overview of biodepro eu project and prion inactivation in ... · overview of biodepro eu project...
TRANSCRIPT
![Page 1: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/1.jpg)
Munich, 11.04.06
M.Mittelbach1, B.Pokits1, H.Müller2, M.Müller1, B.Nebel1, D.Riesner2
1Institute for Chemistry (IFC)Working Group for Renewable Resources
Karl-Franzens-University GrazAustria
2Institute for Physical BiologyHeinrich-Heine University Düsseldorf
Germany
Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process
![Page 2: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/2.jpg)
Munich, 11.04.06
Animal Byproducts as Feedstock
Animals
Food Production Disposal
Food, Tallow Byproducts Rendering
Meat and Bone Meal FatFood, Oleochemistry
SRM – Specified Risk Material
+
+
![Page 3: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/3.jpg)
Munich, 11.04.06
Possible Feedstocks for Biodiesel Production
• Meat and Bone Meal: 10-14 % fat content
In Situ Transesterification Solvent ExtractionColl, Haas, Garcia, Marmer, 2004 Mittelbach, Nebel, 2005
• Animal Fat: 2-step-process: 1) esterification2) transesterification
• SRM Material: Destruction of TSE – prions ?
![Page 4: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/4.jpg)
Munich, 11.04.06
Extracted Fat from Meat and Bone Meal
Parameter Unit Value
Sulfur [mg/kg] 316Nitrogen [wt-%] 0.3Phosphorus [mg/kg] 854Water [mg/kg] 185Free fatty acids [wt-%] 11Unsaponifiables [wt-%] 1.97Total fatty acids [wt-%] 97
![Page 5: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/5.jpg)
Munich, 11.04.06
Biodiesel from MBM Extraction Fat
≤ 5.0< 0.5[mg/kg]EN 14538Group II metals : (Ca+Mg)
≤ 5.0< 0.5[mg/kg]EN 14109Group I metals : K
≤ 5.0< 0.5[mg/kg]EN 14108Group I metals : Na
≤ 10.0< 1[mg/kg]EN 14107Phosporous content
≤ 0.25< 0.5[wt-%]EN 14105Total glycerol
≤ 0.020.058[wt-%]EN 14105Free glycerol
≤ 0.2< 0.001[wt-%]EN 14105Triglyceride content
≤ 0.2< 0.01[wt-%]EN 14105Diglyceride content
≤ 0.8< 0.01[wt-%]EN 14105Monoglyceride content
≥ 6.05.21[h]EN 14112Oxidation stability at 110 °C
≥ 0.50.26[mg KOH/g]EN 14104Acid value
≤ 500140[mg/kg]EN ISO 12937Water content
+5/-2014[°C]EN 116CFPP
≤ 10.08[mg/kg]EN ISO 20846Sulfur content
≥ 120161[°C]DIN EN 22719Flashpoint
3.5-5.04.7[mm2/s]ISO 3104Viscosity at 40 °C
860-900875[kg/m3]ISO 3675Density at 15°C
≥ 96.597.4[wt-%]EN 14103Ester content
EN 14214ValueUnitMethodParameter
![Page 6: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/6.jpg)
Munich, 11.04.06
Prion Protein
![Page 7: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/7.jpg)
Munich, 11.04.06
Prion Protein Deactivation
2 Strategies
Bioassay: Tests with animals+ Absolute results- Time consuming- High number of animals- Injection of biodiesel in brain ??
Analysis of prions: Separation; antibody reaction+ In vitro, no animals necessary- low sensitivity of analysis- no test for infectivity
![Page 8: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/8.jpg)
Munich, 11.04.06
BIODIEPRO„Demonstration of the Production of Biodiesel from Tallow and
Recovered Vegetable Oil (RVO)““Energy, Environment and Sustainable Development – Energy Program”
Contract No.: NNE5/2001/00832Coordinator: ARGENT Energy Ltd., LondonPartner: Institute for Chemistry, KF Uni Graz
Institute for Biotechnology, TU GrazInstitute for Resource Efficient and Sustainable Systems,TU Graz
General plant construction and plant engineering: Biodiesel International (BDI), Graz, Austria
Duration: 1.1.2003 - 31.12.2005Overall costs: € 17.438.593.-EU-funding: € 3.150.000.-
![Page 9: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/9.jpg)
Munich, 11.04.06
Aim of WP 3, R&D
• Investigation whether prion proteins areinactivated and/or destroyed by the Biodiesel-production process
• Spike experiments with model proteins(ApoA1) as well as infectious prion proteins
• New applications for FAME and by-products(glycerol, solids)
![Page 10: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/10.jpg)
Munich, 11.04.06
Work Packages
• WP 1 – Plant Design & Construction• WP 2 – Commissioning & Testing• WP 3 – Research & Development (IFC,
IBTUG)• WP 4 – Analysis & Evaluation (RNSTUG)• WP 5 – Dissemination Activities• WP 6 – Project Management Activities
![Page 11: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/11.jpg)
Munich, 11.04.06
Safety Evaluation for a Biodiesel Process UsingPrion-Contaminated Animal Fat as a Source
B.Seidel, M.Alm, R.Peters, W.Kördel, A.SchäferEnviron.Sci.&Pollut.Res. 13 (2) 2006
Experimental Setup:
• Spiking of fat and biodiesel with Scrapie-infectedhamster brain
• Performing of biodiesel production process• Estimation of degradation via SDS-Gel and Western Blot
![Page 12: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/12.jpg)
Munich, 11.04.06
Safety Evaluation for a Biodiesel Process UsingPrion-Contaminated Animal Fat as a Source
B.Seidel, M.Alm, R.Peters, W.Kördel, A.SchäferEnviron.Sci.&Pollut.Res. 13 (2) 2006
Results:
• Each production step: degradation approx. 104 ID50• Whole Biodiesel production process: 1012 – 1016 ID50• Whole process including sterilisation: 1020 ID50
![Page 13: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/13.jpg)
Munich, 11.04.06
Experimental Design - BIODIEPRO
Model Protein:Apolipoprotein A1 was used for preleminarystudies and for the statistical calculation of the results
Prion Spike Experiments: In respect of the security rules all prion spikeexperiments were done at a S2+ - Laboratory
![Page 14: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/14.jpg)
Munich, 11.04.06
Prion Spike Experiments
Location: Institute of Physical Biology, Heinrich Heine University DuesseldorfHead: Prof. D. Riesner
Safety Precautions:Biochemistry Laboratory S2+
Used Spike Material:„Prion Rods“ 263K (Prep. out of Syrian Golden Hamster Brain)
![Page 15: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/15.jpg)
Munich, 11.04.06
Spike Experiments
Analysis
Waste
GlycerolWater
AnimalTallow PE Fat
-BD
Fat-BD TE Wash
Steps
SpikeProtein
CrudeBD
SpikeProtein
VD
SpikeProtein
HighlyPurified
BD
Analysis
CrudeBD
Analysis
Residue
Analysis
Waste
GlycerolWater
AnimalTallow PE Fat
-BD
Fat-BD TE Wash
Steps
SpikeProteinSpike
Protein
CrudeBD
SpikeProteinSpike
Protein
VD
SpikeProteinSpike
Protein
HighlyPurified
BD
Analysis
CrudeBD
Analysis
Residue
![Page 16: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/16.jpg)
Munich, 11.04.06
SDS Gel Electrophoresis & Western BlotWorking - Laminar Flow Bench
![Page 17: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/17.jpg)
Munich, 11.04.06
Recovery Rate of CHCl3/MeOH- Extraction
45 ng 15 ng 5 ng 45 ng 15 ng 5 ng 45 ng 15 ng 5 ng
PrP solution Extracted from glycerol Extracted from tallow
![Page 18: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/18.jpg)
Munich, 11.04.06
SDS PAGE & Western BlotPre-Esterification
225 150 75 UP1 LP1 225 150 75 ng UP2 LP 2UP: Upper PhaseLP: Lower Phase
PrP-extracted Pr-P-solution
![Page 19: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/19.jpg)
Munich, 11.04.06
SDS PAGE & Western BlotTransesterification 1 & 2
300 150 75 ng UP1 LP1 300 150 75 ng UP2 LP2
PrP-extracted Pr-P-solution
![Page 20: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/20.jpg)
Munich, 11.04.06
SDS PAGE & Western BlotDistillation
300 200 100 ng R1 BD1 300 150 75 ng R2 BD2
PrP-solution Pr-P-extracted
![Page 21: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/21.jpg)
Munich, 11.04.06
Biodiesel-Conversion Step 1Preesterification
Conditions:pH~1, 2 hours, 70°C
• Edible Tallow: 50 g• Spike: 1000 ng PrP• Det. Limit: 10 ng• Degradation Factor:
~ 102
„No PrP - onlyunidentified protein tracesdetectable“
Tallow Fat-Biodiesel
PrPAnalysis
SpikeProtein
„1“
SeparationPre-
esterification
![Page 22: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/22.jpg)
Munich, 11.04.06
Biodiesel-Conversion Step 2Transesterification
Biodiesel
PrPAnalysis
SpikeProtein
„2“
SeparationTrans-esterification
Fat-Biodiesel
Conditions:pH~14, 20 min. 50°C
• Oil/Ester-Mix: 50 g• Spike: 2500 ng PrP• Det. Limit: 10 ng• Degradation Factor:
~ 2,5*102 for each step„No PrP - detectable“
Glycerin+ PrP
![Page 23: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/23.jpg)
Munich, 11.04.06
StandardBiodiesel
VacuumDistillation
SpikeProtein
„3“
High
purified
Biodiesel
PrPAnalysis
• Conditions:Vacuum (<1 mbar), ~150°C, 35 min.
• Standard-Biodiesel: 60gSpike: 10.000 ng PrP
• Det. Limit: 10 ng• Theo. Degr. Factor: 103
„No PrP detectable“
Biodiesel-Conversion Step 3 Distillation
![Page 24: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/24.jpg)
Munich, 11.04.06
Degradation Factors
1033.4*104Distillation – Biodiesel1023.4*104Distillation – Residue
1023.4*104 Merged Wash Phases2.5*1023.4*104 Trans-Est. – Glycerol2.5*1023.4*104 Trans-Est. – Biodiesel
102-Pre-Est. – Water/Acid1023.4*104Pre-Est. – Oil/Ester
PrP-SpikeApo A1-SpikeExp. Step
![Page 25: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/25.jpg)
Munich, 11.04.06
Risk Assessment (Worst Case)
1.0 x 10-4
102
5.0 x 10-12
Rel. efficiency by route of admin.Dose of administration [100 g]Final exposure [ID50 units/p.a.]
1.0 x 10-3Distillation2.5 x10-2; 2.5 x 10-2 Transesterification
1.0 x 10-2Pre-esterification2.6 x 10-4 Prevalence of BSE infection
2.0 x 105Prion infectivity of thecontaminating tissue [ID50/g]
1.5 x 10-3Dose of BSE-infected tissuecontaminating animal fat [g]
Risk ValuesRisk Factors
![Page 26: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/26.jpg)
Munich, 11.04.06
ResultsDetection limit 10 ng prion rods
Recovery of extraction 65 – 85 %
Esterification step no prions det.
Transesterification no prions det. (unident. traces)
Glycerol phase no prions det. (unident. traces)
Distillation no prions det.
Total Degradation > 109
Risk Assessment 5.0 x 10-12 [ID50/p.a.]
Final Risk: 2.5 x 10-12
Background Risk: 1.0 x 10-6
![Page 27: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/27.jpg)
Munich, 11.04.06
European Food Safety Authority(EFSA)
The Scientific Panel on Biological Hazards concludesthat the Biodiesel process as described (BDI)
is considered as safe for treatmentand use of ABP of category 1
![Page 28: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/28.jpg)
Munich, 11.04.06
Biodiesel Plant at Motherwell, ScotlandCapacity: 50.000 t/aRaw material: Recycled Cooking Oil, Tallow, SRM-MaterialInitial Operation: January 2005Construction: Biodiesel International (BDI), Graz, Austria
![Page 29: Overview of BIODEPRO EU Project and Prion Inactivation in ... · Overview of BIODEPRO EU Project and Prion Inactivation in the Biodiesel Process. Munich, 11.04.06 Animal Byproducts](https://reader033.vdocuments.us/reader033/viewer/2022041703/5e42bfdb3a03560595772a4c/html5/thumbnails/29.jpg)
Munich, 11.04.06