Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
SPECTROPHOTOMETRIC STUDIES ABOUT AMYLASE ACTIVITY
IN STARCH HYDROLYSIS REACTION
INTERNAL REPORT
2012
CĂRĂBAN ALINA, POPA VASILICA, MACOCIAN EUGEN, FILIP SANDA
University of Oradea, Romania
Abstract
The experimental work is aimed to determine the influence of some factors
such as cations and anions, the contact surface between starch and amylase and the
amylase extraction depending on the degree of damage to the endosperm using the
DNS method in order to determine the amylase activity in starch hydrolysis.
I.
INTRODUCTION
The amylase activity can be measured following the decrease of the viscosity
of a starch solution, the decrease of the turbidity of a starch suspension, the
decrease of the intensity of a starch-iodine reaction and the increase of the reducing
groups in the reaction medium. The last method is in agreement with the EC-IUB
demands.
The amylase activity is measured using a colorimetric method with DNS
reagent (3,5-dinitrosalicylic acid) after Hosttettler and co., modified by the authors in
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
order to ensure the appropriate conditions for the hydrolyse of starch in baking
industry;
-
The hydrolyse reaction had place at pH 5,5 (the optimum pH for wheat
amylase);
-
As enzymatic activator we used a solution of CaCl2 (Ca ions are activators for
the wheat amylase) in appropriate concentration 0,01M;
-
The hydrolyse reaction occurred at 30ºC, in accordance with that of the dough
fermentation in baking industry.
The starch is hydrolysed on the catalytic action of amylase to fragments, which
can be determined with 3,5-dinitrosalicylic acid, due to their semiacetalic reducer
groups. The formed nitroaminosalycilic acid concentration is corresponded to the
enzymatic activity of amylase.
II.
EXPERIMENTAL
Reagents
Soluble starch supplied by Merck, Darmstadt was used in 1% concentration in
acetate buffer solution at pH 5,5 which contain CaCl2 0,1M.
The DNS reagent (Merck, Darmstadt) was obtained from 1g 3,5-dinitrosalcylic
acid dissolved in 20 ml NaOH 2N, adding 30g double tartrat of natrium and
potassium and completed with distillate water to obtain 100ml solution.
It was used an enzymatic extract of alpha and beta amylase from wheat flour
prepared by extracting 10 g wheat flour in 100 ml distillate water for 30 minute using
a magnetic stirring, than centrifuging at 6000 rpm and the supernatant obtain was
dilute 10 times in distillate water.
Maltose was used as standard solution in the concentration as 100 g/ml.
Starch was used as enzyme substrate in the following forms:
• soluble starch
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL

A1 - Merck soluble starch (1% soluble starch dissolved in phosphate buffer pH
7, containing 0.01 M CaCl2) in order to determine the optimum pH of action of α - and
β - amylase in wheat.

A2 - Merck soluble starch 1% in acetate buffer pH 5.5, used to determine the
influence of the presence of cations and anions in the hydrolysis reaction and the
determination of optimal concentration of calcium ions.

A3 - Merck soluble starch 1% in pH 5.5 buffer containing 0.01 M CaCl2 in
order to determine the optimum temperature of amylase activity in the hydrolysis
reaction;
• granular starch

A4 - granular starch has been obtained from the washings of gluten, which
were collected and centrifuged consecutively for 5 minutes at 6000 r/min and
successive washes, after which it was dried at room temperature for 72 hours.

Granular starch thus obtained (A4) was subjected to a grinding operations
laboratory mill for 5 minutes, 10 minutes, 20 minutes and 25 minutes, achieving
granular starch A5, A6, A7 and A8, with varying degrees damage to grain starch.
The damage degree of the starch granule was revealed under a microscope by
staining with iodine.
Colorimetric analysis
The reaction mixture formed by soluble starch solution, the amylase extract
and the analysed factor was incubated for 5 minutes at 30ºC for the enzymatic
hydrolyse reaction and then the reaction was stopped with DNS reagent and by
boiling the reaction medium for 5 minutes. After cooling, the mixture was colorimeter
at 546 nm related to distillate water.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
To determine the reducing sugars existing in the reaction medium at initial
moment, for all samples were made controls, identically with the tests, except that in
the controls there was no enzyme.
To transform the optical densities read for the tests and controls in moles
maltose it was made a standard curve in concordance with the data from table 1.
The experiment results were calculated as follow: the optical densities for the
controls were subtracted from the test extinction and the results were expressed in
moles maltose enzymatic delivered/1ml/1min.
Table no. 1. Standard curve
Maltose (ml)
0,1
0,3
0,5
0,7
1
-
Distillate water (ml)
0,9
0,7
0,5
0,3
-
1
DNS reagent (ml)
1
1
1
1
1
1
Boiling 5 minutes
Distillate water (ml)
8
8
8
8
8
8
Extinction
0,150
0,305
0,455
0,605
0,765
0,800
Equation
1,0
Adj. R-Square
y = a + b*
0,99126
Value
Optical density
0,8
Standard Error
optical density Intercept
0,09701
0,01964
optical density Slope
0,69037
0,03238
0,6
0,4
0,2
0,0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Maltose (microgram/ml)
Figure 1. The standard curve
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
III.
RESULTS AND DISSCUSIONS
1. The influence of some cations over the amylase activity in starch
hydrolysis reaction
We analyzed the enzymatic activity of  and - amylase from wheat in the
presence of Ca2+, Ba2+, Sr
NH4+, Zn
2+
, Ni2+, K+, Li+, Al
3+
, Sb
3+
, Fe
3+
, Cr
3+
, Mn
2+
, Hg
2+
,
2+
, Co2+, Cd2+ ions using colorimetric method for determination of amylase
activity with DNS reagent, using as substrate soluble starch A2 and as amylase an
aqueous extract E1.
Work Mode No.1
Blank
Sample
Starch (ml) A2
0,5
0,5
DW (ml)
0,3
0,3
-
0,1
Cation (ml)
Enzyme (ml) E1
0,1
0,1
0
Incubation 5 minute at 30 C
DNS (ml)
1
1
Boiling 5 minutes
DW (ml)
8
8
From practical results obtained is noted that the calcium ion is the only cation
activator of amylase activity, other cations studied had different effects as amylase
inhibitory activity [3].
Inhibitory action is embodied in five directions:
1. ions K+ and Al3+ have a weak inhibitory effect;
2. ions Ba2 +, Sr2+, Li +, Ni2+ have a moderate inhibitory effect;
3. ions Zn2 + and Co2+ are stronger inhibitors;
4. ions NH4+, Fe 3+, Cr 3+, are powerful inhibitors;
5. ions Mn 2+, Hg 2+, Cd2+ denaturated amylases.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
Amylase is an enzyme requires the presence of metal ions of calcium in its
molecule for its enzymatic activity. Calcium is bound by ionic bonds with carboxyl
groups in the structure of amylase, amylase molecule so that each atom contains
closely related gram of Ca2 + / molecule. As such, the calcium ion is an activator of
amylase activity [4].
Other cations studied the different effects of inhibition of amylase activity by
altering the conformation space of amylase in varying degrees, to distortion and
precipitation reactions with metals present in the hydrolysis of starch molecules and
destabilize the attraction that the groups OH of starch.
The influence of the presence of metal ions in the hydrolysis of starch with
amylase in wheat flour is shown in Figure 2.
Figure 2. The influence of some cations on the amylase activity
2. The influence of the presence of some anions in the starch hydrolysis
with amylases
It was studied using colorimetric method for determining amylase activity in
DNS the influence of the presence of the following anions HCO3−, CO32−, HSO4−,
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
SO42−, NO2−, NO3−, HCOO−, C6H5O73−, H2PO4−, HPO42−, Cl−, CH3COO−, in the
hydrolysis of starch by amylase of wheat flour using as substrate, soluble starch A2
and the enzyme amylase aqueous extract E1
In parallel, a blank test was performed and containing no anion.
Table no.3 Work Mode no. 3
Blank
Samples
Starch A2(ml)
0,5
0,5
DW (ml)
0,3
0,3
Anion (ml)
-
0,1
Enzyme E1(ml)
0,1
0,1
0
Incubation 5 minutes at 30 C
DNS (ml)
1
1
Boiling 5 minutes
DW (ml)
8
8
Figure 4. The influence of anions on the amylase activity
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
From the results obtained, it appears that carbonate and bicarbonate ions are
moderate inhibitors for amylase activity. The other anions have no influence over the
amylase activity. Therefore the use of bicarbonate ion as additional chemical for
fermented dough causes a decrease of hydrolytic activity of amylase, an aspect that
can be used as a way of correcting the grain quality with high amylase activity (wheat
grass, wheat being attacked by bedbugs wheat and so on).
3. The influence of amylase concentration over the granular starch
hydrolysis
It was studied the influence of amylase concentration over the hydrolysis of
starch granules using the colorimetric method for the determination of amylase
activity with DNS reagent. It was used as enzyme substrate A6 granular starch and
the enzyme amylase aqueous extract of wheat flour E1 in different concentrations.
From the experimental data obtained, it is observed that with increasing
enzyme concentration ("dispersion" lower enzyme) occurs an increase in enzyme
activity. This indicates that not all molecules present in the reaction medium amylase
is active, only those who come in direct contact with the starch granule.
Granular starch is deposited on the bottom so that the area of action of
amylase is limited. If the number of amylase molecules in contact with granular starch
decreases due to the increase in amylase release molecules (low concentration of
amylase solution), the amount of hydrolysis products decreased.
The experimental results are plotted in Figure 5.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
Work Mode no. 4
Sample
Sample
Sample
Sample
Sample
Sample
1
2
3
4
5
6
Starch A6 (g)
1
1
1
1
1
1
DW (ml)
30
25
20
15
10
5
Enzyme (ml)
5
5
5
5
5
5
0
Incubation 15 minutes at 30 C
Prelevation (ml)
5
5
5
5
5
5
DNS (ml)
1
1
1
1
1
1
4
4
4
4
Boiling 5 minutes
DW (ml)
4
4
Figure 5. The influence of amylase concentration over hydrolysis of starch
4. The determination of the contact surface influence between starch
granules and amylase
We observed the influence of surface contact between starch granules and
amylase extract using the working method, colorimetric determination of amylase
activity DNS reagent. We used samples of granular starch A6 1% as E1 enzyme
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
substrate and the enzyme amylase extract the same levels and working conditions,
but placed in beakers of different volumes (based on the different surfaces).
Work Mode no. 5
Sample1
Sample2
Sample3
Starch A6 (g)
1
1
1
DW (ml)
10
10
10
Enzyme (ml)
5
5
5
Incubation 15 minutes
In each sample was taken 5 ml of hydrolysis products over which added 1 ml
reagent DNS, was incubated at boil 5 minutes and then the samples were diluted
with 4 ml of distilled water after that were colorimetry at 546 nm.
The influence of the contact surface granular starch - amylase on hydrolysis is
illustrated in Figure 6.
Figure 6. Influence of the contact surface over granular starch hydrolysis with amylase
It is noted that for a larger contact surface, amylase activity increases. Starch
granules, accumulating at the bottom of the reaction vessel, limiting access only
amylase molecules floor of granular starch - amylase solution so, how this area is
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
higher, the more complex starches - active amylase formats is higher and therefore
the hydrolysis products formed in greater quantity.
5. Determination the influence of substrate condition over amylase activity
It was determined the influence of substrate condition on amylase activity in
wheat flour using colorimetric method for the determination of amylase activity DNS
reagent as No.6 mode.
It was used a biochemical sample containing the enzyme substrate granular
starch A6 (compared to a control performed with Merck soluble starch A3), and the
aqueous extract of amylase enzyme E 1.
From practical results it is observed that soluble starch hydrolysis go much
faster than that of granular starch (about 4 times faster). Granular starch is more
resistant to hydrolysis than the soluble starch.
Work Mode No.6
Blank
Sample
Starch (g)
Starch (A3) 0,5
Starch (A6) 0,5
DW (ml)
20
20
Enzyme E1 (ml)
5
5
0
Incubation 15 minutes at 30 C
Prelevation (ml)
5
5
DNS (ml)
1
1
Boiling 5 minutes
DW (ml)
4
4
The influence of substrate condition on hydrolysis of starch with amylase is
illustrated in Figure 7.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
Figure 7. The influence of substrate state over amylase activity
6. The determination of the degree of starch deterioration over amylase
activity
We determined the influence of the damage degree of grain starch on amylase
activity in wheat flour using samples containing granular starch with different degrees
of damage to the starch granule, A7, A8, A9 and A10 and amylase in wheat flour
extract E1 after mode 7, using colorimetric method for the determination of amylase
activity DNS reagent.
Of practical results it is observed that the damage degree of the starch granule
is higher, the hydrolysis reaction is easier due to amylase release from granule. This
can be used as a means of correcting the baking quality of wheat.
Work Mode no. 7
SampleA7
SampleA8
SampleA9
SampleA10
Starch (g)
1
1
1
1
DW (ml)
8
8
8
8
Enzyme (ml)
1
1
1
1
0
Incubation 15 minutes at 30 C
Prelevation (ml)
5
5
5
5
DW (ml)
1
1
1
1
4
4
Boiling 5 minutes
DW (ml)
http://www.huro-cbc.eu/
4
4
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
III.
CONCLUSIONS
Amylase activity of wheat flour is influenced by a number of exo- and
endogenous factors .The presence in the hydrolysis medium of a certain substances,
determine the growth rate of starch hydrolysis reaction. This can be used practically
to correct the quality of fermentative processes with low endogenous amylase by use
of these substances in the production process.
On the other hand, various metal ions (Ba2+, Sr2+, Ni2+, K+, Li+, Al3+, Sb3+, Fe3+,
Cr3+, Mn2+, Hg2+, NH4+, Zn2+, Co2+, Cd2+), anions (carbonate and dicarbonate), lowers
the rate of hydrolysis of starch with amylase endogenous, so that factors can be used
to improve grain quality with high amylase activity.
Status of starch, the amylase concentration and the optimum conditions for
amylase action are also factors that are influencing the rate of starch hydrolysis.
Study of factors that alter or speed the reaction of amylase endogenous wheat
flour, which involved a large number of experimental measurements was performed
using colorimetric method for the determination of amylase activity using DNS
reagent, modified by the authors, which was priced very well analyzes.
Using this method requires, however, the performance of predeterminations
for finding optimal working concentrations and volumes and some "training" regarding
mode.
By knowing the various factors influencing the hydrolytic activity of amylase in
grains can be made practical working conditions (pH, temperature, presence or
absence of effectors), which increase or inhibit the amylase activity.
The fundamental objective is was to study the hydrolysis of starch amylase by
analyzing the influence of exogenous and endogenous factors, frequently present in
fermentative industry.
The presence of cations and anions in the hydrolysis can activate or inhibit
amylase activity until canceled. Calcium ion is an enzyme activator. Its concentration
optimal for the hydrolysis reaction is 0.01 M.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
Ions of mercury, lead, manganese and antimony totally inactivates the
enzyme. The presence of anions in the hydrolysis does not change amylase activity
except carbonate and sodium bicarbonate are moderate inhibitors for amylase. As
such, the use of sodium bicarbonate as additional raising agent in bakery products
obtained with yeast, causes a lower rate of hydrolysis of starch in dough
fermentation.
Status of starch (soluble or granular) has a major effect on enzymatic
hydrolysis. Thus, soluble starch hydrolysis goes much faster than that of granular
starch, which is influenced by the degree of damage to the starch granule, in the
sense that the degree of deterioration of grain is higher, the rate of hydrolysis
increases.
REFERENCES
[1]. Kimura, A.; Robyt, J. F. (1995) Reaction of enzymes with starch
granules: kinetics and products of the reaction with glucoamylase.
Carbohydr. Res. 277, 87-107.
[2]. Cărăban A., Bungău S, Fodor A, Stănăşel O., (2006), Influenţa acidului
ascorbic, tiaminei şi riboflavinei asupra reacţiei de hidroliză a
amidonului cu amilaze endogene, utilizând interferometria laser,
Revista de Chimie, 57, pp 607-609
[3]. Cǎrăban A, Stǎnǎsel O, Gavriş G, Badea G, (2008), Studies about the
influence of some food lipids over the starch hydrolysis reaction using
laser interferometry technics, Proceedings of Indian Conference,
Impending approaches to Environmental Menace (IMAEM) 2008 TBML
College, Tamil Nadu, 25-26th September, South-INDIA, pp.191-193.
[4]. Hill, G.A., Macdonald, D.G. and Land, X., (1977) Alfa amylase inhibition
and inactivation in barley malt during cold starch hydrolysis,
Biotechologlogy Letters, 19 (11), pp. 1139-1141.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.
Two countries, one goal, joint success
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
[5]. Iordăchescu D., Dumitru I.F.(1988) Biochimie practică, Bucuresti, pp
161-168.
[6]. [Rouau X., Moreau S., Schoch B., Oosten K., (1993) - Cereal
Chemistry, 70, no.6, 626
[7]. McGregor A., Knutson V.(1993) - Starch/Starke , 51,127.
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.