1. Enzymatic depolymerisation of oat β-glucan
Results and discussion
Abstract
The aim of the present study was to modify the molecular weight and viscosity properties of β-glucan in oat bran by hydrolytic enzymatic treatment. Water content, enzyme type, dosage and
reaction time were used as variables in studying and optimizing the reaction conditions for β-glucan hydrolysis. Oat bran fractions with 20.0 and 28.5 % of β-glucan were treated with commercial
enzyme mixtures and with purified Trichoderma reesei endo-glucanase II. After the hydrolysis reaction, β-glucan was extracted with hot water at 80 ºC. The solids were removed by centrifugation,
and the supernatant stored at 5 ºC. Rheological properties and molecular weight distribution of the enzyme-treated β-glucan, as well as the stability of the solution were then measured. The
tested reaction parameters significantly affected the hydrolysis of β-glucan, suggesting that careful optimization of the reaction conditions is needed when aiming at specific hydrolysis products.
Overall description of the depolymerisation process
Conclusions
This study showed an efficient way to tailor the molecular weight of oat bran β-glucan by
enzymatic hydrolysis at limited water content. The suitable range of β-glucan molecular weight
was obtained when the oat bran concentrate was treated at 50 % moisture content at 50 ºC for
more than 3 hours by hydrolytic Depol 740 L enzyme preparation. The peak average molecular
weight of β-glucan after such hydrolysis was 47 kDa. The highest concentration of β-glucan
which did not convert into gel during the 18 days of storage at 5 ºC was 2.0 %. The low
molecular weight β-glucan could be used as an ingredient when developing new types of dietary
fibre-enriched liquid products.
Aliki-Ilona Niniosa,b, Juhani Sibakovb, Ioanna Mandalaa, Konstantinos Fasseasa, Kaisa Poutanenb, Emilia
Nordlundb, Pekka Lehtinenb
a Department of Food Science and Technology, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Greece
(alikininiou@gmail.com, imandala@aua.gr)
bVTT Technical Research Centre of Finland, P.O.Box 1000, Tietotie 2, Espoo, FI-00244, VTT (juhani.sibakov@vtt.fi)
Rheological properties
Figure 3. Frequency sweep
tests of samples after
different incubation time
(open symbols G’, close
symbols G’’, control
sample, incubated for 5
min, incubated for 4 h)
Raw material B: Oat bran concentrate (OBC), 20 % β-glucan
Raw material A: Oat bran concentrate (OBC), 28.5 % β-glucan
Minutes
16 20 24 28 32 36 40 44 48 52 56 60 64
mV
0
5
10
15
20
25
30
35
40
Depol 740 L
Depol 761 P
Depol 686 L
Veron CP
Celluclast 1.5 L
Spezyme CP
Econase CE
β-Glucan concentration after the enzymatic hydrolysis of 1% β-glucan solution (mg/l)
Incubation time 1 h 4h
Enzyme dosage 100 nkat 1000 nkat 100 nkat 1000 nkat
Depol 740 L 7819 299 2426 <20
Depol 761 P 9050 9212 10295 4986
Depol 686 L 6179 549 1337 108
Veron CP 6770 486 1904 83
T. reesei EG II 500 n.a. 98 n.a.
Celluclast 1,5 L 4727 289 928 48
Spezyme CP 6366 548 1634 121
Econase CE 7035 440 2090 113
Table 1. Concentration of β-glucan (originally ca.10 g / l) after 1 and 4 h enzymatic treatment with two
different dosages of the enzymes (100 and 1000 nkat) at 45ºC. The reference sample (without enzyme) had
β-glucan concentration of 9245 mg/l after 4 h incubation in water.
β-Glucan concentration
At high water content
Viscosity (Pas) Cutting speed 1/24 s
Sample 1 day 3 days 5 days 14 days 18 days
DEPOL 740L
2%,40min,10nkat 0.755 0.699 gel gel gel
2.5%,40min,10nkat 2.416 gel gel gel gel
2%,1h50min,10nkat 0.034 0.033 0.031 gel gel
2.5%,1h50min,10nkat 0.057 0.076 0.39 gel gel
2%,2h50min,10nkat 0.014 0.018 0.048 0.098 0.201
2.5%,2h50min,10nkat 0.022 0.074 0.25 gel gel
2%,4h30min,10nkat 0.015 0.012 0.021 0.062 0.102
2.5%,4h30min,10nkat 0.013 0.043 0.129 0.41 0.31
Table 2 Viscosities of samples stored at 5 ºC for several weeks. With enzyme activity
10nkat, dry matter content 2 or 2.5%, and incubation time 40min to 4h and 30min.
Oat bran concentrate (OBC)
Enzyme
and water
Water
Preconditioning at 40 º C
for 30min, 25 % moisture
Hydrolysis in extruder at
45 º C for 1 –2 min,
50 % moisture
Incubation at 50 ºC for
10 min –4 h , 50% moisture
Extraction
Separation of
solids
Stabilisation &
Pasteurisation
Low viscosity
ß-glucan solution
A
B Suspension of oat
bran concentrate
Mixing of the solution
in flasks at 45 ºC
for 30 – 120 min
Enzyme
Separation of solids
InactivatIon
of enzymes at boiling
water for 10 min
High water content (94%)
Low water content (50%)
Inactivation in extruder
at 115 º C for 1 –2 min
Water
Water
Low viscosity
ß-glucan solution
Oat bran concentrate(OBC)
Enzyme
and water
Water
Preconditioning at 40º C
for 30min, 25 % moisture
Hydrolysis in extruder at
45 º C for 1–2 min,
50 % moisture
Incubation at 50ºC for
10 min–4 h, 50% moisture
Extraction
Separation of
solids
Stabilisation &
Pasteurisation
Low viscosity
ß-glucan solution
A
B Suspension of oat
bran concentrate
Mixing of the solution
in flasks at 45 ºC
for 30– 120 min
Enzyme
Separation of solids
InactivatIon
of enzymes at boiling
water for 10 min
Inactivation in extruder
at 115º C for 1–2 min
Water
Water
Low viscosity
ß-glucan solution
Two oat bran concentrates were used as raw materials. They were manufactured either from non-heat-treated whole oat kernels (OBC-1) or from commercial heat-treated oat bran (OBC-2)
according to Sibakov et al. 2010. The β-glucan concentrations of OBC-1 and OBC-2 were 28.5 % and 20.0 (dw), respectively. The oat bran concentrates were enzymatically hydrolysed either at
50 % (low) or at 94 % (high) water content. The process in low water content was based on an efficient mixing of OBC and enzyme, and subsequent incubation of the dough-like mass at 50 ºC for
10 min – 4 h. The hydrolysis at higher water content was performed by using a continuous mixing of the OBC-enzyme suspension at 45 ºC for 1 or 4 h. After both processes, the enzyme was
inactivated by high temperature (115 or 100 ºC) treatment. Both a commercial enzyme Depol 740 L (Biocatalyst Ltd, Wales, UK) and a purified Trichoderma reesei endo-glucanase II (EGII, VTT,
Finland) were investigated.
Figure 1. The Mw
distributions of raw materials
and enzyme hydrolysed oat
bran samples at high water
content.
The β-glucan concentration of water diluted oat bran was measured by Carlsberg’s Calcoflour
method. First, all the enzymes were tested in a solution with ca. 1 % of β-glucan (10 g of oat bran with
30 % β-glucan was weighed into 300 ml of distilled water). Incubation time 4 h didn’t allow the growth
of unwanted microbes.
0
5
10
15
20
25
30
35
40
Minutes
16 20 24 28 32 36 40 44 48 52 56 60 64
Minutes
16 20 24 28 32 36
Raw material: Oat bran concentrate (OBC)
M
w
320 000
Raw material: Oat bran concentrate (OBC)
M
w
320 000
OBC, Depol 740L , 10 min
M
w
218 000
OBC, Depol 740L , 10 min
M
w
218 000
OBC, Depol 740L, 40 min
M
w
93 000
OBC, Depol 740L, 40 min
M
w
93 000
OBC, Depol 740L, 2 h
M
w
71 000
OBC, Depol 740L, 2 h
M
w
71 000
OBC, Depol 740L, 3 h
M
w
49 000
OBC, Depol 740L, 3 h
M
w
49 000
Figure 2. Mw distributions
of β-glucan that were
obtained by enzymatic
hydrolysis in extrusion
process.
Mw distributions
Structural analysis by SEM-microscopy
At low water content
Figure 4. Electron microscopy pictures of hydrolysed and dried oat bran concentrates: A) Raw
material OBC-1 without enzymatic hydrolysis (28.5 % β-glucan), B) OBC-1 hydrolysed by Depol
740L at low water content for 1 h, C) Sample OBC-1 hydrolysed by T. reesei EGII at low water
content for 1 h. Bar = 10μm.
A B C
At low water contentmV
•T. reesei EGII resulted in a greater decrease of the molecular weight of β-glucan.
The most efficient
commercial enzymes
in terms of β-glucan
degradation were :
•Econase CE
•Depol 740L
Soft gel
Rigid gel
Liquid
The Mw distributions were analysed by Alliance 2690 HPLC-SEC.
OBC-2, Depol 740L
1
10
100
1000
10000
0,1 1 10
Frequency (Hz)
Storage(G')
Loss(G'')
Modules(Pa)