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Bioprocessing as a tool to improve
the functionality of plant proteins
for food application
Emilia Nordlund
NIZO Plant Protein Functionality Conference
October 22, 2020
22/10/2020 VTT – beyond the obvious
22/10/2020 VTT – beyond the obvious 2
Our vision
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We help our customers and society
to grow and renew through applied
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Our strategy
We make an impact through
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excellence
We make impact through science and technology
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of the world’s Gartner
Global 500 companies
have partnered with VTT
2,103
employees
patent families
406
turnover of which 47 %
from abroad
245 M€
Sensory, techno-functional and nutritional
properties of food ingredients are prerequisite
for making delicious foods
VTT – beyond the obvious
Techno-functional properties
• Solubility, foaming, emulsifying, gelling, stability,
texturizing, water and fat binding
At the
moment
dominated by
animal origin
ingredients
22/10/2020
Sensory quality
• Mouthfeel, flavour and odour, stability, visual
appearance
Nutritional quality
• Amino acid profile, protein digestibility, anti-nutrients
3
 Indigenous structural features of proteins
 Extraction and drying conditions can cause heavy denaturation
and aggregation
 Biologically complexed with others in the plant matrix
 Influenced by the many co-passengers
22/10/2020 VTT – beyond the obvious 4
Why plant proteins have poor
functionality?
 Fermentation and
germination
• Several endogenous
enzymes activate, which
influence food quality via
polymer degradation and
metabolite formation
• Fermentation and germination
are natural, but relatively slow
processes, taking day(s)
22/10/2020 VTT – beyond the obvious
Bioprocessing as a tool to improve the
plant protein functionality
 Enzyme-aided
processing
• Typically specific, attacks
single compounds in the plant
matrix
• Can be maybe controlled
better than processing by
fermentation and germination
• Enzyme treatment can usually
be implemented in hours
5
Applying bioprocessing in the different
parts of the food supply chain
Ingredient
production
Ingredient
modification
Food
applications
• Separation
technologies
• Removal of unwanted
components
• Texture, structure and
stability
• Sensory and
nutritional quality
• Technological
functionality
• Sensory and
nutritional quality
22/10/2020 VTT – beyond the obvious 6
Modification of plant
proteins by enzymes
Case studies
22/10/2020 VTT – beyond the obvious
• Denaturated rice endosperm proteins treated by
neutral and acidic endoproteases
• Protease selection influenced the outcome:
• Colloidal stability increased and surface
hydrophobicity decreased with increasing degree
of hydrolysis (DH)
• Foaming capacity and stability increased at pH
7 until the DH values of 1.5% (neutral enz) and
1.9% (acid enz). No stable foams achieved at pH 5
• Foam stability higher with acid protease; however,
the appearance of the DH-1.5% foam more
appealing by neutral protease
Enzymatic HYDROLYSIS of rice proteins as a tool to
improve foam and colloidal stability – rice proteins
NEUTRAL ENZ
ACIDIC ENZ
EU-BBI-PROMINENT
Nisov et al 2019. Food Chem, doi.org/10.1016/j.foodchem.2019.125274
Enzymatic CROSSLINKING of plant proteins as a tool to
improve foam and colloidal stability – oat proteins
 Increased dispersion stability and foaming
ability of transglutaminase-treated oat protein
isolate, likely due to:
• improved electrostatic stability
• intra-particle crosslinking  stability against
dissociation/re-association
• smaller particle size
Atomic force microscopy of interfacial films at pH 7.2
Light microscopy of foams from oat protein isolates
(control and TG-treated 1000nkat/g protein).
Storage stability of oat protein isolate dispersionsStorage stability of oat protein isolate dispersions
(1 month at 4 °C).
Ercili-Cura et al., 2015, Food Hydrocolloids, 44, 183-190
Nivala et al., 2017, Food Chemistry, 231, 87-95
Control Transglutaminase-treated
 Crosslinking of gluten proteins by tyrosinase reinforces the wheat dough
Selinheimo et al., J. Agric. Food Chem. 2007, 55, 6357-
 Combining degradation of fibres and crosslinking of proteins results in maximal
improvement for the gluten free oat breadmaking
Enzyme-aided protein network formation to
improve structure properties in breadmaking
REFERENCE, without enzyme TYROSINASE 30 nkat /g flour
Wheat dough analysis by confocal microscopy, Dried dough samples, 0.01% Congo Red + oil
Improving gluten free oat breadmaking with
tyrosinase and xylanase
10
Flander et al. J. Agric. Food Chem. 2011, 59: 8385-
22/10/2020
VTT – beyond the obvious
Glutaminase-mediated functionalization of plant
proteins: increase in solubility, emulsification & gelling
(Trans)Glutaminase-aided gelation of oat protein
concentrate to improve applicability of oats in various food
matrices (Unpublished data from VTT)
11
Modification of plant matrix and
co-passengers to improve
functionality of protein fractions
Case studies
22/10/2020 VTT – beyond the obvious
Enzyme-aided separation of rapeseed protein as
an option for alkali extraction
Cold-pressed (defatted) rapeseed press cake
Alkaline
extraction
Enzyme-aided water
extraction
Protein extract
Isoelectric
precipitation
Protein precipitate
Protein-sugar extract
Washing for salt
removal
Water extract / Alkaline extractRommi et al. 2014 J. Agric. Food Chem., 62 (32), pp 7989–7997
Rommi et al. 2015 J Agric Food Chem 63(11), 2997-3003.
Concentration
 Enzyme hydrolysing carbohydrates (pectinase)
increased the yield of rapeseed protein 25-70%
 Comparable protein yields (40–41% of tot protein)
from enzyme-aided water extraction (pH 6) and
alkaline extraction (pH10) suggest that protein
can be extracted without exposure to alkali Protein-sugar extract
13
22/10/2020 14
Improving in vitro digestibility of bran
proteins by bioprocessing
Solubleprotein%fromtotalprotein
Time (min)
Bio-processed bran
Native bran
Solubilisation of protein in vitro
Nordlund et al.2013, J Cereal Sci, 58, 200-
PROTEIN STAINED IN RED IN
THE MICROSCOPY IMAGES
 Digestibility of
protein in vitro was
clearly improved by
the degradation of
rye bran cell walls
by hydrolytic
enzymes and
fermentation
Improving in vitro digestibility of faba bean
proteins by phytase treatment
 Phytase enzyme degraded phytic acid and
increased solubility of protein when studied
in vitro at upper gut conditions
 Solubility of the proteins of the phytase-
treated faba bean flour was increased
at acidic conditions
Solubleproteinoftotalprotein(%)
Solubilisation of faba protein in vitro by phytase Solubility curve of faba proteins
Phytic acid content
reduced up to 89%
Rosa-Sibakov et al 2018, JAFC, https://pubs.acs.org/doi/full/10.1021/acs.jafc.8b02948
15
EU-BBI-PROMINENT & EIT-FOOD-PROVE
Rapeseed
Hybrid Ingredient
Gel with 3% dietary
fibre and 5% protein
Rice bran
Hybrid ingredient
Gel with 3% dietary
fibre and 3.4% protein
0
20
40
60
80
100
0
500
1000
1500
2000
2500
0 1000 2000 3000 4000 5000
Temperature(°C)
G'(Pa)
Time (s)
G' control G' phytase-treated
0
20
40
60
80
100
1
10
100
1000
10000
0 2000 4000
Temperature(°C)
G´(Pa)
Time (s)
G' phytase treated G' control
Set up: apply phytase in a spoonable food
model with rice bran and rapeseed materials:
 Phytase treatment at pH 5, 50°C, 2h; Heat-
induced gelation by heating at pH 5, 6.7
and 8; 95°C, 5 min
Results:
 Phytic acid content was reduced from 22%
to 3% in rice bran, and from 6.2% to 1.8%
in rapeseed
 Increase in gel strength and water holding
capacity by phytase at alkaline pH
Phytase improves nutritional and technological
value of rice & rapeseed hybrid ingredients
Kortekangas et al 2020, https://doi.org/10.1016/j.foodhyd.2020.105787
 Oat protein hybrid ingredient (55% protein, 30% starch) with improved emulsion
properties after high temperature α-amylase treatment
22/10/2020 VTT – beyond the obvious
17
Improved technological value of oat protein
concentrate by amylase and heat treatment
 Stable emulsion after
high-temperature α-
amylase treatment
  Modification of
co-passangers, not the
protein!
Native
Treated
 WHY: Legumes contain galacto-oligosachharides (GOS)
that can cause intestinal discomfort
 HOW: We tested GOS removal by α-galactosidases
in faba bean and pea ingredients
 We investigated can we remove GOS
1. When using extruder as a bioreactor for the
enzyme-treated ingredient production
2. In preparation of a spoonable product
18
Enzyme-aided FODMAP removal in protein
containing ingredients
 Set up: 50% pea protein concentrate mixed with enzyme-water solution (100
or 1000 nkat/g PPC) in an extruder
Extrusion parameters: 200 rpm, 40 °C; After extrusion, mixture incubated 30 min, and freeze-dried
Enzyme-aided FODMAP removal in protein containing ingredients
Case 1: Using extruder as a bioreactor for
the low GOS ingredient production
19
GOS (mg/g dm) Raf Sta Ver SUM
Control 2.5 41 36 80
DS 100 nkat/g 9.8 16 19 45
DS 1000 nkat/g 1.5 2.7 3.5 8
NEO 100 nkat/g 20 2.2 7.9 30
NEO 1000 nkat/g 0.2 0.1 0.0 0
Galacto-oligos: Raf=Raffinose, Sta=Stachyose, Ver=Verbascose;
DS=Enzyme DS30 (Amano); NEO=Enzyme Neosartorya (VTT).
Enzyme-aided FODMAP removal in protein containing ingredients
Case 2: GOS reduction in spoonable products
GOS (g/100g) Raf Sta Ver SUM
Control 0.07 0.62 0.60 1.28
DS 100 nkat/g 0.17 0.26 0.38 0.80
DS 1000 nkat/g 0.01 0.03 0.03 0.06
NEO 100 nkat/g 0.24 0.01 0.03 0.28
NEO 1000 nkat/g 0.00 0.00 0.00 0.00
Galacto-oligos (GOS): Raf=Raffinose, Sta=Stachyose, Ver=Verbascose
DS=Enzyme DS30 (Amano); NEO=Enzyme Neosartorya (VTT)
20
For low-FODMAP diet, cut-off value for GOS is
<0.3 g/serving in legumes
Varney et al. 2017, https://doi.org/10.1111/jgh.13698
Set up: 15% pea protein concentrate
(Vestkorn) dispersed in water for 100 g
serving size
 Enzyme treatment (Amano DS-30, VTT
Neosartorya) made in a mixing reactor
(100 and 1000 nkat/g PPC, 40 °C, 30 min)
 After enzyme treatment, dispersion
was heated at 90 °C for 5 min in RVA
Results:
 Reduction of GOS to values below cut-
off value “<0.3 g/serving in legumes“
 Treating the pea protein concentrate
with enzymes did not change the
texture of the product (based on RVA)
 There are many options how to use bioprocessing, especially
enzymes in plant food processing
• Separation, structure, taste, mouthfeel, stability, nutritional quality
 Bioprocessing can affect several parameters simultaneously
• When modifying techno-functionality, nutritional quality and flavour may
change as well
Take home messages (1/2)
Ingredient
production
Ingredient
modification
Food
applications
• Separation technologies
• Removal of unwanted
components
• Texture, structure and
stability
• Sensory and nutritional
quality
• Technological
functionality
• Sensory and nutritional
quality
 Think about the strategy to use - know your tools and plant
materials
• Enzyme type: hydrolytic, crosslinking,..
• Substrate: state (native, denaturated), quality and composition
• Whether to modify the target component or co-passengers
Take home messages (2/2)
Ingredient
production
Ingredient
modification
Food
applications
• Separation technologies
• Removal of unwanted
components
• Texture, structure and
stability
• Sensory and nutritional
quality
• Technological
functionality
• Sensory and nutritional
quality
Emilia Nordlund
emilia.nordlund@vtt.fi
+358 40 504 2963
@VTTFinland
@NordlundEmilia
www.vtt.fi

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VTT's Emilia Nordlund: Bioprocessing as a tool to improve the functionality of plant proteins for food application

  • 1. Bioprocessing as a tool to improve the functionality of plant proteins for food application Emilia Nordlund NIZO Plant Protein Functionality Conference October 22, 2020 22/10/2020 VTT – beyond the obvious
  • 2. 22/10/2020 VTT – beyond the obvious 2 Our vision A brighter future is created through science-based innovations Our mission We help our customers and society to grow and renew through applied research Our strategy We make an impact through scientific and technological excellence We make impact through science and technology 2 9% of the world’s Gartner Global 500 companies have partnered with VTT 2,103 employees patent families 406 turnover of which 47 % from abroad 245 M€
  • 3. Sensory, techno-functional and nutritional properties of food ingredients are prerequisite for making delicious foods VTT – beyond the obvious Techno-functional properties • Solubility, foaming, emulsifying, gelling, stability, texturizing, water and fat binding At the moment dominated by animal origin ingredients 22/10/2020 Sensory quality • Mouthfeel, flavour and odour, stability, visual appearance Nutritional quality • Amino acid profile, protein digestibility, anti-nutrients 3
  • 4.  Indigenous structural features of proteins  Extraction and drying conditions can cause heavy denaturation and aggregation  Biologically complexed with others in the plant matrix  Influenced by the many co-passengers 22/10/2020 VTT – beyond the obvious 4 Why plant proteins have poor functionality?
  • 5.  Fermentation and germination • Several endogenous enzymes activate, which influence food quality via polymer degradation and metabolite formation • Fermentation and germination are natural, but relatively slow processes, taking day(s) 22/10/2020 VTT – beyond the obvious Bioprocessing as a tool to improve the plant protein functionality  Enzyme-aided processing • Typically specific, attacks single compounds in the plant matrix • Can be maybe controlled better than processing by fermentation and germination • Enzyme treatment can usually be implemented in hours 5
  • 6. Applying bioprocessing in the different parts of the food supply chain Ingredient production Ingredient modification Food applications • Separation technologies • Removal of unwanted components • Texture, structure and stability • Sensory and nutritional quality • Technological functionality • Sensory and nutritional quality 22/10/2020 VTT – beyond the obvious 6
  • 7. Modification of plant proteins by enzymes Case studies 22/10/2020 VTT – beyond the obvious
  • 8. • Denaturated rice endosperm proteins treated by neutral and acidic endoproteases • Protease selection influenced the outcome: • Colloidal stability increased and surface hydrophobicity decreased with increasing degree of hydrolysis (DH) • Foaming capacity and stability increased at pH 7 until the DH values of 1.5% (neutral enz) and 1.9% (acid enz). No stable foams achieved at pH 5 • Foam stability higher with acid protease; however, the appearance of the DH-1.5% foam more appealing by neutral protease Enzymatic HYDROLYSIS of rice proteins as a tool to improve foam and colloidal stability – rice proteins NEUTRAL ENZ ACIDIC ENZ EU-BBI-PROMINENT Nisov et al 2019. Food Chem, doi.org/10.1016/j.foodchem.2019.125274
  • 9. Enzymatic CROSSLINKING of plant proteins as a tool to improve foam and colloidal stability – oat proteins  Increased dispersion stability and foaming ability of transglutaminase-treated oat protein isolate, likely due to: • improved electrostatic stability • intra-particle crosslinking  stability against dissociation/re-association • smaller particle size Atomic force microscopy of interfacial films at pH 7.2 Light microscopy of foams from oat protein isolates (control and TG-treated 1000nkat/g protein). Storage stability of oat protein isolate dispersionsStorage stability of oat protein isolate dispersions (1 month at 4 °C). Ercili-Cura et al., 2015, Food Hydrocolloids, 44, 183-190 Nivala et al., 2017, Food Chemistry, 231, 87-95 Control Transglutaminase-treated
  • 10.  Crosslinking of gluten proteins by tyrosinase reinforces the wheat dough Selinheimo et al., J. Agric. Food Chem. 2007, 55, 6357-  Combining degradation of fibres and crosslinking of proteins results in maximal improvement for the gluten free oat breadmaking Enzyme-aided protein network formation to improve structure properties in breadmaking REFERENCE, without enzyme TYROSINASE 30 nkat /g flour Wheat dough analysis by confocal microscopy, Dried dough samples, 0.01% Congo Red + oil Improving gluten free oat breadmaking with tyrosinase and xylanase 10 Flander et al. J. Agric. Food Chem. 2011, 59: 8385-
  • 11. 22/10/2020 VTT – beyond the obvious Glutaminase-mediated functionalization of plant proteins: increase in solubility, emulsification & gelling (Trans)Glutaminase-aided gelation of oat protein concentrate to improve applicability of oats in various food matrices (Unpublished data from VTT) 11
  • 12. Modification of plant matrix and co-passengers to improve functionality of protein fractions Case studies 22/10/2020 VTT – beyond the obvious
  • 13. Enzyme-aided separation of rapeseed protein as an option for alkali extraction Cold-pressed (defatted) rapeseed press cake Alkaline extraction Enzyme-aided water extraction Protein extract Isoelectric precipitation Protein precipitate Protein-sugar extract Washing for salt removal Water extract / Alkaline extractRommi et al. 2014 J. Agric. Food Chem., 62 (32), pp 7989–7997 Rommi et al. 2015 J Agric Food Chem 63(11), 2997-3003. Concentration  Enzyme hydrolysing carbohydrates (pectinase) increased the yield of rapeseed protein 25-70%  Comparable protein yields (40–41% of tot protein) from enzyme-aided water extraction (pH 6) and alkaline extraction (pH10) suggest that protein can be extracted without exposure to alkali Protein-sugar extract 13
  • 14. 22/10/2020 14 Improving in vitro digestibility of bran proteins by bioprocessing Solubleprotein%fromtotalprotein Time (min) Bio-processed bran Native bran Solubilisation of protein in vitro Nordlund et al.2013, J Cereal Sci, 58, 200- PROTEIN STAINED IN RED IN THE MICROSCOPY IMAGES  Digestibility of protein in vitro was clearly improved by the degradation of rye bran cell walls by hydrolytic enzymes and fermentation
  • 15. Improving in vitro digestibility of faba bean proteins by phytase treatment  Phytase enzyme degraded phytic acid and increased solubility of protein when studied in vitro at upper gut conditions  Solubility of the proteins of the phytase- treated faba bean flour was increased at acidic conditions Solubleproteinoftotalprotein(%) Solubilisation of faba protein in vitro by phytase Solubility curve of faba proteins Phytic acid content reduced up to 89% Rosa-Sibakov et al 2018, JAFC, https://pubs.acs.org/doi/full/10.1021/acs.jafc.8b02948 15
  • 16. EU-BBI-PROMINENT & EIT-FOOD-PROVE Rapeseed Hybrid Ingredient Gel with 3% dietary fibre and 5% protein Rice bran Hybrid ingredient Gel with 3% dietary fibre and 3.4% protein 0 20 40 60 80 100 0 500 1000 1500 2000 2500 0 1000 2000 3000 4000 5000 Temperature(°C) G'(Pa) Time (s) G' control G' phytase-treated 0 20 40 60 80 100 1 10 100 1000 10000 0 2000 4000 Temperature(°C) G´(Pa) Time (s) G' phytase treated G' control Set up: apply phytase in a spoonable food model with rice bran and rapeseed materials:  Phytase treatment at pH 5, 50°C, 2h; Heat- induced gelation by heating at pH 5, 6.7 and 8; 95°C, 5 min Results:  Phytic acid content was reduced from 22% to 3% in rice bran, and from 6.2% to 1.8% in rapeseed  Increase in gel strength and water holding capacity by phytase at alkaline pH Phytase improves nutritional and technological value of rice & rapeseed hybrid ingredients Kortekangas et al 2020, https://doi.org/10.1016/j.foodhyd.2020.105787
  • 17.  Oat protein hybrid ingredient (55% protein, 30% starch) with improved emulsion properties after high temperature α-amylase treatment 22/10/2020 VTT – beyond the obvious 17 Improved technological value of oat protein concentrate by amylase and heat treatment  Stable emulsion after high-temperature α- amylase treatment   Modification of co-passangers, not the protein! Native Treated
  • 18.  WHY: Legumes contain galacto-oligosachharides (GOS) that can cause intestinal discomfort  HOW: We tested GOS removal by α-galactosidases in faba bean and pea ingredients  We investigated can we remove GOS 1. When using extruder as a bioreactor for the enzyme-treated ingredient production 2. In preparation of a spoonable product 18 Enzyme-aided FODMAP removal in protein containing ingredients
  • 19.  Set up: 50% pea protein concentrate mixed with enzyme-water solution (100 or 1000 nkat/g PPC) in an extruder Extrusion parameters: 200 rpm, 40 °C; After extrusion, mixture incubated 30 min, and freeze-dried Enzyme-aided FODMAP removal in protein containing ingredients Case 1: Using extruder as a bioreactor for the low GOS ingredient production 19 GOS (mg/g dm) Raf Sta Ver SUM Control 2.5 41 36 80 DS 100 nkat/g 9.8 16 19 45 DS 1000 nkat/g 1.5 2.7 3.5 8 NEO 100 nkat/g 20 2.2 7.9 30 NEO 1000 nkat/g 0.2 0.1 0.0 0 Galacto-oligos: Raf=Raffinose, Sta=Stachyose, Ver=Verbascose; DS=Enzyme DS30 (Amano); NEO=Enzyme Neosartorya (VTT).
  • 20. Enzyme-aided FODMAP removal in protein containing ingredients Case 2: GOS reduction in spoonable products GOS (g/100g) Raf Sta Ver SUM Control 0.07 0.62 0.60 1.28 DS 100 nkat/g 0.17 0.26 0.38 0.80 DS 1000 nkat/g 0.01 0.03 0.03 0.06 NEO 100 nkat/g 0.24 0.01 0.03 0.28 NEO 1000 nkat/g 0.00 0.00 0.00 0.00 Galacto-oligos (GOS): Raf=Raffinose, Sta=Stachyose, Ver=Verbascose DS=Enzyme DS30 (Amano); NEO=Enzyme Neosartorya (VTT) 20 For low-FODMAP diet, cut-off value for GOS is <0.3 g/serving in legumes Varney et al. 2017, https://doi.org/10.1111/jgh.13698 Set up: 15% pea protein concentrate (Vestkorn) dispersed in water for 100 g serving size  Enzyme treatment (Amano DS-30, VTT Neosartorya) made in a mixing reactor (100 and 1000 nkat/g PPC, 40 °C, 30 min)  After enzyme treatment, dispersion was heated at 90 °C for 5 min in RVA Results:  Reduction of GOS to values below cut- off value “<0.3 g/serving in legumes“  Treating the pea protein concentrate with enzymes did not change the texture of the product (based on RVA)
  • 21.  There are many options how to use bioprocessing, especially enzymes in plant food processing • Separation, structure, taste, mouthfeel, stability, nutritional quality  Bioprocessing can affect several parameters simultaneously • When modifying techno-functionality, nutritional quality and flavour may change as well Take home messages (1/2) Ingredient production Ingredient modification Food applications • Separation technologies • Removal of unwanted components • Texture, structure and stability • Sensory and nutritional quality • Technological functionality • Sensory and nutritional quality
  • 22.  Think about the strategy to use - know your tools and plant materials • Enzyme type: hydrolytic, crosslinking,.. • Substrate: state (native, denaturated), quality and composition • Whether to modify the target component or co-passengers Take home messages (2/2) Ingredient production Ingredient modification Food applications • Separation technologies • Removal of unwanted components • Texture, structure and stability • Sensory and nutritional quality • Technological functionality • Sensory and nutritional quality
  • 23. Emilia Nordlund emilia.nordlund@vtt.fi +358 40 504 2963 @VTTFinland @NordlundEmilia www.vtt.fi