy cereal starches are among the most digestible of all
content i.e. directly proportional to waxyness of the sorghum. They have also
Zhang and Hamaker (1998) found that digestibility (using porcine pancreatic
α-amylase) of cooked isolated sorghum starches was markedly higher than starch
from cooked sorghum flours. They observed that pepsin pretreatment before cooking
increase in starch digestibility was seen when pepsin treatment was performed after
cooking. These authors also reported that after cooking with reducing agent, 100 mM
Elkhalifa et al. (1999) also observed that in vitro starch digestibility (IVSD) of the
ascorbic acid; however, at high levels of cysteine or sodium metabisulphite the IVSD
was low. Ezeogu et al. (2005) reported that starch digestibility (using pancreatic
The fact that reducing agents improved sorghum starch digestibility suggests
matrix is responsible for the reduced gelatinisation in sorghum. This is the same
mechanism that has been implicated in the reduced protein digestibility of cooked
sorghum (Duodu et al., 2003). This interpretation is supported by the work of Ezeogu
et al. (2005) who found evidence of disulphide bond cross-linked prolamin proteins in
molecular weight polymers (M. Wt > 100k). Also formation of web-like or sheet-like
mashing or heat moisture treatment is reported by Hamaker and Bugusu (2003) and
Wu et al. (2007). Ezeogu et al. (2005) also observed that pressure cooking the flours
improved starch digestibility of vitreous (hard) and floury (soft) endosperm maize and
sorghum flours and markedly so for sorghum vitreous endosperm flour. They
suggested that pressure cooking could have physically disrupted the protein matrix.
and weather damage. However, tannins inactivate extracted malt amylases (Beta et
al., 2000a–c; Daiber, 1975), significantly reducing starch breakdown and sugar
production during brewing (Daiber, 1975). Tannins are well known for their adverse
(including hydrolytic enzymes), metal ions, and polysaccharides (Wu et al., 2007).
Wu et al. (2007) found that the liquefaction of starch in tannin sorghums was more
difficult and slower than in normal and waxy sorghums. Wu et al. (2007) also
confirmed that tannin contents had a strong adverse effect on conversion efficiency of
sorghum to ethanol. Taylor et al. (2006) have reviewed the use of tannin sorghum in
substrate is no longer cheap and good quality raw material due to its decontrol by
improve the alcohol quality. Hence, since 1990s industries are diverting from use of
government is promoting grain based alcohol. In this section, literature review is only
limited to use of sorghum for ethanol production and not the use other starchy
from sorghum.
fermentation (SSF) system for producing ethanol from damaged sorghum (50% sound
and 50% damage grains). They have reported ethanol yields of 91.5% and 78.6% of
the theoretical ethanol yield with use of VSJ1 strain and standard strain MTCC 170
for damaged sorghum. These authors later utilized a similar SSF method to compare
ethanol production from damaged (50% sound and 50% damaged grains) and high
quality sorghum (Suresh et al., 1999b). It was must be noted that the latter method
involved no cooking step. Raw flour starch was saccharified by Bacillus subtilis
kernels that were broken, cracked, attacked by insects, dirty or discolored. The high-
quality sorghum flour was obtained locally. They found that using a level of 25 %
(w/v) substrate yielded 3.5% (v/v) ethanol from the damaged grain sample. For
comparison, the high-quality sorghum flour yielded 5.0% (v/v) ethanol. The values of
optimum pH and temperature were reported to be 5.8 and 35 °C respectively for SSF
process for damaged sorghum. The damaged grain sample was reported to be ten
times cheaper than high-quality grain and thus may be an economical way to produce
ethanol even though yields were lower. The authors further emphasized that
two different locations were used to produce ethanol. The process included following
concentrations varied relatively narrowly (about 5%) across the 16 samples. Genotype
physical properties of the sorghum used in this study, which in turn significantly
affected ethanol yields. The correlation between ethanol concentration and starch
content was positive, as expected, but low (r = 0.35, P > 0.05), while a much more
distinct negative correlation between ethanol concentration and protein content was
found (r = -0.84, P < 0.001). Since protein and starch content are inversely
proportional, it is not surprising that opposite correlations for these two measures to
ethanol production would be found. But, protein content does not have a significant
that protein had a much stronger relationship to ethanol yield than did starch. More
research is needed to determine exactly what components in the grain, and their
interactions, are responsible for ethanol yields in sorghum. It is possible that during
Hamaker and Bugusu (2003). Under these conditions, some of the starch might be
trapped in these protein webs, and its full gelatinisation and degradation by amylases
might be hampered. Evidence for this is provided by the work of Zhan et al. (2006)
gelatinize the starch. In SCFX supercritical carbon dioxide is used in place of water as
the blowing agent. Using SCFX increased ethanol yields by around 5% compared to
starch was accounted to the release of starch from the protein matrix due to SCFX and
review on ethanol production is summarized in Table 3.9 with reaction conditions and
remarks as parameters.
processing the grain can be used to improve ethanol yields and process efficiency.
the samples up to 12% and increased starch content by 5–16%. Fiber content was
decreased by 49–89%. These changes allowed for a higher starch loading for ethanol
11% for 10% decorticated sorghum and 8–18% for 20% decorticated sorghum. Using
decorticated grain also increased the protein content of the distillers dried grains with
solubles (DDGS) by 11–39% and lowered their fiber content accordingly. Using
decorticated sorghum may be beneficial for ethanol plants as ethanol yield increases
and animal feed quality of the DDGS is improved. The bran removed before
Suresh et 200 mL slurry autoclaved at 121 °C for 30 min and a 3% Damaged sorghum (50% sound and 50% damaged grains
al., 1999a inoculum of A. niger (NCIM 1248) and 7% inoculum of yeast used in this work)
was added to it. 2-12% starch concn, 150 rpm, 30 °C, 5 days
Suresh et 100 ml slurry and 0.3% peptone 0.1% KH2PO4 and 0.1% Optimized conditions being pH 5.8, 35 °C and 25% w/v
al., 1999b (NH4)2SO4; pH 5.8. The crude amylase broth (10 ml) of B. slurry concn. High quality and damaged sorghum produced
subtilis VB2 and 6% S. cerevisiae VSJ4 suspension was added 5% v/v and 3.5% v/v ethanol production with no cooking
to slurry and incubated at 35 °C and 200 rpm for 4 days. step.
Zhan et 100 mL slurry, pH 5.8. Liquefaction: 95 °C for 45 min, 80 °C 16 different varieties of sorghum. Positive correlation
al., 2003 for 30 min (0.01 mL amylase/g of starch in both steps of between ethanol concn and starch content and negative
liquefaction). Saccharification: 60 °C (150 U/g of starch) for correlation between ethanol concn and protein content.
Zhan et 30 min. 50 rpm for all steps. Fermentation: 20 g ground Extrusion could break disulphide protein bonds and disrupt
al., 2006 sorghum, 0.3 g peptone, 0.1 g KH2PO4, and 0.1 g (NH4)2SO4 the protein matrix, gelatinize starch, and make more starch
at pH 3.8. Medium was inoculated with 6% yeast suspension available for enzyme hydrolysis, and consequently,
(1×106 cells/mL) and incubated 200 rpm for 72 h at 30 °C. increase ethanol yield and fermentation efficiency.
Wu et al., 30 g flour was mixed with 100 mL distilled water. 10 µL Ethanol yields varied by 22% and conversion efficiencies
2007 liquozyme was added and slurry was digested for 45 min at 95 by 9% among 70 sorghum samples. Positive effect of
°C. Slurry cooled to 80 °C and second dosage of 10 µL starch content on fermentation efficiency and negative
liquozyme was added and liquefaction was continued for effect of protein, tannin, crude fiber, and ash content on
additional 30 min at 120 rpm. Saccharification: 100 µL fermentation efficiency was observed.
Zhao et Spirizyme, 120 rpm, 60 °C, 30 min. pH adjusted to 4.2 and During mashing cross-linked microstructure, which could
al., 2008 inoculated with 5 mL of 48 h yeast pre-culture. hold starch granules or polysaccharides inside or retard or
prevent the access of enzymes to starch get formed. Severe
cross-linking in mashed sample was most likely because of
a combination of heat-induced cross linking and cross-
linking because of protein-tannin interactions. Tight and
open microstructures were observed with low conversion
sorghum and high conversion sorghum, respectively.
elite hybrids of sorghum using dry grind process and identified factors impacting
ethanol production. They have observed variation in the ethanol yield by 22% and
protein, tannin, crude fiber, and ash content on fermentation efficiency. Protein
digestibility of waxy, normal sorghum (60-68 %) were higher that that for high tannin
sorghum (28%); Higher fermentation efficiencies were observed for waxy, normal
sorghum (89-90%) than those of high tannin sorghum (85%). After mashing sorghum
protein were appeared to produce highly extended, strong web like micro structures
(in accordance with results of Hamaker et al., 2003) into which small starch granules
were tightly trapped. These changes related protein structure during mashing could
endothermic peak (60-80 °C), whereas that of normal sorghum starch showed
higher conversion efficiencies to ethanol than normal sorghum; this mainly happens
amylose-lipid complex.
digestibility, solubility, and microstructure during mashing and to relate those changes
tannin and rest were tannin free. They have observed that protein solubility and in
sorghum proteins to form highly extended, strong web like microstructures during
Formation of web like structure was earlier reported by Hamaker and Bugusu (2003)
and Wu et al. (2007). They reported that the cultivar with the lowest conversion
starch, and severe cross-linking in this sample was most likely because of a
sorghum, Solubility and the SE-HPLC area of proteins extracted from mashed
samples were highly correlated with ethanol fermentation. Since protein cross linking
plays a significant role in the fermentation, it was expected that γ-kafirin (%) would
yield nor conversion efficiency significantly. They concluded that protein cross-
linking does play a role in the production of ethanol from sorghum, albeit through
review of early research on wet milling of sorghum can be found in Munck (1995).
Recently, Taylor et al. (2006) have shortly reviewed literature on starch production
pigments are present in the pericarp and/or glumes, they stain the starch (Beta et al.,
2000a–c).
In recent years several new developments in sorghum wet milling have been
bleaching the starch and found a mixture of sodium hypochlorite and potassium
milling Yang and Seib (1995) developed an abbreviated wet-milling process for
sorghum that required only 1.2 parts fresh water per part of grain and that produced
no waste water. The products of this abbreviated process were isolated starch and a
Buffo et al. (1997) investigated the impact of sulphur dioxide and lactic acid
steeping on the wet-milling properties of sorghum and reported that the amount of
lactic acid used during steeping had the most impacted wet-milling quality
characteristics such as starch yield and recovery. These authors also investigated the
surprisingly, they found that grain factors related to the endosperm protein matrix and
its breakdown and subsequent release of starch granules as important factors in wet-
found that treatment of steeped sorghum and maize with protease increased starch
Wang et al. (2000) optimized the steeping process for wet-milling sorghum
and reported the optimum steeping process to utilize 0.2% sulphur dioxide, 0.5%
of sorghum produced starch with an lightness value (Black and white samples will
chroma meter and normally denoted as L) of 92.7, starch yield of 60.2% (db), and
protein in starch of only 0.49% (db). Beta et al. (2000a–c) found that both polyphenol
Using sorghum grits as the starting material for wet-milling rather than whole
sorghum produced lower yields, but the isolated starch was higher in quality.
Sorghum starch matching the quality of a commercial corn starch was successfully
Xie and Seib (2002) developed a limited wet-milling procedure for sorghum
that involved grinding sorghum with in the presence of 0.3% sodium bisulphite
solution. This procedure produced starch with an L value of 93.7 and a starch
recovery of 78%. Large grain sorghum hybrids wet-milled by this ‘‘no steep’’
procedure were reported to produce high-quality starches with L values from 93.1 to
93.7 (compared to 95.2 for a commercial corn starch). Some of the large grain hybrids
tested showed promise for easy recovery of the germ by flotation in a similar fashion
Park et al. (2006) reported the use of ultrasound to rapidly purify starch from
sorghum. This procedure resulted in very high-purity starch with only 0.06% residual
well as breeding sorghum hybrids with improved wet milling characteristics should be
of benefit for the industrial use of sorghum starch, either directly for the production of
bioethanol or other industrial uses such as the production of activated carbon (Diao et
al., 2002) or isolation of phytosterols from wet-milled fractions (Singh et al., 2003).
polymer composed of glucose units linked by α(1→4) glucosidic bonds and α(1→6)
roughly linear molecule containing ~ 99% α(1→4) and ~ 1% α(1→6) bonds with
and ~ 5% α(1→6) bonds. Linear chains of 12-120 glucose units (linked by α(1→4)
Starch has become a very important biopolymer and is used in many industries
transform starch to useful and value added biochemicals. Sweetener and fermentation
industries are two of the main consumers of the starch. Nutritive sweeteners are
mainly starch hydrolysis products namely maltodextrins, high maltose syrup, maltose,
glucose syrup, dextrose, which are used in food and pharmaceutical industry.
Complete starch hydrolysis results into glucose as a final product. The first
sugar calculated as the dextrose on dry weight basis. Theoretical DE is defined as the
equivalent) produced after complete hydrolysis. Pure starch has DE of zero and
equivalent (DE) less than 20, which consist of maltose, malto-oligosaccharides and
partial hydrolysis of natural starch, their saccharide composition varies with nature
used for hydrolysis. Maltodextrins with the same average DE value can have different
physical and biological functionality, and there are different parameters (like type and
Beverage industries, papermaking industry etc. Maltodextrins are also used as carrier
or bulk agents, texture provider, spray-drying aid, flavor encapsulating aid, fat
replacer, tablet expicient, film former, freeze-control agent, sport beverage, to prevent
crystallization and to supply nutritional value i.e. parenteral and enteral nutrition
products. (Alexander, 1992; Marchal et al., 1999) All these aspects of Maltodextrin
Though partial hydrolysis of starch has traditionally been carried out using
acids, acid hydrolysis is being replaced by enzymatic hydrolysis for the production of
tailor-made maltodextrins (Schenck and Hebeda, 1992; Marchal et al., 1999). The
most widely used enzymes for production of maltodextrins using partial hydrolysis of
chapter 2.
Soluble enzymes are widely used for various industrial applications. However,
these enzymes can be used only once since they can’t be separated from the reaction
mixture. Further, most often the presence of enzyme in the final product is
undesirable. In such cases soluble enzymes must be deactivated or killed, and many
times is to be separated from the product at the end of the process. This is generally
carried out by pH adjustment, using either an acid or a base, which eventually leads to
which can be separated easily from the reaction mixture after the reaction is over.
or the other kind of solid surface is called immobilization. Immobilized enzymes are
region of space with retention of their catalytic activities, and which can be used
repeatedly and continuously” (Chibata, 1978). This can be achieved by using a solid
support on to which the soluble enzyme is ‘confined’ and thus is separated from the
Activity of an enzyme stems from its tertiary structure and surface topology.
Enzyme as a protein has specific structure and any changes in it can affect the
bioactivity of the enzyme. Thus its interaction with any surface or molecule can play
an important part in determining the enzyme activity. By the same rule, surface
important role. Ideal solid support should have the following properties:
a) Non-porous matrices have low surface area therefore immobilization yields are
low. In order to increase the enzyme loading fine particles could be used. However,
it is difficult to separate these particles from the reaction mixture. Small particles
also lead to high pressure drops when used in packed bed modes.
b) Porous supports on the other hand have large surface area for enzyme coupling.
However, the porous support must allow easy accessibility in the pores to substrate
supports. These techniques can be divided into three major classes (Kennedy and
Cabral, 1987):
and maltohexaose using soluble and immobilized alpha amylase. They found that the
hydrolysis product profiles, obtained using soluble and immobilized enzyme differed
significantly. When soluble enzyme was used for starch hydrolysis, considerable
the soluble enzyme. Soluble enzyme could partially hydrolyze maltopentaose into
maltotriose, maltose and glucose, whereas immobilized alpha amylase could not
from maltohexaose and maltopentaose was obtained as the major hydrolysis product.
to free enzyme can be attributed to diffusion limitation, which increases with the
hydrolysis (Tarhan, 1989). The other reason is that, immobilization alters three
dimensional structure of the enzyme; which causes changes in its affinity towards the
substrates, thus increasing its product specificity (Ivanova and Dobreva, 1994).
There are several attempts (Kvesitadze and Dvali, 1982; Tarhan, 1989; Roig et
al., 1993; Ivanova and Dobreva, 1994; Tumturk et al., 2000; Lali et al., 2002;
hydrolyze starch. Small size pores (< 0.1 µm) in typical matrix supports offer high
diffusional resistances to the large starch molecules and limits their accessibility to
active immobilized enzyme sites inside the pores leading to low reaction rates.
use of matrix support with large pores diameters (70-80 nm for Kvesitadze and Dvali,
1982; 7-550 nm for Siso et al., 1990; ∼5000 nm for Lali et al., 2002 and Karandikar,
(in the range of 5.1 and 7.6) and significant influence of temperature on saccharide
profile of starch hydrolysate produced using free BLA (Maxamyl). But there is no
literature available on the effect of pH, temperature and initial starch concn on the
amylase.
4.1.3. Objectives
Hence, in the present work, the effect of different parameters like pH,
temperature, initial starch concn and ratio of concn of enzyme units to initial starch
BLA has been also studied in a batch mode. Thermostability and reusability of
immobilized BLA was also studied. Also, a semiempirical model has been used for a
Also, in this work the possibility of production of glucose using gelatinized sorghum
4.2. Experimental
4.2.1. Materials
chromatography LiChrosolv and other chemicals were purchased from Merck Ltd
(India). Bacillus licheniformis α-amylase (BLA) (EC number 3.2.1.1) was gifted by
indigenously according to patent (Lali and Manudhane, 2003) and made available for
the present work. Properties of the CELBEADS are given in the Table 4.1.
Properties Description
Spherecity 0.7-0.9
4.2.2. Methods
The protein concn of the free enzyme was determined using modified Folin
Lowry method (Lowry et al., 1951) using BSA (0-0.6 mg/mL) as a standard. The
reducing sugar concn was measured using DNSA method (Miller, 1959) using
dextrose (0-1 mg/mL) as a standard. Details of modified Folin Lowry method and
following,
concn of reducing sugar (glucose equiv) after complete hydrolysis using amyloglucosidase
n=
concn of reducing sugar (glucose equiv) in starch hydrolysate
By using above formula, DE of dextrose, maltose and starch can be calculated to be
100, 53 and 0 respectively. The values of DE reported later in the text are those
20 cm × 10 cm TLC sheets (silica gel 60, Merck Ltd, India). The samples were
applied to the TLC sheet (prewashed with MeOH) using applicator AS 30 (DESAGA,
Best resolution was obtained by triple development. Mobile phase MeCN: 0.02 M
Na2HPO4 of composition 70:30 (v/v) was used for 1st and 2nd development; whereas
mobile phase MeCN: 0.01 M Na2HPO4 of composition 80:20 (v/v) was used for 3rd
development. TLC sheet, after triple development, was stained by dipping for 4
diphenylamine, 0.8 mL aniline and 6 mL 85% H3PO4) and then keeping at 120 °C for
Heidelberg, Germany) and computer. Sample densitogram is shown in Fig. 4.1 (A)
and TLC image is shown in the Fig. 4.1. (B) Samples of starch hydrolysate were
quantified by use of external standards of Glucose (G1) and Maltose (G2) from Merck
Maltohexaose (G6) and Maltoheptaose (G7) from Sigma (St Louis, MO, USA).
starch hydrolysate reported later in the text are those, which were determined by using
standard curve of G7. Details of HPTLC method are given in the appendix A.
B
A
G5 G3
G6
G4 G2
G7
G8 G1
G10 G9
15 40 50 70 80 mm
Figure 4.1.
A. Densitogram of starch hydrolysate of DE 10, produced using immobilized BLA at
55 °C, pH 5.2 and [S]0 = 90 mg/mL. G1 is Glucose and G2-G10 are
maltooligosaccharides with degree of polymerization 2-10 respectively.
B. TLC image showing chromatographic separation of bands corresponding to
glucose and malto-oligosaccharides
(ECH), while ethylenediamine (EDA) was used as a spacer arm to prevent the
possible steric hindrances between the immobilized enzyme and the substrate.
Activation and coupling procedures (Lali et al., 2002; Hermanson et al., 1992) were
CELBEADS (10 mL) were washed with 200 mL of distilled water and suction dried
to moist cake on a sintered glass funnel. The wet matrix was then added to a conical
flask containing 2 M NaOH (34.5 mL), NaBH4 (0.1275 g) and ECH (3.75 mL). To
this flask another 2 M NaOH (34.5 mL) and ECH (17 mL) were added in small
portions over a period of 2 h under mild stirring. The flask mixture was shaken on an
orbital shaker overnight at room temperature. The matrix was then filtered on a
sintered glass funnel and washed extensively with 200 mL each of 0.1 M HOAc, 0.2
M NaHCO3 and distilled water sequentially. The washed and suction dried epoxy-
of 0.2 M NaHCO3 (22.5 mL) and EDA (15 mL). The mixture was shaken at 50 °C on
and washed successively with 200 mL each of 0.1 M HOAc, 0.2 M NaHCO3 and the
distilled water.
CELBEADS EP EP-CELBEADS
EP-EDA-GA-CELBEADS
fold diluted solution (15 mg protein) of BLA in 0.1 M phosphate buffer (pH 7.5) and
kept under shaking condition overnight at 5 °C. After immobilization, NaBH4 (0.08
g) was added and kept under shaking condition for further 30 min. The enzyme
immobilized matrix was filtered on a sintered glass funnel and then washed with 200
mL each of phosphate buffer, 1 M NaCl solution and distilled water sequentially. The
protein concn and free enzyme units in the supernatants and washes were determined.
Amount of protein and free enzyme units immobilized on the matrix were calculated
by material balance.
Soluble starch was added to 0.1 M acetate buffer (pH 5.6) to have 9 mg/mL
concn and then gelatinized in a stoppered conical flask by heating in boiling water for
acetate buffer (0.1 M, pH 5.6) and 0.1 mL of 10000 fold diluted free BLA was
water bath for 20 min. The reaction was stopped by adding 1 mL of DNSA reagent.
The variation in the concn of reducing sugar was measured by DNSA method using
dextrose as a standard. One free enzyme unit (FEU) was defined as that required to
liberate one micromole of reducing sugar (glucose equiv) per min under conditions of
assay. Activity of commercial preparation of BLA (FEU/mL) was calculated using the
following formula,
End point assay method has been employed to calculate enzyme activity of
mg/mL (0.1 M acetate buffer, pH 5.2) was incubated with 1 mL immobilized BLA at
55 °C for one h and samples were withdrawn initially and finally. Reducing sugar
concn (glucose equiv) of the samples was measured and the immobilized enzyme
units per mL of CELBEADS were determined. One immobilized enzyme unit (IEU)
was defined as that required to liberate one micromole of reducing sugar (glucose
equiv) per min under the conditions of assay. Enzyme units per ml of CELBEADS
BLA).
(pH 5.2 for immobilized BLA and pH 5.6 for free BLA) of different initial starch
concentrations [S]0 varying in the range of 9-45 mg/mL with suitable enzyme concn
(for immobilized BLA, [IEU] = 0.5 and for free BLA, [FEU] = 0.66 i.e. [IEU equiv] =
0.738) were incubated separately at 55 °C for 2 h. The reaction was carried out in a
shaker at 180 rpm. Samples of starch hydrolysate were withdrawn at regular time
intervals of 0.5 h and analyzed for reducing sugar concn. The initial reaction rate (V)
was calculated from the slope of linear part of reducing sugar concn vs. time plot at
all initial starch concentrations for both immobilized and free BLA. Kinetic constants
(Km and Vmax) were determined for free and immobilized BLA from Eadie-Hofstee
plot of V vs. V/[S]0 with -Km as slope and Vmax as the y-intercept. Since Vmax is not a
fundamental property of the enzyme and is dependent on the enzyme concn, it was
converted to turnover number, kcat (i.e. Vmax/[IEU] for immobilized BLA and
Suspension of soluble starch at desired concn (mg/mL) was prepared with 0.1
M acetate buffer (desired pH) and then gelatinized in a stoppered conical flask by
heating in boiling water for 6 min. Immobilized BLA was added to the freshly
prepared gelatinized starch solution to have a desired [IEU]/[S]0 and kept at desired
temperature for 8 h in the shaker at 180 rpm. Rotational speed of 180 rpm was
selected by using the following criterion: 1. All beads should be always in the
suspended form throughout the batch. 2. Selected speed should be in such a range; in
observed to be beyond the speed of 150 rpm). At regular time intervals (0.5 h up to
reaction time of 3 h and then every 1 h till the end of batch hydrolysis), samples were
then analyzed for reducing sugar concn and immediately frozen. Samples were
varying pH, temperature, [S]0 and [IEU] in the range of 4.4 to 7, 37 to 70 °C, 18 to
solution (pH 5.2) without the presence of soluble starch and kept under shaking
conditions (180 rpm) at 55 °C for 24 h. Similarly free BLA was diluted 10000 fold
with 0.1 M acetate buffer solution (pH 5.6) and kept under shaking conditions (180
and free BLA were taken at various time intervals for the measurement of its activity.
Procedure to carry out reaction for reusability study was same as that
immobilized BLA. Then immobilized BLA was first washed thoroughly with
distilled water and acetate buffer solution sequentially, and then kept under shaking
condition with acetate buffer (amount same as the reaction mixture, pH 5.2) at 55 °C
for 30 min to remove any substrate or product molecules, which could have been
trapped inside the pores. Then acetate buffer was again separated from immobilized
BLA. Fresh acetate buffer solution was added to the immobilized BLA and kept at 6
°C, and the same was used for next batch hydrolysis under same conditions on the
next day. For each batch, samples were collected at regular time intervals and
analyzed for reducing sugar concentration to see the progress of the reaction.
equipped at the two ends with adjustable 14 cm long flow adapters. The adapters were
inserted to touch the matrix bed from both the sides. The lower adapter was connected
to a peristaltic pump that pumped the substrate solution from the mixing tank through
the column at a desired flow rate and re-circulated it back to the mixing tank. 40 mL
starch suspension was prepared in 0.1 M acetate buffer (pH 5.2). Temperature of the
starch solution was maintained at 55°C by circulating hot water through jacket around
it (Fig. 4.3). The starch solution was continuously stirred and kept in the form of
uniform solution using a magnetic stirrer. Temperature of the column was also
maintained at 55°C by circulating hot water through the jacket. Samples were drawn
at regular time intervals from the mixing tank to monitor the progress of starch
E
D
C
A
B
Expanded bed experiments were carried out in much the same way except that
the upper adapter was placed well above the settled matrix instead of touching the
matrix. This provided free board for the settled immobilized CELBEADS to expand
when starch solution was passed up through the column at a flow rate of 1 mL/min. It
was seen that this flow rate expanded the bed about 1.4 times from the settled bed
height. Rest of the experiment was same as that for packed bed experiment. Samples
were drawn at regular time intervals and analyzed for reducing sugar concentration.
Reusability of immobilized BLA was also studied in the batch mode with and
without intermittent washing step, and in the packed and expanded mode without
starch solution was injected in the distilled water flowing through the column at
desired flow rate (1, 2 or 3 mL/min). Eluting fractions were collected at every 15 sec
in test tubes and analyzed for starch concentration using the starch-iodine method
collected (C) was plotted against time (t). The value of mean residence time tm was
earlier. It was observed that by material balance, 90% of the loaded FEUs (i.e. 300
FEUs per mL of CELBEADS) and 56% of the proteins loaded (i.e. 0.83 mg per mL of
reasons;
the active site could be oriented towards the support surface i.e. decreasing the
2. A reactive site in the enzyme molecule may be involved in the binding to the
matrix.
and optimum pH slightly decreased from 5.6 to 5.2 (Fig. 4.4). Shift in the optimum
pH towards acidic side (Kvesitadze and Dvali, 1982; Tumturk et al., 2000; Ivanova et
al., 1998) might be because of the difference in the hydronium ion concn in the bulk
(Tumturk et al., 2000). Fig. 4.5 shows that % relative activity above 55 °C was
marginally better and approximately the same over the temperature range of 55 to 70
to 55-70 °C (Fig. 4.5) upon immobilization. Fig. 4.5 also shows that % relative
activity at temperature less than 55 °C was reduced after immobilization. This could
be because, at low temperature, starch solution is more viscous and hence the
diffusional resistance for the migration of the starch molecules through the
temperature. The increase (Tumturk et al., 2000) or no change (Marchal et al., 1999;
Ivanova et al., 1998) in the optimum temperature may be because of the improvement
Properties of free and immobilized BLA are summarized in the Table 4.2.
Activation energy (Ea) of immobilized BLA (3.18) was higher than that of free BLA
(1.63). Similar increase in the Ea after immobilization is reported and attributed to the
immobilized BLA (15 mg/mL) was 4.5 times of the K mfree (3.3 mg/mL). Higher value
of Km for immobilized BLA indicates less affinity between immobilized BLA and
substrate molecules, which could be because of either similar nature of the charges
carried by the support and the substrate or structural changes in the enzyme occurring
the immobilized BLA due to steric hindrances and still persisting diffusional
times for α-amylase (from porcine pancreas) immobilized on HEMA and styrene-
1998). kcat
app
of immobilized BLA (0.93) was about half of the kcat
free
(1.76). Lower
active enzyme site of the immobilized BLA, which subsequently results into lower
reaction rate. Apparent Vmax is reported to decrease marginally (Tumturk et al., 2000)
for porous support. This is mainly attributed to diffusional limitation that exists in the
105
% relative activity
85
65
45
25 Immobilised BLA, 55 °C
Free BLA, 55 °C
5
4 4.4 4.8 5.2 5.6 6 6.4 6.8 7.2
pH
Figure 4.4. pH-% relative activity profile of free and immobilized BLA
105
% relative activity
85
65
45
Figure 4.5. Temperature-% relative activity profile of free and immobilized BLA
well as maximum hydrolysis rates in the later stages of hydrolysis (Fig. 4.6),
indicating that stability of the immobilized BLA is relatively high at pH 5.2. Hence
pH 5.2 was taken as an optimum operating pH and experiments to account the effect
of temperature, [S]0 and [IEU]/[S]0 were performed at pH 5.2. Initial hydrolysis rates
with unbuffered solution (i.e. soluble starch gelatinized in distilled water) are
comparable to that with pH 5.6; but in the later stages of hydrolysis (i.e. beyond the
compared to that with pH 5.6 (Fig. 4.6). This indicates that though the initial
hydrolysis rate with unbuffered solution is high, the stability of the immobilized BLA
conditions.
It was observed that at low DE (8.5 and 12.5), there are no significant changes
in the composition of maltodextrins (Fig 4.7, A & B). However Fig. 4.8 (A) or Fig
4.8 (D) shows that at DE of 20.5, wt % of G5 and G3 significantly increases from 7.7
to 13 and from 4.7 to 6.7 respectively with an increase in the pH from 4.4 to 7; but
there are marginal increases in the wt % of G1, G2, G4, G6 and G7. It can be also
seen from Fig. 4.8 (A) that wt % of oligosaccharides with DP higher than 7 (which
mainly constitute branched dextrins) decreases from 76.8 to 70.5. It also indicates
35
25
20
15
pH
10 4.4 5
5.2 5.4
5 5.6 6
7 unbuffered
0
0 1 2 3 4 5 6 7 8
Time (h)
1.2 90 3.5
)
3 85
1
85 2.5
2 80
0.6 80
1.5
0.4
1 75
wt percentage of G1-G7
75
0.2 0.5
0 70 0 70
4.3 4.8 5.3 5.8 6.3 6.8 4.3 4.8 5.3 5.8 6.3 6.8
9 85 14 80
C D
8
12 75
7 80
10
6 70
75
5 8
65
4 6
70
3 60
4
2 65
2 55
1
0 60 0 50
4.3 4.8 5.3 5.8 6.3 6.8 4.3 4.8 5.3 5.8 6.3 6.8
pH
16 80 16
A B 75
14 14
75
12 12 70
70
10 10
65
8 65 8
)
6 6 60
wt % of oligosaccharides of DP >7
60
4 4
55 55
2 2
wt % of G1-G7
0 50 0 50
4.3 4.8 5.3 5.8 6.3 6.8 35 40 45 50 55 60 65 70
pH Temperature (°C)
16 80 12 80
C D
14 75 75
10
12 70
70
8
10 65
65
8 6 60
60
6 55
4
55
4 50
50 2
2 45
0 45 0 40
0 45 90 135 180 0.0025 0.0085 0.0145 0.0205
[S]0 [IEU]/[S]0
Fig. 4.8. Effect of pHa, temperatureb, [S]0c and [IEU]/[S]0d on the saccharide
composition at DE 20-21.
□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6, + G7, - oligosaccharides with DP >7.
a
[S]0 = 90 mg/mL, 55 °C and [IEU]/[S]0 = 8.27e-3.
b
[S]0 = 90 mg/mL, pH 5.2 and [IEU]/[S]0 = 6.22e-3.
c
55 °C, pH 5.2 and [IEU]/[S]0 = 3.3e-3.
d
pH 5.2, 55 °C and [S]0 = 90 mg/mL.
increase in V and initial rates at temperature above 60 °C are approximately the same
and from 7 to 4.8 respectively (Fig. 4.8 (B) or 4.10 (D)). At DE 20, wt % of G1, G2,
G4, G6, G7 and oligosaccharides higher than G7 increases from 0.9 to 1.35, from 2.4
to 3.1, from 2.3 to 2.6, from 3.9 to 5.7, from 0.7 to 1 and from 69 to 73.5 respectively
with an increase in the temperature from 36 °C to 70 °C (Fig. 4.8 (B)). This indicates
towards G3 and G5, whereas specificity towards G1, G2, G4, G6 and G7 increases.
with an increase in the temperature from 50 to 90 °C, for free α-amylase (B.
oligosaccharides (of different DP) with increasing temperature. (Marchal et al., 1999)
35
Concn of reducing sugars (mg/mL)
30
25
20
15
Temperature (°C)
10
37 45 50
5 55 60 65
70
0
0 1 2 3 4 5 6 7 8
Time (h)
Figure 4.9. Concentration of reducing sugars vs. time with temperature as parameter
at [S]0 = 90 mg/mL, pH 5.2 and [IEU]/[S]0 = 6.22e-3.
.
2.5 6
A B 90
95
5 85
2
90
4 80
85
1.5
80
0.5
1 65
65
0 60 0 60
35 40 45 50 55 60 65 70 35 40 45 50 55 60 65 70
10 85 14 75
9 C D
12
8 80 70
7 10
6 75 65
8
5
6
4 70 60
3 4
2 65 55
2
1
0 60 0 50
35 40 45 50 55 60 65 70 35 40 45 50 55 60 65 70
Temperature (°C)
[S]0 (varying from 18 to 180 mg/mL), DE vs. time is plotted in Fig. 4.11 (B). Since
the ratio [IEU]/[S]0 was same, it was expected that all curves in Fig. 4.11 (B) would
lie on the same line. But at low value of [S]0 (18 mg/mL), [IEU] was also kept low
i.e. 0.0593 in order to maintain [IEU]/[S]0 constant i.e. 3.3e-3. Hence though the
viscosity of the starch solution was less, due to lesser concentrations of IEU and
starch, probability of contact of enzyme active site on beads with starch molecule
becomes less which result into less increase in DE (5.7) in 3 h of reaction (Fig. 4.11
(B)). But as [S]0 increases, probability of contact of enzyme active site on beads with
starch molecule increases; but due to increase in the viscosity, diffusion resistance for
starch molecule to reach active enzyme site inside pore increases. This could be the
reason for initial increase and then the maxima in the increase in DE in 3 h of reaction
(for [S]0 of 45 and 90 mg/mL, increase in DE was 9.2 and 10 respectively; Fig. 4.11
(B)) with an increase in [S]0. Further increase in the [S]0 results into increase in the
viscosity and the diffusion resistance for starch molecule to reach active site inside
pore. This could be the reason for observed lower increase in DE (8.9), in 3 h of
reaction at high starch concn i.e. 180 mg/mL (Fig. 4.11 (B)). Decrease in the rate of
hydrolysis at high starch concentration is reported for starch hydrolysis using free α-
amylase (B. licheniformis, Termamyl) and attributed to the imposed restriction on the
free movements of starch and enzyme molecules due to viscosity effects and/or
reduced water activity (Komolprasert and Ofoli, 1991), supporting the above
conclusion.
It can be seen from Fig. 4.8 (C) and Fig. 4.12 that wt % of G1, G2 and G4
remains approximately the same with an increase in the [S]0 at any value of DE;
whereas wt % of G3 and G5 decreases (from 6.8 to 5.3 and from 13.5 to 11.1
(from 5.3 to 6.7 and from 11.1 to 14.2 respectively at DE of 20). It can be also seen
from Fig. 4(C) that wt % of G6 and G7 marginally increases (from 4.2 to 4.9 and from
0.3 to 0.7 respectively) with an increase in [S]0 from 18 to 180 mg/mL; whereas wt %
of higher oligosaccharides (> G7) increases (from 68.6 to 72.7 at DE of 20) with an
increase in [S]0 from 18 to 90 mg/mL and then decreases (from 72.7 to 66.7 at DE of
4.13). It can be seen from Fig. 4.8 (D) and Fig. 4.14 that there are marginal changes
45 25
40 [S] 0 (mg/mL)
Conc. of reducing sugars (mg/mL)
18 45 20
35
20
10
[S] 0 (mg/mL)
15
18 45
10 5 90 180
5
0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (h) Time (h)
74
0.5 75
1
0 70 0 70
0 45 90 135 180 0 45 90 135 180
12 85 16 75
C D
80 14
10 70
75 12
8 65
70 10
6 65 8 60
60 6
4 55
55 4
2 50
50 2
0 45 0 45
0 45 90 135 180 0 45 90 135 180
Initial starch concentration (mg/mL)
35
25
20
15
[IEU]/[S]0
10
3.3e-3 8.23e-3
5
13.8e-3 20.7e-3
0
0 1 2 3 4 5 6 7 8
Time (h)
1.8 5 90
A B
1.6 4.5
95
1.4 4 86
)
1.2 90 3.5
3 82
1
85 2.5
75
0.2 0.5
0 70 0 70
0.0025 0.0085 0.0145 0.0205 0.0025 0.0085 0.0145 0.0205
8 85 12 80
C D
7 75
10
80
6 70
8
5 75 65
4 6 60
70
3 55
4
2 50
65
2
1 45
0 60 0 40
0.0025 0.0085 0.0145 0.0205 0.0025 0.0085 0.0145 0.0205
[IEU]/[S]0
DE of gelatinized starch solution was ∼ 5-6. For free BLA, hydrolysis ceased
at DE of around 42-43 because BLA could not hydrolyze more α(1→4) linkages due
could be attributed to steric hindrances for branched dextrins in the vicinity of active
enzyme site. DE of starch hydrolysate can be correlated with reaction time (t) by the
and B is pseudo first order hydrolysis constant (h-1). Hydrolysis constant (B) was
equation.
(4.3)
In case of the free BLA, it can be seen from Fig. 4.15 that concn of G1, G2,
decrease with further increase in DE. G8, G9 and G10 also show similar behavior.
For immobilized BLA, it can be seen from Fig. 4.16 that concn of G1, G2, G3,
G4 and G5 increases with an increase in the hydrolysis time; whereas concn of the G6
further hydrolysis) with further increase in the DE. Data of G8, G9 and G10 are not
shown in Fig. 4.16 because, at any DE their wt % was always lower than 0.3. For
immobilized BLA, at any DE, G5 and G3 are the principal products (Fig. 4.16 and
4.17). Similar high production of G5 and G3 from soluble starch was reported
(Ivanova and Dobreva, 1994) with immobilized BLA, but variation in concn of
useful in deciding the appropriate reaction quenching time to get the final product of
12
14 G1 G2
G3 G4
12 10
G5 G6
concn of G1-G5 (mg/mL)
2
2
0 0
time (h) 0 1 2 3 4 5 6 7 8
DE 4.9 13 20.2 31.3 35.3 38.2 40.1 40.9
Figure 4.15. Change in concn of oligosaccharides w. r. t. time and DE, with
free BLA. [S]0 = 90 mg/mL, [IEU equiv]/[S]0 = 8.3e-3, pH 5.6, 55 °C.
35 4.5
G1 G2 G3 G4 G5 G6 G7
4
30
3.5
20 2.5
15 2
1.5
10
1
5
0.5
0 0
time (h) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5
DE 5.8 13.4 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2
30 DE 13 Free BLA
DE 13 Immobilized BLA
25 DE 20 Free BLA
DE 20 Immobilized BLA
wt % oligosaccharide
DE 36 Free BLA
20
DE 36 Immobilized BLA
15
10
0
1 2 3 4 5 6 7 8 9 10
Degree of polymerisation of oligosaccharide
immobilized BLA were much higher than that produced by free BLA and there was
produced by immobilized BLA were significantly lower than that produced by free
work) is different than the earlier reports (Ivanova and Dobreva, 1994; Marchal et al.,
of strain and different botanical source of starch (i.e. corn, tapioca, potato, wheat etc),
which mainly differ in amylose: amylopectin ratio, average molecular weight etc.
G6 and G7 (Fig. 4.16). Since BLA can hydrolyze linear G6 and G7, and not the
branched one, we can say that immobilized BLA produces linear G6 and G7 from
higher linear or branched dextrins in early stages of hydrolysis (DE<25), which gets
free BLA (Fig. 4.15) indicates that free BLA produces significant amount of branched
G6-G10 than linear one. Being speculative, this could be possibly because in the case
complex with active enzyme site rather than branched part. Reason for this could be
support and extent of branching of starch (Marchal et al., 1999). However there are no
separately using immobilized BLA at 55 °C. It was observed that immobilized BLA
could not hydrolyze G4 and G5. But it completely hydrolyses G6 and principally
produces G5 and G1 (this is in agreement with Ivanova and Dobreva, 1994). It also
completely hydrolyses G7 and produces G5, G2 mainly and also small quantity of G1.
G2, and rest to G6 and G1; thus produced G6 further hydrolyses to G5 and G1.
Action pattern and subsite mapping of free BLA with modified malto-
oligosaccharide substrates have been reported by Kandra et al., 2002. Binding region
and a barrier subsite; which have different binding energies. Free BLA cleaves G5 as
a main product from non reducing end of G6, G7 and G8 with yield or bond cleavage
frequencies of 68%, 84% and 88% respectively. However as the DP of the substrate
increases, attack shifts towards reducing end and BLA cleaves G8, G9 and G10 into a
main product G3 with yield of 88%, 83% and 83% respectively. Our results on
hydrolysis of G6 and G7 using immobilized BLA are much similar to these results,
which are reported for the hydrolysis of G6 and G7 using free BLA. However, yield
of G5 were 80% and 88% for hydrolysis of G6 and G7 respectively, which are higher
than that reported with free BLA usage. (Kandra et al., 2002)
G5 and G3 from soluble starch suggests that action pattern of immobilized BLA is
more like an exoamylase with dual product specificity mainly towards G5 and G3.
Free BLA is also reported (Kandra et al., 2002) to have dual product specificity
mainly to G5 and G3 (due to the existence of the barrier subsite) using results of
not considered. However our results of hydrolysis of soluble starch using free BLA
do not show high specificity towards G5 and G3 (discussed earlier) and this must be
because of the absence of steric hindrance for the formation of productive complex
Fig. 4.18 shows that the thermostability BLA was improved after
immobilization. Relative activity (%) of free BLA decreases from 100 to 75, whereas
it nearly remains same for immobilized BLA over the incubation period.
Unlike free enzyme, immobilized enzyme can be easily separated from the
BLA shows that 100% activity of immobilized BLA was retained even after 8 batch
hydrolysis, which indicates good reusability. For comparison, hydrolysis curves of 1st
120
100
% relative activity
80
Free BLA
Immobilized BLA
60
40
20
0
0 3 6 9 12 15 18 21 24
Time (h)
Figure 4.18. Thermostability of free and immobilized BLA.
30
20
15
10
1st batch hydrolysis
8th batch hydrolysis
5
0
0 1 2 3 4 5 6 7 8
Time (h)
Molecular weight distribution of soluble starch and starch hydrolysate were not
for the concentrations of oligosaccharides (G1-G7) vs. time were used, which are
was observed that plotting rate of formation of oligosaccharides (G1-G5) vs. concn of
oligosaccharides higher than G5 yields a straight line. This indicates that rate of
lower than 6 can not be hydrolyzed by immobilized BLA, there is no need for the
depletion term in the differential equations expressing the time dependent variation of
for i = 1-5,
d[G′i ] 5
= ki [IEU](TDW − ∑ [G′n ]) (4.4)
dt n =1
made dimensionless by dividing Eq. 4.4 with TDW on both sides. Eq. 4.4 thus takes
For i = 1-5,
5
d[G i ]
= ki [IEU](1 − ∑ [G n ]) (4.5)
dt n =1
expressing the time dependent variation for G6 and G7. Differential equation for G6
For i = 6-7,
i
d[G i ]
= ki [IEU](1 − ∑ [G n ]) − ki′[IEU][Gi ] (4.6)
dt n =1
where ki′ is kinetic rate constant (h-1[IEU]-1) for hydrolysis of oligosaccharide with
G10 were always lower than 0.3. Values of kinetic constants k1-k7, k6′ and k7′ were
determined by minimizing the sum of square of the error between predicted (obtained
by simultaneously solving Eq. 4.5 and Eq. 4.6 from t = 0 h to t = time required to
attain equilibrium DE of 36.5, which was calculated using Eq. 4.2) and experimental
[IEU] = 1.86, 55 °C, pH =5.2 and [S]0 = 90 mg/mL; are k1 = 0.0069, k2 = 0.0119, k3 =
conditions is shown in the Fig. 4.20. It can be seen from Fig. 4.20 that predicted wt %
fits well with the experimental values of wt % for G1-G5; whereas for G6-G7
were empirically correlated with [IEU], [S]0 and temperature using the following type
of correlation,
Values of A, B, C and D for kinetic constants k1-k7, k6′ and k7′ calculated by nonlinear
Kinetic Correlation
A B C D
constant coefficient
k1 0.0083 -0.0807 14.58 -0.6237 0.97
k2 0.0086 -0.3975 13.28 -0.4374 0.99
k3 0.0415 -0.4727 7.30 -0.4145 0.99
k4 0.0060 -0.3673 12.42 -0.4160 0.99
k5 0.0899 -0.3777 7.26 -0.4033 0.98
k6 0.3580 0.1086 12.01 -0.9275 0.99
k7 0.0022 -0.0633 14.62 -0.0905 0.80
k'6 102 0.5063 8.84 -1.439 0.93
k'7 7.8800 0.1496 9.36 -0.6200 0.89
35 4.5
4
30
3.5
25
3
wt % of G6 and G7
wt % of G1-G5
20 2.5
15 2
1.5
10
1
5
0.5
0 0
time (h) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
DE 5.8 13.5 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2
modes of operations viz. batch mode (performed in shaker), packed bed, and
expanded bed and their hydrolysis curves are compared in the Fig. 4.21. It can be seen
from Fig. 4.21 that the mode of packed bed mode of starch hydrolysis is faster than
conventional batch mode, packed bed imposes high enzyme to substrate ratio and
Effect of superficial liquid velocity (i.e. flow rate / column cross sectional
packed bed mode at three different superficial velocities viz. 1.27, 2.55, and 3.82
cm/min (i.e. 1, 2 and 3 mL/min flow rates, respectively). It can be observed from Fig.
4.21 that as the superficial velocity increases from 1.27 to 2.55 cm/min, hydrolysis
performance improved. But when it was increased from 2.55 to 3.82 cm/min, initial
Mean residence times were measured to be 13.4, 6.3, and 3.4 min at
superficial liquid velocities of 1.27, 2.55, and 3.82 cm/min, respectively (i.e. 1, 2, 3
mL/min, respectively), for the packed bed of 5 mL of CELBEADS. This indicates that
as the liquid velocity increases, residence time decreases and flushing effect starts to
play a role. At low liquid velocities, substrate solution almost reaches all the pores in
the beads and residence time is large and obviously time required for the product
molecules to come out of pores will also be high. It could be speculated that at low
flow rates, residence time scales may be higher than reaction time scales. This could
be the reason for observing lower hydrolysis performance at low flow rate. As the
liquid velocity increases, residence time decreases and may be become comparable to
the reaction time scales. This will result into faster carriage of substrate molecules to
immobilized enzyme active sites, reaction and fast removal of product molecules from
pores to solution. This could be the reason for improvement in the hydrolysis
performance with increase in the flow rate or liquid velocity. If liquid velocity is
further increased, local residence time scales will be much lower than reaction time
scales. This will result into faster carriage of substrate molecules to immobilized
enzyme active sites, but without getting enough time for reaction they may be
removed from the active enzyme site. This is termed as “flushing effect” in the
reactor. This will obviously result into poor hydrolysis performance. This could be the
reason for poor hydrolysis performance with an increase in the superficial liquid
velocity 2.55 to 3.82 cm/min. Same reason could be attributed to poor hydrolysis
performance in the expanded bed mode hydrolysis. In the expanded bed, voidage inter
between the beads is more. Hence, space inter between beads offers least resistance to
the flow of solution. Hence, significant part of the reactant molecules may be passing
40
30
25
20
Batch mode
15
Packed bed, 1.27 cm/min
10
Packed bed, 2.55 cm/min
starch solution on the reusability of immobilized BLA is shown in the Fig. 4.22.
Hydrodynamic stability
100
80
% initial rate
60
40
20
0
0 1 2 3 4 5 6
Reuse number
washing shows that 100% activity of immobilized BLA was retained even after 8
batches of hydrolysis (Fig. 4.22), which indicates good reusability. But when the same
study was performed without intermittent washing (i.e. washing of beads), reduction
in the % initial reaction rate was observed from 100 to 68 (Fig. 4.22) after 6 batches
initial reaction rate was observed to decrease from 100 to 28 for 1.27 cm/min
superficial liquid velocity and from 100 to 14 for 3.82 cm/min liquid velocity (Fig.
4.22). Agglomeration of the beads was visually observed after completion of the
batch, this effect was more pronounced in the packed and expanded bed than that in
the batch mode. This could be because in the batch mode, liquid velocities in the
vicinity of beads were very high as compared to those in the case of packed bed or
expanded bed. This results into not only agglomeration of beads, but blockage of
pores due to starch molecules also. This result into decrease in the activity of
was done, agglomerated beads separated from each other due to high velocities and
pore blockage also get cleared. Due to this no loss in the activity was observed in the
batch mode with intermittent washing. This suggests that the packed bed mode of
operation should be carried out in a semi continuous manner with intermittent bed
operation of the system, easy separation of product from the enzyme etc.) that
immobilized enzyme have over the free enzyme, it was first decided to develop a
process for the production of glucose from sorghum flour using immobilized
sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of
made using immobilized BLA and amyloglucosidase in the batch mode (using
shaker). It was observed that use of immobilized BLA is not suitable for production of
washing.
9 After mixing the gelatinised sorghum slurry with beads, it was very difficult
enzyme bed in both packed and expanded mode due to viscosities and
Thus, it may be more economically viable to use BLA and other enzymes in
the free form for the production of glucose from sorghum flour. The work of
reported in chapter 5.
4.4. Conclusions
pH, temperature and initial starch concn has significant effect on saccharide
produced by immobilized BLA is different than that produced by free BLA at any
value of DE. At any DE, free BLA principally produces maltotriose, maltopentaose
maltopentaose. Immobilized BLA has better thermostability than free BLA and found
to retain 100% activity even after 8 batches of hydrolysis. Immobilized BLA can be
with variation in pH, temperature and starch concn. Used semiempirical model
data of G1-G5, but over predicts wt % of G6 and G7. Such model can be used for
be taken because values of kinetic constant are likely to vary with change in starch
source.
Nomenclature
cereal crop and fifth largest produced cereal in the world after wheat, rice, barley and
maize. Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons
(19.9%), Nigeria (15.5%), India (11.3%), Mexico (9.8%), Sudan (7%), and Argentina
(5.4%) (www.fas.usda.gov). Sorghum ranks third in the major food grain crops in
India. Sorghum is valued because of its ability to grow in areas with marginal rainfall
and high temperatures (i.e. semi arid tropics and sub tropical regions of the world),
where it is difficult to grow any other cereal, and also because of its relatively short
growing season requirement, thus its suitability for double cropping and crop rotation
proteins, moisture, fibers, lipids, and ash in the sorghum were 70.1, 11.2, 11.6, 1.82,
decreasing with change in the way of living due to increased urbanization, increased
per capita income of the population, and easy availability of other preferred cereals in
of staple food for humans, it also serves as a source of feed for cattle and other
livestock in scarcity of maize, but at lower prices. Also, about 10-20 % of the
production gets wasted due to damage and inadequate transport and storage facilities.
Industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are
available in large quantity at Food Corporation of India (FCI) at 10 times lower rate
than the fresh grains (Suresh et al., 1999a). Damage includes chalky appearance,
cracked, broken, mold, infection etc. These damaged grains are not suitable for
that are harmful to health and productivity of human and animal (Bandyopadhyay et
al., 2000). Hence, damage caused by insect infection and attack of fungus (blackened
sorghum or grain mold) because of wet and humid weather makes sorghum grains
farmers, through value added products. There is very small amount of research done
1996; Aggarwal et al., 2001), production of ethanol (Wu et al., 2007; Suresh et al.,
1999a, 1999b; Zhan et al., 2003; Zhan et al., 2006; Zhao et al., 2008) and isolation of
starch (Yang and Sieb, 1996; Xie and Seib, 2002; Higiro et al., 2003; Perez-Sira and
Amaiz, 2004; Park et al., 2006). The reason for the lower level of industrial
et al., 1978; Rooney and Pflugfelder, 1986; Chandrashekar and Kirleis, 1988; Zhang
and Hamaker, 1998; Elkhalifa et al., 1999; Ezeogu et al., 2005) and reduced protein
of starch from sorghum, and digestibility of sorghum starch and sorghum proteins is
to blackened sorghum.
starch from corn (Zhang et al., 2005), sorghum(Park et al., 2006) and rice (Wang and
Wang 2004), and in dry corn milling ethanol production (Kinley et al., 2006; Khanal
et al., 2007).
In the present work, sorghum flour was used directly for liquefaction and
saccharification rather than isolating starch and using it for liquefaction and
around 50–60% i.e rest part (40–50%) gets wasted or does not fetch much price.
Such methodology of direct hydrolysis was first used by kroyer in 1966 using corn
grits for the production of glucose. Direct hydrolysis of flour of maize (Bos and Norr,
1974; Twisk et al., 1976), broken rice (Tegge and Ritcher, 1982) and sorghum (Tegge
and Ritcher, 1982; Devarajan and Pandit, 1996; Aggarwal et al., 2003) was reported
healthy, blackened and germinated, and to study the effect of ultrasound treatment
processes.
5.2. Experimental
5.2.1. Materials
chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd
amylase (BLA) (EC number 3.2.1.1), Amyloglucosidase (AG) (EC number 3.2.1.3),
and Pullulanase (PL) (EC number 3.2.1.41) were gifted by Advance Enzyme
Technologies Pvt Ltd (India). Healthy sorghum and blackened sorghum were
The protein concentration of the free enzyme was determined using the
modified Folin–Lowry method (Lowry et al., 1951) using BSA (0–0.6 mg/mL) as a
standard. The reducing sugar concentration was measured using the DNSA method
method. Details of modified folin lowry method, DNSA method and HPTLC method
Sorghum flour was kept at 80 °C, till constant weight was obtained and
moisture content in sorghum flour was measured using mass balance. Detailed
Particle size distribution of the ground sorghum flour was determined by using
the Coulter Counter Particle Size Analyzer (LS 230) based on laser light diffraction.
Sorghum grains were finely ground to the flour. Sorghum slurry (1% w/v, pH
4.5, 50 mM acetate buffer) was gelatinized for 10 min in boiling water. Then 200
units of BLA and 180 units of AG were added to gelatinized solution and reaction
mixture was kept under shaking conditions (180 rpm) at 55 °C for 24 h. Starch
content in the sorghum flour was calculated by multiplying the total reducing sugar
0.9.
All enzyme assays were designed by end point assay method. It was ensured
that at the end of incubation time concentration of reducing sugar lies in the linear
was added to reaction mixture to stop the reaction. The resulting solution was heated
in a boiling water bath for 10 min. Then 10 mL distilled water was added to the assay
mixture. Absorbance of the solution was measured against substrate blank. The
was defined as that required to liberate one micromole of reducing sugar (glucose
equiv) per min under the assay conditions. Activity of commercial AG formulation
where CRS = Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL
buffer) was incubated with 0.1 mL of 500 fold diluted commercial pullulanase (PL)
solution at 60 °C for 10 min. Then 1 mL of DNSA reagent was added to the reaction
mixture to stop the reaction. The resulting solution was heated in a boiling water bath
for 10 min. Then 10 mL distilled water was added to the assay mixture. Absorbance
of the solution was measured against substrate blank. The variation in the
glucose as a standard. One unit of pullulanase (PLU) was defined as that required to
liberate one micromole of reducing sugar (glucose equiv) per min under the assay
following equation,
where CRS = Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL
4.5) was incubated at desired temperature under shaking conditions (180 rpm) in the
pullulanase (0–4.5), separately for 1 h at 55 °C. Then samples were withdrawn and
diluted ten times using 0.1 N HCl. These samples were analyzed for reducing sugar
the present experimental work, is shown in the Fig. 5.1. Chemistry of the process can
be also depicted from the Fig. 5.1. Chemistry of liquefaction of starch and
Grain sorghum
G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch
|
Milling G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G
|
G–G–G–G–G–G–G–G–G
Preparation of slurry
G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G
Glucose syrup
Figure 5.1. Process flow sheet for production of glucose syrup from sorghum
Sorghum grains were milled using old fashioned flour mill (two stones of 15″
diameter × 2″ height dimensions) and this flour was used for liquefaction followed by
flask containing 100 mL magnetically stirred sorghum slurry. Experimental set-up for
C
B
D
Liquefaction of sorghum slurry (10– 35% w/v in 0.05 M acetate buffer) was
flask in oil bath). Required quantity of BLA was added to sorghum slurry. Then slurry
was poured into conical flask. Oil bath temperature was maintained at 92 °C to get 85
°C temperature for sorghum slurry. Initially viscosity of the reaction mixture was very
high and as the liquefaction progresses viscosity of reaction mixture decreases; this is
using starch-iodine colorimetric reaction. Few drops of KI-I2 reagent (0.05% w/v I2
and 0.5% w/v KI solution) were added to few drops of diluted sample and color of
above-said mixture: deep blue, bluish violet, violet with tinge of dark red, and dark red
with tinge of violet were observed in sequence. When color becomes dark red with a
tinge of dark violet (DE of liquefact was approximately 15 at this stage), liquefaction
for 10 min and supernatant was analyzed for the concentration of reducing sugar.
Liquefaction process was optimized for the liquefaction time of 1.5 h using buffered
v/w of sorghum flour) and CaCl2 (0–500 ppm), and temperature in the range of 75–95
°C.
Healthy sorghum grains were steeped in the ordinary tap water for 12 h and then
first dried at 50 °C. Germinated and blackened sorghum were milled separately using
old fashioned flour mill. Liquefaction of geminated and blackened sorghum was
Ultrasound horn was dipped into sorghum slurry (30% w/v in 0.05 M acetate
buffer of pH 6, and CaCl2 concentration of 200 ppm) to a depth of 1 cm and slurry was
sonicated for different spans of time and at different ultrasound intensities. Ultrasound
horn (Vibra-cell, Sonics and Materials Inc., USA) having maximum power output of
750 W and operating at a frequency of 20 kHz was used in the study. Diameter of the
probe was 1.3 cm. A short ultrasound treatment of sorghum slurry was followed by the
addition of optimized amount of BLA and then subsequent liquefaction for 1.5 h was
performed at optimized conditions. After liquefaction, reaction mixture (30% w/v) was
centrifuged at 5000 g (high centrifugal force was required because of high slurry
concentration and hence viscosity) for 20 min and then supernatant was collected. This
supernatant was then analyzed for dry wt concentration of reducing sugars and reducing
At the end of liquefaction (i.e. 1.5 h), pH of the reaction mixture was reduced to
4.5 using acetic acid. Then sorghum liquefact was filtered hot using muslin cloth and
filtrate (F1) was collected. Cake obtained after hot filtration was mixed well with 10
mL distilled water and filtered. This second filtrate (F2) was mixed with first filtrate
(F1). Optimized quantity of amyloglucosidase was added to the filtrate (F1) or the
mixture of F1 and F2. Then this reaction mixture was kept under shaking conditions
approximately 1% using 0.1 N HCl solution. Samples (1% w/v) were then centrifuged
at 270 g for 10 min and supernatant was analyzed for the concentration of reducing
Since the calculation of % saccharification is based on the original starch content in the
sorghum flour, the term % saccharification can be also regarded as % yield of glucose.
Washing of cake (obtained after hot filtration) followed by second filtration and mixing
process. The starch content and moisture content of the healthy sorghum flour were
h after liquefaction (85 °C, 10% w/v sorghum slurry) at different values of pH of
buffered slurry; 5.2, 5.6, 6, 6.3 and 6.7 were 11.2, 12.4, 14.2, 13.4, and 11.4
acetate buffer).
It can be seen from Fig. 5.3 that liquefaction of sorghum slurry (25% w/v) got
completed in 60 min with BLA concentration of 0.08% v/w of flour in the absence of
CaCl2 supplementation. It can be also seen from the Fig. 5.3 that when concentration
of reducing sugar was around 35 mg/mL (i.e. DE in the range of 15-17), liquefaction of
the slurry was completed according to starch-iodine reaction. The region above dotted
line in the Fig. 5.3 is liquefied region i.e. at all points in this region, liquefaction was
observed to be completed. As the BLA concentration increased, the time at which the
50
45
liquiefied
35
30
25
20 Concn of BLA
(% v/w of sorghum flour)
15 0.04 0.06
not liquified
0.08 0.1
10
0.12 0.16
5
0
0 15 30 45 60 75 90 105
Time (min)
optimum CaCl2 concentration. It can be seen from Fig. 5.4 that liquefaction of
sorghum slurry (25% w/v) was completed in 60 min and all the hydrolysis curves
corresponding to CaCl2 concentration greater than 200 ppm overlapped each other (Fig.
5.4). This indicates that CaCl2 concentration of 200 ppm was optimum for liquefaction.
v/w with CaCl2 concentration of 200 ppm (Fig. 5.3 and 5.4).
45
35
30
25
20
15
CaCl2 concn (ppm)
10
0 100 200
5 300 400 500
0
0 15 30 45 60 75 90
Time (min)
from 10 to 35 % w/v in the slurry. Maximum concentration of sorghum slurry that can
be used for liquefaction was observed to be 30% w/v, at which mixing of the reaction
mixture was experimentally possible and liquefaction also was successfully completed
within 1.5 h. Mixing and homogenization of the reaction mixture was visually much
less efficient at 35% w/v due to very high viscosity and it took around 2.5–3 h to
75–80 °C (Palmer et al., 1992) and 60–80 °C (Wu et al., 2007). Hence, temperature
was varied in the range of 75–95 °C at 30% w/v sorghum slurry concentration for
starch granules and two, dextrinization of gelatinized starch molecules (Reeve 1992).
As the temperature of liquefaction was increased from 75 to 95 °C, two effects occur
temperatures. Hence, there exists an optimum temperature for the liquefaction process.
sorghum flour, at which liquefaction was completed within 1.5 h (according to starch-
was not completed in 1.5 h (according to starch-iodine reaction). This could be because
at 75 °C and 80 °C, starch granules are not completely gelatinized (hence giving blue
color with iodine reagent). However, higher concentration of reducing sugars could be
60
50
30
20
Temperature (°C)
75 80 85
10
90 95
0
0 15 30 45 60 75 90
Time (min)
Healthy, germinated, and blackened sorghum were found to contain 69–70, 69–
70 and 70–71% starch respectively. High starch content in the blackened sorghum
indicates that fungus has infected only pericarp of sorghum grain and not the
endosperm.
It can be seen from Fig. 5.6 that liquefaction of healthy sorghum was completed
attributed to the enzyme inhibition due to mycotoxins present in the blackened pericarp.
90
H B G1 G2 G3 G4
80
60
50
40
30
20
10
0
0 15 30 45 60 75 90
Time (min)
It can be seen from Fig. 5.6 that as the germination time increases, liquefaction
only. This could be attributed to development of protease (i.e. protein matrix degrading
enzyme) and subsequent loosening of the cage of protein surrounding starch granules
17
16.5
DE of liquefact 16
15.5
15 40% amplitude
50 % amplitude
14.5 100% amplitude
14
0 2 4 6 8 10 12
sonication time (min)
17
16.5
DE of liquefact
16
15.5
15 40% amplitude
50 % amplitude
14.5 100% amplitude
14
0 5000 10000 15000 20000
Power consumption (J)
It can be seen from Fig. 5.7 that as the sonication time increases at constant
ultrasound intensity, DE of liquefact also increases. Fig. 5.8 shows that increase in the
ultrasound intensity used. Since ultrasound treatment consumes large energy, it was
decided to keep sonication time low i.e. 1 min. If 1 min is considered as the optimum
time for ultrasound treatment, 100 % amplitude gives maximum increase in the DE of
the liquefact. The reason for increase in DE of liquefact due to prior ultrasound
% relative activity vs. pH profile and % relative activity vs. temperature profile
of Amyloglucosidase are given in the Fig. 5.9 and 5.10 respectively. Optimum pH and
activity were 4.5 and 65 °C, respectively. Enzyme activity at optimum conditions and
110
100 65 °C
i
90
80
Relative activity (%)
70
60
50
40
30
20
10
0
3 3.5 4 4.5 5 5.5 6 6.5
pH
100 pH 4.5
90
80
% relative activity vs. pH profile and % relative activity vs. temperature profile
of pullulanase are given in the Fig. 5.11 and 5.12 respectively. Optimum pH and
were 4 and 60 °C, respectively. Enzyme activity at optimum conditions and protein
110
100
Temperature = 60 °C
90
80
% relative activity
70
60
50
40
30
20
10
0
3 3.5 4 4.5 5 5.5 6 6.5 7
pH
110
100
90 pH 4
80
% relative activity 70
60
50
40
30
20
10
0
30 35 40 45 50 55 60 65 70 75
Temperature, °C
using AG may not be the same as that obtained using the assay procedure. Hence,
decreased to 5% (Fig. 5.13) at 65 °C within first 3 hrs only. Hence, it was decided to
maltodextrins (DE 15, Sigma Aldrich) at different temperatures were also performed.
It can be seen from Figs. 5.13 and 5.14 that the optimum operating temperature for
110
100 Temperature (°C)
i
90 50 55
80 60 65
Relative activity (%)
70
60
50
40
30
20
10
0
0 3 6 9 12 15 18 21 24
Time (h)
70
50
40
Temparature, °C
50 55
30
60 65
20
0 3 6 9 12 15 18 21 24
Time (h)
Figure 5.14. Change in concn of reducing sugar with time. Reaction conditions: 25 mL
of 10% maltodextrins (DE 15) solution in 50 mM acetate buffer, pH 4.5, 1.1 AGU/mL.
the concentration of reducing sugar was observed due to the addition of optimum
pullulanase, increase in the concentration of reducing sugar was observed in the initial
were the same (Fig. 5.16). Hence, it was decided to use only amyloglucosidase for
pullulanase can be used along with amyloglucosidase, but obviously at the added cost
85
80
% Degree of hydrolysis
75
70
65
60
55
50
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Pullulanase units (PLU)
160
Concn of reducing sugar (mg/mL)
140
120
100
80
60 % v of enzyme / w of saccharides
0.1 AG
40
0.1 AG + 0.13 PL
20
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (h)
240
concn of reducing sugars (mg/mL)
200
160
120
% v of AG / w of starch
80 0.026
0.052
40 0.078
0.104
0
0 4 8 12 16 20 24
Time (h)
After liquefaction, liquefied sorghum slurry was filtered hot using muslin cloth.
Hot filtration was necessary because filtration of liquefied slurry at room temperature
yielded less filtrate volume (65 mL), whereas hot filtration yielded higher volume (72
mL). Also due to lower viscosity of liquefied slurry at higher temperature, filtration
was easy. While preparing sorghum slurry, 30 g of sorghum flour was mixed with 80
8 mL of liquefact was still trapped inside the cake and not available for further
saccharification. Hence, washing of the cake after hot filtration was essential. The
values of % saccharification using AG, attained after 24 h, for all the three varieties of
sorghum slurry before liquefaction, and 2. Washing of cake (obtained after hot
filtration) followed by second filtration and mixing of both the filtrates, which are
Table 5.3. Summary of effect of ultrasound treatment and washing on liquefaction and
saccharification performance
Run BLA concn, CRS, mg/mL F1, F2, F1 + F2, Cf %
No % v/w (DE) mL mL mL mg/mL saccharification
H1 0.086 28.9 (13-14) 72 0 72 240 74 (S N; W N)
H2 0.086 27.7 (13-14) 71 15 86 229 84 (S N; W Y)
H3 0.086 31.2 (14-15) 75 0 75 251 81 (S Y; W N)
H4 0.086 28.5 (14-15) 74 15 89 235 90 (S Y; W Y)
G1* 0.086 29.6 (13-14) 73 0 73 240 75 (S N; W N)
G2* 0.086 26.7 (13-14) 73 15 88 225 85 (S N; W Y)
G3* 0.086 39.4 (16-17) 79 0 79 242 82 (S Y; W N)
G4* 0.086 36.7 (16-17) 79 11 90 232 90 (S Y; W Y)
B1 0.093 23.4 (9-10) 67 0 67 245 69 (S N; W N)
B2 0.093 24.6 (9-10) 65 17 82 231 79 (S N; W Y)
B3 0.093 22.7 (9-10) 70 0 70 240 70 (S Y; W N)
B4 0.093 24.4 (9-10) 69 16 85 226 80 (S Y; W Y)
B5 0.13 33.4 (16-17) 73 0 73 245 74 (S N; W N)
B6 0.13 32.1 (16-17) 73 15 88 233 86 (S N; W Y)
B7 0.13 32.1 (16-17) 75 0 75 239 75 (S Y; W N)
B8 0.13 30.4 (16-17) 75 14 89 234 87 (S Y; W Y)
S N: No ultrasound treatment of sorghum slurry before liquefaction
S Y: Ultrasound treatment of sorghum slurry before liquefaction for 1 min at 100
%amplitude
W N: No washing of cake after first filtration
W Y: Washing of cake after first filtration with 10 mL distilled water, followed by
second filtration
F1: Volume of filtrate after first filtration
F2: Volume of filtrate after second filtration
* Liquefaction time 1 h, Sorghum germinated for 24 h
Particle size distribution of sorghum slurry was compared for three conditions;
1. Without ultrasound treatment, 2. Slurry sonicated for 1 min at 40% amplitude, and 3.
Slurry sonicated for 1 min at 100 % amplitude and is shown in the Fig. 5.18. Average
particle size was found to decrease from 302 µm to 163 µm (Slurry sonicated for 1 min
at 40% amplitude) and 115 µm (Slurry sonicated for 1 min at 100% amplitude) due to
cavitationally induced particle fragmentation. It can be also seen from Fig. 5.18 that
there are three major inflection points in the particle size distribution; which
of ~800 µm must be a function of the milling process and mainly corresponds to large
the particle size distribution due to ultrasound is in agreement with the results reported
for corn slurry (Khanal et al., 2007) and for uranium ore slurry (Balasubrahmanyam et
cavitation (i.e. shock wave propagation and microjet formation) at solid-liquid interface
3.5
Without sonication
3
1 min at 40 %
amplitude
2.5 1 min at 100 %
% population
amplitude
2
1.5
0.5
0
0.1 1 10 100 1000 10000
particle size (µm)
Figure 5.18. Effect of prior ultrasound treatment on the particle size distribution in
Though 10 mL of distilled water was added to the cake (obtained after hot
filtration) for washing, second filtration yielded 15 mL of filtrate (H1 and H2; Table
5.3). This means washing and second filtration, followed by hot filtration has extracted
5 mL (out of 8 mL trapped inside the cake) of 1st filtrate (F1). Also it can be seen from
Table 5.3 (H1 and H2) that % saccharification increased from 74 to 84 (13% increase).
Increase in the % saccharification (which is based on the starch content in the sorghum
trapped inside the cake after hot filtration and were unavailable for further
saccharification. Similar behavior was observed in all the cases (Table 5.3).
It was observed (H1 and H3; Table 5.3) that ultrasound treatment of slurry
due to acoustic cavitation (Fig. 5.18). However, % increases in the volume of F1 and
% saccharification are not the same. This indicates that increase in the %
saccharification due to prior ultrasound treatment is not solely due to increase in the
volume of F1, but, there are few more factors responsible for this as discussed below.
If the step of washing of cake after 1st filtration was also provided, in addition to
observed (H1 and H4; Table 5.3) from 74 to 90 (21% increase). Here it should be
clarified that this increase in the % saccharification (74 to 90) is a combination of two
effects; one, from 74 to 81 (9.5% increase) is due to the step of ultrasound treatment
saccharification, discussed in detail later in the text), and two, from 81 to 90 (11%
from the cake after hot filtration). Similar effect of ultrasound treatment was observed
for germinated sorghum also (Table 5.3; G1, G2, G3, G4).
understand the reason behind the observed increase in the % saccharification due to
ultrasound treatment. Cells of inner floury endosperm are round with round starch
granules; whereas, the cells of the outer corneous endosperm are elongated with
polygonal starch granules and are filled with protein bodies (Chandrashekhar and
Mazhar, 1999). Also, multicellular pericarp of sorghum grain unlike other cereals
consists of small starch granules (Palmer 1992). Starch granules of the floury
endosperm of sorghum are loosely associated with paper like sheets of protein material,
while in the corneous endosperm they are tightly packed within rigid protein matrix
pronase (Zhang and Hamaker, 1998), pepsin (Rooney and Pflugfelder, 1986) have
Hamaker, 1998) and also the rate of starch hydrolysis by amyloglucosidase (Rooney
and Pflugfelder, 1986) due to hydrolysis of protein matrix surrounding starch granules.
Cooking of sorghum flour with reducing agents like sodium metabisulfite (Zhang and
increased starch digestibility using pancreatic α-amylase (Zhang and Hamaker, 1998)
disulphide bonds linking protein surrounding starch granules. Sorghum proteins are
disulphide bonding of prolamins (Ezeogu et al. 2005) and large extended web like
microstructure (Hamaker and Bugusu, 2003; Wu et al., 2007) during the cooking of
sorghum flour, into which small starch granules (~ 5 µm) remain tightly trapped (Wu et
al., 2007). These changes related to protein structure during cooking of sorghum flour
Since the lipid fraction within starch granules is insufficient to saturate entire
quantity of amylose, amylose exists in two forms; free amylose and amylose-lipid
complex (Tester et al., 2004). In the case of normal sorghum, this amylose-lipid
peak and ending of gelatinization) between 90 and 105 °C, whereas the remaining
starch i.e. free amylose and amylopectin has major endotherm between 60 and 80 °C
hydrolysis of micelles, which can clog pores of filter media) (Nebesny et al., 2002).
These findings and the presence of amylose-lipid complex in the hydrolysate (after
calorimetry (DSC) study (Nebesny et al., 2002) indicate that amylose-lipid complex
Hence, it seems that amylose-lipid complex and the starch granules encased in
the protein matrix do not get fully gelatinized, and the ungelatinized fraction of starch
remains inaccessible for action of BLA. However, the cavitation phenomena caused by
disrupting both; the protein matrix encasing starch granules and amylose-lipid complex.
This can also be observed with an increase in the peak area corresponding to the
particle diameter of 10-20 µm (Fig. 5.18); which is the diameter of sorghum starch
granule (Tester et al., 2004). These additional free starch granules get gelatinized and
are available for liquefaction, and further saccharification. This must be the reason for
release using 14% w/v corn slurry (raw and cooked) due to prior sonication for 20 or 40
s at amplitudes ranging from 180 to 299 µm has been reported (Khanal et al., 2007).
The increase in the glucose released of the sonicated samples was attributed to particle
size reduction, better mixing due to micro streaming effects, and the release of
additional lipid bound starch (Khanal et al., 2007). Higher conversion efficiency of
waxy sorghum to ethanol than that of normal sorghum (Wu et al., 2007) also supports
the hypothesis that higher quantity of amylose and hence, amylose-lipid complex
to glucose and hence to ethanol. 1 to 10% increase in the DE of liquefact and ethanol
yield has been reported25 due to cavitation resulting out of sonication for 1-7.5 min
before cooking. It was claimed, but, not experimentally proved (Kinley et al., 2006)
that cavitational forces produced by sonication breaks complex proteins (i.e. proteins
For normal healthy sorghum 90% saccharification has been reported (Aggarwal
et al., 2001). However, it should be clarified that experiments performed (Rooney and
Pflugfelder, 1986) for the production of glucose from sorghum (25% w/v slurry)
without any filtration step after liquefaction, as the process was optimized for
bioethanol production. Whereas, in the present work, filtration was done after
liquefaction, which result into trapping of oligosaccharides in the wet cake even after
were performed in the present work for the production of glucose from healthy
saccharification was observed due to prior ultrasound treatment. It can be seen from
blackened sorghum was 9-10, which was much lesser than expected DE i.e. 15. Hence
BLA concentration was increased by about 40 % to get liquefact DE 16-17 (Table 5.3).
Reason could be attributed to the leaching of mycotoxins and phenolic compound in the
blackened pericarp upon sonication and inhibiting the action of BLA in liquefaction,
produced (Aisen et al., 1983). In case of germinated sorghum (germination time 24 h),
This could be attributed to weakening of cell wall and protein matrix around starch
granules due to attack of endoprotease (Aisen et al., 1983) during germination and
hence effective release of starch granules from protein matrix by ultrasound treatment.
The percentage saccharification, however, didn’t go above 90% even after germination.
different varieties
represents operating expenses, which are of recurring in nature. They have significant
impact on the selling price and ultimately profitability. Operating expenses are incurred
after the plant is commissioned and the production begins. There are mainly two
parameters which being major and have direct impact on the cost of production viz. 1.
Raw materials; 2. Utilities. (Mahajani and Mokashi, 2005) In this section, processing
cost for production of glucose from sorghum was determined by considering cost of
raw materials and utilities. Cost of raw materials and utilities required for production of
glucose from healthy, blackened and germinated sorghum are compared in Table 5.4
and 5.5. Whereas comparison of processing cost is given in the Table 5.6.
Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg
Water 2.67 0.04 0.11 3.81 0.04 0.15 3.81 0.04 0.15 3.81 0.04 0.15
BLA required for 0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2
liquefaction
AG required for 0.0006 330 0.198 0.0006 330 0.198 0.0007 330 0.231 0.0006 330 0.198
saccharification
Filtration &
bleaching and color
- - 0.2 - - 0.429 - - 0.429 - - 0.429
removal4
Note: Steam requirement for the batch process is calculated based on following assumptions
1. 20% of the enthalpy is lost to surroundings per h from reaction mixture
2. steam input is saturated vapor and steam output is saturated liquid
i.e. steam requirement = enthalpy / latent heat of water 540 kcal/kg
3. For evaporating 90% of water produced glucose syrup and Evaporation efficiency = 90%
4. Total cost of both filtrations (i.e. after liquefaction and saccharification) = 0.2 Rs/kg of starch substrate
Where, Q Quantity of material, kg
R Rate of material, Rs/kg
C Cost incurred, Rs
Table 5.6. Comparison of processing cost or cost production of glucose from sorghum
Note: 100 % saccharification for pure starch (due to hydrolytic gain 1 kg starch produces 1.11 kg glucose)
90 % saccharification for health and germinated sorghum
85 % saccharification for blackened sorghum
Market cost of glucose syrup (84% b w) = 24 Rs/kg; Market cost of dry glucose = 30 Rs/kg
From Table 5.6, it can be seen that processing cost or cost of production per kg
of glucose produced from healthy, blackened sorghum is about Rs. 4.5 less than that
from isolated starch. Also this cost is 60% and 51% of market cost of glucose syrup for
healthy sorghum and blackened sorghum, respectively. This means that the production
industrial grade damaged sorghum grains (inclusive of 30-55% sound grains) are
available in large quantity at Food Corporation of India (FCI) at 10 times lower rate
than the fresh grains and it contains around 50% starch. If this industrial grade sorghum
is used as starting material then economy of the process may further improve.
5.4. Conclusions
using amyloglucosidase.
1. In the present work, use of ultrasound in the production of glucose from sorghum
optimized reaction conditions for liquefaction and saccharification) was in the range
slurry before liquefaction, and 2. Washing of cake (obtained after hot filtration)
due to physical effects of acoustic cavitation, like shock wave propagation and
granules and these additional starch granules are made available for further action
4. In this work, blackened and germinated sorghum were also used for the production
of glucose and % saccharification was 85% and 90%, respectively, with ultrasound
5. This means that integration of short ultrasound treatment (about 1 min) in the the
production of glucose from dry milled sorghum and its possible subsequent use in
the bioethanol production will result into increase in the production of glucose and
subsequently ethanol, and hence may improve the economic feasibility of the
process.
6.1. Introduction
In the section 5.1, the necessity to exploit industrial application for normal and
farmers through value added products, has been discussed. A little literature is
ethanol and isolation of starch (discussed in detail in the chapter 3). However there is
syrup.
In the present work, sorghum flour was used directly for liquefaction and
saccharification in the similar fashion that used in the production of glucose from
sorghum. Liquefaction part in the process was the same as that was optimized earlier
and discussed in the chapter 5. Hence, objectives of the present work were to optimize
healthy, blackened and germinated, and to study the effect of ultrasound treatment
processes.
6.2. Experimental
6.2.1. Materials
chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd
amylase (BLA) (EC number 3.2.1.1), Barley β-amylase (BBA) (EC number 3.2.1.2),
and Pullulanase (PL) (EC number 3.2.1.41) were gifted by Advance Enzyme
Technologies Pvt Ltd (India). Healthy sorghum and blackened sorghum were
The protein concentration of the free enzyme was determined using the
modified Folin–Lowry method (Lowry et al. 1951) using BSA (0–0.6 mg/mL) as a
standard. The reducing sugar concentration was measured using the DNSA method
method. Details of modified folin lowry method, DNSA method and HPTLC method
Sorghum flour was kept at 80 °C, till constant weight was obtained and the
moisture content in sorghum flour was measured using mass balance. Detailed
Particle size distribution of the ground sorghum flour was determined by using
the Coulter Counter Particle Size Analyzer (LS 230) based on laser light diffraction.
was incubated with 0.1 mL of 5000 fold diluted barley β-amylase (BBA) solution at
50 °C for 10 min. Then, 1 mL of DNSA reagent was added to the reaction mixture to
stop the reaction. The resulting solution was heated in a boiling water bath for 10
min. The variation in the concentration of reducing sugar was measured by DNSA
method using maltose as a standard. One enzyme unit was defined as that required to
liberate one micromole of reducing sugar (maltose equiv) per min under conditions of
assay.
where CRS = Concn of reducing sugars (maltose equiv) in assay mixture, µg/mL
formulation i.e. 2.5 BBAU/mL, 50 mM, pH 5.5) was incubated at desired temperature
under shaking conditions (180 rpm) in the absence of substrate for 24 h. Samples of
BBA solution were taken at regular time intervals and were for its amylolytic activity.
steps:
which BBA cleaves second α(1→4) linkage from non-reducing end of glucose
polymer and produces maltose. Use of only BBA in saccharification will result into
α(1→6) linkages. If pullulanase is used along with BBA, pullulanase will cleave
α(1→6) linkage and BBA can then attack rest of the chain in the β limit dextrins.
Hence saccharification using BBA and pullulanase will result into a mostly maltose
the present experimental work, is shown in the Fig. 6.1. Chemistry of the process has
also been depicted from the Fig. 6.1. Chemistry of liquefaction of starch and
Grain sorghum
G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch
|
Milling G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G
|
G–G–G–G–G–G–G–G–G
Preparation of slurry
G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G
Figure 6.1. Process flow sheet for production of maltose syrup from sorghum
Sorghum grains were milled using old fashioned flour mill (two stones of 15″
diameter × 2″ height dimensions) and this flour was used for liquefaction followed by
sorghum flour was performed in a 250 mL stoppered conical flask containing 100 mL
sorghum slurry (30% w/v), which was magnetically stirred at the optimized
conditions of pH, temperature, CaCl2 concentration and BLA concentration for 1.5 h
i.e. 6, 85 °C, 200 ppm, and 0.086% v/w of sorghum starch, respectively.
At the end of liquefaction (i.e. 1.5 h), pH of the reaction mixture was reduced
to 5.5 using acetic acid. Then sorghum liquefact was filtered hot using muslin cloth
and filtrate (F1) was collected. Cake obtained after hot filtration was mixed well with
10 mL distilled water for washing of the cake and filtered. This second filtrate (F2)
was mixed with first filtrate (F1). Then this reaction mixture was kept under shaking
conditions (150 rpm) for 24 h under optimized conditions of temperature and enzyme
withdrawn and diluted to approximately 1% w/v using 0.1 N HCl solution. Samples
(1% w/v) were then centrifuged at 270 g for 10 min and supernatant was analyzed for
the basis of original starch content in the sorghum flour is defined as follows;
BBA and PL at 5.5 pH and 50 °C) i.e. (% starch content /100) × 1.05
the sorghum flour, the term % saccharification can be also regarded as % yield of
maltose.
sorghum slurry, 4. Washing of cake (obtained after hot filtration) followed by second
Experiments were performed for the production maltose syrup using healthy,
blackened, and germinated sorghum without or with steps 1 or 4, and without or with
temperature profile of Barley β-amylase are given in the Fig. 6.2 and 6.3,
procedure to measure enzyme activity were 5.5 and 50 °C, respectively. Enzyme
110
100
50 °C
90
% relative activity
80
70
60
50
40
30
20
10
0
3 3.5 4 4.5 5 5.5 6 6.5 7
pH
Figure 6.2. Enzyme activity-pH temperature profile at 50 °C.
110
100
90
% relative activity
80
70
60
50
40
30
20
10
0
30 35 40 45 50 55 60 65 70 75
Temperature °C
Optimum temperature for BBA using the specified assay procedure was found
saccharification using BBA may not be the same as that obtained using the assay
°C. It was observed that % relative activity of BBA decreased to 3% of the original at
absence of substrate. But, Figs. 6.5. and 6.6 shows that BBA is active till 24 h also.
This means that during the saccharification presence of substrate stabilizes BBA and
substrate BBA activity decreases drastically. It can be seen from Figs. 6.5 and 6.6 that
120
100
80
% relative activity
Temperature, °C
60 50
40
40
20
0
0 1 2 3 4 5 6 7 8
Time (h)
Figure 6.4. Thermostability of BBA
Fig. 6.5. and 6.6 also shows that use of pullulanase along with BBA in the
to 204. This happens because pullulanase cleaves α(1→6) linkage and makes linear
180
220
200
Concn of reducing sugar (mg/mL)
180
160
140
120
Temperature, °C
100
80 40 45
60 50
40
20
0
0 4 8 12 16 20 24 28
Time (h)
Figure 6.6. Comparison of hydrolysis curves at different temperatures.
Reaction conditions: 20 mL liquefact, 1.1 BBAU/mL; 0.37 PLU/mL;
5.5 pH and 50 °C
After liquefaction, liquefied sorghum slurry was filtered hot using muslin
cloth. Necessity of hot filtration and washing of the cake after hot filtration is
discussed in the section 5.3.3. The values of % saccharification using BBA, attained
after 24 h, for all the three varieties of sorghum were dependent upon the following
cake (obtained after hot filtration) followed by second filtration and mixing of both
the filtrates, and 3. Use of pullulanase along with BBA during saccharification, which
Effect of washing of cake obtained after hot filtration and ultrasound treatment
chapter 5. Hence will not be detailed here and only brief is provided.
was observed. This increase in the % saccharification (which is based on the starch
which were otherwise trapped inside the cake after hot filtration and were unavailable
for further saccharification. Similar effect of washing of cake after hot filtration was
observed for all healthy, germinated, and blackened sorghum (Table 6.3).
Similar effect of ultrasound treatment was observed for all healthy, germinated, and
washing of cake after hot filtration and without or with ultrasound treatment before
saccharification increases from 69 to 75 (Table 6.3; H2 and H3) due to the use of
H6) due to the use of pullulanase in addition to BBA in saccharification. This also
shows that effect of pullulanase use is more pronounced when ultrasound treatment is
due to sonication.
different varieties
represents operating expenses, which are of recurring in nature. They have significant
impact on the selling price and ultimately profitability. Operating expenses are
incurred after the plant is commissioned and the production begins. There are mainly
two parameters which are major and have direct impact on the cost of production viz.
1. Raw materials cost, and 2. Cost of utilities. (Mahajani and Mokashi, 2005) In this
section, processing cost for the production of maltose syrup from sorghum has been
determined by considering the cost of raw materials and utilities. Cost of raw
materials and utilities required for production of maltose from healthy, blackened and
germinated sorghum are compared in Table 6.4 and 6.5. Whereas comparison of
Here it should be remembered that processing cost has been determined for
crystallization must be done and its cost has not considered here.
Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg
Water 2.67 0.04 0.11 3.81 0.04 0.15 3.81 0.04 0.15 3.81 0.04 0.15
BLA required for 0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2
liquefaction
BBA required for 0.0004 1500 0.6 0.0004 1500 0.6 0.0005 1500 0.75 0.0004 1500 0.6
saccharification
Filtration &
bleaching and color
- - 0.2 - - 0.429 - - 0.429 - - 0.429
removal4
Note: Steam requirement for the batch process is calculated based on following assumptions
1. 20% of the enthalpy is lost to surroundings per h from reaction mixture
2. steam input is saturated vapor and steam output is saturated liquid
i.e. steam requirement = enthalpy / latent heat of water 540 kcal/kg
3. For evaporating 90% of water from produced maltose syrup and Evaporation efficiency = 90%
4. Total cost of both filtrations (i.e. after liquefaction and saccharification) = 0.2 Rs/kg of starch substrate
Where, Q Quantity of material, kg
R Rate of material, Rs/kg
C Cost incurred, Rs
Table 6.6. Comparison of processing cost or cost production of maltose syrup from sorghum
Note: 100 % saccharification for pure starch (due to hydrolytic gain 1 kg starch produces 1.05 kg maltose)
85 % saccharification for healthy, blackened and germinated sorghum
Reducing sugars (i.e. dry solids in maltose syrup) produced by using barley beta-amylase contains 70% maltose-assumption.
Note: While comparing cost of dry maltose, it should be remembered that in the calculation of processing cost
per kg of maltose produced, crystallization cost is not considered.
6.4. Conclusions
Production of maltose syrup from sorghum flour involves two steps viz. 1.
1. In the present work, use of ultrasound in the production of maltose syrup from
filtration) followed by 2nd filtration and mixing of both the filtrates, and 3. Use of
frees starch granules and these additional starch granules are made available for
5. This means that integration of short ultrasound treatment (about 1 min) in the
production of maltose syrup from dry milled sorghum may improve the economic
germinated sorghum has been explored through the production of glucose and maltose
syrup. However, there are other products also, that can be produced by using sorghum
as a starting material. Flow sheet for the production of different products is provided
in the Fig. 6.7. In fact industry can switch from one product to another depending
30). Then the sorghum liquefact needs to be filtered, purified and dried to get
maltodextrins.
Flow sheet to produce glucose and maltose syrup has been already discussed
Glucose syrup (DE > 96) produced can further be processed using glucose
amyloglucosidase to glucose syrup with DE greater than 90. Then, this syrup is
out of this mixture. This product is termed as grain based alcohol or bioethanol.
Remanent stillage can be dried to produce DDGS, which can be used as animal feed
Grain sorghum
Milling
Preparation of slurry
Fermentation
followed by Hot filtration
distillation
DE ~ 15 DE ~ 15
Ethanol
Saccharification using
Saccharification using amyloglucosidase barley β-amylase (BBA)
w/o or with pullulanase w/o or with pullulanase (PU)
Glucose isomerase
Fructose syrup Glucose syrup Maltose syrup
References
Aggarwal N. K.; Nigam P.; Singh D.; Yadav B. S., Process optimization for the
production of sugar for the bioethanol industry from sorghum, a non-conventional
source of starch. World Journal of Microbiology & Biotechnology, 2001, 17, 411-415.
Agu, R.C., Palmer, G.H., 1998. A reassessment of sorghum for lager-beer brewing.
Bioresource Technology, 1998, 66, 253–261.
Baks, T.; Bruins, M. E.; Janssen, A. E. M.; Boom, R. M. Effect of Pressure and
Temperature on the Gelatinization of Starch at Various Starch Concentrations.
Biomacromolecules, 2008, 9, 296–304.
Ball, S.G. Recent reviews on the biosynthesis of the plant starch granule. Trends in
Glycoscience and Glycotechnology, 1995, 7, 405–415.
Ball, S.G.; van de Wal, M.H.B.J.; Visser, G.F. Progress in understanding the
biosynthesis of amylose. Trends in Plant Science, 1998, 3, 462–467.
Balole, T.V. & Legwaila, G.M. Sorghum bicolor (L.) Moench, Internet Record from
Protabase. Jansen, P.C.M. & Cardon, D. (Editors). PROTA (Plant Resources of
Tropical Africa), Wageningen, Netherlands, 2005. < http://database.prota.org>
Belton, P.S.; Delgadillo, I.; Halford, N.G.; Shewry, P.R. Kafirin structure and
functionality. Journal of Cereal Science, 2006, 44, 272–286.
Beta, T., Corke, H., Taylor, J.R.N. Starch properties of Barnard Red, a South African
red sorghum of significance in traditional African Brewing. Starch, 2000a, 52, 467–
470.
Beta, T., Corke, H., Rooney, L.W., Taylor, J.R.N. Starch properties as affected by
sorghum grain chemistry. Journal of the Science of Food and Agriculture, 2000b, 81,
245–251.
Beta, T., Rooney, L.W., Marovatsanga, L.T., Taylor, J.R.N. Effect of chemical
treatments on polyphenols and malt quality in sorghum. J. of Cereal Science, 2000c,
31, 295–302.
Bhosale, S.H.; Rao, M.B.; Deshpande, V.V. Molecular and industrial aspects of
glucose isomerase. Microbiol. Rev., 1996, 60, 280–300.
Bird R. and Hopkins R. H.. The action of some alpha-amylases on amylose. Biochem.
J.., 1954, 56, 86-99.
Bos, C. and Norr, N. J. Experiences with the DDS-Kroyer direct hydrolysis process.
Starch, 1974, 26, 181-185.
Buffo, R.A., Weller, C.L., Parkhurst, A.M. Optimization of sulfur dioxide and lactic
acid steeping concentrations for wet-milling grain sorghum. Transactions of the
American Society of Agricultural Engineers, 1997, 40, 1643–1648.
Buffo, R.A., Weller, C.L., Parkhurst, A.M. Wet-milling factors of sorghum and
relationship to grain quality. Journal of Cereal Science, 1998, 27, 327–334.
Buleon, A.; Colonna, P.; Planchot, V.; Ball, S. Starch granules: structure and
biosynthesis. A mini Review. International Journal of Biological Macromolecules,
1998, 23, 85–112.
Chibata, I. Immobilized enzymes: Research and Development. John Wiley and sons:
New York, 1978; pp 132-134.
Corredor, D.Y.; Bean, S.R.; Schober, T.; Wang, D. Effect of decorticating sorghum
on ethanol production and composition of DDGS. Cereal Chemistry, 2006, 83, 17–21.
Daiber, K.H. Enzyme inhibition by polyphenols of sorghum grain and malt. Journal
of the Science of Food and Agriculture, 1975, 26, 1399–1411.
Deb, U. K.; Bantilan, M. C. S.; Ro, A. D.; Rao P. R. Global Sorghum Production
Scenario in Sorghum genetic enhancement: research process, dissemination and
impacts. Bantilan, M. C. S.; Deb, U. K.; Gowda, C. L. L.; Reddy, B. V. S.; Obilana,
A. B. and Evenson, R. E. Eds. 2004. International Crops Research Institute for the
Semi-Arid Tropics (ICRSAT).
Del Pozo-Insfran, D., Urias-Lugo, D., Hernandez-Brenes, C., Serna Saldivar, S.O.
Effect of amyloglucosidase on wort composition and fermentable carbohydrate
depletion in lager beers. Journal of the Institute of Brewing, 2004, 110, 124–132.
Dendy, D. A. V. Sorghum and Millets Chemistry and Technology. USA, St. Paul,
Minnesota, American Association of Cereal Chemists, Inc., 1995, pp. 406.
Denyer, K., Johnson, P., Zeeman, S., Smith, A.M. The control of amylose synthesis.
Journal of Plant Physiology, 2001, 158, 479–487.
Devarajan B.; Pandit A. B., Sorghum flour as Raw Material for Glucose Production.
J. Maharashtra Agric. Univ., 1996, 21 (1), pg. 86-90.
Diao, Y., Walawender, W., Fan, L. Activated carbons prepared from phosphoric acid
activation of grain sorghum. Biosource Technology, 2002, 81, 45–52.
Dufour, J.P.; Melotte; L., Srebrnik, S. Sorghum malts for the production of a lager
beer. Journal of the American Society of Brewing Chemists, 1992, 111, 110–119.
Duodu, K.G.; Taylor, J.R.N.; Belton, P.S.; Hamaker, B.R. Factors affecting sorghum
protein digestibility. Journal of Cereal Science, 2003, 38, 117–131.
Dykes, L. and Rooney, L. W. Sorghum and millet phenols and antioxidants. Journal
of Cereal Science, 2006, 44, 236–251.
Elkhalifa, A. O.; Chandrashekar, A.; Mohamedc, B.E.; Tinay, A.H. Effect of reducing
agents on the in vitro protein and starch digestibilities of cooked sorghum. Food
Chemistry, 1999, 66, 323-326.
Emes, M.J.; Bowsher, C.G.; Hedley, C.; Burrell, M.M.; Scrase-Field, E.S.F.; Tetlow,
I.J. Starch synthesis and carbon partitioning in developing endosperm. Journal of
Experimental Botany, 2003, 54, 569–575.
Ezeogu, L.I.; Duodua, K.G.; Taylora, J.R.N. Effects of endosperm texture and
cooking conditions on the in vitro starch digestibility of sorghum and maize flours.
Journal of Cereal Science, 2005, 42, 33–44.
Figueroa, J.D.C.; Martinez, B.F.; Rios, E. Effect of sorghum endosperm type on the
quality of adjuncts for the brewing industry. Journal of the American Society of
Brewing Chemists, 1995, 53, 5–9.
Hallgren, L. Lager beers from sorghum. in Sorghum and Millets: Chemistry and
Technology. Dendy, D.A.V. Ed. American Association of Cereal Chemists, St. Paul,
MN, USA, 1995, pp. 283–297.
Hamaker, B. R., and Bugusu, B. A. Overview: Sorghum proteins and food quality. In:
Proc. AFRIPRO Workshop on the Proteins of Sorghum and Millets: Enhancing
Nutritional and Functional Properties for Africa. 2003, P. S. Belton and J. R. N.
Taylor, eds. Available at http://www.afripro.org.uk/papers/Paper08Hamaker.pdf.
Pretoria, South Africa.
Higiro, J., Flores, R.A., Seib, P.A. Starch production from sorghum grits. Journal of
Cereal Science, 2003, 37, 101–109.
Ivanova, V.; Dobreva, E.; Legoy, M. D. Acta Biotechnol. 1998, 18, 339-351.
Kandra, L.; Gyemant, G.; Remenyik, J.; Hovanszki G., Liptak, A. FEBS Lett., 2002,
518, 79-82.
Khanal, S.; Montalbo, M.; Leeuwen, J.; Srinivasan, G.; Grewell, D. Ultrasound
Enhanced Glucose Release From Corn in Ethanol Plants. Biotechnology and
Bioengineering, 2007, 98, 978-985.
Kimber, C. T. Origins of domesticated sorghum and its early diffusion to India and
China in Sorghum: Origin, History, Technology, and Production. Smith, C, W.;
Frederiksen, R. A. Eds. Wiley, New york, 2000.
Kleih, U.; Ravi, S. B.; Rao, B. D. and Yoganand, B. Industrial Utilization of Sorghum
in India. Ejournal.icrisat.org, 3, 2007. This study is based upon fieldwork undertaken
in mid-1998 in the context of the project 'Sorghum in India: Technical, policy,
economic, and social factors affecting improved utilization', which was funded by the
Department for International Development (DFID) and jointly undertaken by
ICRISAT, NRCS, and NRI.
Kuriki, T & Umanaka T. The concept of the α-amylase family: Structural similarity
and common catalytic mechanism. Journal of Bioscience and Bioengineering, 1999,
87, 557-565.
Lowry, O. D.; Roseborough, N. J.; Farr, A. L., Rondall, R. J. J Biol. Chem., 1951,
193, 265-275.
Munck, L. New milling technologies and products: Whole plant utilization by milling
and separation of the botanical and chemical components in Sorghum and Millets:
Chemistry and Technology, Dendy, D.A.V. (Ed.). American Association of Cereal
Chemists, St. Paul, MN, USA, 1995, pp. 223–281.
Murty, D.S., Kumar, K.A. Traditional uses of sorghum and millets in Sorghum and
Millets: Chemistry and Technology, Dendy, D.A.V. (Ed.). American Association of
Cereal Chemists, St. Paul, MN, USA, 1995, pp. 185–221.
Nigam, P.; Singh, D. Enzyme and microbial systems involved in starch processing.
Enzyme Microb. Technol., 1995, 17, 770-778.
Ortega Villicana, M.T.; Serna-Saldivar, S.O. Production of lager from sorghum malt
and waxy grits. Journal of the American Society of Brewing Chemists, 2004, 62, 131–
139.
Owuama, C.I. Brewing beer with sorghum. J. of Institute of Brewing, 1999, 105, 23–
34.
Owuama, C.I. Sorghum: a cereal with lager beer brewing potential. World Journal of
Microbiology and Biotechnology, 1997, 13, 253–260.
Park, S. H.; Bean, S. R.; Wilson, J. D. and Schober, T. J. Rapid Isolation of Sorghum
and Other Cereal Starches Using Sonication. Cereal Chemistry, 2006, 83, 611-616.
Perez Siraa, E. E.; Amaiz, M. L. A laboratory scale method for isolation of starch
from pigmented sorghum. Journal of Food Engineering, 2004, 64, 515–519
Radley, J. A. Starch and Its Derivatives. Chapman and Hall, Ltd., London. 1968
Roig, M. G.; Slade, A.; Kennedy J.F. α-amylase immobilized on plastic supports:
stabilities, pH and temperature profiles and kinetic parameters. Biomater. Artif. Cells
Immob. Biotechnol., 1993, 21, 487-525.
Rooney, L.W., Waniska, R.D. Sorghum food and industrial utilization. in Sorghum:
Origin, History, Technology, and Production, Smith, C.W., Frederiksen, R.A. (Eds.)
Wiley, New York, 2000, pp. 689–729.
Singh, V., Moreau, R.A., Hicks, K.B. Yield and phytosterol composition of oil
extracted from grain sorghum and its wet-milled fractions. Cereal Chemistry, 2003,
80, 126–129.
Smith, A.M.; Denyer, K.; Martin, C. The synthesis of the starch granule. Annual
Review of Plant Physiology and Plant Molecular Biology, 1997, 48, 65–87.
Stahlman, P. W.; Wicks, G. A. Weeds and their control in grain sorghum in Sorghum:
Origin, History, Technology, and Production. Smith, C, W.; Frederiksen, R. A. Eds.
Wiley, New york, 2000.
Suresh, K.; Kiran sree, N. and Venkateswer Rao, L. Utilization of damaged sorghum
and rice grains for ethanol production by simultaneous saccharification and
fermentation. Bioresource Technology, 1999a, 68, 301-304.
Suresh, K.; Kiran sree, N. and Venkateswer Rao, L. Production of ethanol b raw
starch hydrolysis and fermentation of damaged grains of wheat and sorghum.
Bioprocess engineering, 1999b, 21, 165-168.
Taylor, J. R. N.; Schober, T. J.; Bean, S. R. Novel food and non-food uses for
sorghum and millets. Journal of Cereal Science, 2006, 44, 252–271.
Tegge, V. G. and Richter G. Sorghum and broken rice as basic materials for glucose
production. Starch, 1982, 34, 386-390.
Tester, R. F.; Karkalas, J.; Qi, X. Starch structure and digestibility Enzme-Substrate
relationship. World’s Poultry Science Journal, 2004b, 60, 186-195.
Tester, R.F.; Karkalas, J. Starch. In: Steinbuchel, A. (Series Ed.) Vandamme, E.J., De
Baets, S., Steinbu¨chel, A. (vol. Eds.), Biopolymers, vol. 6. Polysaccharides. II.
Polysaccharides from Eukaryotes, Wiley–VCH, Weinheim, 2002, pp. 381–438.
Tumturk, H.; Aksoy, S.; Hasirci, N. Food Chem., 2000, 68, 259-266.
van der Maarel M.J.E.C; van der Veen, B.; Uitdehaag, J. C. M.; Leemhuis, H.;
Dijkhuizen, L. Properties and applications of starch-converting enzymes of the α-
amylase family. Journal of Biotechnology, 2002, 94, 137-155.
Wang, F.C., Chung, D.S., Seib, P.A., Kim, Y.S. Optimum steeping process for wet
milling of sorghum. Cereal Chemistry, 2000, 77, 478–483.
Wu, X.; Zhao, R.; Bean, S. R.; Seib, P. A.; Mclaren, J. S.; Madl, R. L.; Tuinstra, M.;
Lenz, M. C. and Wang, D. Factors Impacting Ethanol Production from Grain
Sorghum in the Dry-Grind Process. Cereal Chemistry, 2007, 84, 130-136.
Xie, X.J. and Seib, P.A. Laboratory wet-milling of grain sorghum with abbreviated
steeping to give two products. Starch/Staerke, 2002, 54, 169–178.
Xie, X.J., Liang, Y.T.S., Seib, P.A., Tuinstra, M.R. Wet-milling of grain sorghum of
varying seed size without steeping. Starch/Staerke, 2006, 58, 353–359.
Yang, P. & Sieb, P. Low-input wet-milling of grain sorghum for readily accessible
starch and animal feed. Cereal Chem., 1996, 73, 751–755.
Yang, R., Seib, P.A. Low-input wet-milling of grain sorghum for readily accessible
starch and animal feed. Cereal Chemistry, 1995, 72, 498–503.
Zhan, X.; Wang, D.; Tuinstra, M.R.; Bean, S.; Seib, P.A. and Sun, X.S. Ethanol
and lactic acid production as affected by sorghum genotype and location. Industrial
Crops and Products, 2003, 18, 245-255.
Zhan, X.; Wanga, D. ; Beanb, S.R.; Moc, X.,; Sunc, X.S. and Boyled, D. Ethanol
production from supercritical-fluid-extrusion cooked sorghum. Industrial Crops and
Products, 2006, 23, 304–310.
Zhang, Z.; Niu, Y.; Exkhoff, S. R. and Feng, H. Sonication enhanced corn starch
separation. Starch, 2005, 57, 240-245.
Zhao, R.; Bean, S.R.; Ioerger, B. P.; Wang, D.; Boyle, D. L. Impact of Mashing on
Sorghum Proteins and Its Relationship to Ethanol Fermentation. J. Agric. Food
Chem., 2008, 56, 946–953.
Ciocalteau phenol reagent with the tyrosine residues of the proteins, although other
chromogenic amino acids such as tryptophan, histidine and cysteine and peptide
linkages are also involved. The method is generally applicable in all cases except for
the proteins that do not contain tyrosine. In the case of the simple Lowry method, the
detergents, which may be used for extraction of proteins from the membranes, or
those, secreted during fermentations, interfere with the determinations and hence
The Biuret reaction with alkaline Cu (II) and the reaction of a complex salt of
intense blue green color with the Biuret complexes of tyrosine and tryptophan. The
¾ Test tubes
¾ Pipettes
¾ UV VIS spectrophotometer
A.1.4 Reagents
¾ Folin-Ciocalteau reagent: Ready reagent has been procured for the analysis.
distilled water
distilled water
water
¾ Alkaline copper reagent: The copper reagent is prepared fresh just before use by
tartarate solution
immediately. The solution was incubated at room temperature (30 °C) for exactly 10
min. To this, 0.1 mL of Folin-Ciocalteau reagent (1:1 diluted with distilled water)
was added and vortexed immediately. This was allowed to stand at 30 °C for exactly
30 min. The blue color thus produced was measured with the help of a UV-VIS
was prepared in the protein concentration range of 0–1 mg/mL of BSA as shown in
Figure A.1.
Note
0.07
y = 0.1628x - 0.0051
1
0.06 2
R = 0.9994
Concn of BSA (mg/mL)
0.05
0.04
0.03
0.02
0.01
0
0 0.1 0.2 0.3 0.4 0.5
O. D
Figure A.1. Standard curve for BSA for modified Folin Lowry method
¾ Petri dishes
¾ Oven
A.2.2. Procedure
Petri dish is weighed. 10 g of sorghum flour was spread on the petri dish.
Then it was kept in the oven at 80 °C for drying till constant weight is obtained after
W1 − W2
% moisture content = × 100
Wf
carbon atom in the sugar) in the hemi-acetal or ketal form i.e. not involved in the
glycosidic linkage. This allows the sugar to act as a reducing agent. Whereas,
nonreducing sugar is a saccharide in which all anomeric carbon atoms are in the acetal
form i.e. involved in the glycosidic linkage. The aldehyde (or keto-) form or
hemiacetal (or ketal) form is available for reducing are responsible for the reducing
power of the sugars. When a sugar is oxidized, its carbonyl group (i.e. aldehyde or
This reducing property of sugar was used as a basis for the analysis of
reacts with reducing sugars and other reducing molecules to form 3-amino-5-
nitrosalicylic acid, which is red brown in color and absorbs light strongly at 540 nm.
¾ Test tubes
¾ Pipettes
¾ UV VIS spectrophotometer
above solution pinch by pinch in dark for dissolution, until the complete quantity (i.e.
1 gm) was added. Then the volume was made up to 100 mL by addition of distilled
water to give Dinitrosalicylic acid reagent (DNSA reagent). The reagent was filtered
A.3.5. Procedure
reagent was added to test tube and the test tubes were kept in a boiling water bath for
exactly 10 min. At end of 10 min all test tubes were immediately kept in the cold
water for cooling. Then 10 mL distilled water was added to each test tube.
540 nm against blank sample. Blank sample means 1 mL distilled water instead of
to 1 mL) of the standard solution of glucose were taken in the different test tubes and
the volume was made up to 1 mL using distilled water. 1 mL of DNSA reagent was
added to each test tube and the test tubes were kept in a boiling water bath for exactly
10 min. At end of 10 min all test tubes were immediately kept in the cold water for
cooling. Then 10 mL distilled water was added to each test tube. Absorbance of the
density) was plotted (Fig. A.2) and used as a standard glucose graph in the form of
the samples.
1.4
0.8
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8
O. D
Figure A.2. Standard curve for glucose for DNSA method
A.4.1. Materials
reference standards of glucose and maltose were purchased from Merck India Ltd.
A.4.2. Procedure
Step1: Prewashing the TLC plate with methanol in the TLC chamber. Drying the plate
for ½ hr at 45°C.
Step 2: Application of samples and standards (i.e. synthetic solution containing known
Step 3: TLC plate is then developed in a pre-saturated (saturation time ½ hr) chamber.
Acetonitrile: 0.02 M Na2HPO4 of composition 70:30 (v/v) was used for 1st
of composition 80:20 (v/v) was used for 3rd development. Intermediate drying
was achieved by atmospheric drying for 15 min and drying at 45°C for 15
minutes.
then kept at 120 °C for 10 min. It resulted into colored spots corresponding to
Step 5: Then the TLC plate is scanned at 546 nm using densitometer to get the
and samples.
Step 6: Peak areas were plotted against the known amounts of sugar (standard)
applied to the TLC plate for glucose (G1), maltose (G2), maltotriose (G3),
0.7
2
G1 y = 1.02E-06x + 5.66E-04x
2
0.6 G2 y = 9.26E-07x + 2.67E-04x
2
G3 y = 9.88E-07x + 1.85E-04x
Concn of sugar (glucose equiv.)
2
0.5 G4 y = 9.56E-07x + 1.36E-04x
2
G5 y = 1.15E-06x + 1.08E-04x
0.4 G6
2
y = 7.66E-07x + 4.19E-04x
G7 2
y = 6.40E-07x + 3.96E-04x
0.3
0.2
0.1
0
0 100 200 300 400 500 600 700 800
Peak area
FigureA.3. Standard curve for glucose and malto-oligosaccharides
A.5.1. Principle
amylopectin. Amylose has strong affinity towards iodine and forms amylose-iodine
complex with it. Color of the amylose-iodine complex depends upon DP (degree of
polymerization) of amylose i.e. no of glucose units present in the chain and how it
Amylopectin have little affinity for iodine and gives red coloration with it.
(Radley 1968). Concentration of soluble starch was determined from a standard plot
KI-I2 reagent was prepared as 0.05% w/v iodine and 0.5% w/v potassium
iodide in distilled water. The KI-I2 reagent was stored in an amber colored bottle.
A.5.4. Method:
Standard starch solution (1% w/v) was prepared by gelatinizing the starch
slurry in boiling water for 5 minutes. Different quantities of starch solution (0 to 1ml)
were taken in different test tubes and the volume was made up to 1 ml using distilled
water. 5 ml of KI-I2 reagent was added to each test tube and the mixture was
incubated for 15 minutes in dark. The color developed was measured at 660nm on the
absorbance (optical density) was plotted (Fig. A.4) and was used as a standard.
0.6
0.5 y = 0.7401x
2
R = 0.9987
Concn of starch (mg/mL)
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8
absorbence
Figure A.4. Standard curve for starch for starch-iodine method
function StarchhydrolysisusingimmobilizedBLA
clc;
A=5.78669519;
C=5.78669519*5.487459631; %C= maximum DE
D=0.528008427; %D=rate constant
E=1.86; So=90; % Run 24
K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01]; %Initial guess
P=[E So A C D];
fid = fopen('Run 24.txt'); % File containing experimental data
texp=fscanf(fid,'%g',[10 1]); % experimental time
GCexp=fscanf(fid,'%g',[10 8]); % experimental change in composition of G1-G7
texp=texp';
GCexp=GCexp';
fclose(fid);
GCexp=GCexp./100;
g=GCexp(:,1);
lb=[0 0 0 0 0 0 0 0 0 ];
ub=[];
ub=[20 20 20 20 20 20 20 20 20 ];
options = optimset('LargeScale', 'off');
kvalues=fmincon(@kinetics,K,[],[],[],[],lb,ub,[],options,P,g,texp,GCexp);
K=kvalues
tend=max(texp);
sol = ode45(@f,[0 tend],g,[],P,K);
G = deval(sol,texp);
GC=G;
G=G';
GC'*100
function dGdt=f(t,G,P,K)
k1=K(1); k2=K(2); k3=K(3); k4=K(4);k5=K(5);k6=K(6);k7=K(7);
kcat6=K(8); kcat7=K(9); %h1=K(10);
h1=0; %value of h1
E=P(1);So=P(2);A=P(3);C=P(4);D=P(5);
dGdt = [ E*(k1*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));
E*(k2*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));
E*(k3*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));
E*(k4*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));
E*(k5*(1-G(1)-G(2)-G(3)-G(4)-G(5)-h1*So/TDW(t,P)));
E*(k6*(1-G(1)-G(2)-G(3)-G(4)-G(5)-G(6)-h1*So/TDW(t,P))-kcat6*G(6));
E*(k7*(1-G(1)-G(2)-G(3)-G(4)-G(5)-G(6)-G(7)-h1*So/TDW(t,P))-kcat7*G(7));
];
SYNOPSIS
OF THE THESIS TO BE SUBMITTED TO
UNIVERSITY OF MUMBAI
FOR THE DEGREE OF
DOCTOR OF PHILOSPHY (TECHNOLOGY)
IN THE SUBJECT OF CHEMICAL ENGINEERING
Registration Number and Date 188 (A); Dated: 29th March, 2004
(Satish D. Shewale)
cereal in world after wheat, rice, barley and maize. Production of sorghum in 2004-2005 in
world was 58 Million Tonnes. India was the second largest producer of sorghum after
U.S.A. with production of 10.8 million Tonnes in 2004-2005. Other sorghum producing
countries are Mexico, Nigeria, and China. Maharashtra is the largest sorghum producing
state in India with production of 5.8 million Tonnes. Sorghum ranks third in the major food
grain crops in India. The plant originated in equatorial Africa and is distributed throughout
the tropical, semi-tropical, arid and semi arid regions of the world. It has a potential to
compete effectively with crops like maize under good environmental and management
conditions. The greatest merit of sorghum is that it has marginal lands under moisture
stress or excessive moisture conditions. It is one of the most widely grown dry land food
A few names of sorghum are milo, jowar, kafir corn, guinea corn, and cholam.
Sorghum is also termed as “Nature-cared crop” because it has strong resistance to harsh
environments such as dry weather and high temperature in comparison to other crops, it is
usually grown as a low-level chemical treatment crop with limited use of pesticides and it
Though sorghum is one of the few cereals that can be grown in the semi-arid
regions of the country, demand for the sorghum is decreasing with enhanced
socioeconomic status of the population in general and easy availability of other preferred
source of staple food for humans, it also serves as an important source of cattle feed and
fodder, but at lower prices. Also about 10-20 % of the production gets wasted due to
blackening of crop and lack of good facilities for storage etc. Hence an industrial
application is needed so that sorghum cultivation becomes economically viable for farmers
through value addition products. There is very small amount of work (Devarajan and
Pandit 1996; Aggarwal et al. 2001) done on value addition to sorghum. Hence, the
objective of the present work was a production of value addition products like glucose and
maltose from different qualities of sorghum i.e. healthy, germinated and blackened. In the
present work, we have used sorghum flour for hydrolysis instead of first isolating starch
and then its subsequent use for liquefaction and saccharification because the yields of
starch isolation from sorghum are around 50-60% i.e rest part (40 - 50%) is getting wasted
Constituents of sorghum are starch, proteins, moisture, fats and oil, fibers and ash
with percentage contents in the range of 65-75, 9-11, 9-13, 1-1.5, 1.5-2 and 1-2
respectively (Owuama 1997). Production of glucose from sorghum flour consists of two
amyloglucosidase (AG). However, enzymes can be utilized in the two forms viz. free and
immobilized. Limitations of free enzymes lies in its only once usability and effluent
problem. Immobilized enzymes are the enzymes which are physically confined or localized
in a certain defined region of space with retention of their catalytic activities and which can
be used repeatedly and continuously. Immobilized enzyme has several advantages over the
free enzyme as follows; reusability of the enzyme, continuous operation of the system,
easy separation of product from the enzyme, less effluent problems, increased stability of
the enzyme and few side products and more favorable refining conditions. However, it has
disadvantages of lower reaction rates due to diffusion limitation of substrate molecules to
enzyme active site and diffusion out of product molecules from active site to bulk
solution.
Hence, firstly it was decided to develop a process for the production of glucose
from sorghum flour using immobilized enzymes. This process constitutes following steps
slurry through the bed of immobilized BLA and AG. But before studying this, it was
necessary to first immobilize BLA on beads and study its catalytic characteristics.
(pore size ∼ 3 µm) cross-linked cellulose matrix (CELBEADS; Lali et al. 2003) by
covalent binding method. After immobilization, it was observed that optimum operational
pH decreased slightly from 5.6 to 5.2 (because of the difference in the hydronium ion
concentration in the bulk solution and the microenvironment in the vicinity of immobilized
enzyme molecule) and optimum temperature changed from 55°C to 55 – 70°C (because of
Activity of free BLA was observed to be 16500 EU/mL at 55°C and 6 pH. Activity of
immobilized BLA was observed to be 18.5 EU/mL at 55°C and 5.2 pH.
of starch hydrolysate. Free BLA is reported to be endo-amylase with random attack action
pattern. However, after immobilization it behaves like exo-amylase with dual specificity
towards maltopentaose and maltotriose. Immobilized BLA was observed to produce
different saccharide profile than free BLA at any value of dextrose equivalent. It was
observed that pH, temperature and initial starch concentration has a significant effect on
the saccharide profile of starch hydrolysate produced using immobilized BLA in batch
mode, whereas ratio of concentration of enzyme units to initial starch concentration has no
influence on the same. For free BLA, hydrolysis ceased at DE of around 42-43 because
BLA could not hydrolyze more α-(1-4) linkages due to the presence of branched dextrins;
marginally low (around 36-37). While checking operation stability of immobilized BLA
without intermittent washing between two subsequent 8 hr batches, it was observed that in
the batch mode operation, the initial rate decreases to 70%, whereas in the packed bed it
decreases to about 20 %. A semi-empirical kinetic model has been used for the prediction
It was observed that the use of immobilized BLA is not suitable for the production
9 After mixing gelatinized sorghum slurry with beads, it was very difficult to
separate beads from the slurry even after the completion of reaction
Thus, it may be more economically viable to use BLA and other enzymes in the free form
Production of glucose from sorghum flour comprises two steps viz. 1. Liquefaction
glycosidic bond from non reducing end and releases glucose molecules. Specialty of AG is
that it can cleave both α-(1-4) and α-(1-6) bonds and enables complete hydrolysis of starch.
Hence, Liquefaction process was first optimized. Flour was made from sorghum grains
using old fashioned flour mill. Liquefaction of sorghum flour was performed in a 250 mL
Liquefaction of sorghum slurry (30% w/v in 0.05 M acetate buffer of pH 6) was performed
liquefaction was monitored using starch-iodine colorimetric reaction. When color becomes
reddish with tinge of violet (at this stage DE of liquefact was around 15), liquefaction is
considered to be completed. Liquefaction process is optimized for the reaction time of 1.5
(0.04-0.16% v/w of sorghum flour), and CaCl2 concentration (0-500 ppm) and temperature
in the range of 75 - 95°C. Optimized values of temperature, pH, slurry concentration, BLA
slurry, 0.06 % v/w of sorghum flour i.e. 0.086 % v/w of sorghum starch and 200 ppm
respectively. Sorghum of different qualities i.e. healthy, blackened and germinated were
used for liquefaction. Liquefaction of healthy and blackened sorghum gets completed in
1.5 h under optimized conditions, but liquefaction rate for blackened sorghum was slightly
lesser than that for healthy sorghum. However liquefaction of germinated sorghum gets
completed in 1 h only. We have also studied the effect of prior sonication on the
the intensity. This must be happening because in the sorghum grain there are three
different types of endosperm viz. floury endosperm (starch granules are loosely associated
with protein material), corneous endosperm (starch granules packed inside protein bodies)
and peripheral endosperm (large amount of protein with less amount of starch); sonication
must be making starch granules free, which otherwise are packed inside protein bodies and
are inaccessible for enzyme action. It was also observed that sonication prior to
the slurry was reduced to 4.5 and was hot filtered using muslin cloth to remove large
pericarp particles, fibers and proteins. Then filtrate was saccharified using AG.
may not be same as the optimum operating temperature. Hence thermo stability of AG was
glucose using AG were pH 4.5, 0.05 %v/ w of starch and saccharification time of 24 h. The
value of % saccharification to glucose using AG was in the range of 70- 90% depending
upon following factors; 1. Sonication of sorghum slurry before liquefaction and 2.
Washing of cake (obtained after hot filtration) followed by 2nd filtration and mixing of both
the filtrates. These experiments were performed for healthy sorghum, germinated sorghum
and blackened sorghum. It was observed that sonication of slurry, prior to liquefaction
BBA solution at pH 5.5 and 50 °C. Optimum pH and temperature for pullulanase (PU)
were 3.8 – 4.4 and 60 °C respectively. Activity of PU was 2950 EU per mL of commercial
glycosidic bond from non reducing end and releases maltose molecules. Limitation of
BBA is that it can neither cleave nor bypass α-(1-6) glycosidic bond. Hence use of BBA
results into the production of maltose and β-limit dextrins. Pullulanase (PU) is an endo-
amylase, which hydrolyses only α-(1-6) glycosidic bond. Hence, the combined use PU
with BBA will produce maltose in major quantity; whereas maltotriose and glucose will
For maltose production, liquefaction part remains the same. Filtrate after
liquefaction was also saccharified to maltose using barley β-amylase with or without
pullulanase. Optimized reaction conditions for the saccharification to maltose were pH 5.5,
50 °C, 0.04 % v/ w of starch and reaction time of 24 h. Experiments were performed on
healthy sorghum, germinated sorghum and blackened sorghum for the production of
maltose. The value of % saccharification to maltose using BBA was in the range of 60 -
86% depending upon the following factors; 1. Sonication of sorghum slurry before
liquefaction ; 2. Washing of cake after hot filtration followed by 2nd filtration and mixing
of both filtrates and 3. Use of pullulanase along with BBA. It was observed that sonication
A cost comparison analysis of these methods have been carried out and it has been
shown that a significant value addition of the sorghum can be achieved by the processes
References
1. Devarajan B.; Pandit A. B., Sorghum flour as Raw Material for Glucose Production.
2. Aggarwal N. K.; Nigam P.; Singh D.; Yadav B. S., Process optimization for the
source of starch. World Journal of Microbiology & Biotechnology, 2001, 17, 411-415.
3. Owuama, C.I., Sorghum: a cereal with lager beer brewing potential. World Journal of