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A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
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
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The content of this paper does not necessarily represent the official
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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 30C, in accordance with that of the dough
fermentation in baking industry.
The starch is hydrolysed on the catalytic action of amylase to fragments, which
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
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The content of this paper does not necessarily represent the official
position of the European Union.
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
influence of the presence of cations and anions in the hydrolysis reaction and the
determination of optimal concentration of calcium ions.
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.
Colorimetric analysis
The reaction mixture formed by soluble starch solution, the amylase extract
and the analysed factor was incubated for 5 minutes at 30C 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.
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The content of this paper does not necessarily represent the official
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A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
0,1
0,3
0,5
0,7
0,9
0,7
0,5
0,3
Boiling 5 minutes
Distillate water (ml)
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
0,09701
0,01964
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)
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The content of this paper does not necessarily represent the official
position of the European Union.
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
III.
2+
3+
, Sb
3+
, Fe
3+
, Cr
3+
, Mn
2+
, Hg
2+
2+
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)
Boiling 5 minutes
DW (ml)
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.
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The content of this paper does not necessarily represent the official
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A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
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The content of this paper does not necessarily represent the official
position of the European Union.
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)
Boiling 5 minutes
DW (ml)
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The content of this paper does not necessarily represent the official
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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).
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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
Starch A6 (g)
DW (ml)
30
25
20
15
10
Enzyme (ml)
Incubation 15 minutes at 30 C
Prelevation (ml)
DNS (ml)
Boiling 5 minutes
DW (ml)
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The content of this paper does not necessarily represent the official
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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)
DW (ml)
10
10
10
Enzyme (ml)
Incubation 15 minutes
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
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The content of this paper does not necessarily represent the official
position of the European Union.
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.
Sample
Starch (g)
DW (ml)
20
20
Enzyme E1 (ml)
5
0
Incubation 15 minutes at 30 C
Prelevation (ml)
DNS (ml)
Boiling 5 minutes
DW (ml)
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The content of this paper does not necessarily represent the official
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A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
SampleA8
SampleA9
SampleA10
Starch (g)
DW (ml)
Enzyme (ml)
Incubation 15 minutes at 30 C
Prelevation (ml)
DW (ml)
Boiling 5 minutes
DW (ml)
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The content of this paper does not necessarily represent the official
position of the European Union.
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
III.
CONCLUSIONS
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The content of this paper does not necessarily represent the official
position of the European Union.
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
REFERENCES
The content of this paper does not necessarily represent the official
position of the European Union.
A new method and system for real time fermentation process monitoring
HURO 1001/121/2.2.2
BIOETHANOL
http://www.huro-cbc.eu/
The content of this paper does not necessarily represent the official
position of the European Union.