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SORGHUM MALT OPTIMIZATION WITH Aspergillus spp FOR ETHANOL

PRODUCTION FROM CASSAVA, Manihot utilisima POHL

BY

UGWUANYI, ANTHONY CHUKWUDI

PG/MSC/02/32543

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA,

NSUKKA

OCTOBER, 2008

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SORGHUM MALT OPTIMIZATION WITH Aspergillus spp FOR ETHANOL

PRODUCTION FROM CASSAVA, Manihot utilisima POHL

BY

UGWUANYI, ANTHONY CHUKWUDI

PG/MSC/02/32543

A THESIS SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY,

UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FUFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE

IN MICROBIOLOGY.

SUPERVISOR: L. I. EZEOGU PhD

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA,

NSUKKA

OCTOBER, 2008

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CERTIFICATION

Ugwuanyi, Anthony Chukwudi, Reg. No. PG/M.Sc/02/32543, a postgraduate student in the

Department of Microbiology, has satisfactorily completed the requirements for the Degree

of Master of Science (M.Sc.) in Microbiology (Industrial).

The work embodied in this thesis is original and has not been submitted in part or full for

other diploma or degree of this or any other University.

__________________________ ___________________________

L. I. Ezeogu Ph.D Prof. J. C. Ogbonna

Supervisor Head, Department of Microbiology

Department of Microbiology, University of Nigeria, Nsukka.

University of Nigeria, Nsukka.

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DEDICATION

This work is dedicated to the family of Ugwuanyiagbonwaonugwuowo especially my

beloved parents, Chief Ochiebo R. E. UgwuanyiAgbo, Mrs M. N. Agbokwor, E. Agbo and

all my brothers and sisters.

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ACKNOWLEDGEMENT

My sincere appreciations go to my supervisor, L. I. Ezeogu Ph.D for the general assistance,

both to my person and towards the completion of this work, thanks a million times.

Heartfelt appreciations are also reserved for Prof. J. A. N. Obeta, Prof. S. K. C. Obi, Prof.

B. N. Okolo, Prof J. C. Ogbonna, Dr C. U. Iroegbu, Prof (Mrs.) J. Okafor, Dr J. O.

Ugwuanyi, Dr A. Moneke, Mr. M. A. Ugwuanyi (late), Dr. C.U. Anyanwu, Mr. J.A.C

Ugonabo, Mr. E Eze, Mr. C. Eze, Mr Arinze Okoli, Rev. Sr. Dr Dibua, Mr Nneji, Mr Eze

Felix, Mr Asibe, Mr. Oyibo, Mrs Nwike (and other non academic staff in Block B) of the

Department for their concern towards the completion of this work.

I also appreciate the assistance, interest, worry and encouragements shown by the

following towards this work: Prof. Obioma Njoku, Dr Chima Nwanguma, Prof Onwurah,

Mr. OGB, Mrs Egwuatu (Biochemistry Department); Dr Bobby Asogwa, Eze B.

(Economics Department); Dr Eyo (Zoology), Dr Aja Nnadi, Dr Nwigwe (Vet. Medicine)

Prof. Agwu--- ka sir (Botany), Dr Ifeanyi Iroha (EBSU).

For their love and necessary ‘distractions’, I am also grateful to my colleagues, senior and

junior (Ph.D, M.Sc. and B.Sc. students), in the laboratory and friends, as follows: Frank Ire,

Awah Nsikak (Chief Awah), Ernest Ugwuidu, Mike Ukwuru, Innocent Ogbonna, Odoh

Jerry, Chukwudi Nnamchi, Emeka Nweze, Eton Nkereuwem, Nkechika Duru, Melanie

Ikeh, Ekong Ubong, Deborah Dasimaka, Eric Agbata, Godwin Oyiwona, Idire Samuel, Ify,

Chinelo, Oluchi, Ben, ‘Bishop’, Sebastine, Chibuike, Chibuzor, Akolo, Omeokachie,

Uzoma, Ijeoma, Innocent, Anthonia, Nkiru, Nnenna, Ndidi, Kate, Charles Nwamba, Uche

Obialor, Uche Nwodo, Parker, Nk baby, Tom, Shimave, Chris, Schijindu, Mrs Ugwu, Ij

Company, Emeka, Maduabuchi, Arinze, Chinedu, Ogechi, Nnenna Stephens, Ann, Chira,

Chika, Ken, Emeka Ugwueze, Emma (NPA), Emma (Council), Esther, Lovinda, Daniel,

Danjuma, Fumilola, Chuma Abosi & Bros, Kevin, Sabina, Engee and Odimnobi.

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I am not forgetting people such as Augustine (Papa Ify), Onyebuchi V-Mobile (Gege), Bar.

Nick Omeye, Hon. Roderick Ugwu, Hon. Charles Ugwu, Hon. Dr Shere, Felly Ugwuanyi,

Marcel Ogerewu, Tonedo, Alpho, Leo, Leon (STB), Engr Agbo (NB Plc), Rev. Fr. Emma

Onuh, Silas Agbo, C. C Attama, Capt. Ugwueze, Capt. Nwamba, Albonaz Ltd and Mr &

Mrs Chijioke Obuna for their special assistance, thanks a lot.

Above all, I give all honour, praises, adoration and glory to the almighty God for His

guidance and mercies, through Jesus Christ our Lord and saviour, Amen.

October, 2008

Ugwuanyi, Anthony Chukwudi

Department of Microbiology,

University of Nigeria, Nsukka.

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TABLE OF CONTENTS

Title page ……………………………………………………….. i

Certification ……………………………………………………….. iii

Dedication ……………………………………………………….. iv

Acknowledgement ……………………………………………………….. v

Table of contents ……………………………………………………….. vii

List of figures ……………………………………………………….. xii

Abstract……………………………………………………………………… xiv

CHAPTER ONE

INTRODUCTION ………………………………………………………… 1

CHAPTER TWO

LITERATURE REVIEW……………………………………………………. 3

Sorghum…………………………………………………………………… 3

ETHANOL: a renewable energy source …………….…………………….. 5

Functional, environmental and strategic benefits of ethanol………………. 6

Biomass ethanol production process ……………………………………. … 7

Pretreatment of biomass…………………………………………………. 7

The role of cellulases ……………………………………………………….. 8

Fermentation to ethanol……………………………………………………. 9

Biomass ethanol coproducts…………………………………………….. 10

MALTING ………………………………………………………………. 11

ENZYMES IN MALTED SORGHUM………………………………… 12

-Amylase……………………………………………………………….. 12

Beta Amylase ……………………………………………………………. 13

-Glucosidase …………………………………………………………… 14

Lipases…………………………………………………………………… 15

Peroxidases………………………………………………………………. 16

Proteinases (endopeptidases) and Carboxypeptidases ……………………. 25

Steeping …………………………………………………………………… 18

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Germination ……………………………………………………………….. 20

Kilning …………………………………………………………………….. 21

Malting Loss…………………………………………………………….. 22

Sorghum Malt Proteins…………………………………………………. 23

Water Extracts ………………………………………………………….. 25

Mashing…………………………………………………………………. 26

CHAPTER THREE

MATERIALS AND METHODS………………………………………… 28

Source of Cassava Raw Starch…………………………………………… 28

Sorghum Variety and Source……………………………………………… 28

Sorting………………………………………………………………………. 28

Decortication………………………………………………………………… 28

Starter Cultures and Inoculum Preparation……………………………….. 29

Malting……………………………………………………………………… 29

Surface Sterilization………………………………………………………… 29

Steeping……………………………………………………………………… 29

Steep schedules for decorticated and undecorticated sorghum malting……... 29

Inoculation………………………………………………………………….. 29

Germination (Solid Substrate Fermentation)……………………………….. 30

Sample Collection and Drying (Kilning)……………………………………… 30

ANALYSES …………………………………………………………………. 30

Root lengths (RL) and malting loss (ML)…………………………………… 30

Moisture content……………………………………………………………… 32

Cold Water Extract…………………………………………………………. 32

Hot Water Extract (H.W.E)………………………………………………… 33

Cold Water Soluble Carbohydrate (CWSC)………………………………… 33

Free -Amino Nitrogen (FAN) Contents of Sorghum Malts…………………. 34

Diastatic Power and Amylase Activities……………………………………… 34

Cold Water Soluble Protein (CWSP)………………………………………… 36

Total Non-Protein Nitrogen (TNPN) Determination…………………………. 37

Mashing………………………………………………………………………. 37

Wort Free Amino Nitrogen Determination (W-FAN)………………………… 38

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Wort Total Soluble Protein (WTSP)……………………………………………. 38

Wort Total Soluble Protein (WTSP)…………………………………………… 38

Liquid Substrate Fermentation ………………………………………………… 38

Glucose level determination…………………………………………………… 38

Ethanol Content Determination of fermented samples………………………… 39

CHAPTER FOUR

RESULTS………………………………………………………………….. 40

Effects of decortication and starter cultures of Aspergillus on Aspergillus

growth during sorghum malting…………………………………………….…. 40

Effects of decortication on Percentage Germinative Energy (GE) and

Water Sensitivity (WS) ………………………………………………………… 43

Effect of decortication and Aspergillus spores on sorghum malt enzyme

developments………………………………………………………………….. 45

-amylase……………………………………………………………………... 45

β–amylase……………………………………………………………………... 48

Diastatic Power (DP)………………………………………………………...... 51

Effects of decortication and inoculation with Aspergillus spores on

sorghum malt free amino nitrogen (FAN) developments……………………… 54

Effects of decortication and inoculation Aspergillus spores on Cold water

Soluble Protein (CWS-P)……………………………………………………… 57

Effects of decortication and inoculation with Aspergillus spores on Cold Water

Extract (CWE) development………………………………………………….. 60

Effects of decortication and inoculation with Aspergillus spores on Hot

Water Extract (HWE) development…………………………………………… 63


sorghum malt Wort Free Amino Nitrogen…………………………………. …. 66

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Effects of decortication on sorghum malt Wort Total Soluble Protein………… 68


sorghum malt Cold Water Soluble Carbohydrate (CWS-CHO)………………… 70


sorghum malt Total Non Protein Nitrogen (TNPN)…………………………….. 72


malting loss (ML)……………………………………………………………….. 74


wort extract glucose levels…………………………………………………….. 77

Effects of decortication and inoculation with Aspergillus spores on the

time course of yeast growth on sorghum malt extract………………………… 79


ethanol levels in fermented sorghum malts……………………………………. 82


glucose levels of raw cassava starch hydrolysate hydrolysed with

sorghum malt crude enzymes ………………………………………………….. 84


glucose levels of gelatinized cassava starch hydrolysate using sorghum

malt crude enzymes……………………………………………………………. 87


time course of yeast growth on gelatinized cassava hydrolysate hydrolysed

with decorticated and undecorticated sorghum malt enzymes………………… 90

Effects of decortication and inoculation with Aspergillus spores on the time

course of yeast growth on raw cassava hydrolysate hydrolysed with

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decorticated and undecorticated sorghum malt enzymes…………………….. 93


ethanol production in gelatinized cassava hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt enzymes……………………… 96


ethanol production in raw cassava hydrolysate hydrolysed with decorticated

and undecorticated sorghum malt enzymes……………………………………. 99

CHAPTER FIVE

DISCUSSION………………………………………………………………….. 102

Fungal Count1…………………………………………………………………. 102

GE & WS………………………………………………………………………. 102

Root Length and Malting Loss………………………………………………... 103

Enzymes ………………………………………………………………………. 104

Free Amino Nitrogen (FAN) and Protein Mobilisation……………………...... 105

Water Extracts …………………………………………………………………. 106

CONCLUSION………………………………………………………………… 108

REFERENCES…………………………………………………………………. 109

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LIST OF FIGURES………………………………………………………….. 40

Effects of decortication and starter cultures of Aspergillus on Aspergillus

growth during sorghum malting…………………………………………….…. 40

Effects of decortication on Percentage Germinative Energy (GE) and

Water Sensitivity (WS) ………………………………………………………… 43

Effect of decortication and Aspergillus spores on sorghum malt enzyme

developments………………………………………………………………….. 45

-amylase……………………………………………………………………... 45

β–amylase……………………………………………………………………... 48

Diastatic Power (DP)………………………………………………………...... 51


sorghum malt free amino nitrogen (FAN) developments……………………… 54

Effects of decortication and inoculation Aspergillus spores on Cold water

Soluble Protein (CWS-P)……………………………………………………… 57


Extract (CWE) development………………………………………………….. 60

Effects of decortication and inoculation with Aspergillus spores on Hot

Water Extract (HWE) development…………………………………………… 63


sorghum malt Wort Free Amino Nitrogen…………………………………. …. 66

Effects of decortication on sorghum malt Wort Total Soluble Protein………… 68


sorghum malt Cold Water Soluble Carbohydrate (CWS-CHO)………………… 70


sorghum malt Total Non Protein Nitrogen (TNPN)…………………………….. 72


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malting loss (ML)……………………………………………………………….. 74


wort extract glucose levels…………………………………………………….. 77


time course of yeast growth on sorghum malt extract………………………… 79


ethanol levels in fermented sorghum malts……………………………………. 82


glucose levels of raw cassava starch hydrolysate hydrolysed with

sorghum malt crude enzymes ………………………………………………….. 84


glucose levels of gelatinized cassava starch hydrolysate using sorghum

malt crude enzymes……………………………………………………………. 87


time course of yeast growth on gelatinized cassava hydrolysate hydrolysed

with decorticated and undecorticated sorghum malt enzymes………………… 90

Effects of decortication and inoculation with Aspergillus spores on the time

course of yeast growth on raw cassava hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt enzymes…………………….. 93


ethanol production in gelatinized cassava hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt enzymes……………………… 96


ethanol production in raw cassava hydrolysate hydrolysed with decorticated

and undecorticated sorghum malt enzymes……………………………………. 99

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ABSTRACT

Bioethanol production process from cassava by means of enzymes produced in Aspergillus-

treated, decorticated and undecorticated malted sorghum (koji-like sorghum products)

using a yeast stock culture originally isolated from palm wine was studied. The effects of

the Aspergillus species used as starter cultures and decortication of sorghum grains on

ethanol production and other sorghum malt quality development parameters were

investigated. The results showed that there was a progressive increase in fungal spores

(starter cultures) as determined through serially diluted plate counts, corresponding to the

increase in germination time: Aspergillus awamori-treated decorticated malted sorghum

having the highest fungal count, followed by decorticated malted sorghum treated with

Aspergillus carbonarius, undecorticated malted sorghum treated with Aspergillus awamori,

and undecorticated malted sorghum treated with Aspergillus carbonarius in that order.

Enzyme levels notably α- and β-amylases were more markedly affected by decortication

treatment than treatment due to the Aspergillus species. The amino acid level in form of

free amino nitrogen (FAN) determined by Ninhydrin method was significant (p<0.05) with

respect to the decortication and Aspergillus treatments compared to the controls. Similarly

all other sorghum malt quality indicators such as Cold water extract (CWE), Hot water

extract (HWE), Cold Water Soluble Protein (CWSP), Cold Water Soluble carbohydrate

(CWSC), Total Non-Protein Nitrogen (TNPN), Total Soluble Protein (TSP) and Ethanol

levels of sorghum malts extracts and cassava hydrolysates were in one way or the other

affected by the decortication and Aspergillus treatments. These effects probably resulted

from the increase in endosperm modification of sorghum malts as a result of decortication

and thereby facilitating starter culture invasion.

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INTRODUCTION

Ethanol is an emerging environmentally friendly source of energy in the automobile

industry. It is also useful in food, pharmaceutical and other industrial sectors (Zhan et al,

2003). Commercial production of ethanol requires the use of cheap and readily available

raw materials since the cost of raw materials represents over 60% of the total production

cost. Cassava (Manihot esculentum) starch is a potential raw material for ethanol

production. It is vastly distributed and relatively cheap in Nigeria. To realize this potential,

cassava starch in its native state has to be hydrolysed into simple sugars. Chemical

hydrolysis is expensive and involves corrosive chemicals while biological hydrolysis, using

hydrolytic enzymes is cheaper and safer. Malted sorghum is a cheap source of hydrolytic

enzyme for cassava starch hydrolysis (Zhan et al., 2003; Ezeogu et al., 2005).

The use of starter cultures during malting to improve malt quality has been

investigated. The greenhouse effect caused by increased carbon dioxide in the atmosphere

is chiefly attributed to the fact that petroleum, a fossil fuel, is used as an energy source and

reducing the use of petroleum directly decreases the amount of carbon dioxide released and

thus contributes to alleviating the greenhouse effect (Ohara 2003). As fossilised petroleum

supplies continues to decline, the use of renewable biomass as substrate for production of

petrochemicals is becoming increasingly important and ethanol (bioethanol), one of these

products is emerging as a ‘clean’ substitute for direct use as fuel which can ease both

natural resource limitation and reduce environmental pollution (Zhan et al, 2003). The use

of biomass such as sugarcane and corn as raw materials for the production of ethanol and

other bio-products has become a common practice in Brazil and the United States (Ohara

2003). In Africa and some parts of Asia however, most cheap sources of biomass are

mainly obtained from tubers (cassava wastes) and cereals (sorghum) and because they have

high starch contents (≥70%) are potential raw materials for industrial applications in those

places where they are easily cultivated. (Zhan et al., 2003; Ezeogu et al., 2005). Use of

saccharifying enzymes has replaced the conventional method of starch hydrolysis using

acid. Bioethanol is an alcohol mainly produced by fermentation of sugar- and starch-

containing organic materials such as cassava.

Cassava is a perennial vegetatively propagated shrub cultivated throughout the

lowland tropics for its starchy roots. It is of great economic importance to several tropical

countries of Africa where its consumption (in terms of carbohydrate content) exceeds those

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of other crops (Oboh, 2005) Cassava processing plants generate large amounts of cassava

solid waste. A small amount may be utilized for animal feed but most of it is discharged

with seemingly deleterious effects to the environment. Malted grains are vital source of

saccharifying enzymes. Due to its environmental benefits, bioethanol is considered a

promising biofuel for the industrial sector. Thus it is necessary to reduce its production

costs by using new alternative biomass feedstock and processes (Zhan et al., 2003; Ezeogu

et al., 2005). The present study was carried out to determine how sorghum malts could be

optimized for cassava starch hydrolysis and ethanol production using Aspergillus awamori

and Aspergillus carbonarius as starter cultures.

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LITERATURE REVIEW

SORGHUM

Sorghum has been an important staple in the semi-arid tropics of Asia and Africa

for centuries. These crops are still the principal sources of energy, protein, vitamins and

minerals for millions of the poorest people in these regions. Sorghum is grown in a harsh

environment where other crops grow or yield poorly. They are grown with limited water

resources and usually without application of any fertilizers or other inputs by a multitude of

smallholder farmers in many countries. Therefore, and because they are mostly consumed

by disadvantaged groups, they are often referred to as "coarse grain" or "poor people's

crops". They are not usually traded in the international markets or even in local markets in

many countries. The farmers seldom, therefore, have an assured market in the event of

surplus production.

Sorghum has, however, gradually become very important as a source of raw

material not only in brewing (Ezeogu et al., 2001) and traditional beverage production,

flour industries, ethanol-producing industries et cetera, but also as an important tool for

solid state fermentations. The rising interests in sorghum utilization are perhaps due to the

availability, versatility, suitability, economical, and technical improvements with regard to

sorghum physiology, biochemistry, and applications (Evans and Taylor 1990; Duffour et

al. 1992). As a result of the above reasons, sorghum has replaced much of the imported

barley with its attendant costs in Nigeria, as the primary source of extracts in the brewing

industries (Okolo et al. 1996). Sorghum malting (modification) involves sorting, surface

sterilization, steeping, germination and kilning (controlled drying). It is an important aspect

of brewing which determines the success of the end product – malt and subsequently the

alcohol that originates from the malt (Duffour et al. 1992). Various manipulations and

variations in the malting processes such as the use of a particular liquor temperature for

steeping or incorporation of chemical additives (Palmer et al. 1989) are all aimed at

improving the malt quality towards actualizing superior modified malts at reduced time and

general cost. The latter is however not profitable with sorghum as it is with barley, as a

result unsuitable for its intended use and shows that what obtains for barley might be

different for a different cereal (Palmer et al. 1989). The above information prompted the

use of the technique of solid state fermentation (SSF), a technology which has emerged as a

front runner in the management of agro-industrial products and residues because of its high

value addition, using decorticated and undecorticated sorghum grains that had been

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previously treated differently with amylolytic fungi namely: Aspergillus awamori and

Aspergillus carbonarius just before germination or at the final steeping period.

This became justifiable and necessary with the previous knowledge and report that

sorghum has low starch-degrading enzyme levels and poor solubility (Subramanian et al.

1995). Increased and adequate enzyme elaboration in the sorghum malts through

inoculation of Aspergillus species is therefore necessary as part of our interest concerns the

hydrolysis of milled cassava chaff/bagasse/residue obtained from garri processors to

fermentable level for the purposes of bioethanol production through fermentation processes

using enzymes from A. awamori and A. carbonarius treated malted sorghum. This is in a

way an efficient use of agro-industrial residue providing not just an alternative substrate for

yeast (microbial) growth and subsequent superior product delivery but also aids in reducing

pollution problems. Moreover, cassava has been an important source of food and dietary

calories in the tropics, as a result the disposal/accumulation of unused cassava materials in

the environment is certain and (serving as interesting raw materials) in part prompted this

investigation (Subramanian et al. 1995).

ETHANOL: A RENEWABLE ENERGY SOURCE

Ethyl alcohol, or ethanol, C2H5OH, is a clear, colorless liquid, with a burning taste

and characteristic, agreeable odor. Ethanol is the alcohol in such beverages as beer, wine,

and brandy. Because of its low freezing point, it has been used as the fluid in thermometers

for temperatures below -40° C (-104° F), the freezing point of mercury, and for other

special low-temperature purposes, such as for antifreeze in automobile radiators. Ethanol is

normally concentrated by distillation of dilute solutions. Commercial ethanol contains 95

percent by volume of ethanol and 5 percent of water. Dehydrating agents remove the

remaining water and produce absolute ethanol. Ethanol melts at -114.1° C (-173.4° F),

boils at 78.5° C (173.3° F), and has a specific gravity of 0.789 at 20° C (68° F). Ethanol has

been made since ancient times by the fermentation of sugars. All beverage ethanol and

more than half of industrial ethanol is still made by this process. Starch from potatoes,

cassava, corn, or other cereals can be the raw material. The yeast enzyme, zymase, changes

the simple sugars obtained from starch into ethanol and carbon dioxide. The fermentation

reaction, represented by the simple equation is actually very complex because impure

cultures of yeast produce varying amounts of other substances, including fusel oil, glycerin,

and various organic acids (Lynd, 1996).

C6H12O6→ 2C2 H5OH + 2CO2

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The fermented liquid, containing from 7 to 12 percent ethanol, is concentrated to 95

percent by a series of distillations. In the production of beverages such as whiskey and

brandy, some of the impurities, which supply the flavor, are of great value. Much ethanol

not intended for drinking is now made synthetically, either from acetaldehyde made from

acetylene, or from ethylene made from petroleum. A small amount is made from wood

pulp (Lynd, 1996). Ethanol can be oxidized to form first acetaldehyde and then acetic acid.

It can be dehydrated to form ether. Other products made from ethanol include butadiene,

used in making synthetic rubber; ethyl choride, used as a local anesthetic; and many other

organic chemicals. Ethanol can also be mixed with gasoline to form the automobile fuel

called gasohol. Ethanol is miscible (mixable) in all proportions with water and with most

organic solvents. It is an excellent solvent for many substances and is used in making such

products as perfumes, lacquer, celluloid, and explosives. Alcoholic solutions of nonvolatile

substances are called tinctures; if the solute is volatile, the solution is called a spirit. Most

industrial ethanol is denatured to prevent its use as a beverage. Denaturing involves mixing

ethanol with small amounts of poisonous or unpleasant substances to make the ethanol

undrinkable. The removal of all these substances would involve a series of treatments more

expensive than the federal excise tax on alcoholic beverages.

Depletion of the world’s petroleum supply has led to an increasing worldwide

interest in alternative, non-petroleum-based sources of energy (Kerr 1998). As petroleum

supplies 97% of the energy consumed for transportation, industry and governments

worldwide have been actively identifying, developing and commercializing technology for

alternative transportation fuels. A growing, yet controversial, source of transportation fuel

is fermentation-derived ethanol whose production cost requires significant subsidy to

permit producers to remain in business. Nearly all fuel ethanol is produced by fermentation

of corn glucose in the United States or sucrose in Brazil (Rosillo-Calle and Cortez, 1998),

but any country with a significant agronomy-based economy can use current technology for

fuel ethanol production. This is possible because, during the last two decades, technology

for ethanol production from non-food-plant sources has been developed to the point at

which large-scale production will be a reality in the next few years. Therefore, agronomic

residues such as corn stover (corn cobs and stalks), sugar cane waste, wheat or rice straw,

forestry and paper mill discards, the paper portion of municipal waste, and dedicated

energy crops—collectively termed ‘biomass’—can be converted to fuel ethanol.

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Functional, environmental and strategic benefits of ethanol

All automobile manufacturers produce vehicles that can readily use 10% ethanol or

85% ethanol (E85) blends for fuel, and ethanol can replace diesel in heavy vehicles as well.

About 12.5 billion liters of ethanol is produced from cane sugar in Brazil, and is used as

either 22% blends with gasoline or as neat ethanol fuels containing 100% ethanol. Blending

oxygenates such as ethanol and methyl tertiary butyl ether (MTBE) are well recognized for

causing reduced carbon monoxide levels by improving overall combustion (oxidation) of

the fuel. However, MTBE is being phased out of use because it contaminates domestic

wells (Blackburn et al. 1999). Ethanol is being evaluated as a replacement and may be

readily accepted because of the environmental consciousness, their huge diverse

agricultural economy, and the lack of any clear alternative oxygenate. However, the fact

that ethanol has approximately 65–69% of the energy density of hydrocarbon fuels (Lynd,

1996) must be considered. Due to global warming, the use of fuel ethanol will significantly

reduce net carbon dioxide emissions when it replaces fossil fuels, because fermentation-

derived ethanol is already part of the global carbon cycle (Wyman, 1994). Finally, as the

current price of gasoline fluctuates at the whim of Oil Producing and Exporting Countries

(OPEC), the development of a domestically produced renewable transportation fuel could

be seen as a strategic alternative (Sheehan, 1994).

Biomass ethanol production process

The primary difficulty for commercialization of ethanol produced by fermentation

is its high cost of production relative to the local cost of gasoline. Recent increases in the

wholesale price of crude oil appear to be helping to close the cost gap between ethanol and

gasoline. The cost of ethanol is linked, in part, to the inescapable loss of half of the carbon

during fermentation of sugars by microorganisms. Although the production of ethanol from

cane sugar is a relatively simple process, complexity increases when ethanol is produced

from corn or wheat starch, as these processes require enzymes to hydrolyze starch to

glucose prior to fermentation. Production of ethanol from biomass requires even more

extensive processing to release the polymeric sugars in cellulose and hemicellulose that

account for 23%–53% and 20%–35% of plant material, respectively. Cellulose is a beta-

linked glucose polymer, whereas hemicellulose is a highly branched chain of xylose and

arabinose that also contains glucose, mannose and galactose (Weislogel et al, 1996).

Hydrolysis of these carbohydrate polymers is usually accomplished by exposure to acid

21

(contributed either by the biomass or added externally) and by enzymes. After hydrolysis,

the acid is separated from the sugars, which are fermented to ethanol.

Pretreatment of biomass

In addition to polymeric carbohydrates, plant matter contains varying amounts of

polyphenolic lignin and other ‘extractables’. The pretreatment process aims to separate the

carbohydrates from the lignin matrix while minimizing chemical destruction of

fermentation sugars required for ethanol production. Development of an ideal pretreatment

process is difficult, given that ‘biomass’ includes such sources as hardwood and softwood

trees, agricultural residues such as cassava waste, corn stover, and non-recyclable paper

waste. These diverse feedstocks have caused researchers to test numerous pretreatment

processes ranging from hot water and steam explosion treatments, to alkaline and solvent

pretreatments, to many useful versions of acid pretreatment (Hsu, 1996). The kinetics and

yields of the various acid-based batch and flow-through processes have been compared

recently, and it is clear that flow-through processes provide higher sugar yields and cause

less sugar destruction, but result in a more dilute sugar solution (Jacobson and Wyman,

2000). A new counter-current flow-through process that yields very high levels of

hydrolyzed cellulose and hemicellulose while using only 0.07% sulfuric acid is being

developed (Lee et al, 2000). This counter-current process results in shrinkage of the

biomass, this being responsible for the critical maintenance of relatively high sugar

concentrations. Eighty-two percent hydrolysis of cellulose and near-total depolymerization

of xylose have been reported to yield a solution containing approximately 4% sugar (Torget

et al, 2000). With these exceptional results, it is speculated that it may be possible to

eliminate or greatly reduce the need for cellulase in the bioethanol process. However, it is

acknowledged that detoxification of acid-hydrolyzed lignin and other ‘extractables’ in the

sugar hydrolysate will present additional costs for the total hydrolysis process, costs that

could be avoided entirely if a fully enzymatic process to be developed is implemented

instead.

The role of cellulases

The pretreatment process is designed to initiate the breakdown of the biomass

structure and partially hydrolyze the carbohydrate polymers, making them accessible to

enzymatic attack (Wyman, 1999). Therefore, enzymatic degradation of cellulose and

hemicellulose has been extensively studied over the past 40 years, with significant

22

progress. As hemicellulose is readily hydrolyzed by mild-acid conditions, the recalcitrant

semi-crystalline cellulose has been the target of most biomass enzyme research (Himmel et

al., 1999). Historically, cellulases were first applied in a sequential process (pretreatment

→ cellulase hydrolysis → ethanol fermentation). However, the simultaneous

saccharification and fermentation (SSF) process provides significant cost reduction because

cellulase hydrolysis occurs during fermentation of the glucose (Wyman, 1999). The

processes currently used include fermentation of all biomass sugars in a simultaneous

saccharification cofermentation (SSCF) process. Even after over 40 years of research on

cellulases (Sheehan, 2001), the costs of these enzymes have remained high, in comparison

to the costs of proteases and amylases, for a few key reasons.

Largely owing to their complex, insoluble semicrystalline substrates, cellulases are

relatively slow catalysts (Esteghlalian et al., 2001). Also, maximal activity requires

multiple, related enzyme activities acting synergistically. Specifically, complete hydrolysis

of cellulose requires exoglucanases, endoglucanases and beta-glucosidases to fully

hydrolyze cellulose to glucose (Himmel et al., 1999). Analyses pinpointing areas in

bioethanol production that require most research have identified four critical areas related

to cellulase research: increased thermostability, improved cellulase binding, increased

specific activity, and reduced nonspecific binding to lignin (Wooley et al., 1999). To

improve cellulase activity, mixtures of bacterial and fungal enzyme were used to determine

the ideal interaction of these enzyme activities, but this superior blend is not available in a

single organism (Baker et al., 1995). With the help of modern genetic technology,

production of a ‘superior’ blend by a single organism is certainly possible. The production

and usage of cellulase enzymes from Trichoderma and other organisms are being improved

continuously. Recent research appears to address many of the aforementioned critical needs

(Irwin et al., 2000). One of the most active cellulases known, endoglucanase E1 from

Acidothermus cellulolyticus, has been expressed in tobacco and potatoes (Irwin et al., 2000;

Hooker et al., 1971) providing a potential source of this enzyme.

Fermentation to ethanol

Fermentation of biomass involves significantly greater challenges, owing to the

necessity of converting pentose as well as multiple hexose sugars to ethanol in a SSCF

step. As with pretreatment, fermentation microorganisms have undergone continuous

improvement, especially with the application of genetic engineering. Both yeasts (such as

Saccharomyces and Pichia species) and bacteria (such as Escherichia coli, Klebsiella and

23

Zymomonas) have been genetically engineered to ferment glucose, xylose and arabinose

sugars (Picataggio and Zhang, 1996; Bothast et al., 1999 and Nigam 2001). Commercially,

BC International Corporation (www.bcintlcorp.com) is using genetically engineered E. coli

that produces ethanol from biomass sugars (Ingram et al., 1987), and Arkenol Inc.

(www.arkenol.com) is evaluating Zymomonas for use in its concentrated-acid process. The

ideal bioethanol producer would ferment all biomass sugars, possess good resistance to

lignin monomers, acetate and other inhibitory byproducts, and produce a synergistic

combination of cellulases needed for full cellulose hydrolysis. Lactobacillus is a legendary,

persistent and resistant contaminant in ethanol fermentation. Recent genetic engineering of

this organism has added the genes for xylose utilization with the aim of enabling it to

produce lactic acid from biomass (Picataggio et al., 1998) and, eventually, for ethanol

production. Others have also successfully introduced cellulase genes into Lactobacillus

although not necessarily for fermentation. Furthermore, various thermophilic Clostridium

and Thermoanaerobium species have been investigated for their potential as ethanol

producers, but have been consistently found to suffer from end-product inhibition and

membrane damage (Ingram, 1990).

Biomass ethanol coproducts

Biomass sugars are valuable fermentation feedstocks for many other products that

can be manufactured along with bioethanol (Lynd et al., 1999). Likely coproducts include

organic acids and other organic alcohols (Tsao et al., 1999 and Borden et al., 2000).

In mashing sorghum, endo-β- 1, 3-glucanase tends to effect limited attack on the

endosperm cell walls, causing some β-glucans to be released during mashing. Even in the

presence of additional quantities of endo β-l,3: 1,4 glucanase and β-amylase enzymes,

which are deficient in the malted sorghum, the malt will not give extracts comparable to

barley malt (Palmer et al., 1989; Okon, 1992). To improve the extract development of

sorghum malt, Palmer (1989) devised a decantation mashing procedure which converted

sorghum malt more efficiently. Whilst the new sorghum malt mashing procedure produced

worts which had starch extracts comparable with those of barley malt (Palmer et al., 1989),

the fermented extracts of the sorghum worts were still lower than those of barley malt.

Blanchflower and Briggs (1991) reported a significant increase in the hot water extract

values of triticales by an extended period of germination and finer milling. Okon (1988)

has also suggested the possible dependence of sugar production in sorghum malt on the

fineness of the grind.

24

MALTING

Malting entails basically steeping, germinating and limiting cereal seedling growth

after the production of enzymes required for degradation of starch and proteins in cereal

grain but before the exhaustion of polysaccharide. Also, the process is designed to produce

grain from which the maximum amount of carbohydrate can be extracted for fermentation

in brewing and production of adequate levels of enzymes. The endosperm of malted

sorghum retains starch compaction and is not as friable as the malted grain of barley

(Palmer et al., 1989). Prior to malting, a small proportion of β-amylase in cereals such as

wheat, rye, barley and sorghum is insoluble (Cook, 1962). In malting barley, the combined

actions of endo β-glucanase and proteases render the hard endosperm friable, and optimal

starch and protein extracts are released during mashing (Palmer, 1989). Conversely, the

percentage of soluble amylases in sorghum appears to be influenced by temperature and

time of storage of the grains. Storing sorghum grains for 2 to 3 years at 12 to 23° C gives

higher level of amylases (between 57 and 73%) while newly harvested grains contain about

25%. Lowering storage temperature to 7oC reduces level of soluble amylases in the grains

to about 3% after 3 years. But storing malts for any period of time seems not to affect

soluble amylase content. Nevertheless, malting yields higher proportions of hydrolytic

enzymes such as α-glycosidase, α and β-amylases, which may be either completely soluble

or largely insoluble (Jayatissa et al. 1980). For example, insoluble amylases and α-

glycosidase have been detected in malts from sweet sorghum and related variety. The

insolubility of these enzymes is apparently due to their strong adhesion to insoluble malt

solids (Dewar, 1997).

Malting causes a decrease in density of caryopsis in sorghum grain (Beta et al.,

1995), lowers the amount of lysine from 0.25% in unmalted sorghum to 0.18% in sorghum

malt (Okoh et al., 1989) and also reduces milling energy (Swanston et al., 1994). Sorghum

endosperm contains both vitreous and mealy regions with the percentage of vitreous

endosperm high correlating with grain hardness. The vitreous part of endosperm seems to

influence grain-milling energy and also malt milling energy since it is largely unmodified

during malting. Thus, there is a positive correlation between grain milling energy and malt

milling energy (Swanston et al., 1992). The loss in milling energy due to starch granule

modification during malting may be responsible for the highly significant correlation

between diastatic power and malt milling energy. However, grain-milling energy shows no

significant correlation with percentage extract in sorghum (Swanston et al., 1992). Protein

apparently plays a minor role in determining the quality of sorghum malt as high protein

25

content in sorghum malt causes no brewing problems. This could be because most of the

high molecular weight proteins are either degraded into simpler compounds during malting

and mashing, or coagulated during wort boiling and removed as protein sediment

(Novellie, 1962a). On the other hand malting quality of sorghum is determined by physical

and biochemical factors such as temperature, time of steeping and germinating of grains

with their inherent enzymic activities, kilning temperature (Novellie, 1962a; Pathirana et

al., 1983) and sorghum cultivar (Subramanian et al., 1995). Malt quality invariably

influences type and character of beer produced.

ENZYMES IN MALTED SORGHUM

-Amylase

Alpha-amylase catalyses random hydrolysis of starch chains at -1, 4 glucosidic

linkages distant from the ends of the chains and from -1, 6 linked branches in the chains

(Hough et al., 1971). During malting significant quantity of -amylase is produced in

embryos of sorghum while -amylase is activated from latent forms in starchy endosperm

(Palmer et al. 1989). Alpha-amylase in sorghum malt may be either completely soluble or

largely insoluble depending on the variety of sorghum (Jayatissa et al., 1980). The

formation of - amylase requires adequate oxygen however this can be prevented in the

presence of excess carbon dioxide. - Amylase activity in sorghum malt is 25 to 183 U/g

depending on the sorghum variety (Beta et al., 1995) and increases with sorghum diastatic

power (measured in sorghum diastatic units (SDU) in cultivars with SDU values greater

than 30 (Lasekan et al., 1995).

It was reported that steeping or germinating conditions influence the inhibition or

enhancement of the synthesis of particular isoforms detectable in cereal grains during

malting (Mundy, 1982). The inhibition of a specific dominant -amylase isotype by native

proteinaceous -amylase inhibitor in sorghum invariably depresses total amylase activity

while inactivation of the inhibitor during alkaline steeping enhances total amylase activity

(Okolo and Ezeogu, 1996a). Alternatively, enhancement of alkaline -amylase activity in

one cultivar but not in another may be attributable to the capacity of alkaline steep liquor to

influence protein-binding properties of tannins and polyphenols, which vary in

concentration and distribution in various sorghum cultivars (Chavan et al., 1981; Daiber,

1975). Tannins (located mainly in pericarp and testa) and polyphenols can bind to proteins

26

including enzymes, and are therefore likely to inactivate enzymes involved in hydrolysis of

endosperm materials (Chavan et al., 1979, 1981).

Beta Amylase

Beta amylase is an important enzyme that was first discovered over 100 years ago

(Novellie, 1962a). Current research continues to elucidate more specific information on its

mechanism of action. There are still many questions that remain about which amino acids

play a role in that function and how they do this. Beta amylase also catalyses the hydrolysis

of second to last -1, 4- glucosidic bond at non-reducing end of polysaccharides causing

the release of maltose. Non-germinated sorghum grain shows virtually no -amylase

activity (Taylor and Robbins, 1993) while -amylase may be either completely soluble or

largely insoluble in malt depending on the variety of sorghum (Jayatissa et al., 1980;

Novellie, 1962a). For example, malts made from sweet sorghum and a related variety, bird

proof kaffir corn usually contain insoluble amylases, which appeared to adsorb steadfastly

to insoluble substances, thus making aqueous extraction impossible (Novellie, 1960).

However, a report by Taylor and Robbins (1993) indicates that -amylase is not bound

since neither reducing agents nor papain treatment affects its activity. It is therefore likely

that the difference in observations reflect variation in sorghum cultivars. -Amylase

activity in sorghum malt was reported to be up to 11 to 41 SDU/g (Sorghum Diastatic

Unit/g) by Beta et al. (1995), Taylor and Robbins (1993) and constitutes 27 to 49% of total

diastatic activity in sorghum as reported by Ezeogu and Okolo, (1996).

Owuama, (1998) reported that -amylase was more labile than -amylase and could

be influenced by germination time and temperature. A rapid increase in -amylase activity

occurred within the first 2 days of germination and subsequently declined in rate of

increase for up to 6.5 days. There were wide variations regarding -amylase activity of

sorghum malt in some reports and this might be due to assumptions that -amylase activity

is the difference between total amylase and -amylase activities, an assumption which

ignores activities of other starch degrading enzymes such as -glucosidase. Nevertheless,

-amylase activity is inversely related to temperature, giving the highest activity at 24oC

over a range of 24 to 32oC (Owuama and Okafor, 1990). More maltose-producing enzyme

( -amylase) is present in sorghum malts made at 25oC and 30

oC, producing 66% more

maltose during mashing than malts made at 20oC (Agu et al., 1995). -amylase activity

also shows significant correlation with malt diastatic power and is completely inactivated

27

in 15 min at 68oC (Novellie, 1962b). However, alkaline steeping with final warm water

steep treatment and air rest result in a decrease in -amylolytic activity in cultivar ICSV

400 but an increase in both cultivars KSY 3 and SK 5912 (Okolo and Ezeogu, 1996a),

because the reduction in -amylase activity in cultivar ICSV 400 may have reflected

repression of the synthesis of a major -amylase isotypes. Novellie (1962a) had reported

that isoelectric focusing indicated that sorghum -amylase had a major and a minor

isoenzyme of approximate pI 4.4 - 4.5. -Amylase heterogeneity is influenced by malting

stage and conditions according to Laberge and Marchylo (1986) and Macgregor and

Matsuo (1982). The activity of -amylase in sorghum malt significantly increased when a

combination of final warm water and air rest cycles were employed during malting and

produced -amylase activity, which is 27-49% of total diastatic activity, depending on

sorghum variety (Ezeogu and Okolo, 1996).

-Glucosidase

Alpha glucosidase or maltase is one of the enzymes involved in starch degradation

during cereal seed germination (Sun and Henson, 1992).

-Glucosidase in germinating

grains catalyses hydrolysis of terminal, non-reducing - (1, 4) glucosidic linkages in both

oligosaccharides and -glucans yielding glucose (Manners, 1974). Other starch degrading

enzymes include -amylase, -amylase and limit dextrinase (Aisien et al., 1983; Dyer &

Novellie, 1966; Etokakpan, and Palmer, 1990; Novellie, 1962b; Okon and Uwaifo, 1985).

The activities of starch degrading enzymes result in the production of a mixture of low

molecular weight dextrins (Dunn, 1974). Although, -glucosidase in sorghum is soluble in

water, it is also active in insoluble state while adhering strongly to insoluble malt solids

(Taylor and Dewar, 1994; Watson and Novellie, 1974). Limited -glucosidase extracted

with sodium chloride under alkaline conditions is enhanced by adding papain (Watson and

Novellie, 1974). -Glucosidase development in sorghum is influenced by germination

period and temperature. Sorghum malt from 5 days germination at 30oC, shows highest -

glucosidase activity in extract with sodium phosphate pH 8 containing L-cysteine at pH

3.75 compared to those of 1 to 4 days (Agu and Palmer, 1996; Taylor and Dewar, 1994;

Watson and Novellie, 1974). The sorghum malt with the highest -glucosidase activity

however produces the lowest glucose levels in wort, suggesting that -glucosidase is not

the dominant glucose- producing enzyme during mashing of sorghum malts (Agu and

28

Palmer, 1996). Malt from germinating sorghum at 30oC show the highest levels of -

glucosidase, -amylase and -amylase as well as the highest maltose to glucose ratio,

relative to 20oC and 25

oC germinated sorghum malt, while the role of each enzyme in the

sugar ratios is unknown (Agu and Palmer, 1996). Mashing at pH 4, near optimum for -

glucosidase yields relatively higher proportion of glucose than at usual mash pH 5-5.5,

which is optimal for -amylase (Botes et al., 1967; Taylor and Dewar, 1994). However, at

pH 5-5.5, both total fermentable sugars and free glucose increase with mashing temperature

to a maximum at 70oC but the proportion of glucose declines with increasing mashing

temperature from 58.6% at 600C to 23.1% at 80

oC. In contrast, mashing at pH 4 produces

fewer amounts of total fermentable sugars and free glucose at 70oC than at 60

oC (Taylor

and Dewar, 1994). Higher amount of glucose is observed in wort from EBC conventionally

mashed malt as against using pre-cooked malt insoluble solids where -glucosidase

inactivation occurs, preventing hydrolysis of maltose to glucose and resulting in high

maltose levels in sorghum worts (Taylor and Dewar, 1994).

Lipases

Lipases (triacylglycerol acylhydrolase) catalyses the hydrolysis of triacylglycerides

to free fatty acids and glycerol (Lin, et al., 1983). Malt lipoxidase catalyses peroxidative

reaction that converts free fatty acids to hydroperoxides and aldehydes, which have

detrimental effects on beer such as poor acceptability and reduced shelf-life (Kobayashi et

al; 1993). A higher level of fatty acid is present in sorghum relative to barley, wheat and

millet (Nwanguma et al., 1995). Sorghum grains contain detectable lipase activity which

varies slightly during 24 h steeping period at 30oC and increases during germination to

about 4-fold after 96 h. However lipase activity varies among different sorghum cultivars

(Nwanguma et al., 1995), thus suggesting variations in lipase synthesis or differences in

endogenous regulators of lipase activity (Chapman, 1987). The lipase activity in plumule,

endosperm and radicle are 68%, 29% and 3% respectively. The optimal pH from sorghum

lipase is 7 although the activity range is between pH 5.5-9. The percentage lipase activity

at pH 5.5, 6, 8 and 9 relative to that at pH 7 are 50%, 95%, 88% and 60% respectively

(Nwanguma et al., 1995). Lipase activity decreases in sorghum malt after kilning at 48

oC

for 24h to between 24% and 66% of total lipase activity in green malt. Nwanguma et al.

(1995) also reported that exposing malt water extract for 10 min to temperatures of 50oC

reduce lipase activity to 57%, 43% and 14% respectively, of the original activity, and total

29

loss of lipase activity resulted from heating extract from 30 min at 50oC. Moreover,

mashing at 65oC results in wort with no lipase activity, indicating that lipase activity is

limited to the malting stage.

Peroxidases

Peroxidase catalyses the reductive destruction of hydrogen peroxide and invariably

contributes to the defence system of living organisms against peroxidation of unsaturated

lipids involving oxygen radicals (Floyd, 1990). Lipid peroxidation is undesirable in

malting and brewing and result in the production of hydroperoxides and their

decomposition products- aldehydes (St. Angelo and Ory, 1975) which affect the

availability of wort nutrients and subsequently possibly interfere with yeast metabolism and

influence the flavour and colloidal stability of the beer (Bamforth et al., 1993; Kobayashi et

al., 1993).

Peroxidase activity increased by about 14-fold during the germination of

sorghum grains steeped at 30oC for 24h. However the levels present showed differences

with different sorghum varieties (Nwanguma, and Eze, 1995). Peroxidase activity of

36.49% is detectable in endosperm while a combined activity of 56-61% occurs in the

acrospire and rootlet. The optimal pH for sorghum peroxidase is 5.5 and kilning at 48oC

for 24h shows no depressing effect on the peroxidase activity (Nwanguma, and Eze, 1995).

In crude extract, sorghum peroxidase activity decreases from 77% to 7.5% after 15 min

exposure to temperatures of 0oC to 80

oC, respectively.

Nevertheless, peroxidase activity declines to 5% in 5 min at 85oC and is completely absent

at higher temperatures. Sorghum peroxidase survives better in wort than crude extract and

about 50% of peroxidase activity is retained in wort after mashing from 1 h at 65oC

(Nwanguma, and Eze, 1995). Since remarkable amounts of lipid oxidation products form

during mashing (Meersche et al., 1983), it is therefore important that sorghum peroxidase

remained active in wort to remove oxygen radicals at the later stages of brewing.

Proteinases (endopeptidases) and Carboxypeptidases

Carboxypeptidases and proteinases are important in protein mobilization during

grain germination. Peptidase formation requires adequate oxygen but is prevented in the

presence of excess carbon dioxide (Weith and Klaushofer, 1963). Carboxypeptidases

specifically hydrolyse solubilised proteins to free alpha-amino nitrogen (FAN) essential for

anabolic functions of germinating seedling and as nutrients for yeast metabolism in wort

(Dale et al., 1990). Germination conditions and sorghum cultivar influences

30

carboxypeptidase activity. For example, carboxypeptidase activity increases with

germination time up to 4 days showing 4 times the activity in resting grains (Evans and

Taylor, 1990).

Also moisture, temperature and germination time significantly affect

carboxypeptidase activity with the highest activity occurring in malt from 4 days

germination under medium moisture at 24oC, and yielding maximum FAN value of 275 g

/5h/g dry malt (Evans and Taylor, 1990; Morrall et al., 1986). Sorghum malts resulting

from different final warm steep treatment periods show poor correlation between the length

of final warm steep treatment and carboxypeptidase activity, whose levels vary with

sorghum cultivars. Also, correlation between sorghum malt FAN and carboxypeptidase

activity can be poor or strong depending on cultivar (Okolo and Ezeogu, 1996b).

Proteolytic enzyme activity in sorghum is influenced by both cultivar and malting

conditions but steeping does not significantly affect proteinase or carboxypeptidase

activity. However, different sorghum cultivars grown and malted under similar conditions

differ significantly in proteinase and carboxypeptidase activity (Evans and Taylor, 1990).

Germination temperature (24-32oC) and moisture have little or no effect on proteinase

activity (Evans and Taylor, 1990). Germinating sorghum for 36 h or 48 h causes a

considerable increase in protease activity in embryo or endopeptidases activity in both

embryo and endosperm (Morrall et al., 1986). Increase in germination time up to 4 days

moderately increases proteinase with a maximal yield of 1604 gN/5h/g dry malt. The

highest proteinase activity differs with sorghum malts resulting from different final warm

steep period and also with various cultivars (Okolo and Ezeogu, 1996b).

Proteinase activity in cultivar ICSV 400 rises from 1224 to 1469 gN/3h/g dry malt

as final warm steep period increases from 1.5 to 3.0 h. However, proteinase activity

declines with increase in final warm steep period beyond 3.0 h suggesting an optimum final

warm steep period similar to that for carboxypeptidase activity. Nevertheless, sorghum

cultivar, KSV 8 attains highest proteinase and carboxypeptidase activities at 6 h final warm

steep period (Okolo and Ezeogu, 1996b). Optimal proteinase and carboxypeptidase

activities occur after 3 h final warm water steep period in cultivar ICSV 400 but after 6 h

final warm water steep in cultivar kSV8 (Okolo and Ezeogu, 1996b). However, higher

proteinase activity occurs in cultivar KSV 8 in relation to cultivar ICSV 400, although with

lower CWS-protein in KSV 8. This apparent contradiction can be attributed to qualitative

differences in complexity and structure of endosperm proteins of various sorghum cultivars

and/or differences in the nature of the major proteinase isoforms in grains (Okolo and

31

Ezeogu, 1996b). Apparently, the highest proteinase and carboxypeptidase activities occur

in the same final warm water treatment period for given sorghum cultivars (Okolo and

Ezeogu, 1996b). Varying sorghum cultivars and air rest periods from 1 to 4 h during

steeping with 6 h final warm water (40oC) steep, greatly influence CWS-protein, total cold

water soluble, cold water soluble protein modification index, total free alpha amino acid

nitrogen, and carboxypeptidase and proteinase activities of malt (Okolo and Ezeogu,

1995b).

Steeping

Steeping involves soaking of grains in water until the attainment of an acceptable

moisture level. During steeping certain physical and biochemical changes occur, such as,

swelling of grains, degradation of soluble carbohydrates and removal of some pigments,

micro-organisms and bitter substances from grain. Steeping is essentially regulated to

achieve a suitable moisture level and avoid oversteeping or reaching a saturation point,

which usually results in killing of seed germ (Owuama and Asbeno, 1994). Suitable steep

moisture varies with sorghum grain variety, steeping time and temperature (Owuama and

Okafor, 1987) and steep moisture of grain directly affects sorghum malt quality (Dewar et.

al., 1997). Steeping sorghum grains at temperatures of 10 to 30oC causes as increase in

steep moisture with apparently no appreciably effect on diastatic power of malts (Novellie,

1962b). Nevertheless, steep treatment of sorghum grains influences amylases development.

Steep moisture affects extract, reducing sugar, diastatic power of malt and level of amino

acids in wort. Steeping sorghum at 30oC for 18 to 22 h results in steep moisture of 44-48%

which is optimal for enzymic activity (Owuama and Asbeno, 1994) while steep moisture of

35-40% seems to encourage rapid germination at a temperature of 22oC, in the dark (Aisien

and Ghosh, 1978). Apparently, increase in steep moisture with steeping time from 12 to 20

h at 30oC is directly proportional to diastatic power of malt and consequently an increase in

reducing sugar, cold and hot water extracts (Owuama and Asbeno, 1994). However, steep

moisture level beyond the optimum, leads to a decrease in extract (Pathirana et al., 1983)

and diastatic power of malt (Novellie, 1962b). Steeping methods, with or without change of

water, have virtually no effect on sorghum malt (Novellie, 1962b). Steeping sorghum with

increasing air rest periods of 1 to 4 h at 30oC for 48 h to attain steep moisture of 40-42%,

germinating for 4 days and kilning at 50oC result in (a) a decrease in average main rootlet

length (b) decrease in malting loss from 14.1-18.1% to 9.5-3.6% and (c) an increase in malt

32

diastatic power (including -and -amylases) up to 3 h followed by a decrease after 4 h.

However, variations occur among sorghum cultivars for example, the optima for -and -

amylase activities in cultivar KSV 400 occur at air rest periods of 3 h and 1 h, respectively

but at 2 h and 3 h air rest periods for cultivar KSV 8 (Ezeogu and Okolo, 1996).

-Amylase activity constitutes 36-50% of total diastatic activity in cultivar KSV

400 but 27-49% of total diastatic activity in cultivar KSV 400 but 27-49% in cultivar KSV

8 while cold and hot water extracts gave highest values for KSV 400 and KSV 8 after air

rest of 3 and 4 h, respectively (Ezeogu and Okolo, 1996). Increase in steeping time plus

aeration and steep water temperature enhance diastatic power. Steeping grains plus aeration

at 30oC for 40 h yield maximum diastatic power of 42.6 SDU/g. Steeping at 25

oC for 40 h

under air rest condition produce maximum malt FAN (119.8 mg/100g) while 24 h steeping

with aeration yield highest malt extract (62.5%) (Dewar et al., 1997). Varying the duration

of final warm water steep at 40oC between 1.5 h and 7.5 h and germinating for 4 days at

30oC cause (a) malting loss and decrease in average warm water steep and (b) increase in

diastatic activity, - and -amylolytic activities, and extract yield as the final warm water

steep period increases up to 3 h and thereafter declines. However, these observations vary

with sorghum cultivars (Okolo and Ezeogu, 1995a). The highest -amylolytic activity

occurs at relatively shorter duration of final warm water steep for example, 3 h for KSV 8

and 1.5 h for KSV 400 while peak -amylases activity result after 3 h and 7.5 h final warm

water periods for KSV 400 and KSV 8 respectively. However, diastatic activity for KSV 8

attains another peak, albeit a smaller one, after 7.5 h of final warm water steep, thus

suggesting the involvement of at least another -amylase component. A marked reduction

in average main root length of 53% and 25% occur after 1.5 h and 3 h final warm water

steep for KSV 400 and KSV 8 respectively (Okolo and Ezeogu, 1995a).

Steeping sorghum in 0.1N ammonia solution up to 18h increasingly reduced

enzyme development, cold and hot water extracts, and malting losses (by suppressing the

growth), but does not prevent mouldiness (Ilori and Adewusi, 1991). Steeping sorghum

continuously in alkaline liquor (0.1% Na0H in water) and germinating for 4 days at 30oC

cause repression in germinability (3-34%), root length and malting loss. However, steeping

sorghum cultivar SK 5912 continuously in alkaline liquor plus a final warm water steep

enhance malt diastatic activity (50-250%) and -and -amylase activity. -Amylase

activity constitutes over 70% of the total diastatic activity in alkaline steeped cultivar KSV

400 malts (Okolo and Ezeogu, 1996a). In contrast, alkaline steeping of ICSV 400 with air

33

rest and final warm water treatment repress diastatic activity by 9% although similar

treatment significantly enhance diastatic power and -amylase development in cultivars

KSV 8 and SK 5912 (Okolo and Ezeogu, 1995b). Nevertheless, cultivar SK 5912

produced relatively low HWE although it had improved amylolytic activity (Okolo and

Ezeogu, 1996a).

Germination

Germination involves outgrowth of plumule and radicle of seeding until the

production of adequate enzyme for the malt but prior to the exhaustion of seed nutrients.

During seed germination, storage proteins within endosperm are hydrolyzed by enzymes to

provide nitrogenous compounds for grain outgrowth (Payne and Walker–Smith, 1987).

Small peptides and products of partial protein hydrolysis in endosperm are translocated

across scutellum to embryo where peptides are degraded by peptidases to release amino

acids for plant structure and enzymes synthesis (Aisien and Ghosh, 1978). Germinating

sorghum grains at optimal temperatures of 25 to 30oC for 3 to 7 days, depending on the

grain variety, leads to rapid growth of radicle, a reduction in adequate germination period

and the production of well modified malts, where horny grain endosperm has changed to

powdery, chalky state, with high diastatic power (Pathirana et al., 1983) hot water extract,

sugar contents (Lasekan et al., 1995) and free amino nitrogen (Morrall et al., 1986). The

optimum germination period varies with sorghum grain varieties and germination

conditions such as, illumination and steep moisture. Three days of germination of sorghum

grains steeped in the dark for 18 h, produced malts with higher diastatic power than those

steeped from 32 h (Pathirana et al., 1983). Increasing germination period from 2 to 6 d at

30oC results in an increase in diastatic power, reducing sugar, cold water and hot water

(Lasekan et al., 1995), as well as protein content of sorghum malt (Okoh et al., 1989). In

contrast, germinating sorghum at a relatively higher temperature of 35oC or lower

temperatures of between 15 and 20oC slows down amylase formation and invariably reduce

diastatic power (Morrall et al., 1986).

Diastatic power, which largely measures the combined activity of - and -

amylases, is of greater importance in sorghum malt than extract (Novellie, 1962b) and

seems to be directly proportional to its reducing sugar content (Lasekan et al., 1995).

Generally diastatic power, free -amino nitrogen extract and malting loss increase with

germination time (Morrall et al., 1986). High moisture level in the early stages, within 8

days of germination, usually results in a high diastatic power and consequently early

34

enzymic hydrolysis and transfer of solubilised products to embryo. The diastatic power

subsequently slows down but may in some cases, increase slowly to the end of the

germination periods (Novellie, 1962b). Diastatic activity of malts range from 32.3 to 150.9

SDU/g (Subramanian et al., 1995) and over 50% of -glucan is digested by enzymes after 2

days of germination (Ogbonna and Egunwu 1994). However, diastatic power of 60 to 80

SDU/g is recommended for sorghum grain to be considered for commercial malting

(Novellie, 1962b).

Germination of sorghum grains steeped with air rest at 25-26oC for 6 days, produce

malt whose percentage extract has highly significant correlation with the diastatic power

(Swanston, 1992). Germination temperatures of 24 and 28oC are both equally good for the

development of diastatic power, FAN and extract but higher temperature are progressively

worse. Germination of sorghum grains for 6 days under high (77%), medium (60.9%) and

low (42.8%) moisture conditions affect the diastatic power, FAN, extract and malting loss

and moisture content of green malt (Morrall et al., 1986). Morrall et al., (1986) also

reported a maximum diastatic power of 46.6 SDU/g occuring within 5 days of germination

at 24oC under medium moisture and maximum FAN of 180mg FAN/100g malt produced

under high moisture after 6 days germination at 32oC. Treatment of sorghum with thiram

(0.2%) plus carbendazim (0.1%) improves seed germination by 8 to 40% and reduces seed

mycoflora (Ingle et al., 1994). Sorghum grains heavily infected with mould produce malts

with slightly higher amylase activity (Kumar et al., 1992), thus suggesting that fungi

contribute towards the increase in amylase activity. Seed mycoflora of sorghum species

include Aspergillus flavus, Curvularia, sp, Cladosporium cladosporoides, Fusarium

moniliforme, Rhizopus sp, Alternaria sp, Penicillium sp., Dreschlera sp, and Neurospora

sp. (Kumar et al., 1992)

Kilning

In kilning, green malt is dried in a kiln or oven at a relatively high temperature until

the rootlets become friable or brittle. Kilning contributes to colour development which is

influenced by the extent of modification, duration and levels of temperature-time sequence

of kilning cycle and moisture content of green malt at different stages of the cycle

(Pathirana et al., 1983) Sorghum malts are kilned at elevated temperature of 45 to

100oC(Pathirana et al., 1983), essential for removing raw flavour of green malt and

promoting chemical reactions for the formation of components which impart characteristic

35

flavour to malt (Briggs et al., 1981). Commercially produced sorghum malts for brewing

are usually dried at moderate temperatures up to 50oC (Novellie, 1962b). Varying kilning

process produces malts of differing characteristics. Kilning malts in two stages, exposing

green malt initially to 55oC and subsequently to 65

oC, produce malts with higher sugar

content than kilning at a single temperature of 65oC (Owuama and Asbeno, 1994). In two-

stage treatment, initial exposure to 55oC for some time, considerably reduces moisture

content of green malt before final temperature (65oC) treatment (Owuama and Asbeno,

1994), a process which apparently encourages greater survival of hydrolytic enzymes while

malt acquires characteristic flavour. Higher kilning temperature causes a relatively smaller

decrease in reducing sugar and diastatic power of malts than on hot water extract and

liquefying power. This is apparently due to inactivation of saccharifying amylase, -

amylase to a greater extent than liquefying amylase, -amylase (Pathirana et al., 1983).

During kilning, reducing sugars decrease in quantity while sucrose level often increases

(Owuama and Asbeno, 1994) possibly because of a reversal in the action of hydrolytic

enzymes (Briggs et al., 1981) that appears not to have a direct relationship with amylase

content in sorghum malt (Owuama and Asbeno, 1994), suggesting the involvement of other

enzymes, with varying contributions in different sorghum varieties Owuama and Asbeno,

1994)

Malting Loss

Malting loss is the summation of leaching/steeping, metabolic/respiration and

vegetative/sprout losses (Malleshi and Desikachar, 1986). Basically, it is the loss in weight

of grains after malting. However, malting loss in commercial kaffircorn malts are only due

to metabolic and leaching losses, since roots and shoots are not usually removed but milled

in with the berry (Novellie, 1962b). Factors which influence malting losses include

germination period, germination temperature, steep moisture, kilning temperature and

sorghum variety. Malting losses generally vary with temperature and increase with

germination period. Malting loss is higher at 25oC (8.4%) and 30

oC (10.9%) than at 20

oC

(6.5%) (Agu and Palmer, 1996) and malts produced at 30oC over 1 to 6 days show losses of

3 to 34% depending on sorghum variety (Beta et al., 1995). Germination temperatures of

25 to 30oC are optimal for amylase and diastatic power development in sorghum malt, and

encourage vigorous respiration and high malting losses (Novellie, 1962b). High steep out

moisture of grains and watering during malting, enhance the rate of germination and

36

malting loss while reducing malting loss by lowering temperature or moisture level causes

a marked decrease in diastatic power (Novellie, 1962b). Thus, the attainment of a good

diastatic power in sorghum malt may be linked to high malting loss. A

respiration/metabolic loss of 10 to 15% is expected in a well-malted sorghum with good

diastatic power (Novellie, 1962b).

Sorghum Malt Proteins

Amorphous storage proteins associate with starch granules within endosperm of

barley and sorghum, and during grain germination, malt proteolytic enzymes initiate the

modification of grain reserve in endosperm by hydrolyzing proteins associated with starch

granules, thereby exposing the starch and increasing its susceptibility to amylolysis

(Palmer, et al., 1989). The hydrolysis of insoluble reserve protein in germinating grain

provides amino acids necessary for the synthesis of hydrolytic enzymes and grain structural

materials in growing tissues of seedlings (Mikola and Kobelmainen, 1972). Nevertheless,

malts show lower protein than unmalted grains and malts from sorghum cultivars with

diastatic activity exhibit high levels of an albumin-globulin fraction (Subramanian et al.,

1995). Sorghum malts obtained by steeping grains for 22 h followed with 4 h air rest and

further 24 h wet steep at 20oC (giving steep moisture of 34-35%) and subsequently

germinated for 5 days at 20oC and 30

oC showed higher quantities of nitrogen from

endosperm to embryos than malting at 20oC and 25

oC, but less amino acids and peptides

are transferred to root during malting at 30oC than at 20

oC. Nitrogen may also move from

root to embryo by physiological mechanisms (Agu and Palmer, 1996). Steeping regime and

sorghum cultivar significantly influence FAN values. Generally, exposing sorghum grains

to a steep regime incorporating air rest cycles and final warm water steep result in the

highest FAN level in ICSV 400 and KSV 8 varieties while continuous steep regime without

final warm water steep produce the lowest FAN values.

Cultivar and duration of final warm water (40oC) steep highly influence protein

modification indices viz., soluble protein of cold water extract (CWS-protein), total non-

protein nitrogen (TNPN), a small peptide accumulation, free alpha amino nitrogen,

carboxypeptidase and proteinase activities (Okolo and Ezeogu, 1996b). The application of

final warm water steep without air rest stimulates FAN development in cultivars ICSV 400

and KSV 8 but significantly represses FAN development in SK 5912. Nevertheless,

significant improvement of FAN values occur in all sorghum varieties after all application

of air rest cycles during steeping although the FAN levels vary with cultivar (Ezeogu and

37

Okolo, 1995). Apparently these differences reflect variations in grain protein structure and

degradability (Riggs et al., 1983), amino acid transport processes (Mikola and

Kobelmainen, 1972), and probably differences in enzyme characteristics. ICSV 400 shows

higher FAN, CWS-protein solubilizing activity and accumulation, and better protein

modification potential than KSV 8. However, lower TNPN and TNPN-FAN differences in

ICSV 400 contrasts with its high FAN, thus suggesting superior anabolic protein turnover

apparently from efficient peptide translocation process. Nevertheless, the levels of

nitrogenous substances are inconsistent with the proteolytic activities suggesting the

involvement of factors other than proteolysis in protein modification (Okolo and Ezeogu,

1996b). Remarkably, KSV 8 records lower FAN although it generally expresses high

carboxypeptidase activity in relation to ICSV 400. This suggests a variation in the rate of

protein synthesis from FAN and thus a possible higher rate of anabolic protein turnover in

KSV 8 and lower FAN accumulation (Okolo and Ezeogu, 1996b). Four days of

germination of sorghum cultivars steeped in alkaline liquor (0.1% NaOH solution) for 48 h

at 30oC under different steeping regimes, reveal that steep regime, steep liquor and

sorghum cultivar highly and significantly influences the protein modification index, TNPN,

peptide accumulation, FAN, endo- and exo-protease activities. Alkaline steeping causes a

highly significant increase in sorghum malt FAN (Okolo and Ezeogu, 1996b). FAN in

malt is a net balance of amino acids and peptides resulting from storage protein degradation

and those utilized for synthesizing new proteins in roots and shoots of growing plant

(Taylor and Boyd, 1986). FAN developments vary among cultivars probably because of

differences in major enzyme characteristics and rate of protein variations in grain protein

structure and degradability (Riggs et al., 1983) amino acid and peptide transport processes

(Mikola and Kobelmainen, 1972). Nevertheless other miscellaneous cultivar-dependent

factors also play a role in the control and modulation of protein degradation and synthesis

in germinating plant seeds (Shutov and Vaintroaub, 1987). Free alpha amino nitrogen

development in malt is important in brewing as it constitutes about 70% of total FAN in

wort (Taylor and Boyd, 1986).

In general sorghum malts from grains steeped with air rest period and steepout

moisture of 33-35% show increases in diastatic power, FAN, extract and malting loss with

germination time. Germination temperature of 24 and 28oC are equally good for the

development of diastatic power, FAN and extract. Diastatic power, FAN, and extract and

malting loss increase with high moisture during germination (Morrall et al., 1986).

Germination at 32oC under high moisture shows similar FAN level in malt at 3.0-4.5 days,

38

before increasing further to a maximum of 180mg FAN/100 g malt after 6 days (Morrall et

al., 1986).

Water Extracts

Cold-water extracts (CWE) are soluble products of enzyme hydrolysis from malting

process and these include readily available sugars and amino acids within endosperm of

malts (Holmes, 1991). CWE and hot water extracts (HWE) are influenced by cultivar,

steeping conditions and steep liquor. CWE is generally enhanced in certain cultivars by

alkaline steep with final warm water steep but depressed in others apparently due to

alkaline steep repression of certain malt properties like diastatic power and -amylase

activity (Okolo and Ezeogu, 1996b). A combination of air resting and final warm water

steep at 40oC reduces kernel growth and malting loss but significantly improves CWE,

HWE, diastatic power, -and -amylase activities. But final warm water steep without air

resting causes a decrease in extract recovery and enzyme activity (Ezeogu and Okolo,

1991). Steeping sorghum grains in alkaline liquor generally enhances HWE of malts in

cultivar ICSV 400 but reduce HWE in cultivar SK 5912 albeit with an increase in - and -

amylolytic activities. This suggests possible effect of other native enzymes contributing to

endosperm cell wall structure solubilisation such as exo-and endo-proteases and -

glucanase, and consequent prevention of amylase access to starch granules for efficient

conversion (Okolo and Ezeogu, 1996b). Generally, malting increases water extract (WE),

water extractable protein (WEP), HWE, and hot water extractable protein (HWEP) of

sorghum grains by 3.0-, 3.4-, 2.3-and 2.0-fold respectively (Subramanian et al., 1995).

Diastatic activity correlates significantly and positively with WEP and water-extractable

contents of malt produced at 30oC. Percentage WEP as a proportion of total protein varies

between 11.0 and 36.0% and HWEP range from 19.3 to 44.1% (Subramanian et al., 1995).

CWS-protein in grains steeped with aeration at 30oC and final warm water steep at 40

oC for

6 h is significantly higher than those steeped without air cycle. This may be due to an

increase in protein solubilisation in response to improved enzyme synthesis or better

hydration of endosperm and enzymes mobility (Ezeogu and Okolo, 1995). The CWS-

protein yield varies with sorghum cultivar in both protein solubilisation activity and CWS-

protein accommodation. For example, CWS-protein value from cultivar SK 5912 (1680

mg% dry malt) is significantly higher than those for ICSV 400 (1030mg% dry malt) and

KSV 8 (1280 mg% dry malt), (Ezeogu and Okolo, 1995).

39

Mashing

Mashing in conventional brewing is basically by two methods, viz, decoction and

infusion processes (Hough et al., 1971). During mashing water soluble substances

dissolve, enzymes hydrolyse gelatinized starch and solubilised proteins and to a lesser

extent other high molecular weight substances essential for the type and character of beer,

and finally dissolved substances are separated. Hydrolyses of substances involve enzymes

such as amylases, proteases, peptidases, transglucosidases and phosphorylases which are

regulated by factors like temperature pH, time and concentration of the wort (Manners,

1974). Mashing extracts about 80% of the dry matter from the malt while cold water

extracts about 15% (Wainwright, 1971).

Mashing sorghum malt by decoction process and infusion methods are influenced

by temperature-time regimes, sorghum variety and produce worts of varying composition

(Okafor and Aniche, 1980). In three-stage decoction, about 70% of mash is boiled to

gelatinize starch for greater amylolytic activity while creating plenty of opportunity for

proteolytic enzyme action and minimizing scope for the development of lactic acid bacteria

(Skinner, 1976). Mashing of sorghum malt at 65oC and 70

oC for 30 min each, at second

and third stages respectively, of three stage-decoction process, provides wort with complete

hydrolysis (Okafor and Aniche, 1980). A longer incubation time at saccharifying

temperature (65oC) than dextrinising temperature (70

oC) gives wort higher in reducing

sugar levels (Ownama and Okafor, 1987). Reducing sugars and proteins in wort increase as

concentration of sorghum malt rises from 15 to 25% (Owuama and Okafor, 1987),

apparently because of a simple increase in mash concentration and stability to enzymes

(Hoyrup, 1964). Infusion mashing at 65oC releases higher levels of peptides but lower

quantities of -amino nitrogen and total soluble nitrogen than decantation mash in which

decanted enzymically active wort is used to mash gelatinished sorghum starch at

65oC(Manners, 1974). Mashing sorghum malt by the European Brewing Convention (EBC)

congress procedure (European Brewing Convention, 1997), which involves hydrolysis of

pre-cooked malt insoluble solids using an enzymic malt extract, yield wort with

approximately equal amounts of maltose and glucose (Taylor and Dewar, 1994).

Nevertheless, both treatments give the same quantity of total fermentable sugars and wort

extract. Infusion mashing of 13.8 dry weight of total cereal content, (composed of 21%

sorghum malt (diastatic power ca 38 SDU/g) with cooked adjunct of 70% maize grit and

8% sorghum malt), at 60oC for 2 h at pH 4 in 200 ppm calcium ions result in almost

40

complete conservation of diastatic activity, increase in extract, maximum yield of reducing

sugar in wort, and the detection of -amylase activity which appears to be lacking in the

absence of calcium ion (Taylor and Daiber,1998). A relatively high level of starch extracts

and low level of fermentable extracts have been obtained by using a non-conventional

mashing procedure, decanting active enzyme wort after mashing sorghum malt at 45oC for

30 min, and gelatinizing starchy grist residue at 80-199oC before mixing with wort, to

achieve a saccharifying temperature of 65oC (Palmer, 1989). Palmer (1989) attributed the

result to smaller quantities of -amylase in the wort.

Wort filtration volume is produced in small quantity in mashes containing raw

sorghum than in all malt mashes. Adding external enzyme during mashing of sorghum

malt increases extract yields and free amino nitrogen in wort (Agu et al., 1995).

Introducing industrial enzyme preparations containing -amylase and -glucanase to

mashes with raw sorghum yield higher values of extract recovery in relation to untreated

mashes. Moreover, adding enzyme preparations containing neutral proteinase increases

wort total nitrogen and free amino nitrogen while enzyme preparations with -glucanase or

cellulase decrease wort viscosity relative to untreated mashes (Dale et al., 1990). Also a

20% (w/v) sweet potato flour substitution for sorghum malt increases maltose level in wort,

apparently because of the presence of -glucanase (limiting in sorghum) in sweet potato

(Etim and Etokakpan, 1992). Mashes composed of 50% malt and 50% raw sorghum and

supplemented with enzyme preparations show an increase in wort filtration volume relative

to similar mashes without enzyme supplements (Dale et al., 1990). Wort produced by

double mashing regime from 20% malt and 80% raw sorghum supplemented with

industrial enzyme show filtration and result in sweet and turbid wort. Apparently, this

reflects low malt content of grist and lack of suitable material to form mash filter bed (Dale

et al., 1990).

41

MATERIALS AND METHODS

Source of Cassava Raw Starch

Cassava chaff was obtained from a garri processing plant in Nsukka locality, sun-

dried, ground into a fine powder and stored in a cool dry place.

Sorghum Variety and Source

Local red sorghum grains were obtained from Ogige market, Nsukka.

Sorting

This involved the manual separation of mature healthy sorghum grains from

unhealthy ones and other unwanted materials such as stones, husks, insects and broken

kernel.

Decortication

Fifty percent sulphuric acid was used for the purposes of curticle removal. The

diluted sulphuric acid was allowed to cool and kept in the refrigerator afterwards for further

cooling. About 1- 1.5kg of sorted sorghum grains were introduced into the cold 50%

sulphuric acid, stirred every 5 minutes. After 30 minutes the grains were thoroughly rinsed

in tap water and finally rinsed with distilled water to achieve a pH of 7. Decorticated

(treated) samples were dried at 50°C for 24 hours and stored in a clean airtight container.

Starter Cultures and Inoculum Preparation

The enzyme-producing strains of Aspergillus awamori and A. carbonarius IMI

366159 were used. The fungi were grown on Potato Dextrose Agar (PDA) for 7 days at 28-

30oC. For inoculum preparation, spores of these cultures were harvested by flooding with

sterile physiological saline [0.9 % (w/v) sodium chloride] containing 0.05% Tween 80

followed by gently rubbing with a sterile spatula. A spore count of the suspension was then

determined microscopically using a Thoma Haemacytometer and or spectrophotometer.

Suspended spores (5 × 107/ml) were transferred to 20 ml sterile, acidified (pH 4.0)

Tryptone Soya Broth (TSB) and incubated with gentle shaking for 5-6 h at 42oC. These

spores were harvested by centrifugation (1600g, 15 min), washed and suspended in normal

saline and used for inoculation on decorticated and non decorticated sorghum grains.

42

Malting

Malting was carried out for both treated (decorticated) and untreated

(undecorticated) sorghum samples as follows:

Surface Sterilization

Surface sterilization was carried out for only untreated samples. Following the

manual sorting of the sorghum grains, 300g of the healthy grains were differently surface

sterilized by soaking and stirring regularly in sodium hypochlorate (NaOCl) solution

(commercial bleach), containing 1% available chlorine for 20 minutes. Subsequently the

grains in various 3L transparent plastic bowls, were drained and rinsed three times as

described by Morrall and co-workers (1986) with distilled water

Steeping

The same conditions of steeping were carried out for both treated and untreated

sorghum samples. Following surface sterilization (of untreated or undecorticated sorghum

samples), 600 ml, distilled water was dispensed into each of the plastic bowls, containing

the 300g surface sterilized undecorticated sorghum grains. The same treatment was carried

out for treated or decorticated sorghum samples.

Steep schedules for decorticated and undecorticated sorghum malting

Six hour wet period x 4; 3h air rest period x 4; 1h-wet period (inoculation): the

distilled water was left in the bowls with the grains for 6 hours, called the wet period. At

the end of this 6h-wet period the steeping liquid was decanted. The sorghum grains were

spread on a clean surface thereafter and left for 3 hours. This is the air rest period. The wet

period was repeated followed by air rest regime for a total of 37 hours as shown below.

Inoculation: One-week-old culture of the respective Aspergillus species (Aspergillus

awamori and Aspergillus carbonarius) was used for inoculation. Sterile normal saline (100

ml) mixed with 2 drops of Tween 80 before sterilization by autoclaving at 121 C for 15

min was used to wash out the spores of the Aspergilli from Roux culture bottle into a sterile

Erlenmeyer flask.

43

The Optical density (OD) of the Aspergillus spores suspension in the sterile flask were

adjusted to 1.0, 0.8, 0.6, 0.4, and 0.2 at 600nm. The respective fungal counts of the various

spore concentrations were also determined as follows:

1.0 -- 4.55 x 106 cfu/ml

0.8 -- 3.64 x 106 cfu/ml

0.6 -- 2.73 x 106 cfu/ml

0.4 -- 1.82 x 106 cfu/ml

0.2 -- 9.10 x 105 cfu/ml

Germination (Solid Substrate Fermentation)

Wooden or plastic boxes that have an open top and covered below with plastic

mesh having tiny holes not to allow the leakage of the grains spread inside it, were

provided for sorghum germination. This box was also surface sterilized using sodium

hypochlorate. The germination was carried out in a clean enclosure. Distilled water was

sprayed over the germinating grains while turning the grains for even distribution of water

on the grains 12 hourly for 5 days and humidified by keeping bowl of water in the

enclosure.

Sample Collection and Drying (Kilning)

Approximately 40g of the germinating sorghum grains were withdrawn every 24

hours from each box. They were then transferred into the dryer and allowed to stay for 24 h

at 50 C. One gram was thereafter removed from each of the dried samples and preserved

for analysis of the extent of fungal growth. The remaining malt samples were stripped of

their roots and shoots by rubbing them against a plastic net or sieve, gently ground in an

enamel mortar, transferred to a homogenizer for further blending and finally stored in clean

dry plastic film cups with air-tight lid after proper labeling. Ground malted sorghum

samples were placed in the refrigerator at 4 C till they were needed for the various malt

analyses.

44

ANALYSES

Germination energy (GE) and Water sensitivity (WS)

The IOB (1982) method was used for GE and WS determination. A filter paper

(No. 1, 9 cm diameter) was placed at the bottom of a 9 cm diameter Petri dish and 4 ml of

distilled water to evenly wet the filter paper. Exactly 100 sorghum grains from each of the

varieties were subsequently placed on the paper so that each had good contact with the

moist filter paper; the lid of the Petri dish was put in place and the grains allowed to

germinate. Periodically, at 24 h intervals for 72 h chitted grains were removed and counted.

GE was determined as the % of the grains, which had germinated, by the end of 72 h of

steeping.

To assay for WS the procedure was same as that of GE except that 8 mL of distilled

water was used instead of 4 mL. WS was calculated as the difference between the number

of grains that had germinated after 72 h in the 4 mL and 8 mL tests, respectively.

Root lengths (RL) and malting loss (ML)

At the end of germination, 20 kernels each of the germinated sorghum varieties

were randomly selected and their root length measured using the method described by Ilori

and Adewusi (1991). Malting loss was determined using published methods of the Institute

of Brewing (IOB, 1982). Sorghum grains (50) were counted and weighed before and after

malting. The loss in weight was obtained as the difference between the unmalted grains and

malted grains (i.e. after the removal of roots and shoots). ML was then calculated as

follows:

% ML = W1-W2 x 100%

W1

Where W1 = weight of unmalted sorghum

W2 = weight of malted sorghum.

Moisture content

The methods of the Association of Official and Analytical Chemists (AOC,1980)

were used. Samples of ground malts (2g) were weighed into preweighed moisture glass

dishes, and kept in an oven adjusted to 105 C for 6 h. The glass dishes were then removed

from the oven, cooled in a desiccator and final weight determined. Moisture contents of

samples were therefore calculated as follows:

45

M = W1-W2 x 100%

W1

Where M = moisture content

W1 = weight of sample before drying

W2 = weight of sample after drying.

Specific Gravity (S.G.) Of Worts

This was determined using the AOAC method, (1980). A 10 ml pycnometer

(density bottle) was filled with distilled water at 20 C to get to the 10 ml mark. This was

closed with a thinly perforated glass stopper to stop the excess water or malt sample (wort)

from leaking. The body of the glass was wiped with a dry and clean tissue paper. Same

method was followed for the sorghum malt extracts also attemperated to 20 C followed by

specific gravity determinnation as follows:

S.G. = S

W

Where:

S = weight of the sample, (weight of sample + bottle) – (weight of empty bottle)

W = weight of an equivalent volume of distilled water, (weight of distilled water+ bottle) –

(weight of empty bottle)

Cold Water Extract

A modification of IOB method No 2.5 (Glennie Holmes, 1992) was used. Malt

flour samples (0.75g) were weighed into a 30ml capacity clean plastic centrifuge tubes.

Solutions for extraction (15ml) containing 0.006N NH4OH were subsequently introduced

into each of the tubes. Extraction of the malt grains was carried out using a wrist-action

shaker continuously for 3 hours. The extract was recovered by centrifugation at 3000g for

15min.

The percentage cold water extracts (CWE) was determined by gravimetric method as

outlined in the IOB method No. 2.5 (IOB, 1982). The calculation of CWE was as follows:

46

% CWE = S.G. excess x 100 x 20

3.86

Where S.G. = specific gravity of the extract, and

SG excess = specific gravity of extract – 1.000

Hot Water Extract (H.W.E)

For the H.W.E determination, the procedure of Etok Akpan (1992) was used. Two

grams of sorghum malt flour was extracted in 18 ml of 0.5% NaCl using a wrist-action

shaker continuously for 1 hour. At the end of the shaking the mixture was allowed to settle

for 15 min and the cloudy supernatant carefully decanted into another flask and stored. The

residual grain mash was boiled (100 C) for 10 min to enable the contents gelatinize. This

was cooled to 50 – 55 C. Afterwards the decanted (stored) supernatant was mixed with the

boiled grain mash and kept at this temperature(50 – 55 C) and stirred every 15 min for 1 h

(mashing). The mixture was then cooled to 25 C and made up to 20.6 ml with 0.5% NaCl

and centrifuged for 15 min. Finally the supernatant was measured gravimetrically using

density bottles as outlined in the IOB methods of analysis (1982). The malt H.W.E. was

calculated as follows:

H.W.E. = G x 10.13

Where G = excess gravity denoted mathematically as G = 1000 (SG – 1)

Cold Water Soluble Carbohydrate (CWSC)

The modified method for CWE determination (Glennine Holmes, 1991) described

above was employed. Malt sample (1.25g) was weighed into a 50 ml screw-cap plastic tube

containing 20 ml distilled water, 2.5 mL (0.2N) Ba(OH)2 and 2.5 mL (0.2N) ZnSO4. This

was followed by a 3 h vigorous shaking using a wrist action shaker at 30 C and

centrifugation thereafter. The clear supernatant obtained was weighed to determine the

specific gravity using a density bottle and the CWSC calculated from the value obtained as

was done for CWE.

47

Free -Amino Nitrogen (FAN) Contents of Sorghum Malts

Preparation of samples for FAN content analysis of sorghum malts was carried out

according to the method described by Taylor and Boyd (1986). Two hundred and fifty

milligram malt samples were each extracted in 10ml 5% trichloroacetic acid (TCA) using a

wrist-action shaker at 30 C for 1 h. This was followed by a 10 min centrifugation at 3000

rpm. Exactly 1ml of clear supernatant was diluted 25 times with distilled water and the

FAN content measured using the E.B.C ninhydrin method as outlined in the IOB methods

of analysis (1982).

The procedure for FAN determination of the diluted extract was as follows. One

millilitre of diluted extract was added to 0.5ml of colour reagent, consisting in gram per

liter of Na2 HPO4.12H2O, 100g; KH2PO4, 60g; ninhydrin, 5g; and fructose, 3g. The mixture

was incubated for 16 min in a boiling water bath (100 C) and finally cooled at 20 C for 20-

30 min. and then united with 5ml of diluting solution made up of KIO3, 2g; ethanol, 400ml

and distilled water 600ml. The absorbance was read at 570nm using LKB Nonaspec

spectrophotometer. Distilled water was used as blank. Glycine standards containing 2mg/L

of the amino acid each were also incorporated in the FAN analysis. The FAN content in

mg/L of the TCA extract of the sorghum malt samples was calculated as:

Absorbance of test solution at 570nm x 2 x sample dilution

Mean absorbance of glycine standards.

Diastatic Power and Amylase Activities

Diastatic power and amylase activity were determined using the diamylase

procedure of EtokAkpan and Palmer (1990) in which ß-amylase was selectively inhibited

with HgCl2. Enzyme extract was obtained by extracting milled sorghum malt sample

(0.16g) with 10mL sodium acetate-acetic acid buffer (pH 5.7) containing 50mM sodium

acetate, 100mM NaCl and 10mM CaCl2 for 2.5 hours at 30 C using a wrist action shaker.

The extract was centrifuged at 3000 rpm for 10 min. The supernatant (crude enzyme) was

diluted 10 times and used for enzyme assay. Exactly 0.5 ml of the diluted extracts was

added into two tubes (tubes A and B) of three separate tubes. The third tube, the enzyme

control (tube C) received 0.5 mL distilled water in place of the diluted enzyme extract. To

tube A in addition to the enzyme was added 0.25 ml HgCl2 (0.0001%) or (10-3

mg/mL).

Both tubes B and C, received 0.25 ml of distilled water each in place of HgCl2. The three

tubes were then thoroughly mixed and left for about 15 min, after which tubes A and B

48

were each added 0.25 mL 1% starch acetate buffer (pH 4.6) solution while tube C received

the distilled water equivalent. Again the three tubes were mixed and allowed to stay for 15

min at 30 C. They were shaken after every 5 minutes. This was followed by the addition

into test tubes A, B and C of 1.0 mL mixed Nelson reagents made up of four (4) parts of

Nelson I (sodium potassium tartarate, 12g; sodium carbonate, 24g; sodium bicarbonate,

16g; sodium sulphate, 144g; and 800mL distilled water) and one (1) part of Nelson II

(copper sulphate, 4g; sodium sulphate, 36g; and 200ml distilled water). A blank was also

prepared. It contained 1ml of distilled water and the Nelson reagents only. All tubes (A, B,

C and blank) were boiled (100 C) in a water bath for 20-25 min. Following boiling these

tubes were cooled and each, including the blank received 1.0 mL Nelson arsenomolybdate

solution, prepared by dissolving ammonium molybdate tetra hydrate (25g) in 450ml of

distilled water that was added 21 mL concentrated sulphuric acid and 25 mL sodium

arsenate (3g dissolved and made up to 25 mL with distilled water).

The Nelson’s arsenomolybdate solution was incubated at 50 C for 5 h for enhanced colour

development. This last one-milliliter Nelson’s arsenomolybdate addition brought the

volume in each test tube to approximately 3 mL. The absorbance was subsequently

determined using LKB Novaspec spectrophotometer set at 625nm against the blank.

Reducing sugar levels were determined by the Somogyi-Nelson methods

(Somogyi, 1952). Sorghum malt total reducing sugar level in the HgCl2 containing tube

(tube A) gave the malt α-amylase activity while the reducing sugar level formed in the

second tube (tube B) gave the total diastatic power. The difference between the diastatic

power and α-amylase activity gave the sorghum malt ß-amylase activity. One unit of α-

amylase activity was defined as any amount of enzyme that was capable of releasing 1µg

glucose equivalent per minute under the assay conditions, assuming Hg does not inhibit a-

amylase activity of sorghum malts.

Cold Water Soluble Protein (CWSP)

CWE samples extracted as earlier described were used for CWSP determination.

The soluble protein fraction of the CWE was determined by the Comassie Brilliant Blue

(CBB) method as was used by Lewis et al. (1979) and modified by Glennie Holmes (1992).

The extract (0.5 mL) was vortex-mixed in a clean test tube with 2.5 ml of CBB regent,

prepared by dissolving 120mg of CBB G-250 (Sigma pfs B-1131) in 50 ml absolute

49

ethanol and mixed with 900 mL distilled water. This was followed by the addition of 36 ml

concentrated perchloric acid (HClO4) and thoroughly mixed. The colour which developed

immediately after mixing of the extract and the CBB-G was read off within 40 min in

LKB-Fonaspec spectrophotometer at 620 nm against distilled water based blank. The tests

were done in duplicates. Protein levels in the various CWE of the various sorghum samples

were determined by reading off from a standard calibration curve (appendix) for protein

prepared with bovine serum albumen (BSA) in the range of 0–500µg.

Total Non-Protein Nitrogen (TNPN) Determination

This was done according to the methods of Institute of Brewing (1982). About 0.5g

of sorghum sample was added in a Kjedahl’s digestion flask containing a catalyst mixture

(1:20, CuSO4: N2SO4) and 20 ml concentrated sulphuric acid. This was placed on a

preheated electric hot plate and heated until the mixture turned from black to brown and

finally to light blue. This was carried out in a fume chamber to guide the smokes and fumes

away appropriately. The digest was allowed to cool and was then made up to 100 ml with

distilled water. Exactly, 10-15 ml of this digest dilution was made alkaline using 15 ml

40% NaOH and was transferred to a steam-out apparatus. The ammonia steam was distilled

into 10 ml of 2% boric acid solution containing 0.2 ml of methyl indicator (0.016% methyl

red and 0.033% bromocresol green in ethanol) for 10 min or until about 20-30 ml of

distillate was obtained in the receiving 150 ml conical flask. The distillate was allowed to

stand for about 5 min and titrated with 0.01 M H2SO4 to the end point. Nitrogen in malt

samples was calculated as follows:

TNPN (N) = 0.0014 x titre value

Protein content = N x 6.25

Mashing

Exactly 15g of milled sorghum malt samples were variously put in 250 ml conical

flasks containing 60 ml of 0.05 M NaAc. (Sodium acetate) buffer (pH 5.6) and gently and

but thoroughly mixed. This mixture was then put in a water bath shaker set at 45 C and

incubated with gentle shaking (65-70 rpm) for 1 h. Samples were allowed to stand for 20

min and the cloudy supernatant decanted into a separate clean container. The residue in the

flask was boiled (100ºC) (after adding 5 ml of 0.05 M NaAc (pH 5.6 buffer) to gelatinize. It

was then cooled to 60ºC before being reunited with the decanted supernatant and gently

50

incubated in the shaker (also set at 60ºC) for 1.5 h. Following this final shaking, the

contents in the flask were allowed to cool and then centrifuged. The clear supernatant

(wort) of the centrifuged samples was used subsequently to assay for wort (malt mash

extract) FAN, wort total soluble protein, wort total soluble carbohydrate and glucose

determination.

Wort Free Amino Nitrogen Determination (W-FAN)

A 1:50 dilution of the wort sample (sorghum malt mash extract, obtained as

described above) was prepared by mixing one part of the wort with 49 parts of distilled

water. Wort-FAN was thereafter determined the same way as with sorghum malt. Exactly 1

millilitre of the diluted wort sample was used for the FAN determination.

Wort Total Soluble Protein (WTSP)

Undiluted wort extract (0.5ml) was used in the determination of the wort total

soluble protein. The procedure for CWSP determination was used.

Liquid Substrate Fermentation

Appropriate hydrolysates were placed into 250ml, clean autoclaved (121 C for 15

min) conical flasks, and inoculated with 0.2ml of washed and standardized (1.0 at 600nm/

107 cfu) S. cerevisiae. The microorganisms were then allowed to ferment the hydrolysates

for 72 hours. The growth of yeast cells was monitored spectrophotometrically at 600nm

every four hours.

Total Carbohydrate determination

Phenol-sulphuric acid method of Dubois et al. (1956) was employed for the

determination of carbohydrate levels in fermenting wort samples and enzyme-hydrolyzed

cassava hydrolysate. To a 1 ml volume of appropriately diluted test or control sample in a

clean test tube, 1 ml of 5% (w/v) phenol solution was added. After thorough mixing, 5 ml

of concentrated sulphuric acid was then added using fast flowing pipette. The test tubes

were incubated at room temperature for 10 min, followed by shaking in a water bath

incubator set at 30 C for 15 min. The optical density of the developed colour was thereafter

read at 490nm using a Novaspec spectrophotometer.

51

Ethanol Content Determination of fermented samples

The boiling/titrimetric method of Sg-Gwarr (1987) was used. Into a 50 ml flat

bottom flask, 10 ml of de-ionized water and 10 ml of 10% solution of K2SO4 were

pipetted. Into another receiving test tube (180 – 250 mm x 12 mm), 10 ml of K2 Cr2 O7

(42.5g/l) and 10 ml of a 1:1 dilution of concentrated H2SO4 were delivered. The boiling

flask was mounted on a tripod stand, covered with a rubber bung carrying a delivery glass

tube joined by Teflon tubing to another piece of glass tubing leading into the receiving test

tube (inserted half way into tap water to reduce heat and evaporation) held in position by a

clamp. A low flame was ignited under the round bottom flask and adjusted so that the

liquid in the flask boils with little frothing until about half of the contents have distilled into

the receiving test tube. The contents of the test tube was made up to 60 ml and transferred

into a 250 ml conical flask containing 5 ml of KI (10%) solution, allowed to stand for 2-

minutes, cooled to room temperature and 2 ml of 0.4% starch solution added and mixed.

The mixture was then titrated against a 1 M solution of Na2S2O3.5H2O from a burette until

a blue colour just appeared. Test was done in duplicate and average titre obtained. This titre

which was 8.0 ml represents the blank titre. For test samples, the same procedure was

adopted except that 10 ml of an appropriate dilution of the test sample was used instead of

the 10 ml of de-ionized water in the round bottom flask. Alcohol content of the sample was

then read out by reference to a standard table ( see index). Where a = blank titre and b= test

titre. The level of ethanol from the table was multiplied by the dilution factor to get the

percentage (v/v) ethanol in the fermentation medium.

52

RESULTS

Effects of decortication and starter cultures of Aspergillus on Aspergillus growth

during sorghum malting

Fungal loads on the germinating sorghum grains determined as colony forming

units per millilitre suspension (cfu/ml) is shown in figure 1. Decorticated sorghum malts

irrespective of the type of Aspergilli treatment gave higher mean fungal loads than

undecorticated sorghum malts treated with A. awamori and A. carbonarius. However, A.

awamori-treated decorticated sorghum malts showed higher fungal counts with a mean

value of 1.5 x 105 spores/g when compared to its counterpart treated with A. carbonarius

(1.4 x 104 spores /g). On the other hand undecorticated sorghum malts treated with A.

awamori gave higher Aspergillus growth (9.7 x 103 spores/g) than undecorticated sorghum

malt inoculated with A. carbonarius with mean fungal load of 4.3 x 102 cfu/g.

54

Generally fungal load increased in proportion with the initial amount of inoculum

beginning from the first day of germination to the fifth and final days, indicating an

exponential pattern of growth. Furthermore, there was no growth on the uninoculated

germinated sorghum grain (control), while growth was least on the one with the least

inoculation and highest on the most inoculated sorghum grains accordingly (0.00,

67509.58, 1349070.00 spores/ml). Analysis of variance of their different mean growth

indicated that the treatment due to decortication significantly (P<0.05) affected Aspergillus

growth. The number of Aspergilli spores had level of significance of P=0.055 and an

interaction effect of P=0.134. The less Aspergilli growth on undecorticated sorghum may

be attributed to competing microbial flora on and interference by components of the

pericarp of the sorghum such as tannin.

55

Effects of decortication on Percentage Germinative Energy (GE) and Water

Sensitivity (WS)

The result of the effects of decortications on % GE and WS, shown in figure 2 ,

showed that both decorticated and undecorticated grains recorded high germinative

energies (98% and 96%) and low water sensitivities (3% and 4%), respectively. Generally,

fungal load increased with the initial amount of spore inoculum right from the first day of

germination to the fifth and final day, indicating an exponential pattern of growth.

Similarly, there was no fungal growth on the uninoculated control, though the sorghum

grain germinated, and likewise the least and the highest inoculated sorghum grains gave the

least and highest spores per gram (6.75 x 105 and 1.3 x 10

6 spores/g).

56

0

20

40

60

80

100

120

%GE %WS

% G

rain

ge

rmin

ati

on

Figure 2: Effects of decortication on Percentage Germinative Energy (GE) and

Water Sensitivity (WS)

Decorticated Undecorticated

57

Effect of decortication and Aspergillus spores on sorghum malt enzyme developments

-amylase

The -amylase activities of sorghum malts treated with A. awamori are shown in

figure 3 while that for sorghum malts treated with A. carbonarius are shown in figure 4.

The results showed that decorticated malts treated with A. awamori recorded mean -

amylases higher than the control. Furthermore, the overall mean -amylase activity for

decorticated sorghum malt treated with A. awamori in addition to the control (2720 U/mL)

is greater than that of undecorticated sorghum malts treated with Aspergillus awamori in

addition with its own control (1628 U/mL)

On the other hand -amylase activity in sorghum malts treated with Aspergillus

carbonarius also gave a mean activity value that is greater than that of the control.

However, the mean -amylase activity of undecorticated sorghum malts treated with A.

carbonarius (1758 U/mL) is greater than those of decorticated sorghum malts treated with

A. carbonarius (1021 U/mL). As a matter of fact, a general increase was observed during

the course of germination from the first day to fifth in the case of sorghum malts treated

with A. awamori unlike those treated with A. carbonarius which did not show similarly

definitive results. In addition, sorghum grains treated with either of the Aspergilli produced

higher mean (1842.49 U/mL) -amylases than untreated controls (1481.40 U/mL).

Analysis of variance among the activity means showed a significant difference (p < 0.05)

probably due to the positive effect decortication treatment had on the levels of -amylase

development. The level of significance on -amylase production due to inoculated

Aspergillus spores was P=0.895 and their interactions was P=0.698.

58

Figure 3: Effect of initial A. awamori load and decortication on α –

amylase activity of sorghum malts over time

Decorticated sorghum malts

treated with A. awamori Undecorticated sorghum malts

treated with A. awamori

CONTROL; 9.1X105 ; 1.82X10

6 ; 2.73X10

6 ; 3.6X10

6 ; 4.55X10

6

0

2000

4000

6000

8000

10000

12000

14000

1 2 3 4 5 0 1 2 3 4 5

α –

Am

yla

se a

cti

vit

y (

µg

eq

uiv

ale

nt

of

glu

co

se

)

Germination time (days)

59

3.6X106;

0

500

1000

1500

2000

2500

3000

1 2 3 4 5 0 1 2 3 4 5


α-

Am

yla

se a

cti

vit

y (

µg

eq

uiv

ale

nt

of

glu

co

se)


treated with A. cabonarius Undecorticated sorghum malts treated

with A. cabonarius

Figure 4: Effect of initial A. carbonarius load and decortication on α – amylase activity of sorghum malts over time

CONTROL;

9.1X10; 1.82X106; 2.73X106

; 4.55X106

60

β–Amylase

β-Amylases are exo-enzymes that release maltose molecules from the non-reducing

ends of amylase and amylopectin. β-Amylase activities in both decorticated and

undecorticated sorghum malts that were treated with A. awamori are shown in figure 5.

Mean β-amylase recorded by undecorticated malts treated with A. awamori (U/mL) is

greater than the mean value obtained with decorticated sorghum malts treated with the

same organism (1315 U/mL). There was a relatively progressive increase from the first day

of germination to the third followed by a less definite pattern in the increase in both

decorticated and undecorticated sorghum malts treated with A. awamori afterwards. On the

other hand, in respect of decorticated and undecorticated sorghum malts treated with A.

carbonarius (figure 6) a higher mean β-amylase activity was recorded by the

undecorticated (3157µg glucose equivalent) sorghum malts compared to that of its

decorticated counterpart (1359 U/mL). During the course of germination, the decorticated

sorghum malts treated with A. carbonarius recorded an increase in β-amylase from the first

day to the third day followed by a slight decline towards the final day of germination. On

its part, the undecorticated sorghum malts treated with A. carbonarius showed a generally

progressive increase from the first day of germination to the last day of germination.

Aspergillus-treated sorghum grains produced lower mean (2349.20 U/mL) β-amylases than

untreated controls (2965.66 U/mL) Analysis of variance, however, showed a significant

effect (p < 0.05) on the levels of β-amylase production due to the decortication treatments

but not with β-amylase production due to inoculation with Aspergillus spores (P=0.503)

and their interactions (P=0.908)

61

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5 0 1 2 3 4 5 β

-Am

yla

se a

cti

vit

y (

µg

eq

uiv

ale

nt

of

glu

co

se)


Figure 5: Effect of initial A. awamori load and decortication on β – amylase activity of sorghum malts over time




CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106 4.55X106

62

0

2000

4000

6000

8000

10000

12000

1 2 3 4 5 0 1 2 3 4 5

β-A

my

lase a

cti

vit

y (

µg

eq

uiv

ale

nt

of

glu

co

se

)




with A. cabonarius

Figure 6: Effect of initial A. carbonarius load and decortication on β –

amylase activity of sorghum malts over time

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

63

Diastatic Power (DP)

The diastatic power generally indicates the extent of expression of carbohydrate

utilizing enzymes during malting. The DP for decorticated and undecorticated sorghum

malts treated with A. awamori is shown in figure 7. The figure showed that undecorticated

sorghum malts treated with A. awamori recorded a mean DP (5629. U/mL) which is higher

than the mean DP (2899.80 U/mL) produced by decorticated sorghum malts treated with

the same organism. During the course of germination as shown in the figure, increase in

DP levels were observed right from the first day of germination to the fifth day in the case

of decorticated malt. However, this increase though similar, was not definite after the third

day of germination in the case of undecorticated malts. The next figure (8) shows the DP

levels in sorghum malts treated with A. carbonarius. The figure indicated that more DP

(4960.32 U/mL) was produced by undecorticated sorghum malts treated with A.

carbonarius than that of its decorticated counterpart inoculated with the same organism

(1987.10 U/mL). During the course of germination as shown in the figure, there was an

initial increase, followed by a decline in the DP levels of the decorticated malts; while a

progressive increase from the first day of germination to the fifth day characterized that of

undecorticated malts treated with A. carbonarius. Also Aspergillus-treated sorghum grains

produced lower mean (3775.45 U/mL) DP than untreated controls (4337.65µg U/mL).

Analysis of variance determined for DP showed that DP development in sorghum malts

was significantly (p<0.05) affected by the decortication treatment but not with DP

production due to inoculation with Aspergillus spores (P=0.662) and their interactions

(P=0.855).

64

0

2000

4000

6000

8000

10000

0 1 2 3 4 5 0 1 2 3 4 5

12000


Figure 7: Effect of initial A. awamori load and decortication on Diastatic power of sorghum malts over time

CONTROL; 9.1X105; 1.82X10; 2.73X10

6; 3.6X10

6; 4.55X1

6




Dia

sta

tic

po

we

r (µ

g e

qu

iva

len

t o

f g

luco

se)

65

0

2000

4000

6000

8000

10000

12000

14000

1 2 3 4 5 0 1 2 3 4 5

D

ias

tati

c p

ow

er

(µg

eq

uiv

ale

nt

of

glu

co

se

)




with A. cabonarius

Figure 8: Effect of initial A. carbonarius load and decortication on Diastatic power of sorghum malts over time

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

66

Effects of decortication and inoculation with Aspergillus spores on sorghum malt free

amino nitrogen (FAN) developments

Free α-amino nitrogen development in decorticated and undecorticated sorghum

malts treated with A. awamori during the course of germination is shown in Figure 9.

Decorticated sorghum malts treated with A. awamori recorded higher mean (32.87 mg/ml)

FAN than the undecorticated counterpart (21.82 mg/ml) treated with the same organism.

During the course of germination there was a progressive increase in FAN development in

both decorticated and undecorticated sorghum malts treated with A. awamori. Figure 10 on

the other hand shows the FAN development levels in decorticated and undecorticated

sorghum malts treated with A. carbonarius. The figure shows undecorticated sorghum

malts recording a higher mean FAN value (10.88 mg/ml) than the decorticated counterpart

treated with the same organism (8.09 mg/ml). FAN development during the germination

period increased gradually from the first day of germination to the fifth day. In addition

Aspergillus-treated sorghum grains produced higher mean FAN (19.30mg/ml) than the

untreated controls (14.00mg/ml). FAN development was significantly affected (P<0.05) by

the decortication treatment, unlike FAN production due to inoculation with Aspergillus

spores (P=0.832) and their interactions (P=0.994). The analysis was carried out using the

Univariate analysis of variance method of SPSS (SPSS version 11).

67

0

2

0

4

0

6

0

8

0

10

0

12

0

0 1 2 3 4 5 0 1 2 3 4 5

FA

N(m

g/m

l)

(((m

g/m

l)




Undecorticated sorghum malts


CONTROL; 9.1X105; 1.82X10

6; 2.73X106

;

3.6X106; 4.55X10

6

Figure 9: Effect of initial A. awamori load and decortication on Free Amino Acid (FAN) development in sorghum malts over time

68

CONTROL; 9.1X105; 1.82X10

6; 2.73X106; 3.6X10

6

;

4.55X106

Figure 10: Effect of initial A. carbonarius load and decortication on Free

AminoAcid (FAN) development in sorghum malts over time

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 0 1 2 3 4 5

FA

N (

mg

/ml)



treated with A. cabonarius Undecorticated sorghum malts

treated with A. cabonarius

69

Effects of decortication and inoculation Aspergillus spores on Cold water Soluble

Protein (CWS-P)

Cold water soluble protein in decorticated and undecorticated sorghum malts

treated with A. awamori is shown in Figure 11. Undecorticated sorghum malts treated with

A. awamori gave a slightly higher mean CWS-P (0.91 mg/ml) than the decorticated

counterpart (0.90 mg/ml) treated with the same organism. During the course of germination

progressive increases in CWS-P were recorded by both decorticated and undecorticated

sorghum malts treated with A. awamori. The next figure (12) shows the CWS-P

development in decorticated and undecorticated sorghum malts treated with A.

carbonarius. There was also a progressive increase in CWS-P levels in both decorticated

and undecorticated sorghum malts treated with A. carbonarius; however, the mean CWS-P

of undecorticated (0.88 mg/ml) showed a slightly higher value than that shown by the

decorticated sorghum malts treated with A. carbonarius (0.795 mg/ml). Aspergillus-treated

sorghum grains produced higher mean (0.88mg/ml) CWS-P than untreated controls

(0.85mg/ml). CWS-P development was significantly (P<0.05) affected by the decortication

treatment but not by Aspergillus spores treatment (P=0.951) or their interactions (0.999).

70

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3 4 5 0 1 2 3 4 5


CW

SP

(m

g/m

l)




Figure 11: Effect of initial A. awamori load and decortication on Cold-water

soluble protein (CWSP) production in sorghum malts over time

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

71

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 0 1 2 3 4 5


CW

SP

(m

g/m

l)




Figure 12: Effect of initial A. carbonarius load and decortication on Cold-water

soluble protein (CWSP) production in sorghum malts over time

CONTROL; 9.1X105; 1.82X10

6; 2.73X106; 3.6X106

; 4.55X10

6

72


Extract (CWE) development

Cold Water Extract (CWE) in decorticated and undecorticated sorghum malts

treated with A. awamori is shown in Figure 13. Decorticated sorghum malts treated with A.

awamori recorded a higher mean CWE (14.27 %) than the undecorticated counterpart (9.77

mg/ml) treated with the same organism. During the course of germination progressive

increases in CWE were given by both decorticated and undecorticated sorghum malts

treated with A. awamori. Figure 14 shows the CWE development in decorticated and

undecorticated sorghum malts treated with A. carbonarius. There was also a progressive

increase in CWE levels in both decorticated and undecorticated sorghum malts treated with

A. carbonarius; however, the mean CWE of undecorticated malts (12.19 %) showed a

higher value than that given by the decorticated sorghum malts treated with A. carbonarius

(10.42 %). Aspergillus-treated sorghum grains produced higher mean CWE (12.33%) than

untreated controls (10.53%). Analysis of variance showed that CWE development was

significantly (P<0.05) affected by the decortication treatment but not by Aspergillus

treatment (P=0.951) and their interactions (P=0.951).

73

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 0 1 2 3 4 5


CW

E (

%)




Figure 13: Effect of initial A. awamori load and decortication on % Cold- water

extract (CWE) in sorghum malts over time

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

74

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5 0 1 2 3 4 5


CW

E (

%)




Figure 14: Effect of initial A. carbonarius load and decortication on % Cold-water

extract (CWE) in sorghum malts over time

CONTROL; 9.1X105; 1.82X10

6; 2.73X106; 3.6X10

6; 4.55X10

6

75

Effects of decortication and inoculation with Aspergillus spores on Hot Water Extract

(HWE) development

This is the extractable material from malts during mashing at appropriate

temperatures HWE levels determined for decorticated and undecorticated sorghum malts

treated with A. awamori is shown in Figure 15. The figure shows that during the course of

germination, from the first day to the fifth, there was a progressive increase in HWE

development, except with the decorticated sorghum malts control that slightly declined on

the third day of germination. Moreover, the HWE development in decorticated sorghum

malt samples treated with A. awamori showed a mean HWE value of 228.67 % while that

of the undecorticated counterpart gave 245.92 %. On the other hand, the HWE

development in decorticated and undecorticated sorghum malts treated with A. carbonarius

(figure 16), shows that the decorticated sorghum malts recorded higher mean HWE (252.45

%) than their counterpart that was not decorticated (251.92 %) but treated with the same

organism. During the course of germination, a progressive increase in HWE development

characterized both decorticated and undecorticated sorghum malts treated with A.

carbonarius except with the decorticated malts which were all deflected downwards after

the third day of germination. Furthermore, Aspergillus-treated sorghum grains produced

lower mean HWE (245.37%) than untreated controls (252.85%). HWE development was

not significantly (P > 0.05) affected by the decortication treatment given to sorghum malt

samples.

76

0

50

100

150

200

250

300

350

0 1 2 3 4 5 0 1 2 3 4 5


HW

E (

%)




Figure 15: Effect of initial A. awamori load and decortication on % Hot- water

extract (HWE) in sorghum malts over time

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

77


0

50

100

150

200

250

300

350

0 1 2 3 4 0 1 2 3 4 5

HW

E (

%)




Figure 16: Effect of initial A. carbonarius load and decortication on % Hot-water

extract (HWE) in sorghum malts over time

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

78

Effects of decortication and inoculation with Aspergillus spores on sorghum malt

Wort Free Amino Nitrogen

Assimilable nitrogen levels in malt extracts in form of FAN were determined for

the decorticated and undecorticated sorghum malt samples. The results as obtained from

sorghum malt from the final day samples are as shown in figure 17. The figure shows a

higher wort FAN in decorticated sorghum malts treated with A. awamori with a mean value

of 12.26 mg/ml. This was distantly followed by undecorticated sorghum malts treated with

A. carbonarius (5.699 mg/ml); undecorticated sorghum malts treated with A. awamori

(5.60 mg/ml) and lastly decorticated sorghum malts treated with A. carbonarius (4.35

mg/ml). The figure also indicated that the mean wort FAN levels of the controls are lower

than the wort FAN levels obtained for Aspergillus-treated malt samples. One way analysis

of variance determined for the wort FAN level showed a significant difference (P < 0.05)

on the wort FAN level due to the decortication treatment.

79

Decorticated sorghum malts treated with A. carbonarius

0

2

4

6

8

10

12

14

16

18

Number of spores (CFU)

W-F

AN

Le

ve

ls (

mg

/ml)

Decorticated sorghum malts treated with A.

awamori;

Undecorticated sorghum malts treated with A. awamori;

Undecorticated sorghum malts treated with A. carbonarius

Figure 17: Effect of initial A. awamori and A. carbonarius loads and decortication on Wort-Free Amino Acid (W-FAN) levels in sorghum malts

CONTROL 9.1X105 1.82X10

6 2.73X106 3.6X10

6 4.55X106

80

Effects of decortication on sorghum malt Wort Total Soluble Protein

The wort total soluble protein of decorticated and undecorticated sorghum malts for

final day samples are shown in figure 18. Decorticated sorghum malts treated with A.

awamori recorded higher wort-TSP (1.56 mg/ml) followed by decorticated sorghum malts

treated with A. carbonarius (0.93 mg/ml); undecorticated sorghum samples treated with A.

awamori gave 0.86 mg/ml and finally undecorticated sorghum malts treated with A.

carbonarius had 0.759 mg/ml. The control samples however recorded higher mean values

than undecorticated sorghum malts treated with A. carbonarius. Furthermore, one way

ANOVA showed that a significant difference (P < 0.05) occurred in wort total soluble

protein development due to the decorticated treatment.

81

0

0.5

1

1.5

2

2.5

CONTROL 9.1X105 1.82X10

6 2.73X106 3.6X10

6 4.55X106

Number of Spores (CFU)

TS

P (

mg

/ml)


awamori;



carbonarius

Undecorticated sorghum malts treated with A.

carbonarius

Figure 18: Effect of initial A. awamori and A. carbonarius loads and decortication on

Wort-Total Soluble Protein (W-TSP) levels in sorghum malts

82

Effects of decortication and inoculation with Aspergillus spores on sorghum malt Cold

Water Soluble Carbohydrate (CWS-CHO)

The Cold Water Soluble Carbohydrate (CWS-CHO) is the soluble product of

carbohydrate origin from enzyme hydrolysis during malting. CWS-CHO development in

decorticated and undecorticated sorghum malts and those treated with A. awamori and A.

carbonarius from final day malt samples is shown in figure 19. The figure shows

decorticated control recording lower CWS-CHO than the undecorticated control, whereas

sorghum malt samples treated with A. awamori and A. carbonarius recorded higher CWS-

CHO (24.83 % and 18.86 %, respectively) than the undecorticated sorghum malts treated

A. awamori and A. carbonarius (7.33 % and 9.78 %, respectively). One way ANOVA

showed a significant difference (P < 0.05) in the level of CWS-CHO in the analysed

sorghum malt samples due to decortication treatments.

83

0

5

10

15

20

25

30

35

Number of Spores (CFU)

CW

SC

(%

)

Decorticated sorghum malts treated with A. awamori;


Decorticated sorghum malts treated with A. carbonarius

Undecorticated sorghum malts treated with A.

carbonarius

Figure 19: Effect of initial A. awamori and A. carbonarius loads and decortication on

Cold water soluble carbohydrate (CWSC) levels in sorghum malts

CONTROL 9.1X105 1.82X10

6 2.73X106 3.6X10

6 4.55X106

84

Effects of decortication and inoculation with Aspergillus spores on sorghum malt

Total Non Protein Nitrogen (TNPN)

The amount of TNPN in decorticated and undecorticated sorghum malt controls as

well as those treated with A. awamori and A. carbonarius is shown in figure 20. The figure

indicated that decorticated sorghum malts treated with A. awamori gave higher (12.11 %)

TNPN, followed by undecorticated sorghum malts treated with A. awamori (11.38 %),

decorticated sorghum malts treated with A. carbonarius (10.94 %) and undecorticated

sorghum malts treated with A. carbonarius (9.48 %). The decorticated and undecorticated

sorghum malt controls, however recorded higher mean TNPN than undecorticated sorghum

malts treated with A. carbonarius. One way ANOVA showed a significant difference (P <

0.05) in TNPN due to the decortication treatments on sorghum grains.

85

0

2

4

6

8

10

12

14

16

18

CONTROL 9.1X105 1.82X10

6 2.73X106 3.6X10

6 4.55X106

Number of spores (Cfu)

TN

PN

(%

)


awamori; Undecorticated sorghum malts treated with A. awamori;

Decorticated sorghum malts treated with A. carbonarius Undecorticated sorghum malts treated with A. carbonarius

Figure 20: Effect of initial A. awamori and A. carbonarius loads and decortication on Total non-protein nitrogen (TNPN) levels in sorghum malts

86

Effects of decortication and inoculation with Aspergillus spores on malting loss (ML)

Malting loss for decorticated and undecorticated sorghum malts and sorghum malts

treated with A. awamori is shown in figure 21. The figure shows the control recording

lower ML when compared with A. awamori-treated sorghum malts. The figure also showed

that decorticated sorghum malts treated with A. awamori recorded higher ML (23.46%)

than their undecorticated counterpart treated with the same organism (16.17%). During the

course of germination from the first to fifth day, a progressive increase in ML was

observed. The next figure (22) shows the ML of decorticated and undecorticated sorghum

malts treated with A. carbonarius and their controls. The mean ML for the controls was

lower than that of the treated sorghum malt samples. Moreover, decorticated sorghum malt

samples treated with A. carbonarius recorded lower ML (16.06%) than the undecorticated

counterpart treated with the same organism (17.53%). Also a progressive increase in ML

corresponding to the germination time was observed. Aspergillus-treated sorghum grains

produced higher mean ML (19.05%) than untreated controls (14.63%).There was

significant differences (p<0.05) in the ML of sorghum malts due to the decortication

treatments, Aspergillus treatments and their interactions.

87

Figure 21: Effect of initial A. awamori load and decortication on the

Malting Loss (ML) of sorghum malts over time

0

5

10

15

20

25

30

35

40

45

50

1 2 3 4 5 1 2 3 4 5


% M

L

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

Decorticated sorghum malts treated with A. awamori

Undecorticated sorghum malts treated with A. awamori

88

%M

L

0

5

10

15

20

25

30

35

1 2 3 4 5 1 2 3 4 5

Germination time (days) Figure 22: Effect of initial A. carbonarius load and decortication on

the Malting Loss (ML) of sorghum malts over time

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6

; 3.6X10

6

; 4.55X10

6




89

Effects of decortication and inoculation with Aspergillus spores on wort extract

glucose levels

The wort extract glucose level for decorticated and undecorticated sorghum malts

variously treated with A. awamori and A. carbonarius is shown in figure 23. Final day

germinated controls and treated sorghum samples were used. The figure shows

undecorticated sorghum malts treated with A. awamori recording higher glucose levels

(34.08mg/ml) followed by decorticated sorghum malts treated with A. carbonarius (33.75

mg/ml), undecorticated sorghum malts treated with A. carbonarius (31.70 mg/ml) and

decorticated sorghum malts treated with A. awamori (26.75 mg/ml). The control

nevertheless gave lower glucose than the lowest above. One-way ANOVA showed no

significant difference (P>0.05) on the sorghum malt extract glucose levels due to

decortication treatment.

90

Number of spores (cfu)

0

5

10

15

20

25

30

35

40

45

50

Glu

co

se

le

vel

(mg

/ml)

Decorticated sorghum malts treated with A. awamori; Undecorticated sorghum malts treated with A. awamori;

Decorticated sorghum malts treated with A. carbonarius Undecorticated sorghum malts treated with A. carbonarius

Figure 23: Effect of initial A. awamori load and decortication

on the glucose levels in sorghum malt extract (wort)

CONTROL 9.1X105 1.82X106 2.73X106 3.6X106 4.55 X 106

91

Effects of decortication and inoculation with Aspergillus spores on the time course of

yeast growth on sorghum malt extract

Figure 24 shows the time course of yeast growth on malt extract of decorticated and

undecorticated sorghum malts treated with A. awamori. The figure shows the extract of

decorticated sorghum malts treated with A. awamori giving a higher mean yeast growth

(8.34 OD600) than the extract of the undecorticated counterpart treated with the same

organism (6.37 OD600). During the course of growth a typical growth curve was maintained

by both media up till the stationary level. Figure 25 shows the time course of yeast growth

on malt extract of decorticated and undecorticated sorghum malts treated with A.

carbonarius. The figure shows the extract of decorticated sorghum malts treated with A.

carbonarius giving a lower mean optical density of yeast growth (6.12 OD600) than on the

extract of the counterpart treated with the same organism (6.58 OD600). Aspergillus-treated

sorghum grains produced higher mean (7.00 OD600) optical density of yeast growth than

untreated controls (6.14 OD600). There was a significant difference (P<0.05) on the yeast

growth on sorghum extract due to decortication treatment, Aspergillus treatment and their

interactions. (Where OD600 = Optical Density of spores at 600nm)

92

4.55X106



CONTROL; 9.1X105; 1.82X106; 2.73X106

; 3.6X106

;

Fermentation time (h)

Bio

ma

ss

(O

D600)

0

2

4

6

8

10

12

14

12 24 36 48 60 72 0 12 24 36 48 60 72

Uncorticated sorghum malts treated with A. awamori

Figure 24: Time course of yeast growth on decorticated and undecorticated

sorghum malt extracts treated with different levels of A. awamori

93


treated with A. carbonarius

Undecorticated sorghum malts

treated with A. crabonarius

0

2

4

6

8

10

12


Bio

ma

ss

OD

600

12 24 36 48 60 72 0 12 24 36 48 60 72

Figure 25: Time course of yeast growth on decorticated and undecorticated sorghum malt extracts treated with different levels of A. carbonarius

CONTROL;

9.1X105; 1.82X106; 2.73X106

; 3.6X106

;

0

4.55X106

94

Effects of decortication and inoculation with Aspergillus spores on ethanol levels in

fermented sorghum malts

Ethanol levels in fermented sorghum malt extracts of both controls and treated

sorghum samples is shown in figure 26. The figure shows that fermented extracts from

decorticated sorghum malts treated with A. awamori produced higher ethanol levels

(4.18%) followed by fermented extracts of undecorticated sorghum malts treated with A.

awamori (3.59%), fermented extract of undecorticated sorghum malts treated with A.

carbonarius (3.14%) and fermented extract of decorticated sorghum malts treated with A.

carbonarius producing the lowest (3.08%). Ethanol levels of fermented extracts from

undecorticated control were higher than those from the fermented extract from decorticated

control. One-way ANOVA determined for the ethanol levels in fermented sorghum malts

extracts showed a significant difference (P<0.05) due to decortication treatments.

95

Decorticated sorghum malts treated with A. awamori,

Undecorticated sorghum malts treated with A. awamori,

Decorticated sorghum malts and treated with A. carbonarius,

Undecorticated sorghum malts treated with A. carbonarius,

Figure 26: Ethanol Levels in fermented decorticated and undecorticated

sorghum malt extracts treated with Aspergillus sp

Number of spores (CFU)

Eth

an

ol le

ve

l (%

)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

CONTROL 9.1X105 1.82X10

6 2.73X106 3.6X10

6 4.55X106

96

Effects of decortication and inoculation with Aspergillus spores on glucose levels of

raw cassava starch hydrolysate hydrolysed with sorghum malt crude enzymes

The level of glucose in raw cassava starch hydrolysate after hydrolysis with

decorticated and undecorticated sorghum malt (enzymes) treated with A. awamori during

fermentation is shown in Figure 27. The figure indicated that during the course of

fermentation, there was a progressive decrease in glucose levels of the fermenting cassava

starch hydrolysate. It was also observed that the control produced lower glucose levels than

the treated samples. Mean glucose levels during the course of fermentation by cassava

hydrolysate hydrolysed with decorticated malts that was treated with A. awamori is 3.4

mg/ml while its counterpart hydrolysed with undecorticated sorghum malts; treated with

the same organism gave 2.68 mg/ml. On the other hand, a progressive decrease was also

noted with fermenting cassava hydrolysate, hydrolysed with decorticated and

undecorticated sorghum malt crude enzymes as shown in figure 28. Mean glucose levels

during the course of fermentation for cassava hydrolysate hydrolysed with decorticated

sorghum malts treated with A. carbonarius is 1.97 mg/ml while that of undecorticated

sorghum malts treated with the same organism gave 1.59 mg/ml. Aspergillus-treated

sorghum grains produced higher mean (2.50 mg/ml) optical density of yeast growth than

untreated controls (2.05 mg/ml). There was a significant difference on glucose levels of

raw cassava starch hydrolysis (P<0.05) due to decortication treatment but not with

Aspergillus treatments and their intera ctions

97

Raw milled cassava hydrolyzed with decorticated sorghum malts

(enzymes) treated with A. awamori

Raw cassava milled hydrolyzed with undecorticated sorghum malts

(enzymes) treated with A. awamori

0

1

2

3

4

5

6

0 12 24 36 48 60 72 0 12 24 36 48 60 72


Glu

co

se l

evel (m

g/m

l)

Figure 27: Effect of decortication and inoculation with A. carbonarius on levels of glucose produced from raw cassava starch hydrolysate A. awamori CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

98

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 12 24 36 48 60 72 0 12 24 36 48 60


Glu

co

se l

ev

els

(m

g/m

l)



Raw milled cassava hydrolyzed with undecorticated sorghum malts treated

with A. carbonarius

Figure 28: Effect of decortication and inoculation with A. carbonarius on levels

of glucose of produced from raw cassava starch hydrolysate

72

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

99

Effects of decortication and inoculation with Aspergillus spores on glucose levels of

gelatinized cassava starch hydrolysate using sorghum malt crude enzymes.

The glucose levels of gelatinized cassava starch hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt (enzymes) and treated with A. awamori

during fermentation is shown in figure 29. A progressive decrease in mean glucose levels

was recorded during the course of fermentation for both gelatinized cassava hydrolysate

hydrolysed with decorticated sorghum malts treated with A. awamori (3.42 mg/ml) and the

counterpart, hydrolysed with undecorticated sorghum malt (enzymes) treated with the same

organism (2.60 mg/ml). On the other hand, glucose levels in fermenting cassava starch

hydrolysate hydrolysed with decorticated and undecorticated sorghum malt (enzymes)

treated with A. carbonarius is shown in figure 30. Progressive decrease in the glucose

levels was also recorded. The mean glucose levels during the course of fermentation of the

gelatinized cassava starch hydrolysate hydrolysed with decorticated and undecorticated

sorghum malt enzymes were 2.09 and 2.38 mg/ml, respectively. Aspergillus-treated

sorghum grains produced higher mean (2.77 mg/ml) optical density of yeast growth than

untreated controls (1.90 mg/ml). There was a significant difference (p<0.05) on the glucose

levels due to decortication treatments on sorghum malts.

100

.

Figure 29: Effect of decortication and inoculation with A. awamori on the

levels of glucose produced from gelatinised cassava hydrolysate

Gelatinised milled cassava

hydrolysed with decorticated sorghum malts treated with A. awamori


hydrolysed with undecorticated sorghum malts treated with A. awamori

0

1

2

3

4

5

6

0 12 24 36 48 60 72 0 12 24 36 48 60 72


CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

Glu

co

se l

ev

els

(m

g/m

l)

101

Figure 30: Effect of decortication and inoculation with A. carbonarius on the levels of glucose produced from gelatinised cassava starch hydrolysate

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 12 24 36 48 60 72 0 12 24 36 48 60 72


Glu

cose

lev

el (

mg/m

l)

Gelatinised milled cassava hydrolysed

with decorticated sorghum malts treated

with A. carbonarius

Gelatinised milled cassava hydrolysed

with undecorticated sorghum malts


CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

102


yeast growth on gelatinized cassava hydrolysate hydrolysed with decorticated and

undecorticated sorghum malt enzymes

The time course of yeast growth on gelatinized cassava hydrolysate hydrolysed

with decorticated and undecorticated sorghum malt (enzymes) treated with A. awamori is

shown in figure 31. The figure shows the gelatinized hydrolysate of cassava starch

hydrolysed with decorticated sorghum malt (enzymes) and treated with A. awamori giving

higher mean yeast optical density (16.96 OD600) than the counterpart hydrolysed with

undecorticated sorghum malt (enzymes) treated with the same organism (12.69 OD600).

During the course of fermentation there was a progressive increase in optical density

corresponding to yeast growth in both hydrolysates up to the 36th

hour. This was followed

by a slight decrease in the case of decorticated sorghum malt medium while in the other

undecorticated sorghum malt medium; there was a steady stationary growth. On the other

hand, time course of yeast growth on gelatinized cassava hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt (enzymes) treated with A. carbonarius is

shown in figure 32. The figure indicated that the optical density of yeast growth on cassava

hydrolysate using decorticated sorghum malt (enzymes) treated with A. carbonarius was

slightly higher (10.61 OD600) than the counterpart hydrolysed with undecorticated sorghum

malt (enzymes) treated with the same organism (10.48 OD600). During the course of

fermentation there was a progressive increase in the optical cell density (yeast cell) in both

gelatinized cassava starch hydrolysates medium that had been hydrolysed differently with

decorticated and undecorticated sorghum malt (enzymes) treated with A. carbonarius. This

increase was followed by a slight decrease and steady stationary growth. Aspergillus-

treated sorghum grains produced higher mean (12.86 OD600) optical density of yeast

growth than untreated controls (11.86 OD600). There was a significant difference(P<0.05)

in the optical density of yeast growth on gelatinized cassava starch hydrolysate due to the

decortication treatment, Aspergillus treatments and their interactions on sorghum samples

used as source of enzyme for cassava hydrolysis.

103

.

Fermentation time

(h)

0

5

10

15

20

25

6 12 18 24 30 36 42 48 54 60 66 72 6 12 18 24 30 36 42 48 54 60 66 72

Bio

ma

ss (

OD

600

nm

)

Gelatinised milled cassava hydrolysed with

decorticated sorghum malts treated with

A. awamori

Gelatinised milled cassava hydrolysed with

undecorticated sorghum malts treated

with A. awamori

Figure 31: Effects of decortication and inoculation with A. awamori spores on the time course of yeast growth on gelatinized cassava hydrolysate.

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

104

0

1

2

3

4

5

6

7

8

9

10

6 12 18 24 30 36 42 48 54 60 66 72 6 12 18 24 30 36 42 48 54 60 66 72


Bio

ma

ss

OD

600n

m

Figure 32: Effects of decortication and inoculation with A. Awamori spores on the time course of yeast growth on raw cassava hydrolysate

Raw milled cassava hydrolyzed with undecorticated sorghum malts

treated with A.carbonarius



CONTROL; 9.1X105; 1.82X106; 2.73X106

; 3.6X106; 4.55X106

105


yeast growth on raw cassava hydrolysate hydrolysed with decorticated and


Time course of yeast growth on raw cassava hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt (enzymes) treated with A. awamori is shown

in figure 33. As shown in the figure the mean optical density of yeast growth was higher

(7.50 OD600) in fermented cassava starch hydrolysate hydrolysed with decorticated

sorghum malt than the counterpart hydrolysed with undecorticated sorghum malt

(enzymes) though both were treated with the same organism (6.78 OD600). During the

course of fermentation there was an initial increase in the optical density of yeast cells in

both cassava hydrolysates followed by a relatively steady stationary growth. On the other

hand time course of yeast growth on raw cassava hydrolysate hydrolysed with decorticated

and undecorticated sorghum malt (enzymes) treated with A. carbonarius is shown in figure

34. The figure showed that a higher mean optical density of yeast cells (6.44 OD600) was

recorded in the fermented cassava starch hydrolysate hydrolysed with decorticated

sorghum malt (enzymes) treated with A. carbonarius than the counterpart that was

hydrolysed with undecorticated sorghum malt (enzymes) treated with the same organism.

During the course of fermentation, both medium had an initial progressive increase in yeast

cell optical density followed by a relatively inconsistent steady growth. Aspergillus-treated

sorghum grains produced higher mean (6.70 OD600) optical density of yeast growth than

untreated controls (6.05 OD600). There was, however, a significant difference (p<0.05) in

the optical yeast density on raw cassava starch hydrolysate due to decortication treatments,

Aspergillus treatments and their interactions on malted sorghum samples.

106

Raw milled cassava hydrolyzed with undecorticated

sorghum malts treated with A. awamori

Raw milled cassava hydrolyzed with decorticated


0

1

2

3

4

5

6

7

8

6 12 18 24 30 36 42 48 54 60 66 72 6 12 18 24 30 36 42 48 54 60 66 72


Bio

ma

ss

OD

600n

m

Figure 33: Effects of decortication and inoculation with A. awamori spores on the time course of yeast growth on raw cassava hydrolysate

CONTROL; 9.1X105; 1.82X106; 2.73X106

; 3.6X106; 4.55X106

107

Raw milled cassava hydrolyzed with undecorticated

sorghum malts treated with A. carbonarius

Raw milled cassava hydrolyzed with decorticated


0

1

2

3

4

5

6

7

8

6 12 18 24 30 36 42 48 54 60 66 72 6 12 18 24 30 36 42 48 54 60 66 72


Bio

ma

ss

OD

600n

m

Figure 34: Effects of decortication and inoculation with A. carbonarius spores

on the time course of yeast growth on raw cassava hydrolysate

CONTROL; 9.1X105; 1.82X106; 2.73X106

; 3.6X106; 4.55X106

108

Effects of decortication and inoculation with Aspergillus spores on the ethanol

production in gelatinized cassava hydrolysate hydrolysed with decorticated and


Ethanol levels in fermented gelatinized cassava starch hydrolysate that had been

hydrolysed with decorticated and undecorticated sorghum malt (enzymes) treated with A.

awamori is shown in figure 35. The figure indicated that fermented gelatinized cassava

hydrolysate hydrolysed with decorticated sorghum malt (enzymes) treated with A. awamori

produced more ethanol (3.87%) than the counterpart that was hydrolysed with

undecorticated sorghum malt (enzymes) treated with the same organism (3.28%).This was

confirmed by the Duncan’s new multiple range test for pair wise comparison of means.

During the course of fermentation, a relatively progressive increase in ethanol production

characterized the fermentation durations. Figure 36 shows ethanol production in gelatinized

cassava starch hydrolysate that had been hydrolysed with decorticated and undecorticated

sorghum malt (enzymes) treated with A. carbonarius. The figure shows that there was a

higher mean ethanol (2.95%) produced in fermented gelatinized cassava hydrolysate

hydrolysed with undecorticated sorghum malt (enzymes) than that produced by the

counterpart that was hydrolysed with decorticated sorghum (enzymes) treated with the

same organism (2.79%). This was again confirmed using Duncan’s new multiple range test

for pair wise comparison of means. During the course of fermentation there was a

progressive increase in the mean ethanol level produced in both cassava hydrolysate media.

Aspergillus-treated sorghum grains produced higher mean (3.32 %) optical density of yeast

growth than untreated controls (2.73 %). There was also a significant difference (P<0.05)

in ethanol production in fermented gelatinized cassava hydrolysate due to decortication

treatments, Aspergillus treatments but not with their interactions on the source of enzymes

used for hydrolysis (malted sorghum).

109

Gelatinised milled cassava hydrolyzed with undecorticated sorghum malts treated with A. awamori

Gelatinised milled cassava hydrolyzed with decorticated sorghum malts treated with A. awamori


0

1

2

3

4

5

6

1 2 3 4 5 1 2 3 4 5

% E

than

ol

Figure 35: Ethanol level in fermented gelatinized cassava hydrolysate hydrolyzed with sorghum malts treated with different levels of A.awamori

CONTROL; 9.1X105; 1.82X106; 2.73X106

; 3.6X106

; 4.55X106

110

Gelatinised milled cassava hydrolysed with undecorticated


Gelatinised milled cassava hydrolysed with decorticated



% E

tha

no

l

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 1 2 3 4 5

Figure 36: Ethanol level in fermented gelatinized cassava hydrolysate hydrolyzed with sorghum malts treated with different levels of A. carbonarius

CONTROL; 9.1X105; 1.82X106; 2.73X106; 3.6X106; 4.55X106

111

Effects of decortication and inoculation with Aspergillus spores on the ethanol

production in raw cassava hydrolysate hydrolysed with decorticated and


Ethanol levels in fermented raw cassava starch hydrolysate hydrolysed with

decorticated and undecorticated sorghum malt (enzymes) treated with A. awamori is shown

in figure 37. The figure shows that the mean ethanol levels in fermented raw cassava

hydrolysate hydrolysed with decorticated sorghum malt (enzymes) treated with A. awamori

recorded higher ethanol levels (3.39%) than the counterpart hydrolysed with undecorticated

sorghum malt (enzymes) treated with the same organism (2.79%). During the course of

fermentation there was a progressive increase in ethanol production using the two cassava

hydrolysate media. On the other hand, the ethanol levels in fermented raw cassava starch

hydrolysate hydrolysed with decorticated and undecorticated sorghum malt (enzymes)

treated with A. carbonarius shown in figure 38, indicated that the mean amount of ethanol

produced by samples treated with decorticated malt was slightly higher (2.51%) than the

counterpart produced by the undecorticated malt(2.41%). A progressive increase in ethanol

production during the course of fermentation was observed. Aspergillus-treated sorghum

grains produced higher mean (3.00 %) optical density of yeast growth than untreated

controls (1.80 %).There was also a significant difference (P<0.05) in ethanol production

using raw cassava starch hydrolysate due to decortication treatments, Aspergillus

treatments and their interactions on sorghum samples.

112


Figure 37: Ethanol level in fermented gelatinised cassava hydrolysate hydrolyzed with sorghum malts treated with different levels of A. awamori

Gelatinised milled cassava hydrolysed with undecorticated



hydrolysed with decorticated


% E

than

ol

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

1 2 3 4 5 1 2 3 4 5

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

113

CONTROL; 9.1X105; 1.82X10

6; 2.73X10

6; 3.6X10

6; 4.55X10

6

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 1 2 3 4 5


% E

than

ol

Raw milled cassava hydrolysed with

undecorticated sorghum malts treated with A. carbonarius

Raw milled cassava hydrolysed with

decorticated sorghum malts treated with A. carbonarius

Figure 38: Ethanol level in fermented raw cassava hydrolysate hydrolyzed with sorghum malts treated with different levels of A. carbonarius.

114

DISCUSSION

Decorticated sorghum malts treated with Aspergillus awamori and Aspergillus

carbonarius could have had more fungal growth apparently due to the absence of

inhibitory barrier to substrate (endosperm reserves) for the inoculated organisms. Thus the

tannin on the undecorticated sorghum surfaces could have delayed the growth of fungi

inoculant on their surfaces. This is similar to the findings of Shibuya and Iwasaki (1978).

Furthermore, the higher growth recorded by Aspergillus awamori over Aspergillus

carbonarius on both decorticated and undecorticated sorghum malts could be attributed to

species difference, possession of unique metabolic pathway and excretion of more specific

or general hydrolytic enzymes that aided in the starchy endosperm hydrolysis to utilizable

and fermentable monosaccharides, similar to the earlier report by Fukuda et al. (2001).

Values obtained were within acceptable range for malting as earlier reported by

Hough et al., (1971). There may have been more germination by decorticated sorghum,

probably if there were less interference. Recall that the sorghum grains had previously been

treated with acid which could have reduced the amount of oxygen availabile to the embryo

of the grains. The suitability of grains for malting is determined by parameters such as

germinative energy and water sensitivity. This is because these parameters show how

rapidly and uniformly grains would sprout, grow and subsequently bring about uniformity

in the finished products – malts.

Both decorticated and undecorticated grains recorded high germinative energies,

98% and 96%, and low water sensitivities, 3% and 4%, respectively. Values obtained are

within acceptable range for malting as earlier reported by Hough et al. (1971). Previous

studies on malting of sorghum showed that a germinative energy at 99% level could be

achieved with some sorghum varieties (Agu and Palmer, 1997). This suggests that even

germination will occur during malting of sorghum. Germination of grains is an essential

part of the malting process because when grains do not germinate, or germinate poorly,

they do not contribute to the enzyme development of the malt and uneven modification of

the malt occurs. Sufficient enzyme modification of the endosperm substrates will not be

achieved and will result in sub-optimal extract development. Also important is that

115

ungerminated grains have been shown to be ready sources of microbial infection during

malting of grains (Agu and Palmer, 1997). This could lead to the production of malt with

potentials to develop aflatoxins during the brewing process (Agu and Palmer, 1997). There

may have been more germination by decorticated sorghum, probably due to less

interference having been treated with acid that could have reduced oxygen availability to

the embryo of probably infected undecorticated sorghum samples.

Germinative Energy (GE) of decorticated sorghum malts was slightly higher than

those of undecorticated sorghum samples due probably to the quick accessibility of

moisture that aided the mobilization of the endosperm and embryo reserves as the

decorticated samples no longer possessed outer coverings due to the sulphuric acid

treatment which perhaps had further reduced probable contaminating indigenous

microflora that could have lead to reduction in oxygen availability to the embryo due to

competition. On the other hand excess moisture level might have accounted for the lower

water sensitivity (WS) exhibited by decorticated sorghum samples over those of

undecorticated samples due probably to suffocation or anoxia due to oxygen unavailability

leading to reduced number of grains that germinated.

Rootlet growth during germination of cereal grains has been associated with

malting loss (Ilori and Adewusi, 1991). Growth is dependent on the availability of

sufficient moisture absorption during steeping prior to germination. Therefore growth is

associated with reduction in dry matter, lost as carbon dioxide and water (respiration) and

rootlets leading to malting loss i.e. dry weight loss in converting sorghum grains to malts.

Thus a balance must be struck – enough growth must be allowed to produce well-modified

malt, not allowing malting losses to become excessive. This was the basis of withdrawal of

part of the germinated samples every 24h - in order to monitor the extent of modification.

The presence of rootlets is therefore an outward evidence of the ongoing modification

within the germinating sorghum endosperm following sufficient uptake of moisture. Initial

higher root length shown by decorticated sorghum malts could be as a result of easier

imbibition of moisture and access to more aeration during steeping and air-rest

respectively. The undecorticated sorghum samples recording higher root lengths towards

the end of the germination period could be attributed to the fact that they were able to

116

imbibe and retain relatively moderate moisture and had lesser moulds competing for

nutrients and moisture unlike the counterpart that were stuffed with the starter cultures and

possibly impairing nutrient availability processed for germination purposes.

-Amylases are endo-enzymes which randomly act on the -1-4 links of starch

releasing small dextrins and fermentable sugars. One of the major objectives of this study

concerned the quality improvement of sorghum malt using the two Aspergillus stater

cultures. These qualities among others are amylase mobilization and generation in the

malted sorghum malts. Noots et al. (2001) had reported the improvement of malt

modification by the use of Rhizopus VII as stater cultutre. The results of this study with

respect to amylase activity are in agreement with this report. Alpha amylase also catalyses

random hydrolysis of starch chain at -1, 4 glucosidic linkages distant from the ends of the

chain and from -1, 6 linked branches in the chains (Hough et al., 1971); its formation at

the embryo requiring adequate oxygen (Palmer et al., 1989a)

β-Amylases (exo-enzymes that release maltose molecules from the non-reducing

ends of amylose and amylopectin) was however higher in non- inoculated sorghum malts

than inoculated sorghum malts probably indicating some kind of inhibitory effects on β-

amylases by inoculated organisms in inoculated malts. Taylor et al. 1993 had reported that

non- germinated sorghum grains showed no β–amylase.

Diastatic power (DP) of malts represents to what extent carbohydrate utilising

enzymes in general are expressed during malting. Contrary to earlier report by Etok Akpan

(1992) that β-amylase contributes less DP in malted sorghum than -amylase unlike in

malted barley, β-amylase of inoculated sorghum malts in this study actually gave higher β-

amylase than -amylase. This novel observation would go a long way in presenting

sorghum malts as a suitable substitute as raw material requiring abundance of β-amylase, a

very important saccharifying enzyme employed for starch hydrolysis (Norris & Lewis,

1985)

Free Amino Nitrogen (FAN) levels represent the major source of assimilable

nitrogen for brewing yeasts (Enari and Sopanen, 1986) mostly derived from proteolysis

during malting (Jones, 1969; Taylor 1986; Pickerell, 1986; Taylor and Boyd, 1986). The

117

rates of amino acid and protein metabolism during grain germination as major factors in

FAN determination in sorghum malt had been identified (Taylor, 1983). Being a product of

hydrolysis of solubilised proteins, FAN is essential for anabolic functions of germinating

seedling and as nutrients for yeast metabolism in wort (Dale et al., 1990).FAN levels along

with levels of simple sugars are parameters indicating the extent of enzyme availability and

hydrolysis of endosperm reserve in malted grains such as sorghum malts. This determines

to a great extent the level of CWE and HWE realizable from malts during mashing

(Holmes, 1991). Factors considered in this study exerted significant effects on sorghum

malt FAN as inoculated sorghum malts recorded very high FAN levels compared to non

inoculated controls indicating that extra enzyme delivery into the endosperm obviously

took place due to the establishment of starter cultures on the inoculated sorghum grains.

Physiological differences, efficiency and difference in major enzyme characteristics and

rate of protein variations in grain protein structure and degradability (Riggs et al., 1983)

could explain why A. awamori inoculated sorghum grains recorded higher FAN levels than

A. carbonarius inoculated grains. This same reason could be attributed to different

improved levels of FAN due to decortication – because there must have been better peptide

translocation process supporting better FAN development (Okolo and Ezeogu, 1996b).

Other protein related indices determined such as cold water soluble protein (CWSP)

and total non protein nitrogen (TNPN) all showed highly significant improvements

apparently brought about by the starter cultures inoculated on sorghum grains at the onset

of malting as well as decortication, both factors ostensibly encouraging higher rate of

anabolic protein turnover, translocation and accumulation (Okolo and Ezeogu, 1996b)

through increased initial moisture uptake and better modification in the endosperm reserve.

Cold Water Extracts (CWE) are soluble products of enzyme hydrolysis from

malting process and these include readily available sugars and amino acids within the

endosperm of malts (Holmes, 1991). Also, malt extracts are dissolved materials present in

wort derived from grist according to Briggs et al. (1986). Malt modification brought about

by hydrolytic enzymes during malting has been shown to be enhanced in sorghum malts

using starter cultures (Noots 2001). This enhancement among other parameters treated

above also very importantly include the level of soluble products of sugars and amino acids

118

solubilised with the aid of improved hydrolytic enzymes which accumulated in the grain’s

endosperm. Cold water extract (CWE) measures part of the soluble products utilised by the

embryo of sorghum grains during germination. Factors studied greatly contributed to

increased CWE indicating better modification in inoculated sorghum malts than non

inoculated ones and subsequent availability of higher soluble products.

Hot water extract (HWE) being the analytical measure of the quantity of dissolved

solids in sweet wort however gave a slightly higher HWE in non inoculated controls than

wort of inoculated malts due probably to variations in the enzyme complements contributed

to the mash by the malt, or starter cultures; mashing temperature regime and duration of

mashing.

Cold-water soluble carbohydrate (CWSC) is a soluble product of CHO origin from

enzyme hydrolysis during malting. CWSC recording highly significant improvement in

sorghum malts inoculated with the starter cultures as well as ones given decortication

treatments is an indication that there was increased enzyme generation from starter cultures

in the treated grains.

The combined actions of alpha amylase, beta amylase and other hydrolytic enzymes

present in the malt extract that had been consequently enhanced and improved by factors

studied must have lead to higher glucose mobilisation during mashing in both sorghum

malts and cassava starch prior to fermentation. It is only natural, therefore that high glucose

realization from starch reserves of sorghum malts and cassava starch using enhanced

enzyme extracts would lead to higher ethanol production during fermentation as was

observed and recorded in this study.

Cold water soluble protein modification index demonstrates to what extent malt

proteolytic enzymes initiate the mobilization of grain endosperm reserves, by hydrolysing

endosperm matrix proteins and thus exposing the embedded starch granules to amylosis

(Palmer, 1989a). This is also analogous to Kobalch index.

In conclusion, this study has as a matter of fact, therefore given further support to

the possibility of improving malt quality by the use of starter cultures and decortication as

factors necessary in this respect and consequently enhanced starch hydrolysis using

enzyme extracts from these improved sorghum malts bringing about subsequent enhanced

119

ethanol production from hydrolysed fermented cassava starch hydrolysate. This research

work has therefore, comprehensively shown that decortications and treatment with A.

awamori and A. carbonarius significantly influenced the most of the parameters studied.

120

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