World Journal of Pharmaceutical ReseaRch

Suribabu et al.

World Journal of Pharmaceutical Research

Volume 3, Issue 3, 4958-4964.

Research Article

ISSN 2277 7105

APPLICATION OF PARTIALLY PURIFIED -AMYLASE PRODUCED

BY BREVIBACILLUS BORSTELENSIS R1 IN FOOD INDUSTRIES
1*

K.Suribabu, 1T.Lalitha Govardhan and 2K.P.J Hemalatha

PG Department of Microbiology and Research Centre, Dr.Lankapalli Bullayya Postgraduate College, Visakhapatnam-530 013, A.P, India.

Department Microbiology, Andhra University, Visakhapatnam-530 003, A.P, India.

Article Received on
12 March 2014,
Revised on 05 April 2014,
Accepted on 28 April 2014

ABSTRACT
The -amylase produced by Brevibacillus borstelensis R1 has a
number of applications in many fields such as bakery industry, food
preparations, automation dishwashing, ethyl alcohol dual fermentation,
sago and rice industrial effluent treatment, sewage water treatment,

*Correspondence for
Author

fodder production, laundry and textile industries. The objective of the

study is to test the application of partially purified -amylase in

Dr. K. Suribabu
PG Department of

increasing the shelf life of bread and in preparations of puri and

Microbiology and Research

chapatti. In bakery industry, the anti-staling effect of bread and buns

Centre, Dr.Lankapalli Bullayya

was increased to 11days when compared with normal shelf life of

Post-graduate College,

5days. In preparation of puri and chapatti, dough mixed with -

Visakhapatnam, A.P, India -

amylase has improved taste, flavor, crust color, softness and toasting

530 013.

qualities than normally prepared dough with water. This is only a pilotscale experimentation, further quantitative experiments are awaited.
There is a need to conduct trials which are safe to health.
Keywords: Brevibacillus borstelensis R1, -Amylase, bakery industry, preparation of puri
and chapatti.
INTRODUCTION
Today in the global enzyme market, -amylases account for about 30% [1, 2]. Alpha-amylase
plays an important role in the synthesis of starch conversion products. The presence of
polysaccharides in sugarcane creates problem throughout the sugar manufacturing process,
which was eliminated by the action of amylase. The high quality products depend upon the
efficiency of the amylase which leads to reduction of cost [3].

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Couto & Sanromn [4] described how amylases were employed in processed-food industry
such as fruit juices, starch syrups, baking, production of cakes, brewing, and preparation of
digestive aids. Alpha-amylases have been used in the bakery industry for over more than
hundred

years.

Commercially,

thermostable

maltogenic

amylase

of

Bacillus

stearothermophilus is being used in the bakery industry [5].

Amylases added to the dough of bread to degrade the starch in the flour into smaller dextrins,
which are subsequently fermented by the yeast. The addition of -amylase to the dough
results in enhancing the rate of fermentation and the reduction of the viscosity of dough, for
the improvement in the volume and texture of the product. Moreover, it generates additional
sugar in the dough, which improves the taste, toasting qualities of the bread and crust colour.
In addition to generating fermentable compounds, -amylases improve the moisture retention
of the crumb, enhancing softness, anti-staling effect, shelf life and freshness [6-13].
The heat stable bacterial -amylases have been useful in anti-staling additives by delaying the
starch retrogradation [14]. Stickiness can be solved by using maltogenic amylases to produce
linear oligosaccharides of 2-6 glucose residues. Alpha-amylase induces dextrinization of
starch granules reducing its ability to immobilize water as free water increases dough
mobility [15]. Mesophilic bacterial amylases have been confirmed to be useful in anti-staling
by retarding the starch retrogradation. A thermostable maltogenic amylase of Bacillus
stearothermophilus has been used commercially in the bakery industry [16-20]. Bacterial amylases have higher thermal stability and survive the baking temperatures [21, 22].
MATERIALS AND METHODS
Estimation of amylase by DNS method
Maltose produced by the hydrolytic activity of -amylase on -1, 4 linkages present in
polysaccharides, reduce 3, 5 dinitro salicylate to an orange red colored 5-nitro 3-amino
salicylate which can be measured at 520nm. The starch substrate [0.5ml of 0.5% in 0.1M
phosphate buffer (pH 6.8)] was mixed with 1% (0.2ml) NaCl in a test tube and pre incubated
at 370C for 10 minutes. The supernatant collected from the centrifugation of the production
media was used as enzyme source, 0.5ml of this was added to the reaction mixture. The
reaction was terminated by the addition of 1.0 ml of 3, 5-dinitrosalicylic acid reagent [1.0 gm
DNS in 0.8% NaOH, 60% Na K tartrate] after incubation at 370C for 15 minutes. The
contents were mixed well and kept in boiling water bath for 10 minutes. Then they were
cooled and diluted with 10 ml of distilled H2O. The color developed was read at 520nm. One
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unit of enzyme activity is defined as the amount of enzyme that releases 1.0 mmol of
reducing sugar (maltose) per minute under the assay conditions (Miller, 1959).
Bakery industry
The ingredients of milk bread are Maida (Vijay Gold Brand) 10kg, salt 170gm, Bakers yeast
(Activated by dissolving in one liter of luke warm water) 35gm, sugar 2.3Kg, milk
(Pasteurized) 1.0L and Vanilla powder 25gm.The ingredients were dumped in a molish
machine (Manf. Newton, India) and thoroughly mixed for 30 minutes. The mixed dough was
allowed to stay for 20minutes for the action of yeast. The 100gms dough lumps without
amylase were used as control. Partially purified -amylase (100U/mg protein) was added to
the dough as test samples and allowed the enzyme action for 15minutes. Later on, dough
lumps were prepared for buns and bread and baked in an oven at 2000C for 15minutes.
The control and test buns and bread were stored at room temperature to observe the shelf life
and antistaling nature of the amylase on the products for two weeks.
Puri and Chapatti preparation
The wheat flour was thoroughly mixed with water for dough preparation. For test samples,
the wheat flour was thoroughly mixed with partially purified -amylase (100U/mg protein).
The dough without enzyme was used as control. Then the dough lumps were incubated for 30
minutes at 250C and used to prepare puri and chapatti. The colour, texture, softness and oil
absorption qualities were tested.
Statistical analysis
All the experiments were conducted in triplicate. The results were given as mean value
standard deviation. The conditions were analysed to determine the singnificant difference
between the variables by one way ANOVA, two way ANOVA and correlation analysis by
using the scientific graph pad (Prism 6.1version software).
Analysis of variance (ANOVA) refers to the examination of differences among the sample
means. It is used to examine the significance of the difference amongst more than two sample
means at the same time.

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RESULTS
Bakery industry
In bakery industry, the application of -amylase along with yeast, reduced the thickness of
dough thereby improved taste, crust color, softness and toasting qualities of the bread than
normally baked bread. The shelf life of bread and buns was increased to 11days when
compared with 5days of normal shelf life (Fig. 1a-d).
This is only a pilot-scale experimentation, further quantative experiments are awaited. There
is a need to conduct experiments which are safe to health.

Fig: 1 Shelf life of bread

a, control without -amylase and test with -amylase (100 U/mg protein) after 5 days;
b, control without -amylase and test with -amylase (100 U/mg protein) after 7 days;
c, control without -amylase and test with -amylase (100 U/mg protein) after 11 days
and d, control without -amylase and test with -amylase (100 U/mg protein) shelf life
of bun after 11 days.
Puri and Chapatti preparation
Application of -amylase (100U/mg protein) in Puri and Chapatti dough preparation
improved taste, flavor, crust color, softness and toasting qualities than normally prepared
dough with water (Fig. 2a-h). This is only a pilot-scale experimentation, further quantative

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experiments are needed. There is a necessity to conduct (trials) experiments that are safe to
health.
a

g
e

Fig: 2 Puri preparations: a, dough with -amylase; b, dough with water;

c, puri with -amylase and d, puri with water.
Chapatti preparation: e, dough with -amylase; f, dough with water;
g, chapatti with -amylase and h, chapatti with water.
DISCUSSION
In the present study, the application of -amylase produced from Brevibacillus borostelensis
R1 had improved taste, crust color, softness and toasting qualities of the bread. In bakery
industry, the shelf life of bread and buns was increased from 5 days to 11days. Similar work
on - amylase produced by Bacillus spp. was reported in improving the quality and shelf life
of the bread in bakery industry by Hebeda et al. (1991), Van Damme et al.(1992), Hamer
(1995), Armero & Collar (1996), Qi Si (1996), Sahlstrom & Brathen (1997), Christophersen
et al. (1998), Dauter et al. (1999), Martinez et al. (1999), Nielsen & Borchert (2000) and
Van der Maarel et al. (2002).

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CONCLUSION
In bakery industry, the anti-staling effect of bread and buns was increased to 11days when
compared with normal shelf life of 5days. In preparation of puri and chapatti, dough mixed
with -amylase has improved taste, flavor, crust color, softness and toasting qualities than
normally prepared dough with water. This is only a pilot-scale experimentation, further
quantitative experiments are awaited. There is a need to conduct trials which are safe to
health.
ACKNOWLEDGEMENT
We thank Management of Dr.Lankapalli Bullayya College, Visakhapatnam for the financial
support and facilities provided to make this work possible.
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