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

Each algae species requires different disruption technology

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

http://www.google.nl/url?sa=i&source=images&cd=&cad=rja&docid=WiGJS5Wjv4L4SM&tbnid=lEudUCRUSe5loM:&ved=0CAgQjRwwAA&url=http://www.intlawgrrls.com/2010/08/beets-banned.html&ei=LmujUaf7AsrL0QX_sICwCQ&psig=AFQjCNGk0wGEZW5aX-UhWNx33VgM29ximg&ust=1369750702099124

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…?