NIZO Plant Protein Functionality Conference on October 21-22 gathered around 450 attendees to discuss the recent findings and innovations on plant proteins. Research team leader Emilia Nordlund gave a keynote presentation on bioprocessing technologies to improve the plant protein functionality.

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
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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