Beruflich Dokumente
Kultur Dokumente
204
Ghidini S., Ianieri A., Zanardi E., Conter M., Boschetti T.*, Iacumin P.*, Bracchi. P. G.
Introduction
Stable isotopes
Stable isotopes are those isotopes of an element which are stable and that do
not decay through radioactive processes over time. Most elements consist of more
193
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
than one stable isotope. For instance, hydrogen exists as two stable isotopes 1H and
2
H (called deuterium), carbon exists as two stable isotopes, 12C and 13C and oxygen
exists as three stable isotopes, 16O, 17O, and 18O. In most cases the more abundant sta-
ble isotope species typically contains the fewest number of neutrons for that element.
Stable isotopes have to be distinguished from radioactive isotopes of an element.
Radioactive isotopes have finite life times and undergo a decay to form a different
element. The time required for this decay may vary widely ranging from fractions of
a second to thousands of years. For instance, carbon has six radioactive isotopes (9C,
10
C, 11C, 14C, 15C, and 16C) of which 14C (which half life is 5730 years) is certainly the
best known because of its use in dating biological materials.
Isotope abundances
A brief listing of the stable isotopes and their abundances for the elements
most commonly used in food authentication can be seen in Table 1.
Table 1: Stable isotopes and their abundances for the elements most commonly used in food authentica-
tion research.
Analytical methods
194
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
components of organic matter. In any case the majority of the elements has stable
isotopes that could be useful for this purpose. The use of heavier elements is limited
because of the analytical approach usually adopted.
The light elements are typically measured using a gas isotope rationing mass
spectrometer. The mass spectrometer consists of a source to ionise the gas, a flight tube
with a magnet to deflect the path of the ionised gas, and a detector system at the end
of the flight tube to measure the different isotopic species. First, the element of interest
must be converted to a gaseous form in order to be analysed into the mass spectrometer.
The most commonly used approaches involve introducing hydrogen as H2, carbon as
CO, nitrogen as N2, oxygen as CO2 and sulphur as SO2. Mass spectrometers can analyse
only ions therefore, before the gas is introduced, it is ionised by removal of an electron.
This process is achieved in that part of the spectrometer usually called ion source. Then
as the ionised gas travels down the flight tube (under vacuum or carried by helium), the
paths of light and heavy isotopic species are deflected by the magnet of an angle which
is direct function of their mass over charge ratio. Detectors are positioned at the end of
the flight tube to measure the abundance ratios of the heavy and light isotopic species.
The main limitation of this approach is the impossibility to determine the isotopic pro-
file of heavier elements that could be very useful in food authentication. For instance
strontium proved to be very useful for the authentication of some foods (Fortunato G.
et al.; 2004), but many other elements could be useful. ICP-MS with a time of flight or
magnetic field sector spectrometer could be a solution for the analytical problem. Un-
fortunately both cost and technical complexity still limit the use of these techniques.
where R is the molar ratio of the heavy to light isotopes, e.g., Equation (2):
For the most commonly used light isotopes, the internationally recognised standards
are shown in Table 2.
Table 2: Internationally recognised standards for the most commonly used light isotopes.
195
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
196
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
ratio is more difficult to interpret due to the numerous factors influencing its nature of
soil, industrial emissions and sulphur-containing fertilisers (Rossmann, 2001).
The 87Sr/86Sr ratio depends only on the types of rocks and soils, and not on
human activity, climate or season of production (Rossmann et al., 2000). 87Sr is pro-
duced from 87Rb by radioactive b-decay, whereas the abundance of primordial 86Sr
remains virtually constant in a given rock. Old acidic rocks such as granite show the
highest ratios, mafic and carbonate-rich rocks the lowest.
General food
Olive oil (together with wine) is one of the most studied food items with
regards to commercial frauds and adulteration. Angerosa et al. (1999) made measure-
ments of 13C and 18O of the whole oil and some of its fractions in order to gain
information about the geographical origin of olive oil produced in Greece, Morocco,
Spain, Italy, Tunisia, and Turkey. By applying statistical procedures they demonstrat-
ed that oil samples show the trend to cluster according to the different climatic areas
of growing environment of fruits. Some confusion were observed for samples com-
ing from neighbouring countries having similar climates. Kelly and Rhodes (2002)
highlighted emergent techniques such as compound and position specific-isotope
mass spectrometry. These latter developments offer the potential to provide more
197
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
As already reported isotopic ratios in animals are mainly due to the origin
and nature of the feed. This thing make the technique very interesting for typical pro-
ductions especially for those in which the animals have to follow a particular dietary
198
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
199
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
More recently Knobbe et al. (2006) reinforced these concepts and enriched
the knowledge about the influence of the feeding regimen on isotope ratios in ani-
mals. They determined the stable carbon and nitrogen isotopic composition of urine
and milk samples from cattle under different feeding regimes. The 13C values of
milk and urine were dependent on different feeding regimes based on C3 or C4 plants.
The 13C values are more negative under grass feeding than under maize feeding.
The 13C values of milk are more negative compared to urine. Under grass feeding
the analysed milk and urine samples are enriched in 13C relative to the feed, whereas
under maize feeding the 13C/ 12C ratio of urine is in the same range and milk is de-
pleted in 13C relative to the diet. The difference between the 15N/14N ratios for the two
feeding regimes is less pronounced than the 13C/12C ratios. The 15N values in urine
require more time to reach the new equilibrium, whereas the milk samples show no
significant differences between feeding regimes. Brescia et al. (2003) analysed milk
samples from 2 geographical areas of the Apulia region, to determine whether their
chemical composition could be used to identify their geographical origin. Metal con-
centrations (Ba, Mn, Zn, Fe an Cu) were determined together with C and N isotopic
ratios. Isotopic ratios and Ba concentrations were found to be the most discriminant
variables. Ritz et al. (2005) demonstrated that also the breed of cow influences the
isotopic enrichments of milk. The effect is, however, of small magnitude and unlikely
to diminish the capacity of 18O measurements to discriminate between different
diets and production sites. Renou et al. (2004) used of a combination of NMR and
IRMS for identification of the geographical origin of milk. They showed that feeds
had an influence on fatty acid composition of the milk, whereas production area af-
fected IRMS results. Discriminant analysis results showed that high resolution NMR
and IRMS were complementary techniques for the authentication of milk in relation
to geographic origin and the feeds. In this study 18O was the most discriminant
parameter for both geographic origin and the animal diet.
While there are already many studies on isotope fractions in milk there are
very few papers regarding isotopic ratios in cheese. Manca et al. (2001) tried a char-
acterisation of the geographical origin of Pecorino Sardo Cheese by casein stable
isotope (13C/12C and 15N/14N) ratios together with free amino acid ratios. They tested
cheese from Sardinia, Sicily, and Apulia. Multivariate data treatments revealed good
discrimination possibilities for cheeses according to place of origin.
The 87Sr/86Sr isotope abundance ratio seems to be extremely powerful the
determination of cheese origin. Fortunato et al. (2004) developed an analytical meth-
od based on ICP-MS and used it to differentiate cheeses originating from different
regions (alpine, pre-alpine, Bretagne, Finland, Canada, Australia) accorded to local
geological properties. While carbon and oxygen isotopic determination in cheese are
usually performed on casein fractions in this case no difference was found between
casein-bound and whole-cheese Sr isotope, abundance ratios. Also Pillonel et
al. (2003) found the 87Sr/86Sr isotope abundance ratio to be the most discriminating
variable within the ones they considered to differentiate Emmenthal cheeses from six
European regions.
200
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
Fish
Between foods of animal origin fish is probably the item requiring more
authentication techniques than others due to the need of the certification of origin in
labels (EU Regulation 104/2000) and to the great number of commercial frauds in
this field. Nevertheless only Dempson and Power (2004) used IRMS in fish authenti-
cation. They examined stable isotopes of carbon and nitrogen in wild and aquaculture
origin Atlantic salmon, to evaluate their utility to identify farmed fish. Samples of
muscle tissue obtained from wild salmon were significantly more enriched in nitro-
gen ( 15N: mean = 12.75 0.38) but depleted in lipid corrected carbon ( 13C:
mean = -20.51 0.23) by comparison with aquaculture specimens ( 15N = 10.96
0.19; 13C = -19.25 0.17 ) resulting in a complete separation of the two
groups.
The research of Sweeting et al. (2007) could be very useful to understand
nitrogen isotopic variations in fishes since they assessed the effects of body size,
experimental duration and environmental conditions on fish tissue. Two populations
of European sea bass (Dicentrarchus labrax) were reared on constant diets of dab
(Limanda limanda) muscle or sandeel (Ammodytes marinus) for 2 years under nat-
ural light and temperature regimes. Bass were sampled at approximately monthly
intervals to determine 15N for muscle, heart and liver tissue. Mean values of
15
N were 3.83, 3.54, 2.05 (sandeel diet) and 3.98, 3.32, 1.95 (dab diet)
for muscle, heart and liver tissue respectively. The assumption that fractionation was
independent of body mass was upheld for muscle and heart tissue, but not for liver.
Heart and liver 15N were also affected by temperature probably reflecting the meta-
bolic functions of these tissues and their associated rates of turnover. However in
heart the explanatory power of temperature appeared tied to that of time.
Honey
Anklam (1998) in his review of the analytical methods to determine the geo-
graphical and botanical origin of honey already pointed at IRMS as the most reliable
technique for the detection of the geographical origin of honey. The technique can
also be used for the detection of adulterations in milk. In particular the 13C/12C value
can be useful to detect whether honey has been enriched in exogenous sugars. Pa-
dovan et al. (2003) used the techniques for this purpose and fixed refence parameters.
In particular it was stated that the range of values 13C found for bee-produced honey
was -21.96 to -30.47 for C3 plants and -11.82 to -19.00 for C4 plants. For
cane sugar it was -11.33 to -11.78.
Conclusions
201
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
to the application of the technique in origin assignation is the lack of large databases
of isotopic abundances in food items.
References
202
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
19 (2): 227-234.
15. Fortunato G., Mumic K., Wunderli S., Pillonel L., Bosset J. O., Gremaud G.
(2004) Application of strontium isotope abundance ratios measured by MC-
ICP-MS for food authentication. Journal of Analytical Atomic Spectrometry,
19, 227-234.
16. Gonzalez Martin I., Gonzalez-Perez C., Hernandez Mendez J., Marques Ma-
cias E., Sanz Poveda F. (1999) Use of isotope analysis to characterize meat
from Iberian-breed swine. Meat Science, 52, 437-441.
17. Gonzalez Martin I., Gonzalez-Perez C., Hernandez Mendez J., Sanchez
Gonzalez C. (2001) Differentiation of dietary regimen of Iberian swine by
means of isotopic analysis of carbon and sulphur in hepatic tissue. Meat Sci-
ence, 58, 25-30.
18. Gremaud G., Quaile S., Piantini U., Pfammatter E., Corvi C. (2004) Charac-
terization of Swiss vineyards using isotopic data in combination with trace
elements and classical parameters. European Food Research and Technol-
ogy, 219, 97-104.
19. Hargin, K. D. (1996) Authenticity issues in meat and meat products. Meat
Science, 43 (supplement), 277-289.
20. Kelly S. D., Rhodes C. (2002) Emerging techniques in vegetable oil analysis
using stable isotope ratio mass spectrometry. Grasas y Aceites, 53, 34-44.
21. Knobbe N., Vogl J., Pritzkow W., Panne U., Fry H., Lochotzke H.M., Preiss-
Weigert A. (2006) C and N stable isotope variation in urine and milk of cattle
depending on the diet, Analytical and Bioanalytical Chemistry, 386 (1), 104-
108.
22. Kornexl, B. E., Werner, T., Rossmann, A., & Schmidt, H.L. (1997) Measure-
ment of stable isotope abundances in milk and milk ingredients: a possible
tool for origin assignment and quality control. Zeitschrift Fur Lebensmittel-
Untersuchung Und-Forschung, 205(1), 19-24.
23. Manca G., Camin F., Coloru G. C., Del Caro A., Depentori D., Franco M. A.,
Versini G, (2001) Characterization of the Geographical Origin of Pecorino
Sardo Cheese by Casein Stable Isotope (13C/12C and 15N/14N) Ratios and Free
Amino Acid Ratios. Journal of Agricultural and Food Chemistry, 49, 1404-
1409.
24. Padovan G.J., De Jong D., Rodrigues L.P., Marchini J.S. (2003) Detection
of adulteration of commercial honey samples by the 13C/12C isotopic ratio.
Food Chemistry, 82, 633-636.
25. Piasentier E., Valusso R., Camin F., Versini G. (2003) Stable isotope ratio
analysis for authentication of lamb meat. Meat Science, 64, 239-247.
26. Pillonel L., Badertscher R., Froidevaux P., Haberhauer G., Holzl S., Horn
P., Jakob A., Pfammatter E., Piantini U., Rossmann A., Tabacchi R., Bosset
J.O. (2003) Stable isotope ratios, major, trace and radioactive elements in
emmental cheeses of different origins. Lebensm.-Wiss.u.-Technol., 36, 615-
623.
27. Pupin A. M., Dennis M. J., Parker I., Kelly S., Bigwood T., Toledo M. C. F.
(1998) Use of isotopic analyses to determine the authenticity of Brazilian
203
Ann. Fac. Medic. Vet. di Parma (Vol. XXVI, 2006) pag. 193 - pag. 204
204