University of Nigeria Research Publications

Aut

hor

UGWUIDU, Ernest Ejiofor

PG/M.Sc/99/26310

Title

Evaluation of the Influence of Steeping Conditions on Sorghum Malt and Wort Quality

Parameters

Facu

lty

Biological Sciences

Dep

artm

ent

Microbiology

Dat

e March, 2006

Sign

atur

e

A THESIS SUBh~IIrIrIED TO '11-1. E DEI'A1<1'iVlENT OF Wl lCROBIOI,OC~Y,

F A C U L T Y 01: BIOL0C)CAL SCIENCES UNIVEIISITY OF NICI3IXIA,

NSUI<I<A

IN I'Al<'I'1,41, I ; ~ J I , I ; I 1,h.I k:Nrl' 0 1 ; 'l'H LC IiEQUI I<I<hl Kh'l' t:oU T H E AM A R D 01; iVIASrI'EIX 01; SCIENCE DLGIIEE (MSc)

IN IIIIC'ROBIOI~OGY (B1<11\1'ING SCIENC'IS AND ' l I ~ C I ~ N O l , O G Y ) , LJNIV1<RSlTI3' OF 5 I G E R l A

NSIJ I<I<A

CERTIFICATION

UGWUIDU ERNEST EJIOFOR, REG. NO. PG/MSd99/26310, a postgraduate

student in the Department of Microbiology, has satisfactorily completed the

requirement for the award of Master of Science Degree (MSc) in Microbiology

(Brewing Science and Technology).

The work embodied in this thesis is original and has not been submitted in

part or in full for any other diploma or degree of this or any other university.

- - ------------------------------------------- PROF. B.N. OKOLO Supervisor Department of Microbiology

- - e m - cb*w- - .. -. . - -- - - -- - - - - - - - DR, J.O. UGWUANYI Supervisor Department of Microbiology

MARCH, 2006

DEDICATION

This Dissertation is dedicated to members of UGWUIDU'S family.

ACKNOWLEDGEMENT

I am very grateful to Omniscient God for making this thesis a great

success. My sincere gratitude goes Prof. B.N. Okolo and Dr. Ugwuanyi, J.O. my

research supervisors who laboured conscientiously through moral instruction,

academic and physical endeavours to bring this work to a success.

I acknowledge with gratitude the laboratory assistance received from Dr. .

L.I. Ezeogu and Dr. I.N.E. Onwurah (Department of Biochemistry) for providing

some of the facilities and thrust used in this work.

I must not fail to express my immense appreciation to all the lecturers in

the Department of Microbiology for their academic help and upbringing. Many

thanks go to my departmental colleagues whose encouragement and support

necessitated the completion of this work. They are Awah Nsikak, Ukwuru

Michael, Ire Francis, Nweze Emeka and Eze Emmanuel,

Also the financial support of my beloved parents, Mr. and Mrs. C.O.C.

Ugwuidu that saw me through this work is highly appreciated.

May God bless them, Amen.

UG WUlDU E. E.

TABLE OF CONTENTS

Title page - -

Certification - -

Dedication - -

Acknowledgement -

T a b k of Contents -

List of tables -

List of figures- -

Abstract -

CHAPTERONE - - - Introduction and Rei~iew of Literatures -

CHAPTERTWO - - Materials and Methods -

I 1,

' 1

CHAPTER THREE - - - Results - - - -

\ C

\

CHAPTER FOUR - Discussion - - -

CHAPTER FIVE - Conclusion -

Appendix I -

Appendix II -

References -

LIST OF TABLES

1. Proximate Analysis of Sorghum grain- -

2. ANOVA table for the proximate analysis of sorghum grains.

3. Proximate Analysis of sorghum malt -

4. ANOVA table for the proximate analysis of sorghum malt. - -

5. ANOVA table for the development of moisture content during

Air-rest ar;d Continuous steep. - -

6. ANOVA table for the development of cold water extract during

Air-rest and Continuous steep. - -

7. ANOVA table for the clevelopment of cold water soluble carboliycfrate

during Air-rest and Continuous steep. - - - -

8. ANOVA table for the development of Hot water Extract during

Air-rest and Continuous steep. - - - - -

9. ANOVA table for the development of Hot water Extract proteir

during Air-rest and Continuoirs steep.

10. ANOVA table for the development of Total Non-protein Nitrogen

during Air-rest and Continu0u.s steep. - - - -

11 .ANOVA table for the development of Free Alpha Amino Nitrogen

during Air-rest and continuous steep. - - - - -

12.ANOVA table for the development of Proteinase Activity during

Air-rest and Continuous steep.

13.ANOVA table for the development of Carboxypeptidase Activity

during Air-rest and Continuous steep. - - - - 14.ANOVA table for the development of Glucanase Activity during

Air-rest and Continuous steep. -

15. ANOVA table for the development of Diastatic Power during

Air-rest and Continuous steep. -

1G.ANOVA table for the development of Alpha Amylase Activity

during Air-rest and Contin~lous steep. - - - -

17.ANOVA table for the development of [;eta Amylase Activity during

- Air-rest and Contmuous steep. - - - - 59

18. ANOVA ta tie for the development of Wort fer-mentabil~ty dur~ng

Air-rest and Continuoi~s steep. - - - 60

19.ANOVA table for the development of Wort fermentable Extract

during Air-rest and Continuous steep. - - 6 1

LIST OF FIGURES

Development of t-noisture content of the three sorghum cultivars

during steepmg using Air-rest and Continuous sleep Reg~rnes. -

Development of Cold water Extract of the three sorghum cult~vars

during steeping using Air-rest and Continuous steep Regimes. -

Development of cold water soluble carbohydrate of the

three sorghum cultivalrs during steeping using Air-rest and

Continuous steep Regimes. -

Development of Hot water Extract of the three sorghum

cultivars during steeping using Air-rest and Continuous

steep Regimes. - - -

Development of Hot water Extract protein of the three

sorghum cultivars during steeping using Air-rest and

Continuous steep Regimes. -

Development of Total Non-Protein Nitrogen of the three

sorghum cultivars during steeping using Air-rest and

Continuotis steep Regimes. - - -

Development of Free Alpha Amino Nitrogen of the three

sorghum cultivars during steeping using Air-rest and

Continuous steep Regimes. -

Development of Proteinase Activity of the three sorghum

cultivars during steeping using Air-rest and Continuous

steep Regimes. - - - - - -

Development of Carboxypeptidase Activity of the three

sorghum cultivars during steeping using Air-rest and

Continuous steep Regimes. - - - - -

10. Development of Glucanase Activity of the three sorghum

cultivars during steeping using Air-rest and Continuous

steep Regimes. - - - - - - -

11. Development of Diastatic Power- of the three sorghum

cultivars during steeping using Air-rest and Continuous

steep Regimes. - - - - - - -

12. Develonrnent of A l ~ h a Amvlase Activitv of the three sorahuni

cultivars during steeping using Air-rest and Cont~nuous

- steep Regimes. - - - - - - 58

13. Development of [;eta Amylase Activlty of the three sorghum

cultivars during sleeping uslng Air-rest and Continuous

steep Regimes. -- - - - - - - 59

14. Development of Wort Fermentability of the three sorghum cultivars

during steeplng using Air-rest and Continuous steep Reg~rnes. - 60

15. Development of Wort Fermentable Extract of the three sorghum

cultivars dur~ng steeping uslng Air-rest and Continuous

steep Reg~rnes - 6 1

ABSTRACT

The influence of steeping regimes on some sorghum malts and wort quality

properties were studied for three improved Nigerian sorghum cultivars; lCSV 400, , . SK 5912 and KSV 8. Steeping was carried out with distilled water at 30•‹C, After

6h of steeping, half of the grains were removed from the water and their steeping

interrupted with air-rest period. This batch was designated as air-rest samples; ,

with cycle time 6hr wet 3hr air rest for 45hr followed by 6hr final warm water

steep at 40•‹C. The remaining half had their steeping water changed and returned

back to water and was designated as continuously steeped samples. The

continuously steeped grains were completely immersed in water throughout the . #

51 h steeping period at 30•‹C. Samples were collected every 6h in both air-rest

and continuous steep. Grains were germinated for 5 days and the malt kilned at

50•‹C for 24h. Parameters evaluated include: proximate analysis of the grains;

Cold Water Extract (CWE); Cold Water Soluble Carbohydrate (CWS-COH), Hot . '.

Water Extract (HWE), Hot Water Extract Protein (HWE-Protein); Free alpha

amino nitrogen (FAN); Total Non-protein Nitrogen (TNPN); Carboxypeptidase

and Proteinase activity, Glucanase, Diastatic power and Amylase activity, Wort

fermentability and Wort Fermentable Extract. Two-way analysis of variance

(ANOVA) revealed that: CWE, CWE-OH, HWE-Protein, TNPN,

Carboxypeptidase, Proteinase, Glucanase, Diastatic power and Amylase activity

were significantly (Pe 0.05) influenced by steep treatment and cultivar.

Proteinase, Carboxypeptidase, Glucanase, Diastatic power and Amylase

activities were also influenced by the possible pair-wise interaction of cultivars

and steep treatments. After 48h of fermentation, both wort fermentability and wort

fermentable Extract were significantly (PC 0.05) influenced by steep treatment

cultivar and their pair-wise possible interaction.

CHAPTER ONE

lNTRODUCTlON AND LITERATURE REVIEW

The grass family, gramineae bears many genera of cereal plants which

includes wheat, barley, cwn, rye, mallet, oat, and sorghum. Members of this family

are all monocotyledonous plants. The embryo of the grain contains only one

cotyledon that is catled scutellurn (Palmer et a/., 1989). Plants in this Gramineae

family are both of industrial (commercial) use as well as providing food for domestic

use. To this family belongs the cereal grain sorghum, which has gained wide

oapularity in tropical brewing industries. Two best known species of sorghum are

Sorghum vulgare and Sorghum bicokor (Palmer et a/., 1989). Sorghum grains vary

from red, black, and brown to fawn, yellow and white. The polyphenolic materials

responsible for this colour variation are located mainly in the pericarp-testa region

(Palmer et al, 1989).

Sorghum grains produced in Nigeria are classified into two, namely the Kano

race - the Sorghum caudafum sub species coffars and Sorghum yuieanses. Other

indigenous varieties of sorghum include Guinea, Kaura, Farafara, and Chadrace.

The commercial value of sorghum lies in its use as a food source for human

and animals and as potential brewing and distilling material for the production of

regular beers and spirits. Sorghum has become the grain of choice as possible

replacement of barley in lager beer brewing in most developing countries of the

tropics, e.g, Nigeria for the following reasons:

I . The tropical climate is suitable for growth and mass production of the plant

(Palmer et a/. , 1985)

2. The species and varieties have been found able to withstand the tropical

pests and disease (Palmer et al., 1985).

3. For economic reasons and to reduce and conserve foreign income and

financial losses. (Ezeogu and Okolo, 1994)

4. The increasing demands and high cost of barley by most European brewing

industries have forced the use of local cereals particularly sorghum to become

the malting and brewing grain for most tropical countries. (Ezeogu and Okolo,

1994).

This has led to the development of a local malting industry of which sorghum

malt sprout is a significant by-product. Unfortunately, this by-product, which promises

to be a major source of wastage and environmental problem has been little studied

with respect to its composition and possible usage (Palmer, 1989).

Malting, briefly defined as controlled germination (Enari and Sopanen, 1986)

i's essentially a biological process in which the germination of sorghum is carried out

in controlled environments. It involves three processes namely, steeping,

germination and kilning. There is now increasing interest in the malting of sorghum

but the observed differences in grain composition, physiology and biochemistry of

germination have necessitated the development of unique malting regimes for

sorghum {Ezeogu and Okolo, l994;1995).

The main purpose of malting is to modify storage protein reserve in favour of

assimilable nitrogenous materials essential for adequate yeast growth and

fermentation (Baxter, 1980; Jones, 1985, and Pierce. 1982). However, the use of air

rest cycles in combination with warm water steep has been found to produce good

quality sorghum malt suitable for brewing industries (Ezeogu and Okdo, 1994,

Pierce, 1982).

It is the desired intension of the researcher to find the effects of air rest

periods, steeping conditions and final warm steep on other parameters such as

percentage moisture contents; root and shoot developments; germination energy

and water sensitivity; hot and cold water extracts; total non-protein nitrogen; free

alpha-amino nitrogen; cold water soluble carbohydrate; hot water extract protein

fraction; diastatic power; alpha and beta amylase activity; glucanase activity;

proteinase and carboxypeptidase activity, wort fermentability and fermentable

extracts

Three improved Nigerian sorghum cultivars KSV 8, SK 5912 and ICSV 400

were used. This research work will be useful in the improvement on the use of

sorghum as a brewing raw material and a guide in minimizing malting losses, as well

as serving as a stepping stone in further research in the brewing industry.

1.1 Sorghum Species

The grass family, gramineae is composed of many genera to which the genus

sorghum is one of them. This genus has the species S. vulgare and S. bicolor as the

two most important species that have gained a wide popularity and use in brewing

industry. Sorghum is the fifth most widely grown cereal crop in the world with an

annual production of about 65 million tonnes (Palmer ef a!., 1989). Sorghum grows

well in the semiarid regions, Tropical countries as well as in Oceania. Because of

this, sorghum is ubiquitous in production. The main sorghum producing countries are

the USA, China, India, Nigeria, Mexico and Australia to mention just a few

(Pomeranz, 1989). These countries produce over three quarter of the total crop.

Annual production in the U.S.A , is about 21 million tonnes and Nigeria it is about 3

million tonnes.

Hoseney and Davis (1979) examined the structure of sorghum grains using

samples that represent a wide genetic type with scanning electron microscopy. They

found that sorghum grain has both hard and soft endosperms. The soft or opaque

endosperm was characterized by relatively large intergranular air spaces. The starch

was observed to be essentially round and covered with a thin sheath of protein. They

observed that relatively large spherical portion was characterized by a tightly packed

structure with no air spaces. They found that starch granules were polygonal and

covered with a thin protein matrix.

Aisien et a/. (1986) studied the parenchyma tissue of ungerminated and

germinated sorghum. They found out that the grains contain abundant fat and

protein deposits which undergo various metabolic changes during germination and

seedling growth, The presence of these and other organelles are characteristics of

the mature plant cell. During early period of germination process in sorghum (72h)

there is rapid starch degradation.

-.. - 'I,

1 .I .2 Grain Properties

Sorghum grain is a caryopsis, i.e. a dry fruit. The pericarp is the fruit wall and

the testa - the seed coat. Thus cereal grains are fruits and not seeds, (Palmer eta).,

1989). Harvested sorghum grain does not have a husk. The grain is round, and its

pericarp-testa has a higher level of polyphenol plgrnentation. These polyphenol

pigments of some sorghum cultivars are unpalatable to some birds. Such grains are

referred to as bird proof. These pigments are often referred to as tannin and it

comprises flavanoids and anthocyanidins which cause haze in finished beer.

The aleurone layer of sorghum comprises a single layer of cell that contains

storage lipids and proteins, as well as small amount of phosphorus and potassium,

(Palmer et a/., 1989). Sorghum aleurone layer also contains less phytic acid

(phosphate) and because of this, it is less likely to cause chelation of metal ions

when consumed.

The embryos of sorghum grains are large and contain most of the lipids,

which are located in the scutellum of the embryo. The grain contains more

unsaturated lipids. (e.g. linoleic) than the saturated ones. Sucrose is the main sugar

sucrose and starch are synthesized in the scutellum during subsequent seedling

growth: Amylolytic action in the starchy endosperm of sorghum during malting

produces mainly glucose which is converted in the embryo to sucrose (Aisien, 1982;

Aisien and Palmer, 1983). Sorghum grain contains the B group vitamins such as

thiamin, niacin and riboflavin which are presumed to be located mainly in the embryo

and the aleurone layer (Pomeranz, 1989).

The starchy endosperm of sorghum is the largest tissue and it consists mainly

of starch granules; storage proteins and cell wall materials. The storage proteins

(prolamms and glutelins) and starchy granules are enclosed in the endosperm cells,

the prolamins being more abundant and soluble in alcohol (Hoseney, 7986;

Pomeranz, 1989; Palmer, 1989). The inner layer of this sorghum endosperm is

loosely packed and is said to be meaiy (opaque) while the outer layer is tightly

packed and is said to be steely (vitreous). The starchy granules are about lOmm En

diameter and are associated with proteins and this makes them look amorphous in

nature. One of the most important physical properties of the starch granules of

sorghum is its gelatinization temperature. The starch granules of sorghum (about

10mm) gelatinize at about 75% and this influences the rate at which starch

solubilizes and get hydrolysed by amylolytic enzymes during mashing. Even in

excess of m-amylase and p-amylase, sorghum starch conversion was sub-optimal

below 8Q•‹C (Igyor, 1987). Here it is clear that sugar production in the malting grains

gives no indication of potential of enzymatic conversion of starch during mashing.

The endosperm eel! wall of sorghum contain about 4% pentosan, 28% p-D gtucans

and about 62% adhering proteins. Sorghum grains vary widely in compositicm ar~d in

properties depending on variety, location and climate (Wall and Ross, 1970).

The average chemical composition of sorghum is as follows:

Protein 1 1 .2%

Fats 3.7%

Reducing sugar 1.8%

Crude fibre 2.6%

Wax 0.3%

Ash 1 .S0/o

Starch 74.1%

Tannin 0.1 O/O

Pentosan 2.5%

Moisture 16.5%

(Wall and Rose, 1970).

Sorghum is a source of calcium and small amount of iron to the body. It has

two main types of starch; amylase which is a polymer of glucose units linked by

(alpha) a-1-4 linkages and amylopectin, which has in addition to 01-1-4 linkages

about 55% of a-1-6 linkages that gives a branch structure - Amylose content of

starch in sorghum is about 23% - 28%, the rest is amylopectin. Sugar content of

mature sorghum grain is about 0.9% - 2% (Wall and Ross, 1970).

Chavan et a1 (1979) in their work reported that the polyphenols in the form of

condensed tannin protected the grain against degradation by birds but could reduce

the nutritional quality of the grain.

Narziss and Sumrell (1980), analysed for the relative tannin contents of the

whole seed and the mineral content of the five varieties. They found that many of

essential minerals were located in the pericarp aleurone area of the seed. However,

Heerden (-I9871 anatysed commercially brewed sorghum beer for tannin, riboflavin,

nicotinic acid, minerals and protein. They found out that sorghum beers have high

thiamin, riboflavin and minerals. Also they found that sorghum beer can make

significant contribution to the nutrient requirement of the consumer's diet particularly

when whole sorghum is used as adjunct.

1.2 Sorghum Enzymes

Enzymes present in sorghum are alpha amylase, beta amylase,

amyloglucosidase, alpha (a) glucosidase, dextrinase, pullulanase, protease,

cellulase and endo beta glucanase (Aisien and Palmer, 1983; Etokakpan and

Palmer, 1989).

Physiologically in sorghum, a-amylase and carboxypeptidases are produced

by the scutellum. Endo P-1,3, glucanase, limit dextrinase and endoprotease develop

in the starchy ~ndosperm (Aisim et a/., 1986; Palmer, 1989) Malted sorqhim L. qrains

do not have high level of the endo P-I, 3:1:4- glucanase and P-amylase since there

is no evidence that the aleurone of sorghum grains can produce and secrete

endosperm - degrading enzyme during malting (Daiber et a/., 1973; Aisien et a!.,

1986; Glennie and Weight, 1986; Etokakpan, 1990; Palmer, 1989).

Another important physiological feature of sorghum grain is that it contains

low level of the important diastatic power enzymes - pamylase - possibly because of

the low level of sallt soluble proteins (Aisien, 1988; Palmer, 1989; Etokakpan and

Palmer, 1990). This sorghum malt therefore produces worts, which contains

significantly low level of maltose and are less fermentable.

The functions of these different enzymes during malting include the following:

1.2.1 Amylases - These are principally of two enzyme types namely, alpha (a )

amylase and beta @)-amylase.

Sharnber et at. (1989) carried out a research on enzyme and acid hydrolysis

of sorghum. The grains were steeped at 25•‹C for various time, then germinated at

10'C - 30•‹C for 24 hours, followed by steeping for 15 min. The unmafted and malted

grains were analysed for moisture, amino acid and carbohydrates. They also studied

enzyme and acid hydrolysis of the grains. They found that germination after 24h at

20•‹C - 25OC was goad for malting and the optimum temperature far amylase activity

was 50•‹C - 75•‹C for sorghum. They concluded that enzymic hydrolysis resulted in a

low yield of sugar while acid hydrolysis resulted in a higher yield of glucose.

Ezeogu and Okolo (1994) in their study on the effect of air-rest and warm

steep on malt quality of sorghum cultivars reported that or and 0-amylases activity

were significantly affected by length of air rest. They found out that optimum a and P-

amylolytic activities were achieved when the air rest period is 3 hours. Their

observation further confirms the finding that combination of final warm steep and air

rest cycle can significantly increase the saccharifying contribution of 13-amylase to

the total reducing power of the malted sorghum, thereby concluding that sorghum

malts possessed little or no Ij-amylase activity. Ezeogu and Okolo (1994) in their

work using two sorghum varieties found out that combined effect of warm steep and

air rest period have a significant effect on both cold water extract and hot water

extract. Cold water extract was highest when the air rest period was 3 hours and so

was that at the hot water extract. They opined that careful selection of steeping

regime could lead to improved sorghum malt diastatic power, a- and 0-amylase

activity, extract yield and other important malt properties while at the same time

reducing malting loss and rootlet growth.

Alpha (a)-Amylase - is involved in carbohydrate breakdown (Sanwo and

Demazon, 1992). It is an endoenzyme which randomly attacks the u-1,4 linkages in

the inner region of the substrate liberating glucose and oligosaccharides. The

oligosaccharides are then further degraded into a mixture of maltose, glucose,

maitotriose and dextrins. The a-amylase by-passes a-I, 6 linkages during its

hydrolysis and rapidly decreases the turbidity and viscosity of the starch.

Beta @)-amylase - is an exo enzyme that attacks the penultimate a-1,4 bonds at

the non-reducing end of the substrate, releasing p-maltose. It cannot hydrolyse a - I ,

6 linkages and therefore leaves a residual p-limit dextrins and slowly decreases the

turbidity and viscosity of the starch, (Wall and Ross, 1970). No (-l-amylase is found in

ungerminated sorghum grain but a-amylase is found in small amount as both free

and bound enzymes (Wall and Ross, 1970). The bound a-amylase requires protease

activity to release it into solution. According to Wall and Ross 1970, sorghum grains

have less cr-amylase activity than barley in the ungerminated state. Malted sorghum

has more u-amylase than unmalted one. The a-amylase is stable to heat up to 70•‹C.

Its optimum pH is 4.6 from 20•‹C to 40•‹C and requires calcium ions for its activities.

Dyer and Novelie (1986) studied the distribution and activity of alpha and beta

amylase in germinating sorghum grain. They showed that both a- and P-amylase

were produced during the germination of sorghum and that in any particular malting

trial, the enzymes ratio to one another remained approximately constant throughout

the trial. They found that the actual value at which the ratio remained constant

depends on the temperature of the malting and variety but not on the watering

treatment given during malting. The amylase concentration in the embryo was

observed to be usually higher than in the endosperm.

1.2.2 Diastatic Power

Marrall et a/. (1986) studied the effect of germination time, temperature and

moisture on malting of sorghum. They observed that diastatic power of malt

increases with germination time. A rapid increase occurred up to day 4 of

germination and then the diastatic power increased slowly with germination time or

reached a peak, then declined slightly. They showed that free fatty acid (FAN) and

malting loss all increased with increased germination time. They also found that

germination temperature of 24'C and 28' are both equally good for development of

diastatic power, FAN and extract; but higher temperatures are progressively worse.

Germination temperature over the range of 24OC - 36'C have no effect on malting

loss but malt diastatic power. FAN and malting loss, are all in general increased by

higher germination moisture. They further observed that higher moisture has a

negative effect on diastatic power towards the end of malting and the optimum

moisture content for the rapid germination of sorghum is 35% - 40%. They concluded

that to get a sorghum malt with a high diastatic power, FAN, and extract, a

germination temperature of 24OC - 28OC should be used.

Etokakpan (1992) examined the arnylolytic potential wort fermentability of

Nigerian sorghum, and found out that, the diastatic power of four 'improved' Nigerian

sorghum cultivars were produced principally by a-amylolytic activity. Free amino

nitrogen levels and extracts were high when the sorghum was mashed in a modified

procedure in which the separated active wort was added to the gelatinized (and

cooled) sorghum starch of the mash. The percentage fermentability of the sorghum

wort ranged from 76% to 79%. Maltose level in the wort was about ?.5mglmL.

Etokakpan reported that in all sorghum varieties, the reducing power resulting from

(L-amylase activity and 0-amylase is either absent from sorghum malts or present in

very low amount. Etokakpan therefore opined that the low diastatic power of

sorghum is not as a result of restricted protein and soluble nitrogen development.

1.2.2 Glucosidases

These are of two types namely: the amyloglucosidase and alpha (u-)-

glucosidase.

Amyloglucosidase - acts as a mashing aid. This enzyme can degrade both the a-1,4

and a-1,6 linkages in starch to produce mainly glucose.

Alpha (a) glucosidase - acts as secondary enzyme because it hydrolyses only

dextrins released by 0,-amylolytic attack of the starch granules which are highly

insoluble in water. Alpha-glucosidase is responsible for high amount of glucose

formed when sorghum malt is mashed {Zhuotoasum and Henson, 1991).

1.2.3 Endo Beta Glucanases

Glucanases are groups of enzymes that act and perform similar functions

collectively. This group of enzymes collectively or individually degrade different types

of p-glucans to 1, 3, D-glucose (Bamforth and Martin, 1981).

Bamforth and Martin (1981), outlined three different categories of these

enzymes namely (a) Endo-p-1,3, glucanase, (b) Endo p-1,4 glucanase and (c) Endo

barley beta gtucanase.

p-1,3 Glucanase as the name implies is specific in the degradation of 1;-1,3

glucan, and therefore, play a major role in digesting the walls of these central

endosperms of malts. They, however, have no action on the P-glucans extractable

from flakes and denatured barley. Their main role is to hydrolyse the 13-glucans

which dissolve early in malting (Bathgate, 1982).

P-Glucan is one of the structural polysaccharide in sorghum arid is derived

fiom the polymerization of p-0-glucose monomers. The three types of linkages

established were p-(1+3), [1-+4) and (1+6). Sorghum 0-glucan account for only

about 0.2-0.5% of the water soluble endosperm starches while p-O-glucan in barley

is about 2.7-3.8%. These p-glucans are of central focus in the brewing of

conventional beer since their degradation affects the diffusion of catabolic enzymes

during malting processes

Yeast P-glucans are composed of glucose in p-(1+4) and (1+6) linkages

while those in Basidomycetes OM 806 mycelial wall contain glucose unit joint by a

mixture of p-(1-+6), (1 -4) and (1 -3) linkages.

The P-glucan in sorghum, though far less than that found in barley, is not well

degraded by the catabolic enzymes 'in vivo' thereby causing the poor filtration

problem in the brew house.

Sorghum P-glucan is built on two distinct types of polysaccharides chains x

and y. The x-chains are shorter and highly branched in a manner reminiscent of

amylopectin with configuration of ( 7 -3) linkages of b-D-gtucopyranosyl residues and

about 7% of the P-glucan. The y-chains are longer with less branches and a

configuration structure of (1-4) linkages of P-D-glucopyranosyl residues and is

diiuul W , d the I I - ~ I U L ~ I L The rtiulai I& (18.3) d (C1, C3, CG) a ~ ~ d (C1, C4. CG)

bearing the glucosidic bonds suggest that the polysaccharides chains (x) contains

85-86% of the (1 -6) bonds at the branch points while the y-chain bears only about

3 4 % of (1+6) bonds at the branch points, respectively. A protein named friabilin

appears to be an integral part of the polymer. This protein, which may be enzyme

are involved in the biosynthesis of relevant cell wall materials and its integration with

the p-glucan chains may further impedes enzymes activity that may result in poor

mobilization and modification of sorghum grains during malting and mashing

processes of beer brewing

The presence of two isoforms of (3-glucanase in the white sorghum may

suggest two distinct structural polysaccharide chains in P-glucan of white sorghum. .

Palmer (1992) in his work pointed out that it is quite possible that these partly

degraded p-glucans are extracted into the wort during mashing. Such molecules

could clog the filter bed. Wort filtration problem may naturally arise from various

features: viz incomplete saccharification of the mash due to a high starch

gelatinization temperature, low starch susceptibility to amylases, low starch

dispersibility, and low amylolytic activities.

Poor wort separation again may be attributed to high wort separation to which

several factors might contribute including a high level of non-starchy polysaccharide .

(9-glucan, pentosan) of polyphenal-protein complexes or of cysteine-rich proteins,

the presence of incompletely dispersed gelatinized starch and/or a high dextrin

content (Dufour and Melotte, 1992).

Limited cell wall breakdown in some sorghum cultivafs could be attributed to

the high level of proteins andlor to the low level of cell wall hydrolyzing enzymes,

especially endo-13-1,3, -1,4 glucanase (Onwurah, 2001).

I .2.4 Dextrinases

Owing to their limited action, limit dextrinase enzymes attack gelatinized

starch very slowly, unlike the amylase. They hydrolyse the ~ 1 , 6 linkages in starch.

Combined action of limit dextrinases and amylases degrade completely starch to

fermentable sugars. However, the limit dextrinases of malt are very thermolabile.

They are destroyed by heat at about 65OC.

4.2.5 Proteinases

These are group of enzymes that breakdown protein into amino acids and

they can further be classified on the basis of their functions.

(a) Endo-protease cleaves the internal peptide bonds of protein thereby

increasing their solubility.

(b) Exo-peptidases cleave the terminal peptide bonds thereby releasing amino

acids.

(c) Carboxypeptidases cleave peptide bonds to release those terminal amino

acids which had a free COOH group.

(d) Amino peptidases release amino acids by cleaving those terminal peptide

bonds (1) carrying a free NHZ group.

Proteases are very heat labile, but carboxypeptidases of malt are more thermostable

than the endopeptidases (Wall and Ross, 1970).

Okolo and Ezeogu (1995) in their work correlated the relationship between

proteinase activity in sorghum malt and air rest period, final warm steeping and grain

cultivar. According to their findings, increase in air rest period to 3 hours gives the

highest proteinase activity beyond which there is a decrease. According to their

findings, CWS-proteins, of mainly higher molecular weight polypeptides are major

products of proteinase activity, and that variation of proteinase activity with air rest

period and warm steep in sarghum grains suggest that isoforms of these enzymes

exist or that qualitative differences exists in the endosperrn proteins at the various

sorghum cultivars.

Carboxypeptidases

Okolo and Ezeogu (1995) in their work found that increase in air rest period

up to 4 hours enhances the ratio of proteolysis over amylolysis during malting. They

also observed that exposure of grains to longer period caused progressive increase

in grain metabolism and invariably amino acid utilizing processes thus resulting in

low FAN. This opinion was further strengthened by the increase in CWS protein

modification indices for the malt. In their work they also opined that

carboxypeptidase activity in sorghum malt is both steep treatment and cuitivar

dependent. Carboxypeptidases are responsible for the release of FAN from

solubilized protein during malting (Rank et a/., 1990). However, owing to the variation

in carboxypeptidase activity, Okolo and Ezeogu (1995) further suggested the

existence of carboxypeptidase enzymes in isoforms. Thus from their work, they

stressed that air rest period allowed to the grains in combination with final warm

steep would probably cause the expression of a particular carboxypeptidase isoforrn

best suited by the steeping condition.

Taylor and Evans (1989) in their work defined proteinase and

carboxypeptidase as the term used to describe the proteolytic activities measured in

terms of solubilization of total nitrogen and FAN respectively. Of the various

exopeptidases in germinating cereal seeds, the carboxypeptidases almost

exclusively have acid pH optima (Mikola, 2986) from their findings, they stressed that

using water as an extractant, the solubilization of proteolytic activity was found to

increase with temperature up to an optimum of 40•‹C, while using 4.25M sodium chloride,

the temperature optimum was 30% They also found that greater proteinase and

carboxypeptidase activity was extracted at neutral pH than at acid~c pH.

1.2.6 Lipoxidases

Fat modification index in sorghum is the availability of free fatty acids (FAA) -

released from saturated and unsaturated fats and oils (lipids) during lipolysis by

lipoxidases. During malting, lipoxidases have been found to be active in the

degradation of saturated and unsaturated fatty acids e.g, linoleic acid, linolenic acid

with the production of free fatty acids (FFAs) (Palmer et al., 1989). The enzyme is

stable at 40•‹C while temperature of 60•‹C inactivates 50-80% of it at 30 minutes

(Chikezie, 1 999).

1.3.1 Malting

Matt production is called malting and is essentially a bidogical process in

which the germination of sorghum is carried out in a controlled environment. The

technically important features of germination are the synthesis of hydrolytic enzymes

and the degradation of grain structures (Enari and Sopanen, 1986). Malting consists

of three stages; Steeping, Germination and Kilning.

Glennie and Wight (1986) in their work examined dextrin in sorghum malt,

wort and beer by means of High Performance Liquid Chromatography (HPLC). They

found out that throughout the malting period of 10 days approximately 5%

fermentable sugars and only trace amount of dextrin could be detected and the

pattern remained constant. They continued further that the use of sorghum in beer

makmg contained similar amount of dextrin containing 4 to 9 glucose unit, that are

branched as their concentration was reduced to a great extent by the action of

pullulanase.

1.3.2 Steeping - This involves the immersion of the selected grains in water until a

selected level of moisture content is reached. The main objective of steeping is to

initiate uniform germination and to hydrate the endosperm to a level, which is

suitable for modification, Steeping also washes away from the husk substances

undesirable in brewing and cleans the surface from microorganisms. Steeping also

ellminates carbon dioxide and allows oxygen to have contact with the grains for

effective aerobic respiration. Correct steeping procedures are Important for good

quality malt (Ezeogu and Okolo, 1994).

When the grains have absorbed sufficient water that will enhance moderate .

endosperm modification, the grains are said to be steep-ripe. Steep ripeness of

grains is dependent on the time taken to attain excellent modification, the initial

moisture content of the grain, the water temperature, the grain size and variety as

well as the protein content (Hough et a/., 1971; Enari and Sopanen, 1985; Ezeogu

and Okolo, 1994). The removal of the grains out of water is termed steep-out. Steep-

out is necessary to avoid excess water absorption by the grains, which will result in

over modification and profile germination and high malting loss. Moisture content of

r' about 42% - 46% is sufficient to support growth and biochemical alteration rn the

grain without allowing excessive growth.

Warm steep is used in sorghum malting to moderate rootlet and shootlet

growth. Also the use of air rest in combination with final warm steep significantly

reduces sorghum malting loss and rootlet growth while at the same time increases

extract yield and key enzyme development (Ezeogu and Okolo, 1994).

1.3.3 Germination - The steeped grains are grown under controlled conditions and

the intensity of the germination processes is controlled by grain. The main aim of

germination is to maximize fermentable extract by promoting both endospexm

modification and development of amylolytic enzymes. Chitting marks the beginning

of germination (Hough et a/., 1971). Subsequent embryonic develogment leads to

production of rootlet and growth of the shoot. Grains' variety, nitrogen content, corn

size, and condition of steeping all affect the rate of malting {Hough et al., 1970). For

good and even germination, maintenance of correct oxygenicarbondioxide tension

by proper aeration, maintenance of humidity throughout germination period and

correct germination temperature is of vital importance. Amylases development is

very sensitive to anaerobiosis white proteolysis is favoured by anaerobic conditions

at low temperature (Isebgert, 1964).

Von Holdt and Brand (1960) studied the changes in the carbohydrates of

sorghum during malting. They identified the sugar present in ungerminated sorghum

malt as maltose and lower maltose oligosaccharides. During the malting of sorghum,

the starch content of the grain was found to decrease by 43% from 17.7 to 10.1g1100

corns. They observed that the extent of the starch breakdown in sorghum was

considerably greater than what was recorded in other works (Von Holdt and Brand,

1960). This was attributed to higher temperature used in malting and more to the

vigorous growth of the malt. They showed that sorghum germination was

accompanied by a steady increase in fructose and sucrose which was apparent from

day one and continued until day five, after which there was virtually no change.

Aisien (1982) reported the utilization of soluble carbohydrate during sorghum

germination and seedling growth. He determined the level of sucrose, raffinose,

glucose and fructose in the scutellum of the intact grain embryo, The sucrose and

raffinose levels declined sharply over the germination phase but increased at the

post germination phase; as hexose sugars from the modifying endosperm pass Into

the scutellum. Aisien (1982) opined that while sucrose and raffinose also declined in

geminating excised embryo scutellum, the former recovered at post germination

where as the latter remained low. He further observed that maltose, rnaltotriose and

glucose were the main products of the enzyme modification of the endosperm during

seedling development, which is a post germinative events.

1.3.4 Kilning - Kilning process simply involves drying the germinated grains in hot .

air at a specific temperature within a period usually ?hour. Kilning produces a dry

storable product and further development of biological activities wilt only commence

during mashing. Kilning effects a degree of sterilization and makes rootlet and

shootlet removal easier. Kilning also accelerates milling of the grain. Unwanted

flavour components are removed while desirable ones are introduced either from

existing precursors or from extraneous sources such as from smoke during peat

kilning. Colour is imparted to the product, enzyme current is reduced and other

chemical composition of malt is modified (Hough et a!., 1971: Ezeogu and Okolo,

1 994).

Ilorry et al (1988) studied the environmental effect on the biochemical phase

of malt kilning. They reported that during the phase of kilning, the enzymes, enzyme-

catalyzed reactions, accelerate as malt temperatures are increased and moisture

level are substantial. Also enzyme system in malt vary in their sensitivity to moisture,

temperature, time relationship of kiln environment, air flow rate and kiln depth. They

further observed that some factors lead to differences in hot water extract w~ th~n a

kiln; and the degree of modification of starch, protein and cell wall during maltmg,

and subsequent extraction of these components during mashing. Friability also

allows amylolytic enzymes ready access to cr-glucan substrates and the direct

contribution of TSN. They also reported that cold-water extract is principally derived

from starch cell wall material and protein.

Novellie (1960) studies on kaffir corn (sorghum) observed that kilning at 70•‹C

caused a marked loss in diastatic power. Kilning at range of 40•‹C - 60•‹C caused

only negligible destruction, and therefore recommended a drying temperature of

5OUC in all malting studies to avoid the loss of other perhaps more sensitive

enzymes. Novellie (1960) also studied the comparison of total amylase activity with .

diastatic power for the malting, and deduced that increased in diastatic power during

malting is generally due to an actual Increase in the quality of amylase present in the

grain and not due to malting loss. In particular, the increase in diastatic power

attained at higher moisture content is a genuine increase in a-amylase activity and is

not caused by the heavier malting loss resulting from heavier watering.

Dufour and Jaeger (1987) carried out an experiment on the role of the rnethyl-

tranferase activity on the synthesis of S-methyl methionine, Dirnethylsulphide {DMS) .

precursor during germination and kilning. They studied the S-methyl rnethionine

(SMM) synthesis during sorghum germination, an enzyme extract from green malt

was prepared under sterile conditions, and they reported that the enzyme involved

seemed to be totally resistant to kilning and it is characterized by a broad

temperature tolerance. They concluded that these specific properties might be the

origin of large increase in SMM in the early stage of kilning.

CHAPTER TWO

MATERIALS AND METHODS

2.0 Plant Materials

Three sorghum varieties KSV8, SK 5912, and ICSV 400 were used. All were

obtained from National Seed Service, Zaria, Nigeria

2.1 Malting of Sorghum

Broken kernels, stones, and other fragments were removed by manual sorting

2.7.1 Steeping

Five hundred grams (500g) of each cultivar was weighed, surfaced sterilized

subsequently by immersion in 1% sodium hypochloride solution for 20 minutes;

drained and washed three times with deionized water. The grains were immersed in .

water at 3 0 ' ~ and after 6hr of steeping, half of this batch was removed from water

and their steeping interrupted with air-rest periods. This batch was designated as air-

rest samples with final warm steep (FWS) whereas the other remaining half has the

steeping water changed and returned back to water. This batch was designated as

continuously steeped samples (CS). This steeping method was applied to the three

cultivars. The continuous steeped grains were completely immersed in water

throughout the 51h steeping period at 3 0 ' ~ . The air rest samples had their steeping

cycles as follows; 6h wet and 3h air-rest for 45h, followed by a 6h final warm steep at

4 0 ' ~ .

2.7.2 Germination

At the end of the steep cycles, grains were germinated for 5 days. They were

spread in a shallow germination tray with a fine mesh bottom to germinate for 5 days

In a humidified dark germination box. Grains were turned every 12h and sprinkled

with 1 Om1 of distilled water per 100g of the grain.

2.1.3 Kilning

Germination was arrested by kilning at 50•‹C for 24h in a forced drought oven. The

rootlets and shootlets were removed from the malted grains. Malts were

subsequently milled for 30s in a cooled waring blender at high speed. The malts .

were stored in the refrigerator in air-tight container and used for analysis.

2.2 Germinating Energy and Water Sensitivity

This was determined according to the Recommended method of the Institute

of Brewing (1989). Two tests were carried out using 4ml and 8ml of deionized water.

The 4ml test was for the germinative energy of the sample and the 8ml test for water

sensitivity.

Two filter papers were placed on the bottom of each of the two Petri-dishes,

. 4ml of deionized water added to one Petri dish and 8ml to the other. One hundred

(100) grains were counted and placed on the filter papers in each Petri dish so that

each grain made good contact with the filter paper. In the 8ml test, only the ventral

side was allows to touch the paper to avoid drowning the embryo. The dish was

covered with the lid and good seal was assured. This was incubated in cabin and the

grains which chitted at 24h, 48h, and 72h intervals from the beginning of the

steeping were removed.

Calculation:

Germinative Energy (GE) = GE (4ml)Oh

Water sensitivity (WS) = WS (8mI)%

2.3 Length of Shootlets and Rootlets

After germination, 20 grains were taken at random from each sample. The

lengths of the shoots and roots were measured and their average taken in

centimeter (cm).

2.4 DETERMINATION OF MOISTURE CONTENT

This was determined according to the official method of the Institute of -

Brewinq (108, 1989). The misfure conten) was ci?.~ied CU!. every 6h dun'ng steeping.

Sorghum (5g) was finely grinded in a mortar and mix thoroughly. The weight of the

crucible was noted. Then 3g of the sample was put on the crucible and heated in an

oven at 105OC to a constant weight. The crucible with the sample was weighed again

after drying it in a dessicatar for 20 minutes.

Calculation - The moisture percentage (M) of the sample =

Where W = weight of the sample before drying

W2 = weight of the sample after drying

The average value was reported as percentage moisture content.

2.5 Cold Water Extracts (CWE)

This was determined according to method of Glennie and Holmes (1992). The

ground grist (1.25g) was added to 25ml of deionized water containing 1.5ml of

0.006N ammonia solution in a conical flask. This was shaken with a Gallenkamp

shaker for I h and centrifuged at 5000xg for 10 minutes. The specific gravity of the .

supernatant was determined.

The cold water extract of the malt samples was calculated from the equation.

SG E-xcess x 20 x 1000 %CWE = -

3.8G

where SG is the excess gravity of filtrate over 1000.

2.6 Cold Water Soluble Carbohydrate (CWS-COH)

This was determined according to the method of Glennie and Holmes (1992).

The malt sample (1.259) was mixed in snap-top 30ml centrifuge tube containing

12.8ml of deion~zed water, 1.6ml of 0.2N ZnS04 (malt extractant) and 1.6mI at 0.2N

Ba(OH)2. This was extracted with continuous shaking in a Gallenkamp shaker at 120 -.

oscillation per minute, at room temperature for I h, then centrifuged at 5000xg for 10

minutes. The soluble fraction in the clear supernatant was used for the specific

gravity determination and the CWS-COH, which is the soluble carbohydrate fraction

of the total CWE was calculated.

SG Excess x 20 Calculation O/O CWE-COH =

3.86

2.7 Hot Water Extract (HWE)

This was determined according to the method of Etokakpan (1992). Extract

f

was obtained by shaking 2.59 of sorghum malt flour with 22.5mI of 0.5% sodium

chloride for I h at room temperature in Gallenkamp shaker at 120 oscillations per

minute. The mixture was allowed to stand for 15 minutes and the cloudy supernatant

decanted into 25ml another flask and stored. The residue was boiled for 10 minutes

with occasional shaking until it gelatinized. The supernatant was added and cooled

to 50bC and held for 1 h in a water bath with manual stirring every 15 minutes. [This

volume was made up to 27.7ml with sodium chloride solution and centrifuged at

3000 x g for 10 minutes]. The specific gravity of the wort was measured using a 10ml

SG bottle.

Calculation HWE = G x 10.13

G = 1 0 0 0 ( S G - I )

G = weight of the wort.

2.8 Hot Water Extract Protein (HWE-protein)

This was determined according to the method of Institute of Brewing (1989).

Twenty five milliliter (25ml) of wort obtained from HWE was pipetted into a 500ml

Kjeldahl flask. To this was added 4 drops of concentrated H2SO4 and gently

evaporated to dryness with minimal charring. After this, 20ml of conc. H2SO4 and l o g .

of catalyst (see appendix I ) were added into the flask to wet and mix the content.

This was then boiled until the brown colour disappeared. Stronger heating was

continued for another 20 minutes. The digest was then allowed to cool.

The digest was diluted with 250ml of distilled water followed by the addition of 70ml

of 40% sodium hydroxide solution. The flask containing the digest above was then

connected to a distillation apparatus; whose condenser exit unit tube was dipped

below the surface of 25ml 2% Boric acid solution containing 0.5ml of screened

indicator (see Appendix 1). One hundred and eighty milliliter (180ml) of the distillate .

was collected and titrated against 0.1 N hydrochloric acid to a grey end point.

The moisture content of the sample were also determined.

Calculation:

Titt-c x 100x Normdi ty of HCI x 0.014 % N2 =

Weight of sample

2.9 Free Alpha-Amino Nitrogen (FAN)

This was determined according to the method of Taylor (1983). The ninhydrin

coloured reagent comprises disodium hydrogen phosphate 25g; potassium

dihydrogen phosphate 15g; ninhydrin 1.25g and Fructose 0.75g in 1 litre.

One (1) gram of malt sample was extracted with 40ml of 5% trichloroacetic

acid (TCA) at 30•‹C for I h. This was centrifuged for 25 minutes at 4000xg. One

milliliter (Irnl) of the clear supernatant was diluted to 25ml with distilled water. Two

milliliter of the diluted supernatant was placed in a test tube and I m l of colouring

reagent was added to it. This was stoppered with a glass ball and placed in a boiling

water bath for 16 minutes and then cooled in a water bath at 20•‹C for 20 minutes.

Thereafter, 5mI of diluting solution (see appendix) was added and the optical density

(OD) of the mixture determined with a spectrophotometer at 570nm in a 10mm cell

against a reference blank prepared from the reagent plus 2ml of distilled water in

place of the diluted wort. .

Calculation:

FAN in m g ~ " = Absorbance at 570nm of test solution x 2 x Dilution Mean absorbance of standard glycine.

From the above the value of FAN, in mg% of malt was calculated as:

Malt FAN (mg%) = FAN in TCA Extract (mgL-') x 100 of distilled water

Diluting solution comprises: potassium iodate 0.5g, 96% (vlv) Ethanol 100ml and

150ml o dionized water.

. 2.1 0 Total Non-Protein Nitrogen (TNPN)

This was determined according to the Method of Institute of Brewing (1989)

The malt sample 1.59 was extracted with 40ml of 5% trichloroacetic acid (TCA) at

30•‹C for I h. This was centrifuged for 25 minutes at 4000xg. The clear supernatant .

was collected, Twenty five milliliter (25ml) of the supernatant was placed in a 50OmL

Kjedahl flask. Four drops of concentrated sulphuric acid was added to it and this was

gently evaporated to dryness with minimal charring.

Thereafter, 20ml of conc. H2S04 acid and log of catalyst were added into the

flask to wet and mix the content. This was then boiled until the brown colour

disappeared. Stronger heating was continued for another 20 minutes. The digest

was then allowed to cool.

The digest was diluted with 250ml of distilled water. To this, 70ml of 40% .

sodium hydroxide solution was added. This flask was then connected to a d~stillation

apparatus whose condenser exist unit tube was dipped below the surface of 2%

25ml Boric acid solution containing 0.5ml of screened indicator (see appendix 1).

One hundred and eighty milliliter (180ml) of the distillate was collected and titrated

against 0.1 N HCI to a grey end point. The moisture content of the samples was also

determined.

Calculation:

Titre x 100 x Nomality of HCI x 0.01 4 . TNPN % N2 = mg I '10

Weight of sample

2.1 1 Proteinases and Carboxypeptidases Activity Assay

This was determined as described by Okolo and Ezeogu (1995). Sorghum

malt (29) was extracted with 30ml of 0.1 M citrate phosphate buffer pH 7.0 containing

3.33mM of Dithiothretol and 73mM NaCl solution for 2h at 30•‹C using a Gallenkarnp

shaker at 25 oscillation per minute. The extracts were then dialyzed against the

same buffer and the residue assayed for proteolytic activity using bovine serum .

albumin substrate (BSA). The reaction mixture comprises: enzyme extract or residue

suspension (2ml), BSA substrate (5mg protein) and 0.3M citrate phosphate buffer pH

4.5 (2ml).

Assay Procedures

BSA (0,039) was measured and put in each of the tubes (both sample and

control), followed by the addition of I m l of 0.3M citrate phosphate buffer pH 4.5. One

milliliter of enzyme extract was pipetted to each of the tubes (sample and control

tubes). One milliliter of 15% trichloroacetic acid (TCA) was added to each of the

control tubes. The sample tubes were incubated in 50•‹C water bath for 5h with

shaking at every 15th minutes intervals. The control tubes were placed in the

refrigerator for the same 5h. At the expiration of the 5h, I ml of 15% T.C.A was added . to the sample tubes to terminate the reaction. They were also removed from the

water bath, while the controls were also removed from the refrigerator.

The reaction mixtures were immediately centrifuged at 4000xg for 30 minutes.

The supernatant was subjected to further centrifugation to obtain a clearer

supernatant (3 times). This clearer supernatant was then used for the determination

of proteinase and carboxypeptidase activities.

Carboxypeptidase Activity Determination

One millimeter each of the clear supernatant from both the sample and control

tubes was diluted with 25ml of distilled wafer. Two millimeter of the diluted (samples

and control) materials were placed in a test tube and I m l of coloured reagent (see

appendix I ) was added to it. This was stoppered with a glass ball and placed in a

boiling water bath for 16 minutes and then cooled in a water bath at 20•‹C for 20

minutes.

After this duration, 5ml of diluting solution (see appendix 1) was added to

each of the tubes (sample and control) mixed and measured in a spectrophotometer

at an absorbance of 570nm En a 10ml cell against a reference sample prepared from

the reagent plus 2ml of distilled water in place of the diluted wort.

Calculation

FAN in mg0/0= Absorbance of test solution x 2 x Dilution Mean absorbance of standard.

Carboxypeptidase was calculated as the difference between the FAN content of

supernatant from 5h and Oh incubating reaction mixture.

Proteinase Activity Determination

Two millititre (2ml) of the clear supernatant (from sample and control)

obtained after the last centrifuging was placed in a 500ml Kjeldahl flask. Four drops

of conc. H 2 S 0 4 was added to it and this was gently evaporated to dryness with

minimal charring. .

After this, 4ml of conc. H2S04 and 29 of powdered catalyst was added to the

flask to wet and mix the content. This was then heated until the brown colour has

disappeared. Stronger heating was continued for another 20 minutes. The digest

was then allowed to cool.

The cooled digest was diluted with 25ml of distilled water and 10ml of 40%

sodium hydroxide (NaOH) solution was added to it. This was then connected to a

distilling apparatus whose condenser exit tube unit was dipped below the surface of

2% (IOml) of Boric acid solution containing 0.5rnl of screened indicator. About 40ml

of the distillate was collected and titrated against 0.1 N sulphuric acid to the grey end

point. The moisture content of the samples was also determined as earlier .

described.

Calculation

%N2 = Titre x I 0 0 Normality of H 2 a 4 - - x 0.014 Weight of sample

Proteinase was calculated as the difference between nitrogen content of the

supernatant from 5h and Oh incubation reaction mixture.

Glucan Extraction

Sorghum malt flour (jg) was weighed into a 250ml conical flask and 20ml of . 4% {wh) sodium hydroxide solution containing 1% (wiv) of sodium borohydride ta

remove any reducing sugar present was added, It was shaken for 2'/2h in a

Gallenkamp shaker at 150rpm at room temperature. The hamogenate was

neutralised using acetone. It removed the excess sodium borohydrate, It was then

dried and used for the assay of peta glucan

Glucanase Level

This was determined according to the method of Etokakpan (1490).

Extraction and Assay of Glucanase

The malt flour (0.5g) was extracted in 10ml of 50mM acetate buffer containing

100mM Sodium chloride, 10mM calcium chloride, and 50mM sodium actetate pH 5.7

for 2'12h in an orbital shaker at 150 rpm. The clear supernatant obtained after

centrifugation (300g for I 0 minutes) was assayed for glucanase activity

Endo-P-glucanase was assayed with P-glucan extracted from sorghum

prepared by a slight modification of the method of Palmer ef a/. (1985). The digest

contained 1% sorghum p-glucan in IOOmM sodium chloride, 1 OmM calcium chloride

and 5OmM sodium acetate buffer pH 5.7 (lrnl) and the mixture was incubated at

40•‹C for 30 minutes. The reducing sugar produced was determined according to

Nelson-Somogyi method (1 952). .

A unit of enzyme activity was defined as the amount of the enzyme which was

capable of releasing l t tg glucose equivalent (in reducing activity) per minute under

the assay condition.

Diastatic Power and Amylase Activity

This was determined according to the method of Etokakpan and Palmer

(1 WO),

The enzyme extract was prepared by extracting I g of milled malted grains .

using 12.5ml of 0.1M sodium acetate buffer molarity pH 5.7 for 2'12h at room

temperature with constant shaking (Gallenkamp shaker). The extract was

centrifuged at 50009 for 10 minutes. The supernatant was diluted with sodium

acetate buffer pH 5,7 to obtain concentration from 1-8mg of grain per ml.

ASSAY - 0.2 ml of malt enzyme extract was mixed with 0.2ml buffer (sodium acetate

pH 4.6) in three separate test tubes. To the first and third test tubes, 0.2ml buffer

was added while 0.2171 HgCI2 1 0 ~ m ~ l r n l was added to the second test tube. Another

0.2mI of 1% starch solution was added to the first and second test tube.

They were mixed together and after 10 minutes, the reaction was stopped by

adding 0.8ml of Nelson copper reagents I and 2 and boiled for 25 minutes in a water

bath. The test tubes were cooled and 0.8ml of Arsenomolybdate solutlon was added

and finally 10ml of distilkd water was added. The test tubes were shaken for proper

mixing of the contents and absorbance measured at 565nm. The first test tube was

tor diastatic power activity and the second test tube for a-amylase activity. The third

was for enzyme control.

The P-amylase was calculated by subtracting the a-amylase from the diastatic power

activity.

One unit of the enzyme activity was defined as the amount of enzyme that

released 1 pg of glucose equivalent per minute.

Production of Brewers Yeast

Brewer's yeast was obtained from PAL Brewery Ltd, Oko, Anambra state,

Nigeria. The yeast was tested for viability using Methylene Blue staining method

(IOB recommended method of Analysis 1989) and was found to be viable with a

viable cell count of 760 cells per 1000. The yeast was multiplied by inoculating it on

yeast extract agar supplemented with 10% glucose. Incubation was at room

temperature for 4 t h . The yeast was harvested using normal saline. This was then

centrifuged at 50009 for 20 minutes. The supernatant was discarded and the deposit .

was resuspended repeatedly (3 times) with norma! saline and centrifuged again as

before.

Wort Preparation and Fermentation

Mashing was according to the decantation method of Etokakpan and Palmer

(1990). One part (259) of the malt to 4 par-! (100mI) of the mashing solution was used

(10mM CaCI2 buffer pH 5.0). The pH of each sample was adjusted separately (pH

5.0). The mash was incubated at 35'C in a water bath for lT /?h and the supernatant

was decanted and stored. The residue was boiled in the water bath with occasional

shaking until it gelatinised. This was allowed to cool to 50•‹C and the decanted

supernatant was added and mixed properly, It was incubated in a water bath at 50•‹C

for 1112h; while shaking at every 15 minutes. The temperature was further raised to

65OC for I h . The wort was filtered warm, and sterilized by pasteurisation for I h at

65OC.

Fermentation

The pasteurised wort was cooled to 10•‹C and 20mI of the wort was aseptically

pitched with I g m of brewer yeast. This was incubated in an orbital shaker at 10rpm

at a temperature of 15OC for 48h.

Fermentation Analysis

Wort Specific Gravity

The specific gravity of the wort was measured before and after fermentation

by using a 10ml specific gravity bottle.

Percentage Fermentability of Wort

This was measured after 48h fermentation period using the specific gravity

bottle. This was calculated as follows:

Percentage Fermentability % = OG - FG x 81.9 OG - 100

where OG = Specific gravity of unfermented wort - 1

FG = Specific gravity of fermented wort - 1

Wort Fermentation Extract

This was measured after 48h fermentation period using the specific gravity bottle.

This was calculated as follows:

Fermentable Extract (Lit/deg/g)= OG - FG x 81.9 % x 75 OG - 1.00 100

where OG = Specific gravity of unfermented wort - 1

FG = Specific gravity of fermented wort - 1

CHAPTER THREE

RESULTS

Proxrmate analyses of the sorghum grains prior to subjecting the grains to

maltrng treatments and conditions were carried out. Table 1 shows the proximate .

analysts of the sorghum grains in the three different cultivars. All the cultivars namely

KSV 8, SK 5912, and ICSV 400 have high germinative energy and moisture content.

Their endosperm reserves were high especially the carbohydrate and crude protein

contents. Ash, fats and fibre constituents of the three cultivars, although vary, were

all enough to sustain malting. Generally, the proximate analysis of the grain cultivars

showed that all the three cultivars used were viable and possess the characteristic

qualities enough to break dormancy, sustain malting and release enough endosperm

reserves. ..

Tables 2 and 3 equally showed the proximate analysis of the malt after

subjecting the grains to malting conditions and treatment. Results obtained show that

the malting-losses were minimal in the three cultivars as compared to that of the

grains prior to malting.

Cereal grains endosperm solubilization, mobilization and modification is an

rnducible property of the grain, which is cultivar dependent. Grain water inbibition

during steeping is time-temperature dependent, and steep-ripeness especially prior

to steep-out is a measure of the grains moisture content. Figure I shows the

development of the moisture content of the three sorghum cultivars during steeping

using air-rest and continuous steep regimes. At 42h steeping period, continuous

Steep grains were due for steep-out with moisture content in the range of 35% to

40%, whereas their air-rest counterpart attained steep-ripeness with an elevated

moisture content in t h e range of 40% to 44%, respectively.

Analysis of variance (ANOVA) (Table 5) reveals that both the steeping

regimes, cultivars and their interactions were significant (Pe0.05) in influencing the

observed variations in moisture content development of t h e grains and their cultivars

during steeping.

At the end of the malting, the malts were analysed for some malt quality

parameters. Figure 2 show the development of Cold Water Extract of the three

sorghum cultivars during Air-rest and continuous steep regimes. Grains treated with

air-rest periods show higher CWE values as follows; SK 5912 at 33h (49%), KSV

400 at 42h (42%) and KSV 8 at 51 h (33%); than their continuous steep counterpart;

SK 5912 at 33h (37%), ICSV 400 at 42h (40%) and KSV 8 at 51h (30•‹/~),

respectively.

Analysis of variance (Table 6) revealed that steeping regimes, cultivars and

their possible interactions were significant (Pc0.05) in influencing the observed . variation in cold water extract development of the sorghum grains and their cultivars.

Cold Water Soluble Carbohydrate (CWO-COH) is sometimes used as a

measure of grain modification during malting. Figure 3 show the development of

CWS-COH of the three sorghum cultivars during steeping using air-rest periods and

continuous steep regimes. Grains treated with air-rest period show higher CWS-

COH values; SK 5912 at 33h (48%), ICSV 400 at 42h (45%) and KSV 8 at 33h

(34%); than their continuous steep counterpart, SK 5912 at 24h (45'/0), ICSV 400 at

33h (4j0/'0) and KSV 8 at 33h (36%), respectively.

ANOVA of data (Table 7) revealed that steeping regimes, cultivars and their

pair-wise interactions were significant (Pc0.05) in influencing the observed variation .

in the development of CWS-COH among the cultivars and their batches during

steeping.

Hot Water Extract (HWE) of the cultivars were also determined. The

development of the HWE of the three sorghum cultivars during steeping using air-

rest and continuous steep regimes is shown in Figure 4. Grains treated with air-rest

steep regimes showed higher HWE with values as follows: SK 5912 at 51h (294'/0),

lCSV 400 at 51 h (252%) and KSV 8 at 5 ? h (238%) whereas in continuous steep

samples, SK 5912 at 33h (270%). ICSV 400 at 24h (290%) and KSV 8 at 33h .

(275%), respectively. However, more significant was the behaviour of air-rest

samples. From 24h to 51h, there was a gradual but steady increase in HWE in the

cultivars with both samples of SK 5912 and KSV 8 attaining their peak values at 51 h,

Analysis of variance of the above data (Table 8) revealed that both cultivars,

rtceplnq rcgimes 2nd their possible pairwiso interaction were significant (P4 .05) in - -

influencing the development of HWE among the cultivars and their batches during

steeping.

Hot Water Extract Protein (HWE-protein) development of the three sorghum .

cultivars during steeping using Air-rest and continuous steep regimes were also

determined and the results presented in percentage Nitrogen IohN2) are shown in

figure 5. Samples treated with air-rest periods show elevated HWE-protein

development as follows; at 33h, both KSV 8 and SK 5912 attained their peak values

of 2.B0/0N2 and 2.7%N2 as it peak value. In continuous steep samples, SK5912 at

33h (1.4%) and KSV 8 at 51 h (1 .7%N2), respectively.

ANOVA Table 9 reveals that steeping regimes, cultivars and their possible

pair-wise interactions were significant (P<0.05) in influencing the above observed

variation in the development of HWE-protein among the cultivars during steeping

Total Non-protein Nitrogen (TNPN) development of the three sorghum

cultivars during steeping using Air-rest and continuous steep regimes were also .

evaluated and results presented in mg % FAN are shown in figure 6. The pattern of

TNPN development in both continuous and air-rest samples was not definite. There

was a gradual rise-and-fall pattern which was not synonymous to increase in

steeping time. However, air-rest samples has its highest peak values in the cultivars

as follows: SK 591 2 at 24h (700), ICSV 400 at 42h (900) and KSV 8 at 42h (740) as

against values recorded for continuous steep samples of SK 5912 at 42h (750),

ICSV 400 at 42h (800) and KSV at 51 h (880). Analysis of variance (Table 10)

. reveals that steeping regimes, cultivars and their possible interactions were

significant (Pc0.05) in influencing the above observed variation in the development

of Total non-protein nitrogen among the cultivars and their batches during steeping.

Figure 7 shows in mg % the development of free Alpha-Amino Nitrogen (FAN)

of the three sorghum cultivars during steeping using air-rest and continuous steep

regimes. Both samples attained their peak values at 24h of steeping, but air-rest

samples recorded higher FAN values than the continuous steep samples. More

spectacular was the behaviour of KSV 8 air-rest samples, which show a plateau at

33h to 42h steeping time.

ANOVA (Table 11) revealed that steeping regimes, cultivars and their

possible pair-wise interaction were significant (PC 0.05) in influencing the observed

cultivars and their samples during steeping.

Proteinase activity of the samples and other cultivars were also determined.

Results presented in mglNI5hlg were shown in figure 8. Samples treated with a~r-rest

period show a gradual Increase in proteinase activity till 51h steep out time

especially in cultivars SK 5912 and KSV 8, whereas ICSV 400 has its peak values at

24h (750mglN15hlg) and gradually decfine till 51 h steep out. Similarly, continuous

steep samples show a gradual increase in proteinase activity especially in KSV 8

which recorded a linear increase up to 51h steep out time. Hawever, SK 5912 and

ICSV 400 has their peak values at 33h (800 rnglN/5hlg} and 24h (750 mglNl5hlg)

respectively beyond which, there was a gradual drop till the 51 h steep out time.

ANOVA (Table 12) reveals that steeping regime, cultivars and their possible

interactions were significant (P < 0.05) in influencing the observed variation in the

development of proteinases among the cultivars and their batches during steeping.

Carboxypeptidase activity of the three sorghum cultivars during steeping

using Air-rest and continuous steep regimes were evaluated and results presented in

mglFAN15hlg was shown in figure 9. Just like the TNPN results, the pattern of .

carboxypeptidase development in both continuous steep and air-rest samples was

undefined; having two peak values of high and low ebbs. However, there was a

linear rise-and-fall pattern up till 51R steeping time in both air-rest and continuous

steep samples. In air-rest samples peak values were obtained at 24h (950

mglFANIShlg), 42h and at 51h (860 mglFAN/Shlg) for ICSV 400, SK 5912 at 33h

(800 mglFAN/5hlg) and KSV 8 at 51 h (800 mglFANl5hlg) respectively. Similarly, in

continuous steep samples, ICSV 400 at 42h (1000 mglFAN/5hlg), SK 5912 at 33h

(TOO mglFANl5hlg) and KSV 8 at 51 h (700 mglFANI5hlg) carboxypeptidase activity

respectively.

Analysis of Variance (Table 13) reveals that steeping regimes, cultivars and

their possible interactions were significant (P-4.05) in influencing the . carb~xypeptidase activity as observed among the cultivars and their batches.

13-Glueanase activity of the three sorghum eultivars during steeping using air-

rest and continuous steep regimes were also evaluated and results presented in

mglglucose are shown in figure 10. Air-rest samples attained their peak values at

33h of steeping as follows SK 5972 (90mgfgfucose), ICSV 400 (6Ornglglucose) and

KSV 8 (80rng/glucose) as compared with lower values obtained in the continuous

steep counterparts at the same 33h in which SK 5912 (80mg/glucose), ICSV 400

(58mg!glucose) and KSV 8 (69mglglucose) respectively. The results also show that . there was steady decrease in glucanase activity beyond this 33h steeping time up till

51h steep-out. ANOVA as depicted in Table 14 reveals that steeping regimes,

cultivars and their possible interactions were significant (Pc0.05) in influencing the

glucanase activity as observed among the cultivars and their samples.

The Diastatic power of the three sorghum cultivars and their samples were

also determined and results presented in mglglucose were shown in figure 11. Air-

rest samples attain their peak values at 33h of steeping after an initial time lag of 15h

with values as follows: SK 591 2 (235), ICSV 400 (1 50) and KSV 8 (21 5) as follows: .

SK 5912 (230), ICSV 400 (145) and KSV 8 (205), respectively.

Analysis of variance (Table 15) reveals that steeping regimes, cultivars, and

their pair-wise possible interactions were significant (Pc0.05) in influencing the

development of diastatic power as was obtained among the cultivars and their

samples.

Figure 12 shows the development of alpha amylase activity of the three

sorghum cultivars during steeping using Air-rest and continuous steep regimes. Both

batches attain their peak values at 33h of steeping; Air-rest samples have the

following values presented in mglglucose as follows: SK 5912 (100mg/glucose),

lCSV 400 (26mglglucose) and KSV 8 (24mg/glucose) as compared to their .

continuous steep counterparts: SK 591 2 (96mglglucose), ICSV 400 (21 mg/glucose)

and KSV 8 (20mglglucose) respectively.

ANOVA reveals (Table 16) that steeping regimes, cultivars and their pair-wise

possible interaction were significant (P<0.05) in influencing the development of

diastatic power as were observed among the cultivars and their batches.

peta amylase activity of the three sorghum cultivars during air-rest and

continuous steep regimes were also determined and results presented in mglglucose

are shown in figure 13. Grains treated with air-rest periods show higher peta . amylase activity values as folfows: SK 5912 at 42h (35mg/glucose), ICSV 400 at 33h

(llrngiglucose) and KSV 8 at 33h (27mglglucose); than their continuous steep

counterpart SK 5912 at 42h (32rnglglucase), lCSV 400 at 33 (9mglglucose) and KSV

8 at 33 (24mg/glucose), respectively. Beyond these peak values there was a steady

and gradual decrease in amylase activity in both the cultivars and their batches.

Analysis of Variance (Table 17) reveals that both steeping regimes and

cultivars as well as their possible interactions significantly (P<0.05) influenced the

development of peta amylase in the cvltivars and their respective batches. . Results of the devebpment of wort fermentability of the three sorghum

cultivars during steeping using air-rest and continuous steep regimes presented in

percentage (%) are shown in Figure 14. The response of the cultivars to treatments

varies. Air-rest sarnpl es recarded their peak values as follol

44

. ws; SK 5912 at 24h

(65%), ICSV 400 at 33h (55%) and KSV 8 at 42h (55%) whereas in continuaus steep

samples SK 5912 at 33h (55%), ICSV 400 at 51 h (58%) and KSV 8 at 51 h (60•‹h),

respectively. Analysis of Variance (Table 18) reveals that steeping regimes, cultivars

and their possible pair-wise interactions significantly (Pc0.05) influenced the

development of wort fermentability as was observed among the cultivars and their

batches.

Results of the development of Wort Fermentable Extracts of the three <

sorghum cultivars during steeping using Air-rest and continuous steep regimes

presented in Litnerldegreelgram are shown in figure 15. Samples treated with air-

rest periods attained their peak values as follows: SK 5912 at 51h (550Litldeglgm),

ICSV 400 at 24h (545 Litldeglgrn) and KSV 8 at 33h (420 Litldeglgrn) as compared

to their continuous steep counterparts with values: SK 5912 at 33h (420 Litldeglgm),

ICSV 400 at 42h (430 Litldeglgrn) and KSV 8 at 42h (360 Litldeglgm), respectively.

ANOVA reveals (Table 18) that steeping regimes, cultivars as well as their pair-wise

possible interactions were significant (Pc0.05) in influencing the variation observed .

in the development of Wort Fermentable Extracts among the cultivars and their

respective batches.

T\ - Sorghum Grains

Parameter (%I) ICSV 400 KSV 8 SK 5912

Germinative energy 98.1 95.2 98.2

Water sensitivity 33.1 25.1 15.1

Moisture Content 10.2 10.4 9. I

Carbohydrate 63.6 68.2 68.3

Fat 4.3 4.1 3.8

Ash 7.2 4.7 5.4

Fibre 6.4 4.3 3.3

Crude protein (N x 6.25) 9.7 9.6 10.4 -

Table 2: ANOVA for the Proximate Analysis of Sorghum Grains Sources of variation SS DF MS F-ratio F-tab (5%)

- Cultivar 72.12 2 36.06 10.33 4.2G (8.02)

Proximate composition 120.37 5 24.07 6.90 3.48 (6.06)

C u l t i w x Prox. Comp. 167.53 8 20,94 6.00 3.23 (5.37)

Treatment total 39 1.44 24 163 1 4.67 2.90 (4.73)

Residrml 3 1.42 9 3.49

Volr~cs ill pcr~-e~~tl~esis are bused ot? I % Confidence level

Table 3: Proxirn* Sorghum malt

Parameter (YO) lCSV 400 KSV 8 S K 5912

Moisture Content 12.1 12.2 12.6

Carbohydrate 60.4 58.6 56.3

Fat

Ash

Fibre

Crude protein (N x 6.25) 9.2 10.3 12.1

Table 4: ANOVA for the Proximate Analysis of Sorghum Malt SS DF MS F-ratio F-tab (5%) Sources of variation

Cultival- 184.00 2 92.22 29.56 3.74 (6.5 1)

Germination time 283.71 6 47.29 15.1 G 2.85 (4.46)

Treatment total 5 1 1.66 2 2 23.26 7.46 2.35 (3.13)

Residual 43.52 14 3.12

Valucs in parenthesis are based on 1% Confidence level

I

I-+lCSV 400 1 I....* KSV 8 CS

0 4 . - --.-- I I I - - -

0 6 15 2 4 33 42 5 1

Steeping Time (h)

Figure I : Developnterzt of kloisture Con tent of three Sorgl~zrm Czrltivnrs drrri~tg Steepirlg using air-rest ( - ) and Continr~oris (---- Steep Regirnes

Tddc 5: A N 0 VA Table for the & ? ~ d ~ p n l e i l t of Moisture Content Dirring Air-Rest

and Corrti~~uous Steep

SS df MS F Source of Variation P-value F crit .

Steeping Regime 2780.05 6 463.4916 235.104 0.05 2.996 1 17

Varieties 8.9662 1 2 4.483105 2.274034 0.05 3.88529

S R x Vnnety 48224.7 2 24112.3 373.8 0.05 6.425 1

Error 23.65719 12 1.971433

Total 2813.573 22

O m - - - - - - -. -- - - -

0 6 15 24 33 42 5 1 . .

Steeping Time (h)

Figure 2: Development of Cold Wuter Extract of three Sorgh~r~n Cultivtm dlrrirlg Steeping using air-rest ) tmd Co~rtinuoils ( - - - - ) Steep Regirues

Tuble (j: ANOVA Tuble for tlze Developi~ieizt o f Cold Wuter Extract Duriiig Ais-Rest

aild Coiltiiluocrs Steep

Source of I'nricltior~ SS df MS F P-value F crit - Steeping Regime 3723.348 6 620.558 38.26575 0.05 2.9961 17

Varieties 339.608 1 2 169.804 10.4707 0.05 3.88529

SR s Variety 13951.0 2 6975.0 1701.3 0.05 4.9527

Error 194.6047 12 16.2 1706

Total 4257.561 22

-- + KSV 8 I

+SK 5912

-+ lCSV 400

- - - & - - K S V 8 C S

- - -1, - -SK 5912 CS ! - - - -ICSV 400 CS ;

0 , --

0 6 15 24 33 42 51

Steeping Time (h)

Figure 3: Development of Cold-Water Soluble Carbohydrate of' three Sorgham Cultivar during Steeping using Air-Rest ( - ) and Continuous ( ------ ) Steep Regime

Tnble 7: ANOVA Tnble for the Developnrertt of Cold J?'<lter Solrrhle Crrrbulr-vdrate *

during A ir-Rest urld Coniinrrozts Steep

Source of Variation SS df MS F P-value F crit

Steeping Rcgilne 3049.223 6 508.2038 75.65 187 0.05 2.996 1 17

Varieties 44.56464 2 22.28232 3.3 16975 0.05 3.88529

SR x Variety 18396.1 2 9184.5 159.325 0.05 6.2391

Error 80.61 196 12 6.717663

Total 3174.399 22

+SK 5912

+ ICSV 400

- - 4 - -KSV 8 CS I I - - .x- - -SK 5912 CS

'

- - ICSV 400 CS pp - --

0 . - ... r - r I I I

0 6 15 24 3 3 42 5 1 Steeping Time (h)

Figure 4: Dewlopment of Hot Water Extract of three Sorghum Cultivars during Steeping Steeping using Air-Rest ( ) and Continrrorrs ( ------ ) Steep Regime

Tc~ble 8: ANOVA Tablr for the Development of Hot Wnter Extrtrct drrrirrg Air-Rest

a~rd Corttirtlrorrs S f e q

Source of Variation SS df MS F P-value F crit

Steeping Regime 8935.74 6 1489.29 2.349012 0.05 2.9961 17

Varieties 4068.866 2 2034.433 3.208849 0.05 3.88529

SR x Variety 137538.35 2 68769.1 8.17489 0.05 6.45271

Error 7608,085 12 634.0071

Total 20612.69 22

.$.r

0 I

0 '- - - -. I

0 6 15 24 33 42 5 1

Steeping Time (h)

Figure 5: Development of Hot Water Extract Protein of three Sorghum Cultivars during Steeping using

Air-Rest (- )and Continuous ( ---- ) Steep Regimes.

Tuhle 9: ANOVA Trrbl~ for the Develojment of Hof Wnter Extruct Protein drrrirlg

,4 ir-Rest uird Continrlorrs Steep

Soiii-ce of I'nri~ltio~t SS df MS F P-vnluc! F crit -- Steeping Regime 1.5794 6 0.2632 1.3894 0.05 2.9961

Varieties 5.1385 2 2.5692 13.561 0.05 3.8853

SR rc Variety 7.5391 2 3.5632 15.861 0.05 6.9851

Error 2.2735 12 0.1895

Total 8.9913 22

Steeping Time (h) Figure 6: Development of Total Non-Protein Nitrogen of

three Sorghum Cultivars during Steeping using Air-Rest ( - ) and Continuous ( ------ ) Steep

Regimes.

T(lbk 10: ANOVA Tcible for ?lie Developnterrt of Totnl Nott-Proteirt Nitrogert drwiitg

Air-Rest mid Cortfin~rom Steep

Sorr rce of Vurintion SS df M S F P-value F crit -- Steeping Regime 264309 G 44052 1.0671 0.05 2.9961

Varieties 9240.4 2 4620.2 0.1 119 0.05 3.8853

SR x Variety 5958581 2 99310.9 2.9555 0.05 5.9863

Error 495400 12 41283

Total 768949 22

0 . - ~- - - - - . -

0 6 15 24 33 4 2 5 1

Steeping Time (h)

Figure 7: Development of Free Alpha Amino Nitrogen of three Sorghum Cultivars during Steeping using Air-Rest

( - )and Continuous (----- )Steep Regimes

Tdde 11: A N 0 VA Table for the Develop~~rerlt of Free Alphcr Arnilzo Ni f rupr

during Air-Rest n~zd Co~ztiirriorrs Steep

Source of Variation SS df R4S F P-value F w i t

Steeping Regime 264309 6 44052 1.0671 0.05 2.9961

Varieties 9240.4 2 4020.2 0.1 119 0.05 3.8853

SR x Variety 639023.3 2 31951.6 2.161 l 0.05 5.1231

Eiror 495400 12 41283

0 - - - --- 7-- I I I I

0 6 15 24 33 42 5 1

Steeping Time (h)

Figure 8: Development of Proteinase Activity of three Sorghum Cultivars during Steeping using Air-Rest (- ) and Continuous ( ---- ) Steep Regimes.

Trrhle 12: ANOYA Tnble for tlte Developrer~f of Proteirme Acf iv i t~ ! driririg Air-Rcst

m d Corltin~rorrs Steep

S o ~ i r c e of Variation SS df MS F P-value F crit

Steepins Regime 689714 6 114952 20.107 0.05 2.9961

Varieties 31076 2 15538 2.7178 0.05 3.8853

SR x Variety 634484 2 105747 7.3045 0.05 5.9863

Error 6SGOG 12 5717.1

Total 789395 22

I

0 . . . .- 7 - -

0 6 15 24 33

Steeping Time (h)

Figure 9: Development of Carboxypeptidase Activity of three Sorghum Cultivars during Steeping using Air-Rest

( - ) and Continuous ( - - - - ) Steep Regimes.

Table 13: ANOVA Table for the Developrnerrt of Cmboxypeptidase Activity dwirig

Air-Rest and Corztirruous Steep

Sorrrcc~ of Vcrrintion SS (if MS F P-valrre F crit

Steeping Regime IE+OG 6 18G631 12.288 0.05 2.9961

Varieties 99619 2 49809 3.2795 0.05 3.8853

SR x Variety 777216 2 386081 15.2356 0.05 5.9863

Error 182256 12 15188

0 - - - - - - - 1 --r --- - -

0 6 15 24 33 42 5 1

Steeping Time (h)

Figure 10:Development of Glucanase Activity of three Sorghum Cultivars during Steeping using Air-Rest

( ----

) and Continuous ( ) Steep Regimes.

Table 14: A N 0 VA Tcr ble for tlte Developmcirf of Glircancrse Activi[y durirtg A ir-Rc.sf

and Coi~t i~i irom Steep

Sorrrce of Vdcrf ion SS df IWS F P-value F crit

Steeping Regime 8539.5 6 1423.3 45.404 0.05 2.9961

Varieties 988.21 2 494.11 15.763 0.05 3.8853

SR s Variety 64884.4 2 10814.4 91.705 0.05 5.9863

Total 9903.9 22

- - - ~ - -

6 15 24 33 42 5 1

Steeping Time (h) Figure 11: Development of Diastatic Power of three Sorghum Cultivars during Steeping using Air-Rest ( -) and Continuous ( ---- ) Steep Regimes.

Tuble IS : A N 0 VA Tc~ble for the Developi~lurit of Diastritic Power drrri~rg Air-Rest

a~zd Corttirrrrorrs Steep -

Soirrce of P'm-intion ss df fils F P-value F crit

Steeping Regime 6735.4 G 1122.G 2.6473 0.05 2.9961

Varieties 11301 2 5650.6 13.326 0.05 3.8853

SR x Varicty 44368.8 2 73946.0 18.165 0.05 8.9863

Error 5088.6 12 424.05

Total 23125 22

0 6 15 24 33 42 5 1

Steeping Time (h)

Figure 12: Development of Alpha Amylase Activity of three Sorghum Cultivars during Steeping using Air-Rest ( - )

and Continuous ( ------ ) Steep Regimes.

Tr~ble 16: A N 0 VA Tub Ie for the Development of Alplta Arnylrise Activity d i t r i f~g Air-

Rest crtirl Contir~rrorls Steep

Source of Variation SS df MS F P-vnlue Fcrit

Steeping Regime 55010 6 9168.4 24.729 0.05 2,9901

Varieties 9928.2 2 4964.1 13.389 0.05 3.3853

SR x Variety 61636.2 2 10273.3 67.789 0.05 5.9871

E 1-1-0 r 4449.1 12 370.75

Total 69387 22

0 6 15 24 3 3 42 5 1

Steeping Time (h)

Figure 13: Development of Beta-amylase Activity of three Sorghum Cultivars during Steeping using Air-Rest ( +nd

Continuous ( ---)Steep Regimes.

Tuble 17: ANOKA Tclble for the Developrnerit of Beta-Anij71cise Activity during Air-

Rest n r d Co~rtirtrro~rs Steep

- - - - - - - - -

Source of Variation SS df MS F P-value F crit

Steeping Rcgime 1827.8 6 304.63 9.6167 0.05 2.9961

Varieties 396.04 2 198.02 6.2512 0.05 3.8853

SR x Variety

E rro I.

Total 2603.9 22

0 ! . ---v I , 7-

0 6 15 24 33 42 5 1

Steeping Time (h)

Figure 14: Development of Wort Fermentability of three Sorghum Cultivars during Steeping using Air-Rest ( --) and Continuous ( ---- ) Steep Regimes.

- Ti~blt. 18: AN0 IYA Trrhle for tire Developnwnt of Wort Fern~errfcrbiiity drrrirrg Air-

Steep

Varie

Total

- - - - . - - - - - - - - KSV 8 +SK 5912 + ICSV 400 - - .x. - - KSV 8 CS

Steeping Time (h)

Figure 15: Development of Wort Fermentable Extract of three Sorghum Cultivars during Steeping using Air-Rest ( ) and Continuous ( - - - - - - ) Steep Regimes.

#

Trrble I 9: A NO VA Tuble for the Developmzeirt of Wort Ferr~zerrtuBle Extract drrring

Air-Rest ( r i d Coiltiillrolrs Steep

Source of Variation SS df MS F P-value F crit

Steeping regime 90423 6 15071 2.4092 0.05 2.9961

Vwicties 23459 2 11729 1.8751 0.05 3.8853

SR x Variety 600901 2 100154 4.2507 0.05 5.9863

E I-1.0 r 75064 12 6255.4

Total 188946 22

CHAPTER FOUR

DISCUSSION

The behaviour of grains during germination is an inducible property which

may be influenced by any of the several environmental variables found during grain

steeping (Pollock 1964, Briggs ef al., 1981, Okolo and Ezeogu, 1996a). Proximate

analysis of the sorghum grain (Table 1) revealed that carbohydrate content is in the

range of 63 - 68%, low protein content (9 - 10%), low fat content (3 - 4%). Ash

ranges from 4 - 7% while fibre is between (3 - 6%). Cereal grains endosperm

solubilization, mobilization and modification depend on several factors including

adequate aeration, good contact between enzymes and substrate, substrate

complexity and genetic make-up of the grains. (Glennie et a/., 1984; Okolo and

Ezeogu, 1996b).

Sorghum Grain Moisture Content, Water Sensitivity and Germinative Energy

Water sensitivity has for long attracted attention to malting technicians and

scientist as one of the most important factors affecting germination of grain, .

especially as it affects air-rest periods and the use of addictives. The possibility for

the replacement of barley by sorghum has recently received a boost with results

which have sought to match the grains physiology to its maltability (Palmer et a/.,

1989; Okolo and Ezeogu, 1994: Ezeogu and Okolo, 1995; Owuama, 1999).

The production of sorghum malt of improved variety contrast results obtained

with other varieties, a fraction which in this report was correlated significantly with

differences in germinability (grain germinative energy potential) and water sensitivity,

between different cultivars of sorghum under different environmental condition of .

steeping (Okolo and Ezeogu, 1996). In this report, the germinative behaviour of the

three cultivars of sorghum; SK 5912, KSV 8 and ICSV 400 were studied by

subjecting the grains to different steep treatments. The condition of steeping

influenced the inducibility of grains water sensitivity. All the parameters studied,

steep regime, air- rest period, final warm steep temperature and all their possible

interactions exhibited very mean statistics effect (Pc0.05) on the water sensitivity of

the grain for the three cultivars. Cultivars which were steeped for shorter period (36h)

prior to the application of final warm steep produces significantly (p< 0.05) higher

water sensitivity values compared to grains steeped continuously without air-rest

period and final warm water steep (Tables 1 and 2).

This implies that the steep period (prior to final warm steep) will possibly

improve germinability and potential vigour. This result, however, contrasts with .

earlrer observation made in barley malting by several workers including Briggs et a\.

(1981) whereby low grain germinative vigour was associated with grain steeped for

longer period prior to the application of final warm steep.

In an attempt to find explanation for the above observation, the inducibility of

water sensitivity in the sorghum grains as it relates to their moisture content just prior

to final warm steep was studied. An inverse relationship seemed to exist between

grain moisture content prior to final warm water steep and water sensitivity values

(Figure 1). This was later confirmed to be significant by statistical evaluation of the

relationships, thus establishing that sorghum grain water content prior to final warm

water steep may be complementary/mediator in the complex tripartite relationship

existing between the grain steeping regime (also read as steeping time), temperature

and germinative behaviours. Comparing grain moisture content before the final warm

steep, one additionally observed that the more sensitive grains were, there exist

lower moisture content value before final warm steep. This suggests the existence of

a moisture content threshold value below which the grain would exhibit heightened

water sensitivity as a response to the application d a final warm water steep.

Seed germination is the process of initiating growth in previously quiescent or

dormant embryo. For most seeds, it begins with the imbibition of water, Imbibition of .

water itself has been described as a triphasic process involving a period of rapid

initial water uptake (phase I), which is followed immediately by a plateau phase with

little change in water content (phase 11) and a subsequent increase in water content

coincident with radical growth (phase Ill). The second phase of this process has

been described as the most critical to germination process since it is the phase at

which the seed is most sensitive to environmental factors such as temperature.

Considering that most of the treatments that gave rise to heightened water sensitivity

produces grains which, while visibly beyond the rapid phase of moisture uptake, had .

not reached the final phase (as suggested by the level of moisture uptake upon final

warm steep), one may therefore suggest that it is necessary that from study reached

in the third phase of moisture uptake, that for successful germination to occur, the

use of re-steeping with the application of final warm steep is necessary (Okolo and

Ezeogu, 1995).

In addition to environmental factors, the role played by grain genetic factors in

malting has also received considerable attention with regard to the relationship

between the grain and the graln germinative vigour during malting [Munck, 1989). As

can be seen from the results of the experiment grain from SK 5912 is more water

sensitive than their lCSV 400 and KSV 8 counterpart (SK 591 2 lCSV 400 > KSV 8)

a factor which may be related to the lower seed vitality and low rate of water

6 5

absorption noted for SK 5912 as compared to that of lCSV 400 and KSV 8. The

above two factors and especially the latter would ensure that seed of SK 5912

stayed longer in the second phase of water absorption and as a consequence, gives

rise to more water sensitive grain upon final warm steeping.

Kernels of SK 5912 were more water sensitive than kernel of KSV 8 and ICSV

400 (74.0<92.0<93.0) for continuously steeped grains as compared with that

steeped with air-rest period and final warm-steeped grains (80.0497.048.0)

respectively (Table 3 and 4). Results also showed that temperature of final warm ..

r- steep is an important function regulating seed germinative vigor and water

sensitivity; as values obtained in the continuously steeped grain at 30" was less

than that obtained when final warm steep ( 40 '~ ) was applied.

Development of Extracts in the Sorghum Malt During Steeping

Cold Water Extract (CWE) - This represents the cold water soluble products of

enzymtc hydrolysis during the malting processes. These products are readily

available sugars and amino acids located in the endosperm of the malt (Glennie and

Holmes, 'I 992).

Figure 2 shows the development of cold water extract during steeping. The

pattern of development of CWE during steeping as deduced from the treatments and

their possible pair-wise interaction is highly significant (p -= 0.05) for the three

sorghum cultivars (Table 6). As shown in Figure 2, the CWE yield peak values for

air-rest samples were as follows; SK 5972at 33h (49%), ICSV 400 at 42h (42%) and

KSV 8 at 51 h (32%). In continuous steep sample SK 5912 at 33h (37%), lCSV 400

at 42h (40%) and KSV 8 at 51h /30%), respectively.

Correlating CWE development with moisture content and steeping time, one could

observe that the percentage extract solubilized during steeping increased with .

increase in moisture content (of grain steeped with air-rest). However, extract

development in continuously steeped samples were low, probably because of

inhibition of malt protease and p-glucanase development as a result of submerged

and continuous water supply to the grains as suggested by Okolo and Ezeogu

(1 996b).

Cold Water Soluble Carbohydrate (CWE-COH)

Cold Water Soluble Carbohydrate represents the soluble carbohydrate

fraction in the cold water extract. It is used to measure the level of carbohydrate .

mobilization during malting. Results are presented in figure 3 and Table 7

respectively. Results show that the CWS-COH values for the three sorghum cultivars

SK 5912, ICSV 400 and KSV 8 differed markedly. In air-rest samples, extract peak

values were as follows: SK 5912 at 3371 (48%), ICSV400 at 42h (45%) and KSV 8 at

33h (34%). As compared to that of continuous steeped samples whose results are:

SK 5912at 24h (45%), ICSV 400 at 3% (41•‹/0) and KSV 8 at 33h (36%), respectively.

The apparent enhancement of CWS-COH in malted grain in this work is not

surprising considering that, the activity of proteases resprsnsible for speedy .

endosperm mobilization during malting is reduced by continuously submerging the

grains in water. (Glennie et a/., 1984; Palmer 1989; Palmer et al. 1989; Ezeogu and

Okolo 1994). In addition, the activity of a-amylase required to hydrolyze the starch

and liberate carbohydrates is reduced due to reduced metabolism caused by lack of

oxygen in continuously submerged grains (Palmer ef a/,, 1989).

Temperature (final warm steep), steep regime (interrupted with air-rest periods)

grains cultivar and their possible pair wise interaction significantly (p < 0.05)

influenced CWS-COH development during sorghum steeping as observed in this

work.

Hot Water Extract (HWE)

This represents the hot water saluble products of enzymic hydrolysis during malting.

These products were readily available sugars and amino acids. The effect of steep

treatments on the development of malt HWE was also studied. The results were

presented in figure 4 and Table 8. Extract peak values were obtained in the samples

and their cu'ftivars as follows: In air-rest samples: SK 5912 at 51 h (2941, ICSV 400 at

51 h (252 and KSV 8 at 51 h (238). In continuous steep sample SK 5912 at J3h (270),

ICSV 400 at 24h (290) and KSV 8 at 33h (275), respectively. The study show that

there is a linear relationship between HWE development and the grain moisture

content also read as steeping time, from 15 to 51 h of steeping as shown in figure 4.

Also steeping temperature [deduced as application of final warm steep) affected the

HWE yield: with a corresponding appreciable increase in the yield (Figure 4). The

development of cereal grains HWE is influenced by a number of factors notably

condition of steeping (air-rest period, final warm steep) (Okolo and Ezeogu, 1995);

cereal grain genetic factor, amylase and diastatic powers (Briggs et al, 'l981). One of

the objectives of malting is to increase the level of soluble solids extractable from

cereal grains. Briggs et %I (1981) observed that HWE development is the result of

interplay in proteases, amylases and diastases activities present in cereal grain

endosperm. These enzymes bring about the degradation and further solwbilization of

the cereal grain endosperrn during malting, thus liberating the endosperm

components to give rise to higher yield in HWE. In this work, the condition for these

enzymes to work was potentiated by the use of air-rest periods and final warm steep

as against the control (continuous steep), Irrespective of the cultivar, all the grains

showed a significant increase in HWE yield thus supparting an earlier observation

made by Briggs et a/. (1981) and Okolo and Ezeogu (1995).

Further investigation on the relationship between the steep treatment, grain

var~ety and temperature and their effect on HWE yield as well as their pair wise

possible interaction was deduced statistically as shown in table 8.0. On the basis of - r Analysis of variance (ANOVA), it was shown that the effect of the above factors on

HWE yield is highiy significant (p < 0.05)

The correlation between the HWE yield with the Diastatic power and amylase

activities is shown in the appendix 2. There Is a strong positive correlation, significant

at the 0.05 and D.OA level between HWE and amylase development. This goes

further to support the fact that protease, amylase and glucanase activity resulting in

the high yield of extracts.

Hot Water Extract Protein (HWE-PROTEIN)

This measures the amino acids and peptides soluble in hot water which are

products of enzymic hydrolysis during malting (Glennie and Holmes, 1992; lrvine

1987). The degree of modulation in sorghum is an important malt characteristics as

both carbohydrates and proteins modification affects extract yield. Traditionally, the

extent of protein solubilization during malting as represented by the Kolbach index

(soluble nitrogen (N) of Hot Water ExtractITotal nitrogen (N)) of grain has been used

as an index of grain maltability and modulation during malting. In this work, the level of

protein left in solution in the Hot Water extracted sorghum malt was determined and

the results are presented in figure 5 and Table 9 respectively. . .

Results show that the HWE-protein differed significantly (p < 0.05) with

respect to the final warm steep treatment at 40•‹c, which solubilized markedly were

protein as compared to those of continuously steeped grains. SK 5912 and KSV and

ICSV 400 showed an increase in HWE-protein with the application of final warm

steep as against those of continuously steeped grains, that showed a decrease;

whereas the reverse was the case of KSV 8 continuous sample.

Air-rest samples have their peak values as follows SK 591 2 at 51 h (294%),

ICSV 400 at 51h (252OJo) and KSV at 51h {238%). In continuous steep sample SK .

5912 at 33h (270), ICSV 400 at 24h (290%) and KSV 8 at 33h (275%). Using

Analysis of Variance (ANOVA) Table 9, it showed that significant relationship (p <

0.05) exist with both steeping condition. Temperature (as final warm steep at 40%)

and grain variety, in influencing the level of HWE-protein development during

steeping.

Several factors are involved in protein solubilization during malting. From this

- work, it could be seen that a fall in protein solubilization accompanied an increase in

final warm samples of SK 5912 and ICSV 400 but not for KSV 8; while no specific .

explanation may be preferred for this phenomenon, it is possible as has been

observed for some barley hydrolases by several workers (Nishiyana 1986) that the

physiological stress caused by rise in final warm steep temperature could have

engendered an increase in the protein resulting in the synthesis of protease isoforms

with either a reduced activity for malt protein, or with lowered stability at the

temperature of the malt. This, therefore, suggests a possible temperature

dependence of protease synthesis in sorghum grain similar to what has been

described for several barley hydrolase.

Free Alpha (a) Amino Nitrogen (FAN)

FAN is the balance of catabolic processes which degrade the storage proteins

~nto amino acids and the anabolic processes responsible for the synthesis of these

degradation products into new proteins in the growing tissues of the shoots and

roots. (Marrall et al. 1986; Shutov ef a!. 1984). However. the rate of anabolic

processes is the determining factor in FAN. (Taylor, 1983). Wort a-amino nitrogen

represents the major source of assimilable nitrogen for brewing yeast (Taylor and

Evans,1989). The influence of steeping (interrupted by air-rest periods continuously

submerged grains and final warm steep) on sorghum malt FAN development was

studied and results presented in rng % values are shown in figure 7 and Table 11.

Peak values in FAN development were obtained in the samples and their cultivars as

fobws: in air-rest samples, SK 5912 at 24h (280mg%), ICSV 400 at 24h (28mgoh)

and KSV 8 at 33-42h (230mg%). In continuows steep samples SK 5912 at 24h

(250mg%), ICSV 400 at 24h (270mg%) and KSV 8 at 24h (250mg%), respectively.

The sorghum malt amino acid level were significantly (p < 0.05) influenced by all the

factors under study, as well as through their various interactions. Malt FAN level was

found to be markedly influenced by grain cultivar. Grains of ICSV 400 produced the

highest FAN level followed by SK 5912 while the least is KSV 8. Malt FAN

development displayed a significant sensitivity to final warm steep in all the cultivars

as against those steeped continuwsly, In figure 7, there was a decrease in FAN

level in all the cultivars after the application of final warm steep. In continuous steep

samples, there was a gradual decrease in FAN level after 24h of steeping.

This phenomenon is explicable by the possible differences in the genetic

make up of the three cultivars and this confirms the results of earlier workers who

sought to relate malting performance to the inate property of grains (Munch et a/.,

1989; Okolo and Ezeogu, l996a).

Endosperm solubilization at steep-ripeness enhances FAN production. In this

work, there was a linear relationship between grain moisture content (deduced as

steeping time) and FAN release up to the application of the final warm steep in all

the cultivars of the air-rest samples, before the FAN released began to decline: This

phenomenon was observed with the period of the steeping regime. The mean time

for opt~mal FAN release was 24h. That this pattern of FAN release occurred more

significantly among the different steep regimes, and also' among the cultivars, further

suggests that the timing of the final warm steep treatment may have a significant

effect on the physiological processes responsible for the development of FAN in

malting sorghum grains as had earlier been observed in barley (Baxter and O'Farrell,

1980).

It IS also possible that the FAN behaviour of grain steeped at 4 0 ' ~ final warm

steep was a reflection of the moisture content of the grain just before the final warm

steep. Using Analysis of Variance (Table 1 I ) , it could be observed that grain cultivar,

steeping regimes, air-rest period and application of final warm steep and their

possible interactions significantly (p < 0.05) affected FAN development during

steeping. Correlating FAN development and carboxypeptidase activity showed a

strong correlation significant at r = 0.01 and r = 0.05 confident level in both

continuous steep and air-rest samples of the cultivars (Data shown in appendix 2).

Total Non Protein Nitrogen (TNPN)

Total non-protein nitrogen are product of storage protein hydrolysis, soluble in .

Trichloroacetic acid and comprising amino acids and peptides containing 2 - 70

amino acids units (Ratsogi and Oaks, 1986). Like FAN, TNPN represents a balance

in its release and its removal to new tissues in the germinating seedling. Ail the

factors studied, as well as their various Interactions had significant effect on malt

Trichloracetic acid (TCA) soluble nitrogen development. In this work, there was a

steady increase in TNPN development in all the cultivars prior to the application of

r. final warm steep (as shown in figure 6) in all the cultivars and their samples.

However, continuous steeped samples had higher TNPN development than their air- .

rest counterparts, with peak values at 42h of steeping. Beyond this period, there was

a decline in TNPN development in all the cultivars. Also air-rest samples had the~r

peak values at 42h of steeping in ICSV 400 and KSV 8 whereas SK 5912 peak value

was at 33h.

Dewer et a/. (1996; 1997) have commented on the importance of grain steep

out moisture in sorghum malt quality development. In this work, sorghum malt TNPN

levels in relation to the steep out moisture content just prior to the application of final

warm steep were studied, and results obtained shown in figure 6 and Table 10. An

inverse relationship exists between the moisture content and malt TNPN level for all

the cultivars and their samples after the application of the final warm steep especially

for SK 5912 and ICSV 400. Cereal grain endosperm solubilization is dependent on

the moisture content of the grain prior to steep out. There exists steady linear

relationship between the grain moisture content (also read as steeping time) and

TPNP release prior to final warm steep in all the cultivars. The steeping temperature

has also a similar effect since at ~o'c, there was a steady increase in TPNP release

which declined when the temperature was increased to 4 0 ' ~ .

Seed germination is attended by the hydrolysis of endosperm storage protein

by proteolytic enzymes to provide nitrogen compound for the sustenance of growth -

in the vegetative part of the young plant. It has been suggested that the level of

TNPN in a germinating grain at any time was the product of a balance of activities on

non- protein nitrogen utilization process on the one hand and TNPN-releasing

process on the other hand. (Mikola and Kolehmainen, 1972; Ezeogu and Okolo,

1996). The result presented in this work with respect to both FAN and TNPN may

also represent the effects, which condition of malting as used in this work had on

these two processes.

Analysis of Variance (Table 10) show that all the factors studied; viz variety, steeping .

conditions cultivar and their possible pair-wise interaction had significant effect

(Pc0.05) in influencing TNPN development during steeping.

Proteinase Activity

Endoproteasdproteinase is the key enzyme in the mobilization of endosperm

proteins during malting. It catalyses the initial step in the hydrolysis of endosperm

reserves through the solubilization of endosperm matrix proteins and the release of

other endosperm polymers for hydrolysis by their respective enzymes (Lewis et a/.

1979, Siebert and Kundson 1989; Glennie and Holmes, 1992). They are responsible .

for the release of higher molecular weight polypeptide tagged cold water soluble

proteins. Figure 8 and Table 12 show the development of prateinases during

steeping among the samples and their cultivars. Results were presented in

mg/N215hfg. In air-rest samples, peak values were obtained as follows: SK5912 at

51 h (8000) lCSV at 24h (750) and K8V8 at 5h(800) in continuous steeped samples,

SK59?2 at 42h (700$, ICSV at 24h (700) and KSV8 at 51hj (1000) respectively.

ANOVA (Table 12) revealed that, cultivar, steeping regime and their pairwise

possible interaction had a significant effect (Pc0.05) in the malt proteinase

development during steeping, There was nu correlation between proteinase activity

and extract yield during grain malting. [Data in Appendix 2). It is also remarkable in

this work there is relationship between FAN release and proteinase activity (Data

Appendix). It is possible that this, inconsistency in proteinase activity suggest that

qualitative differences exist in the endosperrn proteins in the various cultivars and lor

that differences exist in the nature of the major isoforms of enbo-proteases released

by these grains andior that endo proteases in sorghum are triggered off at different

temperature in different grains eultivars and in different timefperiod, inducible by the

grain. This concurred with earlier observation made by Okolo and Ezeogu (1995) on

sorghum grains and Riggs et a1 (1983) with barley grains. Thus wide differences in

the grain protein character would invariably affect protein susceptibility to enzymic .

hydrolysis.

Carboxypeptidase Activity

Exo-proteaseslcarbdxy peptldase is the key enzyme in the mobilization of

endosperm protein during malting. It catalyses the initial step in the hydrolysis of

endosperm reserves through the solubilization of endosperm matrix and the release

of other endosperm polymers for hydrolysis by their respective enzymes. They are

responsible for the release of FAN from solubilized protein during malting. (Ronki et .

a/. 1990; Palmer 1989). Carboxypeptidase development In all the cultivars was

affected by all the factors under study. Results shown in figure 9 and Table 13

present carboxypeptidase activity in all the samples and their cultivars. As shown

peak values were obtained in air-rest samples as follows (results are in mg

FANI5hlg) SK 5912, at 33h (800) KSV at 24h (950) and ICSV 8 at 51h (800), in

continuous steep samples, SK at 33h (700) ICSV at 42h (1000) and KSV8 at 51h

(700) respectively. ANOVA (Table 12) revealed that cultivar, steeping regimes and

their pair-wise possible interaction significantly (Pc0.05) influenced carboxy peptidase

development during steeping. However, thls was not so with increase in temperature

(deduced as application of final warm steep at 40'6) which decreases

carboxypeptidase development. Cereal grain endosperm solubilization in moisture .

content (water sensitivity) dependent.

Figure 9 shows a linear relationship between cultivar moisture content (also

deduced steeping time) and carboxypeptidase activity for the cultivars. However,

inverse relationship was obtained with elevated temperature of 40•‹C final warm

steep. The above phenomenon may be explained by correlating the

carboxypeptidase activity with Extract release (FAN) during malting. Appendix 2

shows h e a r correlation (r = 0.01; r = 0.05) significant level between FAN release

and carboxypeptidase activity for the cultivars. It is then apparent that the rate of

anabolism that supercedes catabolism during grain malting is possible determining

factor in malt carboxypeptidase release. The pattern of variation in carboxypeptidase

development with different cultivars in response to the above treatments (air-rest

period and elevated final warm steep at 40•‹C) suggest possible presence of multiple

forms of this enzymes and in this work, this assumption agrees reasonably with

observation advanced by various worker (Enari ei al. 1986; Mikola and Kolehmairren,

1972; Ranki et al. 1990; Okolo and Ezeogu 1995) on the presence of multiple

carboxypeptidase isoforms in malting barley; presuming same to exist in sorghum.

Glucanase Activity

Endo peta 1, 3 and 1, 4 glucanase are enzymes responsible for the hydrolysis

of high molecular weight non-starchy pdysaccharides (P-glucans and pentosans) or

polyphenol-protein complexes or cysteine-rich proteins into low molecular weight at

1, 3 and 1, 4 bonds. (Dufour et al. 1992; Etokakpan, 1992). Figure 10 and Table 14

show the development of glucanase activity during steeping in both samples and

their cultivars. Peak values were obtained as follows; in air-rest samples all the

cultivars at 33h of steeping attained peak values. SK5912 (go), ICSV 400 (60) and

KSV 8 (80). Similar effect was obtained with continuous steep samples at 33h of

steeping; SK5912 (80), ICSV 400 (58) and KSV8 (69) respectively. Beyond this 33h

steeping time, there was a steady decline in glucanase development in the samples

as well as their cultivars. AII the factors under test and their pair wise possible

interaction were highly significant (p < 0.05) in influencing glucanase activity during

sorghum malting as shown in table 14.

Physiologically, in sorghum, P-glucanase develops in the starchy endosperm

during malting (Aisien et at, 1983: Palmer, 1989) where they affect limited attack on

the endosperm cell wall thus causing some p-glucans to be released during malting.

The solubilization of sorghum endosperm by glucanase during malting depends on

the endosperm water (Fig. 10). There is a linear relationship between p-glucanase

release and moisture content (also read as steeping time) of the grain for cultivars till

their peak values of 33h steeping, beyond which there was an inverse relationship till

the 51h steepout time. Similar inverse relationship was obtained with elevated

temperature (deduced as application of final warm steep at 4 0 ' ~ ) among the

samples and their cultivars.

Diastatic Power

Diastatases is collective name given to the combined action of group of

enzymes responsible for complete starch hydrolysis into simple sugar during malting

in cereal grains. The amylolytic enzymes (a and P - amylase) are included in this ..

group (Taylor ef a!. 1993; Demuyakor et a!. 1992; Etokakpan and Palmer, 1990).

Diastatic power and amylase activity in some cases are used as an index of

modification of starch during malting in cereal grains. Malt diastatic power

development was affected in a significant manner (P < 0.05) by all the treatment

parameters as well as their possible pairwise interactions in this study. Grain

diastatic power increased along steeping time up to 33h of steeping in both samples

and their cultivars. Peak values were obtained at 33h steeping time beyond which

there was a decline in diastatic power among the samples and their cultivars. .

.

Diastases release in cereal endosperm during malting is moisture content and water

sensitivity dependent since both are necessary for endosperm solubilization,

mobilization and modification. Increase in moisture content (also deduced as

steeping time) has a linear relationship with diastatase development up to its peak

value beyond which there exists an inverse relationship. Temperature (also read as

application of final warm steep) has a similar effect, since there was a decline in

diastatases with the application of 4 0 ' ~ final warm water steep.

These observed irregular expression in diastatic activity of the cultivars may

be attributed to the fact that certain germinating grain endosperm enzymes lack

enzyme cofactors e.g. calcium ions and Gibberillic acid. (Dewer et a!. 1997; Buckee

et a/ . , 1989). The cofactors are associated with the enhancement of the production

and secretion of amylases, the pace-setting enzyme in the endosperm starch

modification in cereal grains (Dewer et a/., 1996). The differences observed in this

work in diastase response to steep treatments may be both genetical and

physiological. These differences may perhaps glve rise to the inability of the grains to

activate their amylase isoforms when subjected to specific steep treatment or the

inability of the grain to activate their amylase inhibitor substances or both.

Alpha (a) Amylase Activity

Alpha (a ) amylase catalyzes the random hydrolysis of starch chains at o: (1,4)

glucosidic linkages distant from the end of chain and from a (1,6) linked branches in

the chain releasing sugar. (Demuyakor et a/, 1992; Palmer, 1989; Hough et a/,

1971). Its formation in the embryo and release at the endosperm is both oxygen and

carbon dioxide dependent (Etokakpan and Palmer, 1990).

The effects of steep treatment on malt a-amylase development are presented

In f~gure 12 and Table 16. All the factors studied significantly (p -= 0.05) affected a-

amylase development during steeping. In the samples and their cultivars, steeping

the grain with air-rest period has a linear relationship with a-amylase development till

33h which corresponds to its peak value. Same effect was obtained in continuous

steeped samples. However, beyond this peak value, there was an inverse

relationship with grain moisture uptake and a-amylase development in all the

cultivars and their samples. Application of final warm steep further decreased or-

amylase development in all the cultivars and their samples. Sorghum endosperm

solubilization and mobilization during malting is grain moisture content and water

sensitive dependent. (Figure 12). Correlating a-amylase development with extract

yield (HWE and CWE) data in Appendix 2, show that all the cultivars and their

samples had a linear significant relationship (r = 0.05: r = 0.01) with extract yield.

The possible explanation to the above phenomenon may be attributed to u-

amylase polymorphism (Owuama, 1999). Earlier workers have shown that steep

treatments influences the inhibition or enhancement of the synthesis of particular

iscrforms detectable in cereal grains during malting (Jones et a!. 1983; Mc Gregor

1982; Dufour et a/. 1992). The results of this work are in agreement with results

obtained by earlier researchers on a-amylase development in sorghum grains during

malting. Also inhibition of a specific dominant a-amylase isotypes by natural

proteinaceous u-amylase inhibitors in sorghum (Mc Gregor, 1982) invariably

depresses total a-amylase activity as obtained in this work.

Beta (p) Amylase Activity

Beta-amylase catalyses the hydrolysis of penultimate cc (1,4) glucosidic bond

at non-reducing end of polysaccharides causing the release of maltose. They are

activated from latent form in the starchy endosperm during malting (Palmer, 1989).

The effect of steep treatment on the sorghum malt P-amylase are shown in

figure 13 and Table 17. There was a decrease in p-amylase activity in all the

cultivars and their samples. Results obtained show that in both samples and .

cultlvars, peak values were obtained at 33h of steeping in ICSV 400 and KSV 8,

while that of SK 5912 was at 42h. (Figure 13). The effect of moisture content and

temperature on p-amylase development were also evaluated. There was a linear

relationship between p-amylase development and malt moisture content {deduced

as steeping time) up to 33h of steeping and inverse relationship beyond this peak

value for samples of ICSV 400 and KSV 8; while that of SK 5912 was at 42h of

steeping. Temperature (deduced in this work as application of final warm steep at

4 0 ' ~ ) decreased p-amylase devebpment in all the cultivars and their batches.

Novellie (1960) reported in barley that the development of p-amylase activity is

simultaneous to that of a-amylase and therefore calculated P-amylase as the ..

difference between total amylase and a-amylase. Novellie (1960) further reported

that (3-amylase in sorghum grain was slow due to some inhibitory factors inherent in

€he grain.

Dufour and Melotte (1992) in their comparative work on sorghum and barley

maintained that p-amylase developed in sorghum alongside a-amylase during

germination. They still calculated p-amylase as the difference between total amylase

activity and a-amylase. They further stated that p-chloromercuribenzoic acid inhibit

the development of P-amylase during malting.

Taylor (1983) reported that P-amylase was virtually inactive in non-

germinating sorghum grain and further suggested that P-amylase may be completely

soluble or insoluble in sorghum malts depending on the variety. This suggestion has

earlier been pointed out by Novellie (1962) and Jayatissa e l al (1980) in which they

suggested that P-amylase was adsorbed tenaciously to insoluble substances present

in the malt.

Taylor and Robbins (1993) still not satisfied with their reports, treated

sorghum malt with enzyme releasing substances like papain and still recorded low P-

amylase activity, they then concluded that P-amylase was not bond to any insaluble

substances present in the malt. However, they still calculated a-amylase as the -

difference between total amylase and a-amylase.

Owuama (1999) reported that p-amylase was embedded on the matrix of

other starch degrading enzymes such as a-glucosidase and the release of these

enzymes during malting triggered the activity of p-amylase, and therefore stated as

erroneous and misleading, the assumption by many workers and researchers that @-

amylase was the difference between total amylase and a-amylase, since this

assumption does not take into cognizance the effect of other starch degrading

enzymes such as a-glucosidases and dextrinases. Onwuarna (1999) still calculated .

p-amylase as the difference between total amylase and a-amylase activity during

malting.

The variation of P-amylase development in this work may be attributable to

the repression of the synthesis of a major p-amylase isotypes, since isoelectric

focusing has indicated that sorghum 0-amylase has a major and a minor isoenzyme

of approximate PI 4.4 - 4.5 (Taylor and Robbins, 1993; Owuama 1999) or due to p-

amylase heterogeneity which is influenced by malting mnditions and stages. (Mc

Gregor et a/. , 1984).

In this work, owing to the various manipulations carried out in our laboratory

during malting, of sorghum, it could be possible that the various techniques used

could trigger the synthesis and development of P-amylase activity during steeping.

Analysis of variance (ANOVA) (Table 17) revealed that all the parameters studied

(steep treatments, steep condition, cultivars and all their possible interactions were

highly significant (p < 0.05) in influencing P-amylase development during steeping.

Correlation between p-amylase activity and extract development (HWE) data in

Appendix 2, show a strong significant correlation (r = 0.05, r = 0.01) confident level

existing between HWE yield and p-amylase activity among the cultivars and their

samples. .

Wort Fermentability

This measures the fermentable sugars present in the wort. These sugars

include fructose, glucose, sucrose, maltose and maltotriose (Dufour and Melotte

1992). Results of wort fermentability studies after 48hours are shown in figure 14

and Table 18 respectively. Peak values were obtained in the samples and their

cultivars as follows: in air-rest samples; SK 5912 at 24h (65%) lCSV 400 at 33h

(55%) and KSV 8 at 42h (55%). Continuous steep samples, SK 5912 at 33h (55%}, .

ICSV 400 at 51 h (58%) and KSV 8 at 51 h (60%) respectively.

Steep treatments and grain variety together with their possible pair-wise

interaction is highly significant (p < 0.05) in affecting wort fermentability as shown in

ANOVA (Table 18). Wort fermentability is an index of the ability of the fermenting

yeast to effect almost complete attenuation, within a given total brewing time

(Owuama, 1999). This is dependent on yeast viability, and presencelavailability of

fermentable substrates in the wort, These fermentable substrates are products of

malting and mashing. These products are also derived from the cereal grain and -

other brewing raw material/adjuncts (Owuama, 1999).

Wort Fermentable Extracts

This refers to the total extracts present in the wort for yeast growth. These

extracts include sugars, amino acids and peptides and free fatty acids and glycerols.

The fermentable extracts of the war€ was also determined after 48h and results

obtained from the study were in figure 15 and Table 19. From the results air-rest

sample had the highest fermentable extract than the continuous steep samples.

Peak values were obtained as follows: SK 5912 at 51h (550), ICSV 400 at 24h (545)

and KSV 8 at 33h (420). In continuous steep samples, SK 5912 at 33h (4201, ICSV

400 at 42h (430) and KSV 8 at 42h (360) respectively. ANOVA revealed that there is . a highly significant relation (p < 0.05) between the parameters studied (steep

treatment, cultivar, and their pair-wise interactions) and wort fermentable extract

development (Table 19).

The extent of fermentation is dependent on many variables; including the

viability of the yeastlyeast strain, wort fermentable extracts available, fermentation

pH and time (Owuama, 1999). In this work, wort fermentable extract after 48h

yielded higher extract, with the application of final warm steep in samples KSV 400

and KSV 8 and their cultivars except in SK 5912 air-rest sample. It could be possible

that wort fermentable extract in this work, could reflect, differences in the above

observed contributory variables in fermentation.

CHAPTER FIVE

CONCLUSION

Steep treatments (air-rest period, final warm water steep at 4 0 ' ~ ) have a

considerable effect on both malt extract yield, and enzyme development. In this

work, steeping sorghum grains in distilled water for 51 hours with interrupted air-rest

periods of 3 hours (after every 6h steep and a final warm steep at 40% for 6 h

increased endosperm solubilization, mobilization and modification resulting in higher

extract yield as against the continuous steep grains (grains completely immersed in

distilled water for 51 h) in the cultivars, SK 5912, ICSV 400 and KSV 8. Sorghum

endosperm modification indices like: cold water extract [CWE), Hot water extract

(HWE), Cold water soluble carbohydrate (CWS-COH), Hot water protein (HWE-

protein), free a amino nitrogen (FAN'), Total non protein nitrogen (TNPN) were all

higher in samples steeped with air-rest periods and final warm steep than in samples

steeped continuously at the 51 h steeping period.

However, the reverse was the case for enzyme development. Glucanase,

Diastatic power, alpha and beta amylases showed linear increase with steeping

period up to 33h of steeping in both air-rest and continuous steep samples, with

higher values obtained in air-rest samples. Application of final warm steep decreased

enzyme development in all the cultivars (SK 5912, ICSV 400 and KSV 8). In

continuous steep samples from 42h of steeping, enzyme development decreased in

all the cultivars (SK 5912, ICSV 400 and KSV 8).

Proteinase and carboxypeptidase activity showed exceptional behaviour. In

proteinases, continuous steeped cultivars of KSV 8 recorded the highest activity as

against the other cultivars. This was not so in carb~xypeptidases where ICSV 400

recorded the highest activity in continuous steeped cultivars. Application of final

warm steep, increased both proteinases activity in cultivars; SK 591 2 and KSV 8 and

decreased that of ICSV 400. Carboxypeptidase activity in cultivars; KSV 8 was .

increased with application of final warm steep, but decreases were obtained in

cultivars ICSV 400 and SK 5912.

Wort fermentability and wort fermentable extracts were also affected by the

steep treatments and steep conditions. Air-rest samples recorded lower wort

fermentability than their continuous steep counterpart in the cultivars; KSV 8 and

ICSV 400, but not in SK 5912 at 51h steep out whereas application of final warm

steep at 4 0 ' ~ decreased wort fermentability in cultivars SK 5912 and KSV 8, that of

ICSV was increased in continuous steep cultivars, beyond 42h steeping. Wort

fermentability values increased in SK 5912 and ICSV 400, but decreased in KSV 8.

in wort fermentable extract, application of final warm steep decreased wort

fermentable extract in cultivars ICSV 400 and KSV 8, but increased that of SK 5912,

Also beyond 42h of steeping, in continuous steep samples, all the cultivars recorded

decreased wort fermentable extract.

From the results obtained in this work, under the prevailing malting conditions

and their pair-wise possible interactions, one can affirm that steep treated grains

yielded more malt extracts and less enzyme development which in this work is .

cultivar dependent. Considering the elevated modification parameters and because

of the natural heterogeneous endosperm storage reserves, in the different cultivars,

hydrolyse were differently affected by the hydrolytic enzymes. Mareso, most of the

enzymes are either enzyme complexes or enzyme complex with rnultiplelcornplex

isoforms. In this case, steep treatments/conditions, can either activate or inhibit their

synthesis, secretions or release, which will affect the level of endosperm reserve

solubilization and modification with a concomitant increase or decrease in extract

yield as well as in enzyme development as obtained in some of the evaluated

parameters in this work.

APPENDIX I

PREPARATION OF ANALYTICAL REAGENTS

The following analytical reagents were prepared as follows:

A RSENOMOLYBDATE

Ammonium molybdate (259) was dissolved in 450ml of deionized water.

Sodium hydroxide Arsenate (3g) was also dissolved in 25ml of deionized water and

the two mixed together with 25mI of concentrated sulphoric acid. The mixture was

incubated at 40•‹C for 45h.

,- SODIUM ACETATE EXTRCTION BUFFER pH 5.7

The was prepared by dissolving I .47g of calcium chloride, 3.096g of sodium

acetate, 2.9229 of sodium chloride in a 500ml volumetric flask and made up to

volume with double deionized water. The pH was adjusted to 5.7 with acetic acid.

SODIUM ACETATE (ASSAY) BUFFER pH 4.6

Sodium acetate (0.669) was dissolved in 200ml volumetric flask and made up

to volume with double deionized water. The pH was adjusted with acetic acid.

7% STARCH SOLUTION

A slurry of I g of starch was prepared and added to a boiling assay buffer pH .

4.6. This was boiled for 1 minute and allowed to cool and made up to volume

(1 00mI) with the buffer.

MERCURIC CHLORIDE SOLUTION ( 7 0 ~ r n ~ / r n / }

This was prepared by adding 0.001g of mercuric chloride into ? litre of

deionized water.

0.2N BARlUM HYDDROXIDE

This was prepared by adding 6.319 of barium hydroxide in 200ml of deionized

water.

FAN COLOUR REAGENTS

Disodium hydrogen phosphate (25g), potassium dihydrogen phosphate 15g,

Ninhydrin 1.25g and fructose 0 . 7 5 ~ ~ were dissolved in deionized water and made up

to 100ml.

FAN DILUTING SOLUTION

Potassium iodate (0.59) was dissolved in 1 %bt l deionized water and 100rnl of

96% Ethanol added.

GLYCINE STNDARD SOLUTION .

Glycine (0.1 Oi'Zg) was dissolved in 100ml deionized water for use. I rnl was

diluted to 100ml with deionized water to give 2mg amino nitrogen per litre.

0. ? M CITRA TE PHOSPHATE BUFFER pH 7.0

This was prepared by dissolving 1 . 6 8 ~ ~ citric acid, 2.18g sodium hydrogen

phosphate and 0.159g sodium chloride in TOOrnl of deionized water,

O.OO6N AMMONIA SOLUTION

This was prepared by dissolving 5.1 7g of Ammonia in 100ml deionized water.

METAL CATALYST . This was prepared by mixing K2SO4, CUS04.5H20 and selenium dioxide

(Se02) in the ratio of 100:3:3.

7OmM CALCIUM CHLORIDE

This was prepared by dissolving 3.675g of CaCI2 in 1000ml of deionized

water.

BROMOCRESOL GREEN-METHYL RED INDlCATOR

This was prepared by mixing separately O.lwlv Bromocresol green in 50ml of

95O/0 Ethanol and 0 . 1 ~ 1 ~ methyl red in 50ml of 95% Ethanol. The two solutions re

mixed again in the ratio of 10:4 to give 0.059 Brornocresol green and 0.02g methyl

red in 100ml of 95% Ethanol.

0. I N H2S04 SOLUTION

This was prepared by dissolving 2.72m1 of concentrated H2S04 in 100rnl of

distilled water,

40% wlv SODlUM HYDROXIDE SOLUTION

This was prepared by dissolving 40g of NaOH in 100ml of distilled water.

2% wlv BORIC ACID SOLUTION

This was prepared by dissolving 29 of Boric acid in 100ml of distilled water.

CARBOXYPEPTIDASE AND PROTEINASE EXTRACTION ASSAY REAGENTS

0. ?M ClTRA TE PHOSPHATE BUFFER pH 7.0

This was prepared by dissolving 3.21g of Na2HP04 1,250~1 of distilled water.

0.3M CITRATE PHOSPHATE 8UFFER Ph 4.6

This was prepared by dissolving 2.189 of either K2HP04 pr Na2HP04 in 250ml

of distilled water.

3.33mM DITHROTHREITOL

This was prepared by dissolving 0.067g of Dithrothreitol in 25Qrnl of distilled

water.

MASHING SOLUTION (SODIUM ACETATE BUFFER pH 5.7)

This was prepared by dissolving 18.57g of sodium acetate, 8.449 of calcium chloride

and 1.76g of sodium chloride in 1 OOml of deionized water,

0.05M GLACIAL ACETIC ACID SOLUTlON

This was prepared by dissolving 2.875ml of glacial acetic acid in 500ml of

distilled water. Make up to 1 litre.

0.05M SODIUM ACETATE BUFFER pH 5.7

This was prepared by dissolving 6.802~~ of sodium acetate in 1 litre of distilled

water.

50mM SODIUM ACETATE BUFFER pH 4.6

This was prepared by dissolving 490ml of 0.05M sodium acetate in 510ml of .

0.05M Glacial acetic acid.

73mM SODIUM CHLORIDE SOLUT/ON

This was prepared by dissolving 0.15g of sodium chloride in 250ml of distilled

water.

2% SOLUBLE STARCH SOLUTION pH 4.6

29 of starch was added to 100ml of buffer solution pH 4.6 and was boiled for 2

minutes and cooled. The soluble starch was prepared daily as required.

0.2N ZINC SULPHATE

This was prepared by adding 5 .75~~ of ZnS04 in 200ml of deionized water.

SOMOGYI REAGENT

The following salts were dissolved in distilled water and make up to 800ml.

Rochelle salt (sodium potassium tartrate) 129, sodium carbonate 24g, sodium

bicarbonate 16g and sodium sulphate 1449. This was labeled solution I.

These salts were dissolved in distilled water and made up to 200ml.

7 Hydrated copper sulphate Reptahydrate 4g, sodium sulphate 36g. This was labeled

solution 1 1 . Solut~on I and II were then mixed in the ratio of 4 : l and labeled solution 1 1 1

(Somogyi Reagent A).

NELSON REAGENT

This was prepared by dissoIving 259 of Ammonium molybdate tetrahydrate in

450ml of distilled water and with cuntinclous stirring. 21mI of concentrated H 2 S 0 4

was added. Then 3g of sodium arsenomolybdate was dissolved in 25ml of distilkd

water. The two solution were mixed and labeled solution IV (Nelson reagent B). This

r mixture was then incubated in a water bath at 40•‹C for 24-48h. The reagent was

filtered after incubation.

The Nelson-Somogyi Reagent was always prepared fresh by mixing in the ratio of

4:l of Reagent A and 6.

NELSON-SOMOGYI METHOD

One milliliter of the test solution was put in a test tube. This was mixed with

Im l of Somogyi reagent. The tube were covered and heated on a boiling water bath

for 15 minutes, The tubes were then cooled in cord water and I ml of Nelson reagent

r was added. This was left to stand for a minute until the effervescence disappeared. It

was then diluted to a final volume of 10mI and the absorbance was read at 500nm

on a spectrometer.

A unit of the enzyme activity was defined as any amount of the enzyme which

was capable of releasing 1pg glucose equivalent in reducing activity per minute

under the assay condition.

*-

METHYLENE BLUE STAINING METHOD

Reagent Methylene Blue Preparation

Dissolve 0.01g methylene blue in l0ml distilled water, add 2g sodium citrate

dihydrate and stir until it dissolve. Filter and make up the filtrate to 100rnl with

distilled water.

METHOD: Mix the dye solution with an equal volume of a suspension of the yeast .

sample under examination and place on a microscopic slide. Adjust the

concentration of the yeast suspension so that 40 - 60 cells are present per

r microscopic field after mixing with the dye solution. Count approximately 1000 cells

and report the number of unstained cells as a percentage of the total, for this

purpose, ignore buds unless their size is greater than one half of the parent cell.

This is based on the assumption that only viable cells reduces methylene blue to

violetlpink colour.

(According to Institute of Brewing Recommended Method of Analysis, 1982).

Correlations

ksv8 (hot water

extract-co ntinuous)

-534 ,217

7

I

ksv8 (hot water Pearson Correlation extract-air rest) Sig. (2-tailed)

sk5972 (hot water Pearson Correlation extract-air rest) Sig. (2-tailed)

N

ksv8 (hot water

extract-air rest)

1.000

7 526 .225

7 -.I34 ,775

7

-534 .217

7 ,134 .775

7

sk5912 (hot water

extract-air rest)

526 .225

7 1.000

7 ,480 ,275

7

-.359 .429

7 .649 .115

7

icsv 400 (hot water extract-air

rest) , -.I34

.775 7

.480

.275 7

1 .ooo

7

.561

.I 90 7

.802*

.030 7

icsv 400 (hot water Pearson Correlation extract-air rest) Sig. (2-tailed)

N

ksv8 (hot water Pearson Correlation extract-continuous) Sig. (2-tailed)

N sk591 2 (hot water Pearson Correlation extract-continuous) Sig. (2-tailed)

N

Ksv8 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N

icsv 400 (hot water Pearson Correlation extract-continuous)

sk5912 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N I

Sig. (2-tailed) N

icsv (diastatic poweq - Pearson Correlation conthuous) I

I Sig. (2-tailed)

Ksv8 (diastatic powdr) Pearson Correlation I Sig. (2-tailed) I I N

sk5912 (diastatic power) Pearson Correlation Sig. (2-tailed) N

icsv (diastatic power) Pearson Correlation Sig. (2-tailed) N

Ksv8 (amylase activ N

ty) Pearson Correlation Sig. (2-tailed) I

I N sk5912 (amylase activity) Pearson Correlation

Sig. (2-tailed) N

icsv (amylase activity) Pearson Correlation Sig. (2-tailed) N

Ksv8 (amylase activity - Pearson Cosrelation continuous) Sig. (2-tailed)

N

Correlations

I

;sv8 (hot water I Pearson Correlation :xtract-air rest) I Sig. (2-tailed)

sk%I 2 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N

;k5912 (hot water zxtract-air rest)

tsv8 (hot water Pearson Correlation 2xtract-continuous) Sig. (2-tailed)

N jk5912 (hot water Pearson Correlation 2xtract-continuous) Sig. (2-tailed)

N csv 400 (hol water Pearson Correlation zxtract-continuous) Sig. (2-tailed)

I N

icsv (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N Pearson Correlation Sig. (2-tailed)

r(sv8 (diastatic power)

sk59l2 hot water ?xtract-co ntinuous)

,134 .775

7 .649 .I 15

7 ,802' ,030

7

I I N

csv 400 (hot water I Pearson Correlation :xtract-air rest) Sig. (2-tailed)

I N

Pearson Correlation Sig. (2-tailed) N

icsv 400 (hot water extract-co ntinuous)

-.645 .I 18

7 -.024 .960

7 ,819' .024

7

.839*

.018 7

.564

.I88 7

sk5912 (diastatic Pearson Correlation Sig. (2-tailed) N

~csv (diastatic powei) Pearson Correlation Sig. (2-tailed) N

Ksv8 Idlastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 {diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (diastatic power\ - Pearson Correlation continuous) I Sig. (2-tailed)

! Ksv8 (amylase activ ty) Pearson Correlation

Sig. (2-tailed)

sk5912 -+- (amylase ac ivity) Pearson Sig. 42-tailed) Correlation

N icsv (amylase activity) Pearson Correlation

Sig. (2-ta~led] N

Ksv8 (amylase activity - Pearson Correlation continuous) Sig. (2-:ailed)

N

Ksv8 (diastatic power)

-.805* .029

7 -.311 .497

7 .477 .279

7

.867 ,011

7 .358 .431

7

sk5912 (diastatic power)

-.669 ,100

7

Correlations

ksv8 (hot water Pearson Correlation extract-air rest) Sig. (2-tailed)

N sk5912 (hot ~ a t e r Pearson Correlation extract-air rest) Sig. (2-tailed)

N icsv 400 (hot water / Pearson Correlation extract-air rest) Sig. (2-tailed)

N

ksv8 (hot water Pearson Correlation extract-contincrous) Sig. (2-tailed)

N sk5912 (hot water Pearson Correlation extract-continuous) ,, Sig. (2-tailed)

i N icsv 400 (hot water Pearson Correlation extract-continuous) Sig. (2-tailed)

N Ksv8 (diastatic power) Pearson Correlation

Sig. (2-tailed) N

sk5912 (diastatic poier) Pearson Correlation Sig. (2-tailed)

1 N Pearson Correlation Sig. (2-tailed)

continuous) Sig. (2-tailed)

N sk5912 (diastatic p o ~ e r - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (diastatic-power - Pearson Correlation continuous) Sig. (2-tailed)

N Ksv8 (amylase activity) Pearson Correlation

Sig. (2-tailed) N

s k S l 2 (amylase activity) Pearson Correlation Sig. (2-tailed)

I N icsv (amylase activit Pearson Correlation

Sig. (2-tailed)

power) ] continuous) I continuous) -.854' 1 -.802* 1 -.615

sk5912 (diastatic power - icsv Ediastalic

Ksv8 (amylase activlty - Pearson Correlation continuous) 1 Sig. (2-tailed)

I N sk5912 (amylase activity - Pearson Correlation continuous) I

Sig. (2-tailed)

-

-

KsvS (diastatic power -

-

- -

- -

- -

-.

-.

-.

-.

-.

-

-

-

-

-

-

-

-

.- N

icsv (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N

7 .433 ,324

7

7 ,614 .I43

7

7 .819' .024

7

Correlations

1

ksv8 (hot water Pearson Correlation extract-air rest) Sig. (2-tailed)

N sk5912 (hot water :. Pearson Correlation extract-air rest) Sig. (2-tailed)

1 N i cw 400 (hot water Pearsun Correlation extract-air rest) 1 Sig. (2-tailed)

icsv (diastatic power -

continuous) -.858*

-

Ksv8 (amylase activity)

-.527 ,224

7 -.090 348

7 .719 .069

7

,947"' -00 1

7 ,638 ,123

7

ksv8 (hot water extract-continuous)

- - -

sk5912 (amylase activity)

.044

.926 7

Pearson Correlation .810' Sig. (2-tailed) .027 N 7

icsv (amylase activity)

-.508

sk5912 (hot water i Pearson Correlation ,079 extract-continuous) Sig. (2-tailed) 367

N 7 icsv 400 (hot water Pearson Correlation ,680 extract-continuous) Sig. (2-tailed) ,093

N 7 Ksv8 (diastatic power) Pearson Correlation ,937'

Sig. (2-tailed) ,002 N 7

sk5912 (diastatic power) Pearson Correlation B25'

icsv (diastatic power)

Ksv8 (diaslatic power continuous)

Sig. (2-tailed) .022 N 7 Pearson Correlation ,997 Sig. (2-tailed) .OOO N 7

- Pearson Correlation ,948 Sig. (2-tailed) -00 1 N 7

sk5912 (diastatic poker - Pearson Correlation .775 continuous) Sig. (2-tailed) .04f

N 7 icsv Idiastatic power - Pearson Correlation 1 ,000 continuous) Sig. {2-tailed)

N 7 Ksv8 (amylase activity) Pearson Correlation .763

Sig. (2-tailed) .046 N 7

sk5912 (amylase activity) Pearson Correlation .I90 I Sig. (2-tailed) 6 3 3

icsv (amylase activitd) N 7 Pearson Correlation ,780 Sig. (2-tailed) .038 N 7

Ksv8 (amylase activ~ty - Pearson Correlation ,784 contirluous) 1 Sig. (2-tailed) .037

I N 7 sk5912 (amylase activity - Pearson Correlation ,192 continuous) Sig. (2-tailed) .679

N 7 icsv (amylase activity - Pearson Correlation .408 continuous) Sig. (2-tailed) .363

N 7

Correlations

Ksv8 (amylase activity -

sk59l2 (amylase activity -

continuous) .020 ,967

7 .484 ,271

7

icsv (amylase activity -

continuous

7 I

I continuous) tsv8 (hot water I Pearson Correlation 1 -.565 ?xtracl-air rest) Sig. (2-tailed)

N ;k5912 [hot water Pearson Correlation %tract-air rest) Sig. (2-tailed)

N csv 400 (hot water Pearson correlation ?%tract-air rest) Sig. (2-tailed) I tsv8 (hot water Pearson Correlation ,975" 3xtract-continuous) Sig. (2-tailed)

2xtract-continuous) Sig. (2-tailed)

N 7

csv 400 (hot water Pearson Currelalion ~xtract-continuous) Sig. (2-tailed) I N Ksv8 (diastatic powkr) Pearson Correlation

Sig. (2-tailed) N

sk5912 (diastatie power) Pearson Correlation Sig. (2-tailed) N

~csv (diastatic power) Pearson Correlation .8OOb Sig. (2-tailed) .03 1 N 7

KsvR (diastatic power - Pearson Correlation .879' continuous) I

1 Sig. (2-tailed) .009 N 7

sk5912 (diastatic - Peawon Correlation .916* continuous) I Sig. (2-tailed) .004

N 7 icsv (diastatic power - Pearson Correlation .784* continuous) Sig. (2-tailed j ,037

N 7

Ksv8 (amylase activity) Pearson Correlation .986' Sig. (2-tailed) .OOO N 7

sk5912 (amylase activity) Pearson Correlation ,735 Sig. (2-tailed) .060

- N 7 icsv (amylase activity) Pearson Correlation ,976'

Sig. (2-tailed) .OM) N 7

Ksv8 (amylase activity - Pearson Correlation 1 .OOO continuous) I Sig. (2-tailed)

N 7 sk591 2 (amylase ac:ivity - Pearson Correlation .726 continuous) Sig. (2-tailed) ,065

N 7 icsv (amylase activity - Pearson Correlation .869' continuous) 1 Sig. (2-tailed) .0 1 1

I I N I 7

*. Correlation is significant at the 0.05 level (2-tailed).

**. Correlation is significant at the 0.01 level (2-tailed).

Correlations :

I Correlations

1 ksv8 (free amino Pearson Correlation nitrcgen-air rest) Sig. (2-tailed)

N sk5912 (free amino Pearson Correlation nitrogen-air rest) Sig. (2-tailed)

N icsv400(free amino Pearson Correlation nitrogen-air rest) Sig. (2-tailed)

N

ksvO (carboxypeptidase Pearson Correlation -air rest) I Sig. (2-tailed)

N sk5912 I Pearson Correlation (CarDOXypeplidaSe-& Sig. (2-Iailed)

I rest) N icsv400(fcarboxypedtida Pearson Correlation se-air rest) 1 Sig. (2-tailed)

I N ksv8 (free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

N sk5912 (free amino Pearson Correlation nitrogen-conlinuous) Sig. (2-tailed)

N icsv40O(free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

N ksv8 (carboxypeplidhse Pearson Correlation -continuous) I Sig. (2-tailed)

ksv8 (free amino

nitrogen-air rest)

sk5912 (carboxypeptidase- nuous)

N Pearson Correlation

3nti Sig, (2-tailed) N

sk59l 2 (free amino

nitrogen-air rest)

icsv400(fcarboxype~tida Pearson Correlation se-continuous) ! Sig. (2-tailed)

N

icsv400(free amino

nitrogen-air rest)

.877*'

.009 7

.905*'

.005 7

1 .OOO

7

-.099 .833

7 .118

' .800 7

.157

1.000

7

.910** ,004

7

.252 7

,641 ,121

7 .939'* ,002

7 .792* .034

7 .474 .282

7 -341 ,454

7 ,381 ,399

7

.910" 1.000

.004 7 7

.877"* ,009

7

,655 7

.905** ,005

7

.737 7

.I20 ,798 ,

7 ,482 ,273

7 ,501

,387 ,

.392 7

-. 156 .738

7 -102 .828

7 ,207

.278

.546 7

.891* .775' ,007 ,041

7 7 .854* .014

7

.750 ,052

7 .I21 .I05 ,796

7 323

7 .a22 ,963

7

-.025 .958

7 ,056 .905

7

.012 , ,980

7

Correlations

ksv8 (carboxypeptidase Pearson Correlation -air rest) Sig. (2-tailed)

N sk5912 Pearson Correlation

ksv8 (free amino Pearson Correlation nitrogen-air rest) Sig. (2-tailed)

I N sk5912 (free amino Pearson Correlation nitrogen-air rest) j Sig, (?-tailed)

icsv400(fcarboxypeptida Pearson Correlation se-air rest) Sig. (2-tailed)

N ksv8 (free amino Pearson Correlation nitrogen-continuous Sig. (2-tailed)

N sk5912 (free amino Pearson Correlation nitrogen-continuous Sig. (2-tailed)

icsv400(free amino Pearson Correlation nitrogen-continuous Sig. (2-tailed) -: ! N

ksv8 (carboxypeptidase Pearson Correlstion -continuous) Sig. (2-tailed)

N sk5912 Pearson Correlation

icsv400(free amino nitrogen-air rest)

(carboxypeptidase-conti Sig. (2-tailed) nuous) N

N Pearson Correlation Sig. (2-tailed)

. - icsv400(fcarboxypeptida Pearson Correlation se-continuous) Sig. (2-tailed)

ksv8 :carboxypeptid ase -air rest)

,120 ,798

7

icsv400(fcarb oxypeptidase-

air rest) ,501 .252

7

sk5912 (carboxypeptid

ase-air resl) ,482 .273

7

ksv8 (free amino

nitrogen-co ntinuous)

.641

.I21 7

-.156 .I02 ,738 ,828

7 7 -.OW .I 18 ,833 .800

.207 .387 ,392

Correlations

ksv8 (free amino Pearson Correlation nitrogen-air rest) Sig. (2-tailed)

N sk5912 (free amino Pearson Correlation n~trogen-air rest) Sig. (2-tailed)

N icsv400(lree amino ' Pearson Correlation nitrogen-air rest) ; Sig. {2-tailed)

sk5912 (free amino

nitrogen-con tinuous)

,939' .002

7 w' .891*

,007 7

.775' I I .041

icsv400(fre e amino

nitrogen-co ntinuous)

,792" .034

7

ksv8 (carboxypeptidbse Pearson Correlation I .474 1 235

.240 -. 106 ,605 .820

7 7 .486 .094 .269 ,842

7 7 .625 ,075 .I33 .873

7 7 ,566 .301 .I86 .512

7 7 1.000 592

.085 7 7

,692 1 .OOO .085

7 7

I

(carboxypeptidase-chti Sig, (2-tailed) nuous)

( ,524 ( .666 I N 7 7

ksv8 (carboxypeptidase -air rest)

sk5912 (carboxypeptidase-a!r rest)

-continuous)

sk5912

Peafson Correlation Sig. (2-tailed) N Pearson Correlation Sig. (2-tailed) N

ksv8 (carboxypepti

dase

icsv400(fcarboxypeptida Pearson Correlation se-air rest) Sig. (2-ta~led)

N ksv8 (free amino . Pearson Cmelation nitrogen-continuous) Sig. (2-tailed)

N sk.5912 (free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

N icsv400(free amino Pearson Correlation nitrogen-continuousl

I Sig. (2-tailed)

I N

Sig. (2-tailed) N Pearson Correlation

I

icsv400(fcarboxypeptida Pearson Correlation se-continuous) Sig. (2-tailed)

N

.282 -612 7 7

,293 .201

.495 -.024

.258 .960 7 7

Correlations

. e I . . '

iitrogen-air rest) Sig. (2-tailed)

I N sk5912 (free amino 1 Pearson Correlation iitrogen-air rest) Sig. (2-tailed)

N csv4OO(free amino Pearson Correlation iitrogen-air rest) Sig. (2-tailed)

N

ksv8 (carboxypeptidase Pearson Correlation -air rest) Sig. (2-tailed)

N -

sk5912 Pearson Correlation (carboxypeptidsse-dir Sign (Ztailed) rest) I N

Sig. (2-tailed)

ksv8 (free amino Pearson Correlation nitrogen-continuous Sig. (2-tailed)

sk5912 (free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

N icsv400(free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

A

N ksv8 (carboxypeptidase Pearson Correlation -continuous) Sig. (2-tailed)

N sk5912 Pearson Correlation (carb~xypeptidase-cbnti Sig, (Z-tailed) nuous) N icsv400(fcarboxype~tida Pearson Correlation se-continuous) Sig, (*-tailed)

sk5912 I (carboxype icsv400(fcarbo I

3tidase-con xypeptidass-co tinuous) I nlinuous)

.341 1 .381

**. Correlation is sibnificant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

Correlations Correlations

ksv8 (free amino

nitrogen-air rest)

ksv8 (free amino Pearson Correlation 1.000 nitrogen-air rest) Sig. (2-tailed)

N 7 sk5912 (free amino Pearson Correlation ,910*' nitrogen-air rest) Sig. (2-tailed) ,004

N 7 icsv400(free amino , Pearson Correlation 377" nitrogen-air rest) , , Sig. (2-tailed} ,009

N I

7

ksv8 (free amino pearson Correlation -641 nitrogen-continuous: Sig. (2-tailed) .I21

N , 7 sk5912 (free amino Pearson Correlation .939* nitrogen-continuous;

I I Sig. (2-tailed) .002 N 7

icsv400(free amino Pearson Correlation .792' nitrogen-continuous) ; Sig. (2-tailed) .034

N 7 ksv8 (proteinase Pearson Correlation ,406 activity-air rest) Sig. (2-tailed) 367

N 7 sk5912 (proteinase-air Pearson Correlation .4 1 1 rest) Sig. (2-tailed) .359

N 7 icsv 400 (proteinaseiair Pearson Correlation .656 rest) I Sig. (2-tailed) ,110

N 7 ksv8 (proteinase ' Pearson Correlation . -270 activity-continuo~s) j Sig. (2-lailed) .557

N 7 sk5912 Pearson Correlation ,305 (proteinase-continu ,ug) Sig. (2-tailed)

I

N I

icsv 400 I Pearson Correlation (proteir~ase-continuoud) Sig..(Z-tailed)

sk5912 (free icsv400(free ksv8 (free amino amino amino

nitrogen-air nitrogen-air nitrogen-co rest) I rest) I nlinuous)

.910*j .877'*1 ,641

Correlations

sk5912 (free icsv400(fre ksv8 amino e amino (proteinase sk59.12

nitrogen-con nitrogen-co activity-air (proteinase tinuous) ntinuous) rest) -air rest)

ksv8 (free amino i Pearson Correlation .939*+ .792* .406 ,411 nitrogen-air rest) i

I Sig. (2-tailed) ,002 ,034 .367 .359

I N 7 7 7 7 sk5912 (free amino Pearson Correlation .891** .854' .067 .080 nitrogen-air rest) Sig. (2-tailed) ,007 ,014 387 .865

N 7 7 7 7 icsv400(free amino Pearson Correlation .775' .750 .092 .081 nitrogen-air rest) Sig. (2-tailed) .041 .052 .a44 ,863

N 7 7 7 7

N 7 7 7 :sv40fl(free amino Bearson Correlation ,692 1 .OOO -.091 iitrogen-continuous: Sig. (2-tailed) .085 .845

N 7 7 7 sv8 (proteinase Pearson Correlation ,496 -.091 1 .OOO ztivity-air rest) I Sig. (2-tailed) .258 .845

N 7 7 7 k5912 (proteinase-air Pearson Correlation .512 -.032 .992* est) Sig. (2-tailed) .240 ,945 ,000

N 7 7 7 :sv 400 {proteinase-air Pearson Correlation .678 .457 .793* esl) Sig. (2-tailed) ,094 .303 .034

N 7 7 7 sv8 (proteinase Pearson Correlation ,425 -.I44 .964* ~ctivity-continuous) Sig. (2-tailed) .342 ,758 .OOO

I N 7 7 7 k5912 Pearson Correlation .302 -.249 .89It proteinase-continucus) Sig. (2-tailed) .511 59 1 .007

N 7 7 7 :sv 400 Pearson Correlation 339 .454 .778* proteinase-continucus) Sig. (Z-tailedJ .I22 306 .039

N 7 7 7 I

icsv400(free amino nitrogen-air rest)

ksv8 (free amino Pearson Correlation n~trogen-air rest) 1 Sig. (2-tailed)

/ N

Pearson Correlation Sig. (2-tailed) N

sk5912 (free amino nitrogen-air rest)

ksv8 (free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

N

Pearson Correlation ~ i $ . (2-tailed) N

ksv8 (proteinase Pearson Correlation acti~ity-continuous) Sig. (2-tailed)

N sk5912 Pearson Correlation (proteinase-continuaus) Sig. (2-tailed)

N icsv 400 Pearson Correlation (proteinase-continuous) Sig. (2-tailed)

N

sk5912 (free amino Pearson Correlation ni trogen-continuous) Sig. (2-tailed)

N icsv400(free amino Pearson Correlation nitrogen-continuous) Sig. (2-tailed)

I N ksv8 (proteinase i Pearson CorreTation activity-air rest) 1 Sig. (Nailed)

Correlations

sk5912 (proteinase-

ksv8 icsv 400 (proteinase sk5912

:proteinase activity-conti (proteinase-c -air rest) nuous) ontinuous)

,656 .270 .305 . I10 .557 .506

7 7 7 .373 -.028 -.064 ,410 .952 391

7 7 7 ,301 -.I252 ,006 -512 912 .990

7 7 7

N air Pearson Correlation

'*. Correlation is significant at the 0.01 level (2-tailed).

'. Correlation is si&ificant at the 0.05 level (2-tailed).

icsv 400 (proteinase-c ontinuom

rest) Sig. (2-tailed)

icsv 400 (proteinase-air rest)

N Pearson Correlation Sig. (2-tailed]

I N

I I

Correlations 1 Correlations

Ksv8 (cold water extract)

lsv8 (cold water extract) Pearson Correlation 1.000 Sig. (2-tailed) N 7

;k5912 (cold water Pearson Correlation .933* :xtract) Sig. (2-tailed) .002

N 7

Ksv8 icsv (cold I Idiaslatic sk5912 (cold

water extract) ,933"

water extract -+&++?%

csv (cold water extrbct) Pearson Ccrrelation I

.977*

i Sig. (2-tailed) .OOO N 7

(sv8 (diastatic power) Peafson Coirelation ,259 Sig. (2-tailed) 575 i N 7

;k5912 (diastatic pdwer) Pearson Correlation I

.426

i Sig. (2-tailed) .340 N 7

csv (diastnlic power) Pearson Correlation .066 Sig. (2-tai!ed) .888 N 7

(SV8 (cold water extract- Pearson Correlation .953' :ontinuous) Sig. (2-tailed) .OO 1

I N 7 jk5912 (cold water ' Pearson Correlation .985' ~xtract- continuous)! Sig. (2-tailed) .OOO

N 7 csv (cold water extract- Pearson Correlation .964" zontinuous) / I Sig. (2-tailed) ,000

I I ; I N 7

Ksv8 (diastatic powe- - Pearson Correlation ,232 continuous) Sig. (2-tailed) 617

N 7 sk.5912 (diastatic poher - Pearson Correlation .476 continuous)

I Sig. (2-tailed) ,281 N 7

icsv (diastatic power - Pearson Correlation ,037 continuous) Sig. (2-tailed) .937

N 7 Ksv8 (amylase activity) Pearson Correlation .635

Sig. (2-tailed) .126

I N 7 sk5912 (amylase activity) Pearson Correlation .934'

Sig. (2-tailed) .002 N 7

icsv (amylase activity) Pearson Correlation I

.625 Sig. (2-tailed) .133

I I N 7

Ksv8 (amylase activ/t$ - Pearson Correlation ,612 continuous) I Sig. (2-tailed) .I44

, I N 7

sk5912 (amylase activity - Pearson Correlatjon .906 con!inuous) I Sig. (2-tailed) .005

I N 7 icsv (amylase activity - Pearson Correlation ,891 continuous) Sig. (2-tailed) .007

N 7

Correlations

(diastatic sk5912 1 icsv (diastatic ! (sv8 (cold water extract) pearson Correlation

I Sig. (2-tailed) N

power) I power) I

,428 [ .066

;k5912 (cold water

Ksv8 (cold water extract-

Pearson Correlalion

continuous) 4 .953'

'xtract) Sig. (2-tailed) N

csv (cold water extract) Pearson Correlation Sig. (2-tailed) N

<sv8 (diastatic power) Pearson Correlation Sig. (2-tailed) N

;k5972 (diastatic power) Pearson Correlation Sig. p-tailed)

1 N csv (diastatic power!) Pearson Correlation

Sig. (2-tailed)

zontinuous)

N Ksv8 (cold water ex ract- Pearson Correlation \ Sig. (2-tailed)

i N sk5912 (cold water / Pearson Correlatisn extract- continuous) I Sig. (2-tailed)

N icsv (cold water extract- Pearson Correlation continuous) Sig. (2-tailed)

N Ksv8 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (diastatic power -

I Pearson Correlation

continuous) I Sig. (2-tailed) I N .

Ksv8 (amylase activ

sk5912 (amylase

ty) Pearson Correlation Sig. (2-tailed) N

ac;ivity) Pearson Correlation Sig. (2-tailed) N

icsv (amylase activity) Pearson Correlation Sig. (2-tailed) N

Ksv8 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

I N icsv (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

I N

Correlations

icsv (cold (diastatic water extract- power - I sk5912 (cold

water extract- continuous) ) continuous)

.964'? .232 1 continuous)

<sv8 (cold water extract) Pearson Correlation 1 .985*' Sig. (2-tailed) 1 ,000 N 7

jk5912 (cold water Pearson Correlation ,872" extract) Sig. (2-tailed) .011

N 7 csv (cord water extract) Pearson Correlation .932*'

Sig. (2-tailed) ,002 N 7

Ksv8 (diastatic power) Pearson Correlation ,236 I Slg. (2-tailed) .610 I

N 7 sk5912 (diastatic power) Pearson Correlation ,381

Sig. (2-tailed) ,400 N 7

rcsv (d~astatic power) Pearson Correlation ,083 Sig. (2-tailed) ,860 N 7

Ksv8 (cold water extract- Pearson Correlation .927* continuous) Sig. (2-tailed) ,003

N 7 sk5912 (cold water Pearson Correlation 1 .OOO extract- cont~nuous) Sig. (2-ta~led)

N 7 icsv (cold water extract- Pearson Correlation .913* continuous) Sig. (2-tailed) .004

N 7 Ksv8 (diastatic pow& - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N Ksv8 (amylase activity) Pearson Correlation

Sig. (2-tailed) I

I N sk5912 (amylase activity) Pearson Correlation

Sig. (2-tailed)

Sig. (2-tailed) N 7

Ksv8 (amylase activity - Pearson Correlation ,609 continuous) Sig. (2-tailed) .I47

N 7 sk5912 (amylase activity - Pearson Correlation ,833' continuous) Sig. (2-tailed) ,020

N 7 icsv (amylase activity - Pearson Correlation ,851' continuous) Sig. (2-tailed) .015

N 7

<sv8 (cold water extract) Pearson Correlation Sig. (2-tailed) N

;k5912 (cold water Pearson Correlation 2xtract) Sig. (2-tailed)

N csv (cold water extract) Pearson Correlation

Sig. (2-tailed)

Ksv8 (diastatic pow r) Pearson Correlation Sig. (2-tailed)

sk5912 (diastatic po er) Pearson Correlation Sig. (2-tailed)

i N icsv (diastatic power$ Pearson Correlation

Sig. (2-tailed) N

Ksv8 (cold water extract- Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (cold water Pearson Correlation extract- continuous) Sig. (2-tailed)

N icsv (cold water extract- Pearson Correlation continuous) I Sig. (2-tailed)

I N Ksv8 (diastatic - Pearson Correlation contmuous) Sig. (2-tailed)

Correlations

sk5912 (diastatic continuous)

icsv (amylase activity) Pearson Correlation

-

sk5912 (diastatic I icsv (diaslatic

N poNer - Pearson Correlation

Sig. (2-tailed) N

Ksv8 (amylase activ continuous)

sk59l2 (amylase continuous)

cdntinuous) I continuous) .476 ( .037

icsv (diastatic powed - Pearson Correlation continuous) Sig. (2-tailed)

N Ksv8 (amylase activity) Pearsm Correlation

Sig. (2-tailed) N

sk5912 (amylase activity) Pearson Correlation Sig. (2-tailed) N

Sig. (2-tailed) N

ty - Pearson Correlation Sig. (2-tailed) N

ac:ivity - Pearson Correlation Sig. (2-tailed) N

icsv (amylase activitg - Pearson Correlation continuous) Sig. (2-tailed)

N

Ksv8 (amylase activity)

.635

sk5912 (amylase activity)

.934* .I26 ,002

7 7 .783 1 .00Ot .037 ,000

7 7 .681 .967* .092 .OOO

7 7 .910** .47 1

Correlations i

Ksv8 (amylase activity -

continuous) 512 .144

7 .741 ,057

7 ,636 .I24

7

1

Ksv8 (cold water ext'ract) Pearson Correlation I 1 Sig. (2-tailed)

N sk5912 (cold water ! Pearson Correlation extract) Sig. (2-tailed)

N icsv (cold water extract) Pearson Correlation

Sig. (2-tailed) N

icsv (amylase activity)

,625 133

7 ,757' ,049

7 ,655 .I 10

7 Ksv8 (diastatic power) Pearson Correlation

Sig. (2-tailed) N

sk5912 (diastatic power) Pearson Correlation Sig. (2-tailed)

I I N

icsv (diastatic power)

,906' .899* ,005 .006

7 7 353" '921 .OO 1 .003

7 7 Pearson Correlation Sig. (2-tailed) N

-799" .031

7

.800*

.031 7

Ksv8 (cold water ex ract- Pearson Correlalion f continuous) I Sig. (2-tailed) ! N

sk5912 (cold water : Pearson Correlation extract- continuous) Sig. (2-tailed)

N icsv (cold water extract- Pearson Correlation continuous) Sig. (2-tailed)

N Ksv8 (diastatic power - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (diastatic power - Pearson Correlation

,748 .053

7 512 ,744

7 .681 .092

7 .895'* .007

7

continuous)

icsv (diastatic power continuous)

Ksv8 (amylase activ

.772*

.042 7

.609

.I47 7

6 7 9 ,094

7 .879* ,009

7

Sig. (2-tailed) N

- Pearson Correlation Sig. (2-tailed) N

ty) Pearson Correlation

.954"*

.IS0 1 7

.780* ,038

7 ,993'* .OOO

7 .755* .050

7 1.000

7 .976** ,000

7 .753 ,051

7 .886** .008

7

Sig. (2-tailed) ! N sk5912 (amylase activity) Pearson Correlation

Sig. (2-tailed) N

icsv (amylase activity) Pearson Correlation Sig. (2-tailed) N

Ksv8 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

1 N icsv (amylase activity - Pearson Correlation

.916*

.004 7

.784*

.037 7

.986*

.OOO 7

.735

.060 7

.976'

.OOO 7

1.000

7 .726 .065

7 .869' ,011

7

continuous) Sig. (2-tailed) N

Correlations

:sv8 (cold water extract) Pearson Correlation

! Sig. (2-tailed) N

,k5912 (cold water Pearson Correlation !xtract) Sig. (2-failed)

N :sv (cold water extract) Pearson Correlaticn

1 Sig. (2-tailed) 1 N

(sv8 (diastatic power) Pearson Correlation Sig. (2-ta~fed) N

ik5912 (d~astatic power) Pearson Correlation Sig. (2-tailed) N

csv (diastatic power) Pearson Correlation Sig. (2-tailed) N

<sv8 (cold water extract- Pearson Correlation :ontinuous) Sig. (2-tailed)

I N ;k5912 (:old water Pearson Correlation zxtract- continuous) Sig. (2-tailed)

:ontinuous) Sig. (2-tailed)

Ksv8 (diaslatic power - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (diastatic power - Pearson Correlation mntinuous) Sig. (2-tailed)

N rcsv (diastalic power - Pearson Correlation conlinuous) Sig. (2-tailed)

N Ksv8 (amylase activity) Pearson Correlation

I Sig. (2-tailed)

sk5912 (amylase ac ivity) Pearson Correlation Sig. (2-tailed)

icsv (arr,ylase activit ) Pearson Correlation / Sig. (2-tailed) N

Ksv8 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N sk5912 (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N icsv (amylase activity - Pearson Correlation continuous) Sig. (2-tailed)

N -

continuous) I continuous) .906'*1 ,891''

sk5912 (amylase activity -

". Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

I

icsv (amylase activity -

REFERENCES

Aisien, A.O. (1 982). Utilization of Soluble Carbohydrate during Sorghum Germination

and Seedling Growth. Joumalofthe Institute of Brewing 88, Val 4 3 -7.

Ai,?i~fl, A,o, and Palmer, G.A. (1983). The Sorghum Embryo in Relation to the

Hydrolysis of the Endosperm during Germination a r d Seedling GKM[h. Journal of the Science of Food and Agriculture 4,345 - 349.

Aisien, A,(), Palmer, G.A. and Shark, J.K (1986). The Infrastructure of Germinating ,/~l,ina/ Q/ /he kdrl~le of ilmvm~ 12 (2) I65 -

Aisien, A.O. (1988). Sorghum Cultivation and Morphology. Brewing and Distilling International, 3 , 21 - 23.

Association of Official and Analytical Chemists, Official Method of Analysis .

Washinton, D.C. (1980).

Baxter, E D . (1982). Lipoxidases in Malting and Mashing. Journal of the Instifute of Brewing 89, 390 - 395.

Baxter, E.D. and Ofarrell, D.D. (1980). Effects of raised Temperature during Steeping and Germination on Proteolysis during Malting. Journal of the Institute of Brewing 89, 291 - 295.

Bathgate, G.N. (t982). The determination of endo p-glucanase activity in malt. Journal of the institute of Brewing 85,92 - 94.

Bramforth, C.W, and Martin, H.L. (1981). p-glucan and f3-glucan solubilized in malting and mashing. Journal ofthe Institute of Brewing 87, 364 - 371.

Briggs, D.E. (1964). Origin and Distribution of a-amylase in malt. Journal of the Institute of Brewing 70, 14 - 16.

Briggs, D.E., Haugh, J.S., Stevens, R. and Young, T.W. (1981). Brewing water in malting and brewing Sciences Vol 1 (2" ed.) Chapman and Hill. London. Pp. 281 - 289.

Buckee, G.K. (1990). Estimation of Iso-Alpha Acids in Barley by HPLC Collaborative Trial. Journal lnstitute of Brewing. 96(3): 143-149.

Buckee, G.K. and Baker, C.D. (1989). Estimation of fermentable carbohydrate. Collaborative Trial. Journal Institute of Brewing. 95(2): 11 1-1 15.

Chavan, J.H., Kadam, S.S., Ghanskar, C.P. and Salunkhe, D.K, (1979). Removal of Tannins and Improvements of Vitro Protein Digestibility of Sorghum Seeds by Soaking in Alkali. Journal of Food and Science 44, 131 9 - 1321.

Chavan, J.H., Kadam, S.S., and Salunkhe, D.K. (1981). Changes in Tanin FAN, Reducing Sugar and Starch during Seed Germination of Low and High Tanin Cullivars in Sorghum. Journal of Food and Science. 46, 638 - 639.

Chikezie, 1.0 (1 999). Brewing Beer with Sorghum. Journal of the Institute of Brewing 1 05.23-34. .

Diaber, K.H. (1975). Enzyme Inhibition Polyphenol of Sorghum Grain and Malt. Journal of the Science of Food and Agriculture. 26, (7). 1399 - 1402.

Diaber, K.H. and Novellie, L. (1968). Kafficorn Malting and Brewing Studies. XiX Gibberellic acid and Amylase formation in Kaffircon. Journal of the Science of Foad and Agr/cuffure. 19, 87 - 90.

Diaber, K.H.. Malherbe, L, and Novellie, L. (1973). Sorghum Malting and Brewing Studies Part 22. The Modification of Sorghum Malt. Breuwissen Chaff 26, 220 - 225,245 - 251.

Dewar, J., Orovan, E, and Taylor, J.R.N. (1996). Effect of Alkaline Steeping on Water Uptake and Malt Quality in Sorghum, Journal of the Institute of Brewing 103,383 - 285.

Dewar, J., Taylor, J.R.N, and Berjak, P. (1997). Effect of Germination Condition with Optimized Steeping on Sorghum Malt Quality with Particular Reference to Free Amino Nitrogen. Journal of the hstitute of Brewing 103, 171 - 173.

Dufour, J.P. and Jaeger, B.D. (1987). Role of the Methyltransferase Activity in the Synthesis of S rnethylrnethionine (DMS - precursor) during Germination and Kilning. Proceedings of European Brewing Convention 4, 289 - 296.

Dufour, J.P. and Melotte, L. (1992). Sorghum Malt for the Production of a Larger Beer. Journal of American Society of Brewing Chemist. 50 (3) 11 0 - 11 9. .-

Dyer, T.A. and Novellie, L. (1986). Kaffir-corn malting and Brewing Studies. XVI. The Distribution and Activities of u and p-amylase in Germination of Kaffircorn. Journal of the Science of Food and Agriculture. 103, 283 - 285.

Demuyakor, B. and Ohta, Y. (1992). Malt characteristics of Sorghum vulgare - varieties from Ghana. Journal of the Science of Food and Agriculture. 96, 89 - 91.

Enari, T.M. and Sopanen, T. (1986). Mobilization of Endospermal Reserves during the Germination of Barley. Journal of the lnstitute of Brewing, 92, 25 - 31.

Etokapkan, 0 .U and Palmer, G.H. (1990b). Simple Diamylase procedure for the Estimation of a-amylase and Diastatic Activity. Journal of the lnstitc~te of Brewing, 96(2), 89 - 93.

Etokakpan, O.U. and Palmer, G.H. (1990). Comparative studies of the development of Endosperm-degrading enzymes in malting sorghum and Barley, World Journal of Microbiology and Biotechnology 6, 408 - 41 7.

Etokakpan, O.U. (1992). Amylolytic potential of wort fermenting component of Nigerian Sorghum and Barley wort. Journal of Microbiology and Biotechnology 8, 287 - 289.

Ezeogu, L.1. and Okolo, B.N. (1994). Effect of final warm water seep and air-rest cycles on malt properties of three improved Nigerian sorghum cultivars. Journal of the Institute of Brewing, 11 0 335 - 338. .

Ezeogu, L.I. and Okolo, B.N. (1995). Effect of Air-rest periods on malting Response to final warm water steep. Journal of the lnstitute of Brewing, 101, 39 - 45.

Glennie, C.W. and Holmes, M. (1992). Studies on cold water Extract of malt. Soluble carbohydrate and malting behaviour. Journal of the lnstitute of Brewing, 98, 47 51.

Glennie, C.W. and Wight, A.W. (1986). Dextrin in sorghum Beer. Journal of the lnstitute of Brewing, 92, 384 - 386.

Glennie, C.W., Garris, J. and Liebenberg, N.V.D.W. ('1 984). Endosperm modification in Germinating sorghum grains ll. Journal of Cereal Chemistry 61, 37 - 42.

Heerden, l.V Van (1987). In Defence of sorghum. Food Review 1 (15), 83 - 84.

Hough, J.S., Briggs, D.E. and Steven, R. (1971). Malting and Brewing Science. Chapman and Hall. Pp. 71 - 77, I 6 0 - 169;

Hoseney, R.C. (1986). Principles of Cereal Sciences and Technology. St. Paul Minnesota. American Association of Cereal Chemist. 1 1 6, 1 6 - 1 1 8.

Hoseney, R.C. and Davis, A.B. (1979). Grain sorghum condensed Tannins 1. Isolation, estimation and selective adsorption by starch. Journal of Cereal Chemistry, 56, 310 - 312.

lnstitute of Brewing (1 989). Recommended method of Analysis.

Igyor, M.A. (1987). Studies on malting and mashing with sorghum. MSc Thesis Hariot- Walt University Edinburgh, 23 - 26.

Igyor, M.A., Ogbonna, A.C and palmer, G.H. (1989). Effect of Malting Temperature and Time on Enzyme Development and Sorghum wort properties. Journal of the lnstitute of Brewing, 1 04, 1 01 - 104.

Ilony, W.T.N. (1 988). Environmental Effects on the Biochemical phase of malting and kilning. Journal of the American Society of Brewing Chemists 46, 8 - 13.

Irwin, A.J. and Thompson, D.J. (1987). A rapid method for the extraction and analysis of beer flavour compounds. Journal lnstitute of Brewing. 93(2), 11 3- 116.

Isebgert, L. (1964). Essential factors for the production of quality malt. Journal of the lnstitute of Brewing, 70, 167 - 169.

Jayatissa, P.M., Pathirana, R.A. and Sivayogasunderam, K. (1980). Malting Quality of Sri Lankan varieties of sorghum. Journal of the lnstitute of Brewing, 86, 18 - 20.

Jones, R.L. (1985). Protein synthesis and secretion by the Barley Aleurone: A perspective. Israel Journal of Botany, 34, 377 - 395.

Jones, R.L. and Jacobson, J.V. (1983). Calcium regulation of the secretion of or- amylase isoenzymes and 'other proteins from Barley Aleurone layer. Planta 158, 1-9.

Lewis, M.J., Krumland, S.C., Mu hleman, D.J. (1 979). Dye-binding method for measuring protein in wort and Beer. Journal of American Society of Brewing Chemist. 38, 37 - 41.

MacGregor, H.W., Lenour, P., Moll, M. and ~ a u s i a n t , J. (1984). Identification of debrnching enzymes from Barley and malt by lsoelectric focusing. Journal of Cereal Research Academy of Science, 298, 243 - 248.

MacGregor, H.W. (1982). Action of malt amylase on Barley starch granules. Technical Quarterly of the Master brewers&sociation of American 17, 21 5 - 221.

MacGregor, H.W. and Matsuo, R.R. (1986). Malting and Brewing sciences. Challenges and Opportunities. Journal lnstitute of Brewing 102(2), 93-97.

MacGregor, H.W., Dushnicky, L.S., Shroeder, S.W. and Balance, G,M. (1994). Changes in Barley Endosperm during Early Stages of Germination. Journal lnstitute of Brewing 1 00(2), 88-91 .

Morral, P., Boyd, H.K., and Paylor, J.R.N. (1986). Effects of Germination Time, Temperature and Moisture on Sorghum. Journal of lnstitute of Brewing, 92, 439 - 445.

Mikola, J., and Kolehmainen, L. (1972). localization and Activity of various peptidase in Germinating Barley. Planta 104, 167 - 177.

Munch, L., Jorgenson, K.G., Ruud-Hansen, L. and Hansen, K.T. (1989). Methods for the Determination of High Molecular Weight P-Glucan in Barley, Malt, Wort and Beer. Journal of lnstitute of Brewing, 95 (2), 79 - 83.

Nelson-Somogyi, M. (1 952). Notes on Sugar Determination Quantitative Determination of Sugar. Journal of Biological Chemistry 195, 19 - 35.

Narziss, T.N. and Sumrell, G. (1980). Chemical composition o different varieties of grain sorghum. Journal of Agriculture and Food Chemistry. 28, 19 - 21 .

Nishiyana, N. and Kamada, K. (1986). Varietal Differentiation of malt by Resazurin staining. Journal of Institute of Brewing, 92 (3), 250 - 253 . .

Novellie, L. (1962). Kaffircorn Malting and Brewing studies XIII. Variation of Diastatic power with variety, season, maturity and age of grain. Journal of Science of Food and Agricult~~re. 13, 124 - 127.

Novellie, L. (1960). Kaffircorn malting and Brewing V. Occurrence of P-amylase in Kaffircorn malt. Journal of Science of Food and Agriculture 11 (8) 460 - 475.

Oteh, U.C. (1987). Studies on the possible use of maize (Zea mays) as an Alternative Raw materials in Brewing. PGD Project MCB, UNN, 24 - 41.

Okolo, B.N. and Ezeogu, L.I. (1995). Effect of Air-rest periods on the mobilization of sorghum reserve proteins. Journal of the lnstitute of Brewing, 101 463 - 468.

Okolo, B.N. and Ezeogu, L.I. (1996a). ~nhancement of Amylolytic potentials of sorghum malt by Alkaline Steep Treatment. Journal of the lnstitute of Brewing, 102, 79 - 82.

Okolo, B.N. and Ezeogu, L.I. (1996b). Promoting Sorghum Reserve Protein Mobilization by Steeping in Alkaline Liquar. Jpurnal of the lnstitute of Brewing, 102, 277 - 284.

Onwuama, C.1. (1999). Brewing Beer with Sorghum. Journal of the lnstitute of Brewing. 105: 23-34.

Onwurah, I .N.E (2001 ). Crystallinity and polysaccharide chains of fJ-glucan in white sorghum SK 591 1 2. International Journal of Biological Macromolecules, 29, 281 -286.

Palmer, G.H. (1989). Cereal in malting and Brewing. In Cereal Science and ~ e c h n o l o ~ ~ . Ed. Palmer, G.H. ~ b e r d e e n university Press, Aberdeen UK. 61 - 242.

Palmer, G.H. (1991). Enzymic degradation of the endosperm cell walls of germinated sorghum. World Journal of Microbiology and Biotechnology. 7 , 17 - 21.

Palmer, G.H., Etokakpan, O.U. and Igyor, M.A. (1989). Review: Sorghum as Brewing Material. MIRCEEN Journal 5.

Palmer, G.H., Gernah, D.I., Mekernan, G., Nimmo, D.H. and Laycock, G. (1985). Influence of enzyme distribution on endosperm breakdown (modification) during malting. Journal o f the American Society o f Brewing Chemist. 43, 1 7 - 28.

Pierce, J.S. (1982). The Margret Jones memorial lecture. Amino acid in malting and Brewing. Journal of the lnstitute of Brewing, 88, 228 - 233.

Pollock, J.R.A. (1964). "The nature of the malting process". In Barley and malt. Ed. A.H. Cook. Academic Press, London & N.Y 303 - 307

. . Pomeranz, Y. (1989). Modern Cereal Science and Technology. New York. V.C.A

Recommended Method of Analysis of the lnstitute of Brewing 1977. Revised 1986 London. lnstitute of Brewing.

Price, M.L., Stromberg, A.M. and Butler, L.G. (1979). Tannin content as a fraction of grain maturity and drying conditions in several varieties of sorghum bicolar (L) moench. Journal of Agriculture and Food Chemistry 29, 1270 - 1275.

Rank, H, Sopanen, T and Vontilainen, N. (1990). Localization of carboxypeptidase I in germinating barley grins. Plant Physiology 93 1449 - 1452.

Rastigo, V. and Oaks, A. (1986). Hydrolysis of storag'e proteins in Barley endosperm. Journal o f Plant Physiology 81, 901 - 906.

Riggs, T.L., Sanada, M., Morgan, A.C. and Smith, B. (1983). Use of acid gel electrophoresis on the characterization of P-hardien protein in relation to . malting quality and mildew resistance barley. Journal of Food and Agriculture. 34, 576 - 586.

Sanwo, M.M. and Demazon, D.A. (1992). Characteristics of a-amylase during germination of two high sugar sweet corn cultivar of Zea mays, Journal of Plant Physiology. 99, 1 184 - 1 192.

Shamber, T., Vonir, N. and Gambo, E. (1989). Enzymes and acid hydrolysis of malted millet (Penniseton typhoides) and Sorghum (Sorghum bicolar). Journal o f the lnstitute o f Brewing, 95, 13 - 16.

Siebert, K.J, and Kundsen, E.T. (1989). The relationship of Beer high molecular weight protein and foam. Masters Brewers Association American Technology Quarterly 26, 139 - 146,

Shutov, A.D., Beltei, N.K. and Vaintraub, I.A. (1984). A cysteine protease from .

Germinating wheat seed; Partial purification and Hydrolysis of Gluten. Biochemical Journal. 49, 1004 - 101 0.

Somogyi, M. (1952). Notes on Sugar Determination. Quantitative Determination of Sugar. Journal of Biological Chemistry, 1 95, 1 9-35.

Taylor, J.R.N. and Evans, D.J. (1989). Action of Sorghum proteinase on the protein bodies of sorghum starchy Endosperm. Journal of Experimental Botany. 40, 763 - 768.

Taylor, J.R.N. and Robbins, D.J. (1993). Factors influencing oeta amylase activity in sorghum malt. Journal of the lnstitute of Brewing, 99, 41 3 - 41 6.

Taylor, J.R.N. and Dewer, J. (1994). Role of a-glucosidase in the fermentable sugar composition of sorghum malt mashes. Journal of the lnstitute of Brewing, 100, 417 - 419.

Taylor, J.R.N. (1983). Effect of malting on protein and FAN composition of sorghum. Journal of the lnstitute of Brewing, 89, 885 - 892.

Taylor, J.R.N. and Diaber, K.H. (1988). Effect of calcium ions in sorghum Beer . I.

mashing. Journal of the lnstitute of Brewing, 94, 68 - 73.

Von-Holdt, M.M and Brand, J.C. (1960). Kaffircorn mal'ting and Brewing studies in changes in the carbohydrate of kaffircorn during malting. Journal of Food Science and Agriculture 11, 467 - 479.

Wall, J.S. and Ross, W.M. (1970). Sorghum and utilization. Major feed and food in Agriculture and Food series. A.V.1 Publishing Company Inc. West Port Connecticut 1 - 17, 580 - 589.

Wrobel, R, and Jones, B.L. (1992). Electropheretic study of substrate and pH dependence of endoproteolytic enzymes ip malts. Journal of the lnstitute of Brewing, 98, 471 - 478.

Zhuotaosun, C.A. and Henson, H. (1991). A Quantitative Assessment of the Importance of Barley seed a-amylase, P-amylase, Debranching enzymes, a- glucosidase in starch Degradation. Archives of Biochemistry and Biophysics. 284,298 - 305.