POSSIBILITIES OF USING STABLE ISOTOPES FOR IDENTIFICATION OF GEOGRAPHICAL ORIGIN OF MEAT AND MEAT PRODUCTS. A REVIEW

Identification of geographical origin of raw materials and animal-derived products to exclude a possibility of their falsification is quite a complex task, which requires searching for reliable criteria of identification that account for an effect of the geoclimatic and anthropogenic factors. One of the methods for revealing falsification of geographical origin of food including meat products is a method of analysis of stable isotopes (2H/1H, 18O/16O, 15N/14N, 13C/12C) and several other elements. The review describes the main theoretical provisions of stable isotope analysis and their fractionation, presents the results of the investigation of the isotopic composition of meat raw materials and products of animal origin for verification of their geographical origin and feeding systems that differ largely in the content of C3 and C4 plants in the animal diet. Analysis of the C, N and S stable isotope ratio has a significant potential for authentication of meat raw materials and verification of the origin. In addition, it can be used to detect differences between very similar agricultural production systems, even if the underlying mechanisms are not fully elucidated. УДК/UDC 637.5.06:54.027:581.9 DOI 10.21323/2414–438X-2018–3–1–46–58 Для цитирования: Горбунова н.а. возможности использования стабильных изотопов для идентификации географического происхождения мяса и мясных продуктов. обзор. Теория и практика переработки мяса. 2018;3(1):46–58. DOI:10.21323/2414–438X‐2018–3–1–46–58 FOr cItatIOn: Gorbunova n.a. Possibilities of using stable isotopes for identification of geographical origin of meat and meat products. a review. Theory and practice of meat processing. 2018;3(1):46–58. (In russ.) DOI:10.21323/2414–438X‐2018–3–1–46–58 Горбунова Н.А. Федеральный научный центр пищевых систем им. В.М. Горбатова РАН, Москва, Россия Обзор Nataliya A. Gorbunova V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia


Introduction
The task of revealing falsification of animal-derived products linked with a change (distortion) of the information about product geographical origin (country, region) is rather complicated but quite topical.The problem of a certain product authentication or revelation of the fact of its falsification has been acquiring an increasing importance not only for consumers but also for responsible producers and distributors [1,2,3,4,5,6].
An interest of consumers in a solution to the problem of product authenticity regarding the foods they buy is especially increased in case of products from the premium segment, which includes organic foods, products corresponding to the principles of fair trade as well as products with protected designation of origin (PDO).
Local and traditional products are perceived as healthier and tastier, and find a growing demand among consumers [1].
Identification of the regional origin of animal raw materials is quite a complicated task and requires a search for reliable identification criteria with consideration for the geoclimatic and anthropogenic factors.The markers are chemical elements and their isotopes, which presence in the animal body and animal products [7]: -is conditioned by the chemical composition of water, environment and feedstuff; -depends on the animal physiology and element redistribution between organs according to the metabolic requirements; -depends on a biogeochemical condition in a certain sub-region.

Protection of geographical terms
Protection of geographical origin of foods (name of regional origin of goods) is caused by a necessity to prevent possible falsification of a product that is distinguished by its specific properties compared to a range of similar products.The European Union legislation that protects such products, supports diversity of agricultural products and economic activation of local population and prevents its outflow from rural areas, assists producers in obtaining decent payments for authentic products and eliminating unfair competition and product deception by sale of falsified or low quality products [1,8].
At present, protection of geographical indications and guarantee of the traditional peculiarities are regulated in the EU by Regulation (EU) No. 1151/2012 of the European Parliament and of the Council of 21 November 2012 on quality schemes for agricultural products and foodstuffs.In Russia, legal relations in this sphere are regulated by the law of trade mark protection, and names of regional origins of goods are recorded in the register of the Russian Federation.
Two protected geographical terms are distinguished in the EU.
Protected designation of origin (PDO) envisions an absolute adherence to a recipe, the use of the strictly specified raw materials and product manufacture exclusively in the designated place of the region, where its production and qualitative characteristics are influenced by the geographic environment, climatic peculiarities and (or) human factors; it is intended to mean the name of a region used for the name of a product produced only in this region.For example, Prosciutto di Parma (Parma Ham) is PDO and according to the established requirements the entire technological process of ham production is to be carried out in the place of its origin (Parma, Italy).
Protected geographical indication (PGI) is largely based on the acknowledged reputation of this product and at least one of the processes of production, processing or preparation of a product is to be carried out in the specified region.For example, Bayonne Ham or Jambon de Bayonne is an air-dried pork ham, which is produced in the area near Bayonne, France.The product was given the PGI status in 1998.

Methodology of stable isotope analysis
Identification of the geographical origin of animal raw materials is quite a complicated task, as not only the geoclimatic but also anthropogenic factors would affect physico-chemical indicators [9].
At present, the methods for analysis of stable isotopes ( 2 H/ 1 H, 18 O/ 16 O, 15 N/ 14 N, 13 C/ 12 C, 34 S/ 32 S and several other elements) are used to detect falsifications of food geographical origin.These methods allow effective and reliable identification of the geographical origin of a product, its raw material source (natural or obtained as a result of chemical, biotechnological or biochemical synthesis) and a method of animal feeding for animal-derived products [2,10,11,12].
The use of the methods of isotopic composition to establish food falsification began in the 1990s.Nowadays, there are several documents acknowledged by the European Committee for Standardization (CEN) and the As-sociation of Official Analytical Chemists for the methods of stable isotope analysis, for example, in honey (AOAC -No.991.41), juices (AOAC -No.982.21;JAOAC79 -No. 1, 1996;ENV 12142:1996) and others [13].
The isotopic composition is the relative abundances of isotopes of a given element usually expressed as a ratio of low abundant isotope to more abundant (D/H ( 2 H/ 1 H), 18 O/ 16 O, 15 N/ 14 N, 13 C/ 12 C and so on).
Distribution of stable isotopes of light elements in different biological and abiotic systems is significantly different.The peculiarities of distribution are linked with the processes of fractionation; that is, with changes in the isotope ratios in the course of many several biochemical and geochemical processes [14,15].An ability of thermodynamically ordered isotope distribution in complex organic compounds is a specific property of living systems; therefore, the isotope ratio is quite a reliable criterion for discrimination of biogenic and abiogenic compounds.
Current investigations prove that isotopes are memory carriers regarding the birth and transformation of molecules and isotope fractionation is a chemical history of a substance [16].Stable isotopes can be used as isotopic indicators in two cases: 1) as an «external marker» upon entering into a living organism in micro-quantities with food, water, air or medicines; 2) when detecting the ratio of own isotopes of the body, which are intra-molecular phenomenon (so-called «internal marker»).
The fractionation of isotopes is a consequence of their physico-chemical differences, which can affect the velocities of processes or the energy state of a substance system [16].
For food analysis, the most important is fractionation of carbon isotopes upon photosynthesis, fractionation of carbon and nitrogen isotopes upon biochemical (microbial) transformation of the organic matter in soil and accumulation of 15 N (and to a lesser degree 13 C) in the trophic chains, local distribution of oxygen and hydrogen isotopes in water reservoirs [2].
For notation of the isotopic composition, the δ value is used, which is a deviation (usually in parts per thousand (‰) (permille) from the relative standard [2,3,17]: Where, E -is a chemical element; R 1 -is the molar ratio of the heavy to light isotopes in the studied object; R 2 -is the molar ratio of the heavy to light isotopes in the standard; ‰ -permille.
It can be seen from the equation that if the ratio of isotopes (R 1 ) in a sample is lower than in the standard (that is a sample contains less heavy isotopes) than the variation of the isotopic composition δ has a negative value; and, on the contrary, when R 1 is higher than in a standard, the variation is positive.
The generally accepted international standard samples for isotopic analysis are given in Table 1.

Fractionation of stable isotopes
Fractionation of stable isotopes of oxygen and hydrogen takes place upon water circulation in nature.The ocean water has the maximum value of the heavy isotopes 2 Н и 18 O.During evaporation, it is saturated with the light isotopes due to their higher mobility, while the opposite process of water enrichment in the heavy isotopes is observed upon partial condensation [19].
Fractionation of the carbon stable isotopes is mainly associated with the type of plant photosynthesis that differs in the level of fractionation of 12 C and 13 C isotopes (С 3 , С 4 , and CAM photosynthesis types are distinguished).With that, carbon of biological objects is enriched in the light isotope, 12 С, compared to abiotic ones [2,20], which allows answering a question whether the main diet of a human or animal consisted largely of С 3 -plants (grass, hey, rice, wheat, soybeans, potato) or С 4 -plants (corn, sorgo or beef from cattle fed with corn).
Fractionation of the nitrogen isotopes is conditioned by the live activities of soil nitrifying microorganisms, processes of nitrification and ammonification in soil.Quite intensive transfer of nitrogen in the trophic chains is a cause of significant (in tens of promille) differences in δ 15 N in living organisms [2].
The ratio of stable isotopes of elements, which are constituents of all biological tissues (such as the muscle and adipose tissues) depends on many factors; however, several of them are closely linked with their geographical origin [3].For example, 18 О/ 16 О and 2 H/ 1 H ratio in water depends on the elevation above sea level, distance from the oceans and climate of a certain region.Isotopic 15 N/ 14 N, 13 C/ 12 C, 34 S/ 32 S composition depends on the composition of the organic substances of soil and fertilizers.
One of methods for studying the food isotopic composition is mass-spectrometry, which makes it possible to precisely differentiate masses of different isotopes of chemical elements and their ratio, and as a result, to detect product geographical origin according to them.

Studies of the isotopic composition of meat raw materials and animal-derived products
Analysis of the natural stable isotopes of carbon, nitrogen and sulfur is one of the potential tools for verification of the geographical origin and history of cattle feeding, which is linked with the fact that plants and non-migratory animals that eat those plants potentially have region-specific isotopic compositions influenced by climatic and environmental conditions.However, isotopic authentication of animal-derived products is quite a complicated task as farm animals can eat feeds of different origin and, in addition, can be raised on different farms during their lives.Moreover, numerous studies on meat from wildlife species show that the majority of biological and physiological factors that affect the isotopic composition of animal tissues are still inadequately interpreted [4].
Recent studies on authentication of meat raw materials using isotopes of light chemical elements, in general, rely on two main approaches: -analysis of the 18 О/ 16 О and 2 H/ 1 H isotope ratio is used for identification of regional origin associated with climatic conditions of a certain region [10]; -analysis of the 15 N/ 14 N and 13 C/ 12 C isotope ratio is largely applied for determination of dietary components, such as corn or concentrates [4].Denadai et al. (2009) analyzed eggs from two producers in the area of Bastos, São Paulo State (Brasil) and came to a conclusion that analysis of the stable carbon and nitrogen (δ 13 C and δ 15 N) isotopes allows monitoring the inclusion of animalderived components into the diets of laying hens by their detection in egg albumen.In analysis of egg albumen, they found that one manufacturer used only plant-based products, while another 1.5 % of bovine meat and bone meal [11].
To assess the possibility of ascertaining the geographical origin, the Chinese scientists studied the changes in the carbon and nitrogen stable isotope ratios in cattle tissues from various Chinese provinces.To this end, they analyzed 59 samples of beef, cattle crude fat and tail hair from Jilin, Ningxia, Guizhou and Hebei provinces using isotope ratio mass spectrometry (IRMS).The results of discriminant analysis demonstrated that the δ 13 C ratio was the most acceptable indicator for cattle origin traceability compared to δ 15 N for all analyzed tissue samples.The success of classification could be significantly improved by combining the results of the analysis of the C and N stable isotopes [5].
The following research established that analysis of the ratio of stable isotopes 2 H/ 1 H, 15 N/ 14 N, 13 C/ 12 C, 34 S/ 32 S in cattle tail hair can be used as an analytical tool for identification of geographical origin.For example, to classify beef from different Chinese regions, the δ 13 C, δ 15 N and δ 2 H values were measured in 167 cattle tail hair samples from 7 subregions in four beef producing regions, which showed significant differences.An overall rate of correct classification was 82.6 %, a rate of cross-validation was 79.6 % for four beef producing regions compared to 70.7 % and 70.1 %, respectively, for sev-en subregions, which suggest the potential for using stable isotope analysis of cattle tail hair samples in order to create beef traceability database by regions [21].
The effectiveness of δ 13 C and δ 15 N isotope analysis in lipid and residual fractions of lamb protein was verified [22].The study was carried out on 120 samples of lamb produced in different regions of different countries: the United Kingdom, Spain, France, Greece, Island and Italy.To evaluate the region of meat origin, canonical discriminant analysis was used.The differentiation was carried out by stable isotope ratios.Initially, 79.2 % of lamb samples were classified correctly, while cross-validation of the discriminant model reduced the number of samples that were correctly identified by geographical origin to 67 %.
The South African researchers used the potential of stable isotopes 13 C/ 12 C and 15 N/ 14 N as a marker for authentication of lamb from Karoo sheep raised in the Karoo region of South Africa [23].Consumers highly value Karoo lamb for its quality and unique organoleptic characteristics (grassy aroma and taste), which are thought to be conditioned by the fact that sheep are raised in the free-range systems and eat aromatic Karoo plants.Seven farms, which had unique vegetation, were included into the study.
For analysis, 10 lamb meat samples were taken from each of the seven farms.In addition, the isotopic composition of vegetation from the grazing places were studied to precisely determine their influence on the animal tissues when fractioning isotopes in the course of metabolism, which leads to formation of different isotope ratios in different animal tissues.Using the discriminant analysis, the researchers were able to correctly classify 97.62 % and 96.43 % meat samples by δ 13 C and δ 15 N, respectively, depending on a type of vegetation used in animal feeding.The study confirmed that analysis of the stable isotope ratio in meat is a promising analytical tool for authentication of lamb meat and for meat assessment by the type of the animal diet.
The expediency of measuring carbon and nitrogen isotopes for differentiation of beef from Japan, Australia, and the USA, as well as beef from Europe was confirmed by several researchers [24].
The aim of the cooperative research of the scientists from Ireland and the UK was to study the C, N and S stable isotopes as potential markers of geographical origin and the method of feeding (conventional or organic) for beef cattle [10].
To identify the regional origin, beef samples from Belgium (n = 2), the Netherlands (n = 3), France (n = 2), Germany (n = 5), Italy (n = 1), Spain (n=5) and Brazil (n = 10), as well as the US samples (two lots, n = 11 and 12) were analyzed.In addition, samples of beef striploin or round steak from Irish conventional (n = 17) and organic (n = 15) feeding systems were also included into this study.
It was established that European beef, including conventional Irish beef, differed significantly from American beef according to the analysis of the C and N isotopic compositions.The considerable difference in δ 13 C established for European and American beef was explained by differ-ent proportions of plants with types of photosynthesis in the cattle diets.Mean δ 13 C values found in the samples of conventional Irish and other European beef (-24.5 ± 0.7 ‰ and -21.6 ‰ ± 1.0 ‰, respectively) were attributed to a predominance of C 3 plants as ingredients of the diet, while less negative δ 13 C values in the US and Brazilian beef samples (-12.3 ± 0.1 ‰ and -10.0 ± 0.6 ‰, respectively) were explained by the almost exclusive use of C 4 feed, such as corn or (sub)tropical pasture grasses.
According to these results, δ 13 C was identified as a single marker that allowed differentiation of American beef from European.This is likely more relevant to northern Europe, in particular, Ireland and Britain, were pastoral beef producing systems are dominant, while the use of the C 4 crop, corn, is not common.This hypothesis is confirmed by the published independent δ 13 C measurements in muscles and hair from British beef cattle.However, the study does not reflect the existence in central and southern Europe of beef producing systems, which use the diets with the high content of corn.For example, δ 13 C in cattle muscles were in a range from -24 ‰ to -13 ‰ in an analysis of beef from 23 farms in southern Germany; however, in beef cattle with controlled grass feeding, the δ 13 C mean values were -27 ‰.
It is interesting to note that the δ 13 C and δ 15 N ratio in the samples of Irish conventional beef differed significantly from these indicators in other European beef, which shows that authentication of beef origin based on the isotopic analysis can be accomplished on the smaller geographical scale [10].
In this study, the combined analysis of the C, N and S isotopic composition allowed differentiation of Irish beef produced in the conventional and organic cattle feeding systems.For example, conventional Irish beef had less negative values of the isotopic composition and more variable δ 13 C values compared to organic beef (-24.5 ± 0.7 ‰ and -26.0 ± 0.2 ‰, respectively), which confirms that the conventional cattle feeding system uses concentrated feed in contrast to the organic farming system oriented toward grass feed with more negative δ 13 C values compared to concentrates.
Conventional beef had higher δ 15 N values compared to organic beef, which were at the level of 7.8 ± 0.4 ‰ and 6.6 ± 0.4 ‰, respectively.The differences in the isotopic composition between organic and conventional beef, which are partly conditioned by the differences in the consumed feed type (grass or concentrate), are confirmed by the results of the other studies [10,12].It was hypothesized that the obtained results are indicative of the cumulative 15 N enrichment in the plant-soil system due to mineral fertilizers introduced into soil, where conventionally raised animals graze.Also, there can be alternative explanations, such as higher legume content in feeds in the organic farming systems.
In addition, a small increase in the δ 34 S value was observed in the samples of organic Irish beef compared to conventional (7.9 ± 0.6 ‰ and 7.2 ± 0.4 ‰, respectively).The reasons for this increase are not fully understood and it does not correspond to the documented long-term chang-es in the δ 34 S ratio in English soils upon using fertilizers [25].This result can possibly reflect the use of seaweed with higher 34 S content compared to terrestrial sources, which are applied for feed enrichment or as fertilizer in the organic farm systems.
The study of the effect of seasonal changes on the composition of stable isotopes C, N and S in organic and conventional Irish beef (127 organic and 115 conventional samples) demonstrated that the time series of the δ 13 C ratio in the samples of conventional beef were significantly nonrandom and had a clear seasonal positive shift of more than 2 ‰ in the period from December to June, while the δ 13 C value in organic beef was less changeable and significantly lower.In conventional beef, the δ 15 N was surprisingly invariant (remaining close to 7 ‰) during the whole year; organic beef was more variable and was distinguished by lower δ 15 N value.The sulfur isotope composition (δ 34 S) demonstrated complex seasonal dynamics in both beef types [12].
Thus, there can be seasonal patterns in the beef isotopic composition, which, possibly, reflect changes in the ways of animal feeding and are conditioned by the rate of their tissue turnover.Therefore, it is necessary to take into account seasonal changes upon authentication of beef and other animal-derived products with the use of isotopic analysis.
To study the peculiarities of stable isotope composition in meat, a large-scale research was done in South Korea with investigation of 599 pork samples of different origin from 14 countries: 335 samples from South Korea, 264 from South and North America (Canada, USA, Mexico, Chile), 9 from European countries (Austria, the Netherlands, Denmark, France, Belgium, Finland, Poland, Hungary and Spain).The ratio of stable isotopes 13 С/ 12 С and 15 N/ 14 N in proteins of defatted dry pork mass was studied.Analysis demonstrated clear separation of meat from three regions according to its origin: South Korea, America and Europe.Moreover, the researchers revealed close values of δ 13 С for the USA and Mexico (-14.78 ‰ and -14.81 ‰, respectively) and for the Netherlands and Denmark (-25.57‰ and -25.24 ‰, respectively), which can, possibly, be explained by their geographical closeness [26].
The scientists of the East Siberia State University of Technology and the Institute of General and Experimental Biology of the Siberian Branch of the RAS carried out an analysis of the carbon and nitrogen stable isotope content in beef, a study of the trophic relation and an investigation of the carbon isotopic composition in vegetation and soil of eight regions of the Transbaikal Territory.The stable carbon isotope content in grass was -27.16 to -27.86 ‰, which corresponded to the range for biomass of terrestrial C 3 plants [27].
The obtained data demonstrated that the stable carbon isotope content in the muscle tissue from cattle at 2-3 years of age from eight different regions of the Transbaikal Territory (Kabansky, Bichurcky, Dzhidinsinky, Zakamensky, Kizhigincky, Khorinsky, Zaigraevsky and Aginsky) was in a range from -24.28 ‰ to -25.84 ‰.This indicates that all meat tissue samples can be attributed to local raw materials as the δ 13 С values did not exceed the level of -24.0 ‰.The nitrogen stable isotope content in the studied samples did not differ significantly and was from 5.85 ‰ to 7.84 ‰, which is in a range of values that indicate largely natural animal feed.Generalization of the experimental results allowed establishing that all analyzed meat samples presented in the regional retail chain were produced locally and were classified as natural raw materials [27].
It is possible to identify the country of origin of meat raw materials in finished products.For example, the Brazilian and US scientists measured the ratio of the δ 13 С and δ 15 N stable isotopes in the meat component of Big Mac ® patties, which are regarded as a product of «globalization», from 26 countries (Argentina, Australia, Austria, Brazil, Canada, China, Israel, Germany, USA, Japan, Malaysia, Turkey, Sweden, South Africa, Portugal, France, UK and others) [28].
The researchers took into consideration that the δ 13 С stable isotope ratio in Big Mac ® patties would vary in a range from -11 ‰ (cattle were fed exclusively with corn or tropical grasses) to -25 ‰ (cattle were fed with C 3 plants: wheat, soybeans and grasses of the temperate belt).The intermediate values between these extreme values showed that cattle were fed with plants of both С 3 and С 4 groups.
It was established that δ 13 С varied from -25.4 ‰ to -11.1 ‰ for the analyzed meat part of the Big Mac ® patties; the overall median δ 13 С value for all countries was -16.2 ‰.The lowest δ 13 С values were in the sample from the United Kingdom (England and Scotland) and the highest in the samples from Brazil.Quite high differences in the δ 13 С values within a country were found in Australia; for example, the median δ 13 С values in the Big Mac ® patties from Perth and Sydney were -14.0 ‰ (n = 4) and -19.6 ‰ (n = 4), respectively.The carbon stable isotope content in the second batch of Big Mac ® patty samples from a different McDonald's outlets in Sydney was on average -22.7 ‰ (n = 3).
Japanese patties had higher δ 13 С values than was expected based on the analysis of the country's agriculture with plants of C 3 type; however, Japan imports beef from Australia, where plants of C 4 type of photosynthesis are common.As a rule, higher δ 13 С values were found in the samples from the lower latitude countries compared to those from the higher latitude countries, which reflects wider distribution of C 4 plants in the warm regions.
The δ 15 N values in the studied samples of Big Mac ® patties were in a range from 4.2 ‰ to 9.2 ‰ (the median value of 6.6 ‰).The content of 15 N isotopes was the highest in the Japanese patty samples and the lowest in the Chinese samples.The samples from Australia showed a significant variability of the δ 15 N values (in a range from 4.2 ‰ to 8.5 ‰), which was similar to the results of δ 13 С measurement.No statistically significant differences were found in the δ 15 N value in the Big Mac ® patties bought in different countries and grouped by geographical latitude.
Taking into consideration the analysis of the FAO data on beef import, the researchers established that locally produced beef was used for production of Big Mac ® patties in most countries included in this study except six countries presented in the order of an increase in beef import: Sweden, Israel, Portugal, Japan, Malaysia and the Netherlands.

Conclusion
At present, large-scale studies on the use of the method of stable isotope analysis are carried out in several countries of the world to identify the geographical origin of meat and meat products.Analysis of the performed research indicates that the isotopic composition of animal-derived products is determined by the climatic and geographical conditions (the geographical latitude of a region, distance from the sea), cattle feeding base as well as seasonal changes in feeding regimes.Analysis of the stable isotope ratio of C, N and S has a potential as one of the tools for meat authentication.