Deteksi Keamanan Pangan Transgenik

Deteksi Keamanan Pangan
Transgenik
Dahrul Syah
Agenda
Pengantar
Substantial Equivalen
Beyond Substantial Equivalen
Pro – Kontra
Pengaturan di Indonesia
Teknologi Pangan - IPB
1
Biotech Products in
Food Supply
1. Fermentation products
– Yeasts and molds used to make cheese,
bread, wine, beer, soy sauce
2. Enzymes
– Lactase tablets to breakdown lactose in
milk - for allergies
– Chymosin (rennet) for cheese
manufacture
– Amylase in beer
3. Hormones
Bovine growth hormone
(rbst)
Vitamin D
added milk
Insulin
2
4. Whole foods/crops
– FLAVR SAVR,
Endless Summer
tomatoes
– Virus – resistant
squash
– Insect resistant
potatoes, corn
– Herbicide resistant
soybeans, canola
3
Acceptance of Technologies
• Public accepts new technologies with personal
benefits
rInsulin
vs
rBST
Benefits of many
bioengineered
products unknown or not
perceived as personal
Platform of “omics”
AGRONOMICS
PHENOTYPICS
GENOMICS
PROTEOMICS
METABOLOMICS
STATISTICS
Structure
Gene
Protein Metabolite
Function expression expression
expression
Data
integration
Unintended effects?
DIFFERENCES
COMPONENTS
PROTEINS
DNA/mRNAs
TISSUE
PLANT
4
Potential Food Safety Effects
•
•
•
•
•
Toxicants
New Substances
Nutrients
Allergenicity
Other Effects - Unintended
-- IFT Expert Report on Biotechnology and Foods, 2000
Considerations in Safety
Evaluations
• Substantial Equivalence – based on
comparison of recombinant product with
traditional product
– Not an absolute guarantee of safety
– Process to establish that no NEW hazards have
been introduced into the plant or product
• Toxins?
• Nutrient Effects?
Proteins, fats, carbohydrates,
vitamins, minerals
• Allergens?
5
Equivalency testing
Selection of Traditional
Counterpart
History of “safe use”
Genetic characterisation
Agronomic characterisation
Comparison
with Traditional
Counterpart
Chemical characterisation
•nutrients
•toxicants
•allergens
Dietary patterns
Kesetaraan Substansial
Asam Lemak
Allergens
☺ Nutrients:
•
•
•
•
•
Carbohydrates
Proteins
Lipids
Vitamins
Minerals
Asam Amino
Tempe yang dibuat dari kedele import dan kedele lokal
6
Three Levels of Equivalence
1.Equivalent
Oil
No further testing
Oil
2.Equivalence except for
predictable, well-defined traits
Focused evaluation on limited
differences
3.Lack of equivalence, multiple
complex differences or no
traditional counterpart
Rigorous case-by-case safety
assessment
Kesetaraan; Proksimat
60
Mean
Mean
50
50
40
40
% BB
% BB
60
30
30
20
20
10
10
0
Air
Abu
Protein
Lemak
0
Air
Tempe Kedele Import
Abu
Protein
Lemak
Tempe Kedele Lokal
Perbedaan signifikan : kadar air
Sumber: Syah (2005)
7
Chemical Characterisation
Fatty Acid Analysis of Seeds
9 field sites in 1992
50
30
%
%
40
20
10
0
C16:0
(palmitic)
C18:0
(stearic)
C18:1 cis
(oleic)
C18:2
(linoleic)
C18:3
(linolenic)
*No significant differences from the control line were observed
Sumber: Hugget (2001)
% AL/Total AL
Komposisi Asam Lemak (% AL/Total AL)
100
90
80
70
60
50
40
30
20
10
0
Palm itat
Stearat
Arakhidat
Dodekanoat
Tempe Kedele Import
Oleat
Linoleat
Linolenat
Tempe Kedele Lokal
8
Chemical Characterisation
Amino Acid Analysis of Seeds
9 field sites in 1992
% Dry Weight
3
2
1
tryptophan
methionine
cysteine
arginine
lysine
histidine
tyrosine
leucine
0
phenylalanine
H
Control seeds
Literature low
RRS seeds
Literature high
*No significant differences from the control line were observed
Sumber: Hugget (2001)
Kesetaraan : Komposisi Asam Amino
6
% BB
5
4
3
2
1
0
Asp
Glu
Ser
His
Gly
Thr
Arg
Tempe Kedele Import
Ala
Tyr
Met
Val
Phe
Leu
Lys
Tempe Kedele Lokal
9
Polar Chart Asam Amino (%AA/AA Total)
Lys
Leu
Phe
25
20
15
10
5
0
Glu
Ser
Spektrum
Asam Amino
yang identik
His
Val
Gly
Met
Thr
Tyr
Ala
Tempe Kedele Import
Arg
Tempe Kedele Lokal
Toxicant Analysis of Seeds from
Roundup Ready Soybeans
100
9 field sites in 1992
% Dry weight
80
60
40
20
0
Trypsin inhibitor
TIU / mg DW
Lectin
H.U./mg protein extracted
Control seeds
Literature low
RRS seeds
Literature high
*No significant differences from the control line were observed Sumber: Hugget (2001)
10
Kesetaraan : Tripsin Inhibitor
60000
40000
20000
0
Kedele
Tempe
Import
Lokal
Kesetaraan : Asam Fitat
0,28
mg / g
TIU / g
80000
0,27
0,26
0,25
Kedele
Tempe
Import Lokal
11
Kesetaraan : Hemaglutinin
Tempe Kedele Import (1681 HU/g)
Tempe Kedele Lokal (1648 HU/g)
No Changes in Endogenous
Allergens
Soyabean Sensitive
Serum
Total Protein
Non-sensitive
Serum
RR1: GTS line 61-67-1
RR2: Parental line A5403
RR3: GTS line 40-3-2
C1: Cargill control 1
C2: Cargill control 2
C3: ADM control
106 kD
80 kD
49.5 kD
32.5 kD
27.5 kD
18.5 kD
Control 4
Control 3
Control 2
RR-3
RR-1
RR-2
Control 4
Control 3
Control 2
RR-3
RR-2
RR-1
Control 3
Control 4
RR-3
Control 2
RR-1
RR-2
12
Chemical Characterisation - Summary
Component
Proximate analysis
Amino acids
Fatty acids
Trypsin inhibitors
Lectins
Phytoestrogens
Urease
Stachyose, raffinose
Phytate
Known allergens
Beans
Meal
Flour
Isolate
Conc.
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Oil
=
=
=
=
• Based on assessment of over 400 components in 2000 independent analyses J. Nutr. (1996) 126:702-716
• Confirmed with soybeans treated with Roundup herbicide - J. Agri. Food Chem.
1999, 47:4469-4473
• No changes in endogenous soya allergens - J. Allergy Clin. Immunol. 1996,
96:1008-1010
GM Soybeans vs Traditional Varieties
Component
Changed
Unchanged
Nutritional/Toxicant composition
Animal feed performance
Processing
Dietary use
Tolerance to glyphosate
X
One additional protein / gene
X
Sumber: Hugget (2001)
13
Agenda
Pengantar
Substantial Equivalen
Beyond Substantial Equivalen
Pro – Kontra
Pengaturan di Indonesia
Teknologi Pangan - IPB
Keamanan seluruh Komponen;
gen sisipan, pembawa dan inang
Tomato DNA
Fish DNA
Tomato cell
Fish cell
Desired gene
Replicated DNA
Fish
Tomato
Denucleated fish egg
Tomato-fish
Credit: Owen Koo
14
Safety of Genetic Material
Inserted
Characterization of
• Source
• Size
• Number
• Location of insertion
• Identification of sequences in the plant
Safety of DNA itself is not in question
Safety of CTP-EPSPS
•
No toxicity associated with CTP-EPSPS gene
•
No toxicity associated with CTP-EPSPS protein
- EPSPS family of proteins have a long history of safe
consumption
- No amino acid sequence homology to known toxins
or allergens
- No toxicity observed in animal studies
- Shows none of the properties of allergens
J. Nutrition, 1996, 126:728-740
15
CP4 EPSPS - Toxicity Assessment
Mean body weight (g)
35
30
25
20
15
10
5
0
Males, pre-test
Males, day 7,
Females, pre-test
Vehicle control
49 mg/kg CP4 EPSPS
363 mg/kg BSA control
154 mg/kg CP4 EPSPS
Females, day 7
572 mg/kg CP4 EPSPS
Digestion of CP4 EPSPS in vitro
Gastric fluid
C
0
15
30 60 120
Seconds
Intestinal fluid
C
0
10
32 100 270 1181
Minutes
• CP4 EPSPS does not have the stability
characteristics of known food allergens
16
Comparison of CP4 EPSPS to known
allergenic proteins
Allergen
Molecular weight (10-70 kD)
High concentration in food
Heat stable
pH stable
Protease stable
Glycosylated
Sequence homology
+
+
+
+
+
+
+
CP4 EPSPS
+
-
“no significant concern”
Unintended Effects:
“… the unintended expression of some
unknown or unexpected toxic or
antinutrient factor, or the otherwise
unintended enhanced production of
known toxic constituents.” - (Royal Society,
1998)
Evidence of such effects has not been
found as result of bioengineering.
Effects are less likely in bioengineered
than in conventional products.
17
Food Allergens:
• All food allergens are proteins: only a small
fraction of food proteins cause allergic
response.
• Common foods with allergenic proteins:
peanuts, milk, seafoods
• Potential allergenicity of genetic material
(proteins) introduced into a plant is an
important part of safety assessment.
Allergenicity Assessment
• Follows a decision-tree process
• Includes:
– Source of the gene
– Sequence homology of newly introduced
protein
– Reactivity of newly introduced proteins with
Immunoglobulin E (IgE) antibodies from
people with known allergies to source
material
– Other properties, such as digestibility of the
protein
18
Metode : Pengujian Alergenitas
•
Test Passive Cutaneous Anaphylaxis.
- Sensitisasi mencit dengan pemberian ransum tempe
selama 2 bulan.
- Mencit dianestasi, darahnya diambil, diperoleh serum
mencit anti-alergen
- Antiserum diinjeksikan secara intradermal dipunggung
mencit
- Mencit diinjeksi lagi dengan sampel dan evans blue
melalui ekor
- Sampel menyebabkan alergi jika terlihat area biru pada
punggung
Allergenitas ; PCA
Negatif
untuk
kedua
tempe
19
Allergenitas ; Ig E
Hasil
(IU/ml)
No
Perlakuan
1
Standar
2
Ovalbumin (1%)
3
Protein tempe kedelai import (1%)
0
4
Protein tempe kedelai import (3%)
0.2
5
Protein tempe kedelai lokal (1%)
0
6
Protein tempe kedelai lokal (3%)
0
0
0.4
Pro – Kontra (1)
Assumption based
risk assessment
Substantial
Equivalence
SCIENCE/Evidence
based
risk assessment
Tox Testing
20
Pro – Kontra (2)
(Unproved) model
testing risk
assessment
Tox Testing
with Protein
(from E.coli)
Test as consumed
risk assessment
EVENT-Specific
Whole Food
Tox testing
GM
Labelling
21
Pengaturan di Indonesia
UU 7 Tahun 1996 dan PP turunannya
Pasal dan Ayat Khusus
Labelling
Pengaturan
Komisi Keamanan Hayati dan Keamanan Pangan
Komisi Teknis Keamanan Hayati dan Keamanan
Pangan
Teknologi Pangan - IPB
22