Stephanie BLASER Marc ANTON, Eisabeth DAVID- BRIAND, Thibault LOISELEUX 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  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 Blender40 g seeds2 x 10 sec Wet Grinding Polytron Blender300 mg ground seeds2 x 15 sec8000 rpm Centrifugation 1 Carbonate Buffer (O.4 M sucrose) 4° C10000xg30 min Centrifugation 2 Carbonate Buffer (0.6 M sucrose) 4° C10000xg30 min Centrifugation 3 Phosphate Buffer4° C10000xg30 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 seeds2 x 15 sec8000 rpm Centrifugation 1 Carbonate Buffer (0.4 M sucrose) 4° C10000xg30 min Centrifugation 2 Carbonate Buffer (0.6 M sucrose) 4° C10000xg30 min Centrifugation 3 Phosphate Buffer 4° C10000xg30 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

MethodSample Size Average Cream Weights (mg)Standard Deviation ,8415, ,0315,88 CREAM WEIGHTS

Method 3Method 5 LIGHT MICROSCOPE 63x

Method 3 CONFOCAL MICROSCOPE

GRANULOMETER

RegularFreeze-dried NANOSIZER – METHOD 3

RegularFreeze-dried NANOSIZER – METHOD 5

MethodTreatment Sample Size Average Lipid (mg) Standard Deviation 3 Regular77,191,10 Freeze-Dried with Buffer25,70- Freeze-Dried with Water27,00- Overall116,892,22 5 Regular26,35- Freeze-Dried with Buffer26,84- Freeze-Dried with Water410,282,06 Overall88,432,40 LIPID QUANTIFICATION

PROTEIN QUANTIFICATION MethodTreatment Sample Size Average Concentration (mg/ml) Standard Deviation 3 Regular32,46,51 Freeze-Dried with Buffer11,32- Freeze-Dried with Water11,36- Overall52,01,71 5 Regular15,01- Freeze-Dried with Buffer11,84- Freeze-Dried with Water23,62- Overall43,521,39

MethodTreatment Sample Size Average Percent Dry Matter (%) Standard Deviation 3 Dry Heat452,181,80 Freeze-Drying with Buffer255,59- Freeze-Drying with Water255,26- Overall854,351,88 5 Freeze-Drying with Buffer158,63- Freeze-Drying with Water344,843,17 Overall448,297,36 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 firm 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 buffer  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, PhD Marc Anton, PhD Elisabeth David-Briand Thibault Loiseleux THANK YOU