Studies in the Enzymatic depolymerisation of

natural polysaccharides

A THESIS SUBMITTED
TO THE
UNIVERSITY OF MUMBAI

FOR THE


Ph. D. (Technology) DEGREE
IN
CHEMICAL ENGINEERING


SUBMITTED BY
SATISH DASHARATH SHEWALE


UNDER THE GUIDENCE OF
Prof. A. B. Pandit


INSTITUTE OF CHEMICAL TECHNOLOGY
UNIVERSITY OF MUMBAI
MATUNGA, MUMBAI-400 019.
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,



Prof. Aniruddha B. Pandit
(Research supervisor)
UGC Scientist ‘C’ (Professor’s Grade),
Chemical Engineering Division,
Institute of Chemical Technology,
University of Mumbai,
Matunga, Mumbai – 400 019.
Date:
Place: Matunga, Mumbai – 400 019



CERTIFICATE

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.




Prof. Aniruddha B. Pandit
(Research Supervisor)
UGC Scientist ‘C’ (Professor’s Grade)
Chemical Engineering Division,
Institute of Chemical Technology,
University of Mumbai,,
Matunga, Mumbai – 400 019.


Date:
Place: Matunga, Mumbai-400 019.







List of publications

1. Satish D. Shewale; Aniruddha B. Pandit “Hydrolysis of soluble starch using B.
licheniformis α-amylase immobilized on superporous CELBEADS”,
Carbohydrate Research, 2007, 342 (8), Pg. 997-1008.
2. Satish D. Shewale; Aniruddha B. Pandit “Enzymatic production of glucose
from different qualities of sorghum and application of ultrasound to enhance
the yield”, Carbohydrate Research, Forwarded for publication.
3. Satish D. Shewale; Aniruddha B. Pandit “Enzymatic production of maltose
from different qualities of sorghum and application of ultrasound to enhance
the yield”, Carbohydrate Research, To be Forwarded for publication.







































Dedicated to my family . . . .



Acknowledgement

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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 express my sincere gratitude to my research supervisor Prof. Aniruddha B.
Pandit for his constant encouragement, bearing with me and supporting me
in all my downtimes, not spoon feeding me in the research work and bringing
the best in me. I always amazed by his knowledge, his way of thinking, calm
nature, and switching gears from one research area to other in the lab
meeting. His enthusiasm always motivated me and compelled me to do at my
best. I have been enormously fortunate to have a supervisor who has always
had the time and patience to answer the many (often quite stupid and tedious
I think) questions I have had. I am also thankful to him for checking several
versions of the article that needs to be forwarded for publication, reports,
chapters of thesis etc. without getting bore of mistakes in them.
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.
I am grateful to UGC for providing research fellowship during tenure of Ph D
work.
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.
Special thanks to my dear friends, Atul (Ghatotkach) from Physics lab,
Mahendra (Mamu) from BNT lab, Anup, Rajesh, Santosh from GDY lab for
their wonderful company.
Special thanks to my Masters group Sunil (Barakya), Yoko, Ninad, Balu,
Randheer (Bhaiyya) and Shreenivas for their constant encouragement and
timely help.
I also thank Chindarkar, Subhash, and Parab for their cooperation during my
work and their always helping nature.
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

2. Overview of starch and starch hydrolysis products 7
2.1. Starch 8
2.1.1. Starch composition 11
2.1.1.1. Amylose 11
2.1.1.2. Amylopectin 12
2.1.1.3. Other minor components 14
2.1.2. Starch granule structure 15
2.1.3. Gelatinization of starch 18
2.1.4. Starch production and applications 20
2.2. Starch hydrolysis products 21
2.2.1. Starch hydrolyzing enzymes 22
2.2.1.1. Bacterial α-amylase 27
2.2.1.2. Fungal α-amylase 28
2.2.1.3. Glucoamylase 29
2.2.1.4. β-amylase 29
2.2.1.5. Pullulanase 30
2.2.1.6. Isoamylase 30
2.2.1.7. Glucose isomerase 30
2.2.2. Maltodextrins 31
2.2.2.1. Production 31
2.2.2.2. Application 34
2.2.3. Glucose syrup 35
2.2.3.1. Production 35
2.2.3.2. Applications 39
2.2.4. Dextrose hydrolysate, crystalline dextrose and liquid dextrose 40
2.2.4.1. Production 40
2.2.4.2. Applications 44
2.2.5. Maltose syrup 45
2.2.5.1. Production 45
2.2.5.2. Applications 46
2.2.6. Fructose syrup 47
2.2.5.1. Production 47
2.2.5.2. Applications 48

3. Sorghum: Literature Review 49
3.1. Introduction, Origin and Geographical distribution of sorghum 50
3.2. Taxonomy 52
3.3. Production, cultivation area and yield of sorghum 55
3.3.1. Trends in production, cultivation area and yield in the world 55
3.3.2. Trend in cultivation area, production and yield of sorghum 62
in India and different states of India
3.4. Plant anatomy and growth 66
3.4.1. Botanical parts of sorghum plant 66
3.4.2. Growth of sorghum plant 69
3.4.3. Optimum growth requirement of sorghum plant 71
3.5. Grain morphology 73
3.6. Utilization of sorghum 80
3.6.1. Food use 80
3.6.2. Industrial utilization 85
3.6.2.1. Animal feed 85
3.6.2.2. Alcohol industry 87
3.6.2.3. Starch industry 89
3.6.2.4. Other industries 90
3.7. Insect pests, Diseases and weeds on sorghum 91
3.8. Factors affecting industrial utilization of sorghum 91
3.8.1. Gelatinization of starch 92
3.8.2. Protein digestibility 94
3.8.3. Starch digestibility 95
3.8.4. Tannin content in sorghum 99
3.9. Production of ethanol from sorghum: Literature review 99
3.10. Production of starch from sorghum: Literature review 105


4. Hydrolysis of soluble starch using B. licheniformis α-amylase 108
immobilized on superporous CELBEADS
4.1. Introduction and literature review 109
4.1.1. Immobilized enzymes 111
4.1.2. Immobilization of bacterial α-amylase 113
4.1.3. Objectives 114
4.2. Experimental 115
4.2.1. Materials 115
4.2.2. Methods 116
4.2.2.1. Measurement of protein concentration and 116
reducing sugar concentration.
4.2.2.2. HPTLC analysis. 116
4.2.2.3. Immobilization of B. licheniformis α-amylase (BLA) 118
on CELBEADS.
4.2.2.4. Amylolytic activity measurement. 120
4.2.2.4. A. Free BLA. 120
4.2.2.4. B. Immobilized BLA. 120
4.2.2.5. Measurement of kinetic constants of free 121
and immobilized BLA.
4.2.2.6. Hydrolysis of soluble starch using immobilized BLA 122
in batch mode.
4.2.2.7. Thermostability and reusability of immobilized BLA. 123
4.2.2.8. Hydrolysis of soluble starch using immobilized BLA 123
in packed bed or expanded bed mode.
4.2.2.9. Measurement of residence time in the packed bed. 125
4.3. Result and Discussion 125
4.3.1. Immobilization of bacterial α-amylase on CELBEADS. 125
4.3.2. pH and temperature dependence of activity of free and 126
immobilized BLA, and their catalytic properties
4.3.3. Effect of reaction conditions on hydrolysis of soluble 129
starch using immobilized BLA and saccharide composition
4.3.3.1. Effect of pH. 129
4.3.3.2. Effect of temperature. 132
4.3.3.3. Effect of initial starch concentration, [S]
0
and [IEU]/[S]
0
. 134
4.3.4. Comparison of saccharide composition of starch hydrolysate 138
using free and immobilized BLA
4.3.5. Thermostability and reusability of immobilized BLA 142
4.3.6. Semiempirical model for prediction of saccharide composition 144
4.3.7. Effect of mode of operation on hydrolysis of soluble starch 147
4.3.8. Hydrodynamic stability of immobilized BLA 149
4.3.9. Hydrolysis of sorghum slurry using immobilized BLA. 151
4.4. Conclusions 152

5. Enzymatic production of glucose from sorghum 154
5.1. Introduction and literature review 154
5.2. Experimental 157
5.2.1. Materials 157
5.2.2. Analytical methods 158
5.2.2.1. Measurement of protein concentration, reducing sugar 158
concentration and concentrations of malto-oligosaccharides.
5.2.2.2. Measurement of moisture content of sorghum flour 158
5.2.2.3. Measurement of particle size distribution of 158
sorghum flour
5.2.2.4. Measurement of starch content of sorghum flour 158
5.2.3. Amylolytic activity measurement 159
5.2.3.1. Free bacterial α-amylase (BLA) 159
5.2.3.2. Free amyloglucosidase (AG) 159
5.2.3.3. Free pullulanase (PL) 160
5.2.4. Thermostability study of amyloglucosidase (AG) 160
5.2.5. Optimization of AG: PL ratio for saccharification 161
5.2.6. Experimental work for production of glucose from sorghum 161
5.2.6.1. Optimization of liquefaction of sorghum flour 163
5.2.6.2. Liquefaction of sorghum of different varieties 164
5.2.6.3. Effect of prior ultrasound treatment on the 165
liquefaction of sorghum
5.2.6.4. Optimization of saccharification. 165
5.3. Results and Discussion 167
5.3.1. Studies in the liquefaction process 167
5.3.1.1. Optimization of liquefaction process 167
5.3.1.1. A. Effect of pH. 167
5.3.1.1. B. Effect of BLA concentration. 167
5.3.1.1. C. Effect of CaCl2 concentration. 168
5.3.1.1. D. Effect of sorghum slurry concentration 169
5.3.1.1. E. Effect of liquefaction temperature. 170
5.3.1.1. F. Liquefaction of sorghum of different varieties 171
5.3.1.2. Effect of prior ultrasound treatment on liquefaction. 173
5.3.2. Optimization of saccharification 174
5.3.2.1. Properties of free amyloglucosidase and pullulanase 174
5.3.2.2. Thermostability of amyloglucosidase and 176
optimization of operating temperature for saccharification
5.3.2.3. Optimum ratio of amyloglucosidase units to 178
pullulanase units for saccharification
5.3.2.4. Optimization of amyloglucosidase concentration 180
5.3.3. Saccharification of sorghum liquefact 181
5.3.4. Effect of ultrasound treatment on particle size distribution 183
5.3.5. Studies on effect of different process parameters on 184
% saccharification
5.3.5.1. Effect of washing of cake obtained after hot filtration 184
5.3.5.2. Effect of ultrasound treatment on % saccharification 185
5.3.6. Economics of the process of production of glucose from 190
sorghum of different varieties
5.4. Conclusions 194

6. Enzymatic production of maltose syrup from sorghum 196
6.1. Introduction and literature review 197
6.2. Experimental 197
6.2.1. Materials 197
6.2.2. Analytical methods 198
6.2.2.1. Measurement of protein concentration, reducing sugar 198
concentration and concentrations of malto-oligosaccharides.
6.2.2.2. Measurement of moisture content of sorghum flour 198
6.2.2.3. Measurement of particle size distribution of 198
sorghum flour
6.2.2.4. Measurement of starch content of sorghum flour 198
6.2.3. Amylolytic activity measurement 199
6.2.3.1. Free bacterial α-amylase (BLA) 199
6.2.3.2. Free Barley β-amylase (BBA) 199
6.2.3.3. Free pullulanase (PL) 199
6.2.4. Thermostability study of Free Barley β-amylase (BBA) 200
6.2.5. Production of glucose from sorghum: Experimental work 201
6.2.5.1. Liquefaction of sorghum flour 202
6.2.5.2. Optimization of saccharification. 202
6.3. Results and Discussion 203
6.3.1. Optimization of saccharification 203
6.3.1.1. Properties of free Barley β-amylase (BBA) 203
and pullulanase
6.3.1.2. Thermostability of Barley β-amylase and 205
optimization of operating temperature for saccharification
6.3.2. Saccharification of sorghum liquefact 208
6.3.3. Studies on effect of different process parameters on 210
% saccharification
6.3.4. Economics of the process of production of maltose syrup 211
from sorghum of different varieties
6.4. Conclusions 215
6.5. Alternative approaches for value addition to sorghum 216

References 218
Appendix A. Analytical methods 233
Appendix B. Matlab code to find kinetic constants 243
Synopsis
Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
1
















1. Introduction






Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
2
Sorghum (Sorghum bicolor L. Moench) is an important drought resistant
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
(www.fas.usda.gov). Leading sorghum producing countries were United States
(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.
Sorghum ranks third in the major food grain crops in India.
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 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
rotation systems (Smith and Frederiksen, 2000).
Though, production of sorghum is high in India, demand for the sorghum is
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
Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
3
sufficient quantities at affordable prices. Hence, in addition to being a major source
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
human consumption. Several mold-causing fungi are producers of potent mycotoxins
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
even unfit for animal consumption.
Hence, an industrial application is needed to be exploited for normal and
blackened sorghums in order to make sorghum cultivation economically viable for
farmers, through value added products. There is very small amount of research done
on value addition to sorghum through; production of glucose (Devarajan and Pandit,
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
attributed to reduced sorghum starch digestibility (Lichtenwalner 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 digestibility (Duodu et
Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
4
al., 2003) after cooking i.e. heat-moisture treatment of sorghum flour. There is no
literature available on value addition products using blackened sorghum.

Research aim
Objective of the present work was a production of value addition products:
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
saccharification as the yields of starch isolation from sorghum were reported to be
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.

Thesis Outline
Starch structure and chemistry, action of different types of enzymes used for
starch hydrolysis are described in chapter 2. Production, properties and application of
different starch hydrolysis products are also discussed in Chapter 2. Attempt is made
to cover almost everything related to sorghum in chapter 3. In the chapter 3, origin,
geographical distribution, taxonomy, plant anatomy, plant growth and grain
morphology of sorghum are described. Trends in production, cultivation area and
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
(Gelatinization of sorghum starch, protein digestibility, starch digestibility and tannin
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
Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
5
sorghum is reviewed in chapter 3.
Due to several advantages (like reusability of the enzyme, continuous
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
enzymes. This process constitutes following steps viz. 1. Gelatinization of 15 %
sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of
immobilized B. licheniformis α-amylase (BLA) and amyloglucosidase (AG). But
before studying this, it was necessary to first immobilize BLA on beads and study its
catalytic characteristics. Chapter 4 covers studies in the immobilization of BLA on
rigid superporous (pore size ∼ 3 µm) cross-linked cellulose matrix (CELBEADS) and
hydrolysis of soluble starch using immobilized BLA.
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
intensify glucose production (yield) by means of ultrasound treatment. Liquefaction
(using B. licheniformis α-amylase) and saccharification (using amyloglucosidase)
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
prior to liquefaction was studied on the process of liquefaction and saccharification at
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
intensify its production (yield) by means of ultrasound treatment. Liquefaction part
remains same as that in the chapter 5. Saccharification (using β-amylase and/or
pullulanase) process was optimized with the use of normal sorghum flour as a starting
Chapter 1: Introduction
Studies in the Enzymatic depolymerisation of natural polysaccharides
6
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
on the process of saccharification at optimized conditions.
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
also reported in the appendix B.

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
7















2. Overview of starch and
starch hydrolysis products








Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
8
2.1. Starch
Starch is an abundant carbohydrate distributed worldwide in green plants,
where it accumulates as microscopic granules. Starch is a food reserve that sustains
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
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 products of enzymatic hydrolysis of starch,
namely maltodextrins, high maltose syrup, maltose, glucose syrup, dextrose, and
fructose, which are used in food and pharmaceutical industry.
Starch biosynthesis is a complex process (Ball, 1995, 1998; Buleon et al.,
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
to uridine diphosphate glucose (UDP-glucose) and fructose by sucrose synthase, the
UDP-glucose being subsequently converted to glucose-1-phosphate (G-1-P) in the
presence of pyrophosphate (PPi) by UDP-glucose pyrophosphorylase. Then this is
itself converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase. The G-6-P
is translocated across the amyloplast (the intra-cellular organelle responsible for
starch biosynthesis in storage tissues) membrane by specific translocators and is
converted to G-1-P by phosphoglucomutase. There is some evidence that, in cereals at
least, G-1-P may be (a) translocated directly into the amyloplast or (b) be converted
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
9
to, and translocated as, adenosine diphosphate glucose (ADP-glucose) generated as a
consequence of cytosol based adenosine diphosphate (ADP)-glucose
pyrophosphorylase activity in the presence of adenosine triphosphate (ATP). Using
amyloplast located ADP-glucose pyrophosphorylase, G-1-P within the amyloplast is
(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
molecules. Granule bound starch synthase can elongate maltooligosaccharides to form
amylose and is considered to be responsible for the synthesis of this polymer. Soluble
starch synthase is considered to be responsible for the synthesis of unit chains of
amylopectin. Starch branching enzyme creates branching in amylopectin by linking
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
many plant species. Characteristics of starch granule viz. size (~1–100 µm in
diameter), shape (round, lenticular, polygonal), size distribution (uni- or bi-modal),
association as individual (simple) or granule clusters (compound) and composition
(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
and potatoes contributing the major proportion in the world.




Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
10
Table 2.1. Characteristics of starch granules from different botanical sources
(Tester et al., 2004a).

Starch Type Shape Distribution Size (µm)
Barley Cereal Lenticular (A-type), Bimodal 15–25, 2–5
spherical (B-type)
Maize Cereal Spherical/polyhedral Unimodal 2–30
(waxy and normal)
Amylomaize Cereal Irregular Unimodal 2–30
Millet Cereal Polyhedral Unimodal 4–12
Oat Cereal Polyhedral Unimodal 3–10(single)
80 (compound)
Pea Legume Rentiform (single) Unimodal 5–10
Potato Tuber Lenticular Unimodal 5–100
Rice Cereal Polyhedral Unimodal 3–8 (single)
150 (compound)
Rye Cereal Lenticular (A-type) Bimodal 10–40
Spherical (B-type) 5–10
Sorghum Cereal Spherical Unimodal 5–20
Tapioca Root Spherical/Lenticular Unimodal 5–45
Triticale Cereal Spherical Unimodal 1–30
Sago Cereal Oval Unimodal 20–40
Wheat Cereal Lenticular (A-type) Bimodal 15–35
Spherical (B-type) 2–10








Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
11
2.1.1. Starch composition
The chemical composition of starch granules, which depends on its biological
origin has a considerable impact on starch processing technologies. Starch granules
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
approximately 98–99% of the dry weight. Percentage content of amylose and
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
exceeds ~36% (Tester et al., 2004a).
2.1.1.1. Amylose
Amylose is a relatively long, linear α-glucan containing around 99% α(1→4)
and 1% α(1→6) linkages and differs in size and structure depending on botanical
origin. Amylose has a molecular weight of approximately 1 × 10
5
– 1 × 10
6
, a degree
of polymerisation (DP) of 324–4920 with around 9–20 branch points equivalent to 3–
11 chains per molecule. Each chain contains approximately 200–700 glucose residues
equivalent to a molecular weight of 32,400–113,400. (Tester et al., 2004a) Structure
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.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
12

Figure 2.1. Basic structure of amylose and amylopectin. Adapted from Tester et al.
(2004a)
2.1.1.2. Amylopectin
Amylopectin is a much larger molecule than amylose with a molecular weight
of 1 × 10
7
– 1 × 10
9
and a heavily branched structure built from about 95% α(1→4)
and 5% α(1→6) linkages with DP typically within the range 9600–15,900. In
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
broad distribution profile. (Tester et al., 2004a) Structure of amylopectin is shown in
the Fig. 2.1.
The individual chains can be specifically classified in terms of their lengths
(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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
13
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
to the structure of amylopectin (Fig. 2.2), the A-chains of amylopectin are α - (1 → 6)
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).
Depending on the CL and correspondingly the number of (radial) clusters traversed
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)

Figure 2.2. Schematic representation of a section of amylopectin indicating the
branching pattern of unit α - (1 →4) chains (A, B1–B3) joined together by α - (1 →6)
linkages (branch points). (Hizukuri, 1986 as cited in Tester et al., 2004a)




Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
14
2.1.1.3. Other minor components
Minor components associated with starches correspond to three categories of
materials: (i) particulate material, composed mainly of cell-wall fragments; (ii)
surface components, removable by extraction procedures; and (iii) internal
components. The main constituents of surface components are proteins, enzymes,
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
form of phospholipids. (Buleon et al., 1998)
Lipids represent the most important fraction associated with the starch
granules. According to Morrison (1981) three categories of cereal lipids can be
distinguished, i.e. nonstarch, lipids on surface of starch granule and intragranular
lipids. The non-starch lipids comprise triacylglycerols, diacylgalactosylglycerols,
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
amylose and amylose-lipid complex (Tester et al., 2004a). Amylose-lipid complex is
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).
Lipids on the surface of starch granules comprise triglycerides, glycolipids,
phospholipids and free fatty acids derived from the amyloplast membrane and non-
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
15
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
no internal ones (Morrisson, 1981, 1988; Nebesny et al., 2002).
Monoacyl lipids will induce the formation of amylose–lipid complexes during
gelatinization. They will restrict swelling, dispersion of the starch granules and
solubilization of amylose, thus generating opaque pastes with reduced viscosity and
increased pasting temperatures. (Buleon et al., 1998)
In commercially available purified starches, moisture content ranges from 10
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
of starch. (Tester et al., 2004b)

2.1.2. Starch granule structure
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
roughly spherical. These granules are synthesized by plants as semi-crystalline
matrices, where crystallinity is generated by registration of amylopectin double
helices into crystalline lamellae interspersed with amorphous lamellae comprising α-
(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
alternating crystalline-amorphous lamellae provide the basis of semi-crystalline
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
16
(‘dark’) growth rings which comprise about 16 crystalline lamellae, which are of
approximately the same width as the interspersed amorphous (‘light’) growth rings
(~140nm). (Buleon et al., 1998; Tester et al., 2004a, 2004b)
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
the types of starches (Sajilata et al., 2006) are as follows.
Type A. The type A structure has amylopectin of chain lengths of 23 to 29 glucose
units. The hydrogen bonding between the hydroxyl groups of the chains of
amylopectin molecules results in the formation of outer double helical structure. In
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.
Type B. The type B structure consists of amylopectin of chain lengths of 30 to 44
glucose molecules with water inter-spread. This is the usual pattern of starches in raw
potato and banana.
Type C. The type C structure is made up of amylopectin of chain lengths of 26 to 29
glucose molecules, a combination of type A and type B, which is typical of peas and
beans.
Schematic representation of starch granule architecture is given in the Fig. 2.3.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
17


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)

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
18
Literature till now hasn’t completely understood the precise location of
amylose in the starch granule. Amylose may be located in bundles between
amylopectin clusters and randomly interspersed among clusters in both crystalline and
amorphous regions. In high-amylose starches amylose probably forms double helices
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
starch it may be partly co-crystallized with amylopectin. Large amylose molecules
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
able to leach from the granule (Oates, 1997).
2.1.3. Gelatinization of starch
Gelatinization of starch is crucial step for many starch based industrial and
food applications. For example, gelatinization of starch is required prior to enzymatic
hydrolysis to make starch accessible for the amylolytic enzymes. Industrial
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.
When the temperature of an aqueous starch-water suspension is increased,
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
19
include loss of birefringence, change in x-ray diffraction pattern, absorption of water
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%)
is formed. At the molecular level, the gelatinization event is initiated by water
‘plasticising’ amorphous rearrangements and ultimately hydrating double helices as
they unravel as a consequence of the elevated temperature. Gelatinization temperature
of starch is mainly dependent on its source or botanical origin. The gelatinisation
process is shown schematically in Figure 2.4. Gelatinization of starch is affected by
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;
Baks et al., 2008)

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).

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
20
Starch can also be gelatinized by the application of high pressures and or
temperatures in the presence of water. During high-pressure gelatinization,
disintegration of the granules is less (or the granules even remain intact), amylose
solubilization is less, and swelling of the granules is limited as compared to thermal
gelatinization. (Baks et al., 2008)
2.1.4. Starch production and applications
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
for hydrolysate manufacture due to increased refining cost. (Zobel, 1992)
Applications of starch and modified starches are summarized in Table 2.2.
Physical and chemical modifications of starch resulted into wide range of
additional products. Physical modification includes pregelatinization, grinding, and
solvent treatment; these modifications impart some degree of granule dispersibility in
cold water. Since swelling occurs in cold water, these starches are used in instant food
mixes as a thickening agent. Chemical modifications of starch include reactions of
substitution or derivatization, cross-linking, oxidation, and hydrolysis. Derivatized
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.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
21
Cross linking is most commonly accomplished using adipic acid and phosphorous
oxychloride. It is common practice to combine both derivatization and crosslinking in
granules to achieve needed functionality. Oxidized starch, thin boiling starch and
dextrins provide examples of starches that have been depolymerized to varying
degrees. (Zobel, 1992)
Table 2.2. Applications of starch (www.starch.dk)

Food
Beverage Animal Feed Plastic Pharmacy Building
Mayonnaise Soft drinks Pellets
Biodegradable
plastic
Tablets
Mineral fibre
tiles
Baby food Beer By products
Dusting
powder
Gypsum board
Bread Alcohol Concrete
Buns Coffee Gypsum plaster
Confectionery Agriculture Textile Paper Various
Meat
sausages
Jelly gums Seed coating Warp
Corrugated
board
Foundries
Meat rolls and
loaves
High-boiled
sweets
Fertiliser Fabrics
Water
treatment
Ketchup Jellies Yarns Cardboard Coal
Marchmallows
Soups Marmalade Paper Detergent
Snacks Jam Fermentation Non-Wowen
Printing
paper
Oil drilling
Pizza sauces Ice cream Vinegar
Hygienic
diapers
Stain remover
Sauces Dairy cream Enzymes Baby diapers
Packaging
material
Glue
Low fat foods Fruit fillings
Sanitary
napkins
Foamed starch

2.2. Starch hydrolysis products
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
22
2.2. Three steps in which enzymes are used in starch hydrolysis and processing are as
follows:
1. Liquefaction of starch (combination of starch gelatinization and dextrinization of
gelatinized starch)
2. Saccharification of starch liquefact (containing oligosaccharides) to glucose or
maltose
3. Isomerization of glucose
Table 2.3. Different types of nutritive sweetener
Nutritive sweetener DE Class (Degree of polymerization)
Maltodextrins <20 Malto-oligosaccharides (3-9)
High maltose syrup 20-50 Maltose, glucose, Malto-oligosaccharides
Maltose 53 Sugar (2)
Glucose syrup 20-95 Glucose, maltose, malto-oligosaccharides
High fructose syrup - Fructose, glucose
Dextrose 100 Sugar (1)

2.2.1. Starch hydrolyzing enzymes
Starch is a polymer of glucose units, in which glucose units are linked together
by α(1→4) and α(1→6) glucosidic linkages. Glucose (monomer), maltose (dimer),
fructose (isomer of glucose) and malto-oligosaccharides are useful in many
applications. Hence these become commercially and industrially important products.
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
hydrolysis or enzymatic hydrolysis.
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
23
catalyze reaction under moderate conditions of pH and their use has led to better
controlled processes making fewer by-products. (Teague and Brumm, 1992)
Processes involving biocatalysts are potentially energy saving and are very well suited
for a wide range of industrial applications. Moreover enzymes can be produced
industrially in an ecologically safe way and are renewable, degradable and non-toxic
(Uhlig, 1998).
Enzymes (i.e. proteinaceous catalyst) are used throughout industries to convert
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
breakdown of biomaterials into simpler compounds. Most biocatalysts have limited
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
reactions; and (2) enzymes act on specific molecules, referred to as substrates, to
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
catalyzed (Copeland, 2000) into following:
EC 1 Oxidoreductases: catalyze oxidation/reduction reactions
EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)
EC 3 Hydrolases: catalyze the hydrolysis of various bonds
EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidation
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
24
EC 5 Isomerases: catalyze isomerization changes within a single molecule
EC 6 Ligases: join two molecules with covalent bonds
An exceptionally significant field, in which enzymes have proved to be of
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
1960, a breakthrough occurred through the application of enzyme glucoamylase that
caused a complete break down of starch to glucose (Blanch, 1996).
Starch degrading enzymes i.e. “Amylases” can be generally defined as the
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
product specificities, including exo/endo specificity, preference for hydrolysis or
transglycosylation, α(1→4) or α(1→6) glycosidic bond specificity and glucan
synthesizing activity. Plants, animals and microorganism use starch as source of
energy and carbon. Microorganisms mainly bacteria, fungi produce various starch
hydrolyzing enzymes in different environmental niche in order to degrade these large
macromolecule (Vorgias and Antranikian, 1997).
Enzymes of interest to starch hydrolysis industry are hydrolases like α-
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
reviewed amylolytic enzymes involved in starch processing and products derived
from starch. Van der Maarel et al. (2002) have reviewed properties and applications
of starch-converting enzymes of the α-amylase family. The starch hydrolytic enzymes
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
25
comprise 30% of the world’s enzyme consumption (Van der Maarel et al., 2002). Use
of the enzymes in the starch processing is shown in the Fig. 2.6.
Table 2.4. Summary of starch hydrolyzing enzymes
Enzyme
Source
Action pattern and
optimum reaction conditions Product
Bacillus
amyloliquefaciens
(i.e. B. Substilus)
Cleaves internal α(1→4) glucosidic
bonds to give α-dextrins and mainly
maltose (G2), G3, G6 and G7
oligosaccharides. Neutral pH and 70 °C
B. licheniformis
Cleaves internal α(1→4) glucosidic
bonds to give α-dextrins and produces
mainly G5 – G9 and small amounts of
G3, G4 and G5 oligosaccharides.
6 - 7 pH and 85 °C
Bacterial
α-Amylase
EC 3.2.1.1.
Endo-enzyme
B.
Stearothermophilus
Cleaves internal α(1→4) glucosidic
bonds to give α-dextrins and major
products are G2, G3, G5 and G6.
5.5–6.5 pH and 80 °C
Maltodext
rins
Fungal
α-amylase
EC 3.2.1.1.
Exo-enzyme
Aspergillus niger,
Aspergillus oryzae,
Rhizopus oryzae
Cleaves internal α(1→4) glucosidic
bonds and predominantly produces
maltose from starch with significant
quantities of glucose and maltotriose.
For Aspergillus, 4 pH and 60 °C
For Rhizopus, 5-5.2 pH and 40 °C
Maltose
syrup,
glucose
syrup
β-Amylase
EC 3.2.1.2
Exo-enzyme
Barley malt,
Soybean, wheat
Cleaves second α(1→4) glucosidic
bond from non reducing end and give
β-limit dextrins and β-maltose.
5.2 pH, 50-60 °C
Maltose
syrup,
maltose
Glucoamylase
EC 3.2.1.3
Exo-enzyme
Aspergillus niger,
Rhizopus niveus,
Rhizopus oryzae,
Rhizopus sp.
Cleaves first α(1→4) or α(1→6)
glucosidic bond from non reducing end
to give β-glucose.
For A. Niger, 4-5 pH, 60 °C
Glucose
syrup,
glucose
Pullulanase
EC 3.2.1.41

B. acidopullulyticus
Klebsiella planticola
Only α(1→6) glucosidic bond are
cleaved to give straight chain
maltodextrins.
For K. planticola, 6.5 pH and 50 °C
For B. acidopullulyticus, 4.5-5 pH and 60
°C
Linear
dextrins,
Maltose
syrup
Isoamylase
EC 3.2.1.68
Pseudomonas
amyloderamosa
Cleaves α(1→6) glucosidic bond.
pH 3-4 and 45-55 °C
Linear
dextrins,
maltose
Glucose
isomerase
EC 5.3.1.18
Actinoplanes missouriensis
B. coagulans
Microbacterium arborescen
Isomerizes glucose into fructose
7 – 8 pH and 60 °C
High
fructose
syrup

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
26

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)
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
27

Figure 2.6. The use of enzymes in processing starch (www.lsbu.ac.uk)
2.2.1.1. Bacterial α-amylase
Bacterial α-amylase (1, 4-α-D-glucan glucanohydrolases, EC 3.2.1.1,
glycogenase) is an endo-hydrolase which cleave any internal α(1→4) glucosidic
bonds. As the name indicates oligosaccharides produced by hydrolysis of starch using
bacterial α-amylase have reducing group in α configuration. It can bypass but cannot
cleave α(1→6) glucosidic branch points. It can be produced commercially from
following three sources viz. B. Amyloliquefaciens, B. Licheniformis, and B.
Stearothermophilus. Bacterial α-amylase is a thermostable enzyme and work up to
110 °C in presence of substrate. Since this is an organometallic enzyme, it requires
35% in cold water
pH 6, 40 ppm Ca
2+

Starch slurry
Bacterial α-amylase, 1500 U/kg,
105 °C, 5 min
Gelatinization
Gelatinized Starch (< 1 DE)
95 °C, 2 h Liquefaction
Liquefied starch (11-15 DE)
0.3% D-glucose
2.0% maltose
97.7% oligosaccharides
Starch granules
Saccharification
pH 4.5
glucoamylase, 150 U/kg
pullulanase, 100 U/kg
60 °C, 72 h
Glucose syrup (99 DE)
97% D-glucose
1.5% maltose
0.5% isomaltose
1% other oligosaccharides
Maltose syrup (44 DE)
4% D-glucose
56% maltose
28% maltotriose
12% other oligosaccharides
pH 5.5
fungal α-amylase,
2000 U/kg
50 ppm Ca
2+
,
60 °C, 72 h
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
28
supplementation of Ca
2+
ions during liquefaction.
Bacterial α-amylase is particularly used in the liquefaction of starch and
production of maltodextrins. Liquefied starch by means of bacterial α-amylase is
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
of reaction mixture and increase in the DE which is a measure of degree of starch
hydrolysis.
The maximum DE obtainable during liquefaction starch using bacterial α-
amylase is around 40, but prolonged treatment leads to the formation of maltulose (4-
α-D-glucopyranosyl-D-fructose), which is resistant to hydrolysis by glucoamylase
and α-amylases (www.lsbu.ac.uk/biology/enztech/starch.html). DE values of 10-16
are used in most commercial processes, where liquefact is supposed to be
saccharified. Liquefaction till DE of 10-16 is required to reduce the viscosity of the
gelatinized starch to ease subsequent processing and also to avoid further possible
retrogradation of gelatinized starch. Recently, Haki and Rakshit (2003) have
discussed the source microorganisms and properties of bacterial α-amylase in the
context of calcium independence, pullulanase and β-amylase in the context of
thermostability. The industrial needs for such specific thermostable enzyme and
improvements required to maximize their application in the future are also suggested.
(Teague and Brumm, 1992; Olsen, 1995)
2.2.1.2. Fungal α-amylase
Fungal α-amylase (1, 4-α-D-glucan glucanohydrolases, EC 3.2.1.1,
glycogenase) is an exo-amylase which hydrolyzes α(1→4) linkages and
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
29
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
maltose. (Teague and Brumm, 1992; Olsen, 1995)
2.2.1.3. Glucoamylase
Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3, sacchorogenic
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
bonds. At low starch concentrations, glucoamylase completely degrades starch to
glucose and this fact has been used for development of starch assay. At high
concentration of glucose, it catalyzes polymerization of glucose into higher polymers
generally referred as condensation or reversion products. Reversion products include
isomaltose, isomaltotriose, isomaltotetraose, kohibiose, nigerose, maltose, α,β-
trehalose, and panose. As practically complete conversion of starch to glucose is
possible using amyloglucosidase, it is also referred as saccharifying amylase. It is
normally used in the production of glucose syrup and dextrose. (Teague and Brumm,
1992; Olsen, 1995)
2.2.1.4. β-amylase
β-amylase (1,4-α-D-glucan maltohydrolase, EC 3.2.1.2) is a exo-acting
enzyme. It cleaves second α(1→4) glucosidic bond from non reducing end of starch
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
30
or glycogen polymer and releases β-maltose unit. β-amylase can neither cleave
α(1→6) glucosidic bond (unlike glucoamylase) nor bypass it (unlike fungal α-
amylase). Hence hydrolysis of starch by β-amylase results into production of maltose
and β-limit dextrins. (Teague and Brumm, 1992)
2.2.1.5. Pullulanase
Pullulanase (α-dextrin endo-1,6-α-glucosidase; EC 3.2.1.41; limit dextrinase,
debranching enzyme, amylopectin 6-glucanohydrolase, pullulan 6-glucanohydrolase)
cleaves α(1→6) glucosidic bonds in amylopectin, pullulan, and glycogen. It
debranches amylopectin to produce linear dextrins and pullulan (a polysaccharide
with a repeating unit of maltotriose that is α(1→6) linked) to produce maltotriose.
Group II pullulanase (α-amylase–pullulanase or amylopullulanase) hydrolyze both
α(1→4) and α(1→6) glucosidic bonds and produces maltose and maltotriose (van der
Maarel et al., 2002).
2.2.1.6. Isoamylase
Isoamylase (Glycogen 6-glucanohydrolase, EC 3.2.1.68, debranching enzyme)
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
Marrel et al., 2002)
2.2.1.7. Glucose isomerase
Glucose isomerase (EC 5.3.1.18) catalyzes Isomerization of D-glucose to D-
fructose. An excellent review on this enzyme and its industrial application has been
written by Bhosale et al., (1996).


Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
31
2.2.2. Maltodextrins
Maltodextrins, which are partially hydrolyzed starch products, have been on
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):
a non-sweet, nutritive saccharide polymer that consists of D-glucose units linked
primarily by α(1→4) bonds and that has a DE (dextrose equivalent) of less than 20.
Maltodextrin is a saccharide mixture that consists of maltose, malto-oligosaccharides
and linear or branched dextrins. Starch hydrolysis products are commonly
characterized by their degree of hydrolysis, expressed as the dextrose equivalent
(DE), which is the percentage of reducing sugar calculated as dextrose on dry-weight
basis. (Alexander, 1992)
Marchal et al., (1999) provides an excellent review article, which focuses on
the production of maltodextrins with more defined saccharide compositions; glucose
and maltose syrups, and cyclodextrins are not considered here. Design of the desired
saccharide composition and production possibilities for maltodextrins have been
discussed in detail by Marchal et al., (1999).
2.2.2.1. Production
Maltodextrin is usually produced by liquefaction process. Liquefaction of
starch is a combination of gelatinization of starch and dextrinization of gelatinized
starch. Liquefaction of starch can be accomplished by the use of acid or enzyme.
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
32
enzyme to a low DE (usually < 3), followed by treatment at high temperature (~120-
130 °C in jet cooker) to ensure complete gelatinization of starch. Second stage
involves hydrolysis using bacterial α-amylase at temperature around 85-105 °C.
(Teague and Brumm, 1992; Alexander, 1992)
Random nature of acid catalyzed hydrolysis limits its utility as a means of
producing low DE products or liquefact suitable for further conversion. Low DE
conversion products of acid hydrolysis contain more glucose than their enzymatically
produced counterparts at the same DE. This leads to increased sweetness,
hygroscopicity, and susceptibility to color development upon heating, generally
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
retrogradation. Hence, 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. (Reeve, 1992; Teague and Brumm, 1992;
Marchal et al., 1999)
Only dextrose equivalent of maltodextrins has been shown to be inadequate to
predict product performance in various applications. Maltodextrins with the same DE
can even have different properties in various applications that reflect differences in
their molecular composition. The saccharide composition of a maltodextrin
determines both its physical and biological functionality. Factors influencing the
saccharide composition are type and source of enzyme, source of starch, starch
concentration, temperature, organic solvents, pressure, immobilization, downstream
processing and extraction of products during hydrolysis (Marchal et al., 1999) and
grouped according to potential and costs in Fig. 2.7.

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
33

Figure 2.7. Different ways to influence the saccharide composition of a starch
hydrolysate grouped according to potential and costs. (Adapted from Marchal et al.,
1999)

Dextrose equivalent
Starch Maltodextrin a
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)

P
o
t
e
n
t
i
a
l

Costs
Enzymes
Temperature
Downstream processing
Source of starch
Concentration Immobilization
Extraction of products
Pressure
Organic solvents
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
34
2.2.2.2. Application
Maltodextrin is a versatile ingredient, which finds numerous applications in
the food processing and pharmaceutical industries. These applications are based on a
wide range of functional properties like freezing point depression, hygroscopicity,
osmolality, prevention of coarse crystals, solubility, sweetness, viscosity, and
absorption by human. The dextrose equivalent (DE) value and saccharide composition
of maltodextrin determines the variation in the effect of the above-mentioned
parameters. (Marchal et al., 1999). Increase or decrease in these properties as function
of DE is illustrated in Fig. 2.8.
Maltodextrins are used as spray drying aid/ flavor encapsulation, bulking
agent, texture provider, fat replacer, tablet expicient, film former, sport beverage,
parenteral and enteral nutrition products (Alexander, 1992; Marchal et al., 1999).
Spray drying aid/ flavor encapsulation. Preparation of dried flavors by encapsulating
flavor or flavor oil.
Carrier / Bulking agent. Used in dry mix products (includes puddings, soup, and
frozen desserts.) and dry mix beverages. Particularly advantageous in dry
soup mixes due to less hygroscopicity.
Nutritional. It is directly used as rehydration / energy beverage, sport drink and as a
carbohydrate source in the nutritional fluids like energy bar, infant formula
(i.e. non milk or non lactose based fluids), enteral products, parental nutrition
product, oral nutritional supplement due to easy digestibility, instant
dispersibility in cold water, and low osmolality.
Medical. Used in production improved excipients for tableting or directly as tablet
excipients, isotonic solution that can be directly infused into veins of patient.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
35
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
spoonable salad dressings
Desserts. Used as ingredient in the preparation.
Dairy. Maltodextrin is used extensively in coffee whiteners, imitation sour creams,
imitation cheeses and whipped toppings.
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
and crystal inhibition. In hard candies, it reduces the hygroscopic
characteristics.

2.2.3. Glucose syrup
Glucose syrup is a purified concentrated aqueous solution of nutritive
saccharides obtained from starch. Glucose syrups are largely composed of glucose
and maltose (concentration of glucose is largest and followed by concentration
maltose) and have DE values between 20 and 80. Major source of starch for
production glucose syrup is corn. (Howling 1992)
2.2.3.1. Production (Reeve, 1992; Teague and Brumm, 1992; Howling, 1992)
Acid conversion
When a suspension of starch in water is heated with acid (normally HCl or
H
2
SO
4
) at a temperature exceeding that required for starch gelatinization (normally
water boiling conditions at atmospheric pressure), rapid hydrolysis takes place, with
breakage of both α(1→4) and α(1→6) glucosidic linkages. It is normally regarded as
a random type of hydrolysis. Rate of hydrolysis is a function of temperature and
concentrations of both acid and starch. Acid catalyzed starch hydrolysis is remarkably
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
36
reproducible. Provided the reaction conditions (i.e. time, temperature, acid
concentration, starch concentration) are kept constant.
Random nature of acid hydrolysis puts important constraint on the glucose
syrups produced. If a DE of less than 30 is obtained, due to random action there
remains a proportion of glucose polymers of sufficient chain length to give rise to
retrogradation, resulting in the formation of insoluble starch particles. Second
limitation is that in the production of glucose syrup with DE above 55, acid promote
dehydration and condensation in addition to hydrolysis that results into undesirable
products like formic acid, gentiobiose, hydroxymethylfurfural, and levulinic acid.
These undesirable products serve as impurities and also impart undesirable color to
syrup.
Hence the range of glucose syrup produced by acid catalyzed hydrolysis is
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).
Typical sugar composition of glucose syrups produced by acid catalyzed hydrolysis
(Howling, 1992) is shown the table 2.5.

Table 2.5. Typical Sugar Composition of Acid Converted Glucose Syrups
(Howling, 1992)
Sugar 30 DE 42 DE 55 DE
Dextrose % on dry basis 10 19 31
Maltose % on dry basis 9 14 18
Trisaccarides % on dry basis 10 11 13
Higher sugars % on dry basis 71 56 48



Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
37
Acid - enzyme conversion
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
catalyzed hydrolysate of 20-40 DE was enzymatically hydrolyzed at temperature of
55-60 °C. Second stage enzymatic hydrolysis can be done using bacterial α-amylase,
amyloglucosidase, fungal α-amylase, β-amylase individually or combination of two
enzymes, depending upon the properties of required glucose syrup i.e. DE value and
saccharide composition (mainly of glucose, maltose and maltotriose). pH of the acid
catalyzed hydrolysate was adjusted to appropriate value (4.5 for amyloglucosidase,
5.5 for fungal α-amylase and 6 for bacterial α-amylase).
Low DE dextrose syrup was produced by hydrolysis of 17-20 DE acid
catalyzed substrate using bacterial α-amylase. Higher end of spectrum, 55-80 DE
were made by hydrolysis of 17-20 DE acid substrate or 40-42 DE acid substrate using
amyloglucosidase alone or in combination with fungal α-amylase. Table 2.6 shows
saccharide composition, enzyme and substrate used for a range of acid-enzyme syrups
(Howling, 1992).
Table 2.6. Composition of acid-enzyme converted glucose syrups. (Howling, 1992).
DE of Acid catalyzed
hydrolysate, 1
st
stage
17 20 40 40
Enzyme used in second
stage
Bacterial
α-amylase
β-amylase fungal α-amylase
+ glucoamylase
β-amylase
+ glucoamylase
DE 26 42 63 63
Dextrose % on db i.e.
dry basis
4 6 36 37
Maltose % on db 4 45 30 32
Higher sugars % on db 92 49 34 31

Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
38
Enzyme-Enzyme conversion
This method was developed after the discovery of thermostable Bacterial α-
amylase. Liquefaction of starch was performed using bacterial α-amylase at 90-100
°C and hydrolysate of 12-15 DE was produced. Then this hydrolysate was further
saccharified using enzyme combinations described in the earlier section on Acid-
enzyme conversion at 55-60 °C to produce glucose syrup of desired DE and
saccharide composition.
Progress in the processes from acid to acid-enzyme to enzyme-enzyme not
only provided better control over saccharide composition but also reduced the
formation of color precursor 5-HMF (5-hydroxymethylfurfural) and the ash content in
the glucose syrup (Howling, 1992).
Refining and evaporation (Howling, 1992).
Refining means the process by which filtered starch hydrolysate product is
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
odor contaminants. In enzyme-enzyme process based glucose syrup, calcium sulphate
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
other starch hydrolysate products also.
After refining, glucose syrups require concentration to about 80% solids for
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
39
shipment. This dry wt concentration is a compromise between minimum water
content, viscosity, resistance to microbial contamination, and ability to pump and
store at temperatures that do not reduce shelf life by color formation. Evaporation is
carried out at as low temperature as possible to minimize color formation.
2.2.3.2. Applications (Howling, 1992)
Applications are based on a wide range of functional properties like freezing
point depression, hygroscopicity, osmolality, prevention of coarse crystals, solubility,
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
applications. In general confectionary area is the dominant worldwide market for
glucose syrups.
Confectionary. Glucose syrups are used in various confectionary products like hard
candies, toffees/ caramel/ fudge, Gums and jellies, fondants, marshmallows, and
chewing gum. Properties of concern are sweetness, hygroscopicity, and viscosity.
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.
Fruit preserves. Due to preservative nature of carbohydrate solids in excess of 67%
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,
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
40
cookies, crackers, pies, and cake.
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.
Alcoholic beverages. Used in alcoholic beverages to provide body, viscosity,
sweetness control and a source of fermentable carbohydrates. High DE syrup is
also used as priming sugars in brewing industry.
Pharmaceutical industry. Used in three area: 1. Fermentations that produce antibiotics
and fine chemicals, 2. Medicated confectionary and 3. as a carrier for liquid cough
mixtures and medicines.

2.2.4. Dextrose hydrolysate, crystalline dextrose and liquid dextrose
Dextrose hydrolysate is one of the most important starch hydrolysis products.
It contains D-glucose in excess of 90% on dry basis (db) with a typical value between
95 and 96 % db D-glucose. Dextrose hydrolysate after refining and concentration
serves as feedstock to dextrose crystallization process. Dextrose hydrolysate is
sometimes also termed as high DE glucose syrup. The products produced from this
feedstock include crystalline dextrose, liquid glucose and high fructose syrup. Liquid
dextrose is a syrup rich in D-glucose that exceeds content of D-glucose in dextrose
hydrolysate and is typically 99% db or greater. (Mulvihill, 1992)
2.2.4.1. Production (Reeve, 1992)
Acid catalyzed starch hydrolysis process is incapable of yielding more than
85-90% dextrose under practical conditions because of concurrent reversion and
dehydration reactions. The acid-enzyme process will yield 93% dextrose, since acid
hydrolysis produces highly branched saccharides resistant to action of glucoamylase.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
41
Hence dextrose hydrolysates are today produced almost exclusively by totally
enzymatic processes (Fig. 2.6), which give dextrose levels of 95-96%.
There are mainly two steps involved in the enzymatic production of Dextrose
hydrolysate from starch:
1. Liquefaction of starch using bacterial α-amylase
2. Saccharification of liquefied starch using amyloglucosidase with or without
pullulanase
Liquefaction
Starch liquefaction is defined as the combination of two processes:
1. Complete gelatinization (including hydration) of starch polymer, ensuring
accessibility to hydrolytic attack.
2. Dextrinization to a degree that prevents retrogradation on further processing.
In the enzymatic liquefaction of starch, bacterial α-amylase is used, which is
thermostable and hydrolyzes only internal α(1→4) linkages in the starch. Most
commonly used bacterial α-amylase is from B. licheniformis. Other sources of
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
using bacterial α-amylase produces malto-oligosaccharides, linear and branched
dextrins. Though enzymatic starch hydrolysate contains high molecular wt glucose
polymers, it is less susceptible to retrogradation because high mol wt fraction in
hydrolysate is more highly branched due to inability of bacterial α-amylase to cleave
α(1→6) linkages.
High pH (6 – 6.5) and calcium requirement (~100-400 ppm) to ensure enzyme
stability are disadvantages of this process. Added calcium salts must be removed at
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
42
the cost of increased refining expenses. Production of maltulose precursors is
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
enzyme liquefaction and autoclaving of starch in the enzyme-enzyme process of
glucose manufacture. The precursors are hydrolyzed during subsequent
saccharification with amyloglucosidase to form maltulose as one of the products. Acid
liquefaction prior to saccharification does not result in maltulose precursor formation.
(Dias and Panchal, 1987)
In the liquefaction, liquefact with DE preferably between 7 and 15 should be
produced under conditions unfavorable to maltulose precursor development. Inability
of amyloglucosidase to hydrolyze maltulose will otherwise lead to a reduction in the
final dextrose level attainable. Liquefaction is usually performed with reaction time of
1-2 h.
Saccharification of liquefied starch
After liquefaction, pH of the reaction mixture is reduced to 4.5 and
temperature decreased to 60 °C. Saccharification of starch liquefact is performed with
or without pullulanase to produce dextrose hydrolysate. Saccharification time is
usually 24-96 h depending upon enzyme dosage. Amyloglucosidase is an exo-
amylase with ability to cleave both α(1→4) and α(1→6) linkages. Hence it seems that
100% dextrose is possible theoretically. Amyloglucosidase produces 100% dextrose
(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
concentration it is 96%). During saccharification at higher concentrations (necessary
for an economic process), the dextrose level rises to the maximum indicated and then
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
43
begins to fall. This occurs due to reverse reactions catalyzed by amyloglucosidase,
which form disaccharides by repolymerization of dextrose. Reversion reactions are
favored by high substrate concentration. These reversion reactions will proceed till
equilibrium is reached. Here, it should be remembered that maximum attainable
dextrose is same, irrespective of amyloglucosidase concentration in the reaction
mixture. But increase in the dosage of amyloglucosidase will also enhance the rate of
reversion and enzyme inactivation by heating to 80 °C can become necessary to avoid
a rapid reduction in dextrose content.
Hydrolysates obtained from 30-35% w/v starch slurry are economically and
technically accepted. After completion of liquefaction and saccharification of starch
slurry (30-35% w/v), a 33-39% w/v solution is obtained with DE of 97-98 and
saccharide composition of 95-96%db dextrose, 1.2-2% maltose, 1-2% isomaltose, 0.4-
0.8% maltotriose and 0.6-1% higher saccharides. Normal refining (carbon, strong
cation exchange) will remove glucoamylase activity before significant reversion
occurs.
Dextrose content in the dextrose hydrolysate can be further increased by using
debranching enzyme i.e. pullulanase along with amyloglucosidase. Pullulanase will
cleave α(1→6) linkages faster (which are cleaved, but relatively slowly by
amyloglucosidase) and make linear chains available for faster hydrolysis by
amyloglucosidase. Use of pullulanase also reduces quantity of glucoamylase resistant
oligosaccharides, which are normally present. By adding pullulanase, dosage of
amyloglucosidase can be reduced to maintain hydrolysis rate. The consequent
reduction in the reversion rate will result into an increase in the dextrose level (>1%).
Choice of pullulanase usage in saccharification in combination with amyloglucosidase
is dictated by process economics and dextrose levels required.
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
44

Dextrose monohydrate (α-D-glucose monohydrate) crystals are produced by
crystallization of saturated D-glucose solutions at temperatures below 50 °C.
Crystallization at higher temperature gives the anhydrous α and β forms. (Mulvihill
1992)
Dissolving crystalline dextrose monohydrate in water (remelting) gives high
purity syrup of 71% ds and 99.5% db D-glucose has been the usual method of
production. Liquid chromatographic separation using ion-exchange resins produces a
stream rich in D-glucose by separating it from higher saccharides present in
hydrolysate streams. The chromatographically produced liquid dextrose is nearly
equivalent to remelt dextrose in D-glucose content and is acceptable in many
applications. (Mulvihill 1992)
2.2.4.2. Applications (Mulvihill 1992)
Dextrose hydrolysate, crystalline dextrose and liquid dextrose are mainly used
in foods, fermentation, industry, and pharmaceutical uses.
Foods. In food applications are similar as mentioned for glucose syrup.
Industrial Applications. Liquid glucose and solids are used in adhesives, building
materials, chemical (mainly sorbitol) manufacture, fermentation, tobacco, and
leather.
Pharmaceutical applications. Anhydrous dextrose recrystallized from dissolved
monohydrate crystal and treated to remove pyrogens is utilized in intravenous
solutions. Used as excipients in making direct compression tablets.



Chapter 2: Overview of starch and starch hydrolysis products
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45
2.2.5. Maltose syrup
Maltose syrup is a purified concentrated aqueous solution of nutritive
saccharides obtained from starch, containing maltose as principal sugar. Maltose
syrup is classified into high maltose, extra high maltose, and high conversion syrup.
Composition of maltose syrups is shown in the Table 2.7.
Table 2.7. Typical composition of maltose syrups (Reeve 1992)
Maltose syrup DE Glucose concn.
%db
Maltose concn.
%db
High maltose 40-46 <4 45-55
Extra high maltose 50-55 <4 70-80
High conversion 60-70 30-35 30-45

2.2.5.1 Production (Reeve 1992)
Production of maltose syrup from starch involves following two steps:
1. Liquefaction of starch using bacterial α-amylase
2. Saccharification of liquefied starch using fungal α-amylase or β-amylase with or
without pullulanase.
Liquefaction part essentially remains same as discussed in the section 2.2.4.1.
except that liquefaction is allowed to proceed to produce liquefact with DE of 7-10.
High maltose syrups are produced by saccharification of starch liquefact with
fungal α-amylase or β-amylase. Fungal α-amylase is endo-amylase, which cleaves
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
with β-amylase yields maltose and β-limit dextrins. β-amylase is used in
Chapter 2: Overview of starch and starch hydrolysis products
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46
saccharification at 5.5 pH and 60 °C, whereas fungal α-amylase is utilized at 5.5 pH
and 50-55 °C.
For the production of very high maltose syrup with maltose content of 70-
85%, debranching enzyme, pullulanase or isoamylase, must be used along with β-
amylase. Debranching enzyme cleaves α(1→6) linkages and produces linear dextrins,
which are further saccharified by β-amylase to produce maltose. Major constraint on
maltose level attainable is the liquefact DE. As the liquefact DE increases, quantity of
maltose that can be produced reduces. Therefore, liquefact DE should be as low as
possible. Liquefact DE of 5-10 is normally suitable to attain 70-80% maltose level.
Reaction conditions are pH of 5.5 and temperature of 60 °C.
High conversion syrup, usually of 60-70 DE are formulated to be of maximum
sweetness and fermentability, while resisting crystallization at 4 °C and 80-83% dry
wt concentration. Dextrose content is limited to a maximum of 40% db to avoid
crystallization. Maltose is the bulk of remaining saccharides. High conversion syrups
are produced by the combined use of amyloglucosidase and fungal α-amylase or β-
amylase in the saccharification of liquefied starch. Choice between fungal α-amylase
or β-amylase has been done mainly on economic rather than technical basis. Ratio
between amyloglucosidase and maltogenic enzyme must be varied in order to meet
required dextrose and maltose content. Low DE liquefact increases the
maltose/dextrose ratio.
2.2.5.2. Applications
Maltose syrup is increasingly gaining popularity among diabetic and diet-
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
Chapter 2: Overview of starch and starch hydrolysis products
Studies in the Enzymatic depolymerisation of natural polysaccharides
47
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. Fructose syrup
2.2.6.1. Production
High fructose syrup (HFS) is normally produced by enzymatically isomerizing
refined dextrose hydrolysate (containing 94-96% db dextrose). Isomerization is done
by passing the hydrolysate stream through a column of immobilized glucose
isomerase. Dextrose hydrolysate stream with 94-96% dextrose is clarified, refined
with carbon or ion-exchange resins and concentrated to 45% w/v. Clarification is
extremely important as it removes particles in the stream that are detrimental to
column performance, while refining removes calcium ions detrimental to the activity
of immobilized glucose isomerase. Clarified and refined dextrose hydrolysate is then
adjusted to pH of 7-8.5 and supplemented with 0.5-5 mM magnesium ions. Lower
cost is obtained if inlet pH is 7-7.5, whereas maximum throughput is obtained at pH
8.3. The magnesium maximizes and stabilizes the enzyme activity while
counteracting the inhibitory effect of residual calcium ions. The system will tolerate
calcium up to 0.075 mM (3 ppm) as long as magnesium concentration is maintained
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
performed as a continuous process at temperature of 53-61 °C. Feed flow rate is a
function of column age, decrease in activity with time, operating conditions and
immobilization technology employed. It is maintained in order to have 42-45%
fructose in the product stream. This is HFS-42. This HFS-42 is fractionated to
produce HFS-90 containing 90% fructose. HFS-42 and HFS-90 are blended to
Chapter 2: Overview of starch and starch hydrolysis products
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48
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
1992; White 1992)
2.2.6.2. Applications
HFS-55 is approved to replace 50% of the sucrose in carbonated beverages.
Functional attributes of HFS include sweetness, viscosity, humectancy,
fermentability, resistance to crystallization (HFS > 55%), browning/ flavor
development, and colligative properties (e.g. freezing point depression and osmotic
pressure). Extensive list of applications of HFS (White 1992) is as follows:
Alcoholic beverages and Brewing. Beer, brandy, cordials, liqueurs, wine.
Animal feed.
Baking industry and snack foods. Biscuits, breads, cakes, caramel color, cookies,
crackers, doughnuts, extracts and flavors, frosting/icing, pies, potato chips,
pretzels, rolls etc.
Nonalcoholic beverages. Carbonated, fruit drinks/ juices, powdered mixes
Canners and packers. Berries, candied fruits, fruit fillings, fruit pectin, soups, tomato
sauces, vegetables
Cereals. Breakfast cereals
Chemicals, drugs, pharmaceuticals. Acids, amino acids, antibiotics, food and drug
coatings, drugs, enzyme, fermentation processes, lecithin, mannitol, medicinal
syrups, organic acids, shampoo, pharmaceuticals.


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3. Sorghum: Literature Review








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3.1. Introduction, Origin and Geographical distribution of sorghum
Sorghum (Sorghum bicolor L. Moench) is an important drought resistant
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 (www.fas.usda.gov). Leading sorghum producing countries were United States
(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
large quantities of fodder.
Sorghum is believed to be originated in equatorial Africa, where a large
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
Chapter 3: Sorghum: Literature Review
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51
Americas, Europe and Australia between latitudes of up to 50°N in North America
and Russia and 40°S in Argentina. (Kimber et al., 2000; Balole & Legwaila, 2005)
Sorghum is distributed throughout the tropical, semi-tropical, arid and semi
arid regions of the world. Sorghum is also found in temperate regions and at altitudes
of up to 2300 meters in the tropics. It has a potential to compete effectively with
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
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 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
and crop rotation systems (Smith and Frederiksen, 2000).
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,
Kenya, Tanzania, Burundi, and Zambia. It is an important crop in India, Pakistan,
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
different places in the world. Sorghum is known by various names in Africa: as
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
in several countries in Southern Africa. It is called jowar in India, kaolian in China
Chapter 3: Sorghum: Literature Review
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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
perennial species being Sorghum almum (Columbus grass), Sorghum halepense
(Johnson grass) and Sorghum propinquum. Subspecies of Sorghum Bicolor (L.)
Moench are arundinaceum, bicolor and drummondii. (Dahlberg, 2000)
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
hybridization of 2 or more basic races. (Dahlberg, 2000)
The description of the 5 basic races (Dahlberg, 2000) in short is as follows,
1. Bicolor: The most primitive cultivated sorghum, characterized by open
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
are rarely important. They are frequently found in wet conditions.
2. Guinea: It is characterized by usually large, open inflorescences with branches
Chapter 3: Sorghum: Literature Review
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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.
3. Kafir: It is characterized by relatively compact panicles that are often cylindrical in
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
southern savanna from Tanzania to South Africa. Kafir landraces tend to be
insensitive to photoperiod and most commercially important male sterile lines are
derived from kafir type sorghum.
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
‘karan dafi’ by the Hausa people in Nigeria.
5. Durra: It is characterized by compact inflorescences, characteristically flattened
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-
season. Most of them are drought resistant.
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Different intermediate races are guinea-bicolor, caudatum-bicolor, kafir-
bicolor, durra- bicolor, guinea- caudatum, guinea-kafir, guinea-durra, kafir-caudatum,
durra-caudatum, kafir-durra. Hybrid races exhibit various combinations and
intermediate forms of the characteristics of the 5 basic races. Durra-bicolor is found
mainly in Ethiopia, Yemen and India, guinea-caudatum is a major sorghum grown in
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
to brown, red and almost black. Classification and characterization of sorghum is
given briefly in Dahlberg (2000).
Taxonomical hierarchy of Sorghum bicolor (L.) Moench (www.itis.gov) is as
follows:
Kingdom Plantae -- Plants
Subkingdom Tracheobionta -- Vascular plants
Sperdivision Spermatophyta –Seed plants
Division Magnoliophyta -- Flowering plants
Class Liliopsida -- Monocotyledons
Subclass Commelinidae
Order Cyperales
Family Poaceae -- graminées, grass family
Genus Sorghum Moench -- sorghum
Species Sorghum bicolor (L.) Moench -- black amber, broomcorn,
chicken corn, shatter cane, shattercane, sorghum, wild cane
Subspecies (ssp.)
Sorghum bicolor ssp. Arundinaceum – Common wild sorghum
Sorghum bicolor (L.) Moench ssp. bicolor – grain sorghum
Sorghum bicolor ssp. drummondii – Sudangrass
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Synonyms for Sorghum bicolor – Black amber, broom-corn, broomcorn, chicken
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,
Sorghum bicolor var. caffrorum, Sorghum caffrorum, Sorghum cernuum, Sorghum
dochna, Sorghum dochna var. technicum, Sorghum drummondii, Sorghum durra,
Sorghum saccharatum, Sorghum subglabrescens, Sorghum vulgare, Sorghum vulgare
var. caffrorum, Sorghum vulgare var. durr, Sorghum vulgare var. roxburghii,
Sorghum vulgare var. saccharatum and Sorghum vulgare var. technicum.

3.3. Production, cultivation area and yield of sorghum
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
availability is limited and is frequently subjected to drought. About 100 countries
grow sorghum.
3.3.1. Trends in production, cultivation area and yield in the world
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,
the sorghum-growing countries are Botswana, Eritrea, Kenya, Lesotho, Madagascar,
Malawi, Mozambique, Namibia, Somalia, South Africa, Swaziland, Tanzania, Zambia
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and Zimbabwe. In West and Central Africa, the crop is grown in Benin, Burkina
Faso, Burundi, Cameroon, Central African Republic, Chad, Egypt, Gambia, Ghana,
Guinea, Guinea-Bissau, Ivory Coast, Mali, Mauritania, Morocco, Niger, Nigeria,
Rwanda, Senegal, Sierra Leone, Sudan, Togo, Tunisia and Uganda. In Latin America,
the sorghum-growing countries are Argentina, Brazil, Colombia, El Salvador,
Guatemala, Haiti, Honduras, Mexico, Nicaragua, Peru, Uruguay and Venezuela. In
Europe, it is grown in France, Italy, Spain, Albania and Romania. (Deb et al., 2004)














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Figure 3.1. Distribution of sorghum area, 1999-2001. (Source: Deb et al., 2004)


Figure 3.2. Distribution of sorghum production, 1999-2001. (Source: Deb et al.,
2004)

19.9
15.5
11.3
9.8
7
17.9
5.4
5
4.2
4
United States Nigeria
India Mexico
Sudan Others
Argentina Ethiopia
Australia China

Figure 3.3. % production of sorghum country-wise with world production of 64.5
Million Tones in 2007-2008. (Source of data: www.fas.usda.gov)

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Production of sorghum in 2007-2008 in the world was 64 Million Metric Tons
(www.fas.usda.gov). Foremost sorghum producing countries were United States,
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%),
respectively in 2007-2008 (Fig. 3.3) (www.fas.usda.gov).
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 20
th
century, was 42%
higher than in the early 1970s. Niger is at 5
th
position in sorghum cultivation area, but
is at 18
th
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
decrease in sorghum cultivation area.



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Table 3.1. Area, production and yield of sorghum in different countries, 1999-2001.
Rank
Country
Area
('000 Ha)
Production
('000 T)
Yield
(kg/Ha)
Area
wise
Production
wise
Yield
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)




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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

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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
10 million T, whereas in India production of sorghum decreased to 7.3 million T
(Table 3.2). Though, USA is largest sorghum producing country, its annual
production is steadily decreasing mainly because of decrease in the sorghum
cultivation area. In 2007-2008 annual production of sorghum in USA is around 60 %
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
decreasing followed by decrease in the sorghum cultivation area.
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
production 13 400 t in Israel, and 30 ha and 300 t in Jordan.
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3.3.2. Trend in cultivation area, production and yield of sorghum in India and
different states of India
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
sugarcane, cotton etc increases.
0
2
4
6
8
10
12
14
16
18
20
1 11 21 31 41 51
Year
Sorghum cultivation area (million ha)
% coverage under irrigation
1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.4. Trend in sorghum cultivation area (million ha) and % coverage under
irrigation (Source of data: www.agricoop.nic.in)

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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
country is increasing continuously.
0
2
4
6
8
10
12
14
1 11 21 31 41 51
Year
P
r
o
d
u
c
t
i
o
n

(
M
i
l
l
i
o
n

T
o
n
e
s
)
1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.5. Trend in annual sorghum production (Source of data: agricoop.nic.in)

Average annual area under sorghum in India declined from 16 million ha in
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
due to the decrease in area under sorghum.



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Table 3.3. Trend in Area, production and yield of sorghum in different states of India
during 1972 – 2002. (Source of data. Deb et al., 2004)
Average Area ('000 ha) State
1972-75 1981-84 1991-94 1998-2002
Andhra Pradesh 2709.9 2102.2 1057.2 721.6
Gujarat 970.6 956.6 444.6 206.1
Karnataka 2037.3 2205.7 2159.2 1885
Madhya Pradesh 2122.7 2138 1363.9 690.6
Maharashtra 5718 6588.7 5857 5019.8
Rajasthan 971.7 968.3 714.6 588.4
Tamilnadu 665.3 688.7 500.8 402.6
India (Total) 16139.3 16469 12703.5 10012.3
Average Production ('000 Tones)
Andhra Pradesh 1363.9 1326.4 815.6 559.2
Gujarat 321.4 544.7 267.6 190
Karnataka 1578 1726.3 1842.7 1707.7
Madhya Pradesh 1598 1747.7 1277.3 575.9
Maharashtra 2577.7 4740.7 5351.3 4388
Rajasthan 337.3 451.7 243.1 153.8
Tamilnadu 504 492 508.3 403.9
India (Total) 8826.3 11578 10773.3 8272
Average Yield (kg/ha)
Andhra Pradesh 506.7 630 770 779.3
Gujarat 333.3 570 616.7 896.7
Karnataka 763.3 783.3 856.7 906
Madhya Pradesh 750 816.7 936.7 828.7
Maharashtra 436.7 720 906.7 875.3
Rajasthan 350 463.3 330 363.6
Tamilnadu 760 710 1013.3 1001.7
India (Total) 543.3 706.7 846.7 826
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0
200
400
600
800
1000
1200
1 11 21 31 41 51
Year
Y
i
e
l
d

(
k
g
/
H
e
c
t
a
r
e
)
1950-51 1960-61 1970-71 1980-81 1990-91 2000-01

Figure 3.6. Trend in the sorghum yield (Source of data: agricoop.nic.in)

50.2
8.1
7.5
5.9
3.8
3.3
2.20.3 0.1 0.2
18.5
Maharashtra
Karnataka
Andhra Pradesh
Madhya Pradesh
Tamilnadu
Uttar Pradesh
Rajasthan
Gujarat
Haryana
Orissa
Others

Figure 3.7. State wise distribution of sorghum production during 2001-2002
(Source of data: agricoop.nic.in)

Maharashtra was largest sorghum producing state in 2001-2002 with share of
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
sorghum in the late 1990s (1998-2002) declined by 1 to 60% in major sorghum-
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
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sorghum production primarily remains in the two states of Maharashtra and
Karnataka, where area under sorghum production stands at a total of 7 million ha
(Table 3.3).

3.4. Plant anatomy and growth
Sorghum is a self-pollinating plant and its drought resistance is higher than
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.
3.4.1. Botanical parts of sorghum plant
Botanical parts of sorghum plant are shown in the Fig. 3.8. Sorghum plant
consists of following botanical parts (Plessis, 2008):
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
impediments occur, roots can reach a lateral distribution of 1 m and a depth of up to 2
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m early in the life of the plant. The roots are finer and branch approximately twice as
much as roots from maize plants.

Figure 3.8. Botanical parts of sorghum plant
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
number of leaves, which may vary from 8 to 22 leaves per plant.
Stem
The stem of the plant is solid and dry, to succulent and sweet. Stalk is the main
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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
the stem may also develop lateral shoots.
Inflorescence (panicle)
The inflorescence of sorghum, the panicle, may be compact or open. The
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
cross-pollination (approximately 6 %) occurs naturally.
Seed
The ripe seed (grain) of sorghum is usually partially enclosed by glumes,
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
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pericarp and testa results in a dark-brown or red-brown colour. Grain structure is
explained in the section 3.5. i.e. grain morphology.
Better drought tolerance of sorghum than most other grain crops (Plessis
2008) can be attributed to:
1. An exceptionally well-developed and finely branched root system, which is
very efficient in the absorption of water.
2. It has a small leaf area per plant, which limits transpiration.
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
protects the plant form desiccation.
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.

3.4.2. Growth of sorghum plant
Growth of sorghum plant is mainly distributed in following three stages
(Plessis, 2008):
1. Vegetative stage (0 to 30 days after sowing) – Identification of growth stage is
done according to leaf development
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2. Reproductive stage (30 to 60 days after sowing) – Identification of growth
stage is done according to development of grain kernels
3. Grain filling and physiological maturity (60 to 90 days after sowing).
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
in about 30 to 35 days after emergence. This is a critical point in the development of
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
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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
is no longer increasing in dry weight. This physiological maturity is not necessarily
the harvest maturity. At physiological maturity, the grain moisture will be 25 to 40
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)

3.4.3. Environmental requirement of sorghum plant
The optimum growth requirements of sorghum plants, in order to exploit its
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
(20 – 35 °C) and a frost-free period of approximately 120 to 140 days.
Following environmental conditions are necessary for growth of the sorghum
plant (Kimber, 2000):
1. Day length. Sorghum is a short-day plant, which means that the plant requires short
days (long nights) before proceeding to the reproductive stage. Traditional
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
11 hours. Photoperiods longer than 11 to 12 hours stimulate vegetative growth.
The tropical varieties are usually more sensitive to photoperiod than the quick,
short-season varieties. Sorghum plants are most sensitive to photoperiod during
flower initiation.
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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
rainfall conditions varying from 400 mm to 800 mm.
3. Altitude. Sorghum can grow at altitudes from sea level to 3000 m. The high figure
is probably due to humanly controlled selection.
4. Temperature. Sorghum is a warm-weather crop, which requires high temperatures
for good germination and growth. Seeds germinate well at 10 to 35 °C. Optimum
temperature for germination is 30 °C; being a crop of tropics, it is tolerant of high
temperatures. Breeding efforts have extended its range into cooler area. Base
temperatures vary with cultivars. Temperature plays an important role in growth
and development after germination. A temperature of 27 to 30 °C is required for
optimum growth and development. The temperature can, however, be as low as
21 °C, without a dramatic effect on growth and yield. Exceptionally high
temperatures cause a decrease in yield. Flower initiation and the development of
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.
At an age of 1 to 3 weeks, plants may recover if exposed to a temperature of 5 °C
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.
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5. Soil Requirement. Sorghum plants tolerate a wide range of soils. Sorghum is
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
between 10 and 30 % are optimum for sorghum production.

3.5. Grain morphology
The caryopsis (seed) consists of three distinct anatomical components (Fig.
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.
Pericarp thickness ranges from 8 µm to 160 µm and varies within an
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
impedes moisture loss.
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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,
aleurone, and endosperm exhibit a blue autofluorescence, which is mainly due to
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
(spherosomes). The peripheral area is composed of several layers of dense cells
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
and less protein in the peripheral endosperm than regular sorghums.
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
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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
incoming light giving an opaque or chalky appearance.
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.
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Figure 3.9: Diagrammatic representation of sorghum grain (Source: Chandrashekar and Mazhar, 1999)
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Sorghum grain consists of starch, proteins, fibers, lipids etc. Physical (i.e.
distribution in pericarp, germ, and endosperm) and chemical composition of sorghum
grain is given in the Table 3.4.
Table 3.4. Composition of sorghum seed in % (Source: Waniska and Rooney, 2000)
Caryopsis Endosperm Germ Pericarp
Caryopsis
Range
100

84.2
81.7 – 86.5
9.4
8.0 – 10.9
6.5
4.3 – 8.7
Protein
Range
Distribution
11.3
7.3 – 15.6
100
10.5
8.7 – 13.0
80.9
18.4
17.8 – 19.2
14.9
6.0
5.2 – 7.6
4.0
Fiber
Range
Distribution
2.7
1.2 – 6.6
100









Lipid
Range
Distribution
3.4
0.5 – 5.2
100
0.6
0.4 – 0.8
13.2
28.1
26.9 – 30.6
76.2
4.9
3.7 – 6.0
10.6
Ash
Range
Distribution
1.7
1.1 – 2.5
100
0.4
0.3 – 0.4
20.6
10.4

68.6
2.0

10.8
Starch
Range
Distribution
71.8
55.6 – 75.2
100
82.5
81.3 – 83
94.4
13.4

1.8
34.6

3.8

Carbohydrates in sorghum are composed of starch, soluble sugar and fiber
(pentosans, cellulose, and hemicellulose). Starch is most abundant and others have
low content.
Starch
Description of starch is given in detail in the chapter 2. Sorghum starch has
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 NaClO
2
or rinsing with
NaOH or methanol produces acceptable color. Normal sorghum starch contains 23 –
30 % amylose. Average molecular weights of amylose and amylopectin were 1 to 3 ×
10
5
and 8 to 10 × 10
6
kD, respectively. Sorghum that has three recessive wx genes
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produces caryopses that contain mostly amylopectin and are termed as waxy sorghum.
Waxy sorghum consists of 1% amylose. Heterowaxy sorghum (inclusive one or two
wx genes) consists of 5 to 19 % amylose. Lichtenwalner et al. (1978) reported that %
amylose in normal (WxWxWx), heterowaxy1 (WxWxwx i.e. single wx gene),
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
floury endosperm. Gelatinization temperature range of for sorghum starch is 71 – 80
°C. (Waniska and Rooney, 2000)
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
solubility characteristics: albumin (water soluble), globulin (soluble in dilute salt
solution), prolamin (soluble in alcohol) and glutelin (extractable in dilute alkali or
acid solutions).
The structural and functional properties of kafirins are reviewed by Belton et
al. (2006). In common with other cereals, the major storage proteins (kafirins) in the
grain of sorghum are soluble in alcohol–water mixtures and therefore defined as
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
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from the sequences of cloned DNAs but has not been characterised at the protein
level).
Prolamins (kafirins) constitute the major protein fractions in sorghum,
followed by glutelins. Lack of gluten is characteristic of sorghum protein
composition, and traditionally, the bread which cannot be baked from sorghum and
millet is only cake bread. (Leder, 2004)
Fat and Lipids
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.





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3.6. Utilization of Sorghum
Utilization of the sorghum can be classified mainly in following two
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
maize) as feed elsewhere.
3.6.1. Food use
Worldwide, approximately 27 million tons of sorghum was consumed as food
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
consumption of sorghum in rural producing areas is more stable, and usually
considerably higher, than in urban centers. And within these rural areas, consumption
tends to be highest in the poorest, most food-insecure regions. Sorghum is eaten in a
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
four main sorghum- based foods:
• Flat bread, mostly unleavened and prepared from fermented or unfermented dough
in Asia and parts of Africa;
• Thin or thick fermented or unfermented porridge, mainly consumed in Africa;
• Boiled products similar to those prepared from maize grits or rice;
• Preparations deep-fried in oil.
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Table 3.5. Sorghum utilization by type and region (Source: ICRISAT/FAO, 1996)

Human
Food
Animal
Feed
Other
uses
Total
utilization
Per capita
annual Food use

million
tons
million
tons
million
tons
Million
tons kg
1979–81 average
Developing countries 25 14.7 4.4 44.2 7.7
Africa 9 0.8 2.3 12.1 18.8
Asia 15.7 7.4 2 25.1 6.1
Central America an the
caribbean 0.4 7 0.2 7.6 3.6
South America 0.1 3.7 0.3 4.1 0.3
Developed Countries 0.3 20.4 0.6 21.2 0.2
North America 0.1 10.5 0.2 10.8 0.5
Europe 0 2.8 0 2.8 0
USSR (Former) 0 2.5 0 2.5 0
Oceania 0 0.4 0 0.4 0
World 25.3 35.1 5 65.4 5.7
1989–91 average
Developing countries 25.1 14.5 3.7 43.3 6.2
Africa 11.5 0.9 1.8 14.2 18.2
Asia 13.3 6.1 1.6 21 4.6
Central America an the
caribbean 0.4 8.4 0.3 9.1 2.7
South America 0 2.7 0.2 2.9 0.1
Developed Countries 0.4 16.8 0.5 17.7 0.3
North America 0.2 10.9 0.2 11.3 0.8
Europe 0 1.2 1.2 1.4 0
USSR (Former) 0 0.3 0 0.3 0
Oceania 0 0.8 0 0.8 0
World 25.5 31.3 4.2 61.1 4.8
1992–94 average
Developing countries 26.4 14.8 5.5 46.7 6.2
Africa 12.8 1.3 3.2 17.3 18.6
Asia 13.3 5.6 2 20.9 4.1
Central America an the
caribbean 0.4 7.5 0.3 8.3 2.9
South America 0 3.1 0.3 3.4 0.1
Developed Countries 0.3 15.8 0.7 16.8 0.2
North America 0.1 11.1 0.3 11.5 0.5
Europe 0 1.1 0.2 1.3 0
CIS 0 0.1 0 0.1 0
Oceania 0 0.8 0 0.8 0
World 26.7 30.6 6.2 63.5 4.8





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Per capita annual consumption of sorghum and its importance as a food
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
consumption is over 70 kg). (ICRISAT/FAO, 1996)
However, both production and food utilization have fallen during the 1980s
and early 1990s, because of shifting consumer preferences. As incomes rise,
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
porridge. In all cultures traditionally depending on cereals, a range of treatments of
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
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is consumed in Nigeria and parts of Ghana is ogi. In much of Northern Africa a
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
prepare couscous, although pearl millet is preferred. Sorghum noodles are an
important food product in China. Sorghum is used for tortilla preparation either alone
or in combination with maize in Honduras, Nicaragua, Guatemala, El Salvador and
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,
building materials, and fuel. By applying hydrothermic technologies (flaking,
puffing, extrusion, micronizing) new sorghum and millet products of good quality and
good taste can be produced. (Leder, 2004)
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
from wheat, barley, or rye. Coeliac disease is a syndrome characterised by damage to
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
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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
snacks and pasta. (Taylor et al., 2006)
Sorghum can contain substantial levels of a wide range of phenolic
compounds, which have health promoting properties, in particular their antioxidant
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
alcohols, aldehydes and acids are
interconverted in cellular metabolism, so that all three classes might lower
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
sterols and policosanols, lowered cholesterol absorption and plasma non-HDL
cholesterol in hamsters. (Taylor et al., 2006).
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-
Chapter 3: Sorghum: Literature Review
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free bread making), Cakes and cookies, Tortillas, snack foods, parboiled sorghum,
and noodles.

3.6.2. Industrial Utilization
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
be used as industrial raw material. Statistics on industrial demand (‘000 t) for
sorghum in India is summarized in Table 3.6.
3.6.2.1. Animal feed
About 48 percent of world sorghum grain production was fed to livestock
(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
sorghum is generally regarded as an inferior cereal when consumed as food, the
income elasticities for livestock products (and hence the derived demand for feed) are
generally positive and high.
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
livestock industry is correspondingly intensive. Over 85 percent of the use of sorghum
as animal feed occurs in Developed countries. (ICRISAT/ FAO)
Another important factor is consumer preference for meat colour. Maize
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,
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consumers generally prefer white-coloured meat. Therefore, sorghum is a valued
ingredient in some compound feed rations (for poultry, pigs and some breeds of beef
cattle). In contrast, sorghum is discounted by producers in India because consumers
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).
Industry 1998
1
2010
2

Poultry feed
Broilers 86-129 570-1150
Layers 312-468 1100-1830
Others 20-30 156-234
Dairy feed 160-240 290-570
Alcohol 90-100 200-500
Starch 50 30-80
Total 718-1017 2346-4364
1. These figures reflect the average utilization of sorghum during the 1990s,
based on past inclusion rates and current requirements of raw material, rather
than on specific data for 1998. The poultry and starch industries use sorghum
only when maize is expensive or not readily available
2. Figures are projected ones

The limited inclusion of sorghum in poultry feed and its relatively low status
as a raw material can be accounted to disadvantages of sorghum as given in the Table
3.7.




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Table 3.7. Industry-perceived advantages and disadvantages of using sorghum in
poultry feed (Kleih et al., 2007).
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

3.6.2.2. Alcohol industries
Sorghum has potential for being used in the production of bio-industrial
products, including bioethanol. Sorghum is a starch-rich grain with similar
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
Chapter 3: Sorghum: Literature Review
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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.
Nowadays government policies on licensing alcohol production and trade are
changing and also government is promoting production of grain based alcohol.
Hence, present scenario is providing an opportunity for sorghum to gain greater
acceptability as a raw material in the alcohol industry. Some distillers indicated a
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
producers and industrial users. (Kleih et al., 2007)
Maharashtra Government is in favor of promoting grain-based alcohol
production to create a demand for rainy-season sorghum. It must be remembered in
this context that rain-damaged or blackened sorghum could be a favorable raw
material for alcohol production because of its lower market price. Maharashtra, the
Chapter 3: Sorghum: Literature Review
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main producer of rainy season sorghum, regularly faces the problem of finding
suitable users of blackened sorghum which constitutes 40-60% of its produce,
depending on the rainfall pattern during grain maturity. Advantages and
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.

Table 3.8. Advantages and disadvantages of using sorghum in alcohol production
(Kleih et al., 2007)
Advantages Disadvantages
• No major technical constraints with • Sorghum is a food grain, and may not be
modern technology available for alcohol production in times of
• Causes least pollution food shortages
• Good quality alcohol free from sulphates • Some producers in Maharashtra face
and aldehydes present in molasses based difficulties in selling grain-based alcohol,
alcohol largely due to the State-imposed export pass
• Can create demand for damaged grain fee. This difficulty is localized.
• Possible regular sourcing of grain from • Cost of molasses based alcohol is lower than
rainy-season crop grain based alcohol
• Byproduct of grain alcohol production
can be used as animal feed
.
3.6.2.3. Starch industry
In general the wet milling of sorghum is similar to that of maize. A thorough
review of early research on wet milling of sorghum can be found in Munck (1995).
However, a problem particular to sorghum is the presence of polyphenolic pigments
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 production is reviewed briefly in section 3.10.
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In 2004, starch production in the world was around 60 million t. 70 % of the
starch was produced from corn. Other raw materials being wheat, sweat potato,
cassava and potato. (www.starch.dk/isi/stat/rawmaterial.html) In India, 0.7 million t
of starch was produced in 1998, out of which 0.6 million t was produced from maize
and 0.1 million t was from cassava. (Kleih et al., 2007)

3.6.2.4. Other industries
Malting and brewing
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
or as an adjunct. (Kleih et al., 2007)
There has been extensive research and development work and several
excellent reviews published covering enzymes in sorghum malting, sorghum malting,
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).

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3.7. Insect pests, Diseases and weeds on sorghum
Chandrashekar and Satyanarayana, 2006 have reviewed available information
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, and Weeds and their control in grain sorghum, respectively.

3.8. Problem areas and factors affecting them in industrial utilization of
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
economy as it contributes to rural household food security. (Duodu et al., 2003)
However there are few problem areas in the utilization of sorghum food as well as
industrial utilization and are discussed here.

3.8.1. Gelatinization of starch
Gelatinization of the starch is most important initial step, due to which starch
becomes more susceptible to enzyme action and completely digestible by starch
hydrolyzing enzymes. Phenomenon of starch gelatinization is discussed briefly in the
section 2.1.3.
Sorghum starch gelatinization temperature ranges were reported to be 67–73
°C and 71–81 °C for sorghums grown in southern Africa and in India, respectively
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(Taylor et al., 2006). Gelatinization temperature of sorghum starch is reported to be
in the range of 75–80 °C (Palmer, 1992) and 60–80 °C (Wu et al., 2007).
Gelatinization temperature of sorghum starch is far higher than the range
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,
commercial enzymes or a combination of the two.
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,
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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.
Chandrashekar and Kirleis (1988) showed that the degree of starch
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
cooking markedly increased the degree of gelatinisation. However, increase in the
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
seemed to be surrounded by numerous protein bodies. In contrast, in the soft grains,
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,
whereas soft grains contains large amount of strand protein.

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3.8.2. Protein digestibility
Sorghum plays a crucial role in the world food economy as it contributes to
rural household food security (ICRISAT/ FAO). A nutritional constraint to the use of
sorghum as food is the poor digestibility of sorghum proteins on cooking.
Digestibility may be used as an indicator of protein availability. It is essentially a
measure of the susceptibility of a protein to proteolysis. A protein with high
digestibility is potentially of better nutritional value than one of low digestibility
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
two broad categories:
Exogenous factors: These refer to factors that arise out of the interaction of sorghum
proteins with non-protein components like polyphenols, non-starch polysaccharides,
starch, phylates and lipids.
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
discussed in detail here.



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3.8.3. Starch digestibility
Starch digestibility is an important parameter from the context of feeding
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
for production of ethanol.
Lichtenwalner et al. (1978) reported that amylose content decreased and in
vitro starch digestibility using amyloglucosidase increased with incremental increases
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
amyloglucosidase. Their data is shown in the Table 3.9.

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


Rooney and Pflugfelder (1986) have reviewed factors affecting starch
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
interactions, the cellular integrity of the starch-containing units, antinutritional factors
Chapter 3: Sorghum: Literature Review
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and the physical form of the feed or food material. Starch exists inside the endosperm
of cereals enmeshed in a protein matrix, which is particularly strong in sorghum (Fig.
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
due to restrictions in accessibility to starch caused by endosperm proteins.
High-amylose corn (amylomaize) has poor digestibility in both raw and
cooked forms, while waxy cereal starches are among the most digestible of all
starches. Digestibility of a starch is generally inversely proportional to amylose
content i.e. directly proportional to waxyness of the sorghum. They have also
explained briefly the reasons behind lower digestibility of sorghum as compared to
corn. (Rooney and Pflugfelder, 1986)

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Figure 3.10. SEM images of intermediate texture sorghum kernels. A) Endosperm
cross section (P = pericarp, AL = aleurone cell layer, PE = peripheral endosperm, CE
= corneous endosperm; approx 200X). B) Corneous endosperm area (SV = starch
void, SG = starch granule; approx 1,000X). C) Protein and starch of corneous
endosperm (PM = protein matrix, PB = protein bodies, SG = starch granule; approx
2,000X). D) Starch of floury endosperm (approx 4,000X). (Source: Rooney and
Pflugfelder, 1986)



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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
increased the starch digestibility of sorghum flour by 7–14%, but no significant
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
sodium metabisulfite, starch digestibility of sorghum flours increased significantly.
Elkhalifa et al. (1999) also observed that in vitro starch digestibility (IVSD) of the
treated gruel initially increased in the presence of cysteine, sodium metabisulphite or
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
porcine α-amylase) was significantly higher in floury sorghum endosperm than
vitreous endosperm and cooking with reducing agent, 2-mercaptoethanol, increased
starch digestibility in sorghum, and more with vitreous endosperm flours.
The fact that reducing agents improved sorghum starch digestibility suggests
that disulphide bond cross-linking within the kafirin-containing endosperm protein
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
high proportion and extensive polymerization through disulphide bonding of
prolamins on cooking of sorghum through SDS-PAGE, with the formation of high
molecular weight polymers (M. Wt > 100k). Also formation of web-like or sheet-like
protein structures due to a disulphide-mediated protein polymerization process during
mashing or heat moisture treatment is reported by Hamaker and Bugusu (2003) and
Chapter 3: Sorghum: Literature Review
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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.

3.8.4. Tannin content in sorghum
Tannin is located in the testa portion of sorghum grain. Tannins confer
valuable agronomic properties on sorghum, including protection against insects, birds
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
effect on starch digestibility because of their ability to interact with proteins
(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
malting and brewing.

3.9. Production of ethanol from sorghum: Literature review
Demand of ethanol is consistently increasing as an alternative energy source.
Ethanol is normally manufactured from sugarcane molasses. This conventional
substrate is no longer cheap and good quality raw material due to its decontrol by
Indian government. Also in order to compete in international market, there is need to
improve the alcohol quality. Hence, since 1990s industries are diverting from use of
Chapter 3: Sorghum: Literature Review
Studies in the Enzymatic depolymerisation of natural polysaccharides
100
molasses to starchy substrates for production of ethanol. Also, due to several
environmental issues associated with production of ethanol from molasses,
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
substrates like corn, wheat, tapioca, rice etc.
Recently, Taylor et al. (2006) has reviewed literature on production of ethanol
from sorghum.
Suresh et al. (1999a) developed a simultaneous saccharification and
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
amylase and fermented by Saccharomyces cerevisiae. The damaged portion included
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
utilization of raw starch (i.e. without cooking) would save energy.
Chapter 3: Sorghum: Literature Review
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Zhan et al. (2003) investigated the impact of genotype and growth
environment on the fermentation quality of sorghum. Eight sorghum hybrids grown in
two different locations were used to produce ethanol. The process included following
steps: heating with thermostable α-amylase at 95 °C and then 80 °C (liquefaction),
incubation with amyloglucosidase at 60 °C (saccharification), inoculation with S.
cerevisiae and fermentation for 72 h at 30 °C. It was found that ethanol
concentrations varied relatively narrowly (about 5%) across the 16 samples. Genotype
and production environment had a significant effect on chemical composition and
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
effect on the percentage of the theoretical ethanol yield. However, it is interesting
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
the initial heating steps, a disulphide-mediated protein polymerization process
occurred, resulting in web-like or sheet-like protein structures, as described by
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)
who investigated cooking sorghum using supercritical-fluid-extrusion (SCFX) to
Chapter 3: Sorghum: Literature Review
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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
non-extrusion cooked sorghum. An improvement in the bioconversion of sorghum
starch was accounted to the release of starch from the protein matrix due to SCFX and
enhancing the availability of starch for conversion to fermentable sugar. Literature
review on ethanol production is summarized in Table 3.9 with reaction conditions and
remarks as parameters.
In addition to breeding sorghum specifically for fermentation quality, pre-
processing the grain can be used to improve ethanol yields and process efficiency.
Corredor et al. (2006) investigated decorticating sorghum prior to starch hydrolysis
and ethanol fermentation. In general, decortication decreased the protein content of
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
fermentation and resulted in increased ethanol production. Ethanol yields increased 3–
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
fermentation could be used as a source of phytochemicals (Awika et al., 2005) or as a
source of kafirin and wax.
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Table 3.9. Summary of literature review on ethanol production from sorghum
Reference Reaction conditions Remarks
Suresh et
al., 1999a
200 mL slurry autoclaved at 121 °C for 30 min and a 3%
inoculum of A. niger (NCIM 1248) and 7% inoculum of yeast
was added to it. 2-12% starch concn, 150 rpm, 30 °C, 5 days
Damaged sorghum (50% sound and 50% damaged grains
used in this work)
Suresh et
al., 1999b
100 ml slurry and 0.3% peptone 0.1% KH
2
PO
4
and 0.1%
(NH4)
2
SO
4
; pH 5.8. The crude amylase broth (10 ml) of B.
subtilis VB2 and 6% S. cerevisiae VSJ4 suspension was added
to slurry and incubated at 35 °C and 200 rpm for 4 days.
Optimized conditions being pH 5.8, 35 °C and 25% w/v
slurry concn. High quality and damaged sorghum produced
5% v/v and 3.5% v/v ethanol production with no cooking
step.
Zhan et
al., 2003
16 different varieties of sorghum. Positive correlation
between ethanol concn and starch content and negative
correlation between ethanol concn and protein content.
Zhan et
al., 2006
100 mL slurry, pH 5.8. Liquefaction: 95 °C for 45 min, 80 °C
for 30 min (0.01 mL amylase/g of starch in both steps of
liquefaction). Saccharification: 60 °C (150 U/g of starch) for
30 min. 50 rpm for all steps. Fermentation: 20 g ground
sorghum, 0.3 g peptone, 0.1 g KH
2
PO
4
, and 0.1 g (NH
4
)
2
SO
4

at pH 3.8. Medium was inoculated with 6% yeast suspension
(1×10
6
cells/mL) and incubated 200 rpm for 72 h at 30 °C.
Extrusion could break disulphide protein bonds and disrupt
the protein matrix, gelatinize starch, and make more starch
available for enzyme hydrolysis, and consequently,
increase ethanol yield and fermentation efficiency.
Wu et al.,
2007
Ethanol yields varied by 22% and conversion efficiencies
by 9% among 70 sorghum samples. Positive effect of
starch content on fermentation efficiency and negative
effect of protein, tannin, crude fiber, and ash content on
fermentation efficiency was observed.
Zhao et
al., 2008
30 g flour was mixed with 100 mL distilled water. 10 µL
liquozyme was added and slurry was digested for 45 min at 95
°C. Slurry cooled to 80 °C and second dosage of 10 µL
liquozyme was added and liquefaction was continued for
additional 30 min at 120 rpm. Saccharification: 100 µL
Spirizyme, 120 rpm, 60 °C, 30 min. pH adjusted to 4.2 and
inoculated with 5 mL of 48 h yeast pre-culture.

During mashing cross-linked microstructure, which could
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.
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104
Wu et al. (2007) have performed ethanol production from 70 genotypes and
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
conversion efficiencies by 9% among 70 sorghum samples, indicating significant
effect of sorghum genotype on fermentation efficiency to ethanol. They reported
positive effect of starch content on fermentation efficiency and negative effect of
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
contribute to incomplete gelatinization and hence hydrolysis of starch and conversion
efficiency to ethanol. DSC thermograms of waxy sorghum starch consists of single
endothermic peak (60-80 °C), whereas that of normal sorghum starch showed
presence of two endothermic peaks; one, in 60-80 °C corresponds to amylopectin, and
second, in 85-105 °C corresponds to amylose-lipid complex. Waxy sorghum gives
higher conversion efficiencies to ethanol than normal sorghum; this mainly happens
due to presence of amylose-lipid complex. Wu et al. (2007) concluded that major
factors adversely affecting conversion efficiency to ethanol being condensed tannin,
high viscosity, low protein digestibility, protein-starch interactions, and amount of
amylose-lipid complex.
Zhao et al. (2008) have characterized the changes in sorghum protein in
digestibility, solubility, and microstructure during mashing and to relate those changes
Chapter 3: Sorghum: Literature Review
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to ethanol fermentation quality of sorghum by using 9 sorghum cultivars (2 contained
tannin and rest were tannin free. They have observed that protein solubility and in
vitro protein digestibility decreased significantly after mashing. Tendency of
sorghum proteins to form highly extended, strong web like microstructures during
mashing was confirmed using CFLSM (confocal laser-scanning microscopy) images.
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
efficiency formed a tightly cross-linked microstructure, which could hold starch
granules or polysaccharides inside or retard or prevent the access of enzymes to
starch, and severe cross-linking in this sample was most likely because of a
combination of heat-induced cross linking and cross-linking because of protein-tannin
interactions. More open web-like microstructures were observed in cultivars with
higher conversion efficiencies upon mashing. Protein digestibility of the unmashed
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
relate significantly with conversion efficiency. But, it neither correlated to ethanol
yield nor conversion efficiency significantly. They concluded that protein cross-
linking does play a role in the production of ethanol from sorghum, albeit through
indirect measures of protein cross-linking (i.e. reduction in protein digestibility after
mashing, which is due to protein cross linking).

3.10. Production of starch from sorghum: Literature review
In general the wet milling of sorghum is similar to that of maize. A thorough
review of early research on wet milling of sorghum can be found in Munck (1995).
Chapter 3: Sorghum: Literature Review
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Recently, Taylor et al. (2006) have shortly reviewed literature on starch production
from sorghum. However, a problem particular to sorghum is that where polyphenolic
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
reported. Perez-Sora and Lares-Amaiz (2004) investigated alkaline reagents for
bleaching the starch and found a mixture of sodium hypochlorite and potassium
hydroxide to be the most effective. To improve the economics of sorghum wet-
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
high moisture fraction that was diverted to animal feed.
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
relationships between sorghum grain quality characteristics and wet-milling
performance in 24 commercial sorghum hybrids (Buffo et al., 1998). Perhaps not
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-
milling of sorghum. Related to this, Mezo-Villanueva and Serna-Saldivar (2004)
found that treatment of steeped sorghum and maize with protease increased starch
yield, with the effect being greater on sorghum than maize.
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%
Chapter 3: Sorghum: Literature Review
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107
lactic acid at a temperature of 50 °C for 36 h. Using this steeping process, wet-milling
of sorghum produced starch with an lightness value (Black and white samples will
have lightness value of 0 and 100 respectively. Lightness value is measured by
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
content and sorghum grain properties influence sorghum starch properties.
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
produced by wet-milling sorghum grits (Higiro et al., 2003).
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
as is done for maize (Xie et al., 2006).
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
protein in the starch. New developments in wet-milling procedures for sorghum as
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).
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
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4. Hydrolysis of soluble starch using
B. licheniformis α-amylase
immobilized on superporous
CELBEADS





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4.1. Introduction and Literature review
Most green plants produce starch as a means of energy storage. Starch is a
polymer composed of glucose units linked by α(1→4) glucosidic bonds and α(1→6)
linkages. It consists of two types of polymers: amylose and amylopectin. Amylose is
roughly linear molecule containing ~ 99% α(1→4) and ~ 1% α(1→6) bonds with
molecular weight of
6 5
10 1 10 1 × − × . Amylopectin is a much larger molecule
(molecular weight of
9 7
10 1 10 1 × − × ) and is a branched polymer with ~ 95% α(1→4)
and ~ 5% α(1→6) bonds. Linear chains of 12-120 glucose units (linked by α(1→4)
glucosidic bonds) are connected by α(1→6) glucosidic linkages.
Starch has become a very important biopolymer and is used in many industries
as a feedstock material. In several industrial processes, enzymes are used to
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
commercial maltodextrin was Frodex 15 (later called Lo-Dex 15), introduced by
American Maize Products Company in 1959 (Alexander, 1992).
Starch hydrolysis products are commonly characterized by their degree of
hydrolysis, expressed as the dextrose equivalent (DE), is the percentage of reducing
sugar calculated as the dextrose on dry weight basis. Theoretical DE is defined as the
percentage of reducing sugar (glucose equivalent) to total reducing sugar (glucose
equivalent) produced after complete hydrolysis. Pure starch has DE of zero and
dextrose has DE of 100.
The term maltodextrins is used for the saccharide mixtures of dextrose
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superporous CELBEADS
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equivalent (DE) less than 20, which consist of maltose, malto-oligosaccharides and
linear or branched dextrins (Alexander, 1992). Since maltodextrins are products of
partial hydrolysis of natural starch, their saccharide composition varies with nature
and concentration of enzyme/s, extent of hydrolysis and botanical origin of starch
used for hydrolysis. Maltodextrins with the same average DE value can have different
saccharide composition (Kennedy and Cabral, 1987). DE value of a maltodextrin has
been shown to be inadequate to predict product performance in various applications
(Chronakis, 1988). The saccharide composition of maltodextrins determines it’s both
physical and biological functionality, and there are different parameters (like type and
source of enzyme, source of starch, starch concn, temperature, organic solvents,
immobilization of enzymes, downstream processing etc.) influencing the saccharide
composition of maltodextrins (Marchal et al., 1999). Design of the desired saccharide
composition and production possibilities for maltodextrins were briefly discussed
(Marchal et al., 1999). Aspects related to the saccharide composition include
hygroscopicity, gelation, sweetness, stability, fermentability in food products,
osmolality, and absorption of maltodextrins by humans (Marchal et al., 1999).
Maltodextrins find applications in various industries like confectionary industry,
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
are discussed in detail in the chapter 2.
Though partial hydrolysis of starch has traditionally been carried out using
acids, acid hydrolysis is being replaced by enzymatic hydrolysis for the production of
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superporous CELBEADS
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tailor-made maltodextrins (Schenck and Hebeda, 1992; Marchal et al., 1999). The
most widely used enzymes for production of maltodextrins using partial hydrolysis of
starch are α-amylase from B. licheniformis, B. stearothermophilus and B.
amyloliquefaciens. Production of maltodextrin from starch is reviewed in detail in the
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
effluent problems. These problems can be overcome by using ‘insoluble’ enzymes,
which can be separated easily from the reaction mixture after the reaction is over.
4.1.1. Immobilized enzymes
The process of making enzymes insoluble through their localization on some
or the other kind of solid surface is called immobilization. Immobilized enzymes are
defined as “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” (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
bulk phase containing substrate and eventually the product.
Advantages of an immobilized enzyme over the soluble enzyme are:
1. Reusability of the enzyme
2. Continuous operation of the system
3. Easy separation of product from the enzyme
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4. Less effluent problems
5. Increased stability and activity of the enzyme in some cases
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
properties of the solid matrix on to which the enzyme is immobilized plays an
important role. Ideal solid support should have the following properties:
1. Large surface area to achieve higher immobilization yields
2. Hydrophilic character to avoid denaturation of enzyme
3. High rigidity to withstand high pressure drop in packed bed columns
4. Chemical, mechanical and thermal stability
5. Resistance to microbial attack
6. Permeability to allow easy diffusion of substrates and products in and out of
the matrix pores
Morphologically, matrices can be classified as a) Porous and b) Non-porous.
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
and product molecules and should minimize internal diffusional resistances.
Various chemistries can be used for the immobilization of enzymes on solid
supports. These techniques can be divided into three major classes (Kennedy and
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superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
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Cabral, 1987):
1. Support binding method
1.1. Physical adsorption
1.2. Ionic binding
1.3. Metal linking method using inorganic supports
1.4. Covalent binding
2. Entrapment method
2.1. Gel entrapment
2.2. Microencapsulation
3. Cross-linking method
4.1.2. Immobilization of bacterial α-amylase
Ivanova and Dobreva (1994) studied the hydrolysis of starch, maltopentaose
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
amounts of low molecular weight saccharides, identified as glucose, maltose and
malto-oligosaccharides up to maltopentaose were produced. Immobilized alpha
amylase on the other hand produced higher quantity of malto-oligosaccharides than
the soluble enzyme. Soluble enzyme could partially hydrolyze maltopentaose into
maltotriose, maltose and glucose, whereas immobilized alpha amylase could not
hydrolyze maltopentaose. Soluble enzyme hydrolyzed maltohexaose completely,
while immobilized alpha amylase exhibited an increase in the production of glucose
from maltohexaose and maltopentaose was obtained as the major hydrolysis product.
Reasons for a different saccharide composition with immobilized enzyme compared
to free enzyme can be attributed to diffusion limitation, which increases with the
degree of polymerization (DP) of oligosaccharide, enhancing the heterogeneous
hydrolysis (Tarhan, 1989). The other reason is that, immobilization alters three
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
114
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;
Karandikar, 2004) to immobilize bacterial α-amylase and application of it to
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.
However these diffusional resistances can be overcame or significantly reduced by
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,
2004) for immobilization of enzymes. Hence in the present work, Bacillus
licheniformis α-amylase (BLA) was immobilized on superporous (pore diameter ∼ 3
µm) CELBEADS, in order to minimize diffusional resistances.
It is reported (Marchal et al., 1999) that there is no significant influence of pH
(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
saccharide composition of starch hydrolysate produced using immobilized bacterial
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
concn on the saccharide profile of starch hydrolysate produced using immobilized
BLA has been also studied in a batch mode. Thermostability and reusability of
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
115
immobilized BLA was also studied. Also, a semiempirical model has been used for a
priori prediction of saccharide composition of starch hydrolysate with respect to time.
Also, in this work the possibility of production of glucose using gelatinized sorghum
slurry has been explored.

4.2. Experimental
4.2.1. Materials
3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for
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
Advance Enzyme Technologies Pvt Ltd (India).
CELBEADS, a rigid superporous cross-linked cellulose matrix, were prepared
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.
Table 4.1. Properties of CELBEADS (Lali and Manudhane, 2003)
Properties Description
Mean Bead size 200 µm (100-350 µm)
Spherecity 0.7-0.9
Nature Rigid aerogel
Average pore size ~ 3 µm
Total volume porosity ~ 57%
Bulk density (water) 1438 kg/m
3




Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
116
4.2.2. Methods
4.2.2.1. Measurement of protein and reducing sugar concentration.
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
DNSA method are provided in the Appendix A.
Theoretical dextrose equivalent (DE) of the starch hydrolysate is defined as
following,
100
18) n (162 e hydrolysat starch of wt mol averaged number
180.6 i.e. glucose anhyd of wt mol
DE ×
+ ×
= (4.1)
where n is the average degree of polymerization (DP) of starch hydrolysate, which
can be calculated by following formula;
e hydrolysat starch in equiv) (glucose sugar reducing of concn
sidase amylogluco using hydrolysis complete after equiv) (glucose sugar reducing of concn
n =
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
calculated using Eq. 4.1.

4.2.2.2. HPTLC analysis.
Oligosaccharide separation of starch hydrolysate samples was performed using
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,
Heidelberg, Germany), equipped with a 10 µL microsyringe (Hamilton, Switzerland).
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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
117
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
seconds in the diphenylamine-aniline-phosphoric acid reagent (40 mL acetone, 0.8 g
diphenylamine, 0.8 mL aniline and 6 mL 85% H3PO4) and then keeping at 120 °C for
10 min. Densitometry was performed using HPTLC densitometer CD 60 (DESAGA,
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
India Ltd and Maltotriose (G3), Maltotetraose (G4), Maltopentaose (G5),
Maltohexaose (G6) and Maltoheptaose (G7) from Sigma (St Louis, MO, USA).
Concentrations of malto-octaose (G8), maltononaose (G9) and maltodecaose (G10) in
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.
15 40 50 70 80 mm
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10

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

A
B
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
118
4.2.2.3. Immobilization of B. licheniformis α-amylase (BLA) on CELBEADS.
Surface hydroxyl groups of CELBEADS were activated using epichlorohydrin
(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
followed for immobilization. Chemistry of the immobilization is shown in Fig. 4.2.
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), NaBH
4
(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 NaHCO
3
and distilled water sequentially. The washed and suction dried epoxy-
activated matrix (ECH-CELBEADS) was then added to a flask containing a mixture
of 0.2 M NaHCO
3
(22.5 mL) and EDA (15 mL). The mixture was shaken at 50 °C on
an orbital shaker for 24 h. The resulting matrix (ECH-EDA-CELBEADS) was filtered
and washed successively with 200 mL each of 0.1 M HOAc, 0.2 M NaHCO
3
and the
distilled water.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
119
CH
2
CH CH
2
O
O
H
2
N CH
2
CH
2
NH
2
CH
OH
CH CH NH
2
O CH
2
N
CH
2
CH
OH
CH CH CH
2
NH
2
O CH
2
HOC CHO ( CH
2
)
3
NaBH
4
CH
OH
NH CH CH
2
O CH
2
NH ( CH
2
)
3
CHO
N
CH
2
H
2
N Enzyme NaBH
4
CH
OH
NH CH
2
CH
2
O CH
2
NH ( CH
2
)
3
CHO CH
2
CH
OH
NH CH
2
CH
2
O CH
2
NH (CH
2
)
3
CH
2
CH
2
NH Enzyme
CH
2
CH CH
2
O
Cl
CH
2
CH CH
2
O
O
OH
+
EP-CELBEADS
EP-EDA-CELBEADS EDA
+
+
EP-EDA-CELBEADS
GA Sodium borohydride
EP-EDA-GA-CELBEADS
EP-EDA-GA-CELBEADS
+
Enzyme
+
Sodium borohydride
Immobilized enzyme
CELBEADS EP
EP-CELBEADS

Figure 4.2. Chemistry of immobilization of enzyme on CELBEADS

The matrix (ECH-EDA-CELBEADS) was then further activated overnight for
enzyme conjugation using 30 mL of 12.5% w/v aqueous glutaraldehyde (GA). The
activated matrix (ECH-EDA-GA-CELBEADS) was washed well with distilled water
to remove traces of glutaraldehyde. Activated matrix was mixed with 20 mL of 100
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, NaBH
4
(0.08
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
120
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.

4.2.2.4. Amylolytic activity measurement.
4.2.2.4. A. Free BLA.
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
6 min. Mixture of 0.5 mL of the gelatinized starch solution (9 mg/mL), 1.4 mL of
acetate buffer (0.1 M, pH 5.6) and 0.1 mL of 10000 fold diluted free BLA was
incubated at 55 °C (optimum enzyme activity temperature; found separately) in a
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,
min 20 mL) 0.1 (i.e. aliquot enzyme of volume (180.6) glucose of wt. mol.
10000) (i.e. aliquot enzyme of factor dilution
g/mL) ( equiv.) (Glucose sugars Reducing of ion Concentrat
FEU/mL
× ×
×
=
µ


4.2.2.4. B. Immobilized BLA.
End point assay method has been employed to calculate enzyme activity of
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
121
immobilized bacterial α-amylase. Gelatinized starch solution (25 mL) of concn 90
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
can be calculated from the following formula,
min 60 (180.6) glucose of wt. mol.
ml 25
g/ml) ( hr 1 in produced ) equivalent (glucose sugar Reducing
CELBEADS of EU/ml
×
×
=
µ

Retained enzyme activity is defined as the ratio of sp activity of immobilized
enzyme to sp activity of free enzyme (calculated by assay procedure of immobilized
BLA).

4.2.2.5. Measurement of kinetic rate constants of free and immobilized BLA.
Gelatinized starch solutions (25 mL each) prepared in 0.1 M acetate buffer
(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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
122
(K
m
and V
max
) were determined for free and immobilized BLA from Eadie-Hofstee
plot of V vs. V/[S]
0
with -K
m
as slope and V
max
as the y-intercept. Since V
max
is not a
fundamental property of the enzyme and is dependent on the enzyme concn, it was
converted to turnover number, k
cat
(i.e. V
max
/[IEU] for immobilized BLA and
V
max
/[IEU equiv] for free BLA).

4.2.2.6. Hydrolysis of soluble starch using immobilized BLA in batch mode.
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
which there is no dependence of hydrolysis curve on the rotational speed (which is
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
withdrawn and diluted to concn of 9 mg/mL to avoid retrogradation. Samples were
then analyzed for reducing sugar concn and immediately frozen. Samples were
thawed and saccharide composition of samples was determined by HPTLC. Effect of
reaction conditions on hydrolysis and oligosaccharide composition was studied by
varying pH, temperature, [S]
0
and [IEU] in the range of 4.4 to 7, 37 to 70 °C, 18 to
180 mg/mL and 0.2964 to 1.86 respectively.


Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
123
4.2.2.7. Thermostability and reusability of immobilized BLA.
Immobilized BLA (5mL) was resuspended in 50 mL of 0.1 M acetate buffer
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
rpm) at 55 °C without the presence of starch for 24 h. Samples of immobilized BLA
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
described in section 4.2.2.6. At end of 8 h, reaction mixture was separated from
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.

4.2.2.8. Hydrolysis of soluble starch using immobilized BLA in packed bed or
expanded bed mode.
Hydrolysis of gelatinized starch solution was carried out with immobilized
BLA (2 mL) packed in a 10 mm diameter and 20 cm long jacketed glass column
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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
124
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
hydrolysis by analyzing the same for reducing sugar.

Figure 4.3. Experimental set-up for hydrolysis of starch using
immobilized α-amylase in packed bed mode.
A Water bath cum circulator
B Peristaltic pump
C Magnetic stirrer
D Jacketed mixing tank
E Packed bed of immobilized α-amylase on CELBEADS
F Jacketed column

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
A
B
C
D
E
F
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
125
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
intermittent washing steps by employing procedure described in the section 4.2.2.7.
4.2.2.9. Measurement of residence time in the packed bed.
5mL of de-aerated underivatized CELBEADS were packed in a 10 mm
diameter and 20 cm long glass column. A pulse of 2 mL of 10% (w/v) gelatinized
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
(described in Appendix A). The Concentration of starch in the eluting fractions
collected (C) was plotted against time (t). The value of mean residence time t
m
was
calculated by the formula (Fogler, 2005): ) ( / ) ( t C t tC t
m
∆ Σ ∆ Σ = .

4.3. Results and Discussion
4.3.1. Immobilization of bacterial α-amylase on CELBEADS.
BLA was immobilized onto CELBEADS by covalent binding as described
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
CELBEADS) got immobilized.
The number of enzyme units immobilized per ml of CELBEADS can be
determined from material balance as mentioned earlier. But when an enzyme is
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
126
immobilized on matrix, activity of enzyme may decrease, or change due to several
reasons;
1. Some immobilized enzyme molecules may be immobilized in such a way that
the active site could be oriented towards the support surface i.e. decreasing the
accessibility of substrate molecule to the enzyme.
2. A reactive site in the enzyme molecule may be involved in the binding to the
matrix.
3. The enzyme molecules on binding may be held in an inactive configuration.
4. The reaction conditions used for immobilization may cause denaturation or
inactivation of the enzyme.
Hence it becomes necessary to determine activity of immobilized enzyme
separately (reported in Table 4.2) in addition to FEUs immobilized per mL of
CELBEADS from material balance.

4.3.2. pH and temperature dependence of activity of free and immobilized BLA
and their catalytic properties.
After immobilization, pH-enzyme activity profile shifted towards acidic side
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
solution and the microenvironment in the vicinity of immobilized enzyme molecule
(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
°C after the immobilization, indicating that optimum temperature changes from 55 °C
to 55-70 °C (Fig. 4.5) upon immobilization. Fig. 4.5 also shows that % relative
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
127
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
macropores is likely to be more at low temperature (<55 °C) as compared to higher
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
in the enzyme rigidity upon immobilization by covalent binding.
Properties of free and immobilized BLA are summarized in the Table 4.2.
Activation energy (E
a
) of immobilized BLA (3.18) was higher than that of free BLA
(1.63). Similar increase in the E
a
after immobilization is reported and attributed to the
change in enzyme structure upon immobilization (Ivanova et al., 1998).
app
m
K

of
immobilized BLA (15 mg/mL) was 4.5 times of the
free
m
K (3.3 mg/mL). Higher value
of K
m
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
upon immobilization or lower accessibility of substrate to the active enzyme site of
the immobilized BLA due to steric hindrances and still persisting diffusional
limitation. The apparent value of K
m
is reported to increase up to 2.6 times and 9
times for α-amylase (from porcine pancreas) immobilized on HEMA and styrene-
HEMA microspheres (Tumturk et al., 2000)

respectively; up to 10 times for α-
amylase (B. licheniformis) immobilized on different types of matrices (Ivanova et al.,
1998).
app
cat
k

of immobilized BLA (0.93) was about half of the
free
cat
k (1.76). Lower
value of k
cat
of immobilized BLA is due to lower accessibility of substrate to the
active enzyme site of the immobilized BLA, which subsequently results into lower
reaction rate. Apparent V
max
is reported to decrease marginally (Tumturk et al., 2000)
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
128
for nonporous support, as well as significantly up to 20 times (Ivanova et al., 1998)

for porous support. This is mainly attributed to diffusional limitation that exists in the
pores of porous support.

Table 4.2. Properties of free and immobilized BLA
Parameter Free BLA Immobilized BLA
Optimum pH 5.6 5.2
Optimum Temperature (°C) 55 55-70
Protein content (mg/mL) 75 0.83
a

Activity of biocatalyst
(FEU/mL)
(IEU/mL)

16500
18450
b


300
a

18.5
Sp activity
(FEU/mg of protein)
(IEU/mg of protein)

220
246.1
b


361
a

22.3
Retained enzyme activity
after immobilization
n.a 9.1%
E
a
(kcal/mol) 1.63
c
3.18
d

K
m
(mg/ml) 3.3 15
V
max
(µmol/(min.mL)) 1.3 0.46
k
cat
(min
-1
) 1.76 0.93
a
calculated using material balance;
b
calculated using assay procedure adapted for immobilized BLA (i.e. IEU
equiv);
c
calculated using arhenius plot over temperature 32-55 °C;
d
calculated using arhenius plot over temperature 37-55 °C.

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
129
5
25
45
65
85
105
4 4.4 4.8 5.2 5.6 6 6.4 6.8 7.2
pH
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
Immobilised BLA, 55 °C
Free BLA, 55 °C

Figure 4.4. pH-% relative activity profile of free and immobilized BLA
5
25
45
65
85
105
30 35 40 45 50 55 60 65 70 75
Temperature (°C)
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
Immobilised BLA, pH 5.2
Free BLA, pH 5.6

Figure 4.5. Temperature-% relative activity profile of free and immobilized BLA

4.3.3. Effect of reaction conditions on hydrolysis of soluble starch using
immobilized BLA and saccharide composition
4.3.3.1. Effect of pH.
Hydrolysis performed at pH 5.2 showed maximum initial hydrolysis rate as
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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
130
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
reducing sugar concn of 20 mg/mL), hydrolysis rate decreases significantly as
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
in unbuffered solution is relatively low due to continued exposure to varying pH
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
that increase in the pH of the reaction mixture up to 7 favors binding of higher
dextrins to immobilized BLA and the subsequent hydrolysis of the same.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
131
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7 8
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
4.4 5
5.2 5.4
5.6 6
7 unbuffered
pH

Figure 4.6. Concentration of reducing sugars vs. time with pH as parameter
at [S]
0
= 90 mg/mL, 55 °C and [IEU]/[S]
0
= 8.27e-3.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
4.3 4.8 5.3 5.8 6.3 6.8
70
75
80
85
90
95
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
4.3 4.8 5.3 5.8 6.3 6.8
70
75
80
85
90
95
B
0
1
2
3
4
5
6
7
8
9
4.3 4.8 5.3 5.8 6.3 6.8
60
65
70
75
80
85
C
0
2
4
6
8
10
12
14
4.3 4.8 5.3 5.8 6.3 6.8
50
55
60
65
70
75
80
D
w
t

p
e
r
c
e
n
t
a
g
e

o
f

G
1
-
G
7















)

w
t

p
e
r
c
e
n
t
a
g
e

o
f

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
s

o
f

D
P

>
7






)
pH

Figure 4.7. Effect of pH on the saccharide composition at reaction conditions same as
Fig. 4.6. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.
□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6, + G7, - oligosaccharides with DP >7.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
132
0
2
4
6
8
10
12
14
16
4.3 4.8 5.3 5.8 6.3 6.8
pH
50
55
60
65
70
75
80
A
0
2
4
6
8
10
12
14
16
35 40 45 50 55 60 65 70
Temperature (°C)
50
55
60
65
70
75
B
0
2
4
6
8
10
12
14
16
0 45 90 135 180
[S]
0
45
50
55
60
65
70
75
80
C
0
2
4
6
8
10
12
0.0025 0.0085 0.0145 0.0205
[IEU]/[S]
0
40
45
50
55
60
65
70
75
80
D w
t

%

o
f

G
1
-
G
7
w
t

%

o
f

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
s

o
f

D
P

>
7







)

Fig. 4.8. Effect of pH
a
, temperature
b
, [S]
0
c
and [IEU]/[S]
0
d
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.

4.3.3.2. Effect of temperature.
Initial rate of reaction (V) increases significantly with an increase in the
temperature from 37 °C to 55 °C; but further increase in temperature results in lesser
increase in V and initial rates at temperature above 60 °C are approximately the same
(Fig. 4.5 and Fig. 4.9).
At DE of 20, wt % of G5 and G3 remains constant with an increase in the
temperature from 36 to 50 °C at 12-13 and 6-7 respectively, but further increase in
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
133
temperature up to 70 °C significantly decreases wt % of G5 and G3 from 12.5 to 8
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
that increase in the temperature decreases product specificity of immobilized BLA
towards G3 and G5, whereas specificity towards G1, G2, G4, G6 and G7 increases.
This results in more homogeneous molecular weight distribution with an increase in
the operating temperature.
Similar decrease in wt % of G5 and G3 and increase in wt % of G2 and G4
with an increase in the temperature from 50 to 90 °C, for free α-amylase (B.
licheniformis, Maxamyl), are reported and is attributed to the combination of the
following aspects: (1) A decrease in product specificity of α-amylase with increasing
temperature. (2) An increase in amount of transglycosylation products with increasing
temperature. (3) A change in the ratio of rate of hydrolysis of different linear
oligosaccharides (of different DP) with increasing temperature. (Marchal et al., 1999)
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7 8
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
37 45 50
55 60 65
70
Temperature (°C)

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.
.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
134
0
0.5
1
1.5
2
2.5
35 40 45 50 55 60 65 70
60
65
70
75
80
85
90
95
A
0
1
2
3
4
5
6
35 40 45 50 55 60 65 70
60
65
70
75
80
85
90
B
0
1
2
3
4
5
6
7
8
9
10
35 40 45 50 55 60 65 70
60
65
70
75
80
85
C
0
2
4
6
8
10
12
14
35 40 45 50 55 60 65 70
50
55
60
65
70
75
D
Temperature (°C)
w
t

p
e
r
c
e
n
t
a
g
e

o
f

G
1
-
G
7
w
t

p
e
r
c
e
n
t
a
g
e

o
f

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
s

o
f

D
P

>

7
)


Figure 4.10. Effect of temperature on the saccharide composition at reaction
conditions same as Fig. 4.9. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.
□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.

4.3.3.3. Effect of initial starch concn, [S]
0
and [IEU]/[S]
0.

In order to compare hydrolysis curves (Fig. 4.11 (A)) at different values of
[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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
135
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
respectively at DE of 20) with an increase in [S]
0
from 18 to 90 mg/mL and further
increase in [S]
0
from 90 to 180 mg/mL results in an increase in wt % of G3 and G5
(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
20) with a further increase in [S]
0
from 90 to 180 mg/mL.
With an increase in [IEU]/[S]
0
obviously hydrolysis takes place fast (Fig.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
136
4.13). It can be seen from Fig. 4.8 (D) and Fig. 4.14 that there are marginal changes
in saccharide composition with an increase in the ratio of [IEU]/[S]
0
.
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6 7 8
Time (h)
C
o
n
c
.

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
18 45
90 180
[S]
0
(mg/mL)
0
5
10
15
20
25
0 1 2 3 4 5 6 7 8
Time (h)
D
e
x
t
r
o
s
e

e
q
u
i
v
a
l
e
n
t

(
D
E
)
18 45
90 180
[S]
0
(mg/mL)

Figure 4.11(A). Figure 4.11(B).
Figure 4.11. A. Concentration of reducing sugars vs. time with [S]
0
as parameter
B. DE vs. time with [S]
0
as parameter; at 55 °C, pH 5.2 and [IEU]/[S]0 = 3.3e-3.
0
0.5
1
1.5
2
2.5
3
3.5
0 45 90 135 180
70
75
80
85
90
95
100
A
0
1
2
3
4
5
6
7
8
0 45 90 135 180
70
74
78
82
86
90
B
0
2
4
6
8
10
12
0 45 90 135 180
45
50
55
60
65
70
75
80
85
C
0
2
4
6
8
10
12
14
16
0 45 90 135 180
45
50
55
60
65
70
75
D
w
t

p
e
r
c
e
n
t
a
g
e

o
f


G
1
-
G
7
w
t

p
e
r
c
e
n
t
a
g
e

o
f

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
s

o
f

D
P
>
7



)
Initial starch concentration (mg/mL)

Figure 4.12. Effect of [S]
0
on saccharide composition at reaction conditions same as
Fig. 4.11. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.
□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
137
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7 8
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
3.3e-3 8.23e-3
13.8e-3 20.7e-3
[IEU]/[S]
0

Figure 4.13. Concentration of reducing sugars vs. time with [IEU]/[S]
0
as
parameter at pH 5.2, 55 °C and [S]
0
= 90 mg/mL.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.0025 0.0085 0.0145 0.0205
70
75
80
85
90
95
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0.0025 0.0085 0.0145 0.0205
70
74
78
82
86
90
B
0
1
2
3
4
5
6
7
8
0.0025 0.0085 0.0145 0.0205
60
65
70
75
80
85
C
0
2
4
6
8
10
12
0.0025 0.0085 0.0145 0.0205
40
45
50
55
60
65
70
75
80
D
[IEU]/[S]
0
w
t

p
e
r
c
e
n
t
a
g
e

o
f

G
1
-
G
7
w
t

p
e
r
c
e
n
t
a
g
e

o
f

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
s

o
f

D
P

>
7














)

Figure 4.14. Effect of [IEU]/[S]
0
on saccharide profile at reaction conditions same
as Fig. 4.13. A, DE 8-9; B, DE 12-13; C, DE16-17; and D, DE 20-21.
□ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and + G7, - oligosaccharides with DP >7.


Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
138
4.3.4. Comparison of saccharide composition of starch hydrolysate using free
and immobilized BLA
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
to the presence of branched dextrins; whereas for immobilized BLA, DE of starch
hydrolysate at hydrolysis equilibrium was marginally low (around 36-37). Reason
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
following exponential equation,
Bt)) exp( A(1 DE DE
0
− − + = (4.2)
in which DE
0
is initial DE of the gelatinized starch solution (∼ 5-6), A + DE
0
is
maximum attainable DE by hydrolysis (which is around 36-37 for immobilized BLA)
and B is pseudo first order hydrolysis constant (h
-1
). Hydrolysis constant (B) was
correlated with operating parameters for immobilized BLA by following empirical
equation.
008314T)) -33.65/(0. 45.18)exp( 0.521[S] (-0.002[S] ) U]/[S] 5.67e4([IE B
0
2
0
0.892
0
+ + =
(4.3)
In case of the free BLA, it can be seen from Fig. 4.15 that concn of G1, G2,
G3, G4 and G5 increases w. r. t. reaction time. Concn of G6 and G7 increases with an
increase in DE and attains maxima at DE of about 37 and then shows a minor
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
and G7 increases up to DE of 25 and 20 respectively and then decreases (due to their
further hydrolysis) with further increase in the DE. Data of G8, G9 and G10 are not
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
139
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
oligosaccharides w. r. t. DE or time has not been reported. This information will be
useful in deciding the appropriate reaction quenching time to get the final product of
desired saccharide composition.
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 time (h)
c
o
n
c
n

o
f

G
1
-
G
5

(
m
g
/
m
L
)
0
2
4
6
8
10
12
c
o
n
c
n

o
f

G
6
-
G
1
0

(
m
g
/
m
L
)
G1 G2
G3 G4
G5 G6
G7 G8
G9 G10
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.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
140
0
5
10
15
20
25
30
35
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5
time (h)
c
o
n
c
n

o
f

G
1
-
G
5

(
m
g
/
m
L
)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
c
o
n
c
n

o
f

G
6

a
n
d

G
7

(
m
g
/
m
L
)
G1 G2 G3 G4 G5 G6 G7
DE 5.8 13.4 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2

Figure 4.16. Change in concn of oligosaccharides w. r. t. time and DE, with
immobilized BLA. [S]
0
= 90 mg/mL, [IEU]/[S]
0
= 20.7e-3, pH 5.2, 55 °C.
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10
Degree of polymerisation of oligosaccharide
w
t

%

o
l
i
g
o
s
a
c
c
h
a
r
i
d
e
DE 13 Free BLA
DE 13 Immobilized BLA
DE 20 Free BLA
DE 20 Immobilized BLA
DE 36 Free BLA
DE 36 Immobilized BLA

Figure 4.17. Comparison of saccharide profile produced by free and immobilized
BLA.
Fig. 4.17 shows that at same DE, wt % of G1, G2, G3 and G5 produced by
immobilized BLA were much higher than that produced by free BLA and there was
no significant change in wt % of G4, whereas wt % of G6, G7, G8, G9 and G10
produced by immobilized BLA were significantly lower than that produced by free
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
141
BLA. Composition of starch hydrolysate produced by free BLA (reported in this
work) is different than the earlier reports (Ivanova and Dobreva, 1994; Marchal et al.,
1999) (produced by α-amylase, B. licheniformis), which could be due to different type
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.
At hydrolysis equilibrium with immobilized BLA, there were only traces of
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
further hydrolyzed leaving only traces of G6 and G7 at hydrolysis equilibrium.
However presence of significant quantities of G6-G10 at hydrolysis equilibrium with
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
of immobilized BLA, linear part of higher dextrins must be forming productive
complex with active enzyme site rather than branched part. Reason for this could be
attributed to steric hindrances, which is a property of both porous nature of the
support and extent of branching of starch (Marchal et al., 1999). However there are no
such steric hindrances in the case of free BLA.
Few experiments were also performed on hydrolysis of G4, G5, G6 and G7
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.
This must be because immobilized BLA hydrolyses major fraction of G7 to G5 and
G2, and rest to G6 and G1; thus produced G6 further hydrolyses to G5 and G1.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
142
Action pattern and subsite mapping of free BLA with modified malto-
oligosaccharide substrates have been reported by Kandra et al., 2002. Binding region
or active site of BLA is composed of five glycone, three aglycone-binding subsites
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)
Major production of G5 by hydrolysis of G6 and G7, and higher production of
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
hydrolysis of linear G6-G10, however effect of branching characteristic of starch was
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
between free BLA and branched dextrins.

4.3.5. Thermostability and reusability of immobilized BLA
Fig. 4.18 shows that the thermostability BLA was improved after
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
143
immobilization. Relative activity (%) of free BLA decreases from 100 to 75, whereas
it nearly remains same for immobilized BLA over the incubation period.
Immobilization of enzyme, by covalent binding, often improves thermostability
(Paolucci-Jeanjean et al., 2000). Reason could be attributed to improvement in
enzyme rigidity after immobilization.
Unlike free enzyme, immobilized enzyme can be easily separated from the
reaction mixture and reused. Hence reusability or operational stability is an important
criterion for industrial use of immobilized enzyme. Reusability study of immobilized
BLA shows that 100% activity of immobilized BLA was retained even after 8 batch
hydrolysis, which indicates good reusability. For comparison, hydrolysis curves of 1
st

and 8
th
batch are shown in Fig. 4.19.
0
20
40
60
80
100
120
0 3 6 9 12 15 18 21 24
Time (h)
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
Free BLA
Immobilized BLA

Figure 4.18. Thermostability of free and immobilized BLA.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
144
0
5
10
15
20
25
30
0 1 2 3 4 5 6 7 8
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)
1st batch hydrolysis
8th batch hydrolysis

Figure 4.19. Reusability of immobilized BLA at pH 5.2, 55 °C,
[IEU]/[S]
0
= 6.13 e-3, [S]
0
= 90 mg/mL.

4.3.6. Semiempirical model for prediction of saccharide composition
Molecular weight distribution of soluble starch and starch hydrolysate were not
calculated in this work. Also, kinetic mechanism of starch hydrolysis is quite
complex due to the presence of multi substrates. Therefore, semiempirical equations
for the concentrations of oligosaccharides (G1-G7) vs. time were used, which are
analogous to reported (Paolucci-Jeanjean et al., 2000) semiempirical equations. It
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
formation of oligosaccharides has an order of reaction one w. r. t. the concn of
oligosaccharides higher than G5. Since, as stated earlier oligosaccharides with DP
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
G1-G5; which are as follows,
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
145
for i = 1-5,
) ] G [ [IEU](TDW
dt
] G d[
5
1 n
n
i

=
′ − =

i
k (4.4)
in which ] G [
i
′ is concn (mg/mL) of oligosaccharide with DP of i, TDW is dry weight
concn of starch hydrolysate (mg/mL) and k
i
is kinetic constant (h
-1
[IEU]
-1
) of the
formation of oligosaccharide with DP of i. Concentration terms in the Eq. 4.4 were
made dimensionless by dividing Eq. 4.4 with TDW on both sides. Eq. 4.4 thus takes
the following form,
For i = 1-5,
) ] [G [IEU](1
dt
] d[G
5
1 n
n
i

=
− =
i
k (4.5)
in which [G
i
] is dimensionless concn or wt fraction of oligosaccharide with DP of i.
As stated earlier, immobilized BLA hydrolyses G6 and G7, so it becomes essential to
add depletion term ( ] [IEU][G
i i
k′ ) while constructing a differential equation
expressing the time dependent variation for G6 and G7. Differential equation for G6
and G7 are as follows,
For i = 6-7,
] [IEU][G ) ] [G [IEU](1
dt
] d[G
i i
i
1 n
n
i
k k
i
′ − − =

=
(4.6)
where
i
k′

is kinetic rate constant (h
-1
[IEU]
-1
) for hydrolysis of oligosaccharide with
DP of i. Differential equations of G8-G10 are not considered because wt % of G8-
G10 were always lower than 0.3. Values of kinetic constants k
1
-k
7
,
6
k′ and
7
k′ 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
wt % of oligosaccharides (G1-G7). This was done by developing a code (Code is
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
146
provided in Appendix B) in MATLAB. Kinetic constants for reaction conditions;
[IEU] = 1.86, 55 °C, pH =5.2 and [S]
0
= 90 mg/mL; are k
1
= 0.0069, k
2
= 0.0119, k
3
=
0.0191, k
4
= 0.0072, k
5
= 0.0508, k
6
= 0.0489, k
7
= 0.0148,
6
k′ = 0.9725 and
7
k′ =
3.2536 (unit of all kinetic constants is h
-1
[IEU]
-1
). Comparison of the experimental
and predicted wt % of oligosaccharides (G1-G7) at above-mentioned reaction
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
predicted wt % lies slightly below the experimental values of wt % up to DE of 20,
but beyond 20 DE model overpredicts wt % of G6-G7. These kinetic rate constants
were empirically correlated with [IEU], [S]
0
and temperature using the following type
of correlation,
D
0
C B
[S] (T/273) A[IEU] = k (4.7)
where A, B, C, and D are correlation constants and T is temperature in K.
Values of A, B, C and D for kinetic constants k
1
-k
7
,
6
k′ and
7
k′ calculated by nonlinear
regression (using POLYMATH) are given in the Table 4.3.
Table 4.3. Values of A, B, C and D for kinetic constants
Kinetic
constant
A B C D
Correlation
coefficient
k
1
0.0083 -0.0807 14.58 -0.6237 0.97
k
2
0.0086 -0.3975 13.28 -0.4374 0.99
k
3
0.0415 -0.4727 7.30 -0.4145 0.99
k
4
0.0060 -0.3673 12.42 -0.4160 0.99
k
5
0.0899 -0.3777 7.26 -0.4033 0.98
k
6
0.3580 0.1086 12.01 -0.9275 0.99
k
7
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

Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
147
0
5
10
15
20
25
30
35
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 time (h)
w
t

%

o
f

G
1
-
G
5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
w
t

%

o
f

G
6

a
n
d

G
7
DE 5.8 13.5 19.5 24.1 25.9 28.3 30.3 33.6 36.1 36.2

Figure 4.20. Comparison of experimental and predicted saccharide composition;
[IEU] = 1.86, 55 °C, pH 5.2, [S]
0
= 90 mg/mL.
Symbols represent experimental data; □ G1, ■ G2, G3, ◊ G4, G5, ♦ G6 and +
G7; continuous lines represent predicted data.

4.3.7. Effect of mode of operation on hydrolysis of soluble starch
Hydrolysis of gelatinized starch solution was performed in three different
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
the batch mode. This must be happening because in contrast to reaction in a
conventional batch mode, packed bed imposes high enzyme to substrate ratio and
promotes greatly increased reaction rates.
Effect of superficial liquid velocity (i.e. flow rate / column cross sectional
area) on the hydrolysis performance was studied by performing experiments in the
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.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
148
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
reaction rates decreased and hydrolysis performance was adversely affected.
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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
149
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
through this voidage and obviously resulting lower reaction rates.
0
5
10
15
20
25
30
35
40
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
Batch mode
Packed bed, 1.27 cm/min
Packed bed, 2.55 cm/min
Packed bed, 3.82 cm/min
Expanded bed, 1.27 cm/min

Figure 4.21. Effect of mode of operation on hydrolysis of soluble starch
at pH 5.2, 55 °C, starch concentration = 10% w/v and [IEU]/[So] = 8.9 e-3

4.3.8. Hydrodynamic stability of immobilized BLA
Reusability of immobilized enzyme is very important aspect in the industrial
utilization of it. Effect of mode of operation in performing hydrolysis of gelatinized
starch solution on the reusability of immobilized BLA is shown in the Fig. 4.22.
When mode of operation is varied, hydrodynamic conditions faced by immobilized
enzyme changes. Hence this reusability is named as Hydrodynamic stability.
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
150
Hydrodynamic stability
0
20
40
60
80
100
0 1 2 3 4 5 6
Reuse number
%

i
n
i
t
i
a
l

r
a
t
e
Batch with intemittent washing
batch without intermittent washing
Packed bed 1 mL/min without intermittent washing
Packed bed 3 mL/min without intermittent washing
Expanded bed 1 mL/min without intermittent washing

Figure 4.22. Effect of mode of operation and intermittent mixing on reusability of
immobilized BLA. pH 5.2, 55 °C, starch conc = 10% w/v, and [IEU]/[So] = 8.9 e-3

Reusability study of immobilized BLA in the batch mode with intermittent
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
of hydrolysis. After 6 batches of hydrolysis in the packed bed mode of operation, %
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
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
151
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
immobilized enzyme after completion of batch. When intermittent washing of beads
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
washing after periodic time intervals.

4.3.9. Hydrolysis of sorghum slurry using immobilized BLA.
Due to several advantages (like reusability of the enzyme, continuous
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
enzymes. This process constitutes following steps viz. 1. Gelatinization of 15 %
sorghum slurry in boiling water for 10 min. 2. Circulating slurry through the bed of
immobilized BLA and amyloglucosidase.
In the present work, attempt of hydrolysis of gelatinized sorghum slurry was
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
glucose from sorghum due to following,
9 Change in the action pattern of BLA due to immobilization
9 Drastic reduction in the enzyme activity in reusability without intermittent
washing.
9 After mixing the gelatinised sorghum slurry with beads, it was very difficult
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
152
to separate beads from slurry after the completion of reaction.
9 Technical difficulty in circulation of sorghum slurry through immobilized
enzyme bed in both packed and expanded mode due to viscosities and
pericarp particles in the slurry.
9 BLA is now cheaply available enzyme at cost of 250 Rs./kg
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
enzymatic production of glucose from sorghum using enzymes in free forms is
reported in chapter 5.

4.4. Conclusions
Bacillus licheniformis α-amylase (BLA) was immobilized on superporous
CELBEADS. After immobilization, optimum pH for the enzyme action marginally
decreases and optimum temperature remains same though thermostability increases.
pH, temperature and initial starch concn has significant effect on saccharide
composition at same value of DE. Saccharide composition of starch hydrolysate
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
and maltohexaose; whereas immobilized BLA principally produces maltotriose and
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
used as an additional tool for production of maltodextrins solely or in combination
with variation in pH, temperature and starch concn. Used semiempirical model
predicts wt % of oligosaccharides (G1-G7) that satisfactorily fits the experimental
data of G1-G5, but over predicts wt % of G6 and G7. Such model can be used for
Chapter 4: Hydrolysis of soluble starch using B. licheniformis α-amylase immobilized on
superporous CELBEADS
Studies in the Enzymatic depolymerisation of natural polysaccharides
153
selecting operational parameters such as temperature, starch concn as design
parameter to obtain desired saccharide profile in maltodextrins. However care should
be taken because values of kinetic constant are likely to vary with change in starch
source.

Nomenclature
A, B, C, D see Eq. 4.7
[G´
i
] concn of oligosaccharide with DP of i (mg/mL)
[G
i
] dimensionless concn or wt fraction of oligosaccharide with DP of i
[FEU] number of free enzyme units per mL of starch solution
[IEU] number of immobilized enzyme units per mL of starch solution
[IEU equiv] number of Eq. immobilized enzyme units per mL of starch solution
[S]
0
initial starch concn (mg/mL)
DE dextrose equivalent of starch hydrolysate
k
1
-k
7
kinetic constant for formation of G1-G7 (h
-1
[IEU]
-1
)
6
k′ kinetic constant for depletion of G6 (h
-1
[IEU]
-1
)
7
k′ kinetic constant for depletion of G7 (h
-1
[IEU]
-1
)
free
cat
k turnover number of free enzyme (min
-1
),
free
max
V /[IEU]
app
cat
k turnover number of immobilized enzyme (min
-1
),
app
max
V /[IEU equiv]
K
m
intrinsic michaelis constant (mg/mL)
free
m
K K
m
of free enzyme (mg/mL)
app
m
K apparent K
m
of immobilized enzyme (mg/mL)
n average degree of polymerization (DP) of starch hydrolysate
T temperature (K)
t reaction time (h)
TDW dry weight concn of starch hydrolysate (mg/mL)
V initial reaction rate
V
max
intrinsic maximum reaction rate
free
max
V V
max
of free enzyme
app
max
V apparent V
max
of immobilized enzyme
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
154

















5. Enzymatic production of glucose
from sorghum







Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
155

5.1. Introduction and literature review
Sorghum (Sorghum bicolor L. Moench) is an important drought resistant
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
(www.fas.usda.gov). Leading sorghum producing countries were United States
(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
systems (Smith and Frederiksen, 2000). Average percentage contents of starch,
proteins, moisture, fibers, lipids, and ash in the sorghum were 70.1, 11.2, 11.6, 1.82,
3.54, and 1.8 respectively (Wu et al., 2007).
Though, production of sorghum is high in India, demand for the sorghum is
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
sufficient quantities at affordable prices. Hence, in addition to being a major source
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
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
156

human consumption. Several mold-causing fungi are producers of potent mycotoxins
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
even unfit for animal consumption.
Hence, an industrial application is needed to be exploited for normal and
blackened sorghums in order to make sorghum cultivation economically viable for
farmers, through value added products. There is very small amount of research done
on value addition to sorghum through; production of glucose (Devarajan and Pandit,
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
exploitation can be attributed to reduced sorghum starch digestibility (Lichtenwalner
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
digestibility (Duodu et al., 2003) after cooking i.e. heat-moisture treatment of
sorghum flour. Literature related to production of production of ethanol and isolation
of starch from sorghum, and digestibility of sorghum starch and sorghum proteins is
reviewed in the chapter 3. There is no literature available on value addition products
to blackened sorghum.
Application of ultrasound is reported in wet milling process for isolation of
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).
Chapter5: Enzymatic production of glucose from sorghum
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In the present work, sorghum flour was used directly for liquefaction and
saccharification rather than isolating starch and using it for liquefaction and
saccharification as the yields of starch isolation from sorghum were reported to be
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
for the production of glucose.
Objectives of the present work were to optimize enzymatic liquefaction and
saccharification processes to produce glucose from three varieties of sorghum i.e.
healthy, blackened and germinated, and to study the effect of ultrasound treatment
prior to liquefaction on the performance of liquefaction and saccharification
processes.

5.2. Experimental
5.2.1. Materials
3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for
chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd
(India). Commercial preparations in liquid formulation of Bacillus licheniformis α-
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
purchased from the local market.

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5.2.2. Analytical Methods
5.2.2.1. Measurement of protein concentration, reducing sugar concentration
and concentrations of malto-oligosaccharides.
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
(Miller 1959) with dextrose (0–1 mg/mL) as a standard. Concentrations of glucose
and malto-oligosaccharides up to maltoheptaose were measured using the HPTLC
method. Details of modified folin lowry method, DNSA method and HPTLC method
are given in the Appendix A.
5.2.2.2. Measurement of moisture content
Sorghum flour was kept at 80 °C, till constant weight was obtained and
moisture content in sorghum flour was measured using mass balance. Detailed
method to measure moisture content is given in the appendix A.
5.2.2.3. Measurement of particle size distribution of sorghum flour.
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.
5.2.2.4. Measurement of starch content of sorghum flour.
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
(glucose equiv) produced upon complete hydrolysis (i.e. at end of 24 h) by a factor of
0.9.
Chapter5: Enzymatic production of glucose from sorghum
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5.2.3. Amylolytic activity measurement methods
5.2.3.1. Free bacterial α-amylase (BLA)
Procedure for measurement of activity of free Bacillus licheniformis α-
amylase (BLA) is detailed in the chapter 4.
5.2.3.2. Free amyloglucosidase (AG)
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
part of the concentration of reducing sugars vs. time curve.
Gelatinized soluble starch solution (0.9 mL, 1% w/v, pH 4.5, 50 mM citrate
buffer) was incubated with 0.1 mL of 10000 fold diluted commercial
amyloglucosidase (AG) solution at 65 °C for 10 min. Then 1 mL of DNSA reagent
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
variation in the concentration of reducing sugar was measured by DNSA method
(Appendix A) using dextrose as a standard. One unit of amyloglucosidase (AGU)
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
was calculated using following equation.
min 10 mL) (0.1 aliquot enzyme of volume 180) (i.e. glucose of wt mol
factor dilution enzyme produced equiv) (glucose sugar reducing of g
EU/mL
× ×
×
=
µ

min 10 mL) (0.1 aliquot enzyme of volume 180) (i.e. glucose of wt mol
(10000) factor dilution enzyme
mL) (1 mixture assay of volume min) 0 at C - min 10 at (C
AGU/mL
RS RS
× ×
×
×
=
where C
RS
= Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL

Chapter5: Enzymatic production of glucose from sorghum
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5.2.3.3. Free pullulanase (PL).
Gelatinized soluble starch solution (0.9 mL, 1% w/v, pH 5.5, 50 mM citrate
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
concentration of reducing sugar was measured by DNSA method (Appendix A) using
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
conditions. Activity of commercial pullulanase formulation was calculated using
following equation,
min 10 mL) (0.1 aliquot enzyme of volume 180) (i.e. glucose of wt mol
factor dilution enzyme produced equiv) (glucose sugar reducing of g
EU/mL
× ×
×
=
µ

min 10 mL) (0.1 aliquot enzyme of volume 180) (i.e. glucose of wt mol
(500) factor dilution enzyme
mL) (1 mixture assay of volume min) 0 at C - min 10 at (C
PLU/mL
RS RS
× ×
×
×
=
where C
RS
= Concn of reducing sugars (glucose equiv) in assay mixture, µg/mL

5.2.4. Thermostability study of free Amyloglucosidase.
Amyloglucosidase solution in acetate buffer (i.e. 3.63 AGU/mL, 50 mM, pH
4.5) was incubated at desired temperature under shaking conditions (180 rpm) in the
absence of substrate for 24 h. Samples of AG solution were taken at various time
intervals and were for its amylolytic activity.


Chapter5: Enzymatic production of glucose from sorghum
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5.2.5. Optimization of AG: PL ratio for saccharification.
Gelatinized soluble starch solution (5 mL, 2% (w/v), pH 4.5, 0.05 M acetate
buffer) was incubated with 18 U of amyloglucosidase with varying units of
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
(glucose equiv) concentration and optimum AG: PL ratio was obtained.

5.2.6. Experimental work for production of glucose from sorghum
Production of glucose from sorghum flour consists of two reaction steps:
1. Liquefaction using B. licheniformis α-amylase (BLA) in which gelatinisation of
free starch granules and dextrinization (depolymerisation) of gelatinized starch take
place simultaneously. This produces mixture of malto-oligosaccharides, linear
and branched dextrins.
2. Saccharification using amyloglucosidase (AG) in which AG cleaves first α(1→4)
linkage from non-reducing end glucose polymer and produces glucose.
Process of production of glucose syrup from sorghum flour, which is used in
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
saccharification of liquefied starch is discussed in detail in the chapter 2.
Chapter5: Enzymatic production of glucose from sorghum
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Figure 5.1. Process flow sheet for production of glucose syrup from sorghum
Gelatinization of free starch
granules Dextrinization of
gelatinized starch molecules.
Removal proteins and fibers to prevent colour
formation due to solubilization of proteins
G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch
|
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

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 produced after saccharification of liquefied starch
G
Saccharification using
Amyloglucosidase (AG)
55 °C, 4.5 pH
Hot filtration
Filtration and Purification
Gl ucose sy r up
Milling
Preparation of slurry
Grain sorghum
Removal of fine particles, lipids, proteins
and unreacted starch gel
Glucose, malto-oligosaccharides, linear and branched dextrins
produced after liquefaction
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–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–G G–G–G–G–G–G–G–G
Liquefaction using
bacterial α-amylase (BLA)
85 °C, 6 pH
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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
its subsequent saccharification.
5.2.6.1. Optimization of liquefaction of sorghum flour.
Liquefaction of sorghum flour was performed in a 250 mL stoppered conical
flask containing 100 mL magnetically stirred sorghum slurry. Experimental set-up for
liquefaction of sorghum flour is shown in the Fig. 5.2.

Figure 5.2. Experimental set up for liquefaction of sorghum flour
A Magnetic stirrer
B Conical flask containing 100 mL reaction mixture
C Silicone oil bath
D Heater
E Stirrer to for mixing in oil bath
F Temperature sensor

A
B
C
D
E
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Liquefaction of sorghum slurry (10– 35% w/v in 0.05 M acetate buffer) was
performed using BLA at desired temperature (maintained by immersing the conical
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
an indication of increase in DE of reaction mixture.
Samples were withdrawn at time interval of 15 min and diluted to
approximately 1% (w/v) using 0.1 N HCl. Progress of liquefaction was monitored
using starch-iodine colorimetric reaction. Few drops of KI-I
2
reagent (0.05% w/v I
2

and 0.5% w/v KI solution) were added to few drops of diluted sample and color of
mixture was observed. As the liquefaction progresses, following different colors 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
was considered to be completed. Samples (~ 1%w/v) were then centrifuged at 270 g
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
slurries of different pH values (5.2–6.7), varying concentrations of BLA (0.04–0.16%
v/w of sorghum flour) and CaCl
2
(0–500 ppm), and temperature in the range of 75–95
°C.

5.2.6.2. Liquefaction of sorghum of different varieties
Healthy sorghum grains were steeped in the ordinary tap water for 12 h and then
germinated for 12 h, 24 h, 36 h, and 48 h separately. Germinated sorghum grains were
Chapter5: Enzymatic production of glucose from sorghum
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first dried at 50 °C. Germinated and blackened sorghum were milled separately using
old fashioned flour mill. Liquefaction of geminated and blackened sorghum was
performed at optimized liquefaction conditions and compared with liquefaction of
normal healthy sorghum.

5.2.6.3. Effect of prior ultrasound treatment on the liquefaction of sorghum.
Ultrasound horn was dipped into sorghum slurry (30% w/v in 0.05 M acetate
buffer of pH 6, and CaCl
2
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
sugar (glucose equiv) concentration. Dextrose equivalent (DE) is defined as following;
100
mg/mL e, hydrolysat starch of concn dry wt
mg/mL e, hydrolysat starch in the C
(DE) quivalent Dextrosee
RS
× = (5.1)
where C
RS
is concentration of reducing sugar (glucose equiv)

5.2.6.4. Optimization of saccharification.
Saccharification process was first optimized for the processing temperature by
performing thermostability study of AG and also performing saccharification of
maltodextrins at different temperatures.
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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
(150 rpm) for 24 h under optimized conditions of temperature and enzyme
concentration. At regular time intervals, samples were withdrawn and diluted to
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
sugar (glucose equiv). Percentage saccharification on the basis of original starch
content in the sorghum flour is defined as follows;
100
Q mg in flour sorghum of amount
mL in mixture reaction of volume mg/mL in C
cation saccharifi %
f
×
×
×
= (5.2)
where C
f
is concentration of reducing sugar (glucose equiv) at steady state (i.e. 24 h)
Q is mg of reducing sugar (glucose equiv) produced per mg of sorghum flour
using method described in 2.2.3. i.e. (% starch content /100) × 1.11
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.
Production of glucose from sorghum flour consists of following five steps in
sequence; 1. Ultrasound treatment on sorghum slurry, 2. Liquefaction of sorghum
slurry using B. licheniformis α-amylase, 3. Hot filtration of liquefied sorghum slurry, 4.
Washing of cake (obtained after hot filtration) followed by second filtration and mixing
of both the filtrates, and 5. Saccharification of filtrate F1 or mixture of F1 and F2 using
amyloglucosidase. Experiments were performed for production glucose using healthy,
Chapter5: Enzymatic production of glucose from sorghum
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blackened, and germinated sorghum without or with steps 1 or 4.

5.3. Results and Discussion
5.3.1. Studies in the liquefaction process
Flour of healthy sorghum grains was used for optimization of liquefaction
process. The starch content and moisture content of the healthy sorghum flour were
estimated to be 69–70% and 10–11% respectively. Average particle size of the
sorghum flour used was 302 µm.
5.3.1.1. Optimization of liquefaction process
5.3.1.1. A. Effect of pH.
Values of the concentration of the reducing sugar (glucose equiv) obtained at 1
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
respectively. This indicates that optimum pH for liquefaction of sorghum flour is 6.
Hence further study on liquefaction of sorghum flour was performed at pH of 6 (0.05 M
acetate buffer).
5.3.1.1. B. Effect of BLA concentration.
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
CaCl
2
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
liquefaction was completed decreased.
Chapter5: Enzymatic production of glucose from sorghum
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0
5
10
15
20
25
30
35
40
45
50
0 15 30 45 60 75 90 105
Time (min)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)
0.04 0.06
0.08 0.1
0.12 0.16
liquiefied
not liquified
Concn of BLA
(% v/w of sorghum flour)

Figure 5.3. Effect of BLA concentration on concentration of reducing sugar (glucose
equiv) versus time curve. Reaction conditions: 25% w/v sorghum slurry, 85 °C, pH 6
and CaCl
2
concn = 0 ppm.
Data points without shadow – blue color on starch-iodine reaction
Data points with shadow – disappearance of blue color (i.e. dark red with tinge of
violet) on starch-iodine reaction

5.3.1.1. C. Effect of CaCl
2
concentration.
It is well known that, since BLA is an organometallic enzyme, supplementation
of Ca
2+
ions improves performance of BLA. Hence, liquefaction was performed at
BLA concentration of 0.06% v/w of flour at different concentrations of CaCl
2
to find
optimum CaCl
2
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 CaCl
2
concentration greater than 200 ppm overlapped each other (Fig.
5.4). This indicates that CaCl
2
concentration of 200 ppm was optimum for liquefaction.
It is worth noting that liquefaction performance at BLA concentration of 0.08% v/w
Chapter5: Enzymatic production of glucose from sorghum
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169

without supplement of CaCl
2
is similar to that obtained at BLA concentration of 0.06%
v/w with CaCl
2
concentration of 200 ppm (Fig. 5.3 and 5.4).
0
5
10
15
20
25
30
35
40
45
0 15 30 45 60 75 90
Time (min)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)
0 100 200
300 400 500
CaCl
2
concn (ppm)

Figure 5.4. Effect of CaCl
2
concentration on concentration of reducing sugar (glucose
equiv) versus time curve. Reaction conditions: 25% w/v sorghum slurry, 85 °C, pH 6
and BLA concn = 0.06% v/w of sorghum flour.
Data points without shadow – blue color on starch-iodine reaction
Data points with shadow – disappearance of blue color (i.e. dark red with tinge of
violet) on starch-iodine reaction

5.3.1.1. D. Effect of sorghum slurry concentration
Liquefaction was performed at various concentrations of sorghum flour varying
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
complete liquefaction. Hence, it was decided to use 30% w/v as optimum
Chapter5: Enzymatic production of glucose from sorghum
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170

concentration of sorghum flour in the slurry for all further experiments.

5.3.1.1. E. Effect of liquefaction temperature.
Gelatinization temperature of sorghum starch is reported to be in the range of
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
liquefaction. Liquefaction is a combination of two processes: one, gelatinization of free
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
simultaneously: one, rate of gelatinization of starch increases, and two, rate of
dextrinization of starch molecules decreases due to enzyme deactivation at elevated
temperatures. Hence, there exists an optimum temperature for the liquefaction process.
Temperature of 85 °C was observed to be optimum (Fig. 5.5) for the liquefaction of
sorghum flour, at which liquefaction was completed within 1.5 h (according to starch-
iodine reaction). Though concentration of reducing sugars produced at temperatures of
75 and 80 °C were higher than that produced at a temperature of 85 °C, liquefaction
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
attributed to continued depolymerisation of gelatinized starch molecules. At
temperatures of 90 °C and 95 °C, liquefaction performance was significantly poorer
than its counterparts at 85 °C (Fig. 5.5). Optimum conditions for liquefaction of
sorghum flour are summarized in the Table 5.2.
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0
10
20
30
40
50
60
0 15 30 45 60 75 90
Time (min)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
75 80 85
90 95
Temperature (°C)

Figure 5.5. Effect of Temperature on concentration of reducing sugar (glucose equiv)
versus time curve. Reaction conditions: 30% w/v sorghum slurry, pH 6, CaCl
2
concn =
200 ppm.
Data points without shadow – blue color on starch-iodine reaction
Data points with shadow – disappearance of blue color (i.e. dark red with tinge of
violet) on starch-iodine reaction

5.3.1.1. F. Liquefaction of sorghum of different varieties
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
in 1.5 h under optimized conditions. However, liquefaction progress of blackened
sorghum is slightly (7%) slower as compared to healthy sorghum. This could be
attributed to the enzyme inhibition due to mycotoxins present in the blackened pericarp.
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0
10
20
30
40
50
60
70
80
90
0 15 30 45 60 75 90
Time (min)
C
o
n
c

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)
H B G1 G2 G3 G4

Figure 5.6. Effect of variety of sorghum on concentration of reducing sugar (glucose
equiv) versus time curve. Reaction conditions: 30% w/v sorghum slurry, 85 °C, pH 6
and CaCl
2
concn = 200 ppm
Data points without shadow – blue color on starch-iodine reaction
Data points with shadow – disappearance of blue color (i.e. dark red with tinge of
violet) on starch-iodine reaction. H - Healthy sorghum; B - Blackened sorghum; G1,
G2, G3, G4 - healthy sorghum germinated for 12 h, 24 h, 36 h, and 48 h, respectively,
after steeping for 12 h in plain water.

It can be seen from Fig. 5.6 that as the germination time increases, liquefaction
performance also improves. However, beyond germination time of 24 h, there is no
significant improvement in the liquefaction performance. However, liquefaction of
germinated sorghum (germination time of 24 h) was observed to be completed in 1 h
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
during the process of germination.


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5.3.1.2. Effect of prior ultrasound treatment on liquefaction.
14
14.5
15
15.5
16
16.5
17
0 2 4 6 8 10 12
sonication time (min)
D
E

o
f

l
i
q
u
e
f
a
c
t
40% amplitude
50 % amplitude
100% amplitude

Figure 5.7. Effect of sonication time and ultrasound intensity on DE of liquefact.

14
14.5
15
15.5
16
16.5
17
0 5000 10000 15000 20000
Power consumption (J)
D
E

o
f

l
i
q
u
e
f
a
c
t
40% amplitude
50 % amplitude
100% amplitude

Figure 5.8. Variation in DE of liquefact with power consumption in sonication.

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
DE is approximately same at constant power consumption, irrespective of the
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
174

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
treatment will be discussed in detail, later in the text.

5.3.2. Optimization of saccharification
5.3.2.1. Properties of free amyloglucosidase and pullulanase
% 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
optimum temperature of amyloglucosidase using assay procedure to measure enzyme
activity were 4.5 and 65 °C, respectively. Enzyme activity at optimum conditions and
protein content of commercial formulation of amyloglucosidase were 36300 AGU/mL
and 370 mg/mL.
65 °C
0
10
20
30
40
50
60
70
80
90
100
110
3 3.5 4 4.5 5 5.5 6 6.5
pH
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

(
%
)










i

Figure 5.9. % relative activity vs. pH profile for amyloglucosidase
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
175

0
10
20
30
40
50
60
70
80
90
100
30 40 50 60 70 80 90 100
Temperature, °C
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

(
%
)
pH 4.5

Figure 5.10. % relative activity vs. temperature profile for amyloglucosidase

% 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
optimum temperature of pullulanase using assay procedure to measure enzyme activity
were 4 and 60 °C, respectively. Enzyme activity at optimum conditions and protein
content of commercial formulation of pullulanase were 2950 PLU/mL and 50 mg/mL.
0
10
20
30
40
50
60
70
80
90
100
110
3 3.5 4 4.5 5 5.5 6 6.5 7
pH
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
Temperature = 60 °C

Figure 5.11. % relative activity vs. pH profile for pullulanase
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
176

0
10
20
30
40
50
60
70
80
90
100
110
30 35 40 45 50 55 60 65 70 75
Temperature, °C
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
pH 4

Figure 5.12. % relative activity vs. temperature profile for pullulanase
Properties of free amyloglucosidase (AG) and pullulanase (PL) are summarized
in the Table 5.1.
Table 5.1. Properties of amyloglucosidase and pullulanase
Parameter Free Amyloglucosidase Free pullulanase
Optimum pH 4.5 3.8–4.3
Optimum temperature (°C) 65 60
Activity 36300 AGU/mL 2950 PLU/mL
Protein content (mg/mL) 370 50
Specific activity 98 (AGU/mg of protein) 59 (PLU/mg of protein)

5.3.2.2. Thermostability of amyloglucosidase and optimization of operating
temperature for saccharification
Optimum temperature of AG using the specified assay procedure was found to
be 65 °C (Table 5.1). However, optimum operating temperature for saccharification
using AG may not be the same as that obtained using the assay procedure. Hence,
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
177

study of thermostability of AG was essential at an optimum pH of 4.5. Thermostability
of AG was first checked at 65 °C. It was observed that % relative activity of AG
decreased to 5% (Fig. 5.13) at 65 °C within first 3 hrs only. Hence, it was decided to
check thermostability of AG at lower temperatures also to find optimum operating
temperature for saccharification of maltodextrins using AG. In addition to
thermostability check at different temperatures, experimental runs utilizing AG and
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
saccharification of maltodextrins to glucose is around 55–60 °C.
0
10
20
30
40
50
60
70
80
90
100
110
0 3 6 9 12 15 18 21 24
Time (h)
R
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

(
%
)












i
50 55
60 65
Temperature (°C)

Figure 5.13. Thermostability of AG at pH 4.5 at different temperatures.
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
178

20
30
40
50
60
70
0 3 6 9 12 15 18 21 24
Time (h)
C
o
n
c
n

r
e
d
u
c
i
n
g

S
u
g
a
r

(
m
g
/
m
L
)



50 55
60 65
Temparature, °C

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.

5.3.2.3. Optimum ratio of amyloglucosidase units to pullulanase units for
saccharification
Optimum ratio of AGU: PLU was found to be 18: 2 and about 25% increase in
the concentration of reducing sugar was observed due to the addition of optimum
quantity of pullulanase (Fig. 5.15) using starch as substrate. During saccharification of
sorghum liquefact using amyloglucosidase with and without optimum quantity of
pullulanase, increase in the concentration of reducing sugar was observed in the initial
stages of reaction. However, the concentration of reducing sugars at the end of 24 h
were the same (Fig. 5.16). Hence, it was decided to use only amyloglucosidase for
saccharification of sorghum liquefact, eliminating the use of pullulanase for
saccharification time of 24 h. If saccharification time needs to be reduced to 8 h,
pullulanase can be used along with amyloglucosidase, but obviously at the added cost
of the new enzyme.
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
179

50
55
60
65
70
75
80
85
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Pullulanase units (PLU)
%

D
e
g
r
e
e

o
f

h
y
d
r
o
l
y
s
i
s

Figure 5.15. Optimization of ratio of AG units to PL units
for hydrolysis of soluble starch.
0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)
0.1 AG
0.1 AG + 0.13 PL
% v of enzyme / w of saccharides

Figure 5.16. Saccharification of sorghum liquefact using amyloglucosidase
with and without pullulanase (14% w/v, pH 4.5, 55 °C)



Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
180

5.3.2.4. Optimization of amyloglucosidase concentration
Saccharification of sorghum liquefact (pH 4.5) was performed at different
concentrations of amyloglucosidase. It can be seen that at AG concentration of 0.052%
v/w of starch saccharification was completed at 24 h and attains concentration of 235
mg/mL which is same as that attained at higher concentrations of AG (Fig. 5.17).
HPTLC of starch hydrolysate at 24 h showed presence of glucose as a principle sugar
with more than 95% selectivity.
0
40
80
120
160
200
240
0 4 8 12 16 20 24
Time (h)
c
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r
s

(
m
g
/
m
L
)





0.026
0.052
0.078
0.104
% v of AG / w of starch

Figure 5.17. Variation in the concentration of reducing sugars versus time
with amyloglucosidase concentration as a parameter.
Optimized conditions for saccharification of sorghum liquefact are summarized
in the Table 5.2.





Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
181

Table 5.2. Optimized parameters for Liquefaction and Saccharification
Parameter Liquefaction Saccharification
Temperature (°C) 85 55
pH 6 4.5
Slurry concentration 30% w of sorghum flour / v of slurry n.a.
BLA concentration 0.06% v/w of sorghum flour i.e.
0.086% v/w of sorghum starch
n.a.
AG concentration n.a. 6 AGU/mL of liquefact i.e.
0.058 % v/ w of starch
CaCl
2
concentration 200 ppm n.a.

5.3.3. Saccharification of sorghum liquefact
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
mL of acetate buffer to produce 100 mL of slurry. However, after hot filtration,
followed by liquefaction, only 72 mL filtrate was recovered. This means approximately
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 were dependent upon the following factors; 1. Ultrasound treatment on
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
summarized in the Table 5.3.

Chapter5: Enzymatic production of glucose from sorghum
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182


Table 5.3. Summary of effect of ultrasound treatment and washing on liquefaction and
saccharification performance
Run
No
BLA concn,
% v/w
C
RS
, mg/mL
(DE)
F1,
mL
F2,
mL
F1 + F2,
mL
C
f

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



Chapter5: Enzymatic production of glucose from sorghum
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183

5.3.4. Effect of ultrasound treatment on particle size distribution
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
corresponds to ~800 µm, 10-20 µm and ~1 µm. Due to ultrasound treatment of
sorghum slurry prior to liquefaction, peak area corresponding to ~800 µm decreases;
whereas, peak areas corresponding to 10-20 µm and ~1 µm increases. Inflection point
of ~800 µm must be a function of the milling process and mainly corresponds to large
pericarp particles. However, inflection point of ~1 µm may correspond to cell debris
produced due to disintegration of the cells of endosperm of sorghum. Such decrease in
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
al., 2006). Particle fragmentation increasing solid-liquid interfacial area and
enhancement in the convective diffusivity of leach solvent through micropores of ore
structure was attributed to microscopic convective motion created by acoustic
cavitation (i.e. shock wave propagation and microjet formation) at solid-liquid interface
and this results into enhancement in the rate of leaching of uranium
(Balasubrahmanyam et al., 2006).
Chapter5: Enzymatic production of glucose from sorghum
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184

0
0.5
1
1.5
2
2.5
3
3.5
0.1 1 10 100 1000 10000
particle size (µm)
%

p
o
p
u
l
a
t
i
o
n
Without sonication
1 min at 40 %
amplitude
1 min at 100 %
amplitude

Figure 5.18. Effect of prior ultrasound treatment on the particle size distribution in
30% w/v sorghum flour slurry.

5.3.5. Studies on effect of different process parameters on % saccharification
5.3.5.1. Effect of washing of cake obtained after hot filtration
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 1
st
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
flour) is mainly through the recovery of oligosaccharides, which were otherwise
trapped inside the cake after hot filtration and were unavailable for further
saccharification. Similar behavior was observed in all the cases (Table 5.3).


Chapter5: Enzymatic production of glucose from sorghum
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185

5.3.5.2. Effect of ultrasound treatment on % saccharification
It was observed (H1 and H3; Table 5.3) that ultrasound treatment of slurry
before liquefaction increased the volume of F1 from 72 to 75 mL (4% increase) and
increased the % saccharification from 74 to 81 (10% increase). This indicates that
ultrasound treatment prior to liquefaction improved filterability of the liquefied slurry
because of either increase in the DE of liquefact (Table 5.3) or particle disintegration
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 1
st
filtration was also provided, in addition to
ultrasound treatment prior to liquefaction, an increase in the % saccharification was
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
(through the availability of additional starch granules for liquefaction and
saccharification, discussed in detail later in the text), and two, from 81 to 90 (11%
increase) is due to step of washing (through the recovery of additional oligosaccharides
from the cake after hot filtration). Similar effect of ultrasound treatment was observed
for germinated sorghum also (Table 5.3; G1, G2, G3, G4).
It is first necessary to understand the sorghum grain structure in order to
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
Chapter5: Enzymatic production of glucose from sorghum
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186

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
(Chandrashekhar and Mazhar, 1999).
Treatment of sorghum flour (before cooking) with proteolytic enzymes like
pronase (Zhang and Hamaker, 1998), pepsin (Rooney and Pflugfelder, 1986) have
shown an increase in the starch digestibility by pancreatic α-amylase (Zhang and
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
Hamaker, 1998) or 2-mercaptoethanol (Chandrashekar and Kirleis, 1988) also
increased starch digestibility using pancreatic α-amylase (Zhang and Hamaker, 1998)
or degree of starch gelatinization (Chandrashekar and Kirleis, 1988) due to cleavage of
disulphide bonds linking protein surrounding starch granules. Sorghum proteins are
also reported to produce high molecular weight polymers by polymerization through
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
with amylase, contribute to subsequent incomplete gelatinization, hydrolysis of starch,
and negative impact of protein content on conversion efficiency of sorghum to ethanol
(Wu et al., 2007).
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
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
187

complex (Tester et al., 2004). In the case of normal sorghum, this amylose-lipid
complex is reported to have endotherm or a temperature range of gelatinization (onset,
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
(Wu et al., 2007). Use of lysophospholipase along with amyloglucosidase in
saccharification resulted into higher degree of degradation of amylose-lipid complex
(due to hydrolysis of lysophospholipids), and an improvement in the rate and yield of
filtration of hydrolysate produced after completion of saccharification (due to partial
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
saccharification for 72 h using amyloglucosidase) proved by differential scanning
calorimetry (DSC) study (Nebesny et al., 2002) indicate that amylose-lipid complex
affects the final degree of saccharification that can be practically achieved.
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
ultrasound treatment prior to liquefaction may be releasing starch granules 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
an increase in the liquefaction performance and increase in the % saccharification due
to ultrasound treatment prior to liquefaction. Similar enhancement in the glucose
release using 14% w/v corn slurry (raw and cooked) due to prior sonication for 20 or 40
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
188

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
affects liquefaction and saccharification, and hence conversion efficiency of sorghum
to glucose and hence to ethanol. 1 to 10% increase in the DE of liquefact and ethanol
yield has been reported
25
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
not susceptible to hydrolysis to amino acids by proteolytic enzymes) to less complex
proteins, which are more bio-available to digestive systems of animals.
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
washing procedure and hence giving lower % saccharification. When experiments
were performed in the present work for the production of glucose from healthy
sorghum without filtration step, values of % saccharification were observed to be 87–
89 and 93–95 without and with ultrasound treatment, respectively.
In the case of blackened sorghum (grain mold), marginal increase in the %
saccharification was observed due to prior ultrasound treatment. It can be seen from
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
189

Table 5.3 that at optimized concentration of BLA, DE of liquefact obtained with
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,
which require additional BLA content for liquefaction and AG content in
saccharification. Thus, lower value of blackened sorghum is partially offset by higher
BLA dosage requirement.
During germination and seedling growth of sorghum, different enzymes
including protease, endoprotease, limit dextrinase, α-amylase and endo-β-gluconase get
produced (Aisen et al., 1983). In case of germinated sorghum (germination time 24 h),
there is significant increase (~30%) in liquefact DE due to prior ultrasound treatment.
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.









Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
190

5.3.6. Economics of the process of production of glucose from sorghum of
different varieties
In the chemical project economics, cost of production plays a key role. It
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.
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
191

Table 5.4. Cost of raw material to process 1 kg of starch
Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg
Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter
Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs
Starch or sorghum

1 16 16 1.43 5 7.1 1.43 3 4.3 1.43 5 7.1
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
liquefaction

0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2
AG required for
saccharification

0.0006 330 0.198 0.0006 330 0.198 0.0007 330 0.231 0.0006 330 0.198
Total - - 16.51 - - 7.72 - - 5 - - 7.65

Where, Q Quantity of material, kg
R Rate of material, Rs/kg
C Cost incurred, Rs
BLA B. licheniformis α-amylase
AG Amyloglucosidase






Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
192

Table 5.5. Cost of utilities to process 1 kg of starch
Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter
Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs
Steam for
liquefaction
1,2

at 85 °C for 1.5 h
0.53 1 0.53 0.76 1 0.76 0.76 1 0.76 0.76 1 0.76
Steam for
saccharification
1,2
,
at 55 °C for 24 h

1.18 1 1.18 1.69 1 1.69 1.69 1 1.69 1.69 1 1.69
Filtration &
bleaching and color
removal
4


- - 0.2 - - 0.429 - - 0.429 - - 0.429
Cost of steam for
evaporation
3

2.67 1 2.67 3.81 1 3.81 3.81 1 3.81 3.81 1 3.81
Total - - 4.58 - - 6.69 - - 6.69 - - 6.69

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

Chapter5: Enzymatic production of glucose from sorghum
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193

Table 5.6. Comparison of processing cost or cost production of glucose from sorghum

Parameter Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum
Cost of Raw material 16.51 7.72 5 7.65
Cost of Utilities 4.58 6.69 6.69 6.69
Total cost 21.09 14.41 11.69 14.34
Glucose produced, kg 1.11 1 0.95 1
Processing cost,
Rs/kg of glucose produced
19 14.41 12.3 14.34

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
Chapter5: Enzymatic production of glucose from sorghum
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194

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
of glucose from sorghum is economically feasible. According to Suresh et al., 1999a,
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
Production of glucose from sorghum flour involves two steps viz. 1.
Liquefaction of flour using Bacillus licheniformis α-amylase and 2. Saccharification
using amyloglucosidase.
1. In the present work, use of ultrasound in the production of glucose from sorghum
flour has been explored.
2. The value of % saccharification to glucose using healthy sorghum flour (at
optimized reaction conditions for liquefaction and saccharification) was in the range
of 70- 90% depending upon following factors; 1. Ultrasound treatment of sorghum
slurry before liquefaction, and 2. Washing of cake (obtained after hot filtration)
followed by 2
nd
filtration and mixing of both the filtrates.
3. Ultrasound treatment to sorghum slurry prior to liquefaction appears to disrupt
hydrophobic protein matrix surrounding starch granules and amylose-lipid complex
due to physical effects of acoustic cavitation, like shock wave propagation and
microjet formation in the vicinity of liquid-solid interface. This frees starch
Chapter5: Enzymatic production of glucose from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
195

granules and these additional starch granules are made available for further action
of α-amylase and amyloglucosidase. A significant increase (~8–10%) in the %
saccharification was observed.
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
treatment before liquefaction.
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.

Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
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6. Enzymatic production of maltose
syrup from sorghum






Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
197

6.1. Introduction
In the section 5.1, the necessity to exploit industrial application for normal and
blackened sorghums, in order to make sorghum cultivation economically viable for
farmers through value added products, has been discussed. A little literature is
available on value addition to sorghum through; production of glucose, production of
ethanol and isolation of starch (discussed in detail in the chapter 3). However there is
no literature available on value addition to sorghum through production of maltose
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
saccharification process to produce maltose from three varieties of sorghum i.e.
healthy, blackened and germinated, and to study the effect of ultrasound treatment
prior to liquefaction on the performance of liquefaction and saccharification
processes.

6.2. Experimental
6.2.1. Materials
3,5-Dinitrosalicylic acid (DNSA), soluble starch, maltose, dextrose, MeCN for
chromatography LiChrosolv and other chemicals were purchased from E. Merck Ltd
(India). Commercial preparations in liquid formulation of Bacillus licheniformis α-
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
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
198

purchased from the local market.

6.2.2. Analytical Methods
6.2.2.1. Measurement of protein concentration and reducing sugar concentration
and concentrations of malto-oligosaccharides.
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
(Miller 1959) with maltose (0–1 mg/mL) as a standard. Concentrations of glucose
and malto-oligosaccharides up to maltoheptaose were measured using the HPTLC
method. Details of modified folin lowry method, DNSA method and HPTLC method
are given in the Appendix A.
6.2.2.2. Measurement of moisture content
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
method to measure the moisture content is given in the Appendix A.
6.2.2.3. Measurement of particle size distribution of sorghum flour.
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.
6.2.2.4. Measurement of starch content of sorghum flour.
Enzymatic method of Measurement of starch content of sorghum flour is
described in the 5.2.2.4.



Chapter 6: Enzymatic production of maltose syrup from sorghum
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199

6.2.3. Amylolytic activity measurement methods
6.2.3.1. Free bacterial α-amylase (BLA)
Procedure for measurement of activity of free Bacillus licheniformis α-
amylase (BLA) is detailed in the chapter 4.
6.2.3.2. Barley β-amylase (BBA)
0.9 mL of 1 % (w/v) gelatinized starch solution (pH 5.5, 50 mM citrate buffer)
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.
min 10 mL) (0.1 aliquot enzyme of volume 180) (i.e. maltose of wt mol
factor dilution enzyme produced equiv) (maltose sugar reducing of g
BBAU/mL
× ×
×
=
µ

min 10 mL) (0.1 aliquot enzyme of volume 362) (i.e. maltose of wt mol
(10000) factor dilution enzyme
mL) (1 mixture assay of volume min) 0 at C - min 10 at (C
BBAU/mL
RS RS
× ×
×
×
=

where C
RS
= Concn of reducing sugars (maltose equiv) in assay mixture, µg/mL
6.2.3.3. Free pullulanase (PL).
Procedure for the measurement of activity of free pullulanase (PL) is detailed
in the section 5.2.3.3.



Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
200

6.2.4. Thermostability study of free Barley β-amylase (BBA).
Solution of barley β-amylase in acetate buffer (500 fold diluted commercial
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.

6.2.5. Production of maltose syrup from sorghum: Experimental work
Production of maltose syrup from sorghum flour consists of two reaction
steps:
1. Liquefaction using B. licheniformis α-amylase (BLA) in which gelatinisation of
free starch granules and dextrinization (depolymerisation) of gelatinized starch take
place simultaneously. This produces a mixture of malto-oligosaccharides, linear
and branched dextrins.
2. Saccharification using barley β-amylase (BBA) with or without pullulanase, in
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
production of maltose and β limit dextrins, due to inability of BBA to bypass
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
with small quantities of glucose and maltotriose.
Process of production of maltose syrup from sorghum flour, which is used in
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
saccharification of liquefied starch is discussed in detail in chapter 2.
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
201


Figure 6.1. Process flow sheet for production of maltose syrup from sorghum
Gelatinization of free starch
granules Dextrinization of
gelatinized starch molecules.
Removal proteins and fibers to prevent colour
formation due to solubilization of proteins
G–G–G–G–G–G–G–G–G–G–G–G–G–G Starch
|
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

G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G–G
Saccharification using
Barley β-amylase (BBA) with
or without pullulanase (PL)
50 °C, 5.5 pH
Hot filtration
Filtration and Purification
Mal t ose sy r up
Milling
Preparation of slurry
Grain sorghum
Removal of fine particles, lipids, proteins
and unreacted starch gel
Glucose, malto-oligosaccharides, linear and branched dextrins
produced after liquefaction
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–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–G G–G–G–G–G–G–G–G
Using BBA only Using BBA and PL
G–G G G–G–G G–G and small
| quantity of glucose
G–G–G–G–G
Maltose, β-limit dextrins

Liquefaction using
bacterial α-amylase (BLA)
85 °C, 6 pH
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
202

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
its subsequent saccharification.
6.2.5.1. Liquefaction of sorghum flour.
Experimental set-up for liquefaction of sorghum flour is shown in the chapter
5 (Fig. 5.2). Process of liquefaction of sorghum slurry is already optimized to
produce sorghum liquefact with DE of around 15 (refer Chapter 5). Liquefaction of
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, CaCl
2
concentration and BLA concentration for 1.5 h
i.e. 6, 85 °C, 200 ppm, and 0.086% v/w of sorghum starch, respectively.
6.2.5.2. Optimization of saccharification.
Saccharification process was first optimized for the processing temperature by
performing thermostability study of BBA and also performing saccharification of
sorghum liquefact at different temperatures.
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
(BBA and / or Pullulanase) concentration. At regular time intervals, samples were
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 concentration of reducing sugar (maltose equiv). Percentage saccharification on
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
203

the basis of original starch content in the sorghum flour is defined as follows;
100
Q mg in flour sorghum of amount
mL in mixture reaction of volume mg/mL in C
cation saccharifi %
f
×
×
×
= (6.1)
where C
f
is concentration of reducing sugar (maltose equiv) at steady state (i.e. 24 h)
Q is mg of reducing sugar (maltose equiv) produced per mg of sorghum flour
using method described in 2.2.3. (Saccharification was performed using
BBA and PL at 5.5 pH and 50 °C) i.e. (% starch content /100) × 1.05
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
maltose.
Production of maltose syrup from sorghum flour consists of following five
steps in sequence; 1. Ultrasound treatment of sorghum slurry, 2. Liquefaction of
sorghum slurry using B. licheniformis α-amylase, 3. Hot filtration of liquefied
sorghum slurry, 4. Washing of cake (obtained after hot filtration) followed by second
filtration and mixing of both the filtrates, and 5. Saccharification of filtrate F1 or
mixture of F1 and F2 using barley β-amylase with or without pullulanase.
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
the use of pullulanase in saccharification.

6.3. Results and Discussion
6.3.1. Optimization of saccharification
6.3.1.1. Properties of free barley β-amylase and pullulanase
Percentage relative activity vs. pH profile and % relative activity vs.
temperature profile of Barley β-amylase are given in the Fig. 6.2 and 6.3,
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
204

respectively. Optimum pH and optimum temperature of barley β-amylase using assay
procedure to measure enzyme activity were 5.5 and 50 °C, respectively. Enzyme
activity at optimum conditions (according to assay procedure) and protein content of
commercial formulation of BBA were 12500 AGU/mL and 74 mg/mL, respectively.
0
10
20
30
40
50
60
70
80
90
100
110
3 3.5 4 4.5 5 5.5 6 6.5 7
pH
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y
50 °C

Figure 6.2. Enzyme activity-pH temperature profile at 50 °C.
0
10
20
30
40
50
60
70
80
90
100
110
30 35 40 45 50 55 60 65 70 75
Temperature °C
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y

Fig 6.3. Enzyme activity-temperature profile at pH of 5.5
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
205

Percentage relative activity vs. pH profile and % relative activity vs.
temperature profile of pullulanase are provided in the chapter 5. Optimum pH and
optimum temperature of pullulanase using assay procedure to measure the enzyme
activity were 4 and 60 °C, respectively. Enzyme activity at optimum conditions
(according to assay procedure) and protein content of commercial formulation of
pullulanase were 2950 PLU/mL and 50 mg/mL, respectively.
Properties of free barley β-amylase (BBA) and pullulanase (PL) are
summarized in the Table 6.1.
Table 6.1. Properties of barley β-amylase and pullulanase
Parameter Free barley β-amylase (BBA) Free pullulanase (PL)
Optimum pH 5.5 3.8–4.3
Optimum temperature (°C) 50 60
Activity 12500 BBAU/mL 2950 PLU/mL
Protein content (mg/mL) 74 50
Specific activity 169 (BBAU/mg of protein) 59 (PLU/mg of protein)

6.3.1.2. Thermostability of barley β-amylase and optimization of operating
temperature for saccharification
Optimum temperature for BBA using the specified assay procedure was found
to be 50 °C (Table 6.1). However, optimum operating temperature for
saccharification using BBA may not be the same as that obtained using the assay
procedure. Hence, study of thermostability of BBA was essential at an optimum pH
of 5.5. In addition to the thermostability check at different temperatures, experimental
runs on saccharification of sorghum liquefact utilizing BBA or BBA + PL at different
temperatures were also performed. Thermostability of BBA was first checked at 50
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
206

°C. It was observed that % relative activity of BBA decreased to 3% of the original at
50 °C and to 20% of the original at 40 °C within first 3 hrs of incubation in the
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
it remains active till 24 h, though in the thermostability study in the absence of
substrate BBA activity decreases drastically. It can be seen from Figs. 6.5 and 6.6 that
the optimum operating temperature for saccharification of sorghum liquefact to
produce maltose is around 50 °C.
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8
Time (h)
%

r
e
l
a
t
i
v
e

a
c
t
i
v
i
t
y







50
40
Temperature, °C

Figure 6.4. Thermostability of BBA

Fig. 6.5. and 6.6 also shows that use of pullulanase along with BBA in the
saccharification increases concentration of reducing sugars (maltose equiv.) from 174
to 204. This happens because pullulanase cleaves α(1→6) linkage and makes linear
part of the dextrin available for action of BBA.
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
207

0
20
40
60
80
100
120
140
160
180
0 4 8 12 16 20 24 28
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)








40 45
50
Temperature, °C

Figure 6.5. Comparison of hydrolysis curves at different temperatures.
Reaction conditions: 20 mL liquefact, 1.1 BBAU/mL; 5.5 pH and 50 °C
0
20
40
60
80
100
120
140
160
180
200
220
0 4 8 12 16 20 24 28
Time (h)
C
o
n
c
n

o
f

r
e
d
u
c
i
n
g

s
u
g
a
r

(
m
g
/
m
L
)





40 45
50
Temperature, °C

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



Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
208

Optimized conditions for liquefaction of sorghum flour and saccharification to
maltose syrup are shown in the Table 6.2.

Table 6.2. Optimized parameters for Liquefaction and Saccharification
Parameter Liquefaction Saccharification
Temperature (°C) 85 50
pH 6 5.5
Slurry concentration 30% w of sorghum flour / v of slurry n.a.
BLA concentration 0.06% v/w of sorghum flour i.e.
0.086% v/w of sorghum starch
n.a.
BBA concentration n.a. 0.04 %v/ w of starch
CaCl
2
concentration 200 ppm n.a.


6.3.2. Saccharification of sorghum liquefact
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
factors; 1. Ultrasound treatment on sorghum slurry before liquefaction, 2. Washing of
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
are summarized in the Table 6.3.




Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
209

Table 6.3. Summary of effect of ultrasound treatment and washing on liquefaction
and saccharification performance.

Run
No
BLA concn,
% v/w
C
RS
,
mg/mL
F1,
mL
F2,
mL
F1 + F2,
mL
C
f

mg/mL % saccharification
H1 0.086
31 72 0 73 185 61 (S N; W N; PU N)
H2 0.086
28 72 15 88 173
69 (S N; W Y; PU N)
H3 0.086
28 72 15 88 188
75 (S N; W Y; PU Y)
H4 0.086
34 75 0 75 195 66 (S Y; W N; PU N)
H5 0.086
32 75 14 89 186 75 (S Y; W Y; PU N)
H6 0.086
32 75 14 89 212 86 (S Y; W Y; PU Y)
G1* 0.086
33 75 0 75 180 61 (S N; W N; PU N)
G2* 0.086
32 75 14 89 172 69 (S N; W Y; PU N)
G3* 0.086
32 75 14 89 195 79 (S N; W Y; PU Y)
G4* 0.086
48 78 0 78 187 66 (S Y; W N; PU N)
G5* 0.086
44 78 12.5 90.5 183.5 75 (S Y; W Y; PU N)
G6* 0.086
44 78 12.5 90.5 209 86 (S Y; W Y; PU Y)
B1
0.125 40 72 0 72 188 61 (S N; W N; PU N)
B2
0.125 37 72 14 86 183 71 (S N; W Y; PU N)
B3
0.125 37 72 14 86 212 83 (S N; W Y; PU Y)
B4
0.125 39 75 0 75 195 66 (S Y; W N; PU N)
B5
0.125 36 75 12.5 87.5 189 75 (S Y; W Y; PU N)
B6
0.125 36 75 12.5 87.5 217 86 (S Y; W Y; PU 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
PL N : Pullulanase not used in saccharification
PL Y : Pullulanase not used in saccharification
pH = 5.5, temperature = 50 °C, BBA concn = 0.04 %v/ w of starch, PL concn =
0.057% v/w of starch, Liquefaction time = 1.5 h, saccharification time = 24 h.

Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
210

6.3.3. Studies on effect of different process parameters on % saccharification
Effect of washing of cake obtained after hot filtration and ultrasound treatment
before liquefaction on % saccharification remains exactly the same as discussed in
chapter 5. Hence will not be detailed here and only brief is provided.
Due to the incorporation of washing step, increase in the % saccharification
was observed. This increase in the % saccharification (which is based on the starch
content in the sorghum flour) is mainly through the recovery of oligosaccharides,
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).
Increase in the % saccharification due to the step of ultrasound treatment is
through the availability of additional starch granules because of the disruption of
hydrophobic protein matrix surrounding starch granules and amylose-lipid complex
for liquefaction and saccharification, discussed in detail earlier in the chapter 5.
Similar effect of ultrasound treatment was observed for all healthy, germinated, and
blackened sorghum (Table 6.3).
Experiments to study effect of use of pullulanase were performed with
washing of cake after hot filtration and without or with ultrasound treatment before
liquefaction. It was observed that in the absence of ultrasound treatment, %
saccharification increases from 69 to 75 (Table 6.3; H2 and H3) due to the use of
pullulanase in addition to BBA in saccharification. Whereas, in the presence of
ultrasound treatment, % saccharification increases from 75 to 86 (Table 6.3; H5 and
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
provided. Reason could be attributed to the availability of additional starch granules
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
211

due to sonication.

6.3.4. Economics of the process of production of maltose syrup from sorghum of
different varieties
In the chemical project economics, cost of production plays a key role. It
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
processing cost is given in the Table 6.6.
Here it should be remembered that processing cost has been determined for
production of dried maltose syrup. In order to obtain pure maltose, selective
crystallization must be done and its cost has not considered here.
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
212

Table 6.4. Cost of raw material to process 1 kg of starch
Sorghum contains approximately 70% starch. Hence starting quantity of sorghum will be 1.43 kg
Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter
Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs
Starch or sorghum

1 16 16 1.43 5 7.1 1.43 3 4.3 1.43 5 7.1
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
liquefaction

0.0008 250 0.2 0.0009 250 0.225 0.0013 250 0.325 0.0008 250 0.2
BBA required for
saccharification

0.0004 1500 0.6 0.0004 1500 0.6 0.0005 1500 0.75 0.0004 1500 0.6
Total - - 16.91 - - 8.12 - - 5.51 - - 8.10

Where, Q Quantity of material, kg
R Rate of material, Rs/kg
C Cost incurred, Rs
BLA B. licheniformis α-amylase
BBA Barley β-amylase






Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
213

Table 6.5. Cost of utilities to process 1 kg of starch
Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum Parameter
Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs Q, kg R, Rs/kg C, Rs
Steam for
liquefaction
1,2

at 85 °C for 1.5 h
0.53 1 0.53 0.76 1 0.76 0.76 1 0.76 0.76 1 0.76
Steam for
saccharification
1,2
,
at 55 °C for 24 h

0.98 1 0.98 1.41 1 1.41 1.41 1 1.41 1.41 1 1.41
Filtration &
bleaching and color
removal
4


- - 0.2 - - 0.429 - - 0.429 - - 0.429
Cost of steam for
evaporation
3

2.67 1 2.67 3.81 1 3.81 3.81 1 3.81 3.81 1 3.81
Total - - 4.38 - - 6.41 - - 6.41 - - 6.41

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

Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
214

Table 6.6. Comparison of processing cost or cost production of maltose syrup from sorghum

Parameter Isolated starch Healthy sorghum Blackened sorghum Germinated sorghum
Cost of Raw material 16.91 8.12 5.51 8.10
Cost of Utilities 4.38 6.41 6.41 6.41
Total cost 21.29 14.53 11.92 14.53
Maltose produced, kg
Reducing sugars produced, kg
0.73
1.05
0.62
0.89
0.62
0.89
0.62
0.89
Processing cost,
Rs/kg of maltose produced
Rs/kg of reducing sugars
produced
29.2
20.3
23.4
16.33
19.2
13.4
23.4
16.3

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.


Market cost of dry maltose (imported from Japan) = 250 Rs/kg

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.
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
215

6.4. Conclusions
Production of maltose syrup from sorghum flour involves two steps viz. 1.
Liquefaction of flour using Bacillus licheniformis α-amylase and 2. Saccharification
using Barley β-amylase without or with pullulanase.
1. In the present work, use of ultrasound in the production of maltose syrup from
sorghum flour has been explored.
2. The value of % saccharification to maltose using healthy sorghum flour (at
optimized reaction conditions for liquefaction and saccharification) was in the
range of 70- 90% depending upon following factors; 1. Ultrasound treatment of
sorghum slurry before liquefaction, 2. Washing of cake (obtained after hot
filtration) followed by 2
nd
filtration and mixing of both the filtrates, and 3. Use of
pullulanase in the saccharification.
3. Ultrasound treatment to sorghum slurry prior to liquefaction appears to disrupt
hydrophobic protein matrix surrounding starch granules and amylose-lipid
complex due to physical effects of acoustic cavitation, like shock wave
propagation and microjet formation in the vicinity of liquid-solid interface. This
frees starch granules and these additional starch granules are made available for
further action of α-amylase and barley β-amylase. A significant increase (~8–
10%) in the % saccharification was observed.
4. Use of pullulanase in the saccharification along with barley β-amylase increased
% saccharification through cleavage of α(1→6) linkages that results into
availability of linear chains for action of barley β- amylase.
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
feasibility of the process.
Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
216

6.5. Alternative approaches for value addition to sorghum
In the present work, possibility of value addition to healthy, blackened and
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
upon the market needs.
First step in the production of any product is liquefaction, i.e. simultaneous
gelatinization of free starch granules and dextrinization of gelatinized starch using
bacterial α-amylase. Liquefaction can be continued to achieve desired DE (15, 20 or
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
in detail in chapter 5 and 6 respectively.
Glucose syrup (DE > 96) produced can further be processed using glucose
isomerase to produce fructose syrup. (Refer Chapter 2)
Sorghum liquefact without any hot filtration can be saccharified using
amyloglucosidase to glucose syrup with DE greater than 90. Then, this syrup is
fermented to produce ethanol. After completion of fermentation, ethanol is distilled
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
or can be anaerobically digested to produce bio-gas.



Chapter 6: Enzymatic production of maltose syrup from sorghum
Studies in the Enzymatic depolymerisation of natural polysaccharides
217


Milling
Liquefaction using
bacterial α-amylase
Hot filtration
Saccharification using
barley β-amylase (BBA)
w/o or with pullulanase (PU)
Filtration and Purification
Maltose syrup
Preparation of slurry
Grain sorghum
Saccharification using amyloglucosidase
w/o or with pullulanase
Filtration and Purification
Glucose syrup
Saccharification of liquefact
to Glucose using
amyloglucosidase
Further filtration and
Purification to produce
Maltodextrins of different
DE 15, 20 or 30
Ethanol
DE ~ 15 DE ~ 15
Fermentation
followed by
distillation
Fructose syrup
Glucose isomerase

Figure 6.7. Production schemes of different products from sorghum
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Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
233
Appendix A : Analytical methods

A.1. Measurement of protein concentration (Lowry et al., 1951)
A.1.1. Principle and mechanism
The method is dependent on the color obtained on reaction of the Folin-
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
modified method have been proposed.
The Biuret reaction with alkaline Cu (II) and the reaction of a complex salt of
phosphomolybdotungstate, called the Folin-Ciocalteau phenol reagent, which gives an
intense blue green color with the Biuret complexes of tyrosine and tryptophan. The
Folin-Lowry method is 10 times more sensitive than UV absorption at 280 nm and
100 times more sensitive than Biuret method.
A.1.2. Apparatus and Equipments
¾ Test tubes
¾ Pipettes
¾ UV VIS spectrophotometer
A.1.3. Chemicals required
Folin-Ciocalteau reagent, Bovine Serum Albumin (BSA), Sodium hydroxide,
Sodium carbonate, Copper sulfate and Sodium potassium tartarate
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
234
A.1.4 Reagents
¾ Folin-Ciocalteau reagent: Ready reagent has been procured for the analysis.
¾ Standard protein solution: Dissolve 100 mg of Bovine Serum Albumin (BSA) in
100 mL of distilled water
¾ Sodium hydroxide solution: Dissolve 2 g of Sodium hydroxide in 500 mL of
distilled water
¾ Sodium carbonate solution: Dissolve 10 g of Sodium carbonate in 500 mL of
distilled water
¾ Copper sulfate solution: Dissolve 1.56 g of copper sulfate in 100 mL of distilled
water
¾ Sodium potassium tartarate: Dissolve 2.37 g of sodium potassium tartarate in
100 mL of distilled water
¾ Alkaline copper reagent: The copper reagent is prepared fresh just before use by
mixing 10 mL of sodium hydroxide solution, 10 mL of sodium carbonate
solution, 0.2 mL of copper sulfate solution and 0.2 mL of sodium potassium
tartarate solution

A.1.5. Procedure (Modified method)
1 mL of alkaline copper reagent was added to 0.1 mL of sample and vortex
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
spectrophotometer at 660 nm against blank sample. The standard calibration curve
was prepared in the protein concentration range of 0–1 mg/mL of BSA as shown in
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
235
Figure A.1.
Note
¾ Reagent with turbidity should be discarded immediately
¾ With every new reagent, new standard curve has to be plotted
y = 0.1628x - 0.0051
R
2
= 0.9994
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0 0.1 0.2 0.3 0.4 0.5
O. D
C
o
n
c
n

o
f

B
S
A

(
m
g
/
m
L
)






1
 
Figure A.1. Standard curve for BSA for modified Folin Lowry method

A.2. Measurement of moisture content in the sorghum flour
A.2.1. Apparatus and Equipments
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
drying. Moisture content is determined by using following formula;
100
W
W W
content moisture %
f
2 1
×

=  
where, W
f
= weight of flour, g 
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
236
W
1
= combined weight of petri dish and flour, g
W
2
= combined weight of petri dish and flour after drying is completed, g

A.3. Measurement of reducing sugar concentration (Miller et al 1951)
A.3.1. Principle and mechanism
A reducing sugar is a saccharide which has an anomeric carbon (carbonyl
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
ketone group) is converted to a carboxyl group.
This reducing property of sugar was used as a basis for the analysis of
reducing sugars. 3, 5-Dinitrosalicylic acid (DNSA) is an aromatic compound that
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.

A.3.2. Apparatus and Equipments
¾ Test tubes
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
237
¾ Pipettes
¾ UV VIS spectrophotometer
A.3.3. Chemicals required
Sodium hydroxide (NaOH), 3, 5-Dinitrosalicylic acid (DNSA), Potassium
sodium tartarate (Rochelle salt)
A.3.4. Preparation of DNSA reagent
30 g Potassium sodium tartarate (Rochelle salt) and 1.6 g sodium hydroxide
were dissolved in 70 mL distilled water. 3, 5-dinitrosalicylic acid was added to the
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
and then stored in an amber colored bottle.
A.3.5. Procedure
1 mL of appropriately diluted sample was added to test tube. 1 mL of DNSA
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.
Absorbance of the solution was then measured using spectrophotometer (Chemito) at
540 nm against blank sample. Blank sample means 1 mL distilled water instead of
sample of solution of reducing sugar.
A standard solution of 1 mg glucose/mL was prepared. Different quantities (0
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
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
238
cooling. Then 10 mL distilled water was added to each test tube. Absorbance of the
solution was then measured using spectrophotometer (Chemito) at 540 nm against
blank sample. A graph of glucose concentration (mg/mL) vs. absorbance (optical
density) was plotted (Fig. A.2) and used as a standard glucose graph in the form of
calibration equation to find out concentration of reducing sugars (glucose equiv) in
the samples.
Note: DNSA reagent with turbidity should be discarded
y = 1.6585x + 0.0281
R
2
= 0.9999
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 0.2 0.4 0.6 0.8
O. D
C
o
n
c
n

o
f

g
l
u
c
o
s
e

(
m
g
/
m
L
)












 
Figure A.2. Standard curve for glucose for DNSA method

A.4. Measurement of concentrations of malto-oligosaccharides using High
Performance Thin Layer Chromatograph (HPTLC)
A.4.1. Materials
Reference standards of maltotriose, maltotetraose, maltopentaose,
maltohexaose and maltoheptaose were purchased from Sigma – Aldrich; whereas
reference standards of glucose and maltose were purchased from Merck India Ltd.
Acetonitrile for chromatography Licrosolv was used for HPTLC.
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
239
20 cm × 10 cm TLC sheets (silica gel 60, Merck Ltd, India).
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
weights of glucose, maltose, maltotriose, maltotetraose, maltopentaose,
maltohexaose and maltoheptaose) on the TLC plate using automatic
application device (DESAGA) and then plate is subjected to atmospheric
drying for 15 minutes.
Step 3: TLC plate is then developed in a pre-saturated (saturation time ½ hr) chamber.
Triple development resulted into good quality of chromatogram. Mobile phase
Acetonitrile: 0.02 M Na
2
HPO
4
of composition 70:30 (v/v) was used for 1st
and 2nd development; whereas Mobile phase Acetonitrile: 0.01 M Na
2
HPO
4

of composition 80:20 (v/v) was used for 3rd development. Intermediate drying
between two development is essential for proper separation of peaks; which
was achieved by atmospheric drying for 15 min and drying at 45°C for 15
minutes.
Step 4: Plate after triple development was dipped in the diphenylamine-aniline-
phosphoric acid reagent (which consists of 40 ml acetone, 0.8gm
diphenylamine, 0.8 ml aniline and 6 ml phosphoric acid) for 4 seconds and
then kept at 120 °C for 10 min. It resulted into colored spots corresponding to
glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltoheptaose.
Step 5: Then the TLC plate is scanned at 546 nm using densitometer to get the
chromatogram and values of peak areas corresponding to glucose, maltose,
Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
240
maltotriose, maltotetraose, maltopentaose, maltoheptaose in standards solution
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),
maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and
maltoheptaose (G7). From these calibration curves (Fig. A.3), concentration of
glucose and malto-oligosaccharides were determined using peak area obtained
for the reaction samples.
y = 1.02E-06x
2
+ 5.66E-04x
y = 9.88E-07x
2
+ 1.85E-04x
y = 9.26E-07x
2
+ 2.67E-04x
y = 7.66E-07x
2
+ 4.19E-04x
y = 9.56E-07x
2
+ 1.36E-04x
y = 1.15E-06x
2
+ 1.08E-04x
y = 6.40E-07x
2
+ 3.96E-04x
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 100 200 300 400 500 600 700 800
Peak area
C
o
n
c
n

o
f

s
u
g
a
r

(
g
l
u
c
o
s
e

e
q
u
i
v
.
)
G1
G2
G3
G4
G5
G6
G7
 
FigureA.3. Standard curve for glucose and malto-oligosaccharides






Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
241
A. 5. Qualitative method to see presence of starch and Quantitative method for
determination of starch concentration
A.5.1. Principle
Starch contains two polysaccharide components viz. amylose and
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
varies with DP of amylose is shown in the following table.
DP (degree of polymerization) Color of amylose-iodine
complex
5-10 (actually called as malto-oligosaccharides) Red
10-25 Violet
Above 25 Deep blue

Amylopectin have little affinity for iodine and gives red coloration with it.
(Radley 1968). Concentration of soluble starch was determined from a standard plot
generated using the starch-iodine method (Bird and Hopkins, 1954).
A.5.2. Chemicals required:
Sublimed I
2
, Potassium iodide
A.5.3. Preparation of KI-I
2
reagent:
KI-I
2
reagent was prepared as 0.05% w/v iodine and 0.5% w/v potassium
iodide in distilled water. The KI-I
2
reagent was stored in an amber colored bottle.


Appendix A: Analytical methods
Studies in the Enzymatic depolymerisation of natural polysaccharides
242
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-I
2
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
Chemito-2300 UV-VIS spectrophotometer. A graph starch concentration (mg/ml) vs.
absorbance (optical density) was plotted (Fig. A.4) and was used as a standard.
y = 0.7401x
R
2
= 0.9987
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.2 0.4 0.6 0.8
absorbence
C
o
n
c
n

o
f

s
t
a
r
c
h

(
m
g
/
m
L
)












 
Figure A.4. Standard curve for starch for starch-iodine method
Appendix B: Code in Matlab to determine kinetic constants
Studies in the Enzymatic depolymerisation of natural polysaccharides
243
Appendix B : Code in Matlab to determine kinetic constants

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=[];

%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

K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01];
ub=[10 10 10 10 10 10 10 10 10 ];
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

Appendix B: Code in Matlab to determine kinetic constants
Studies in the Enzymatic depolymerisation of natural polysaccharides
244
K=[0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01];

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

[t,G] = ode45(@f,[0 tend],g,[],P,K);
plot(t,G(:,1),'.',t,G(:,2),'-',t,G(:,3),'+',t,G(:,4),'*',t,G(:,5),'+',t,G(:,6),'^',t,G(:,7),'--');
t;
G;
for i=1:length(t)
TDW(i)=TDW(t(i),P);
end;
TDW;



function error = kinetics(K,P,g,texp,GCexp)
sol = ode45(@f,[0 10],g,[],P,K);
G = deval(sol,texp); %evaluation of G values at values of
%experimental time.
E=P(1);So=P(2);A=P(3);C=P(4);D=P(5);
G=G';
GC=G;
GCexp=GCexp';
error=[0 0 0 0 0 0 0];
M=length(texp);
for i=1:7
for x = 1:M
error(i)=error(i)+(GC(x,i)-GCexp(x,i))^2;
end;
end;
error=sum(error);
K;
error=error*100;








Appendix B: Code in Matlab to determine kinetic constants
Studies in the Enzymatic depolymerisation of natural polysaccharides
245
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));
];

function TDW=TDW(t,P) %TDW total dry weight
E=P(1);So=P(2);A=P(3);C=P(4);D=P(5);
DE=A+C*(1-exp(-D*t));
acl=(180*100/DE-18)/162;
TDW=162*1.11*100*So/(DE*(180*100/DE-18));
INSTITUTE OF CHEMICAL TECHNOLOGY
UNIVERSITY OF MUMBAI

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

Name of the Candidate

Mr. Satish D. Shewale
Title of the thesis



Studies in the Enzymatic
depolymerisation of natural
polysaccharides

Name and Designation of Guiding
Teacher




Prof. (Dr.) Aniruddha B. Pandit
UGC Scientist ‘C’ (Professor’s Grade)
Chemical Engineering Division,
Institute of Chemical Technology,
Matunga, Mumbai 400 019.
Place of Research Work


Chemical Engineering Division,
Institute of Chemical Technology,
Matunga, Mumbai 400 019.
Registration Number and Date 188 (A); Dated: 29
th
March, 2004
Date of submission of Synopsis 18
th
March, 2008

Signature of the Candidate




(Satish D. Shewale)

Signature of the Guiding Teacher




Prof. (Dr.) Aniruddha B. Pandit
Introduction
Sorghum (Sorghum bicolor L. Moench) is fifth largest produced and an important
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
grains in India. It does well even in low rainfall areas.
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
has a potential to adapt itself to the given natural environment.
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
cereals in sufficient quantities at affordable prices. Hence, in addition to being a major
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
or does not fetch much price.
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
steps viz. 1. Liquefaction using B. licheniformis α-amylase (BLA) 2. Saccharification using
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
viz. 1. Gelatinization of 15 % sorghum slurry in boiling water for 10 min. 2. Circulating
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.

Studies in hydrolysis of soluble starch using immobilized B. licheniformis α-amylase
In this work B. licheniformis α-amylase (BLA) is immobilized on rigid superporous
(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
the improvement in the enzyme rigidity upon immobilization by covalent binding).
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.
A High performance thin layer chromatography (HPTLC) analytical method was
developed to analyze saccharide composition (i.e. concentration of malto-oligosaccharides)
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;
whereas for immobilized BLA, DE of starch hydrolysate at hydrolysis equilibrium was
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
of saccharide composition of starch hydrolysate with respect to time.
It was observed that the use of immobilized BLA is not suitable for the production
of glucose from sorghum flour due to following,
9 Change in action pattern of enzyme giving different product composition profile
9 Drastic reduction in enzyme activity in reusability without intermittent washing
9 After mixing gelatinized sorghum slurry with beads, it was very difficult to
separate beads from the slurry even after the completion of reaction
9 BLA is now cheaply available enzyme at a cost of 250 Rs./kg
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.

Production of Glucose from sorghum flour using B. licheniformis α-amylase (BLA)
and Amyloglucosidase (AG)
Production of glucose from sorghum flour comprises two steps viz. 1. Liquefaction
using B. licheniformis α-amylase (BLA) 2. Saccharification using amyloglucosidase (AG).
In the liquefaction, gelatinization of free starch granules and dextrinization of gelatinized
starch granules occurs simultaneously. In the saccharification, AG cleaves first α-(1-4)
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
stoppered conical flask containing 100 mL magnetically stirred sorghum slurry.
Liquefaction of sorghum slurry (30% w/v in 0.05 M acetate buffer of pH 6) was performed
using BLA at 85 °C (maintained by immersing conical flask in oil bath). Progress of
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
h using buffered slurries of different pH values (5.2-6.7), varying concentrations of BLA
(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
concentration and CaCl2 concentration are 85-90°C, 6, 30 % w of sorghum slurry/v of
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
liquefaction performance. It was observed that sonication of sorghum slurry before
liquefaction improves liquefact DE by 10 – 25 % depending upon the sonication time and
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
liquefaction increases filterability of the slurry after liquefaction. After liquefaction, pH of
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.
Optimum pH and the temperature for amyloglucosidase by using assay procedure
were 4.5 and 65 °C respectively. Activity of AG was observed to be 36300 EU per mL of
commercial AG solution at pH 4.5 and 65 °C. Optimum temperature by assay procedure
may not be same as the optimum operating temperature. Hence thermo stability of AG was
studied at different temperatures and saccharification was also performed at different
temperatures. It was observed that operating optimum temperature for saccharification
using AG was 55-60°C. Other Optimized reaction conditions for saccharification to
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 2
nd
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
improves % saccharification by around 10 % and a similar improvement in %
saccharification was observed due to washing of cake after 1
st
filtration.

Production of Maltose from sorghum flour using B. licheniformis α-amylase (BLA),
Barley β-amylase (BBA) and / or pullulanase
Optimum pH and temperature for barley β-amylase (BBA) were observed to be
5.3-5.7 and 50 °C respectively. Activity of BBA was 12500 EU per mL of commercial
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
PU solution at pH 4 and 60 °C. BBA is an exo-amylase which cleaves 2
nd
α-(1-4)
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
get produced in lesser quantities.
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 2
nd
filtration and mixing
of both filtrates and 3. Use of pullulanase along with BBA. It was observed that sonication
of slurry, prior to liquefaction improves % saccharification by around 10 % and a similar
improvement in % saccharification is observed due to washing of cake after 1
st
filtration.
Use of pullulanase along with BBA increases % saccharification by around 20 – 30%
depending upon whether prior sonication was done or not.
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
described in this work.

References
1. Devarajan B.; Pandit A. B., Sorghum flour as Raw Material for Glucose Production.
J. Maharashtra Agric. Univ., 1996, 21 (1), pg. 86-90.
2. 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.
3. Owuama, C.I., Sorghum: a cereal with lager beer brewing potential. World Journal of
Microbiology & Biotechnology, 1997, 13, 253–260.
4. Lali, A. M.; Manudhane, K. S. Indian Patent Application No., 356/Mum/2003.

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