4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October

11-13, 2016, Penang, Malaysia

Outline
• Introduction
– 3-MCPD and 3-MCPD ester
3-MCPD esters: – Research by others
– Mechanism hypothesis
Current Knowledge and Status – A quick look at refining

Nuzul Amri Ibrahim • Studies in MPOB

Muhamad Roddy Ramli – Adoption of BfR 8 and AOCS cd 29a-13
Raznim Arni Abdul Razak – Mitigation studies
Ainie Kuntom
• Conclusions/ Recommendations
Malaysian Palm Oil Board

3-MCPD & 3-MCPD ester EFSA Report 2016

Food processed contaminants
Compound Effect to health
3-monochloropropanediol
(3-MCPD)
3-monochloropropanediol ester
(3-MCPDE)
q 2- and 3-MCPD esters and glycidyl
esters were detected in vegetable
Possibly carcinogenic to humans1 oils
Nephrotoxic (EFSA Report 2016)
Nephrotoxic (EFSA Report 2016) q 3-MCPD ester and free forms are
nephrotoxic (damage to kidney)
v Esterified and free forms were
assumed to contribute equally
to internal exposures.
v TDI: 0.8 µg/kg bw/day

1 Classification by International Agency for Research on Cancer

4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Chemical structure Studies on 3-MCPD & 3-MCPDE

R
Year Presence of free 3-MCPD/ 3-MCPD ester in food
O O OH 1978-1980 Hydrolysed vegetable protein, e.g soy sauce (free)
1983 Spanish rapeseed oil (ester)
OR1 Cl OH Cl 2004 Processed food: toasted bread, cheese, cooked
meat, salami, infant formula, coffee and creamer
3-MCPD ester 3-MCPD (ester)
2006 Vegetable oils (ester)
2009 Infant and baby food (ester)

3-MCPD esters Formation Mechanism Hypothesis

R
• Family of chloropropanols R O R O R
OH Cl O O
H+ O Cl-
• Categorized as food-processed O O + O +
-H2O
contaminant OR1 OR1 OR1 OR1 Cl
Cyclic acyl
• Mainly in refined oils Acylglycerol
oxonium ion
2- & 3- MCPD ester
– Due to heat and presence of chloride R = alkyl
R1 = H or COR
2-MCPD 3-MCPD

ILSI Report 2009, Brussels

4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Physical refining process

Crude palm oil Phosphoric acid is added

A quick look at physical Degumming

refining process Bleaching clay is added Phosphoric acid and gums

Bleaching

Filter Press Gums/Clays and

phosphoric acid are
removed
Deodorisation

Refined palm oil

Source of H+ and Cl- Studies in MPOB

From acid and From oil, water and clays
acid activated clays 1. Adoption of method of analyses
R
• BfR 8 2010 to 2014
R O R O R
OH
H+ O Cl-
Cl O O • AOCS cd 29a-13 Since 2015
O O + O +
-H2O
OR1 OR1 OR1 OR1 Cl
Cyclic acyl
2. Mitigation measures
Acylglycerol oxonium ion 2- & 3- MCPD esters

R = alkyl
R1 = H or COR
2-MCPD 3-MCPD
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

BfR Method 8 Repeatability of results

100 mg of oil/fat sample Concentration (mg/kg), % Recovery (± SD) RSD (%)
n=6
0.25 97.6 (± 0.022) 0.02
Internal standard (d5-3-MCPD)
1.0 103.8 (± 0.03) 0.03
Acidic transesterification with H2SO4
(hydrolysis for 18h at 40°C) 4.0 107.7 (± 0.128) 0.12

6.0 107.9 (± 0.115) 0.11

Neutralization with NaHCO3

Derivatization with PBA

GC-MS Analysis

Calibration curve GC chromatogram

0.9
Area ratio of 3-MCPD and

0.8
deuterated 3-MCPD

0.7
0.6
0.5
y = 0.1361x + 0.0102
0.4 R² = 0.9995
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7
Spiking level of 3-MCPD (mg/kg)
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Cross check with other

Participation in BfR ring test
laboratories
Values 3-MCPD ester results (mg/kg)
3-MCPD ester levels (mg/kg)
Samples
BfR actual values (range) 0.16 – 3.92
MPOB Unilever Eurofins

Reported values (mean 0.26 – 3.51

Bleached palm 1.048 0.83 2.4

range from six laboratories) oil

RBD palm oil A 4.3 4.37 6.0

MPOB values (range) < 0.25 – 3.25
RBD palm oil B 5.96 5.99 7.9

AOCS Method Cd 29a-13 Repeatability of results

100 mg oil/fat sample
Conc. Day 1 Day 2 Day 3 Mean Std Dev RSD
Add internal standards + THF Level
0.08 70 80 85 78.3 3.2 4.09
Conversion of GE to 3-MBPD monoesters in bromide salt 0.5 85 90 91 88.7 4.1 4.6
2.5 105 109 112 108.7 5.3 4.9
Conversion of 2- and 3-MCPD esters and 3-MBPD
*Percentage recovery for 2-MCPDE & 3-MCPDE (LOD =
esters into free form in acid methanolic solution
0.01)

Extraction of fatty acid methyl esters Conc. Day 1 Day 2 Day 3 Mean Std Dev RSD
Level
Derivatization of 2- and 3-MCPD and 3- 0.1 71 82 84 79 2.5 3.16
MBPD with Phenylboronic acid (PBA) 1.0 86 90 92 89 3.1 3.5
4.0 105 110 115 110 5.7 5.2
GC-MS Analysis
*Percentage recovery for GE (LOD = 0.05)
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Cross check with European labs.

(AOCS 3-in-1 method) Mitigation measures
9
8 Objectives
7
Crude palm • To evaluate the possible factors responsible
6 oil
RBD palm for formation of 3-MCPD esters during
5 oil
refining of CPO.
mg/kg

4 RBD palm
olein
3
RBD palm
2 stearin • To suggest possible solutions/alternatives to
1 minimise the formation.
0
MPOB

MPOB

MPOB
SGS

SGS

Eurofins

SGS
Eurofins

Eurofins

-1

3-MCPD ester levels Glycidol ester levels 2-MCPD ester levels

*MPOB (AOCS Cd 29a); SGS (AOCS Cd 29b); Eurofins

(AOCS Cd 29c)

Pilot-plant scale refining:

200kg/batch refining pilot plant
Condition
Parameter Standard Modified
H3PO4(%) 0.1 0.02-0.2
Acid activated 1 1-2
b/clay(%)
Deod’n temp(°C) 260 220-250
Duration(h) 1.5 3
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Experiments Effect of temperature

Ö Compare Standard refining process with 3.5 8

modified conditions 3 7

3-MCPDE content, mg/kg

2.5
ØEffect of degumming, acid activated clays, 5

Colour, Red
natural clays and deodorization temperature. 2
4
3-MCPDE
Colour
1.5

Ö Checked acidity of clays and chloride 1

3

contents. 0.5
2

Ö Monitor the formation of 3-MCPD ester 0

220 230 240 250 260
0

Deodorization temperature, oC
Results

Effect of deodorization temperature at 230 °C Acidity of clays (pH)

Variables: Phos acid and b/clay
Acid activated pH
3.5
3
260 °C clays
Deod’n temp: 230 °C*
3-MCPDE, mg/kg

2.5 A1 5.8
2
A2 5.8
1.5
1
A3 5.3
0.5 Natural clays
0
N1 6.6
0.1/1 0.1/2 0.2/1 0.2/2 0.1/1
Phos. acid/B. clay, % N2 6.6
N3 7.7
*Residence time: 3 hours
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Correlation between pH of clays

Total chloride contents
and formation of 3-MCPD esters
3.2

Acid degumming experiments • Clays (n=26) 60-152 ppm

3.0
• Water(n=7) 7-38 ppm
3-MCPD esters content (ppm)

2.8 • Phosphoric(n=8) 1-2 ppm

R2 = 0.9759 • Citric acid(n=2) 1 ppm
2.6
• CPO(n=11) 8-29 ppm
2.4

2.2
Analysed by a commercial laboratory (March-April 2012)

2.0
5.0 5.5 6.0 6.5 7.0 7.5 8.0
pH

Effect of rinsing
Effect of Cl in bleaching clay
Deodorization temp: 260 °C
6

3
3-MCPDE content, mg/kg

5
2.8
2
R = 0.9752

3-MCPDE content, mg/kg

2.6
4
2.4
Not rinsed
2.2 3 Rinsed 1X

2 Rinsed 2X

2
1.8
0 10 20 30 40 50 60 70
Level of Cl in bleaching clay, mg/kg 1

0
1 2
The level of Cl in b/clay shows positive correlation
with the formation of the esters
CPO samples
Results
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Deodorisation-time effect
(K. Hrncirik, Berlin, 2010)

Report by other researchers

Formation of 3 –MCPD esters occurs within first 20 mins

Effect of Diacylglycerols
Bertrand Matthaus (2010, AOCS Mtg)
Effect of DAG
Sample DAG,% 3-MCPDE, mg/kg
Feed oil 6.1 4.766 㼼 0.006
TL IM 3h 13.7 4.023 㼼 0.246
TL IM 9h 21.6 3.211 㼼 0.171
TL IM 12 h 22.4 2.480 㼼 0.019
TL IM 24 h 25.5 2.477 㼼 0.057

3-MCPDE
DAG
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Effect of FFA and DAG Observations

FFA, % DAG, % 3-MCPDE, mg/kg • Acid degumming followed by bleaching
Fresh 0.4 3.5 1.62 㼼 0.023 causes formation of 3-MCPD esters in
Day 1 0.8 3.1 0.866 㼼 0.007 bleached oils.
0.6 3.2 1.305 㼼 0.031
• Rinsing of CPO prior to refining could
Day 2 2.0 3.9 1.252 㼼 0.035
reduce the formation of the esters
1.3 3.8 2.584 㼼 0.199
Hydrolysed 56 3.9 <LOD
• FFA and DAG are not directly correlated to
58 5.3 <LOD
the formation of 3-MCPD esters
• High deodorization temperature led to high
Levels of FFA and DAG are not directly formation of the esters
correlated to the formation of the esters

Factors for consideration Conclusion

1. Bleaching Factors that cause the formation of 3-MCPD
Levels of chloride in clays
ester
Acidity of clays
HCl or sulphuric acid activation of clays • Acid degumming
Time and temperature • Total chloride content in bleaching clay
2. Deodorization
Temperature
• pH of bleaching clay
Time • High deodorization temperature
Chloride content in oil
Acidity of oil
• Presence of Cl in CPO
4th Asia-Pacific International Food Safety Conference & 7th Asian Conference on Food and Nutrition Safety, October 11-13, 2016, Penang, Malaysia

Current study
Recommendations Mitigation measures for 3-MCPD esters:
Identification of source of chloride at
plantations and palm oil mills
• Combine acid degumming with water
degumming • To find mitigation measures for eliminating chloride in
CPO
• Use bleaching clay with the lowest total – Midstream: mills operation
chloride content, while the pH should be – Upstream: plantation activities
R
almost neutral to alkaline. R O
OH
R
Cl
O R O O
H+ O Cl-
O
• Reduce deodorization temperature -H2O
O + O +

OR1 OR1 OR1 OR1 Cl

– From 260 °C to 230 °C Acylglycerol
Cyclic acyl
2- & 3- MCPD ester
oxonium ion
• Rinse CPO prior to refining R = alkyl
R1 = H or COR
2-MCPD 3-MCPD

Current situation
• Maximum level has not been regulated
– JECFA will meet in Rome, November 2016

• Nonetheless, Malaysian refineries should

be proactive