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
MATERIALS AND METHODS
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
Carbonate Buffer (0.4 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 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
MATERIALS AND METHODS
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
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
0.1 1 10 100 1000 10000
Num
ber
(%)
Size (d.nm)
Size Distribution by Number
Record 100: SB EX 3.3 1 Record 101: SB EX 3.3 2 Record 102: SB EX 3.3 3
0
20
40
60
80
0.1 1 10 100 1000 10000
Inte
nsity
(%
)
Size (d.nm)
Size Distribution by Intensity
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)
Size Distribution by Volume
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
Num
ber
(%)
Size (d.nm)
Size Distribution by Number
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
Regular Freeze-dried
NANOSIZER – METHOD 5
0
20
40
60
0.1 1 10 100 1000 10000
Inte
nsity
(%
)
Size (d.nm)
Size Distribution by Intensity
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)
Size Distribution by Volume
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
Num
ber
(%)
Size (d.nm)
Size Distribution by Number
Record 109: SB Ex 5.1 Regular 1 Record 110: SB Ex 5.1 Regular 2Record 111: SB Ex 5.1 Regular 3
0
20
40
60
80
0.1 1 10 100 1000 10000
Inte
nsity
(%
)
Size (d.nm)
Size Distribution by Intensity
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)
Size Distribution by Volume
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
Num
ber
(%)
Size (d.nm)
Size Distribution by Number
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
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
Method TreatmentSample Size
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
Method TreatmentSample Size
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