Process Biochemistry 48 (2013) 374379

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Concentration by ultraltration and stabilization of phytase produced by

solid-state fermentation
Daniel E. Rodrguez-Fernndez a, , Jos L. Parada b , Adriane B.P. Medeiros a , Julio C. de Carvalho a ,
Luiz G. Lacerda b , Jos A. Rodrguez-Len b , Carlos R. Soccol a
a
b

Department of Bioprocess Engineering and Biotechnology, Federal University of Paran (UFPR), CEP 81.531-980, Curitiba, PR, Brazil
Department of Biological Sciences, Positivo University, R. Prof. Pedro Viriato Parigot de Souza, 5300, CEP 81.280-330, Curitiba, PR, Brazil

a r t i c l e

i n f o

Article history:
Received 2 October 2012
Received in revised form
19 December 2012
Accepted 29 December 2012
Available online 6 January 2013
Keywords:
Ultraltration
Stabilization
Long-term storage
Phytase
Aspergillus niger

a b s t r a c t
Ultraltration is an attractive downstream processing technique for concentrating enzymes and could
be considered the primary step of purication. However, the efciency of this process is often limited
by protein fouling and shear-induced enzyme inactivation, which decreases permeate ux and results
in the loss of enzyme activity. Although the rejection of phytase was higher than 99%, the loss of the
enzyme activity was 14% during operation, indicating that the shear forces generated in the lter have
signicant inuences on the enzyme activity. Two preparations using glycerol (25% and 35%, v/v) as a
cryo-protecting agent at different temperatures were studied. The preparation containing 35% glycerol
retained 70% of the initial enzyme activity at 70 C after 1 h and had more than 3 and 6 months storage
half-life at 29 C and 4 C, respectively.
2013 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction
The use of enzymes in industrial applications is increasing continuously, and most likely, the number of established biocatalyst
processes will double in the next years. Enzymes are frequently
used in the production of food, feed, detergents, pharmaceuticals
and other products. The enzyme market is rate of 5 billion US dollars
with an annual growth rate approximately 6.510% [13].
Phytases (myo-inositol hexaphosphate phosphohydrolases)
belong to a special class of phosphomonoesterases. These enzymes
are mainly used in the industrial preparation of feed to help pigs,
chickens and other monogastric animals digest the phytate present
in their diets. The addition of phytase to the feed of non-ruminant
animals enhance the release of the phosphate content of phytic
acid to improve animal nutrition. Therefore, the need for phosphate addition to the phytase-supplemented diets is reduced, and
the animals excrete less phosphate [46].
Downstream processes are important to efcient and economical production of enzymes in industry and the improvement of
the stability of these biological products. In this sense, the selection of the correct techniques is essential in preventing the loss of
enzyme activity. The number of studies on the concentration and
partial purication processes of proteins employing membranes

Corresponding author. Tel.: +55 41 3361 3271; fax: +55 41 3361 36 74.
E-mail address: danernesto72@yahoo.com (D.E. Rodrguez-Fernndez).
1359-5113 2013 Elsevier Ltd. Open access under the Elsevier OA license.
http://dx.doi.org/10.1016/j.procbio.2012.12.021

have increased during the last few decades. At present, ultraltration is one of the most widely used techniques for protein
concentration and can be used as a primary purication step. Ultraltration has been used in different biotechnological processes and
applied with success due to the low energetic consumption and the
efcient separation of small molecules of proteins and enzymes.
However, the loss of the enzyme activity has been observed in
many cases, mainly due to the shear force generated in the system and by the removal of ions that have a positive inuence on
the enzyme activity [79]. The process of concentration by ultraltration is inuenced by physicalchemical factors (i.e., pH, enzyme
concentration, temperature and composition), the characteristics
and geometric conguration of the equipment and the type and
pre-treatment of membranes [8].
The resistance of the catalytically active protein structure
towards high temperatures, pH and other denaturing factors are
some of the most important criteria for the commercialization
and industrial application of enzymes [1012]. Different tools have
been developed to reduce the loss of activity in the commercial products, and the efforts to increase the stability of enzymes
are focused mainly on the use of some additives, the genetic
modication of microorganisms and, recently, protein engineering
[2,1315].
Stabilizing an enzyme involves suppressing protein unfolding
and retaining the catalytic activity. Enzyme stability in aqueous
solutions can be maintained by using additives that have a low
molecular weight and are able to create hydrogen bonds with the

D.E. Rodrguez-Fernndez et al. / Process Biochemistry 48 (2013) 374379

enzyme that replace the bonds of water molecules (e.g., glycerol

and trehalose) [1618]. Trehalose, polyols, sucrose and other sugars
have been reported as additives for the long-term stabilization of
proteins. Denaturation of enzyme in water is caused by the hydratation of the peptide chain. Addition of glycerol (components of low
hydrophobicity) increase the stability of the enzyme in solutions
because the glycerol added is preferentially excluded from the surface layer of the protein molecule and the water shell around the
protein molecule is preserved, that means that the expose nonpolar groups remain intact conferring more rigidity to the molecule,
which is essential for protein stabilization. That rigidity prevents
the aggregation of proteins during the refolding of many proteins
[19].
Glycerol is a well-known substance capable of protecting
microorganisms under stress conditions and contributes to achieve
the proper folding of proteins and enzymes, increasing their stability and conservation for long periods of time [2026]. Glycerol is
obtained as by-product from biodiesel production, whose production has increased in the last years.
The increment in biodiesel demand generates a large amount of
cheap glycerol that can be used as feed additive in animal nutrition
as reported by several authors in scientic literature [2729]. In
Brazil the cost of glycerol is lower than other sugar alcohols such
as mannitol and sorbitol. This difference in the price made glycerol
the most attractive alternative to be employed as cryo-protecting
agent for phytase in liquid formulations.
This work addresses ultraltration as a method for concentrating the phytase from Aspergillus niger produced by the solid-state
fermentation (SSF) of citrus peel and the development of a suitable method for enzyme stabilization in phytase formulations to
be applied as feed additives for monogastric animals employing
glycerol. Enzymatic formulations should have the largest shelf life
as possible.
2. Materials and methods
2.1. Microorganism
The A. niger F3 strain, belonging to the Biotechnology and Bioengineering Division of the Federal University of Paran, was used for phytase production. Periodic
reactivation was made in Czapeck medium slants incubated for 7 days at 30 C. The
fungus was maintained in Czapek medium at 48 C.
2.1.1. Inoculum preparation
Remaining sugars (mainly fructose, glucose and sucrose) of juice extraction,
present in the citrus peel, were extracted from 10 g of ground dried citrus peel
mixed with 100 mL of distilled water and heated at 60 C for 15 min. Preparation
of the inoculum using those sugars allows to decrease the lag time that precedes
all fermentative process [30]. The solution was ltered after cooling to room temperature. Then, the solution was diluted 1:10 with distilled water, followed by the
addition of 0.3% NH4 NO3 (v/w). Finally, the pH was adjusted to 5.0. The culture
media was sterilized at 121 C for 20 min. The mycelium of the A. niger F3 was used
to inoculate the medium and was incubated in a shaker at 30 C and 120 rpm for
96 h [31].

375

2.3. Extraction of phytase from the solid fermented matrix

The enzyme was leached from the fermented material (at a solidliquid ratio
1/25 on a dry solid material) in a tank containing 10 L of distilled water. The extraction was performed for 45 min using an FISATOM Model 713D agitator, and the
suspension was centrifuged in a tubular CARL PADBERG modelo Z41 centrifuge at
10,000 rpm [33].
2.4. Concentration of phytase
The phytase present in the extract was concentrated via ultraltration employing a cassette system (Sartorious, Model No. SM 17521, Germany) using a Millipore
membrane with a 10 kDa nominal cut-off. The ow rate in the feeding operation
was xed at 1.8 L min1 , and the pressure drop was 5 psi g. The permeability of
the membrane was 49.2 mL m2 h1 atm1 . The retentate volume in time (VRet ) was
determined as the ratio of feed volume at the beginning of the operation (VFeed ) and
the volumetric concentration factor (VCF). The runs were performed three times. The
volumetric concentration factor was calculated according to the following relation:

VCF =

VFeed
VRet

2.5. Activation and long-term storage stability of phytase

Glycerol was employed for studying the stability of the concentrated phytase
during storage. The study was performed under three conditions: control (without glycerol) or with glycerol at 25% or 35% (v/v). Eighteen 50 mL glass asks were
used to store the preparations: six asks were prepared for the control, six for 25%
glycerol and six for 35% glycerol at different temperatures. Each week, three bottles were chosen at random for enzyme analysis. The preparations were stored at
4 C, 29 C, 37 C or 45 C for four months (16 weeks), and periodic samples were
obtained to determine the phytase activity. To predict the stability of the enzymes,
the inactivation constants were determined using the Arrhenius equation.
2.6. Phytase activity
The phytase activity was determined at 50 C in 350 L of 100 mM sodium
citrate buffer, pH 3.0 containing 875 nmol sodium phytate [34]. The enzymatic reaction began when 50 L of enzyme solution was added to the assay mixture. After
incubating for 10 min at 50 C, the liberated phosphate was measured using to the
ammonium molybdate method. Then, 1.5 mL of a freshly prepared solution of acetone/5 N H2 SO4 /10 mM (NH4 )6 Mo7 O24 (2:1:1, v/v) and 100 L 1.0 M citric acid was
added to the assay mixture. Any opaqueness to the solution was removed by centrifugation prior to measuring the absorbance at 355 nm. To calculate the enzyme
activity, a calibration curve was determined over the range of 5600 nmol phosphate ( = 1.872 cm2 /nmol). The activity units was expressed as 1 mol phosphate
liberated per mL per min (mol mL1 min1 ). The blanks were prepared by adding
the (NH4 )6 Mo7 O24 solution to the assay mixture prior to adding the enzyme.
2.7. Protein concentration
The total protein content was measured using the method of Lowry with bovine
serum albumin (BSA) as the protein standard [35].
2.8. Ionic composition of the extracts
In order to analyse the content of cations in the extracts, several ions (Na+ , NH4 + ,
K+ , Mg2+ and Zn2+ ) were detected using the ionic chromatography methodology (761
Compact IC from Methrom, USA) before and after concentration.

3. Results and discussion

2.2. Solid-state fermentation
Phytase production in citrus peel is regulated by induced mechanism due to
the low concentration of phytic acid (lower than 0.025 mol per gram) present in
that solid residue [32]. Citrus dried peel was used as solid substrate and support,
with 75% of the particles with sizes from 0.8 mm to 2 mm and the other 25% of the
particles with sizes between 2 and 3 mm. The combination of both size particles give
a better mass and heat transfer during fermentative process. The complete medium
for solid-state fermentation was prepared by the addition of the following salts in a
dry state: 0.43% NH4 NO3 , 0.021% NaSO4 , 0.077% MgSO4 7H2 O, 0.042% ZnSO4 7H2 O,
0.162% KCl and 0.011% Ca(OH)2 [29].
Water was added to the medium in order to obtain 60% initial humidity. The
inoculation was made at 1:10 (v/w) of 1 kg of wet solid media. The fermentations
were carried out in a bioreactor at 2 kg for 96 h at 30 C. The moisture content of
the culture media and the Aspergillus-fermented material were determined using
an infrared balance at 105 C until the weight became constant [31]. Fermentation
was carried out in a bioreactor reported by Rodrguez-Fernandez et al. [31].

3.1. Enzyme concentration by ultraltration

Phytase from A. niger F3 were produced via SSF using citrus
peel as substrate. After 120 h, the concentration of the enzymes
in dry basis was 65.32 U/g. At the end of fermentation, the enzymes
were extracted from the solid matrix at an initial concentration of
7667 U/L in aqueous solution.
3.1.1. Concentration by ultraltration
The volume containing the extracted phytase was concentrated
6-fold by ultraltration with a feeding ow rate of 1.8 L min1 and
a pressure drop of 5 psi g. Fig. 1 demonstrates the behaviour of the
permeate ux (PF) as function of time. Two different stages were

376

D.E. Rodrguez-Fernndez et al. / Process Biochemistry 48 (2013) 374379

700

Table 2
Enzyme rejection and yield after 50 min of concentration by ultraltration.

Permeate flux (L m-2 min-1 )

600

Equation

500

200

Yield

Yield (%) =

Phytase
activity
loss

Phytaseact.loss (%) = 100

retent.total.act.
total.act.initial.solution

Values
100

100%

100

 total perm.act.total ret.act 

total feed act.

86.14%
100

13.86%

Table 3
Ionic concentration in the feed and retentate after ultraltration.

100
0

permeate.phyt.act.
retentate.phyt.act.

% Phytaserej = 1

400
300

Enzyme
rejection

10

20

30

40

50

Time (min)
Fig. 1. Permeate ux during the ultraltration up to a 6-fold concentration volume.

observed during ultraltration: the rst stage was a dramatic drop

that occurred within the rst 20 min of the ultraltration process.
When the value of the permeate ux reached half of the initial value,
and the second stage was observed when the variation in the permeate ux was reduced. This reduction in the permeate ux was
mainly due to fouling, and the increment of the viscosity of the feed
was concentrated. The higher ux values led to lower operating
costs. In addition, performing at lower values was not convenient
because phytase, as many other proteins, could be susceptible to
shear stress, which led to a reduction in enzyme activity. Data in
Fig. 1 may be modelled that is expressed by Eq. (1):
PF = 586.65 (t)0.228

(1)
(L m2

min1 )

Ions

Feed (ppm)

Retentate (ppm)

Na+
NH4 +
K+
Mg2+
Zn2+

713.24
99.01
8893.51
1075.93
2236.22

16.67
52.29
96.17
29.31
0.02

to improve enzyme activity. On the other hand, the enzymes

present lost their activity (Table 3). The other ions eliminated during the concentration process had not signicant inuence the
phytase activity. For this reason, it is possible that the loss of phytase activity was due mainly to the shear forces generated in the
membrane.
For enzymes susceptible to shear stress, long process times
lead to a signicant loss of enzyme activity; therefore, 50 min was
dened as an adequate time for concentrating the enzyme. The volume subjected to ultraltration was 9 L, and the nal volume was
1.5 L after 50 min of operation; therefore, the volumetric concentration factor was 6.

and t is time (min).

where PF is the permeate ux
The adjusted model had a regression coefcient (R2 ) of 0.9874
and predicted accurately the permeate ux desired to concentrate
the phytase.
Table 1 presents the values of the initial (7667 U L1 ) and nal
(39,402 U L1 ) enzyme activities of the phytase after concentrating
the initial volume of the extract 5-fold. However, the phytase activity was increased 4.32-fold. The protein concentrations in the feed
(86,730 mg) and in the retentate (13,047 mg) indicate that 85% of
total protein present in the extract was eliminated in the permeate. The reduction in protein content increased the specic activity
from 0.81 to 4.60 U mg1 . During the process of concentration, a
large number of biological molecules smaller than phytase were
probably eliminated.
The total phytase activity in the feed at the beginning of the
ultraltration process was 51,002 U, and at the end of the process,
the total phytase activity was 44,117 U. The enzyme rejection coefcient and yield [36,37] were determined and are shown in Table 2.
The enzyme rejection value (99.95%) indicates that there was no
enzyme in the permeate, and the yield (86.14%) indicates that 14%
of the activity was lost in the process possibly due to shear forces
generated during the concentration process.
Both Zn2+ and Mg2+ ions were reported as having an inhibitory
effect [34], so their elimination in the permeate was hypothesized

3.2. Stabilization of phytase activity

Table 1
Phytase activity and specic activity in the feed and retentate after ultraltration
using a 10 kDa cut-off membrane.

Table 4
Inuence of glycerol on the phytase activity for 25% and 35% (v/v) glycerol addition.

Phytase (U L1 )
Feed
Retentate

5667
24,502

Proteins (mg L1 )
5916
10,653

Specic activity (U mg1 )

0.96
4.60

All biological products used as feed additives must be stable to

resist inactivation by heat from feed processing and storage; at the
same time, its production must be cheap. Another problem with
phytase use as feed additive is that the enzyme loses activity over
time; therefore, it is important to conserve their activities for as
long as possible. In the present work, glycerol was used as a stabilizing agent. Based on preliminary studies of different stabilizers
under different concentrations, three formulations containing the
concentrated extracts were prepared: control (without glycerol) or
with 25% glycerol (v/v) or 35% (v/v) of glycerol.
Table 4 demonstrates the activation effect of glycerol on the
enzyme activity with improvements of 1.43- and 1.34-fold for 25%
glycerol and 35% glycerol preparations, respectively. These increments were calculated considering the volumes of extract added
to the preparations. The activation effect could be due to refolding
of the peptide chains and the stabilization of tertiary and quaternary structures as a result of the hydrogen bonds and other forces
created between the enzyme and the glycerol. Experiments using
higher concentrations of glycerol did not show signicant improvement.
After the inuence of glycerol on the phytase activity was tested,
the inuence of pH and different temperatures was evaluated in the

% Glycerol

Phytases (U mL1 crude extract)

Relative specic activity (%)

Control
25%
35%

39.40
56.33
53.13

100
142.9
136.1

D.E. Rodrguez-Fernndez et al. / Process Biochemistry 48 (2013) 374379

Control

100
90

25% Glycerol

90,0

35% Glycerol

80,0

Residual Activity (%)

70

Res. Act. (%)

R 4C

100,0

80

60
50
40

R 29 C
R 37 C
R 46 C

70,0
60,0
50,0
40,0
30,0
20,0

30

10,0

20

0,0

10
0

377

10

12

14

16

Time (weeks)
1

Fig. 4. Preservation of phytase activity during long-term storage for a 25% glycerol
formulation at different temperatures.

pH
Fig. 2. Phytase activity at different pH values after 1 h at 50 C.

two glycerol preparations. There was no signicant change in the

relative activities (%) with the changes to the pH compared with
the control (Fig. 2) although the differences in activities (expressed
as U mL1 ) remained the same. The optimal conditions for phytase
were in the range of pH 3.04.0. Under all conditions at pH 2.0, the
activity decreased drastically (40%), whereas the activity at pH 6
was 60%.
After a 1 h incubation at 30 C and 40 C, the phytase activity was
39.40 U mL1 , which is 100% of the total relative activity. At 50 C
and 60 C, the activity of the control was 90.9% and 86.6%, respectively. When the temperature was increased to 70 C, the activity
in the sample was 22.8% of that observed at 40 C (Fig. 3). The
presence of glycerol maintained the activity close to 100% at 60 C.
Signicant improvements were observed at 70 C, with 25% glycerol maintaining approximately 58% (22.85 U mL1 ) of the maximal
activity observed at 3040 C. The other preparation with 35% glycerol maintained 75% (29.67 U mL1 ) of the maximal activity.

important for phytase conservation because the growth of a considerable number of microorganisms is restricted under these low
water activities.
Although the mechanisms for stabilizing the effects of additives in enzyme formulations are not fully understood, one may
devise a formulation applicable to many different proteins rather
than just one. On the other hand, the use of a stabilizing agent such
as glycerol greatly enhances stability without chemical or genetic
manipulation of the target protein. This is especially useful in feed
applications where the need to satisfy regulatory requirements
may work against a chemically or genetically modied derivative.
Both preparations (25% and 35% glycerol) were incubated for 4
months at 4, 29, 37 or 46 C. The best temperature for preserving
phytase activity was 4 C, as shown in Figs. 4 and 5. The nal activities were 36.17 U mL1 with 25% glycerol and 27.22 U mL1 with
35% glycerol.
In Figs. 6 and 7, the data for residual activity are plotted and
the linearization of Arrheniuss equation was used to calculate the
values of inactivation constants:

3.3. Long-term storage of phytase preparations

ln

After analysing the effects of pH and temperature, the stability of the enzyme during storage was analysed. The water activity
in the glycerol solutions decreased to 0.939 in the formulation
with 25% glycerol and to 0.902 for 35% glycerol. This observation is

where A is the activity of the phytase formulation at any time of

storage (U mL1 ), A0 is the activity of the phytase formulation at
the beginning of storage (U mL1 ), k is the inactivation constant
(week1 ) and t is time (weeks).

A
A0

= kt

(2)

R 4C

100,0
Control
25% Glicerol
35% Glicerol

100,00

Residual Activity (%)

Res Act. (%)

70,00
60,00
50,00
40,00

70,0
60,0
50,0
40,0
30,0

30,00

20,0

20,00

10,0

10,00
0,00

0,0
30

40

50

60

70

R 37 C
R 46 C

80,0

90,00
80,00

R 29 C

90,0

10

12

14

16

Time (weeks)

Temperature (C)
Fig. 3. Phytase activity at different temperatures after 1 h at the optimal pH 3.0.

Fig. 5. Preservation of phytase activity during long-term storage for a 35% glycerol
formulation at different temperatures.

378

D.E. Rodrguez-Fernndez et al. / Process Biochemistry 48 (2013) 374379

0,5

Table 5
Denaturation constants for 25% and 35% glycerol.

Time (weeks)

25% Glycerol
0
0

10

12

16

R2 = 0.964 1

-1

35% Glycerol

29 C

37 C

46 C

4 C

29 C

37 C

46 C

0.070
10.9

0.193
4.4

0.244
3.8

0.025
28.2

0.047
14.3

0.110
7.1

0.151
5.0

R2 = 0.9783
ln(A/Ao) 4C

Table 6
Denaturation constants obtained from the Arrhenius linearization model for 25%
and 35% glycerol.

ln(A/Ao) 29 C
R2 = 0.974

-1,5

4 C

0.046
Kinactivation
(weeks1 ) 15.5
t1/2
(weeks)

R2 = 0.9533

-0,5

ln(A/Ao)

14

Temp. ( C)

ln(A/Ao) 40 C
ln(A/Ao) 46 C
Linear (ln(A/Ao) 46 C)
Linear (ln(A/Ao) 40 C)

-2

Linear (ln(A/Ao) 29 C)
Linear (ln(A/Ao) 4C)

Temperature ( C)

k25 (sem1 ) 25% glycerol

k35 (sem1 ) 25% glycerol

4 C
29 C
37 C
46 C

0.046
0.071
0.193
0.244

0.025
0.050
0.122
0.152

-2,5

Fig. 6. Linearization of the Arrhenius equation for a 25% glycerol formulation after
4 months.

Straight lines with high regression coefcient values (R2 > 0.95)
demonstrate that the thermal inactivation kinetics for all different
temperatures of storage for both preparations followed a rst-order
kinetic behaviour. The inactivation constants and half-life times are
shown in Table 4. The values of the inactivation constants for 25%
glycerol were at least 1.5 times higher than those for 35% glycerol.
The inactivation of the phytase enzymatic activity could be due to
irreversible chemical degradation processes related to the amount
of free water present in the samples. Phytase produced under similar conditions was also reported to have a half-life of 180 days
[38] but only after a purication process and with a very low initial
activity (4.5 U mL1 ), representing a yield of 6.35% of all recovery
process; this yield was 13.5% less than that obtained prior to the
purication step, so this step resulted in the process being costly
and possibly unfeasible from an economic point of view.
The experiments indicated that the half-life for the 35% glycerol
formulation was higher than that for the 25% glycerol formulations compared at the same temperature. However, it is especially
important to highlight that the value predicted by the model for the
35% glycerol formulation stored at 4 C demonstrated a half-life of
28.2 weeks, and the analysis indicated that the half-life was 26.7
weeks. Furthermore, the 35% glycerol preparations had a half-life
0,2

greater than 6 months as it is shown in Table 5, which is benecial

in preserving enzyme activity in aqueous solution. Several scientic papers have demonstrated that phytase possess a half-life of 20
days or 14 days [38,39] when stored at 4 C. Similar results achieved
in this paper were reported for catalases with a 6-month half-life
at 4 C; for storage at 30 C, the half-life was 2 months [40], which
is 1 month less than that reported in this article.
When analysing the behaviour of the inactivation constant, its
value doubled when the temperature was increased from 4 C
to 29 C; however, the inactivation increased more than 5-fold
when the temperature was increased from 29 C to 46 C as it is
shown in Table 6. These nding demonstrate that glycerol is a good
cryo-protecting agent to be employed under conditions of low temperature. At high temperatures, its ability to protect proteins and
enzymes decreases considerably.
4. Conclusion
Ultraltration allowed concentrating the phytase activity 4.3
times, whereas the volume concentration achieved was 6, which
possibly means that there was a loss of phytase activity by the
action of shear forces in the membrane, limiting the benet of the
elimination of ions and small biological molecules below 10 kDa.
Using 35% glycerol increased the enzyme activity and stability,
retaining over 68% of the initial activity after 16 weeks and providing a half-life time of 6 months.

Time (weeks)

References
0,0
0

10

12

-0,2

14

16

R2 = 0.9517

ln(A/Ao)

-0,4
R2 = 0.963 6

-0,6
-0,8

-1,0
-1,2

-1,4

R2 = 0.971
R2 = 0.9812

ln(A/Ao) 4C
ln(A/Ao) 29 C
ln(A/Ao) 40 C
ln(A/Ao) 46 C
Linear (ln(A/Ao) 46 C)
Linear (ln(A/Ao) 40 C)
Linear (ln(A/Ao) 29 C)
Linear (ln(A/Ao) 4C)

Fig. 7. Linearization of the Arrhenius equation for a 35% glycerol formulation after
4 months.

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