Enzymes in gluten-free bread making
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Prof. Cristina M. Rosell
Enzymes in gluten-free bread making by Cristina M. Rosell is licensed under a Creative Commons
Reconocimiento-CompartirIgual 4.0 Internacional License.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
OUTLINE:
106
0
0.025
0.050
0.075
1.
Role of enzymes in bakery
2.
Gluten free products
3.
Enzymes with structuring action
4.
Enzymes with degrading activity
5.
Enzymes for nutritionally improving gluten free breads
6.
Concluding remarks
7.
Acknowledgments
105
104
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
1. Role of enzymes in bakery
Enzymes
baking
industries
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
Starchdegrading
enzymes
NSP
degrading
enzymes
Protein
enzymes
Lipid
enzymes
Proteases:
Exopeptidase
Endopeptidase
s
Lipases
Phospholipase A
Other
enzymes
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
α-amylase
β-amylase
Glucoamylase
Most commonly used
Pentosanases:
Hemicellulase
Xylanase
Phytase
Glucose oxidase
Lipoxygenase
Laccase
Transglutaminase
Debranching
enzymes:
Isoamylase
Pullulanase
Asparaginase
Transferases:
Amylomaltase
CGTase
Rosell & Dura
(2014). Enzymes in baking industries. In: Enzymes in Food and Beverage. Ed E. Chandrasekeran.
Less commonly used
CRC Press
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
Enzymes
α-amylases
Dough
Improve fermentation
CGT-ases
106
0
0.025
0.050
0.075
Pentosanases
Improve handling
Hemicellulases
Xylanases
Reduce processing time
Increase gluten strength
Improve handling and
fermentative characteristics
Easy mixing
May compromise gas holding ability
Increase hardness
Decrease extensibility
105
104
Heating
Proteases
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
Tyrosinase
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Transglutaminase
Lipases
Phospholipase A
Phytase
Glucose oxidase
Lipoxygenase
Laccase
Rosell & Dura (2014).
CRC Press
Decrease water absorption
Increase stability
Favors tiny gas cells
Improve rheological
properties
Dough strengthener
Improve handling and
suppress stickiness
Shorten fermentation
Bread
Improve volume and
crumb texture
Improve volume
Decrease firmness
Improve volume and
crumb texture
Increase volume
Increase shelf-life
Staling
Anti-staling effect
Slower staling
kinetics
Reduce breadstaling
Increase shelf life
Uneven crumbs and
large pore size
Soft texture and
increase volume
Increase volume and
improve structure
Improve crumb
structure
Flavor enhancement
Increase specific
volume
Increase volume
Antistaling effect
when combined
with α-amylase
Increase specific
volume and soften
crumbs
Improve nutritional
values
Improve quality
Dough strengthener
Improve visco-elasticity
Bleaching agent
Improve stabilization and
Soften crumb
machinability
Enzymes
in strengthener
baking industries. In: Enzymes in Food and Beverage. Ed E. Chandrasekeran.
Dough
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
2. Gluten free products
H
106
0
0.025
0.050
0.075
105
104
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
2000
1500
D
1000
control
HPMC
VS
500
0
0
200
400
600
800
Time (s)
 Gluten forms a viscoelastic matrix necessary for holding the
CO2 released during the fermentation
 1st Challenge: Design gluten free products
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
2. Gluten free products
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Building an internal network:
• Hydrocolloids
• Proteins
• Enzymes: GO, Laccase, PFO, TG
Rosell (2009). Enzymatic manipulation of gluten-free bread. In: Gluten-free Food
Science and Technology. Ed E. Gallagher.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
Mechanism of GO
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
β-D-glucose + O2
Gluconic Acid + H2O2
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Mechanism of TG
Acyl – transfer reaction
Crosslinking reaction
Deamidation
Rosell (2009). Enzymatic manipulation of gluten-free bread. In: Gluten-free Food Science and
Technology. Ed E. Gallagher.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
Glucose Oxidase // Rice bread
106
0
0.025
0.050
0.075
105
Heating
3,0
103
1600
102
20
30
40
50
60
70
80
90
Temperature (C)
0 % HPMC
2 % HPMC
3000
2500
2,5
2000
1400
0 % HPMC
2 % HPMC
1500
Specific volume (cm3/g)
1000
1200
control
HPMC
500
Crumb hardness (g)
Viscosity (cP)
G' (Pa)
Cooling
104
0
0
200
400
2,0
600
800
Time (s)
1,5
1,0
1000
800
600
400
0,5
200
0,0
0
0,00
0,01
0,02
0,03
Enzyme concentration (%,w/w)
0,00
0,01
0,02
0,03
Enzyme concentration (%,w/w)
Gujral and Rosell (2004) Food Res Technol 37, 75-81.
Rosell (2009). Enzymatic manipulation of gluten-free bread. In: Gluten-free Food Science and Technology.
Ed E. Gallagher
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
Glucose Oxidase // Corn bread
106
0
0.025
0.050
0.075
1200
105
Heating
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
Crumb hardness (g)
103
Viscosity (cP)
G' (Pa)
Cooling
104
1000
800
600
400
200
0
Control
GO 0.01
GO 0.02
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
Transglutaminase // Rice bread
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
2000
1500
1000
control
HPMC
500
0
200
400
600
800
Time (s)
0,8
2
5
0
0
a
b
2
0
0
0
3/g)
0,6
c
c
0
%
H
P
M
C
2
%
H
P
M
C
1
5
0
0
0,4
1
0
0
0
Crumbhadnes(cm
0
Free amino groups (µg/mg flour)
Viscosity (cP)
G' (Pa)
Cooling
104
0,2
5
0
0
0,0
0
0,0
0,5
1,0
1,5
Enzyme concentration (%, w/w)
0
,
0
0
,
5
1
,
0
E
n
z
y
m
e
c
o
n
c
e
n
t
r
a
t
i
o
n
(
%
,
w
/
w
)
Gujral, Haros, Rosell (2004) J Cereal Sci 39, 225-230
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
1
,
5
3. Enzymes with structuring action
Proteins: pea, soybean, albumen, casein
Viscoelastic parameters
G΄ (Pa)
G΄΄ (Pa)
Tan 
5372
663
0,176
0
6460b
719b
0,112a
1
8992c
1211c
0,145a
2
10902d
1273c
0,118a
3
382a
77a
0,205b
4
126a
33a
0,298c
0
4412a
611a
0,168a
1
6333b
714a
0,183a
106
0
0.025
0.050
0.075
Level
105
104
Overall mean
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
Protein
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
TG
1
2
3
4
5
1
Values followed by different letters in the same column within each factor are significantly
different (p<0.05).
Protein levels: 0 (without protein isolate), 1 (pea), 2 (soybean), 3 (egg albumen), 4 (whey
protein).
TG levels: 0 (without TG), 1 (1% TG).
Marco and Rosell (2008) J Food Eng 84, 132-139
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
1,8
-TG
100
1,6
1,2
SP 13%-95
SP 25%-95
1,0
SP 25%-110
0,8
SP 13%-110
106
Torque (Nm)
0
0.025
0.050
0.075
105
104
Heating
103
60
40
0,6
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
0,4
20
2500
2000
0,2
1500
1000
control
HPMC
500
0,0
0
800
Time (min)
50
600
40
400
Time (s)
30
200
20
0
10
0
0
Viscosity (cP)
G' (Pa)
Cooling
Temperature ( C)
80
1,4
Rice-Soybean
+TG
Marco, Pérez, León, Rosell (2008) Cereal Chem 85, 59-64.
Marco, Rosell (2008) Eur Food Research Technol. 227, 1205-1213
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
3. Enzymes with structuring action
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Storck et al. (2013). LWT - Food Science and Technology. 53/ 1 (2013) 346–354.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
4. Enzymes with degrading activity
PROTEASE (Neutrase 1.5 MG, Novozymes; PR)
106
0
0.025
0.050
0.075
105
104
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
buckwheat (BW)
sorghum (SG)
corn (CR)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Renzetti and Arendt. Journal of Cereal Science 50, (2009): 22-28
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
4. Enzymes with degrading activity
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
PROTEASES:
- protease A and M from Aspergillus oryzae
- protease P from Aspergillus melleus
- thermoase PC10F from Bacillus stearothermophilus) from Amano Enzyme Inc.
(Nagoya, Japan).
- Thermolysin from Bacillus thermoproteolyticus rokko (Type X) from Sigma
Aldrich
control
HPMC
500
0
0
200
400
600
800
Time (s)
RICE BREAD
Kawamura-Konishi et al. Journal of Cereal Science 58, no. 1 (2013): 45-50.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
4. Enzymes with degrading activity
PROTEASES:
- Alcalase (subtilisine) from Bacillus licheniformis
- iZyme (whey protease) from Novozyme (Denmark)
- Flavourzyme (aminopeptidase) from Novozyme
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
CORN BREAD
4. Enzymes with degrading activity
PROTEASES:
- iZyme (whey protease) from Novozyme
(Denmark)
106
0
0.025
0.050
0.075
105
CORN BREAD
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
4. Enzymes with degrading activity
AMYLASES:
- Fungamyl from Novozyme (Denmark)
- Powerfresh (Dupont)
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
RICE BREAD
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
CONTROL
GFB AM
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
FUTURE
TRENDS
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
Enzymes for
nutritionally
improving
gluten free
breads
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
5. Enzymes for nutritionally improving gluten free breads
SOURDOUGHS:
106
0
0.025
0.050
0.075
- Acidification
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
- production of exopolysaccharides,
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
- activation of enzymes like proteases, amylases and phytases
- production of antimicrobial substances like propionate
 Detoxification of the gluten containing cereals
 Reducing antinutrients
 Improving quality
Moroni, Dal Bello, Arendt. Food microbiology. 2009;26(7):676-84.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
5. Enzymes for nutritionally improving gluten free breads
- GERMINATION:
- Activation of enzyme activities
106
0
0.025
0.050
0.075
105
104
Heating
103
102
20
30
40
50
60
70
80
90
- amaranth, buckwheat, maize, millet, rice, sorghum and
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
quinoa
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
 Reduce anti-nutrients content
 Reduce eGI
Omary, Fong, Rothschild, Finney. Cereal Chemistry. 2012; 89(1):1-14
Cornejo & Rosell (2014) Unpublished results.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
5. Enzymes for nutritionally improving gluten free breads
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
PROLYL ENDOPEPTIDASES from different sources.
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
• Produce gluten-free foods from gluten-containing
raw materials
• Produce an oral therapy for CD
Wieser, Koehler. Journal of Aoac International. 2012, 95(2):356-63.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
FINAL
REMARKS
control
HPMC
500
0
0
200
400
600
800
Time (s)
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
 Enzymes are useful processing aids in gluten
free bread making.
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
 Enzymes’ action is largely dependent on raw
material, thus enzyme type and level must be
optimized for each gluten free recipe.
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
Innovation: re-defining role of enzymes from
“processing aids” to “healthy aids”.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
ACKNOWLEDGEMENTS:
- Hardeep Singh Gujral (Guru Nanak University, India)
- Cristina Marco (IATA-CSIC)
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
- Mª Estela Matos (IATA-CSIC)
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
Time (s)
600
800
- Marcia Arocha Gularte (Universidad de Pelotas, Brasil)
- Fabiola Cornejo (ESPOL, Ecuador)
Funding sources:
• Proyect UE. CRAFT Proyect (QLK1-2002-72162), 2003-2005
• Proyect (AGL2005-05192-C04-01/ALI) by Spanish Ministry of Economy
and Sustainability and CSIC.
• Award from Coelic Association of Madrid in 2009
• Proyect (AGL2008- 00092 ALI , AGL2011-23802) by Spanish Ministry of
Economy and Sustainability and CSIC.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es
106
0
0.025
0.050
0.075
105
Heating
103
102
20
30
40
50
60
70
80
90
Temperature (C)
3000
2500
Viscosity (cP)
G' (Pa)
Cooling
104
2000
1500
1000
control
HPMC
500
0
0
200
400
600
800
Time (s)
For more information:
Web site: http://www.iata.csic.es
e-mail: crosell@iata.csic.es
Webpage: http://www.iata.csic.es/mcristinamolinarosell/
Research ID: http://www.researcherid.com/rid/F-4888-2010
Enzymes in gluten-free bread making by Cristina M. Rosell is licensed under a Creative
Commons Reconocimiento-CompartirIgual 4.0 Internacional License.
Cristina M. Rosell (IATA-CSIC) - crosell@iata.csic.es - www.iata.csic.es