Chlorophyll Reduction in Rape Seeds and its Influence on the Bleaching Earth

Consumption during Refining

Frank PudelThomas Krause

PPM Pilot Pflanzenöltechnologie Magdeburg e.V.

Bernhard GrimmUllrich Eckhardt

Humboldt-Universität zu Berlin

1212thth International Rapeseed CongressInternational Rapeseed CongressMarch 25 March 25 –– 30, 2007, 30, 2007, WuhanWuhan, China, China

• Influence of chlorophyll on rape seed oil quality

• Effect of the chlorophyll content in cruderapeseed oils on bleaching earth consumption

• Chlorophyll reduction strategies

• Conclusions

AgendaAgendaOutlineOutline

Chlorophyll in Rapeseed OilChlorophyll in Rapeseed Oil

Main Source:Rapeseed Oil

Chlorophyll

- is essential for the photosynthesis- occurs in all edible oils- reduces the oxidative stability, taste

and shelf life

Chlorophyll and Chlorophyll and PeroxidePeroxide ValueValue

0

1

2

3

4

5

6

7

8

9

10

0 0,5 1 1,5 2

Chlorophyll after Bleaching in ppm

Pero

xid

e V

alu

e a

fter

Ble

ach

ing

Chlorophyll and Peroxide ValueChlorophyll and Peroxide Value

0.05

Chlorophyll and Oxidation StabilityChlorophyll and Oxidation Stability

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10

Chlorophyll in Crude Oil in ppm

Ra

nci

ma

t Sta

bil

ity

aft

er

Deo

dori

zati

on

in

h

The Rapeseed ProblemThe Rapeseed Problem

Wochen nach Bestäubung4 6 8

rela

tive

Ein

heite

n

ChlorophyllÖla-Tocopherol

Weeks after pollination

rela

tive

un

its

ChlorophyllOila-tocopherol

shorter vegetation periods,aridness, coldness, early winter onset

Chlorophyll Removing by Chemical RefiningChlorophyll Removing by Chemical Refining

Crude Oil

Bleached Oil

Deodorised Oil

Degummed Oil

Neutralised Oil

0 100 %

Chlorophyll

Chlorophyll and Chlorophyll and BleachingBleaching

• Rapeseed production, worldwide*: 49 Mio. t/a

• Rapeseed oil production, worldwide*: 18 Mio. t/a

• Bleaching earth consumption**: ~ 0,11 Mio. t/a

• Reduction of 0,1% bleaching earth consumption leads to

– 16.000 t/a less bleaching earth (consumption and disposal)– 4.800 t/a less oil losses– preservation of minor components (tocopherols, sterols)

Which amount can be saved only by reducing the chlorophyllcontent in the crude oil (resp. rapeseed) ?

* DUSSER, 2007; ** 90% refining, 0,7% bleaching earth

Economic Data for BleachingEconomic Data for Bleaching

Degummed, (neutralised) rapeseed oil

Oil 1degummed,neutralised

Oil 2degummed,neutralised

Oil 3degummed

Peroxide Value 23,20 4,15 1,00

Anisidine Value - 6,87 -

Acid Value 0,13 0,13 1,27

UV–Extinctionat 232 nm

- 1,81 -

Water - 0,07 % -

Carotine - 54,7 ppm -

Chlorophyll a 3,73 ppm 3,50 ppm 4,39 ppm

Phosphorus < 1 ppm 4,9 ppm < 1 ppm

Calcium - 1,21 ppm -

Iron - 0,082 ppm -

Manganese - <0,01 ppm -

Copper - <0,01 ppm -

Magnesium - 0,19 ppm -

Total Tocopherol - 768 ppm -

Test Scheme of Laboratory ExperimentsTest Scheme of Laboratory Experiments

Refinedrapeseed oil

Chlorophyll a

Bleaching

Deodorisation

Analysis

Parameters:

- Bleaching earth consumption - Temperature- Bleaching time- Citric acid concentration

2,065 ppm3,73 ppm5,75 ppm8,08 ppm

20,9 ppm

Chlorophyll:

DerDer ZusammenhangZusammenhang zwischenzwischenBleicherdeeinsatzBleicherdeeinsatz und und RestchlorophyllgehaltRestchlorophyllgehalt

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 0,5 1 1,5 2 2,5

Bleaching Earth Consumption in %

Chlo

rop

hyl

l a

fter

Ble

ach

ing

in

pp

m

Chlorophyll in Crude Oil in ppm20,98,085,753,73

Bleaching BehaviourBleaching Behaviour

0

0,1

0,2

0,3

0,4

0,5

0,6

0 0,5 1 1,5 2

Bleaching Earth Consumption in %

Chlo

rop

hyl

l a

fter

Ble

ach

ing

in

pp

m Chlorophyll in Crude Oil in ppm8,085,753,73

Results: Bleaching Earth ConsumptionResults: Bleaching Earth ConsumptionBleaching Earth ConsumptionBleaching Earth Consumption

Not only very high chlorophyll contents in crude oils can be a problem.

Low chlorophyll in crude oil leads to significant lower bleaching earth consumption !

Strategien zur Strategien zur ChlorophyllreduzierungChlorophyllreduzierung

• Classical plant breeding• Transgenic Plants

- Chlorophyll biosynthesis: Inactivation of chlorophyll producing enzymes

- Chlorophyll catabolism: Overexpression of chlorophyll degrading enzymes

• Use of recombinant proteins to developan enzymatic chlorophyll removal process

Chlorophyll Reduction StrategiesChlorophyll Reduction Strategies

Klassische ZKlassische ZüüchtungchtungChlorophyll Kinetics of Different LinesChlorophyll Kinetics of Different Lines

Tower

0

200

400

600

800

1000

1200

1400

1600

25 35 45 55 65

Tage nach Bestäubung

Ges

amtc

hlor

ophy

ll pr

o Fr

isch

mas

se [µ

g/g

FM]

Cresor C.R.O. (IPK)

0

200

400

600

800

1000

1200

1400

1600

25 35 45 55 65

Tage nach Bestäubung

Ges

amtc

hlor

ophy

ll pr

o Fr

isch

mas

se [µ

g/g

FM]

IPK Golden

0

200

400

600

800

1000

1200

1400

1600

25 35 45 55 65

Tage nach Bestäubung

Ges

amtc

hlor

ophy

ll pr

o Fr

isch

mas

se [µ

g/g

FM]

IPK Bronowski

0

200

400

600

800

1000

1200

1400

1600

25 35 45 55 65

Tage nach Bestäubung

Ges

amtc

hlor

ophy

ll pr

o Fr

isch

mas

se [µ

g/g

FM]

39 winter and 33 summer lines were investigated.

Winter lines contain generally lower chlorophyll.

Here are kinetics of 4 summer lines, which are quite different.

There is a potential for further classical breeding activities !

Glutamat

5-Aminolevulinsäure

Protoporphyrin IX

Protohäm Mg-Protoporphyrin IX

HHäämm

Protochlorophyllid aPhytochromobilin

Chlorophyllid a

Chlorophyll Chlorophyll aa

Mg-Chelatase

Glutamat-1-semialdehyd-aminotransferase (GSA-AT)

Chlorophyll BiosynthesisChlorophyll Biosynthesis

H2O 2 H+ O2 + Fe2+ NADPH

ChlChl ChlidChlid PhaeidPhaeid RCCRCC FCCFCC NCCNCCPhytol Mg2+ Fe3+ NADP+

Chlase Mg-Dechelatase Phaeo-a-Oxygenase RCC-Reduktase

Chlorophyll CatabolismChlorophyll Catabolism

Strategien zur Strategien zur ChlorophyllreduzierungChlorophyllreduzierung

• Inactivation of chlorophyll producing enzymes– GSA-AT, Mg-chelatase

• gene amplification / PCR-isolation• gene constructs by RNA interference• rape hypocotyl transformation by agrobacterium tumefaciens

• Overexpression of chlorophyll degradingenzymes– Chlorophyllase (from citrus clementii)– Phaeophorbide-a-oxygenase (from tomato)– RCC-reductase (delivered by University of Bern)

Genetic Engineering SchemeGenetic Engineering Scheme

Examples of 39 Transgenic PlantsExamples of 39 Transgenic Plants

Gene Construct Expected Effect Results

1-2 (GSA-AT-RNAi) Inhibition of chlorophyll synthesis no

2-1 (Mg-Chelatase-RNAi) Inhibition of chlorophyll synthesis -

6-1 (Chlorophyllase) Acceleration of chlorophyll metabolism in 4 cases +

7-1 (Chlorophyllase without TP) Acceleration of chlorophyll metabolism

In 4 plants with chlorophyllase overexpression gene constructsreduced chlorophyll contents at the 42 day after pollinationwere measured.

• Chlorophyllase• Phaeophorbid-a-oxygenase• RCC-reductase

• Questions:

– Are the enzymes active in oil?– Which are the optimal reaction conditions?– Where the enzymes are to put into the oil?– Are the enzymes able to harm the oil?

Recombinant Proteins for Enzymatic Recombinant Proteins for Enzymatic Chlorophyll Removal from OilChlorophyll Removal from Oil

Crude Rapeseed Oil

Hydratisation

Phosphatides

Degumming

Citric Acid

Neutralization Soap

80 °C

55 °C ; 2h reaction time

Buffer, pH 7.8

Separation ?

Chlorophyll a

Chlorophyllase

Water

Alkaline

Enzymatic Refining TestEnzymatic Refining Test

ConclusionsConclusions

• Recombinant chlorophyllase from Citrus cl. is able to split the chlorophyll into chlorophyllide and phytolunder normal edible oil refining conditions.

• But, for the separation of the hydrophilic chlorophyllidean acetonic system is necessary.

• The activity of recombinant pheaophorbide-a-oxygenase and RCC-reductase is proved in vitro. Because of the need of some co-factors, their application in an edible oil refining process is not to expect.

Results of Enzymatic Refining Test Results of Enzymatic Refining Test

ConclusionsConclusions

• Even low reductions of the chlorophyll content in crude rapeseed oil lead to a considerable decrease in refining expenses, especially to a lower bleaching earth consumption.

• Different chlorophyll reduction strategies were investigated.

• The chlorophyll kinetics of 33 summer and 39 winter rapeseed lines during ripening were measured. Large differences between them indicate a potential for classical plant breeding of rapeseed lines with low chlorophyll content.

• There were 39 transgenic plants generated. 4 of them showed a significantly lower chlorophyll content at the 42. day after pollination.

• An enzymatic refining process by the use of recombinant proteins for chlorophyll removing is not to expect.

ConclusionsConclusions

AcknowlegementAcknowlegement

Humboldt University BerlinBernhard GrimmUlrich EckhardtSridevi DamarajuKersten Träder

PPM MagdeburgFrank PudelThomas KrausePetra König

AcknowlegementAcknowlegement

University GiessenWilfried LühsKarim Zarlhoul

University BernStefan Hörtensteiner

Financial supported by Federal Ministry of Education and Research, InnoRegio, InnoPlanta.

AcknowlegementAcknowlegement

Thank You

For Your Attention.