Influence of Gibberellic acid (GA3) on diastase and cellulase development of Nigerian millet (Pennisetum maiwa) and sorghum (Sorghum bicolor)

Influence of Gibberellic acid (GA3) on diastase and cellulase development of Nigerian millet (Pennisetum maiwa) and sorghum (Sorghum bicolor)

frocrss Biochemisrry 28 (1993) 105-108 Influence of Gibberellic Acid (GA,) on Diastase and Cellulase Development of Nigerian Millet (Pennisetum ma...

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frocrss

Biochemisrry

28 (1993)

105-108

Influence of Gibberellic Acid (GA,) on Diastase and Cellulase Development of Nigerian Millet (Pennisetum maiwa) and Sorghum (Sorghum bicolor) R.C. Agu,“” B.C. Okeke,” S.C. Nwufo,” C.M. Udeb & A.H. OnwumeluC & Brewing, bDepartment of Applied Biochemistry, Industrial Chemistry, Nnamdi Azikiwe University, PMB 5025, Awka, Nigeria

aDepartment of Applied Microbiology

r Department

of

(Received 4 March 1992; revised version received and accepted 14 June 1992)

Gibberellic acid, GA, (O-O.30 mgllitre) was sprinkled at steep-out on maiwa) and sorghum (Sorghum bicolor) during millet (Pennisetum germination and growth at 28 “C. Optimum enzyme levels were obtained when 020 mgllitre of the additive was applied on both cereals. For millet, the highest diastase and cellulase activities were observed on the jifth day of germination (O-20 mg/litre of the GA, applied at steep-out), while sorghum showed highest activities of these enzymes on the fourth day of germination fi)r the same concentration of GA,.

INTRODUCTION

production and release of enzymes which degrade the hemicellulosic-protein-starch complex of the endosperm. Abrasion of the pericarp at the distal end of barley grain allows the grain to malt from both its embryo and non-embryo ends simultaneously.’ For the successful malting of grains with gibberellic acid, the grain’s physiology should be well understood.’ The failure of barley to grow in Nigeria has led to studies on the possibilities of beer brewing from sorghum and millet.3-10 Research into the improvement of the malting qualities of locally grown millet, using malting additives such as potassium bromate, has been investigated.“,12 The present study was therefore undertaken to investigate the effect of gibberellic acid (GA,) on the diastase and cellulase activities of millet (Pennisetum maiwa) and sorghum (Sorghum hicolor) during germination and growth.

The enzyme level of barley malt has earned the malt a unique position as the conventional brewing cereal. The enzymes are present in such an amount that they can completely degrade their endosperm as well as up to 50 % of those of other cereals, such as maize and rice, without the addition of external enzymes. Efforts have been made, without success, to cultivate barley in Nigeria, but it does not thrive well in harsh tropical weather conditions.’ The use of additives in malting is not new and intensive studies over the years have ensured a firm place for gibberellic acid (GA,) in the routine commercial malting of barley.2 Gibberellic acid at low concentration has been shown to accelerate the * Present address: Department of Applied Microbiology and Brewing, Enugu State University PMB 01660, Enugu, Nigeria.

ProcessBiochenzisfr,~

of Science

and Technology,

105 003%9592/93/$6.00

0

1993

Elsevier

Science

Publishers

Ltd.

England

R. C. Agu et al.

106 Table 1. Effect of gibberellic Germination time (dap)

1 2 3 4 5 4 7

on diastase

activity

(U/ml)

Gihberellic 0

0.10

of sorghum

(S) and millet (M) Malts”

acid concentrations

O-15

(mgllitre)

0.20

0.25

0.30

s

M

s

M

S

M

S

M

S

M

s

A4

10.6 12.2 14.8 17.4 17.4 15.9 15.6

9.1 10.2 13.1 14.7 15.0 14.5 12-3

11.2 12.9 151 18.2 17.X 16.2 15.9

10.1 10.7 13-8 14.9 16.7 15.9 13.1

11.8 13.7 15.9 18.9 18.6 16.8 16.3

10.6 11.2 14.1 156 18.1 16.1 13.6

12.0 13-8 19-5 24.1 19-9 17.4 17.0

10.9 11.7 18.3 19.0 22.6 21.9 18.3

10.1 10.6 15.3 21.6 17.4 16.6 15.6

9.7 10.1 15.0 15.9 18.2 16.0 15.1

8.6 9.4 15.0 21.6 IS.3 IS.9 19-O

7.7 8.1 14.2 15.6 15.9 15.1 15.0

a Each value is the average S, Sorghum; M, millet.

MATERIALS

acid (GA,)

of three

replicate

experiments.

AND METHODS

Thoroughly cleaned millet and sorghum samples (6 kg each) were weighed into their respective steeping vessels containing tap water. The grains were soaked for 18 h without change in steep liquor, after which the liquor was drained off, the grains thoroughly washed using a water spray and then air-rested for 5 h. The soaking was continued for another 24 h with changes in the steep liquor at 3 h intervals. At the end of the steeping period, the samples were cast on a cement floor and made into six heaps (for each sample of millet and sorghum). Gibberellic acid, 0.10, 0.15, 0.20, O-25 and 0.30 mg/ litre, was sprinkled on five heaps of each sample in that order, while the sixth sample of each grain heap was sprinkled with tap water. Samples were taken from each heap on a daily basis for seven days and kilned in a forced draught oven at 45 “C for 24 h. Milling was carried out with a Thomas-Wiley mill.

Enzyme extraction Diastase Sorghum and millet malt samples (1 g) in 10 ml of 50 mM sodium acetate buffer @H 5.2) were incubated at 28 k 1 “C for 1 h with constant shaking. The clear extracts which served as sources of enzymes were obtained by centrifuging the malt suspensions at 3000 g for 15 min at 4 “C.

CelluIase These were extracted as described for diastase except that 50 mM sodium acetate buffer (pH 5.0) was used.

Enzyme assay procedure Diastase activity Equal volumes, 0.2 ml each, of the enzyme extracts and 1% soluble starch solution in 50 mM sodium acetate buffer (pH 5.2) contained in different screwcapped tubes (for sorghum and millet), were incubated at 40 “C for 30 min. Nelson and arsenomolybdate reagents were added.15 The enzyme activities were terminated by heating the reaction mixtures in boiling water for 10 min after which they were cooled to room temperature (28 + 1 “C) and the absorbance measured at 500 nm. The reducing sugar equivalents were determined from a glucose standard curve. One unit of diastase activity is defined as the amount of enzyme that releases 1 pmol of glucose reducing sugar equivalent under the assay conditions. CM-cellulase activity This was assayed as described for diastase except that 1% carboxmethyl cellulose suspension in 50 mM sodium acetate buffer (pH 5.0) was used as substrate. One unit of cellulase activity is defined as the amount of enzyme that releases 1 pmol of glucose reducing sugar equivalent under the assay conditions. RESULTS

AND DISCUSSION

The influence of gibberellic acid (GA,) on diastase activity is shown in Table 1, while Table 2 shows the influence of GA, on the cellulase activity. The highest values of both diastase and cellulase activities were observed on the fourth and fifth days of germination for sorghum and millet, respectively, when 0.20 mg/litre of the GA, was sprinkled at steep-out.

GA, Table 2. Effect of gibberellic

acid (GA,)

on enzyme

on ccllulase

development

activity

(U/ml)

of millet

of sorghum

0.10

0

0.15

107

and sorghum

(S) and millet (M) Malts”

Gibberellic acid concentrations

Germination time (days)

1 2 3 4 5 6 7

influence

(mgllitre) @25

0.20

030

S

M

S

M

s

M

s

M

s

M

s

M

8.7 9.7 13.8 14.6 14.0 12.7 IO.7

9.9 10.4 11.7 14.7 14.1 13.6 13.4

9.0 10.7 14.0 15-l 14.7 13.8 Il.3

10.1 10.9 12.1 15.6 16.2 14.1 13.9

9.2 11.0 14.6 16.1 15.6 14.4 14.0

10.3 11.2 13-3 16.4 17.3 15.6 14.7

9.6 11.7 18.3 24.9 18.6 15.3 15.3

10.9 12.0 159 18.5 22.5 13-6 13.3

9.3 IO.1 15.3 23.4 17.1 153 15.0

10.1 10.3 12.6 15.8 168 12.9 126

8.8 9.1 13.8 20.7 16.2 14.7 14.2

9-7 9-9 11.7 19.8 15.6 12.5 12.5

LI Each value is the average S, Sorghum; M, millet.

of three replicate

SifUclUfS

(wMorellic

Sfrucfure

t Pllogibbcric

experiments.

SPruc?urs

I

acid

I

Sffuclufa

m acid

.U

( g&beMenic

1

Fig. 1. Gibberellic

( Gibberic

acid and its degradation

Gibberellins are highly active plant hormones which stimulate the reorganisation of cellular organelles and the synthesis of new proteins including enzymes.’ A gradual rise in diastase and cellulase activities was observed as the concentration of the GA, was increased (for all days of germination). However, for all the concentrations of GA, used in this study, diastase and cellulase activities reached their maximum values on day 4 and day 5 for sorghum and millet, respectively, but dropped towards the end of the germination period. Gibberellic acid (GA,) is slightly soluble in water (pH 34) up to 5 g/litre and has been reported that it degrades gradually into various forms that are

oc;d

)

1p

oekf

1

products.

not as active as gibberellic acid itself (Fig. 1).14 Gibberellic acid (structure I) degrades to gibberellenic acid (structure II) and further to allogibberic acid (structure III) for which activities of only 75 % and 35%, respectively (when compared with gibberellic acid) have been reported.14 Some differences, however, have been reported between the malting properties of sorghum and barley. For example, during malting a-amylase develops in the scutellum of sorghum,15 while other studies revealed that it is the aleurone tissue surrounding the endosperm of barley that is largely, if not entirely, involved in the production of hydrolytic enzymes.2*16 Most important is the report that

108

R. C. Agu

gibberellic acid accelerates enzymic modification of barley malt while this plant hormone has no stimulatory effect on enzyme development or on the malting rate of sorghum malt.” The role of the embryo (in barley) is to secrete gibberellins (for example, gibberellic acid) which stimulate enzyme synthesis and release by the aleurone layer,l’ while added GA, induces the aleurone cells to produce large quantities of a-amylase.l”~‘y However, in sorghum this enzyme is produced in and secreted from the scutellum.15 There is a paucity of information on the mode of enzyme synthesis and action in the millets grown in Nigeria. For diastase activity, values of 24.1 U/ml (0.20 mg/litre) obtained on day 4 of germination for sorghum and 22.6 U/ml ((O-20 mg/litre) for millet on day 5 are high when compared with 17-4 and 150 U/ml for their respective water controls. Similarly, cellulase activity (24.9 U/ml) after four days of germination for sorghum and 22.5 U/ml for millet after five days of germination at 0.20 mg/litre of GA, for both cereals are also high when compared with 14.6 and 14.0 U/ml for their respective water controls. Higher doses of O-25 and 0.30 mg/litre of the additive resulted in a decline in both diastase and cellulase activities, respectively. It is not quite clear if the decline in enzyme activities was due mainly to the high dosage of the additive, but since the enzymes assayed were measured in terms of reducing sugar production, part of the sugars are metabolised by the respiring embryo resulting in depletion of accumulated sugars, hence a drop in enzyme level. Diastase is a collective name for various hydrolytic enzymes. It is possible that any or some of these enzymes may respond to added gibberellic acid during malting of sorghum and millet; further work is in progress.

ACKNOWLEDGEMENT The authors thank Diamond Breweries Limited for supplying some of the materials.

et al.

REFERENCES Jayatissa, P. M., Pathirana, R. A. & Sivayogasunderam, K., Malting quality of Sri Lankan varieties of sorghum. J. Insf. Brewing, 86 (1980) 18-20. 2. Palmer, G. H., The industrial use of gibberellic acid and its scientific basis-A review. J. Inst. Brewing, 80 (1974) 13-30. 3. Okafor, N. & Aniche, G. N., Brewing a lager beer from Nigerian sorghum. Brewing & Distilling Int., 10 (1980) 32-5. 4. Agu, R. C. & Obanu, Z. A., Studies on beer production from Nigerian millet. J. Food Sci. & Technol. (Mvsore). / .I 28(2) (1991) 81-3. 5. Aniche, G. N. & Palmer, G. H., Develonment of amvlolytic activities in sorghum and barley malt J. Insr. Brewing, 96 (1990) 37779. 6. Okafor, N. & Aniche, G. N., Studies on the brewing of lager beer from Nigerian sorghum. J. Food Sci. & Technol. (India), 24 (1987) 1314. 7. Aniche, G. N., Studies on the effect of different mashing methods on sorghum wort composition and beer quality. Nigerian J. Tech. Res.. 2 (1990) 23-7. 8. Aisien, A. 0. & Muts, G: C. J:, Micro-scale malting and brewing studies of some sorghum varieties. J. Znsr. Brewing, 93 (1987) 328-31. 9. Morall, P., Royd, H. K., Taylor, J. R. N. & Van De Walt, W. H., Effects of germination time, temperature and J. Inst. Brewing, 92 moisture on malting of sorghum. (1986)43945. 10. Okafor, N. & Aniche, G. N., Preliminary investigation into the brewing of lager beer from Nigerian sorghum. Brewing & DistiNing Znt., 10 (1980) 21-35. 11. Agu, R. C. & Okeke, B. C.: Studies on the effect of potassium bromatc on some malting properties of Nigerian millet (Pennisetum maiwa). Process Biochemistr);, 26(2) (1991) 89-92. 12. Agu, R. C. & Okeke, B. C., Effect of potassium bromate on diastase, cellulase and hemiccllulase development in Nigerian malted millet (Pennisetum maiwa). Process Biochemistry, (1992), 27, 335-8. 13. Somogyi, M., Notes on sugar determination. J. Biological Chem., 195 (1952) 19-23. 14. Griffiths, C. M., MacWilliam, I. C. 8~ Reynolds, J., Nature, London, 202 (1964) 1026. Cited by Palmer, G. H., Industrial use of gihherellic acid and its scientific basis-A review. J. Inst. Brewing, 80 (1976) 13-30. 15. Aniche, G. N. & Palmer, G. H., hiicroscopic assessment of increasing moisture treatments on endosperm modification in sorghum. Ferment., 3 (1990) 378-80. 16. Palmer, G. H., The morphology and physiology of malting barley. In Cerealsfor Fooak and Beverages, ed. G. Inglett & L. Munck, Academic Press, London, 1980, pp. 301-87. 17. Macleod, A. M., The physiology of malting. In Brewing Science, ed. J. R. A. Pollock, Academic Press, London, 1979, pp. 145-231. 18. Palmer, G. H., Cereals in malting and brewing. In Cereal Science and Technology, Aberdeen University Press, Aberdeen, 1989, pp. 61-242. 19. Palmer, G. H., Responses to cereal grains in gibberellic acid. J. Inst. Brewing, 70 (1976) 378-80. 1