Stephanie BLASER Marc ANTON, Eisabeth DAVID- BRIAND, Thibault LOISELEUX INRA 18/07/2014 VALIDATION STUDY OF TWO RAPESEED OIL BODY EXTRACTION METHODS.

Stephan ie BL ASER

Marc ANTON, E i sabeth DAV ID-BR IAND, Th ibau l t LO ISELEUX

INRA

18/07/2014

VALIDATION STUDY OF TWO RAPESEED OIL BODY

EXTRACTION METHODS

Rapeseed Brassica napus Common uses

Bulk oil - canola oil High protein animal feed Biodiesel

INTRODUCTION

Oil bodies Energy storage structures in plant seeds 0.5- 2.5 µm diameter Components

Triaclyglycerol core Outer phospholipid monolayer Charged surface proteins - oleosins

Structure gives physical and chemical stability

INTRODUCTION

Potential applications in the human food system Deliver stable, preemulsifed oil into appropriate food

systems Create a poorly-digested emulsion, through high

pressure processing, for the increasing obese population Have an oil source extracted without solvents

Objective Find the method that extracts the highest quantity of oil

bodies with the highest purity

INTRODUCTION

Extraction Method 3

MATERIALS AND METHODS

Dry Grinding

IKA Blender

40 g seeds

2 x 10 sec

Wet Grinding

Polytron Blender

300 mg ground seeds

2 x 15 sec

8000 rpm

Centrifugation 1

Carbonate Buffer (O.4 M sucrose)

4° C

10000xg

30 min

Centrifugation 2

Carbonate Buffer (0.6 M sucrose)

4° C

10000xg

30 min

Centrifugation 3

Phosphate Buffer

4° C

10000xg

30 min

Collect Cream

Extraction Method 5


Soak Seeds

40 g seeds + 60 mL water

Refrigerate overnight

Dry Grinding

IKA Blender

2 x 10 sec

Wet Grinding

Polytron Blender

503 mg ground seeds

2 x 15 sec

8000 rpm

Centrifugation 1


4° C

10000xg

30 min

Centrifugation 2


4° C

10000xg

30 min

Centrifugation 3

Phosphate Buffer

4° C

10000xg

30 min

Collect Cream

Extraction 3 and 5 treatments Regular - fresh cream in phosphate buffer (100 mg/ml) Freeze-dried cream in phosphate buffer Freeze-dried cream in water

Analyses Dry Matter Size

Microscope Granulometer Nanosizer

Protein quantification BSA

Lipid quantification Isopropanol/hexane extraction


RESULTS

Method Sample Size

Average Cream Weights (mg)

Standard Deviation

3 28 100,84 15,24

5 14 110,03 15,88

CREAM WEIGHTS

Method 3 Method 5

LIGHT MICROSCOPE

63x 63x

Method 3

CONFOCAL MICROSCOPE

GRANULOMETER

0.1 1 10 100 10000

1

2

3

4

5

6

7

Average Particle Size

Method 3 RegularMethod 3 Freeze Dried with BufferMethod 3 Freeze Dried with WaterMethod 5 RegularMethod 5 Freeze Dried with BufferMethod 5 Freeze Dried with Water

Diamètre des gouttes (µm)

Volu

me (

%)

Regular Freeze-dried

NANOSIZER – METHOD 3

0

10

20

30

40

50

60

0.1 1 10 100 1000 10000

Inte

nsity

(%

)

Size (d.nm)

Size Distribution by Intensity

Record 100: SB EX 3.3 1 Record 101: SB EX 3.3 2 Record 102: SB EX 3.3 3

0

10

20

30

40

50

0.1 1 10 100 1000 10000

Vol

ume

(%)

Size (d.nm)

Size Distribution by Volume


0

10

20

30

40

0.1 1 10 100 1000 10000

Num

ber

(%)

Size (d.nm)

Size Distribution by Number


0

20

40

60

80

0.1 1 10 100 1000 10000

Inte

nsity

(%

)

Size (d.nm)


Record 104: SB Ex 3.3 pow der 2 Record 106: SB Ex 3.3 second try 1Record 107: SB Ex 3.3 second try 2 Record 108: SB Ex 3.3 second try 3

0

10

20

30

40

50

0.1 1 10 100 1000 10000

Volu

me (

%)

Size (d.nm)



0

10

20

30

40

50

0.1 1 10 100 1000 10000

Num

ber

(%)

Size (d.nm)



Regular Freeze-dried

NANOSIZER – METHOD 5

0

20

40

60

0.1 1 10 100 1000 10000

Inte

nsity

(%

)

Size (d.nm)


Record 109: SB Ex 5.1 Regular 1 Record 110: SB Ex 5.1 Regular 2Record 111: SB Ex 5.1 Regular 3

0

10

20

30

40

0.1 1 10 100 1000 10000

Vol

ume

(%)

Size (d.nm)



0

10

20

30

40

0.1 1 10 100 1000 10000

Num

ber

(%)

Size (d.nm)



0

20

40

60

80

0.1 1 10 100 1000 10000

Inte

nsity

(%

)

Size (d.nm)


Record 115: SB EX 5.1 Pow der 1 Record 116: SB EX 5.1 Pow der 2Record 117: SB EX 5.1 Pow der 3

0

10

20

30

40

50

0.1 1 10 100 1000 10000

Vo

lum

e (

%)

Size (d.nm)



0

10

20

30

40

50

0.1 1 10 100 1000 10000

Num

ber

(%)

Size (d.nm)



Method TreatmentSample Size

Average Lipid (mg)

Standard Deviation

3

Regular 7 7,19 1,10

Freeze-Dried with Buffer 2 5,70 -

Freeze-Dried with Water 2 7,00 -

Overall 11 6,89 2,22

5

Regular 2 6,35 -Freeze-Dried with Buffer 2 6,84 -Freeze-Dried with Water 4 10,28 2,06

Overall 8 8,43 2,40

LIPID QUANTIFICATION

PROTEIN QUANTIFICATION


Average Concentration (mg/ml)

Standard Deviation

3

Regular 3 2,46 ,51

Freeze-Dried with Buffer 1 1,32 -

Freeze-Dried with Water 1 1,36 -

Overall 5 2,01 ,71

5

Regular 1 5,01 -Freeze-Dried with Buffer 1 1,84 -Freeze-Dried with Water 2 3,62 -

Overall 4 3,52 1,39


Average Percent Dry Matter (%)

Standard Deviation

3

Dry Heat 4 52,18 1,80Freeze-Drying with Buffer 2 55,59 -Freeze-Drying with Water 2 55,26 -

Overall 8 54,35 1,88

5

Freeze-Drying with Buffer 1 58,63 -Freeze-Drying with Water 3 44,84 3,17

Overall 4 48,29 7,36

DRY MATTER

Cream Collection Weights No major difference between Method 3 and 5

Size Method 3 vs 5

Both displayed similar presence of floculation and particle sizes- granulometer, nanosize, and light microscope

Regular vs. Freeze-dried Light microscope

No major visual differences Granulometer

More floculation present in samples freeze-dried with buffer Nanosizer

Freeze-dried samples showed a wider range of particle sizes Differences in freeze-dried samples potentially due to

destruction of oil bodies during harsh treatment

DISCUSSION

Lipid Quantification Slightly higher collection from Method 3

Protein Quantification Slighly lower concentration from Method 3

Dry Matter Slightly higher Percent Dry Matter from Method 3

DISCUSSION

Challenges Cream collection

Cream can stick to cap of centrifuge tube Cream in Method 3 is, in general, not as fi rm and durable as Method 5

Size measurement instrumentation Granulometer and nanometer have size detection limits that are on both sides

of the oil bodies upper and lower diameter range New equipment arriving next month

Freeze-drying Low volume of final product Time consuming Slightly lower protein content With buff er

Powdery final product - easier to collect Contained phosphate buffer salts

Room for errors in calculations

With water Waxy final product - more difficult to collect

DISCUSSION

Future options Using sustainabiliy grown seeds

2010 Unilever Sustainable Agriculture Code Put into effect in Germany under Cargill

Using the valueable protein for a human food source and not just for animal meal Hurdles: glucosinolates, phenolics, phytates, and high amount of

fiber Benefits: balanced amino acid profile, functional properties

(emulsifying, foaming, and gelling), and new alternative to feed increasing population

Creating industrial scale extraction methods without the use of dangerous solvents

SUSTAINABILITY

Method 3 More consistent Less time consuming Less protein contamination

Method 5 Easier handling of cream Long soak step More protein contamination

Freeze-drying Time consuming Few added benefits

Next step Compare against Thibault’s data Increase collection volume to larger bench scale

CONCLUSION

Stephanie Jung, PhDMarc Anton, PhDElisabeth David-BriandThibault Loiseleux

THANK YOU

Stephanie BLASER Marc ANTON, Eisabeth DAVID- BRIAND, Thibault LOISELEUX INRA 18/07/2014 VALIDATION STUDY OF TWO RAPESEED OIL BODY EXTRACTION METHODS.

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