INTRODUCTION- It is an exo-enzyme use for the
hydrolysis of carbohydrate. Chemically,
it is also known as α- 1,4-glucan glycohydrolase in which glucan refers to
a sequence of connected glucose units and glycohydrolase made up of glycol and
hydrolyse, which mines breakdown of glycosidic linkage present in between two
glucose units. The enzyme identification code allocated to the Commission for
Enzymes EC 3.2.1.3 by IUBM1992. Number 3 denotes the
hydrolysis of large sugar molecule by the addition of water. While
number 2 denote the glycosidase, which breaks the glycoside
linkage present in between two glucose molecule [1].
Wheat bran is part of wheat so it contains many nutrients like protein,
carbohydrate, fat, and fibres etc. the average content of protein, carbohydrate, fat,
fibres and phosphorus are 16.4%, 65%, 4.5%, 6%, and 1.29%,
respectively. The 0.13% trace amount of calcium’s obtained in previous
report, given by the Islam et
al. [2].
The
glucoamylase can be produced by different type of microorganisms such as
bacteria, fungus, and yeast. A. niger can produce different proteolytic enzyme, carbohydrate
enzyme, and hydrolytic enzyme etc [3].
The nutrient content of fibre
can support the growth of the micro-organisms. The present
study deals with the use of cost efficient wheat bran for the solid state
fermentation for the production of glucoamylase and evaluated the production
level.
MATERIALS AND METHODS
The present study was proceed from March 2019 to June 2019 in
the Department of Biochemical Engineering, HBTU Kanpur, India.
Culture of Aspergillus niger- A. niger
culture was procured from National Chemical Laboratory, Pune (Maharashtra),
India. A. niger culture was grown on potato
dextrose agar (PDA) media and incubated at 30°C for 5 days.
Preparation of Inoculum- Spores were used to inoculate production medium. For sporulation,
flasks containing 5 gram moistened substrate was inoculated from 5 days
old slants followed by incubation for 5 days at 30°C
temp. At the end of incubation period, the fermented mass mixed with 50 ml of
sterile saline water, containing 0.1% Twin 80 and then left for 20 min. It
was then filtered to remove unwanted components of medium from spores. Spore
count was performed to adjust the count (5107 spores/ml) in spore suspension.
For counting of spores, spread plate method was used after serial dilution of
filtered containing spores [4].
Glucoamylase
enzyme
Extraction- The glucoamylase enzyme was estimated
by reducing sugar and the reducing sugars liberated were determined by
Dinitrosalicylic acid (DNSA) method that given by Miller [5].
Preparation
of standard graph of Glucose- Taken
different test tube and prepared the 1 ml different concentrations (0, 0.2,
0.4, 0.6, 0.8, and 1 mg/ml) of glucose by the help of distilled water and added
to 1 ml of DNS (Dinitrosalcilic acid) in each test tube and incubated the test
tube in boiling water bath at 100oC for 10 min duration. After that
cool the test tube and taken the OD at 540 nm wavelength [6].
.png)
Fig. 1: Standard
glucose curve of Glucose concentration
Optimization of the fermentation parameters
for glucoamylase production by using wheat bran as a substrate-
Optimization of Carbon source- To
study, the effect of the carbon source on production glucoamylase, media was
prepared by using different 1% carbon source (glucose, fructose, maltose,
starch, and sucrose) and media was inoculated by A. niger and incubated
at 35oC for 5 days duration. The other fermentation parameters
were kept constant.
Optimization
of Nitrogen source- To study, the effect of the different
nitrogen source on the production of glucoamylase, 1% of different nitrogen
source (urea, peptone, yeast extract, malt extract, and ammonium sulphate) were
added in different fermentation medium and media was inoculated by A. niger and incubated at 35oC for 5 days duration. The other
fermentation parameters were kept constant [7].
Optimization
of Temperature -
Effect
of the temperature on glucoamylase production was also investigated to know
the optimized temperature for enzyme production. For this, the inoculated
medium was incubated for 7 days at different temp. (25°C, 30°C,
35°C, 40°C, and 45°C).
The other fermentation parameters were kept constant [8].
Optimization
of pH- Effect
of initial pH on glucoamylase production was investigated to know the optimum
pH for enzyme production. For this study, initial pH of medium was adjusted to
different initial pH value (3, 4, 5, 6, 7, and 8) at the end of the incubation
period (7th days) enzyme activity was determined. The other
fermentation parameters were kept constant [9].
Inoculum age- Inoculum
of different ages (1 to 7 days old) was used to study the effect of inoculum
age on glucoamylase production. The other fermentation parameters were kept
constant [10].
Inoculum
level- Effect of inoculum level on glucoamylase production
was examined to know the inoculum size for best enzyme production. Different
inoculum levels between 5–30 % (5, 10, 15, 20, 25, and 30) were used and
at the end of incubation period (7 days) the enzyme activity was measured. Rest
of the fermentation parameters were maintained
constant [11].
RESULTS
Effect of carbon with wheat bran-
To study, effect of the carbon source on production glucoamylase, media was
prepared by using 1% different carbon source (glucose, fructose, maltose,
starch, and sucrose) and media was inoculated by A. niger and incubated at 35oC for 5
days. As seen from Fig. 2, the maximum enzyme activity (0.450913 U/ml) was
found at fructose as a carbon source. The lowest activity (0.316658 U/ml) was
at starch as carbon source. The enzyme activity of other carbon source glucose,
maltose and sucrose, was 0.387952, 0.339805, and 0.360175 respectively.
.png)
Fig. 2: Production of glucoamylase
at different carbon supplements with wheat bran
Effect of nitrogen with wheat bran-
To study, the effect of the different nitrogen source on the production of
glucoamylase, 1% of different nitrogen source (urea, peptone, yeast extract,
malt extract, and ammonium sulphate) were added in different fermentation
medium and media was inoculated by A. niger and incubated at 35°C for 5 days.
As seen from Fig. 3, the maximum enzyme
activity (0.449987 U/ml) was found at peptone as a nitrogen source. The lowest
activity (0.299992 U/ml) was at urea as a nitrogen source. The enzyme activity
of another nitrogen source yeast extract, malt extract, and ammonium sulphate
were 0.417581, 0.391656, and 0.412026, respectively.
.png)
Fig. 3: Production of glucoamylase
at different nitrogen supplements with wheat bran
Effect
of temperature- As seen from Fig. 4, the maximum
enzyme activity (0.425914 U/ml) was found at temperature 35oC. The enzyme activity (0.2036.98 U/ml) on lower temperature was
observed at 25oC temperature. Between temperatures 25oC to 45oC, the enzyme activities were
(0.361101, 0.333324 and 0.287029 U/ml) at temp. 30oC, 40oC and 45oC respectively.
.png)
Fig. 4: Production of glucoamylase at different Temperature
Effect of pH- As
seen from Fig. 5, the maximum enzyme activity (0.444432 U/ml) was found at pH
6.0. The enzyme activity on lower activity (0.194439 U/ml) was observed to be
at pH 3.0. Between pH 3.0 to 8.0, the enzyme activities were (0.268511,
0.333324, 0.398137, and 0.314806 U/ml) at pH 4, 5, 7 and 8 respectively.
.png)
Fig. 5: Production of glucoamylase at different pH
Effect of inoculum age- As
seen from Fig. 6, the maximum enzyme activity (0.459246 U/ml) was found
inoculum age at 5 days. The lower enzyme activity (0.362027 U/ml) was observed
at 2 days inoculum. Between inoculum ages 1 day to 7 day, the enzyme activities
were 0.327769, 0.348138, 0.399989, 0.412026 and 0.378693 U/ml at inoculum age
1,3,4,6 and 7 day respectively.
.png)
Fig. 6: Production of glucoamylase
at different inoculum age
Effect of inoculum level- As
can be seen from Fig. 7, the maximum enzyme activity (0.450913 U/ml) was found
at inoculum level at 15%. The lowest enzyme activity (0.299992 U/ml) was
found at 5% inoculum level. Between inoculum level 5% to 30%, the enzyme
activities were 0.42221, 0.419433, 0.380545, and 0.3601751 U/ml at inoculum
level 10, 20, 25 and 30% respectively.
.png)
Fig. 7: Production of glucoamylase
at different inoculum level (%)
Production
of glucoamylase at optimized condition- From the above study, we got the best condition for the production of
glucoamylase from A. Niger sp. The production of glucoamylase at
fructose as a carbon source, peptone as a nitrogen source the pH of the media
was 6 and inoculum level 15%. Media was inoculated with 5 day old culture. At
given condition production was observed as 0.6932 U/ml.
DISCUSSION- Solid state fermentation of wheat bran give a clear idea to
utilize food waste for the production of glucoamylase but the amount of
glucoamylase produced during this process was very less. The
additional carbon and nitrogen source was given to support the initial growth
of A. niger. Pavezzi et al. [12] reported that potato
starch as a best substrate for the production of glucoamylase (8.3 U/ml) by A. awamori
in combination with Saccharomyces
cerevisiae, while we have reported, the maximum production of 0.6932 U/ml, there was great difference in
the concentration of glucoamylase, this may be due to different organism or due
to different substrate. Pavezzi et al. [6], Norouzian
et al. [12] and, Adefisoye et. al. [13] had
suggested that pH 6 was taken for the maximum production of glucoamylase. The
current study also suggested similar result.
The
similar study was done by Anto et al. [14] suggested that temperature play a
violet role in the production of glucoamylase, the maximum production of
glucoamylase was at 55oC while current study suggested 35oC
for the maximum production, this may be due to difference in microorganism.
Another previous research study done by Lakshmi et al. [15] that suggested that maximum production of
glucoamylase had produced by nitrogen source i.e. peptone (a organic compound).
Inoculum age and inoculum size change with the change of microorganism
different study suggest 3 day to 7 day inoculum age
and 5 to 25% inoculum size were given by other previous studies i.e. Kumar et al. [16]; Selvakumar et al. [17]; Papagianni et al. [18]; Kumar et al. [19];
Arasaratnam et al. [20]. While this study suggest 5 day inoculum
age and 15% inoculum size, which are similar to the above study.
CONCLUSIONS- The use of wheat bran as substrate for the production of
glucoamylase by A. niger can be a great
idea for the utilization of waste food material. For the grater production of
glucoamylase the solid state media of wheat bran was supplemented with
different carbon source and nitrogen source. The maximum production of
glucoamylase was observed at fructose as a carbon source, peptone at a nitrogen
source. Apart from carbon and nitrogen source some other factor likes pH (6);
inoculum level (15%) and inoculum age (5 day) also played a vital role in the
production glucoamylase.
The present
study revealed that wheat bran can be use as substrate for the A. niger for the
production of glucoamylase but the production was very less, this may be due to
the complex nature of the wheat bran. The production of glucoamylase can be
increase by pre-treatment of wheat bran.
ACKNOWLEDGEMENTS-
We would like to
thank my supervisor Professor S. J. Nagar Sir and all teaching and non-teaching
staff of Department of Biochemical Engineering.
CONTRIBUTION OF AUTHORS
Research concept- S. J. Nagar
Research design- Khushboo Gupta
Supervision- S.
J. Nagar
Materials- Khushboo
Gupta
Data collection- Khushboo
Gupta
Data analysis and Interpretation- Khushboo Gupta,
S. J. Nagar
Literature search- Khushboo Gupta
Writing article- Khushboo Gupta
Critical review- S.
J. Nagar
Article editing- S.
J. Nagar
Final
approval- S. J. Nagar
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