DIFFERENTIATION OF EGG PRODUCTION SYSTEMS
USING STABLE ISOTOPE SIGNATURES
Quek Shu Yi Alicia1, Bay Lian Jie1, Yat Yun Wei2, Joanne Chan Sheot Harn2, Thomas Walczyk1,3
1Department
of Chemistry, Faculty of Science, National University of Singapore, 2Food Safety Division, Health Sciences Authority,
3Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore
INTRODUCTION AND AIMS
Small variations in natural isotopic compositions of carbon, nitrogen and strontium have become an
indispensable tool for food authentication. Food authentication protects consumers from fraudulent
practices of food producers and sellers who try to disguise actual food production methods, quality or
geographical origins. Using eggs as an example, differentiation of food production systems based on
production methods and production origins were the focus of this project. In today’s context, eggs have
become a staple food product in the conventional diet of most individuals. As a result of the inherent
profits and extensive consumption of eggs, there have been numerous incidences of egg frauds. Hence,
this signifies the need for a robust analytical tool for egg authentication.
AIMS OF THE PROJECT:
1) To demonstrate the use of carbon and nitrogen isotope signatures to differentiate between organic and
conventional barn egg production systems.
2) To explore the stability of carbon and nitrogen isotope signatures in egg production systems to aid in
designing optimum sampling strategies and setting up of a local database for egg authentication
programs.
3) To demonstrate the use of carbon, nitrogen and strontium isotope signatures to differentiate between
the geographical origin of eggs, using eggs from Singapore, Malaysia and China as examples.
PRINCIPLES OF THE STUDY
15N
Enrichment
Tertiary
Consumer
MATERIALS AND METHODS
• Animals excrete more urine that is enriched in 14N with every increase of the trophic
level, causing their bodies to be more enriched in 15N content. An increase in 15N
content by 3 - 4 ‰ is observed for each step up the trophic level.
Carbon & Nitrogen Isotope Ratio Analysis
Soil & Substance
Synthetic Fertilizer
Organic Nitrogen
Secondary
Consumer
NH4
Sample Loading
Homogenizing
N2
Effluent/Manure
Primary
Consumer
15N
Enrichment
•
Maize
Wheat
13C
C3 plants
Enrichment
•
C4 plants
Isotope Ratio Mass
Spectrometry
Strontium Isotope Ratio Analysis
87Rb
Primary
Producer
Precise Weighing
t1/2 = 48.8 x 109 years
87Sr
Strontium is able to readily substitute
calcium in minerals and can be
integrated into the organism.
87Sr/86Sr of these organisms would
indicate its diet and geographical
origin.
Homogenizing
MicrowaveAssisted Digestion
Strontium
Separation
Reconstitution
Thermal Ionization
Mass Spectrometry
RESULTS AND DISCUSSION
Correlation between Different Egg Components
δ15N
Delipidised Yolk
Albumin
Delipidised
Yolk
Internal
Membrane
Albumin
Stability of Signatures
External
Membrane
δ15N
1
0.987
4.5
1
Internal Membrane 0.986
0.990
1
External Membrane 0.983
0.985
0.980
Farm 1
Intra-Month Variation:
0.27 ‰ for δ15N
0.14 ‰ for δ13C
(‰)
Inter-month variability
A
A
A
Farm 2
A
B
Intra-month variability
1
2
2
Intra-Month Variation:
0.10 ‰ for δ15N
0.25 ‰ for δ13C
Inter-month variability
A
A
B
• 3 eggs were sampled from each
farm on each occasion.
Farm 3
• t-tests performed between the
intra-month readings and
between
the
inter-month
readings.
Inter-month variability
A
B
A,B
Intra-month variability
1
1
1
4.0
• Data sets with common
alphabets (A-C) and numbers
(1-2) were not statistically
different.
1
3.5
δ13C
Delipidised
Yolk
Albumin
Internal
Membrane
External
Membrane
Delipidised Yolk
1
Albumin
0.987
1
Internal Membrane
0.972
0.973
1
External Membrane
0.966
0.981
0.965
1
Shell
0.926
0.927
0.905
0.900
Shell
3.0
2.5
δ13C
(‰)
A
-15
-16
Inter-month variability
A,B
B,C
A
A,C
Intra-month variability
1
1
1
Inter-month variability
A
A
A
• The
targeted
instrument
measurement reproducibility is
0.3 ‰ for δ15N and δ13C.
Inter-month variability
A
B
A,B
• If magnitude of measured
variability (overall measured
uncertainty) < 0.3 ‰, the
isotope signatures are deemed
to be sufficiently stable.
Intra-month variability
1
1
1
1
-17
•
•
•
All egg components are highly correlated with each other (n = 27).
All Pearson correlations have statistical significance of less than 0.05.
Albumin chosen as the representative component for all carbon and
nitrogen isotope analysis.
-18
Average inter-month variation:
0.19 ‰ for δ15N
0.49 ‰ for δ13C
-19
-20
14/10
19/10 28/10
5/12
14/1
δ13C (‰)
• Organic eggs have higher
δ15N because
• Conventional free range eggs
exhibit high δ15N as well.
1)The chickens are fed organic
feed.
2)The chickens feed on
organisms higher up the
trophic level.
• Primary source of enriched
15N
content stems from
feeding on organisms higher
up the trophic level.
δ15N (‰)
• Carbon isotope ratios are not
able to act as markers for the
differentiation.
• Since the δ13C largely
overlap, it signifies that the
chickens were fed feed of
the same composition.
14/10
19/10
28/10
5/12
14/1
14/2
5/12
14/1
14/2
δ15N is the stable marker.
Differentiation of Egg Production Systems by Production Origins
Differentiation of Egg Production Systems by Production Methods
δ15N (‰)
14/2
•
•
•
δ13C (‰)
Overall carbon and nitrogen isotope signatures of eggs collected from Oct
2012 – Feb 2013.
Multivariate linear discriminant analysis using both carbon and nitrogen
isotope signatures showed that Singaporean and Malaysian eggs can be
separated into their respective countries more than 80 % of the time.
Nitrogen isotope signatures utilized as the main tool for the differentiation of
Malaysian and Singaporean eggs.
87Sr/86Sr
•
•
Strontium (Sr) signatures from
the 3 countries largely overlap.
Signified that the chickens were
probably fed feed produced from
the same region.
CONCLUSION
• Carbon and nitrogen isotope ratio analysis on one egg component
can be taken as a representation of the δ13C and δ15N of the entire
egg.
• It is possible to differentiate organic eggs from conventional barn
eggs using δ15N.
• δ13C and δ15N of eggs are sufficiently stable within a month
across different farms within a country. Across months, δ13C is not
as stable while δ15N is quite constant.
• 13C content can only be used to analyse the feed composition and
can not be used as a marker for the differentiation of eggs by
production methods or origins if the feed does not differ.
• Singaporean and Malaysian eggs can be differentiated due to the
differences in farming practices, which resulted in the enrichment
of 15N content for Malaysian eggs. Results point towards the
feasibility that eggs can be differentiated based on production
origins if the farming practices differ amongst different countries
or regions.
• Strontium isotope ratios could not be used to differentiate eggs
from Singapore, China and Malaysia.