University of Nigeria Research Publications

OKPALANMA, Emeka Felix

Aut

hor

PG/M.Sc/88/6772

Title

Production of Malt-Based Syrups from Sorghum (Sorghum Bicolor) and Millet (Pennisetum

Typhoiodes) Grains

Facu

lty

Agriculture

Dep

artm

ent

Food Science & Technology

Dat

e

May, 1991

Sign

atur

e

I ~ ~ U C T I O N OF MALT-BASED SYRUPS FROM SORGHUM(SORGHUM BICOLOR) AND MILLET (PENNISEWM TYPHOJDES ) GRAINS

OKPALANMA, EMEKA FELIX PG/M .sc./88/6772 '

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY FACULTY OF AGRICULTURE, UNIVERSITY OF NIGERIA,

iJSL'KKA.

I N PARTIAL FULFUS/1ENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE DEGREE (M,SC,) I N

FOOD SCIENCE AND TECHN'OLOGY, UNIVERSITY OF NIGERIA, NSUKKA

MAY, 1991 .

Mr. Okpalamm, Emeka F e l i x , a postgraduate student i n

t h e Department of ~ ' o o d Science and Technology, and with

r e g i s t r a t i o n Number PG/M,~o/88/6772 has s a t i s f a c t o r i l y

completed t h e requirements f o r course and research w r k

f o r t h e degree of master of science i n Food Science and

Technology, The work embodied i n t h i s t h e s i s i s o r f g i n d

and has not been submitted i n part o r f u l l f o r any o t h e r

diploma o r degree of t h i s o r any other unive~sity,

t

Dr. C.C.1 OlQUOHA /&JkhM DR. A e L . IIGXDRONYE

SUPERVISOR.

DEDICATION

his thssis Is dedicated to all widours

who could muster enough courage, patience

to stay and train their children properly

as my mother did.

i r"

iii.

ACKNOWLEDGEMENTS

I want t o express sincere grat i tude and ' d e e p s t appreci~lt ion to

m. A. I. Ihekoronye, my S~p@JXisor, f o r the many hours hg spent i n

b l p i n g m e plan, canplete and evaluate this research project. I also

extend my th&s t o all my lectu~cers i n the department of Food Science

tind Technology. i

I grateful ly acknowledge t h assistance of the departmental

technologists .: Onuchukwu, Onyebuashi , Kalu and Nwokedi of ~ iochemis t ry

department.

I express my personal thanks to Rev. Fr. Eutabasfl i , M., M r . Obadiegu,

M., Dr . Chinyere, P. and Kene Oranu for many things. Also to M r . J. Nwabue

f o r h i s patience and for t i tude i n typing the. manuscript.

Fihally, I owe a special debt of grat i tude to a l l my lec ture rs i n

the University of I f e , during my f i r s t degree programme.

V.

Page TABLE OF CONTENTS

TITLE PAGE

CERTIFICATION

iii DEDICATION . .

ACKNOWLEDGEMENT

TIlBLE OF CONTENTS

LIST OF TABLES

v i i LIST OF FIGURES

v i i i ABSTRACT

INTRODUCTION

LITERATURE REVIEW

Production of Sorghum and Millet; Grains

sorghum grain . . Millet grain . . C h e m i c a l and Biochemical studies on Sorghum and Millet grains . . sorghun grain . . Millet grain . .

' i Malting' Character is t ics of Nigerian Sorghum and Millet VazLeties . . sorghum malt . . ~ i l l e t malt . . ~ l u c o s e syrups . . k f i n i t i ons . . ~e-s of Production of Glucose Syrups

~ c i d conversion . . Acid-enzyme conversion .. Enzymeenzyme conversion 2 2

Refining . . 25 ,

Properties and Functional uses of Glucose @..-..-\a ' C ) C )

Malt Based Syrupm . . Mtuihing method . . Preparation of m a l t syrups

MATERIALS AND METHODS . Materials . . Source of cereal grains .. Methods . . Determination of Malting Characteristics of the Cereal G r a i n s , Sorghum and Mil le t

Determination of moisture content

Determination of percentage foreign seeds and braken kernels , Determination of thousand corn weight

Determination of getminative energy

Determination of germinative capacity

Det@rmination of Optimum Malting conditions of the Cereal Grains ,,

Moisture Content as a function of s teep time

Determination of Optimum steep time

Determination of Optimum g e d n a t i o n period

Effects of ki lning a t 4s0c and varying periods of time on moisture con t e n t of the ma1 t.

Determination of malting losses as a function of gemination periods .. Production of Sorghum, M i l l e t Malts.

Evaluation of M a l t ' s qual i ty charac te r i s t i cs

Determination of Cold Water ex t r ac t

Determination of d i a s t a t i c power

Dekermhation of ~ o t water ex t rac t

Determination of malting l o s s

Studies on Malt's Amylases

Extraction of m a l t anylase

Preparation of 1% buffered s ta rch substra te

. vi i .

3.5.3 Preparatioh of Maltose Calibration curve 43

3.5.4 Datemination of optimum p~ f o r amylase ac t iv i ty . 44

3.5.5 Oetenninationof Optimum temperature fo r amylase ac t i v i t y .. 44 .

3.5-6 s ta rch Extraction £ran the Cereal grains 44

3 7 ~ r o x h a t e / ~ h e m i c a l Analysis. of grains, m a l t t i and starches f ran sorghum and m i l l e t . 47

3.7- 1 Crude Protein determination 47

3.7.2 Fat determination 48

3,.7.3 Crude fibre determination ' 49

3 ..7.4 ~ s h Determination

3.7.5 Total Carbohydratedetennination

3,'7,6 ~ e l s t i n L z a t i o n temperature determination 5 1

3.7.7 Starch determination in s ta rch concantrate by hydrolytic method. 5 3

3.8 Production of M a l t Based Syrups 5 4

3.8.1. wort Preparation by three stage'dacoction mashing method from sorghum malt 54

Effect of varying mash concentrations and aaccharification periods on reducing sugar contents of worts i n a three stage decoction mashing, .. I 54

Effect of varying concentrations of glucomylase and saccharification periods on the reducing sugar content of malt hydrolysates 5 5

M a l t Based Syrup Production 55 .

Glucose Syrup Production 5 6

Determination of some properties of syrups 57

~e t enn ina t ion of spec i f ic gravi ty/l)egtee baume' 57

Percentage reducing sugar content/ Dextrose equivalent(IX1 value determination 58

Determination of Colour 60

CHAPTER 4.

4.1

CHAPTER 5.

RESULTS AND DISCUSSION 6 1

Malting charac te r i s t i cs of the cereal grains, sorghum and millet. . . 61

Optimum malting conditions of ths cereal grains, sorghum and mi l l e t 62

Evaluation of m a l t 1 s qual i ty charac te r i s t i cs 68 Determination of optimum pH and temperature condi t iom f o r malt's amylase act ivi ty . 70 ~roximate/chemical analysis of the sorghum/ m i l l e t gra ins and malts. , . 74

Chemical analyses of starches extracted from millet and sorghum grains. 76 Effects of varying mash, glucoamylase concantra- t tons and sacchar i f icat ion periods on redwing sugar contents of the wort syrup. 7 7

Properties of malt based eyrups and acid enzyme converted glucose syrups. 81 . SUMMARY AND CONaUSICbJS 84

REFERENCES

APPENDIX

LIST OF TABLES

TABLE 1 - Proximete analyses o f .Pear l m i l l i t ( ~ W I 3 ) (meman with . ranges i n Parenthesis where available) $0

1, 2 - Sorghum m a l t proximate analysis a f t e r four days gemination . As

n 3 Checklist of properties and funct ional 'uses of corn syrupta In spec i f ic food products. 30

t* 4 Malting charac te r i s t i cs of the cereal grains, Sorghum and millet . 62

5 valuation of malt * s qual i ty charact@.ristics 70

@ 6 ~roximate/chemical analysis of grains and malts 75

n 7 Analyses of starches from millet and sorghum grains 77

8 Properties of m a l t based syrups 83 -

" 9 Properties of acid-cenzyme converted glucose 83 syrups. . . 4

LIST OF FIGURES

A General manufacturing procedure f o r corn syrups 25

Properties and functional uses of corn syrups 3 1

Flow char t of w e t mill ing operations i n s ta rch production from cereal grains .. 46

Flow char t of acid-enzyme converted glucose syrups production e m 56

Flowchart of malt based syrup production 57 ~t'

Plo t of moisture contents(%) against steeping time(hours) . . 63

Plo t of d i a s t a t i c power(o~) against steeping time (hours) a f t e r 4 days of germination 64

Plo t of d i a s t a t i c power (OL) against gurmination periods(days1 a f t e r Sohours of steeping . 6 6 .

Plo t of malting loss(%) against gemination periods(days) . 6 7

Plo t of moisture cogtent(%) against kilning period(hours) a t 45 C. . , 69

p l o t of mg maltose against p~ . 72 . .

Plo t of maltose ca l ib ra t ion curve 7

p l o t of mg ma1 tose against t e m p e r a t ~ r e ( ~ ~ ) \.73

p l o t of glucose standard curve 7 8

Plo t of mg dextrose against mash concentration(%) and time(hours1 . . 79

Plo t of mg dextrose against ~ 1 ~ = c s - i - ~ ~ L ~ c concentration(%) and time (hours)

xi. ABSTRACT

he s u i t a b i l i t y of two cereals(sorghum and millet) f o r the production

of malt-based slyrup w a s determined.

i'roximate analys is waa c a r r i e d on the grains. The g ra ins were steeped

f o r SO hours, germinated for 5 days a t room temperature and k i lned f o r 48hrs

a t 4s0c. Ma1 t i n g c h a r a c t e r i s t i c s of t h e g ra ins determined include: t h e

germinative energy, germinative capaci ty , 'Hot water e x t r a c t and d i a s t a t i c

power. Starch w a s ex t rac ted from the two g ra ins and used f o r syrup production

Optimum condit ions f o r the ac t ion of m a l t amylases in syrup production were

a l s o determined,

The ma1 tg s qua l i ty c h a r a c t e r i s t i c s analysed showed t h a t sorghum gra in

generated b e t t e r malt. Malted gains contained higher amounts of p ro te in and

crude f i b r e , and lower amounts of f a t , ash, and t o t a l carbohydrates than tha

h a l t e d gains. 1000 g r a i n s of m i l l e t and sorghum weighed 6-8 and 33,3g

respectively. Malting l o s s values w e r e 16-20% f o r m i l l e t and 12016% f o r

sorghum. Germinative capaci ty of the millet g ra ins was 85% while sorghum

gra ins had a germinative capaci ty of 90%. Hot water e x t r a c t values were

1 8 0 . 1 ~ ~ / k ~ f o r m i l l e t and 2 0 3 ~ ~ ~ ~ f o r sorghum, O~timum d i a s t a t i c power of

2 7 O ~ and 3 2 O ~ were obtained f o r m i l l e t and sorghum reepect ive ly , Sorghum

s t a r c h yielded syrup of a b e t t e r quality than millet s tarch . Sorghum s t a r c h

a l s o has a lower g e l a t i n i z a t i o n temperature and lower ash content than

millet s tarch . Optimum pH range f o r alpha amylwe a c t i v i t y i n both m a l t

e x t r a c t s was 6-7, Optimum tempera- range f o r anylase a c t i v i t y was

found t o be 40-50°c f o r m i l l e t and 60-70°c f o r sorghum.

CHAPTER 1

INTRODUCTION

The development of ma1 t based syrup involves th ree fundamental stages:

'(1) Production of m a l t by a process c a l l e d malting.

(ill Preparat ion of wort from t h e malt by d e m t i d n mashing process.

(iii) Further sacchar i f i ca t ion o f the w a r t to malt based ~ y r u p using

external microbial amylases. ,

Glucose syrup i s t r a d i t i o n a l l y produced from corn s t k c h , hence

its name 'Corn syrupr. I t is t h e pur i f ied 'concentra ted aqueous so lu t ion

of n u t r i t i v e saccharides o f Dextrose equivalent(DE) 20 o r more obtained .

by hydrolysis of e d i b l e s t a rches !Whistl.er a t ' al,, 1984). The hydro ly t i c , -1

agents include ac id , microbial amylases, malt amylases and combinations of

these. Fur ia (1968) has described the technology of glucose syrup

production, while M ~ C ;U&istgr(1979) has described t h e ac t ion p a t t e r n s of

enzymes used i n t h e commercial corn syrup production.

Malting is e s s e n t i a l l y a b io log ica l process in which t h e germination

of cereal g ra in i s c a r r i e d o u t in a con t ro l l ed environment. The t echn ica l ly

intportant f e a t u r e s of gemina t ion are t h e synth.gies of hydrolyt ic enzymes

and t h e degradation of t h e g r a i n structure. When both processes have

reached the des i red s tage , t h e germination is in te r rup ted by drying o r

k i ln ing,

Malting s tud ies have been done by severa l workers; Aisien(l983)

invest igated t h e u t i l i z a t i o n of soluble carbohydrates during sorghum

yern~indtion. . Novellie (1960) es tab l i shed t h a t sorghum ma1 t do not posses

t h e b - c u n ~ l c i s e ,protease, c e l l u l a s a and hemi$ellulase a c t i v i t i e s of e i g h t

c u l t i v a r s of process millets.

Decoction mashing t r a d i t i o n a l l y employs malt which is less modified . -. . L

thm t h a t used i n infusion mashing and is only l i g h t l y kilned(Briggs.5: &,

w r i n g t h e k i ln ing process, an optimum temperature is chosen t h a t

s t r i k e s a balance between t h e development.of the c h a r a c t e r i s t i c m a l t

f lavour, colour and the sustenance of high ma1 t d i a s t a t i c power. The 1

colour i s produced dur ing 'k i ln ing through mai l lard reac t ions between the

prote ins and sugars present i n t h e m a l t , (a na tu ra l browning reac t ion) ,

Malt based syrups are used widely i n the food industr ies:

(i) Brewing industry: D i a s t a t i c syrup contr ibutes t o converting o t b r

s tarchy adjuncts to simpler sugars while non-diastat ic mal t syrup

contr ibutes towards t o t a l fermentables.

(11) Malt syrups .are increas ingly being used as na tu ra l food colourants

thereby replacing caramels,

(iii) I n the baking industry, D i a s t a t i c malt syrups may be used i n breads

a s a yedst food t o r e l eas ing sugars n a t u r a l l y and con t r ibu te t o loa f volume

irnd texture. Malt syrup is a l s o used i n brown bread and dark cake manufacture,

breakfas t ce rea l ,and b i s c u i t manufacture . ( i v ) I n thcphannaceut ica l industry, m a l t syrup could be incorporated i n t o

i n f a n t l i q u i d drug mixtures a s sweetening, colouring and f lavour carriers. -

G ~ U C O S ~ syrup is widely used i n confectlonary and baking indus t r i e s ,

i n canning of f r u i t s and vegetables, s o f t drink indus t ry , i n beverages, and

other products requi r ing sweetness. Hoover(1963) has i l l u s t r a t e d t h e

functional p roper t i e s of corn syrups a s they r e l a t e b > t h e type of conversion,

f i g u r e '2 Hoover(1964) has a l s o prepared a c h e c k l i s t of p roper t i e s and

funct ional uses of corn syrups in a wide va r i e ty o f foods, (Table 9)

The value of Niger ia ' s annual consumption of glucose syrup was

estimated atsN80.625 mi l l ion and t h a t of c r y s t a l l i n e glucose In form of

dextrose monohydrate was est imated a t about W60 mil l ion(Federa1 I n s t i t u t e

of I n d u s t r i a l Research, Technical memorandum No, 25, 1970)- These d a t a

derived from the l imi ted market surveys conducted were more l i k e l y to be

an under es t imates than over-estimates.

I n Nigeria, t h e r e l a t i v e abundance o f Sorghum and millet crop8 w i t h

an average m u a l production f i g u r e of 4,8 metric tonnqr and 2.4 metric

t o m e s respectively(Sumraru* miscellanous paper 90, 1979), has prompted

the current research e f f o r t s towards cos t reduction. I n 1982 alone,

Nigeria's import value f o r m a l t was p u t a t about M40 mill ion i n foreign

exchmge. Both wheat and corn are today in Nigeria ,Golden cereals': EF!A, i

hence the quest f o r subs t i tu te and/or blends.

Malting of t h e loca l cereals , sorghum and millet, generate endogenous

malt amylases which augment the imported microbial amylaae used i n the .

sclccharification process of malt symp pr=2utticn, thereby saving cost.

A i m s and Objectives of the Study:

The study was carr ied o u t to prepare malt-based syrups from loca l ly

avail able cereal grains. The spec i f ic objectives were:

1. t o determine which of the two cereals , sorghum and m i l l e t has a higher

ma1 t ing potential .

2. to determine the malting conditions necessary f o r optimizing the ,

sorghum/ m i l l e t malts' d i a s t a t i c power.

3. to determine the leve ls of microbial ainylase and conditions su i tab le

f o r the production o f r e l a t i ve ly cheap malt based syrup.

CIWTER 2

2. LITERATURE REVIEW

2.1 Production of Sorghwn and M i l l e t grains:

2 . 1 Sorghum qrain:

h l though vorghum rank8 f o b # mong c e r e a l s in c u l t i v a t e d area world

wide following wheat, r i c e , and maize, i t i s the most important cereal i n

Nigeria occupying about 46% of t h e total land area devoted t o the growing

of ceredls. The a rea devoted t o sorghum has increased by about 25% over

the l a s t two decades growing from 4.6 mi l l ion hectares i n 1959 t o qin

e ~ t i m a t e d 6.1 mi l l ion hectares i n 1979. Sorghum present ly accounts f o r

about 50% of t h e t o t a l c e r e a l production i n the country. Production has \

gone from 2.5 mi l l ion metric tonnes i n 1960 to 4.8 mi l l ion metric t o i a s

i n 1978(Samaru miscellaneous paper No. 90, 1979). I n 1981/82, production

was 16,192 tonnes and y i e l d per hectare was 841 kilograms (Federal o f f i c e i

of s t a t i s t i c s , Lagos, 1983. Survey of modern Holdings of Agriculture

1981/82, p. 20).

The o r i g i n of t h e crop was traced to o the r regions of Africa, from

Ethiopia across the Sudan (Damon, 1962). . A survey of the indigenous

sorghum v a r i e t i e s reveal four economically important varieties name:ii, I

Guinea, kaura, Farafara , and Chad races (Buntung and C u r t i s , 1970).

2.1.2 ~ i l l e t grain:

Millet although n o t as important a s some of the o the r cereals when

t o t a l world production figures are considered >it is nevertheless t h e 6

6.

bas ic d a i l y d i e t ' o f severa l mi l l ion people i n Africa and I n d i a ( ~ a r t i n e t G., -

There is an average annual production of 2.4 mi l l ion metric t o m e s and

d nat ional average y ie ld of 75Okg/hd (Sanaru miscellanous paper, 90; 1979). - 1n'1981/82 production w a s 9,862 t o m e s , and y i e l d per hectare , 566 kilogram

( ~ e d e r a l Office of s t a t i s t i c s , Lagos, 1983). There are two main types of

millet grown i n Nigeria. These are the Gero and Maiwa types. The Gero

m i l l e t s are of s h o r t e r dura t ion taJcing 75-100days t o mature. Maiwa on the

o the r hand takes between 120-150 days t o mature. T k Gero type, because

of i t s g r e a t e r adap tab i l i ty , is favoured over maiwa and over 8C% of a l l

millet grown i n Nigeria is of the Gero type(Samaru miscellanous paper,

NO', 90, 1979'). Rachie (1974 l i s t e d the canmon and corresponding s c i e n t i f i c

names of t e n v a r i e t i e s of millets grown world over.

2,2 chemical and Biochemical s t u d i e s on Sorghum and Millet grains:

2,2,1 Sorghum grain:

'Neucere and sumrell (19801, s tudied the proximate analys is , f a t t y

dcid composition, f r e e sugars, mineral content and the i id is t r ibut ion of

tannins i n f i v e v a r i e t i e s of sorghum biccdor(L). Moench, They showed t h a t

the va r i e ty wi th predanlnantly f loury endosperm (NSA 740) has t h e h ighes t

prote in content , . Some d i f fe rences i n f a t , ash, carbohydrate, and , f i b r e -

contents w e r e a l s o noted among t h e f i v e v a r i e t i e s . The content of neu t ra l

l i p i d s i n the f i v e lines of g ra in sorghum ranged from 2.66 t o 3.49%. They

a l s o noted s u b s t a n t i a l d i f f e rences in t he mineral uptake of the f i v e

v a r i e t i e s and according to t h e r e s u l t of comparative m a l y s i s of f i v e

sugars i n theee v a r i e t i e s s tudied , nmely; f ruc tose , glucose., sucrose,

m d l toae a d raf f h o s e , f ruc tose and glucose comprised the h ighes t contents

of f r e e sugdrs. m

Rooney a d s u l l i n s (1970) i n t h e i r study, compared the g ra in produced

on d ip lo id ( 2 ~ ) and t e t r a p l o i d (4x1 l i n e s of t h e sorghum, Sorghum b ico lo r

(L) moench, c u l t i v a r T

propert ies . ~ c c o r d i n g

yredter i n kernel size

x 403 f o r physical , morphological and chemical

t o their r e s u l t s , g ra in f r an t e t r a p l o i d was

and p ro te in content and was lower i n s t a r c h content

and test ?eight than g ra in from the d ip lo ids . Mean values of d ip lo id and

t e t r ap lo ids were 12.8 and 15.x prote in , 72.3 and 68.8% s ta rch , 26.8 and

41.4 g/1000 kernels , and 74.3 and 70.0kg/hl respectively. The . resu l t

a l s o showed t h a t endosperm cells of t h e t e t r a p l o i d were l a r g e r than those

of the diploid. Kernel dens i ty and amino acid composition were similar.

Hoseney et &. , (1974 ) examined the s t r u c t u r e of sorghum gra in

samples by scanning e l e c t r o n microscopy. They observed t h a t t h e s o f t o r

opeque endosperm is charac ter ized by r e l a t h e l y l a r g e in te rg ranu la r air

spaces, and showed t h a t its s t a r c h was e s s e n t i a l l y round and covered with

a t h i n sheet of protein. Furthermore, they discovered t h a t t h e hard

endosperm resu l t ed from s t rong adhesion between prote in and s t a r c h and

a lso when t h e hard endosperm was f rac tured , many a ta rch granules were

broken r a t h e r t h k t h e s t a r c h p ro te in i n t e r f a c e being broken. Resul ts

a l s o revealed t h a t a dwarf v'ariety from Sudan. had r e l a t i v e l y few pro te in

bodies i n the endospenn

va r i e ty contained 3.019

and t h a t amino acid analys is confirmed

l y s i n e pe r 1009 prote in , s i g n i f i c a n t l y

t h a t this

more than

normal i n sorghum grain. 1

S u l l i n s and ROOney (1974') compared sorghum gra ins t h a t d i f f e r i n

endospenn t ex tu re and endospenn type i n order to evaluate the usefulness

of microscopy t o account f o r d i f f e rences observed i n the feeding p roper t i e s

of these grains. They observed t h a t t h e waxy.aorghum kernel sec t ions . . .

hdd the smdllevt proportion of per iphera l endospenn a rea of the four

g ra ins excrmined. The waxy sec t ions were a l s o more r a p i d l y so lub i l i zed by

pronase and alpha- amylase enzymes and by buffered .rumen f l u i d than, the

non w a x y sect ions. According t o them, the f indings 'might account f o r

observcltions of feeding trias in which steers fed non-waxy sorghum gra in

d i e t s r equ i re 8 t o 20X more feed t o produce a pound of g ra in than steer

fed waxy sorghum gra in d i e t .

Deyo et &., (1990) determined t h e proximate and amino ac id composition

of mature and immature samples of sorghum grain. Thei r d a t a i n d i c a t e

marked d i f fe rences in amino acid content. ~ h e i observed t h a t crude

p ro te in content of immature and mature sorghum gra in was shilar. The

feeding s t u d i e s which they c a r r i e d o u t showed less ava i l ab le energy fram

immature than mature sorghum grain.

Haikerual and ca hie son( 1971) determined total p ro te in and amino i

acid composition of a number ofsorghum sample including those from two

f i e l d experiments. They showed t h a t t h e germ contained ' t he h ighes t proportion

of prote in , followed by the whole kerzel, the endosperm and the per icarp

a l s o t h a t the amino ,acid c a p o s i t i o n of those p a r t s was d i f f e r e n t , with

higher proportion of lys ine , h i s t i d i n e , arginine, glycine, a sp&t ic acid,

threonine, and val ine i n the g e m , than t h e whole kernel.

2.2.2 Mi l l e t grain;

Bcrdi et_ &.,(I9761 i n their work, found t h a t pea r l millet s t a r c h

ranged i n diarneter from 8 - 12 11, somewhat smaller than corn o r sorghum

atarch. They observed t h a t pas t ing p roper t i e s of m i l l e t s t a r c h were

e h i l a r to those of sorghum s t a r c h , except during the 1 hour holding period

a t 95O~. They showed t h a t m i l l e t s t a r c h contained 1% amylose compared

with 23% i n sorghum s tarch . Arnylograms of m i l l e t f l o u r a l s o gave low peak

v i s c o s i t i e s compared to sorghum f l o u r ind ica t jng an ac t ive alpha-anylase 4)

sys tern.

proximate ana lys i s of millet g ra in has been c a r r i e d out. Shepherd

e t , dl., (1972) reported from E a s t Afr ica the proximate composit ionof - - m i l l e t g ra in ( ~ r y weight bas i s ) . P ro te in ranged between 11.5 and 13.8%

l i p i d ranged f r m 4.8 - 9.2%, f i b r e 1.0 - 3.8% and ash, 1.1 - 2.4%.

Table 2.1, shows t h e proximate ana lys i s a s reported by some authors,

general ly, p ro te in (%I vary from 8.4 - 21.8, l i p i d ( % ) from 2.9 - 7.5,

carbohydrate (%) ' f r a n 53.9 - 83.8, Fibre(%) from 1.2 - 10.7, ash ('961, from

Badi - e t ' -- aL., (1976) showed t h a t pea r l millet &aln endospenn was

composed of both hard( t rans lucent1 and soft(opeque) par ts . . The hard p a r t

h s t i g h t l y packed, polygonal shaped s t a rch - granules and a ma t r ix , p ro te in

containing r e l a t i v e l y l a rge , embedded p ro te in bodies. The s o f t endospenn

Fetu

ga

(19

77

) A

frica

Ga

dr

y and B

ideau

(19

74

) A

frica

nagbail( 1

97

7-~

erso

nd

l comnunication to Hulse

Afric

a

et

al.(1

98

0)

- -

Po

pli and

Sin

gh

(l97

2)

Ind

ia

L'p

rety and A

us

tin (1

97

2)

Range o

f means

Range o

f ra

ng

es

Nig

eria

hds lose ly packed, spher ica l s t a r c h granules c ~ v e r e d with a t h i n shee t

of proteih. The s o f t endosperm conta ins many a i r spaces, and no p ro te in

bodies.

Lorenz a d liinze ( 1976 determined a d c&npared t h e functi&nal

c h u a c t e r i s t i c s of p r o s and fcrxtai l millet s tarches with those of wheat

and rye starches. The m i l l e t starches showed higher water binding

c a p c i t y values and g e l a t b i z a t i o n temperatures than t h e wheat s tarch.

with two exceptions, the millet s t a rches produced swelling power values

d t 90°c which were similar t o those of the wheat s tarch . They observed I

t h a t the s o l u b i l i t i e s of t h e millet s t a rches were lower than those of t h e

wheat s t a r c h , except f o r t h e s t a r c h from one va r i e ty of m i l l e t ; and t h a t

the amylograph v i s c o s i t i e s of m i l l e t s t a rches were higher than those of

the wheat s t a r c h a t a l l re ference points.

l tasule~(1977) - - s tudied t h e weight and composition of m i l l e t parts . He

found t h a t the seed coa t s contained r e l a t i v e l y high percentage of prote ins ,

sugars, and f a t s . The seed c o a t s had high contents of pentosans,

ht?raicelluloses, and f i b r e which are s i g n s o'f low n u t r i t i v e value. Removal . of seed c o a t s from m i l l e t s l e d to a higher n u t r i t i v e value.

Ramachandra - e t ' &.,(1977), found t h a t the t g t a l phenol and tannin

l e v e l s of f i n g e r millet v a r i e t i e s i n d i c a t e wide va r i a t ions i n phenolic

contents. T-hey showed thatqwhite g ra in v a r i e t i e s had lower phenolic

content than, the brown-grain varieties. I n v i t r o p ro te in d i g e s t i b i l i t y

values of low tannin samples were higher than those of t h e high tannin

simples. Dehulling had e f f e c t of removing most o f . t h e phenolics from

f inger , rnille t gra ln w i t h .concomitant increase i n i n v i t r o p ro te in

d i g e s t i b i l i t y .

A U ~ U S t - et A. , ( 1979) , analysed f o r p r a k h , SZL~Z acid c = p ~ s i t i W ,

and mineral assay of 14 inbreed l i n e s of pea r l millet(Pennisetum americanum

(L) ~ e a k e ) from the p lan t ,b reed ing . proyrdn a t Ti f ton , Georgia. T k i i r

d a t a shared t h a t p ro te in con ten t varied 'from 10.7 - 17.1%. Chemical

scores on the amino ac ids showed l y s i n e t o be the l imi t ing amino acid.

hey es tab l i shed t h a t mineral content var ied considerably among the

d i f f e r e n t hybrids. The predominant elements were phosphorus and potassium.

p r u t h i and ~ h a t i a ( l 9 7 0 ) s tudied two improved strains4 of Pennisetmm - i

t ypho ideun(@baj ra@) and were found t o have a l i p i d content of about 5.0%

and bound l i p i d content of about 0.5%. They observed t h a t i n the non-polar

f r a c t i o n , s t e r o l esters, hydrocarbons, xd t r ig lyce r ides , are t he p r inc ipa l

cons t i tuents . They separated polar l i p i d s by twodimentional tNn- layer

chromdtography and l e c i t h i n was found to be t h e major component.

Dorisova et s . , ( 1 9 8 2 ) inves t iga ted the e f f e c t of various s t ages of

t h e tecthology of process m i l l e t on t h e amino acid con ten t of m i l l e t

protein. They s t a t e d t h a t t h e l e v e l s of methionine and ty ros ine in

husked m i l l e t increased by about 1196, whereas those of l y s i n e and g lycine

decreased by 1% and 11% canpared to unhusked millet. Polishing of

husked m i l l e t decreased t h e l e v e l of g lycine by 22.- and t h e l e v e l s of

threonine, tyros ine by 7.1% to 13.8% compared to unpolished husked m i l l e t .

Cooking of polished husked m i l l e t decreased to ta l amino acid content by

2.3 ~ d . t ing chdrac teristics of Nigerian sorghum and Millet var ie t ies :

2.3.1 Sorghum malk

Aisian G,, g., (1978) in a study of the germination behaviour of

Guinea corn, (Sorghum vulgare) inves t iga ted its percentage germination

(germination energy) and length of the ascrospi re ranged between 2-2.5cm,

1 he r e s u l t a l s o showed t h a t the optimum moisture con ten i f o r rapid .

gemina t ion was between 35 and 40%, a t opkhum temperat ire of 22Oc. he

r e s u l t s f o r the rest of t h e germinative capci ty , percentage germination a t

d i f f e r e n t times of gemina t ion are tabulated.

Daiber and ~ o v e l l i e ( 1 9 6 8 ) found t h a t g i b b e r a l l i c acid had l i t t l e e f f e c t

on amylase development in normal k a f f i r corn. They observed t h a t only

immature seeds and very l a r g e g ra ins produced more amylase when t r e a t e d with

g i b b e r a l l i c a c i d , - b u t this e f f e c t was much smaller than t h a t found with

barley. They concluded on f u r t h e r inves t iga t ion t h a t amylase ,formation i n

sorghum appears t o be preponderantly' a function ' , of the embryo.

~ o v e l l i e ( l 9 6 0 ) e s t ab l i shed t h a t sorghum malts are poor i n betzi-amylase

compared with ba r l ey malts( b u t s i m i l a r t o Oat and r a g i ) and do not possess i

high d i a s t a t i c power, H e showed t h a t sorghum malts contained beta-ainylase

i n considerable quan t i t i e s , 18-39% of Ule sdcchslri-k'ying r lct ivi ty being due

t o the beta-amylase. H e found t h a t t h e alpha-and b e t a - amylases developed

a t approximately t h e same r a t e during germination since t h e i r ra t io(which

vAries from 0.22:l to 0.64:1) was p r a c t i c a l l y cons tant throughout the

ma1 t i n g process.

~ i s i e n ( 1 9 8 2 ) i n h i s s t u d i e s found t h a t modification i n t h e sorghum

gra in endosperm during seedling growth and malting was associa ted mainly

w i t h increased a c t i v i t i e s of alphsmylas4endo-b)-4lucanase, l i m i t

dextrincise and endoprotease. H e found t h a t t h e major s t a r c h - degrading

enzyme was alpha-amylase and also observed t h a t t h e a c t i v i t i e s of endo-

/+gluccmase, l i m i t dext r inase a d endoprotease were comparatively higher, in

t h e endosperm than in the embryo during seedling growth.

~ d y l o r ( l Y 8 3 ) i n h i s study, observed t h a t when sorghum i s malted, much

of t h e ni trogen i n the kernel i s t ransfered to the roo t s and shoots. H i s

exanination of Osborn p ro te in f r a c t i o n s ex t rac ted from t h e kernel r evea l s

t t ~ t ds i n the case of ba r l ey t h e prolamins are t h e major source of t h e

ni trogen t ransfer red . E'urthennore, he fouhd t h a t t h e 'two most important

f r e e ac ids of sorghum m a l t appear t o b e asparagine and glutamine, as i n

germinated wheat and maize.

w i l l i a m ( 1983) s tudied t h e e f f e c t s of tannin on malting a s we1 . l a s the

change i n polyphenols during malting of b i rd - res i s t an t and non bird-

r e s i s t a n t c u l t i v a r s , H e observed t h a t no d i f fe rence could be found i n

the percent germination nor in the r o o t and shoot production of t h e m a l t s

of the two c u l t i v a r s , H e found t h a t t h e r e was an. increase i n the antho-

cyanidin content of the r o o t s and shoots during malting.

~waifotl983),9ma-irradi~~dtw0 Nigerian species of sorghum - Sorghum acaUdatum(sk. 5912) and sorghum guineense(HP 3 ) p r i o r t o malting - 9' on a c o b a l t i r r a d i a t o r . H e exposed t h e spec ies to t h e following doses;

0.22, 0.44, 1.76, and 4.95 krd - I n t h e assays f o r d i a s t a t i c power,

/+amylase, and a lpbany l - . W h i l e i n t h e assays f o r germinative energy

and lengths of r o o t l e t s and acrospire,, they were exposed t o a dose of

0-5 krd. H e found t h a t a dose of 1.76 krd ra i sed the d i a s t a t i c 'power,

6-mylase ,g(- dmylclse, germinative energy, and lenghts of roo t l e t s and

crcrospire ~n(utimcrlly r e l a t i ve to those of the unirradiated sorghun is' t%

species studied. H e observed t h a t the e f f ec t of 1.76krd was, however,

higher i n Sk 5912 species than i n HP3 species.

irnfchie( 1982) studied f i ve Nigerian sorghum 'var ie t ies B.E.S., F.F.B.L.

~ ~ 2 1 , ~ ~ 1 4 9 9 , and LRV, and came c u t with results of the proximate analysis i

a f t e r four days of geminat ion as shown on tab le 2.2 below,

TABLE , 2 .

SORGHUM MALT PROXIMATE ANALYSIS (ANICHIE 1982)

-- - -- -- - - -

Ma1 t ing l o s s (%) 25,OO 24.00 22-00 22.50 17-82

log f i l t r a t i o n time 1.2b 2 .01 1.62 1.78 1.52

Ma1 t Nitrogen (%I 1, 70 1.68 1-61 1-54 1- 74

Aisien (1982) investigated the u t i l i z a t i o n of soluble carbohydrates

during sorghum germination and seedling growth, He determined sucrose,

raff inose and fructose leve ls in the scutellvn of i n t a c t and excised

sorghum seedling during growth, H e found t h a t i n the scutellum of the

i n t a c t grain embryo, sucrose and ra f f inose l eve l s declined sharply over

the germination phase but increased a t post-germination ( i e roo t enwgence)

a s hexose sugars from the modifying endosperm passed into t he scutellum.

tie observed t h a t maltose, maltotxiose and glucose were the main products

of t he enzymic modification of t h e endospem during seedling development,

which is a post-germination event atxi therefore concluded t h a t the growing

. -.---z --! cf the embryo, with its higher inver tase a c t i v i t y showed grea te r

cdpdcity f o r sucrose metabolism than t h e scutellum.

2.3.2 Millet Malt: I

opoku et &.,(1981) geminated m i l l e t g ra ins f o r 84h and kilned a t

45Oc to obta in a m a l t product. They conducted analys is of vitamins,

phytate, oxillate, tannins, total phenols, and calcium t o determine t h e

nu t r ' i t iona l value of t h e g ra ins and the malt. hey found t h a t the., . levels

of vitamins were higher i n t h e malt than i n t h e grains. Also t h a t s l i g h t

increases i n p ro te in and tote$ phenol w e x e observed in the m a l t , while

l i p i d , phytase, and oxala te l e v e l s decreased during malting. The r e s u l t

of the proximate analys is w a s given i n a table. . '

Skovron and Lorenz (1979) determined t h e - a?ylase, protease,

ce l lu lase , and hemicullulase a c t i v i t i e s of e i g h t c u l t i v a r of proso

(~anicum miliaceum) m i l l e t s . They found t h a t a l l t h e c u l t i v a r s showed

b-my1 ase, pro tease , c e l l u l a s e and hemicellul ase a c t i v i t i e s with the

exception of one sample that sllowed no hemicellulase ac t iv i ty . The

optimum pH f o r b-amylase a c t i v i t y was found t o be approximately 5.0, and

production of maltose per in'illilitre of e x t r a c t ranged from 0.73 t o 1.93 f i a f t e r l h of incubation a t pH 5.25. Also t h e pH optimum f o r protease

6

a c t i v i t y was near 3.0 and 5.0, production of tyros ine per m i l l i l i t r e of

e x t r a c t ranged f r m 12.5 to 75.5 Yj aftez ii! of h~&i i i io i i ai pH 4.8.

Opokus 3 d., (1983) s tudied the quan t i td t ive and q u c l l i t ~ t i v e changes

i n cdbohydra tes , prote ins , and l i p i d ma te r i a l s during t h e germination of - mil le t . They found t h a t a two-stage metabolism was exhibi ted during

ycrmin~i t i o n a d t h ~ t Y t a c h content d e c r e a o d during germination which

coincided with cir~ increacja i n so lub le carbohydrate and p r t e ins . . They

f u r t h e r observed t h a t tho high l i p i d content of the g r a i n was reduced t o

Ueleia culd arts son-Varriano(198Ib) s tudied the e f f e c t of pea r l millet

dnylases on i n t a c t s t a r c h granules and heated s t a r c h suspensions, Amylases

i n crude m i l l e t e x t r a c t s showed higher amylolytic a c t i v i t y on wheat s t a r c h

than on m i l l e t s t a r c h , both in amylograph determination and s t u d i e s on

hydrolysii of raw starches. According to the r e s u l t s , t he a c t i v i t y p a t t e r n

of m i l l e t alpha amylase was similar t o t h a t of o t h e r cereal alpha amylases

' . with t h e r a t e of appearances of hydrolysis products being dependent on the

p a r t i c u l a r s t a r c h subs t ra t e s ,

Gudisevd e t ' &., ( 1981) screened twelve v a r i e t i e s of sorghum( Sotghwn - b i c o l o r ) , 14 v a r i e t i e s of pea r l millet(Pennisetwn typhoidem), 12 v a r i e t i e s

of s e t a r i a ( s e t a r i a italics), four v a r i e t i e s of ragi(E1eucine coracana),

11 v i d e t i e s o f echinocloa m i l l e t (Echinocloa colona) , 13 v a r i e t i e s of

proso (Panicium meliacem), 11 varieties of kodo ;, (Paspalum scorbiculatum),

did 11 v a r i e t i e s of miliare(Pani.ciwn mi l i a re ) f o r inh ib i to ry a c t i v i t y

aga ins t human s a l i v a r y amylase, Echinocloa, proso, kodo and miliare had

no de tec tab le a c t i v i t y . Two s t r a i n s of sorghum and one s tra ip of pear l

millet d id n o t show .&-anylase i n h i b i t o r y ac t iv i ty . ~ l l o t h e r seeds had

6

ac t iv i ty , the highest being observed i n sorghum. According t o the r e su l t s ,

the inh ib i to rs were non-dialysable and were inactivated by pepsin treatment.

Also s e t a r i a and 8orghum inh ib i to rs vere r o l a u v e i y t inenoiab i le compared

to r a g i and p e u l m i l l e t inhibitors.

Maileshi and Desikacha (1979) evaluated the malting potent ia l of high

yielding var ie t ies of ragi(E1eusine coracana). Three were found t o be of

good malting var ie t ies as they possessed good germinative energy, high

amylase ac t iv i ty , with good y ie lds of malted flour. Gemination conditions

were 24h .teeping and 72h germination a t 25-26O~.

s k o r a i n and ~ag le (1973) in evaluating the use of ba j ra o r pearl

mi l le t fo r malting purposes, compared the beta amylase ac t i v i t y of ba j ra

and barley malts. They found t h a t beta amylase ac t i v i t y of germinated

bajra increased up to 30h and decreased up t o 72h., while t h a t of barley ?

increased continuously up to 72h. They therefore concluded t h a t i f ba j r a

i s t o be used f o r m a l t production, then sho r t malting i s radvocated.

Abdul-Hassan and Varriano-Martson(1982) -s tudied the amylolysis of .

pearl m i l l e t s t a rch and its f rac t ions by pear l m i l l e t alpha amylase.

Gemination resul ted in a 120 fo ld increase i n spec i f ic ac t i v i t y of the

enzyme over t h a t of the alpha amylase f ran mature grain. Results showed

t h a t raw m i l l e t s t a rch w a s r e s i s t a n t to attack by alpha amylase. Fran

germinated mi l l e t , a lso amylase was readi ly hydrolysed by pur i f ied millet

alpha my1 ase, while sane portions of m i l l e t anylopectin were hydrolysed

slowly by alpha amylase.

t ' d et A., ( lgVl6 ) found t h a t decreasing germination temperatures .

fruu 35 t o 2 5 O ~ i n b a j r a and from 25 to 1 5 O ~ i n bar ley , =esul ted i n a '

s i g n i f i c a n t increase in t o t a l amylolytic a c t i v i t y a s w e l l as p r o t e o l y t i c

a c t i v i t y of green malts prepared fram the two cereals.. They observed

t h d t t o t a l m y l o l y t i c a c t i v i t y was mainly due to b e t a amylase i n both

m d l t k 4 , more s o i n b a j r a malt. They suggested t h a t the germination a t low

temperdture l eads t o b e t t e r y i e l d s a s w e l l a s qua l i ty of malt.

Pokhryal e t a l . , (1977) s tudied hybrids of pear l m i l l e t g r a i n s - - (Pennisetum typhoides Linn(Brum) stapt .dnd tiubb), They examined hybrids

f o r t o t a l p ro te in content and amino acid spectra. They found t h a t p ro te in

values ranged from 11.0 - 14.7X. Lysine and threonine which are l i m i t i n g

amino ac ids according t o chemical score showed range of 2 .S6 - 3.46 and

1.99 - 2.44 g / 1 6 g ~ , respectively. ,

L'dl a d., ( 1973) compared various p roper t i e s of m a 1 t from b a j r a with

t h a t of bar ley malt. They observed t h a t proximate analys is r e s u l t s of the

two mal ts showed l i t t i e d i f fe rence i n t h e i r canposition. Both b a j r a and '

8

bar ley malts had comparable amylolytic a s w e l l as p r o t e o l y t i c a c t i v i t i e s .

According t o t h e r e s u l t , there w e r e very few d i f fe rences i n enzymatic

physical p roper t i e s of a good malt though it developed a b i t t e r t a s t e

a f t e r a s h o r t t i m e .

2.4 Glucose Syrups:

Contdolled hydrolysis of s t a r c h with ac id , enzymes, o r combinations

of these y i e l d s severa l s t a r c h hydrolysates which include: glucose syrup,

rnaltodextrinu, high maltose syrup and high fructose corn syrups and the i r

sol ids respectively . s t a c h from corn, sorghum, mi l l e t , potato, tapioca and other p lan t

sources are used i n producing those hydrolysates.

2.4.1 ~ e f initions:

Gl~.~cose syrup(Corn syrup). Is the pur i f ied concentrated aqueous

solution of nu t r i t i ve saccharides of DE 20 o r more'obtained by hydrolysis

of edible starch.

Maltodextrin: Is a mixture of purified nu t r i t i ve saccharides obtained

by hydrolysis of s tarch having a DE of l e s s than 20. r /

~ i g h Maltose syrup: as t h e name implies has a higher than normal

maltose content when compared t o other enzymatically produced sykps.

High fructose corn S~~UE(H.F.C,S): Is corn syrup

additional s tep of enzymic conversion of a portion of

fructose .

produced with the

P.glucose t o D-

Dextrose Equivalent(DE): Is an indication of t o t a l reducing sugars

calculated as D-glucose on a dry-weight basis. The DE value is inversely

re la ted t o the degree of polymerisation,(DP).

~non,(1979) c l a s s i f i ed corn sywps according to method o t conversion;

acid conversion, acid-enzyme conversions and enzyme-enzyme conversion.

2.5 ~ e t h o d s of production of ~ ~ U C O S ~ Syrups:

2.5-1 Acid Conversion: Acid conversion process i s carr ied out i n d: pressure

vessel termed a I c ~ n v e r t e r * ~ # t a r c h is mixed with water t o form a suspension

o r s lurry , containing 3040% dry starch. The required amount of d i l u t e acid,

usually about 0.12%, based on the weight of s tarch, is added and the

tt.rnper*tu~.e rdised by l i v e stem to 140-1600~. The heating continues f o r

12-20 minutes, The cooked o r ge la t in ized s t a r c h i s converted f i r s t t o the

higher polysdccharides. As t h e process proceeds, o the r sugars are produced;

~ c c o r d i n g B e M i l l ~ ( 1 9 6 7 1 , tlle #- D -(1 4 4) l inkages undergo

hydrolysis more e a s i l y than do the 4 - U - (1 3 6 ) linkages. Furthermore,

l inkages nearer the non-reducing end of t h e s t a r c h polymer are hydrolysed

more rapid ly than bonds located in the polymer i n t e r i o r , r e s u l t i n g therefore

i n a random hydrolysis.

H a r ~ e y ~ ( 1 9 8 3 ) observed t h a t acid hydrolysis of g ra in products is

considered t o modify f a t t y and p ro te in cons t i tuen t s , r e s u l t i n g i n off- ,

flavoured materials . According t o . h i m , acid hydrolysis con t r ibu tes to

the }\reduction of miscellaneous sugar products t h a t in t u r n can con t r ibu te

t o va r i ab le f lavour and fennentabi l i ty .

w i t t and Blythe (1976) inves t iga ted the fermentabi l i ty of m a l t worts

s u p & n e n t e d with 35% acid - thinned and 35% enzyna thinned corn syrup

s o l i d s , respectively. Under p i l o t brewing condit ions, t h e worts containing

the acid-thinned syrup showed a slower fermentation r a t e ,

2.5.2 Acid-enzyme conversion:

~cid-enzyme converted corn syrups are produced by m e a n s of a two-

s t age hydrolysis. The f i r s t s t a g e ( l ique fac t ion) is accomplished wi th

ac id , a s described above, and i ts e x t e n t i s determined by the des i red DE

value and carbohydrate cornposition of tine f in i shed syrup. The second

s t age (sacchar i f ic i l t ion) i s c a r r i e d o u t by means of s t a r c h hydrolysing

enzymes, usual ly O(-my l a s e , P-amylase and glucoamylase depending on

t h e required type of corn syrups and its composition. he ac t ion p a t t e r n s

22.

of enzymes used i n canmercial corn aymp manufacture have been described

by Mac All ioter (1979).

he scid-enzyme process according to Ough (1962) tends t o eliminate

carbohydrates degradation products and b-linked reversion products such

as gentiobiose. Hurst and Turner(1964) have described a patented process

for production of highly fermentable, non -~~~~ t&! , I i z i ; ; g corn syrdps with

high leve ls of glucose and maltose contents, w1th.a mixture of gluco-

cirnyldse and fungal d-amylasg . Different r a t i o s of P.glucose to maltose

can be obtained by a l t e r i ng the proportdo& of th. two enzymes, then

concentrations and conversion time.

Alternatively, when high maltose syrups are desired, barley &amylase

i s added and the hydrolysis proceeded u n t i l the required leve l of maltose

i s produced. Maeda and Tsao( 1979) reported the use of microbial 8-amylase *

i n Japan i r rdus t r i d ly ra ther than the p lan t enzyme. Mltsushashi e t al.,

(1974) developed a patent which employs simultaneously, maltorgenic enyme

and pullulanase ( o(-l,6-glucosidasei irr tie prepuration of high maltose

syrups from acid l i qu i f i ed starch.

2.5.3 Enzyme-enzyme conversionr

High conversion hydrolysates are prepared almost exclusively by the

use of eniymes. Mac ~ l l i s t e r ( 1 9 7 9 ) observed t h a t acid-catalyzed

hydrolysis of s ta rch is not capable of giving pract ical hydrolysates with

more than about 90% Pglucose, owing t o acid catalyzed reversion and

dehydration react ions resu l t ing in a s izeable l o s s of D-glucose.

The objective of t h e l i q u e f a ~ t i o n process is to convert a concentrabd

auepenoion of s tarch granules i n t o a solut ion of soluble dextr ins of low

viscosi ty f o r convenient handling in ordinary equipment and f o r easy

converoion to glucose by gluco-amylase. Mac Allister(1979) described the

process. According t o his process, a suspension of s ta rch in water i s

treated with calcium hydroxide (slaked lime) to pH 6-7,q optimal f o r 0( -amylase.

Lime is used, because it serves a s a source of calcium ion needed by most

O( -amylase as act ivator and s tab i l i ze r . A solut ion of bac te r ia l M-amylase

is then added, and the suspension i s pumped i n t o a steam jet where the

temperature is raised inf&antaneoualy to 80-115~~. The s ta rch is immediately

gela t inized a d i n the presence of the amylase, is depolymerised rapidly to

- ilzld ;ass. I r The sacchar i f icat ion process t h a t follows ensures the conversion of I

I I

s ta rch t o D-glucose i n yie lds as high a s possible using glucoamylase. Once

the l iquefaction s tage has'been completed, the resu l t ing solution, containing

a mixture of maltose-oligosaccharides, is transformed to a high conwersion

syrup by holding it f o r 36dOh in a stored tank a t appr6ximately SSOC and

pH 4.3 w i t h glucoamylase.

The amylt.r.se and amylopectin portions of s tarch are converted by

4-anyldse during l iquefaction to a col lect ion of l i n e a r and branched

dextrins. The l i nea r dextr ins are rapidly and almost . to ta l ly converted

to D-glucose by glucoamylase. The branched dextr ins are much less

susceptible t o hydrolysis. Abdul lahe t &.,(1963) observed t h a t t h i s was

due t o the lower r a t e a t which glucoamylase cleaves the o ( - ( l j 6 ) D - glucosidic linkage, as campared to cleavage of theO(-~-(l+41 linkdge.

Tkirpk &.,(1976), using a s ing le enzyme system, produced glucose

syrup md dextrosd fran maize g r i t s . H e found ou t t h a t glucose syrup

production in. a s ing le enzyme e y s w with bac te r ia l 4-amylase a t pH 6.0

and 85 '~ eliminate many disadvantages of tkie d o a l e enzyme system eg.

microbial infection, pH adjustment during the reaction, high enzyme costs ,

proteolysio. The syrup produced has a DE value of 38%. 0.3% ash and 0.03%

nitrogen.

Yoshizawaet &.,(1980) in their s tud ies found t h a t s tarch heated a t

1 2 0 ~ ~ f o r 20 minutes was e a s i l y digested by o(-mylase a t p~ 6.0, while

raw s t a r ch was only p a r t i a l l y digested, Also they discovered that

d i & s t i b i l i t y of l iquef ied corn s ta rch was higher than t h a t of rice.

H i l r s t s &.,(1971) produced s ta rch conversion syrups having a minimum

fermentable ex t rac t s (F.E) value of 7%, a minimum dextrose e q u i v a l e n t ( ~ ~ 1

value of 47% and a max.dextrose content of 47% by sacchar i f icat ion of a

s tarch hydrolysate with an enzyme composition comprising a dias tase ,

glucoamylase and amylo -1,6- glucosidase.

Mandels et &,,(1975) reviewed the enzymic conversion of waste - cel lulose material t o glucose syrups f o r use in the food industry. They

discussed the production of a canplete ce l lu lase complex from Trichodenna

Viride Qu 9414 and pretreatment of substra tes by b a l l mill ing t o produce - maximum saccharification. Ac.cording to the review, sane prunising substra tes

f o r conversion a re listed: milled bagasse gave 42% sacchar i f icat ion in 4h,

milled m i l k cartons 81% saccharification i n 24h a t 50% pH 4.8.

Figure 2.1 shows a general manufacturing.procedure f o r glucose syrups.

Process Ytee

& /znzylne sacchar i f ica t ionf

I

Carbon r e f i n i n 47

Fig. 1. A general manufacturing

2.6 Refininq:

Modern systems are continuous conver ters

F a t and protinaceous impur i t ies p rec ip i t a t e .

Removes major por t ion of insoluble impuri t ies .

x

~emain ing insoluble impur i t ies removed

s o l i d s increased t o 55%

For acid-enzyme hydrolysed syrups enzyme treatment applied a t this stage. .

Powdered o r ac t iva ted granular carbon used.

ion-exchange treatment is opt ional used when ash free, very colour s t a b l e syrup are desired.

Sol ids increases t o 82%

procedure f o r corn syrups.

I n the r e f in ing processes, high qua l i ty corn syrups and s o l i d s o r

c r y s t a l l i n e dextrose demands the removal of:

( a! coloured compounds (b) meta l l i c ions.

( C) Hydroxymethyl fu r fura l

(dl nitrogen containing canpounds introduced with the o r ig ina l s ta rch

or with t h e enzyme preparations used i n the process

( a ) .Orgwic acids which can impart undesirable flavours o r colours to

the various products and

( f : sol-like pa r t i c l e s of unhydrolysed or degraded starch,

Carbon treatment removes most of t he soluble proteinaceous material&

present and subs tan t ia l ly a l l t h e 5-(hydroxy-methyl),- 2 - furaldehyde *

formed during the acid treatment, Also, many. commercidl-ly activated carbon

are ef fec t ive i n removal of heavy metals such as i ron and copper,. t h a t

can a c t a s ca t a ly s t s f o r developing colour, - Most new ' i n s t a l l a t i on observed

Conlee(l971) use counter current applicatiok of iactlvated granular arba an

in cy l indr ica l column because it can be conveniently re-activated,

yielding more favourable economics,

A typical ion-exchange deionization system cons i s t s of s i x fixed bed

columns (three pa i r s of cat ion and anion axcircuiyrt i.tz3in 03 aervice r'or

regeneration), The cat ion exchange r e s i n s used are strong acid exchangers

(sulfonated resins i n the hydrogen fonn) and the anion exchangers uaually

are weak base res ins (tertiary m i n e i n the free base form), The anion

exchange r e s in s removes acids generated by reaction of the , s a l t s i n the

syrup l iquor with the cation-exchange resins.

Bezhal - c t &, (1981) discussed an experimental equipnent used f o r

e l ec t ro - f i l t r a t i on of glucose syrups which achieves separation of various i

foreign substeinces eg. micro-ozganims,pmteinls and other macromolecular

cornpoundrr, fu r the r colouring mat ters , c o l l o i d s etc. The syrup pass

through a l aye r of granulated mater ia l , under the ac t ion of d i r e c t

elecuical current . The undesirable p a r t i c l e s are coagulated and trapped

on the granules.

~ a l d a s s a r i ( 19 71 ) described a process whereby concentrated solLtLons

of sugar are t r ea ted with resins to remove impur i t ies such a s amino acids,

mineral ac ids and salts, organic bases and ac ids etc. by passage through

e s e r i e s of four ion-exchange r e s i n f i l t e r s . The r e s i n s are r edmera ted

with 10-15% H2S04 o r 44% NaOH. I n c o n t r a s t with o the r processes, th is

only needs 2 4 opera t ives , cuts i n d u s t r i a l costs by 80% and gives an

improved pr@uc t.

Hersiczky (1972) constructed a f i l t r a t i o n u n i t f o r continous operat ion,

maximum capaci ty 10,0001/h and s u i t a b l e f o r all types of r e a c t o r and

hydrolysis. With s i n g l e passage operat ion, t h i s p u r i f i c a t i o n u n i t removed

94% of suspended matter f r a n upper, middle and lower regions of the

hydrolysate and increased output of the f i l t e r s t a t i o n by 50%.

2.7 P roper t i e s and Functional u s e s of Glucose syrups. '1

There are severa l types of corn syrups each of which has i t s own sa t

of propert ies . These p roper t i e s are the sun of the c h a r a c t e r i s t i c s of the

components which make up each syrup. These include: Dextrose equivalent

(DE), carbohydrate canposi t ion, a c i d i t y and pH, sulphur dioxide, fermentable

e x t r a c t , Baume

Humectancy and

and s p e c i f i c g rav i ty , Ash, Prote in , colour, v i scos i ty ,

hygroscopicity 'etc.

~ u d v i g et &.,(1975), studied the fac tors responsible f o r the browning

of glucose syrups during storage. They suggested t h a t i n i t i a l s ta rch

materials must not contain 0,6 - Om% protein, with only small mounter

of o i l and fibres. Also after f i l t r a t i o n , a pre-concentration t o 28-32

t3dume' degrees is necessary, followed by a second f i l t r a t i o n . Further more,

the optimal temperature f o r c rys ta l l i za t ion i s important.

Keaslay ( 1978) studied the ca t a ly t i c hydrogenation of glucose syrups

as a means of controll ing hygroscopicity and suscep t ib i l i ty of browning

a d fermentation reactions without changing properties such a s viscosi ty 9

osmotic pressure or sweetness. According to the resu l t s , hydrogenation

s ign i f ican t ly decreased (P 0.05) moisture uptake of syrups a t 100% RHO

Browning decreased with DE before hydrogenation of syrups, and hydrogenation' .

(eg reduction i n DE from 100-76) of a syrup reduced colour development when . the syrup was heated with amino acids. Furthermore, hydrogenation of

syrups decreased % fermentable sugars.

Hoover(1963) has i l l u s t r a t e d the functional properties

as they r e l a t e t o tha type of conversion. This is shown i n

he arrows of increasing s i z e point t o the di rec t ion of the

of ,Corn Syrups

f igure 2.2.

most desi rable

corn syrup t o use f o r a par t i cu la r property, a l l o ther fac tors being the

same. I n select ing the most su i t ab l e corn syrup, the greater the number

of arrows t h a t go in the desired direct ion, the b e t t e r is t h a t corn syrup

for the intended application. For example, i n choosing a corn syrup f o r f I

use i n i c e cream production, the lower coriversion syrups are preferred.

These syrups.increaae the bodying and cohesive e f fec t s , viscosity, and

prevent excessive growth of ice c rys t a l s during freezing.

29.

~oover (1964) has a l s o prepared a c h & l i s t of p roper t i e s and

functional uses of corn syrups i n a wide va r i e ty of foods. Table 3 shows

these data. This table may b e used advantageously by f i r s t determining

the property o r p roper t i e s of a food which may be improved w i t h the use

o f corn syrup and then s e l e c t i n g the most s u i t a b l e syrup. For example, in

bakery products higher conversion corn syrups are prefer red where browning,

f ermen t a b i l i t y , sweetness, and f lavour enhancement are desired. '

2.8 Malt based Syrups.

2.8.1 Mashing Method.

~ a l t is converted t o wort by b a s i c a l l y two methods of mashing; the

decoction mashing method and the single-temperature mashing system c a l l e d

infusion method(Briggs e t a1 1981). I n infus ion muhing process, no p a r t ' '

of the mash i s boi led and returned to t h e main mash, r a t h e r the whole mash

i s gradually heated f r a n mashing-in t o rnashing-off. When the mashing-in

temperature ( 35-SOOC is progressively r a i s e d to mashing-of f temperature

( 75-80°C through the sacchar i f i ca t ion temperature range of 65-70°c, thet

mashing process is c a l l e d upward infus ion, while the downward infus ion

process resrllts when the f i n a l temperature of t h e marsh(65-70°C) is lower

than the i n i t i a l temperature of t h e mash(75-80°c). ~ n f u s i o n mashing is

s u i t a b l e f o r ttie highly modified m a l t . (r"

I n decoctl-on mashing, ' a por t ion of the mash is boi led and returned

to the rest of the mash in t he mash tun. Tradi t ional decoction mashing

employs malt which is less modified than t h a t used i n infus ion mashing

and i s only l i g h t l y k i lned(8r iggs -- e t a1 1981). There are three d i f f e r e n t

kinds of decoction methods. '

IYPF OF CORN SYRUP' PROPFRIY OR IIItJC1IC)NAI L J S F

(ALPllAnEllCAILY) - LOW.CONV. REG.-CONV. IN1ER.-CONV. IiIOII-CONV.

BODYING AGEN.1

BROWNING REACTION

CONFSIVENESS

FERMENTABII.ITY

FLAVOR ENIIAFICEMENT

FlAVOR TRANSFER MEDIUM

FOAM STARILIZFR

)tVMECTANCY

blYGROSCOrlCITY

NUTRlllVE SOLIDS

OSMOTIC TRFSSURE

PRFVENTION 01: COARSE I C E CRYSIALS DURING FRtEZING

5HEEN PRODUCER

Checklist of propert ies

and funclional uses o f corn syrups

in specific food producls

- - -. - -. . - -- -. - - - - . - - - -. -- -. .- - Baby loods - - - - -- -- -- - -- - -- - . - - Bakery producls .. - ....... - -. -. .............. - .. - - .- - ..... Beverages, brewed - - ..... - . ..... - ......-... - .. - . Bcveragcs, carbonated - lion alcoliulic - .- . - -- -- - - - -- - - - - - -. - Breakfast loods --------.--- Catsup, chili sauce, ton~dlo raucr --- -- - .- ... Cereals, prepared - 0--- ---- Cheese spreads and Irluds - - Chewing gunr -- - ------ Chocolate p~oducls -. -- - ... .......

Cilrus juices, dried -..-, -, .. - ............ - ... ---- . '

~ondcnsed milk

Cunlrctionr - - --- Cordials and liqueu~s . - ..... .............................. Eggs, lruzer~ or dried

.---- Extracts and llavors

-----------. -- Frostings and icings - ------ - ----- - Fwi l bullers

Fruit juicer and fruil jirice drinks

(i) The one mash method'

( i l l The double mash method

(iii) The th ree mash methud.

K a r e l (1967) described and i l l u s t r a t e d the production, on a continuous

baaie a 'complete mash' containing almost a l l the so lub le mal t substances

and moat of t h e enzymes. I n h i s study, f i n e l y ground malt was mashed a t 5

65O~(1 1 5) by s t i r r i n g f o r 30 seconds. The mash was then subjected t o a

pressure shock of 294 p.s.i and immediately converted by passing through a .

pressure r e l i e f valve i n t o a s a c c h a r i f i e r , where the temperature was increased

by IOC per minute from '65O t o 70-71°c. This temperature was maintained

u n t i l the iodine test gave a yellow colour. The t o t a l conversion period

w a s 10-20 minutes,

.Barre t and G r i f f i t h s (1966) s tud ied s h e e f f e c t s of malt k i l n i n g on

wort proper t ies , The r e s u l t s s h o w that as colour increased and moisture

decreased the e x t r a c t value of the malts remained e s s e n t i a l l y cons tant I

while t h e d i a s t a t i c power decreased subs tan t i a l ly . ~ h l s i n tu rn was

pa ra l l e l ed by a reduction of fermentabi l i ty in the derived worts. Measurement

of the individual sugars p resen t i n the worts showed t h a t the decrease i n

fermentabi l i ty was associated with a diminution in the percentage of maltose

and an increase 10 the dex t r in content , while the values f o r o the r sugars

were r e l a t i v e l y unaffected.

Desrousseaux and Montreuil(l966) s t u d i e s on commercial mashing showed

tha t , /+amylase ac t ion occured optimally a t about 6 3 O ~ , it slowed down as

the temperature reached 70°c, and w a s i n h i b i t e d a t higher temperatures. The -

optimum f o r 1 hit dextr inase a c t i v i t y w a s 50-63O~, higher t e m p e r a w e s

destroying the enzyme. Alpha-amylase action began above 63Oc, a t ta ined a

maximum around 72Oc and diminished a t higher temperature.

Narzisa and L i tzenburger (1977) , inves'tigated the mashing conditions

and gum contents and thus concluded t h a t it was possible to regulate gum

contents t o some extent by var ie t lon of mashing method but the s t a t e of

modlficetion of the malt plays a much more decisive pa r t than mashing

conditions. Also they added t h a t the a l te ra t ion of the pH of mashing t o

5.5 effected advantageous degradation of ti-glucan only a t high mashing

temperatures.

2.8.2 Preparation of m a l t syrups.

The three mash mmthod involves usshing a i n a t about 35-40°c. After I I

sometime about one th i rd of the mash, the f i r s t mash, is taken l n t o a k e t t l e I and boiled and brought back to the mash tun where the temperature of t he

1 i i

whole mash is ra ised t o 5 0 - 5 5 ~ ~ , t h i s i s kept f o r a period of t h e , about I I

15-30 minutes. Boiling destroys the enzyme i n the boiled portion as i

w e l l as ge la t in izes the starch. Diasta t ic action is thus f a c i l i t a t e d by 1

t h i s process. Then a second mash (again one th i rd portion of the e n t i r e

mash) is boiled and returned to the main mash. T h i s brings the temperature j

t o 6 0 - 6 5 ~ ~ , t he saccharification temperature. It is allowed a t t h i s

temperature fo r 30-60 minutes. Lastly a th i rd is t reated i n the same manner,

and i t r a i s e s tho temperature of the whole mash t o 70-75O~, the mashing-off

3 temperature. This is a l so kept f o r 30-60 minutes.

The resul t ing wort is c l a r i f i e d , theapH adjusted t o 4-5 and appropriate

amount of glycoamylases added. The saccharification is allowed to c o n t h u e

f o r several hours (12-72h) depending on the extent of conversion desired

r~ld th i s i s determined by i t s dextrose equivalent value(DE).

he melt syrup which contains a mixture of saccharides is neutralised

and concentrated by evaporation in multi-effect evaporaba or for

laboratory works, on a boil ing water-bath to about 80% slolids so aa t o I

inhibit microbial spoilage.

CHAPTER 3

MATERIAIS AND METHODS

3.1 MATERIALS

3.1,1 Mi l l e t g r d n s (Pennisetun~ ~ y p h o i d e s ) and sorghum grains(s0rghwn

h lcol o r ) were purchased f ran Orba market , NsulEka.

3.1.2 ~myloglucosidase(AMG), from ~ s p e r g i l l u s n ige r was purchased from

Nove ~ n d u s t t i a l Enzymes Division, Novo Alle DK - 2880 Bagsvaerd, Denmark. I I I

3.2.3 Other Chemicals and reagents were of the pures t ana ly t i ca l grades.

3.2 METHODS - 3 9 2 Determination of malt ing ~ h ~ w a c t e r i s t i c s of t h e c e r e a l qra lns ;

I

Sorqhum and m i l l e b ,

-j 2 .I Determination of moisture content:

The moisture contents of the g r a i n s and malts w e r e determined I i I

&cording to the I n s t i t u t e of Brewery, I,O.B.(1977) method of analys is , i !

dS ~ O ~ ~ O W S :

About 209 sample of g r a i n s were f i n e l y ground in a Thanas Wiley M i l l

Model ED-5, and thoroughly mixed. 5g of the ground sample was placed

in a moisture d i s h which was closed and weighed immediately to 0.001g. i The cover of t h e d i s h w a s removed and placed in a pre-heated oven f o r 1 .

I

exact ly 3h a t I O S ~ C , The l i d was replaced and removed 'from the oven,

then allowed t o cool i n a d e s i c a t o r f o r a t l e a s t 20 minutes t o ,room

temperature. The d i s h was then re-weighed t o 0.001g.

Calculation: %

The moisture percentage(M) of the sample

Wkre W1 - weight of sample before drying

w2 - weight of sample a f t e r drying

3.2r2'. ~ e t e r m i n a t i o n of Percentage Foreign Seeds and broken kernels .

The percentage of fore ign seeds and broken ke rne l s of the ce rea l

g ra ins were determined according t o t h e method of Association of O f f i c i a l

Analytical Chemists, A.OoA.Cm(1980) a s follows:

509 of each of t h e g ra ins was weighed and the fo re ign seeds and broken

kernels were counted out. he g r a i n s were reweighed and the d i f fe rence i n

weight recorded a s a percentage of the o r i g i n a l weight.

3 . 2 . 3 ~ e t e r m i n a t i o n of thousand Corn weight:

A thousand corn weight of t h e g ra in samples were determined according to

the method of l.O.B(l977) a s follows:

20g samples were weighed o u t a f t e r removal of fore ign matter and half

corns. The number of corns i n each sample counted and moisture content

determined. 4;

Calculation:

-The weight of 1000 corns of d r y corns i n gram(g)

Where W - t o t a l weight of c e r e a l g r a i n s taken

DM a D r y matter percentage of t h e g ra ins

N = Tota l number of corns counted.

3.204 a~ The objec t ive of t h i s test was to measure the. p

I

iercen tage of g ra ins .

which cm be expected to germinate f u l l y i f the sample is malted normally s

d t the time of t h i s test. The I,O,Ei.(1977) method of analys is was adopted

as follows:

100 corns f r a n the samples w e r e placed i n a p e t r i d i s h l i n e d with two

f i l t e r pcrpers i n t h e bottom to which 4 m l of water had been added, The

p e t r i d i s h was covered and the g r a i n s allowed t o g e m i n a t e i n a cupboard,

The c h i t t e d corns w e r e removed a t 24, 48, and 72h from the beginning of

steeping,

Percentage of corns chii2ted a s the g e q l n a t i v e energy were ca lcu la ted

thus:

Germinative energy = GE(%)

3.2 , 5 Determination of germinative Capacf t .

The ob jec t ive of tNs test w a s t o measure the percentage of l i v i n g

corns i n the sample. The germinative cap'acity of the g r a i n s was determined

using the hydrogen peroxide method a s described by H o u g h s , &,,(1981)

a s follows:

200 corns were steeped i n 2OOml of 0.75% hydrogen p e r o x i d e ( ~ ~ 0 ~ f o r

48h a t room temperature, The s t e e p l i q u o r w a s replaced with f r e s h hydrogen

peroxide ' so lu t ion and was l e f t f o r f u r t h e r 24h. Gernlinat im capaci ty was

then ca lcu la ted as f o l l w s :

Where n I number of corns t h a t d i d no t g e h i n a t e ,

3 . 3 ~kterminsCiotr of Optinrun Malting Conditions of the Cereal Grains:

? ? .I . . . A i~iois t u r e Content as a function of s teep time:

Eight pe t r id i shes l ined w i t h filter papers a t t h e i r boktoms were ,

provided and f i l l e d with equal volumes of tap water. 20g of the g ra ins were

cledned m d steeped u t roOm temperature i n each of the pe t r id i shes f o r

v x i o n s t i m e s , 10-80h, with e i g h t hourly change of s t eep l iquor . .

A t the end of edch s t eep period, the g ra ins were drained, surface

wdter b lo t t ed with f i l t e r paper, then t h e moisture content determined, as

i n sec t ion 3 - 2 ' 1

3 . 3 . 2 ~ e t e r m i n a t i o n of Opbhum Steep t i m e :

ha gra ins (209) were steeped a t various times, 10-80h as described

d o v e . Each of the e i g h t sets w a s allowed t o gemind ie f o r 4 days i n a

deck cupboard and then k i lned f o r 48h a t 55Oc, after which the mal t ' s

d i a s t a t i c power was determined as i n sec t ion 3.2 -4

3 . 3 . 3 Determination of opthum germination period:

The g ra ins (20g) were steeped f o r 50h and germinated f o r various

periods, 1-7 days i n a dark cupboard, l a t e r ki lned f o r 48h a t 5 5 ' ~ and

the ma1 tm s d i a s t a t i c power determined.

3.3 .4 Effec t s of k i ln ing a t 4 5 ' ~ and varying periods of time on moisture - content of the m a l t : %

Samples of malted g r a i n s a t optimum malting conditions(50h steeping,

and 5 days germination) were Kilned a t various periods(l2h, 24h, 36h, 48h,

60h) a t 45'~. and moisture content determined.

3.3.5 Determination of Maltinq losses a s a function of germination periodr

The g ra ins (209) were steeped f o r 50h and germinated f o r yarious

periods, 1-7 days, then tho r e s u l t i n g malting losses per n t h day o f t -

germination determined as i n sec t ion 3 -4 -4 .

Production of swghwn, Millet Malts:

I k g of each of t h e g ra ins was cleaned and steeped i n ordinary t a p

water f o r 50 hours a t room temperature with 8 hourly change of s t eep

l iquor t o both minimize t h e growth of microbes and provision of more

oxygen t o t h e embryo of the grains. A t t he end of t h i s s teeping period,

t h e g r a i n s were drained and spread on a cleaned f l o o r of a dark cupboard.

Wdter W ~ S sprinkled on the corns when they were v i s i b l y d r i ed , t o ensure

adequate moisture supply throughout the 5 day genningtion period.

Germin.ation was terminated by k i ln ing a t 4 5 O ~ f o r 48h in a hot-air

oven. A t t he exp i ra t ion of t h i s time, the malt became f r i a b l e and the

. , k i ln ing was stopped. .. .

30 4 - Evaluation o f Ma1 t@ s q u a l i t y cha rac te r i s t i c s :

~ e t e r m i n a t i o n of Cqld Water Extract(CwE) 3.4-1

cold water e x t r a c t s of t h e m a l t s were determined according t o t h e

I.O.B.(1977) methods of analys is as follows:

10y ground mal t was d iges ted with 200ml of d i s t i l l e d water containing

121111 of 0 . l ~ ammonia f o r 3h a t 20°c, s t i r r i n g d t ha l f Hourly in te rva l s .

The r e s u l t i n g so lu t ion was f i l t e r e d and the s p e c i f i c g r a v i t y o f . t h e

0 f i l t r a t e measured a t 20 CO

water e x t r a c t (CWE) %

x 20

Cdcu la t ion ;

The Cold

where G r tha excess degrees of g rav i ty of the f i l t r a t e taking water a t

?oOc ds 1000.

i e G = 1000(SG - 1). 3 . 4 . 2 Determindtion of d i a s t a t i c Power (Using F e h l h q ' s T i t r a t i o n ) .

Dias ta t i c Power determination w a s c a r r i e d in accordance with t h e

~ n s t i t u t e of Brewery, I.O.B. (1977) methods of analys is a s follows:

iul u u f i l t e r e d cold water extract of a m a l t in fus ion was prepared and

allowed t o ' s e t t l e . 3ml a l i q u o t o r s u i t a b l e volume of t h e supernatant

l i q u i d was p ipe t t ed into 3 100ml of 2% buffered s t a r c h so lu t ion attemperated

d t 20°c, and contained in 2 0 h l f lask. The f l a s k was shaken and maintained

d t t h i s temperature f o r e m c t l y 1 hour from when the al.iquot was added.

30ml of 0 . l N NaOH so lu t ion was added .to s top the r eac t ion , and made

up t o 2001111 a t 20°c wi th d i s t i l l e d w a t e r . 5ml of mixed Fehlings so lu t ion

was p ipet ted I n t o a 1501111 narrow-necked bo i l ing f lask . The ,digested

s t a r c h so lu t ion was added from a b u r e t t e t o t h e cold Fehlings t o within

l r n l of the f i n a l end point. T@ f l a s k contents was mixed and boi led with

moderate e b u l l i t i o n f o r 2 minutes. The bo i l ing was continued and wi th in

1 minute, 3 drops ,of methylene b lue ind ica to r was added and the t i t r a t i o n

completed.

The end po in t was indica ted by decolor iza t ion ' of tho hethylene b lue

and the r eac t ion l iqu id j u s t becoming' red.

C d c u l atlon:

Dias t a t i c power( DP) expressed i n degree ~ in tne r (O~)

Where X = no of m l of malt e x t r a c t

y I no of m l of converted s t a r c h to reduce 5ml o f Fehlingee.

S = t i t r e f o r s t a r c h blank.

Determination of t i t re f o r s t a r c h blank:

The undiluted 2% s t a r c h so lu t ion was t i t r a t e d aga ins t a mixture of

l m l of mixed Fehlinges so lu t ion and 2ml of F e N i n g e s so lu t ion B, using

t h e technique described under method, with methylene b lue indica tor .

(The blank may be neglected i f i t i s less than 3% of t h e measured

d i a s t a t i c value of the m a l t ) .

3-4.3 ~ e t e r m i n a t i o n of Hot Water Extrac t (Hw~)r

The hot water Extract of t h e mal t w a s determined by the procedure

described i n tho method of I n s t i t u t e o r Brewery I.O.B(l977) as follows:

50g of ground m a l t was mixed with 360ml of d i s t i l l e d water previously

0 heated t o about 68 C s o as t o ensure an i n i t i a l mash mix temperature of

6 5 O ~ with c o n t i n o u s ' s t i r r i n g f o r 10 minutes. The mixture was l e f t a t

6s0c ' f o r 1 hour. The mixture was then quickly cooled t o 20°c(with ice

chips) and t h e volume m d e up t o 515ml with d i s t i l l e d water, The mixture

was f i l t e r e d and t h e s p e c i f i c g r a v i t y of 'the f i l t r a t e was determined a t

20°c with s p e c i f i c g rav i ty k o t t l e within one hour of c o l l e c t i n g t h e sample.

Calculation of Hot Wabr Extract.

~ h t i ~ x t r a c t ( E ) as-is' expressed as l i t r e degrees/kg I G x 10.13.

0 Where (i - excess degrees of gravi ty of the f i l t r a t e taking water a t 20 C

3 .4 -4 ~e te rmina t ion of Malting Loss(%):

The percentage malting l o s s of the malted samples was determined

according to the method described by ~ove l l i e (1962) q$ follows:

A thousand kernel weight of the or ig ina l (unmal.kd) grain was

determined on a dry weight bas i s before malting. After malting,. the

thousand kernel weight of the r 1 1 ~ 1 i ; e c i sanple was a lso determined after

removal of the roots and shoots by hand-threshing and moisture by heating

( d r y weight basis) .

Calculations:

Malting l o s s (%) = 100(Co - Cn) co

where, Co - 1000-kernel weight of the m a l t e d grain.

cn P 1000 - kernel w t , of the m a l t on the nth day of germination.

. .5 s tudies on Malt's amylase:

3.5 ~ x t r a c t i o n of malt amylase: ,

Malt anyrase w a s extracted according t o the mthod of Shanbe 'et. - 2..

(1988) a s follows;

The malted grains (5.0g) (under optimum conditions) were ground

separately i n a morter, and quant i ta t ively transfered i n t o 100ml standard

volumetric f l ask by washhg with d i s t i l l e d water, then made up t o 100ml.

~t was incubated a t 3 7 ' ~ f o r 3h i n 250ml conica l f l a s k and 2.01111 samples

withdrdwn, centr ifuged (8000g, 0.5h) and the supernatant s tored i n a

r e f r i g e r &or.

3.5.2 Preparat ion of 1;Yo buffered s t a r c h substrate:

59 s t a r c h (dry b a s i s ) were made i n t o a pas te with a l i t t l e cold

w a t e r and then poured i n t o 400ml of bo i l ing water, The mixture w a s boiled

f o r 2 minutes and then cooled. Adequate quantity of each of t h e buffer

so lu t ions prepared ealier $see appendix) w a s added and each of the mixtures

was made up t o 500m1 r e s u l t i n g t o 1% buffered s t a r c h s u b s t r a t e so lu t ion of

pH 4, 5 , 6 , 7 and 8 respectively.

3.5 - 3 erepclration of m a 1 tom cal . ibrat ion curve:

A series of maltose so lu t ions were prepared, so t h a t 2ml conta in

0.4 - 2,Omg anhydrous maltose as follows:

I n t o each of the t e n test tubes were added 0.4 - 2.0ml of stock

standard maltose so lu t ion containing 2mg/2ml respect ive ly . The so lu t ions

were respec t ive ly made up to 2ml each by addi t ion of appropriate amount of

d i s t i l l e d water. 1 m l of t h e 1% s t a r c h so lu t ion and 2ml of DNS reagent

were added.

The test- tubes were t r ans fe r red t o a rack i n a bo i l ing water ba th

and heated f o r fi;e minutes and then cooled t o room temperature a f t e r

which t h s content of each tube w a s d i l u t e d t3 2 0 m l with d i s t i l l e d water.

., s u i t a b l e amount of each sample was poured i n t o a 'color imeter cuve.tte f o r

o p t i c a l dens i ty determindtion a t 505 n'!n a g h a s t a reference blank which

.-contain only 2 m l water, Iml s t a r c h and 2ml DNS reagent.

'i'

3 5 .A ~ e t e r m i n a t i o n of optimum p~ f o r amylase a c t i v i t s

2ml of the d i l u t e d amylase e x t r a c t (2ml e x t r a c t i n 2001111 d i s t i l l e d

water) was added to test tubes i n a rack-containing lml each of the 1%

buffered s t a r c h s u b s t r a t e s o l u t i o n a t the pH 4, 5 , 6 , 7, 8 previously

prepared. The tubes were shaken f o r 5 minutes to mix it properly and

incubated i n a thermosta t ica l ly con t ro l l ed water ba th a t 37Oc f o r 10 minutes.

The d i a s t d t i c r eac t ion was stopped by the addi t ion of 2ml D N S colour .

reagent. ~ l l the tubes were heated i n a boi l ing water ba th f o r 5 minutes

and then cooled t o room temperature a f t e r which the .con ten t s of t h e tubes

were d i l u t e d t o 20ml with d i s t i l l e d water, The absorbance was read a t i

505 run agains t a reference blank. The blank was prepared by bo i l ing the

arnylase e x t r a c t f o r 5 minutes before adding to t h e 1% buffered s t a r c h

s u b s t r a t e so lu t ions , The concentrat ion of t h e reducing sugars a s maltose

i n the s t a r c h h ~ d r o l y s a t e was ca lcu la ted by ext rapola t ing i t s absorbance

value from the maltose c a l i b r a t i o n curve.

3.5.5 la terminat ion of optimum temperature f o r amylase a c t i v i t g

2ml of t h e d i l u t e amylase e x t r a c t was added t o 1 m l of 1% s t a r c h

( s u b s t r a t e ) so lu t ion buffered a t optimum p~ range 6-7, and incubated f o r

30 minutes a t various temperatures, 30, 40, 50, 60, 70 and 80Oc.

;!ml of (D*) colour reagent was added(and o the r procedures repeated)

and the concentrat ion of reducing sugars a s maltose calculated.

3.5.6 s t a r c h ex t rac t ion from t h e c e r e a l mains:

The w e t mi l l ing method of Watson(l97Q) was adopted i n t h i s work. The

I process involves cleaning, s teeping, coarse mi l l ing , deyerrning f i n e

~ r d l l i n y , separa t ion of f i b r e , s t a r c h separa t ion f r a n g lu ten , and drying of

3tdrch as shown in the flow char t (Fig . 3.1).

Two kilograms of cleaned g r a i n was placed i n a troqgh, and covered ' t with 3 litres of 0.45% sodium metabisulphite solut ion. The trough was kept

i n a water ba th maintained a t 4 5 O ~ f o r 40h. A t the end of t h e 40h, t h e

g ra ins were drained and washed wi th c l ean water. The g ra in was coarse ly

ground with a manually operated corona handmill, cons i s t ing of two s tud

p l a t e s of wtdch one p l a t e r o t a t e s while the o the r i s s t a t i c . The adjacent

f ~ e ; iif tl-ii plates are studded 'with t ee th , which are s o arranged t h a t the

g ra ins pass i n between the s t a t i o n a r y and moving plates. By adjus t ing the

p l a t e s , it was poss ib le to vary t h e d is tance between t h e t e e t h so a s to :

obta in a des i red p a r t i c l e size. The purpose of this mil l ing s t e p is t o

t e a r the ke rne l s a p a r t i n order t o l i b e r a t e the gems. .Some:endosperm

s td rch was however l i b e r a t e d during degenning. \

The s l u r r y of coarsely ground g r i s t w a s degenned by f l o a t a t i o n i n a

b ig bas in of water. The f l o a w d g e m was scooped with a guaze while the

rest of t h e ma te r i a l s s e t t l e d . The opera t ion was repeated many times by

swir l ing t h e c o n t e n t of the bas in u n t i l a l l the geqn w a s v i r t u a l l y removed.

The d e g e m d g r i s t was next screened on nylon bo l t ing c l o t h of 100 mesh to

remove the bulk of the f r e e , s t a r c h and gluten. The screen t a i l i n g f r a c t i o n

composed l a rge ly of p ieces of honey endosperm and f ib re .

The screen t a i l i n g f r a c t i o n was f i n e l y ground to achieve maximuril

s t a r c h l ibe ra t ion . The f i b m w a s separated from the s t a r c h and glu ten on

, \

screenin I--$

~ i g . 3. Flow chas t of w e t mi l l ing opera t ions i n s t a r c h production from ce rea l grains. . &/"

t h e nylon bol t ing c lo th . .The starch and-gluten f r a c t i o n s obtained from

both coarse mi l l ing and f i n e mi l l ing were mixed. The separa t ion of s t a r c h

. - from gluten was achieved by sedimentation. The s t a r c h with a dens i ty of . -.

1.5 s e t t l e d i n the aquaous s l u r r y a t a f a s t e r r a t e than t h e g lu ten p a r t i c l e s

\

of dens i ty 1.1 (Kent, 1975)., Tim s e t t l e d gluten and s t a r c h were made to

flow down a slop1)ed tray. The g lu ten flowed out f i r s t . The . s t a r c h

f rdc t lon wds fu r the r washed with water and the remaining glugen separated.

h f 'a ir ly gluten-free s t a r c h was obtained.

The s t ixch concentrate was placed between f o l d s of f i l t e r paper a d

0 dried i n an oven kept a t 50 C f o r 48h.

G

3*7 - ~ ~ r o a i m a t e / ~ h e m i c a l ana lys i s of gra ins , ma1 ts and s t a rches from Sorghum and ~ i l l e t :

3.7,1 Crude p ro te in determination:

Crude prote in of the samples was determined i n accordance with t h e

procedure of Association of O f f i c i a l Analyt ical Chemists A.O.A.C.(1980)

as follows:

bout 0.29 of t h e s a p l e w a s weighed o u t accura te ly i n t o a 50ml

k je ldah l f l a sk . The following were then added i n t o t h e f lask . 59

&hydrous sodium sulphate, 1g hydrated cupr ic su lphate and l O m l concentrated

sulphur ic acid. The d iges t ion w a s c a r r i e d o u t by heat ing the f l a s k on an

e l e c t r i c c o i l u n t i l i ts contents becane clear. Heating however was

continued f o r a t l e a s t one hour after the so lu t ion had cleared. ~ f t e r

kwating,the con ten t s was t ransfer red with severa l washing i n t o 25Oml

volun~et r ic f l a s k , and was made up t o the mdrk a f t e r cooling.

D i s t i l l a t i o n apparatuswas set up, and steam was passed through it

f o r 10 minutes. 5rnl of bor i c acid i n d i c a t o r was placed i n 25Qml conica l

f ldsk . The conidal f l a sk was placed under the condenser such t h a t t h e

condenser t i p was placed i n the d i s t i l l a t i o n apparatus and was r insed

'i" down with d i s t i l l e d water.' The cup was closed with t h e rod, and 5ml of

60% N&H was pu t in; t h i s was le t In c a r e f u l l y , leaving 'behind a l i t t l e

to prevent ammonia escaping.Steam w a s then let through f o r about 5 minutes . -

( u n t i l t he amount of l i q u i d in the conica l f l a sk was about twice what it w a s i n

i n the beginning of d i s t i l l a t i o n ) . Then the bor ic acid ind ica to r was t i t r a t e d

wi th 0.0W H c l t o the end point. The t i t re was the number of m l s of 0,OlM

[icl trhdt changes the ind ica to r from green t o pinkish colour.

Calcul &ions: -- -- -

L e t w represent weight of sample

I ,u t T represent ~ n l s of titre 0.0l.M ~ c l

1 l i t r e o f M H C 1 a 14.01gN

1 l i t r e of 0.0lM H c l = 0.4401 g~

Tml of 0.01M H c ~ = 0.0001401 x T g N

250 ---1 of d i g e s t = 0.0001401 x T x 250gN

5 - 5

% crude p ro te in = 0.0001401 x T x 250 x 6.25 x 100 ,w x 5

3,7,2 F a t Determination:

F a t w a s determined according t o the A.O.A.C.(1980) method of a r d y s i s

as follows:

0 A n ex t rac t ion f l a s k was cleaned, d r i ed i n an oven a t 100 C and its

weight determined. 29 of the sample was accura te ly weighed and t r ans fe r red I

i n t o t h e e x t r a c t o r thimble. The thimble with the sample was then pu t into

t h e Soxhlet ex t r ac to r . About three q u a r t e r s of, t h e con ta ine r w a s f i l l e d

with petroleum e the r .

allowed to run f o r about 3h.

~t t h e end of the e x t r a c t i o n , t h e thimble w a s removed and t h e e t h e r

The

to it and

condenser

f l a s k was placed on t h e hea t e r , t h e Soxhle t e x t r a c t o r connected

condenser i n t u r n connected to t h e Soxhlet. The t a p to t h e

was turned on and t h e hea t e r switched on. The e x t r a c t i o n was

e x t r a c t e d o i l weighed.

~ a l c u l a t i o m -

% f a t L W t of o i l x W t . o f sample

recovered. F i n d l y , t h e o i l w i l s dried a t 1 0 0 ~ ~ i n an oven and t h e

3.7 , 3 Crude F i b r e determination:

The A m e r i c a n Assoc ia t ion o f C e r e a l c h e n i s t s , A.~.C.c.,(1976) method

o f a n a l y s i s wds employed i n the determina t ion of c rude f i b r e i n t h e samples

a s follows: ti'

The r e s i d u e s from e'ther e x t r a c t de te rmina t ion or de- fa t ted samples

were t r a n s f e r r e d t o a d i g e s t i o n f l a s k . 200ml of b o i l i n g 1.5% su lphur i c

ac id was added. The d i g e s t i o n f l a s k was t hen connected to a condenser and

heated, The f l a s k was f r equen t ly r o t a t e d u n t i l t h e sanple w a s thoroughly

w e t . The f l a s k was removed after 30 minutes, f i l t e r e d through l i n e n i n

a funnel and washed wi th b o i l i n g water u n t i l the f i l t ra te was no longer

ac id ic . The i n s o l u b l e matter was washed back i n t o the d i g e s t i o n f l a s k

conta in ing b o i l i n g 15% NaOH so lu t ion . The f l a s k was connected to a

r e f l u x condenser and b o i l e d f o r 30 minutes, a f t e r which t h e mixture was

SO.

dlowed t o s t m d f o r b o n e minute. f he contents were then f i l t e r e d through

a cheese c l o t h i n a funnel and residue thoroughly washed with bo i l ing water II

and then with 1% hydrochloric acid, and again with bo i l ing water u n t i l

no lonyer acidic. Then i t w a s wcrshed twice with 95% ethanol , three times

with d i e t h y l e the r , and f i n a l l y t r an fe r red to a crucible. The c ruc ib le

and contents w e r e d r i ed to a constant weight a t 1 0 0 ~ ~ . The content was

then heated over a flame a t red hea t f o r 20 minutes. The c r u c i b l e was

cooled i n d des lcdtor m d weighed. The percentage of crude f i b r e was

ca lcula ted ds follows:

Crude f i b r e (%) 5 l o s s i n w t . x 100 - I w t , o f sanple 1

3.7.4 ~et t : rminat ion of A s t r - --- --

The A.O.A.C.(1980) method of ana lys i s was used f o r ash content

determinations.

About 5g of the stunple was weighed and heated i n a 50ml p e t r i d i s h

a t 1 0 0 ~ ~ u n t i l water was expelled. Few drops of pure o l i v e o i l w a s added

and the mixture heated over flame u n t i l swelling stopped. The d i s h was

then placed on a furnace a t 525Oc and l e f t the re un t i l white ash was

obtained. ,The ash was moistened.with water, d r i ed on a steam ba th and

then on a ho t p l a t e , t h e r e a f t e r re-ashed a t 525 '~ t o cons tant weight.

The ash content w a s ca lcu la ted a s a percentage of t h e o r i g i n a l

weight of the sample as follows: . .

Ash I w t . of ash x 100 w t . of sarnple

3-7.5 Tota l Carbohydrate determination:

0,2g of ground sample w a s mixed wi th 50ml of d i s t i l l e d water i n a

boil ing beaker. 3ml of concentrated .. . I l c l . w d s added and the mixture boi led

u n t i l complete hydrolysis. The content of t h e f l a s k was cooled &::d i

neut rd l i sed with 5N NaO1.I solut ion. The hydrolysate was then t r ans fe r red

i n t o a 100rnl volumetric f l a s k and the volume rnade up t o 100ml wi th

d i s t i l l e d water. 0.2ml of this s o l u t i o n w a s p ipe t t ed and mdde up t o 2ml

(10 fo ld d i l u t i o n ) with water f o r the dctemir?at,ion.

G ~ U C O S ~ was determined using t h e anthrone reagent (Deriaz, 1961). A

stock glucose so lu t ion of O.8mg/ml was prepared. Glucose standards were

prepared by ddding O m l , 5m1, 10m1, 15m1, 20ml, and 25ml of the stock

so lu t ion i n t o s i x d i f f e r e n t l O O m l volumetric f lasks . The volumes were

made ui) t o the mark with d i s t i l l e d wd te r .

l m l of each of the standard so lu t ions and test simples were

respect ive ly p ipe t t ed i n t o test tubes. To each of t h e test tubes, 5ml

of dnthrone r e g e n t wcjs added and properly mi,xed. These were covered ond

inunedidtely p u t i n a bo i l ing water ba th f o r 20 minutes f o r t h e colour to

develop. They were cooled and t h e i r absorbance measured a t 620nm. From

t he standard glucose c a l i b r a t i o n curve plot ted , the concentrat ion of

glucose i n the test sample was read off.

Calculdtion:

% total. carcohydrate P mg of glucose equivalent t o sample absorbance

from graph x conversion fackor(25) x d i l u t i o n fac tor(10) .

3.7.6 Gele t in iza t ion temperature determination:

Ttm g e l e t i n i z a t i o n temperature determination was c a r r i e d o u t in

accordance with t h e rnethod of Novellie ilnd ~ c & t e :(1961).

29 of ground sample w a s pu t i n 40ml of water i n a l a r g e test- tube, and

quickly brought t o the desired tunpera ture with good s t i r r i n g f o r a t l e a s t

5 minutes heating. ~ f t e r g e l e t i n i z a t i o n , * e mixture was cooled t o 30°c

md placed i n a cons tant temperature water bath. Malt e x t r a c t (40rnl) was

added and a f t e r a thorough i n i t i a l mixing, the mixture was s t i r r e d hal f

hourly f o r 3h. A s m p l e 20ml was withdrawn and added t o 30ml of 0,SN '

NaOH so lu t ion i n a 250ml ' v o l u m e t r i ~ f lask . The mixture was made t o volune

asld t he s u g a content detennincd by Benedict 's quan t i t a t ive method(Plummer,

1977). For the blank, equal weight of sample was p u t i n t o 40ml of

w d t e r and e x t r a c t (40ml) added to the ungelat inized s t a r c h a t 30°c f o r 3h,

The reducing sugar w d s s i m i l a r l y determined a f t e r stopping the enzyme act ion

with 30ml of 0.5N NaOH solut ion.

25ml of ~ e n e d i c t l s q u a n t i t a t i v e reagent was measured i n t o a l O O m l

conical f lask . To t h i s was added 3g of anhydrous Na--CU3 and a few pieces

of anti-bumping granules. The mixture was heated over a bunsen flame and

when i t boi led vigorousl.y, the sugar so lu t ion was run i n slowly from a

tiurt:tte. When ~i bulky white p r e c i p i t d t e w d s formed, the s u g a so lu t ion was run

in more slowly u n t i l t h e l a s t t r a c e s of blue had disappeared. The t i k e

which was the volume of sugar requi red was noted.

The concentrdt ion of the sugar was cdlcula ted from the following

fdctors ; 25ml of Benedictcs reagent i s equivalent t o 50mg of glucose, 53mg 8

of f ruc tose , 68.8mg of l ac tose , 74 mg of maltose o r 49mg of hydrolysed

sucrose.

mg s u q a per 100ml

where f P. sugar fdc to r

3 7.7 s t a r c h deterinination i n s t a r c h concentrate by hydrolyt ic method:

A typica l method employing acid hydrolysis f o r the determination of

sterrch i n f-lours is t h a t of Radley(1976) employed i n t h i s s tudy as follows:

2-59 of d ry s t a r c h concentra te w a s mixed with 50ml of cold water and

dllowed t o stand f o r lh. 2011-11 of H c l and 150ml of water were added t o the

suspension. his w a s p u t i n t o a round bottomed f l a s k and r e f l u e d f o r 2h.

The content of the f l a sk was cooled and neut ra l i sed with 5N NaOH. The volume j I

w a s l a t e r mdde up to 2SOml. lml was p ipe t t ed into a 100ml volumetric f l a s k 1

m d the volume made up to the mark with d i s t i l l e d wat . Glucose was t;Jr determined using t h e anthrone reagent(Deria2, 1961). A series of glucose

so lu t ions were prepiired, s o t h a t l m l c o n t d n s 0.04 - 0.2mg, and. these

were used t o c a l i b r a t e the glucose standard curve. I .. ,

I m l

pipe t t r d

-.7,7nC.."l ' - --r -* -y

ba th f o r

of each of the standard so lu t ion and the test sample was respect ive ly

i n t o test- t*&es. To each, 5ml o f anthrone reagent was added and I !

mixed. These were covered and immediately p u t i n a bo i l ing water 1

20 minutes f o r the colour t o develop. They were cooled and t h e i r

absorbance measured a t 620m aga ins t a blank which contained only I m l o f ,

water and 5ml of anthrone reagent. The concentcation of t h e test sample . . .

wcls obtained f r a n t h e & s o r b a x e by e x t r i r p o i a i i a ~ . The mass of glucose d

wclv o b t d n e d by c d c u l d t i o n s involving the concentrat ions and d i l u t i o n s

mdde. The mclss of s t a r c h was con:eq!ently obtained from t h e mass of

j lucosc using t h e r e l a t i o q

~ , i s s of glucose x 0.9 = Mass of s tarch .

production of Malt based Syrups:

3 , ~ . 1 *wort preparat ion by th ree s t age decoction Mashing Method from Sorghum malt.

The method, cu r ren t ly adopted f o r masl~ing bar ley m a l t a s described

by ~ o u g h e t , &.,(1981) was used i n t h i s work as follows: - Sixty grams of Sorghum malt mil led t o 1-2mm p a r t i c l e s i z e ( i n a Thomas

illy ill Model'ED 5 ) was mixed with 32Gml of t ap water, t o give 18.4%

mash. This !as held a t 40°c f o r 30 minutes. One t h i r d por t ion o f mash

was withdrawn, boi led f o r 5 minutes, and qeturned t o the main mash. The

temperature of the mash rose to SOOC and was maintained a t t h i s temperature

f o r 15 minutes a t a pH 6.5. (This was adjusted wi th 2~ Ca(OHI2 ~ 0 1 ~ t i 0 n ) ~

One t h i r d por t ion of the mash was again removed, boi led f o r 5 minutes and

returned t o the main mash. The temperature of the mash was ra i sed t o 60°c,

and t h i s was maintained a t a temperature range of 60-65O~ f o r 30 minutes. .

A f u r t h e r one t h i r d por t ion of the mash was removed, boi led f o r

5 .minutes and returned to the main mash. The temperature rose, and was

maintained a t 70-75O~ ( the m;shing o f f temperature) f o r 30 minutes.

3 w 8 2 Effec t s of varying mash concentrat ions and S x c h a r i f i c a t i o n periods on reducing sugar contents of worts in a t h r e e s t age decoction mashing.

I n the mashing method discussed above, p ro teo lys i s was dllowed a t

4uDc and 50°c. sacchar i f i ca t ion by t h e malt amylases was encouraged by

holding t h e mash a t 60 - 6 5 O ~ rvld 70 - 7s0c temperatures respect ive ly . Mash

concentrat ions of 25%, 35%, and 45%, and t o t a l sacchar i f i ca t ion periods of

Ih , 2h, and 3h were employed i n t h e production of warts. The r e s u l t i n g '

reducing s u g d con ten t s (as glucose) determined a s described under sec t ion "

3 .9 .4 .

3 - 8 - 3 E f f e c t of v x y i n g concentr~tt. jons of a lucomylase and uaccharif i c a t i o n - , -

periods on the reducing sugar contents of wort.

Wort W ~ Y p r e p d e d from a 25% mash. Its pH was adjusted t o 4.3 with a

0 3~ ~ c l so lu t ion , and temperature maintained a t 55 C. Varying concentrat ions

of glucoamyl~sc; 0.09%, 0.10X and 0,15X (dry weight b a s i s of the mash) were

ddded respect ive ly , then sacchar i f i ca t ion c a r r i e d ou t f o r 12h, 24h, and

The r e s u l t i n g hydrolysates were neu t ra l i sed , f i l t e r e d and malysed f o r

reducing sugar content ( as glucose) , as described under sec t ion 3.9.4 '

3.8 -4 Ma1 t bdsed- Syrup production:

50y of sorghum malt mi l led t o 1-2mm p a r t i c l e s i z e ( i n a Thomas wiley

M i l l , Model ED-5) was mixed wi th 2 0 h l of tap water t o g ive 25% mash. The

ptf of the r e s u l t i n g wort w a s adjusted t o 4.3. X t r temperature was maintained

a t 5 5 ' ~ using a thennos ta t i ca l ly con t ro l l ed water bath. 0.15%- glucoamylase

(D.W.B of the mash) was added and incubated f o r 24-72h (with cons tant

shaking) unt i l t h e des i red DE value was at tained.

The m a l t hydrolysate s o l u t i o n produced was neu t ra l i sed with 2M Na2C03

solut ion. A t e n f o l d d i l u t i o n of it was made, f i l t e r e d (wi th f i l t e r paper)

and. f i n a l l y concentrated to a syrupy consistency by evaporation on a

bo i l ing water bath.

/ ~ h r e e s t age decoction mashinq/

1

J /~nzyme sacchar i f i c a t i o n l

1

JI . . h i n i s h e d product ( ~ a l t syrup)/

~ i g . 5. Flow c h a r t of malt based syrup production. . \

3.9 Glucose Symp Production:

The acid-enzyme(dua1-stage) conversion method w a s used i n the glucose

syrup production. I n the f i r s t s tage , which w a s acid l ique fac t ion , s t a r c h

w d s mixed w i t h water t o form a suspension of s i u r r y , conicaininy 25% d r y

s tarch . his was poured i n t o a pyrex f l a s k and i t s pH adjusted to 1.8

with 3M H c l so lu t ion , The f l a s k and its con ten t w a s autoclaved f o r 20 minutes,

The r e s u l t i n g hydrolysate w a s cooled, and neu t ra l i sed t o pH 6.0. T h i s

was l a t e r f i l t e r e d i n t o a flask, and t h e second s t age of the conversion,

enzyme sacchar i f l ca t ion e f fec ted by addi t ion of 0.15% g l u c o a m y l a s e ( 9 ~ ~ o f

s t a r c h ) , and holding i t i n a w a t e r bath(with cons tant a g i t a t i o n ) a t

was determined by the des i red DL level . The r e s u l t i n g s t a r c h hydrolykite

was concentrated by evaporat ion on a boi l ing .water bath,

4 h o n - exchange deionized/

I

Fig. 4. Flow c h a r t of Acid-enzyme converted glucose syrup production.

3.9 Determination of some Proper t i e s of Syrups:

3.9.2 Determination of Spec i f i c q r a v i t y / ~ e g r e e baume of the Syrups.

The s p e c i f i c g r a v i t y of the syrups were determined according t o t h e

procedure described in ~.O,A.C.(1980) method of analysis .

A 50ml s p e c i f i c g r a v i t y b o t t l e was f i l l e d with d i s t i l l e d water,

0 stoppered irnd immersed i n water ba th a t 20 C. Af ter 30 minutes-, t he

s p e c i f i c g rav i ty b o t t l e was removed, d r i ed with f i l t e r paper and weighed.

The sme procedure was repaated with the s m p l e s . The s p e c i f i c g r a v i t y

of the samples were ca lculdted as follows:

SG P weight of l i q u i d held i n SG b o t t l e weight of water held i n SG b o t t l e .

Uegree baurne ( ' Me' 1

This is r e l a t e d t o Speci f ic grdvi ty b y , t h e following fonnula(Corn

r e f i n e r s Assacidtion, 1965) .

3 ,go 3 " Determkidtion of percentage t o t a l s o l i d s of t h e Syrups.

A sample of the syrup w a s t r ans fe r red i n t o a previously d r i ed and

weighed s t a i n l e s s steel dish. The d i s h and content was l a t e r placed on a

boiling water ba th and evaporated to dryness, This was weighed and then

placed i n .an oven and d r i ed f o r 3h a t 1 0 5 ~ ~ . The d i s h was returned t o the

oven and weight checked a t thirty minute i n t e r v a l s u n t i l no f u r t h e r l o s s

i n weight could be detected. The d i s h was cooled i n a d e s i c a t o r f o r

20 minutes and the weight taken. ti'

The percentage t o t a l s o l i d s , was ca lcu la ted thus:- .

% t o t a l s o l i d s = W t of sample a f t = drying x 100 W t of sample bekore 'drying

! ' 3.9.4 J Percentage reducing sugar conten t / ~ e x t r o s e equivalent ( DE.) value determination.

Reducing sugar content (as glucose)/Dextrose eqUivalent(DE) values of

thesyrup s m p l e s were detenninfid &cording t o the I n s t i t u t e of Brewery

I.O.U.(1977) method of analysis as follows:

It% w/v solu t ion was ,prepared by weighing.25,OOg of the syrup i n a . . .

gldss d i sh and dissolving same by.gradua1 s t i r r i n g i n warm water. his

w d s t r a s f e r r e d quan t i t a t ive ly to a 2501111 graduated f l a s k , and a f t e r

0 adjus t ing the temperature t o 20 C, it was mdde up to the mark a t t h a t

temperature. 251111 of t h i s so lu t ion was p ipe t t ed i n t o a 250ml graduated

f l a s k and d i l u t e d t o mark a t .20°c. This was mixed w e l l and f i l t e r e d . I t

wr i s used as the "di lu ted solution".

25ml of mixed Fehlings' so lu t ion was pipetted i n t o a 250rnl S a i l i n g

f lask; and m almost sufficient of the d i l u t e d so lu t ion was added from the

b u r e t t e t o the cold fehl ings ' so lu t ion t o e f f e c t reduction, s o t h a t , i f

poss ib le , n o t more than l m l was required l a t e r to complete the t i t r a t i o n .

The contents of t h e f l a s k was mixed, and heated over a w i r e gauze, k e p t i n

moderate e b u l l i t i o n f o r 2 minutes and th ree drops of methylene b lue ind ica to r

added without removal of the flame, then the t i t r a t i o n completed i n one

minute with continuous e b u l l i t i o n . I

The end point (decolor iza t ion of the methylene blue) was taken as the

volume ' d t which the r eac t ion mixture turned red. The t i t r e was recorded.

Calculations:

The r e s u l t s were,ccilculated a s the most appropriate sugar (glucose o r

maltose) using the appropr ia te f a c t o r from t h e l ane & Eynon table . For the

d i l u t i o n given, the percentage reducing sugar i n the sample ' a s isg

% ~ e d k i n ~ sugar = Lane & Eynon f a c t o r x 100 T i t r e .

Dextrose Z q u i v a l e n t ( ~ ~ ) r % reduclnq sugar (as glucose) x 100 % t o t a l s o l i d s

3 m9.5 . - Deterrnl-nation o f colour of the syrups:

The colour of the syrups were determined by the procedure described

i n A.O.A.C(l980) method of apdysis as follows:-

59 ce l - i t e was added t o 100rnl f i l t e r e d syrup, mixed and f i l t e r e d

through f i l t e r pclper. The f i r s t 40ml f i l t r a t e was returned t o the f i l t e r .

Absorbmce of c l e a r t o t a l f i l t r a t e was determined a t 430m using a

spectrophotorner. Syrup colour was ca lcula ted a s . follows:

colour = 1 0 . x x correc t ion t o 4' cell s i z e . c ~ l o u r was reported to t h e

nea res t 0.05 uni ts .

CHAPTER 4

4. RESULTS AND DISCUSSIONS

4.1 ~ a l t i n g c h a r a c t e r i s t i c s of t h e ce rea l p;rains, sorghum and millet:

Table i shows the malting c h a r a c t e r i s t i c s of the c e r e a l g ra ins

used. The values of 33.30g and 6.8g 1000-kernel weight obtained respect ive ly

f o r sorghum and millet g r a i n s were smaller compared t o 50.9g f o r b u l e y

reported by s ingh and ~ a u r o ( 1 9 7 7 ) . However, s ince these g ra ins are

smaller i n s2ze, it is expected t h a t the weight would be smaller. his

agrees wi th the suggestion made by Nout and Davis(1982) t h a t i n l a r g e

s c a l e malting, modification i n p l a n t cm be ef fec ted t o take care of the

d i f fe rences i n s i z e of the grains.

The percentage fore ign seeds and broken kernels gave 0.32% and 0.56%

re3;)ectively f o r Sorghum and m i l l e t grains. These r e s u l t s show t h a t

broken kernels and foreign seeds are r e l a t i v e l y low from t h e bulk g ra in

samples.

tiough et &., ( 1981) repor ted t h a t f o r good malting q u a l i t y of barley,

minimum germinative energy (G.E) requi red is 96%. However, i n t h i s study,

G.E. of 82% and 76% were obtained respect ive ly f o r sorghum and ' m i l l e t

grains. Aniche(l302) obtained 60%

(G.C. ) f o r L 181 sorghum var ie ty ;

7% germinative energy f o r millet.

C.E. and 98.5% germinative 'capacity

while Singh and Tauro(1977) reported

Germhative capaci ty (G.C.) of 90%

and 85% were obtained f o r sorghum and m i l l e t respect ive ly in t h i s study,

iind s ince G.C is a measure of percentage of l i v i n g corns under aided

condi t ions , i t i s therefore suggested t h a t hydrogen peroxide be used

62.

durlriy 111dlthg of sorghum iuld m i l l e t g r a i n s to enhance germindtivt: cclpacity.

MAIJTING CKARACTERISTICS OF l l i E CERAL GRAINS: SORGHUM AND MILLET

~ h a r a c t e r i s t i c s Sorghum Millet

1000 - kernel weight(g) 33.30 6 .8

% Foreign seeds and broken kernels ' 0.32 0.56

Germinative energy (% ) 82 76

O p t h u m mj l t ing condit ions of the Cereal grains: Sorghum and Millet:

Figures 6 t o 10 present t h e optimumgmalting condi t ions of the

ce red l grains. The v a r i a t i o n o f moisture content(%) aga ins t s t e e p time(hours1

i.s shown i n f i g u r e 6 , The r e s u l t i n d i c a t e a sharp rise i n water uptake

during the f i r s t 1'0 hours. Fur the r s teeping above 20 hours showed marginal

increases. I t i s evident from the r e s u l t s t h a t sorghum gra ins absorbed

moisture fclster than m i l l e t gra ins , t h i s may be due t o i t s r e l a t i v e l a r g e

corn s ize . Dahlstron e t g . , ( 1 9 6 3 ) observed t h a t l a r g e r corns absorb water - - more rap id ly than smaller ones i n i t i a l l y , and the d i f fe rence i n water

absorption a f t e r 24 hours is marginalised. Also Hartong and Kretschmer

(1961) found t h a t samples of g r a i n s t h a t absorbed w a t e r f a s t e r gave b e t t e r

malts than g r a i n s t h a t absorb water more slowly.

0 The r e s u l t obtained from t h e p l o t of d i a s t a t i c power( L) agains t

steep t i m e (hours) f i g u r e ' 3 , showed t h a t the opthum s teep t i m e Was

1 1 I 1 1 I I \ I

10 20 30 40 50 . 60 70 80 Steeplng t i me (hours )

Fig. 6 : Moisture content (%) against s t e e p i n g t i m e ( h o u r s ) . Sorghum grain

i M i l l e t g r a i n

20 ' 30 S t s e p i n g

Fig. 7 : Diastotic power (%) against st;esping tima (hours) a f ter 4 days o.f ge rmina t ion . - .. b-d 8 Sorghum, o-o 8 Mil let '

50 kwurs. A t t h i s time, the d i a s t a t i c powers of the two cerea l s were

mdx.i.mum a f t e r the grains w e r e allowed to germinate f o r four days. Furthermore,

extrapolating the 50 hours optimum steep t h e t o r e s u l t s of the p lo t of

moisture content(%) against time(hours) f igure fj, gave variously 38%

and 33% optimum moisture contents f o r sorghum and m i l l e t grains respectively. I

out and Davis (1982) obtained 45% moisture c o n b n t a f t e r 35 hours and

20 hours of steeping sorghum QAndivo' and sorghum 'igumba, respectively.

The optimum gemination period of 5 days was obtained after steeping '

I the grains fo r 50 hours. This r e s u l t was deducted from the p l o t of

d i a s t a t i c power (OL) against germination periods(days), f igure 8 The

0 r e s u l t a l so indicate t h a t rnJxknum d i a s t a t i c pX!=rs of 32O~ and 27 L were

'

obtained respectively f o r sarghum and m i l l e t malts under the same st ipulated , I

malting conditions. The r e s u l t shows t h a t there was no d i a s t a t i c power I

measurable in ungerminated grain but rises sharply fran 1-3 days reaching

i ts peak a f t e r 5 days of gemination. This suggests t h a t d i a s t a t i c enzymes

absent i n ungerminated grain develops with germination. I t has been established

tha t alpha anylase was not present i n the ungerminated sorghum grain and t h a t

the ac t i v i t y was only measurable a f t e r germination(Aisien et z., 1983). i I

Figure 9 -shows the r e s u l t of malting loss (%I measurements during

the various periods of germination(days). The r e s u l t showed t h a t the

malting l o s s of grains increased with increase i n the period of germinatim.

Signif icant increases i n malting l o s s were recorded between 2-4 days of

germination, which correspond ta t h e periods of s i gn i f i can t drops in

1000 - kernel weights during gemination. The ranges of 12 - 16% and 16.20%

G e r m i n a t i o n p e r i o d ( d a y , ) F i g . 8 : Diastolic power (%I a g a i n s t germination p e r i o d s ( d a y s )

a f t e r 50 hours of steeping. I

H '= Sorghum, M = Mill8 t

I 1 I 1 1 1

2 3 4 5 6 7 Germinat ion pkr iod ( doyo )

9 : Molting loss (%I against perminotion per iod ( d a y s ) . 04 a Millet molt

M Sorghum ma I t

malting losses were obtained respectively f o r sorghum and m i l l e t m a l t s

covering 4 to 7 days of gemination. The malting l o s s f o r barley have

been given by Hough et: ( 1981) ae 6~12%. The high malting l o s s of

the millet grains could be due t o excessive aeration during sixaping

leading t o grains growing uncontrollably dullng germination according to

H o u g h e , &., (1981). Malting l o s s could also r e s u l t from long steeping

period a s materials trend to be leached i n t o the s teep water. .To reduce

malting l o s s , it, i s suggested t h a t the f i l t e r paper8 used should no t be

saturated with water, o r I n the a l te rna t ive , malting f loors should be wed.

Relault of kilning s tudies , ( f igure 10 ) shows t h a t moisture re lease is . ;

reciprocally re la ted to moisture absorption. Signif icant l o s s i n moisture

w a s recorded after 12 hours of ki lning a t 4s0c. With regards to moisture

level i n r e l a t i on to storage qua l i t i e s of malta, optimum ki lning can be

carr ied out a t 45O~ f o r 24-48 hours depending ~n the moist- l eve l of

t h e green malt and the moisture content of the m a l t required. The e f f e c t

of temperature on the m a l t characters was no t investigated, however,

other workers; Nout and Davis (1982) and Novellie,(1962) showed t h a t only

kilning a t .70°c resul ted in a s ign i f ican t l o s s i n d i a s t a t i c ac t iv i ty ,

kilning in the range of 4 0 - 6 0 ~ ~ caused only negligible destruction.

4.3 Evaluation of ma1t;l'squality characterist ics:

The malts* qual i ty charac te r i s t i cs were evaluated and the results

presented on tab le 5 .. Generally, low Cold Water Extract(C.U.E), high

Hot Water ~ x t r a c t ( ~ . W . ~ . ) and la static Pow~(DP). are indicat ive of good

m a l t character is t ics . Hough et; %,(1981) reported ~ a l u e s of 307 ~ O h g

Ki ln ing p e r i o d ( h o u r s ) ig . 1 0 : Moisture content (%) agoins* kilning per iod( hours) at 45OC.

o-a 8 M i l l e t malt, - k + - ? 8 $ o t r g h u m m a l t ,

(HUE , 18.6% (CWE) and 63 '~ (DP ) f o r barley malt!. Tha corresponding values

obtained i n this atudy f o r sorghum and m i l l e t malts( t @ l e f , .' were I

r e l a t i ve ly inadequate to pass fo r good malts. The high C.W.E obtained

may be due t o malting a t high temperature of around 2 8 ' ~ ~ aa against t h a t

0 f o r barley which is usually a t 15 C o r less (Preece, 1954). as w e l l ao a

higher moisture content achieved by sprinkling of water. Improvements In

the malting conditions f o r the grains could considerably reduce the high

C-9-E- , and increase the H.W.E. and D.P. values obtained. However, the

values obtained f o r Sorghum malt i n ' the three, qual i ty charac te r i s t i cs arc

comparatively more encouraging than those of m i l l e t malt.

THE MALT'S C\UALITY CHARACTERISTICS

Character is t ics Sorghum m a l t M i l l e t malt

Cold Water Extract , CWE(%)

~ o t water ex t rac t , HWE ( ~ O / k g )

4.A Determination of Optimum pH and Temperature conditions f o r malt's . - .

amylase act ivi ty .

The r e s u l t s of the optimum conditions (pH and tempersture) f o r mylase

ac t i v i t y of the malts were as pretsentsd i n f igures 12 ' and -13 respectively.

Figure 12 shows t h a t a t optimum pH range of 6-7, the amylase ac t i v i t i=e I

of both sorghum and mi l l e t malts were maximum. Also f igure 13 reveals

t h a t amylase ac t i v i t y of sorghun m a l t peaked a t optimum temperature range

- mg Maltose in 3 m l so lu t ion

Fig . I 1 : .Maltose cal ibrat ion c u r v e . '

40 50 60 70 80 90 T e m p e r a t u r e ( O C )

.IFi g 8 : Optimum ternperoture determination far ornylo~e activity in both millet, ond sotghum ma l t $. I t b

of 60-7o0cI while t h a t of m i l l e t malt b e c q e m a x i m u m a t 40-50°c. The

optimum p~ value rmge of 6-7 obtained i n t h i s etudy agrees with thQ

values reported by ahembe &-,(1988). S i l l s and Stewart(l984) reported

optimum pH range of 5.5-6.5 f o r purified barley alpha amylase. ~ o u g h

e t &.,(1981) points out t h a t pH value of 6.0 is quite su i tab le f o r - 0

anylase ac t iv i ty a t 65 C. The r e s u l t i s also in agreement with the pH

optima of 5.2 (Preeoe, 19541, f o r /3-amylase, and 7.O(Worthington, 1979)

f o r alpha amylase enzymes in barley.

shembe 6 &. , (1988) reported 35-45O~ temperature optima f o r m i l l e t

and 50-75Oc $or sorghum m a l t s @ amylase ac t iv i t i es . Also S i l l s and Stewarts

(1984) reported values of 3 5 - 4 0 ~ ~ f o r pur i f ied barley alpha amylase. A

Chandraskhara and swminikhan ( 1958) showed optimum d P e r a t u r e f o r both

alpha and b-anylases in pearl m i l l e t t o be 60°c. The &mporature optima in

both cases are below gelat inizat ion temperature ranges of millet(70-80°c)

and sorghum (68-80°c) obtained i n t h i s study; a necessary prerequis i te fo r

e f fec t ive degradation of s ta rch by amylases. T h general poor malting

qual i ty of sorghum and m i l l e t ! gra ins as a lso reported by other authors is

thus

4.5

f a t ,

pa r t ly due to this fact .

~roxim&/~hemica~. analyses of the sorghum/millet grains and maltst

The r e s u l t s of proximate analyses (moisture content, crude protein,

crude f i b r e , and Ash) t o t a l carbohydrate. and 'gel a t l n i zation temperature

ranges of the grains and m a l t s used i n producing the syrup8 are

i n . t a b l e 6 , , he proximata cmposi t ion values obtained f o r mi l le t grains

and its malt w e r e c lose to those reported by Opdrtu et . , 1 9 8 1 Also

Aniche (1982) proximate composition values reported f o r Sorghum malts

( t ab le 2 ) caipclred f avourably with the values obtained i n t h i s work.

The Sorghum values f o r protein, 10.40%, c l o s e l y agree with 10.9% reported

by Hough et . , 1 9 8 1 . The r e s u l t show t h a t sorghum has lower values

of f a t , and higher values of carbohydrate and protein than mille t . Grains

used f o r syrup production should be low i n fat and protein but r i c h i n

to tdl carbohydrate especially starch. Cereals with higher f a u p r o t e i n

contents are considered unsuitable f o r glucose syrup manufacture since

they have detrimental e f f ec t s on flavour ar\d contribute t o colour v i a maillard

reaction. I n malt based syrup production, where colour is r a the r required,

the high protein content f ac to r i s no more necessary, therefore sorghum

grain is bes t su i ted f o r the malt syrup production.

TARLE 6.

CHEMICAL/PROXIMATE ANALYSES OF SORGHUM/MLLLET GRAINS AND MALTS

~ i l l e t Sorghum

Grain Malt Grain Malt

Moisture content(%) 9-10 ' 6.50 10.12 7.50

Crude protein (%I 8.30 10.50 10.40 11.30

4.6 Chemical analyses of starches extracted from mi l l e t and sorghum, grains:

Starches were extracted from mi l l e t and sorghum grains and subsequently

subjected t o chemical analyses. The r e su l t s obtained are presented on

t51e 7. The lower s ta rch contents of 76*40(millet) and 83.40(sorghum)

d-.-.P.-.. r e l a t i ve to values obtained by Brenner - et - & . , \ L Y Q O ~ and nuisr &, &.,

(1980) may be due t o the unsophisticated equipment which was used during

the s ta rch extract ion process. The pr incipal parameter i n the refined

s ta rch concentrate which a f fec t s both the qual i ty and volume of glucose

syrup is the s ta rch content. The higher the s tarch content, the greater

the reducing sugars obtained on hydrolysis., The comparatively higher protein

values of 2.1% ( m i l l e t s t a rch) and 2.4%(sorghum starch) a s against 0.2016

reported by Hulse & &.,(1980) may equally be due to inadequate separation i

of gluten from starch. The leve ls of f a t 0.83% ( m i l l e t s t a rch) and 0.66%

(sorghum s ta rch) are also higher than 0.003% reported by B r e n n e r c 2.. (1968). Therefore, a more sophisticaited equipment than tk one used i n

t h i s work is suggested f o r s ta rch extract ion i f glucose syrup with a near

water-white colour i s desired.

Table 7 shows t h a t the gelat inizat ion temperature ranges of the

extracted' refined starches are lower than t h a t of the corresponding grains

( t ab l e 6 ) thereby rendering the refined s ta rch more susceptible t o

amylalytic degradation. However, sorghum starch, having a lower gela t inizat ion

temperature range, lwer f a t and ash contents than m i l l e t s t a rch is preferred

i n glucose syrup production.

TAJ3LE. 7.

CtiEMlCAL ANALYSES OF STARCIIES FROM MILLET AND SORGHUM GRAINS

Millet s ta rch Sorghum s ta rch

~ o i s ture content (%I 9.2 8.6 b ;'

Crude protein (76) ' 2.1 2 .4

Fat (%)

Crude f i b r e (%I 0.71 0.27

Ash (%I 0.60 0.40

Starch content (%) 76.40 83 .40

4.7 Effects of varying mash, glucoamylase concentrations and saccharificc~tion - periods on reducing sugar contents of the wort syrup:

Figures 15 and 16 show respectively the r e s u l t s of the e f f ec t s of

varying mash concentrations and time; glucoamylase concentrations and time;

on t h e reducing sugar content of mort syrup. Results show t h a t makimum

conversion was a t ta ined a t the lowest substra te concentration, 25% mash;

highest enzyme-substrate r a t i o , 0.15% glucoamylase and a t a longer

sacchar i f icat ion time. However, there are feas ib le ranges of in teract ing

variables during the sacchar i f icat ion process of syrup production. The

25-35s subs t ra te concentra$lon range struck a b a l e e b e t w e e n the cos t of

removing excess water from hydrolysates against higher conversfon t o glucose

a t ta inable a t low substra te concentration, Also, 0.10 - O,f i .% enzyme-

subs t ra te r a t i o is reasonable s ince cos t of enzyme balanced against

sacchar i f icat ion and equipment required f o r long saccharification. The

0 ~08 0.1 2 0.16 G l u c o s e conc. (mq/ml )

F i g . 1 4 Glucose s tandard c u r v e . .. .

T i m e < h o u r s ) . Fig . I5 Effect o f vary ing mash concenirot.ion and t ime on

reducing sugar .;content of the wort a

= 45% mash, H = 3S0/, mash , )++( 25'10 mash

Fig. 16 : E f f e c t of w r y i n g concentrat ions of g l u c o a m y l a r e , . ~ n d time on the reducing sugar content o f the wort syrup.

5 0 0 -

480-

460-

% 440- 0 0 L Q,

a 420- Q) Ln 2 C

M

g400- u' E

380-

360

= 0.05 % of glucoarnylase M = 0 10 % of giucoamylase w, = 0.15 O k of glucoamylase

,

J

0 12 Ti me ( hours 24 36

sacchar i f icat ion range of 12-72 hours chosen f o r the experiment w a s based

on enzyme-substrate r a t i o used and extent of corrverlion cle,aircd, The

optimum temperature range of glucoarnylase ac t i v i t y is S O - ~ ~ O C , where the

upper l im i t i s dependent on glycoamylasestability, and lower l i m i t on neid

t o i nh ib i t microbial contamination during long saccherrification periods.

Furthennore, the pH 4-5-0 is dependent on aource of glucoanylase, and a l so

eug& are most s t ab l e a t this pH range.

4.8 Properties of malt based syrups and acid-enzyme converted glucose syrups: !

Tables 8 and g show the r e s u l t s of analyses i n some properties of i I

malt based syrups and glucose syrups produced i n this study. The r e s u l t s !

show, three c lasses of syrups; Regular ( 4 2 ~ ~ ) , intermediate ( 5 2 ~ ~ 1 ,

and High (69~~j. Conversion syrups were produced f o r malt and glucose

syrups respectively.

The values of 4 3 ' ~ ~ and 42OF3ef density ware obtainad f o r m a l t syrups

and glucose syrups respectively, The higher value f o r malt syrup may be

due t o its r e l a t i ve ly higher content of cellulosfic materials.

The r e s u l t a l so show t h a t the ash content of m a l t syrup was 0,4% while

that of glucose syrup was 0,65%. Junk and Pancoast,(l973) noted t ha t the

normal concentration range of ash i n corn syrup was from 0.1 - 0.3%. The

higher ash content of glucose syrup obtained may be as a r e s u l t of sod im

chloride, which is derived primarily fran the neutra l izat ion of hydrochloric

acid w i t h sodium carbonate during the acid l iquefaction process. '

Colour values of 5.1 un i t s and 2.2 un i t s were obtained f o r m a l t ant!

glucose syrups respectively. Junk and Pancoast(l973) a l so noted t h a t the

colour of newly refined'corn syr-p is usually @water-whlte( and has a

value of 0.25 units. The higher value of malt syrup colour i s a t t r ibu ted

t o the colour developed during the ki lning process of malting. However,

the colour of the glucose syrup could be reduced through carbon &d ion-

exchang~ ref ining processes.

he carbohydrate composition as glucose(%) and maltose(%) r e s u l t s

show a s imilar trend on both tables. Generally, the values increase

proportionately across the three c lasses of syrups, Also, mare g l u c o s ~

are i n each case produced than maltose, this is expected since the

glucoamylase preparation from fermentations of Aspergillus niger contain

r e l a t i ve ly l e s s amounts of 8-amylase.

The lower glucose yie ld i n m a l t syrup r e l a t i v e t o glucose syrup i n

the three c lasses of syrups may be due to poor d i a s t a s i s of the m a l t s

0 amylases whose temperature optima, 60-70 C, is lower than the m a l t ' s

s t a rch gela t inizat ion temperature range of 68-F30°c; Consequently, generating 8

more l i m i t branched dextrins which are less susceptible t o glucoamylase

than the l i nea r dextrins(Abdul1ah A., 1963)

Results .also indicate t h a t maltose contents of malt based syrups are

s l i g h t l y higher than thoge of glucose syrups{ this trend might be traced

to the r e l a t i ve . quanti ty of p-anylase which is a major component of malt

amylases and a minor consti tuent of glucoamylase enzyme preparations from

Aspergillus niger. -

TABLE. 8.

PROPERTIES OF MALT BASED SYRUPS

Property Regular Intermediate High

Dextrose equivalent (DE ) 42 52 69

0 Commercial Bauma( B e ) 43O 43O 43O

~ l u c o s e (961 2 0 28 4 1 I

Maltose (96) 15 19 2 2

TABLE, 9.

PROPERTIES OF ACID ENZYME CONVERTED GLUCOSE SYRUP

Dextrose equivalent (DE) 42 5 2 69

0 Canmercial Baume ( Be' 42 42 42

Solids (%) 80 . 82.2 80

. ~ s ' h ( % I 0.65 0.65 0.65

Glucose (%) 23 3 1 45

Maltose (%) 13 13 2 0

CHAPTER 5

5. SUMMARY AND CONCLUSIONS

ConvenUonally, malt syrups used by food industr ies are produced fran

b u l e y . he broad objective of the present investigation was t o produce

rnalt Byrups pr ic ipa l ly fran sorghum and millet grains which would be used

by food industries. The Nigerian ecosystan does not 8upj$?rt th0 a g r i c u l t w a l

production of barley grains, thus jus t i fying thL8 objective. The r e s u l t s

prcrduced fran the invesaga t ion are summarized as follows:

1. Both the malting and m a l t ' s qual i ty charac te r i s t i cs of the grains

studied indicate t h a t aarghum generates be t t e r m a l t than millet .

2. 50 hours of steeping and 5 days of gemination a t room temperature gave,

A*&.- - - L a --.... ,.., YYLY16U111 d i a s t a t i c power development i n sorghum and m i l l e t grains. b

3. Feasible ranges of 25-35% substra te and 0.10-0.15% enzyme concentrations

are bes t sui ted f o r syrup production.

4. Sorghum s ta rch is more adequate f o r glucose syrup prodyction. High

colour value of the syrup obtained could be reduded when it is fur ther

refined with carbon and ion-exchange de-ionized processes.

One would therefore suggest fu r the r research in comection with,theae

r e su l t s as follows:

. More malting 'study is advocated in order t o f ind b e t t e r favourable Y

malting conditions f o r mi l le t ' e spec ia l ly i n the area of malting loss reduction.

b. The control and measurement of colour/flsvol.~,r acquired by sorghum m d l b

during the ki lning process.

c. Isola t ion, pur i f icat ion ,and characterization of sorghum malts alpha

. 85,

amylase and i ts t iultabil i ty i n starch liquefaction process of glucose syrup

production comparad with that o f a microbial alpha amylase.

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APPENDIX

Preparation of Analytical reagents, Dini t rosal icyl ic acid(DNS) reagent .

I g of 3 ' , 58 - d in i t ro sa l i cy l i c acid w a s disolved In 20ml ob 2N NaOH, T h i s was made up to 50ml with d i s t i l l e d water. 3 0 9 of potassium sodiumtartarate (Rochelle S a l t ) was then dissolved i n the resu l t ing solut ion, and made up t o 10Oml with d i s t i l l e d water and i s ca l led DNS reagent.

Anthrone Reayen t.

7601111 of sulphuric acid (98% W/W H2SC4) Y" &d=d ', 336n1 of w ~ t e r . with s t i r r i n g a f t e r cooling, 1g of thiourea and l g of anthroha were added and s t i r r e d u n t i l dissolved. The resu l t ing solut ion was s tored i n a refr igerator .

Hydrogen ~ e r o x i d e ( H ~ S O ~ ) , - 0.75% Solution:

A f resh solut ion was prepared each time by d i lu t ing S m l of 30% 4 0 2 (100 vol) to 200 m l with d i s t i l l e d water, The resul t ing solution was- stored i n a refr igerator .

Methylene blue, 1%

1g of the pure.dye was dissolved i n l O O m l of d i s t i l l e d water.

Ihver t sugar standard s o l u t i o ~

To 9.50g of pure sucrose was added 5ml of hydrochloric acid and di luted with d i s t i l l e d water to 100ml. This w a s s tored a t room temperature f a r several days, and then made up to 2501111. When required, 25ml of the stock solution was neutralised with ocdi-at hydroxide and made up to 200ml t o give a 0.5% inve r t solution.

Fehlinaos Solution A and Br

Commercial Fehling's solutions A and B for laboratory analysis were purchased from BDH company. Before each analysis an equal mixture of the two were made up to 5ml f o r d i a s t a t i c power determination and 25ml f o r reducing/~extrose equivalent determination.

Benedictc s reagent

1739 of sodium c i t r a t e and lOOg of sodium carbonate were'dissolved i n 8001111 of w m water, f i l t e r e d and made up to 850ml with water. ~eanwhi le , 17,3g of Copper Sulphate was dissolved in about l O O m l of water and made up t o 150ml. The f i r s t solut ion was poured i n t o a 2- l i t re beaker and the Copper Sulphate solut ion added slowly with stlrrlng.

Acetic acid 1 .ON:

28.65m1 g l ac i a l acetic acid a t 20O~ was di luted t o 5001nl with d i s t i l l e d water.

Acctate buffer s o l u t i ~ n , pH 4.6;

689 odium acetat. (CH CalNa.3H20) was dissolved i n 3 0 h l s f L O N ace t ic acid and made up to ane l i t r a with d i s t i l l e d water a t 20 C.

Sodium hydroxide, 0. Wt

4-09 of N ~ O H was dissolved i n one litre of d i s t i l l e d water. oy"

2% s ta rch solution, buffered a t pH 4.6:

A 109 cieam of soluble s t a r ch was made with d i s t i l l e d water.and poured i n t o about, 400ml of boi l ing d i s t i l l e d water s t i r r e d constantly, he solution was boiled fo r 2 minutes and cooled under cover, t o avoid skin formation t o 20'~. l O m l of aceta te buffer w a s then added and the volume made up to 500ml. This w a s prepared daily.

0. W ammonia solution:

6.7 m l of the concentrat& ammonia was di luted to l l i tre with d i s t i l l e d water.

Buffer Preparations:

Buffer were prepared according to the method described by Sidney and '

Nathan( 1955 ) as follows:

From the stock solutions of A t 0.l.M ace t ic acid (5.76m1 i n l Q O O m l water) and 8: 0.1M sodium aceta te 8.2g In l O O O m l water). Acetate buffer were prepared thus: pH 4 t 41.0ml A + 901111 B di luted to l O O m l

pH 5 t 14.8ml A + 35.2m1 B d i lu ted to l O O m l i

Also fran the stock solut ion of A : O.lM monobasic sodium phosphate (13.99 i n l O O O m l water) and B, 0.lM d ibas ic sod im phosphate 27.83g i n 1000ml water). Phosphate buffers were prepared thus:

pH 6 s 87.Tml A + 12.3ml B d i lu ted to l O O m l

pH 7 : 39.01111 A + 61.0ml B di luted to l O 0 m l . ,* pH 8 t 5.3ml A + 94.7ml B di luted to 100ml. !,- . --+

, ' "

,- ! . I : > * \ + b