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Future potential of our single biggest protein source: Rubisco
Latest insights on value-chain analysis, processing and functional properties
Corjan van den Berg, Anneke Martin, Maaike Nieuwland, Aard de Jong, Peter Geerdink, Maurits Burgering, Ronald Visschers
USP TNO on protein
Protein Structure &
conformation
processing
positive effects
Extraction/Isolation/purification Structure-function relation Modification Ingredient interaction
adverse effects
functionality/structure
(tiny)TIM Bioactivity Satiety Protein as carrier/encapsulation
Selective separation: biorefinery Innovative processing: SHS, UHP, rapid manufacturing, electrospinning, 3D-printing
Application in food / models: bread, meat, confectionary, sauces etc.
Allergenicity Digestibility (TIM) Predictive models New protein sources
Peptide Design: In silico bioactivity
screening of theoretical hydrolysates
in relation to target receptor
Production and engineering of
hydrolysates / peptides (digestion,
fermentation, TNO Intestinal Model (TIM))
LC-MS quantitative identification
& sequencing of peptides
Bioavailability and efficacy of hydrolysates
and selected peptides (in vivo/vitro).
“Matching”
peptide
Protein Valorisation Platform Creating added value towards bioactives
Receptor
Pick your personal favourite: Important global trends
increasing population
reducing CO2 emissions
environmental concern
food security
less fossil-based resources
sustainability
The European protein value-chain…(or lack thereof)
1 from EU seeds 2 European feed producers (FEFAC), 2007
• Alternative/sustainable sources of protein are needed
• Food security: Locally produced/less import dependent
protein value chain & Functionality
• Currently soy based protein
• Imported mainly, unsustainable
• Look at the whole value-chain
1Pie chart: European feed producers (FEFAC), 2007
European protein meal import for feed1
Key characteristic:
Protein functionality Replacing cattle protein with plant protein:
5 kg soy protein 1 kg cattle protein
5 kg of plant protein 1 kg of cattle protein
or
1 kg of plant protein same as 1 kg of cattle protein?
No lignin makes a crop suitable for mild biorefining of protein
no lignin in algae/leaves
Mild cell disruption possible
proteins remains functional!
New feedstocks/resources in The Netherlands
sugar beet potato algae
Area harvested (ha) 73329 159233 <10*
Total quantity (kton) 5858 7333 <1*
Leafs yield (ton DS/ha/y) 5,0 3,1 n.a.
Netto biomass production (ton DS/ha/y) 18,6 11,9 18,3
Netto protein production (ton/ha/y) 1,1 0,7 9,2
Total potential production NL (kton/yr) 80 111 ?
Others:
Duckweed, seaweed, etc
Biochemistry 101: Rubisco did what again?!?
Ribulose 1,5-bisphosphate carboxylase/oxygenase
First step of calvin cycle
(carbon fixation)
Most abundant protein on
the planet
part of photosynthesis reaction
RuBisCo: a highly conserved enzyme plant code reviewed sequence No. AA Identical (no.) Identical (%)
Spinach (spinacia oleracea) P00875 yes 475 475 100.0
Sugar beet (beta vulgaris) Q4PLI7 no 475 465 97.9
Potato (Solanum tuberosum) P25079 Yes 477 444 93.5
Desmodesmus serratus D2KAG0 no 290 261 90.0
Chlorella Vulgaris QP12466 yes 475 419 88.2
Spinach RuBisCO:
8 large and 8 small chains complex with
substrate ribulose-1,5- bisphosphate
Rubisco has excellent properties for food
Nutritional value
Very well digestible
Well balanced amino acid profile
Non allergenic !!!
Functionality
Excellent gelling
High foam performance
Good emulsification properties
High solubility (pH dependent)
Main issues to be tackled in algae/leaves biorefinery
disruption Mild separation
functional properties
Our dream scenario: fully integrated biorefinery
separation
fermentation Carb/protein
separation
protein
separation
Purge
Clean-up
phosphate
nitrate
disruption
algae
protein
carbohydrates
lipid
growing
algae
nutrients
nucleic acids
other
Picture taken from Beer et al, 2009
Carbohydrate
Protein (Rubisco)
Lipids
Nucleic acids
Our biorefinery philosophy:
Keep ingredient functionality and cell physiology in mind
Mild disruption for optimal value creation
Methods tested on desmodesmus
Dyno mill +
Homogeniser +
Ultrasonic (small scale) +
Ultrasonic (large scale) -
Microcutter -
High pressure (4000 bar) -
PEF -
Enzymatic -
And combinations
Energy costs: ca 2.0 kWh per kg dry weight
17
Algal cell disruption technologies
Introducing VALORIE: Versatile ALgae On-site
Raw Ingredient Extractor C
ultiv
ation s
ite s
ize (
Ha)
AlgaePARC 0
1
10
Mobile aquatic biomass biorefinery
(1 kg DS/hr => ~0,5 Ha cultivation needed)
VALORIE operating range
Rubisco from sugar beet leaves
• Input: Juice produced by partners
• Capacity 1 m3/hr
• Output: dry powder
• Theoretical production 10 kg/hr
Sugar beet leaves: Harvesting
• Leaves were harvested manually and mechanically
• Harvester capacity 20 ton/hr
• Leaves harvested without stems and dirt
Production of juice from leaves (Logistics)
Extruder
• Pressing of sugar beet leaves complex
• Ideal press method determined
• 1st step: extruder for cell disruption
• 2nd step: screw press for fluid production
• Yield: >70% juice / leaves
600 litre juice produced
Lessons learned 1: leaves & juice storage
Leaves unstable:
- Bacterial / enzymatic decay
- Loss of fluid
Juice unstable:
- Enzymatic and non-enzymatic oxidation
- Binding of phenolics with protein
Solution: • Stabilization juice by addition of sodium meta-bisulphite and CaCl2
• Addition of salts in large amount undesirable
• Meta bisulphite not necessary for algae case!
• Fast processing (<12 hr) crucial for producing functional protein
Lessons learned 2: decolourization of protein
Precipitation of chlorophyll (pending patent)
• Heat/cooling regime induce precipitation of membrane proteins
• Clarification done by:
• Decanter centrifuge, capacity 200 – 1000 l/hr
• Separator centrifuge, capacity 200 – 1000 l/hr
• Optional methods for the removal of remaining chlorophyll:
• Microfiltration: pore size 0,5 µm (eliminated based on pilot trials)
• Column chromatography: off-flavours / colorants removal
• Flocculation results in efficient removal of chlorophyll
• Application in the food production chain questionable
• Concentration performed with ultrafiltration
• 30 times concentrated
• Spray drying performed at 180 – 200 °C
• Air temperature outlet: 85°C
Concentration and drying of the protein (Process)
Gelation properties (globular proteins)
Heat treatment unfolding aggregation gelation
Gelation kinetics, type of gel and gel strength are a.o. influenced by:
- temperature-time
- pH
- presence of salts
- protein concentration
NOT every protein denatures and forms gels!
Mechanisms of network formation
Heat-induced gels
High Temperature (whey, soy, egg white)
Low temperature (gelatin)
Cold-set gels, pre-heat treatment followed by:
Acidification (yoghurt)
Enzyme induced, e.g. rennet (cheese)
Addition of salts, e.g. Ca2+ (tofu)
High pressure induced
Combination of pressure and temperature
Type of network:
• fine/coarse
stranded
• particle
determines rheological
and eating properties
Methods: from molecule to food product
Chromatography
Thermal analysis
Circular dichroism
SDS Page
< 10 nm 20-500 nm 1-500 mm mm-cm >mm-cm
Information on:
-structure
-unfolding vs. native
-denaturation temp.
Light scattering
Electron microscopy
Information on:
-aggregation
-size of structures
Confocal microscopy
Light microscopy
Rheology
Information on:
-size of structures
-properties of network
at small deformation
-ingredient interaction
Texture analyzer
Microscopy
Texture analyzer
Sensory panel
Information on:
-properties of
network at large
deformation related
to eating properties
-microstructure
Information on:
-properties of
network at large
deformation
-sensory
properties, liking
Typical methods
Texture Analyzer – large deformation – eating properties
Rheometer – small deformation – gelation kinetics
Comparison with other proteins
RBC-S gels at lower
concentrations compared
to WPI and EWP
Gels with higher G’ are
formed for RBC-S
Legumes & Soy data low
gel performance
WPI
EWP
RBC-S
Texture analysis – large deformation properties
10% RBC-S
10% WPI + 0.2 M NaCl
10% EWP
10% EWP + 0.2 M NaCl
RBC
10% WPI +
0.2 M NaCl 10% EWP 10% EWP +
0.2 M NaCl 10% SPI 15% WPI
Particulate versus stranded gels
Stranded gels: no visible structures meaning aggregates < 20 mm
Concluding remarks
Rubisco is THE protein, which could help us in achieving a truly
sustainable protein value chain
Several sources of rubisco are locally available
Rubisco has a wide variety of functional properties
No history of allergy
Next steps…
Scale up
new sources
Valorise plant protein sources
Anything in it for you…?