The use of stable isotopes for food web analysis

General aspects in isotope biogeochemistry was summarized with emphasis on delta 15N and delta 13C contents in plants and animals in natural ecosystems. In the estuary, the variation of isotope ratios were principally governed by the mixing of land-derived organic matter, marine phytoplankton, and seagrasses. A clear cut linear relationship between animal delta 15N and its trophic level was obtained in the Antarctic food chain system. Several current efforts to use the stable isotopes for food web analysis were demonstrated for some terrestrial and marine systems as well as human food web.     

Principles and limitations of stable isotopes in differentiating organic and conventional foodstuffs: 2. Animal products

In this review, we examine the variation in stable isotope signatures of the lighter elements (δ²H, δ¹³C, δ¹⁵N, δ¹⁸O, and δ³⁴S) of tissues and excreta of domesticated animals, the factors affecting the isotopic composition of animal tissues, and whether stable isotopes may be used to differentiate organic and conventional modes of animal husbandry. The main factors affecting the δ¹³C signatures of livestock are the C3/C4 composition of the diet, the relative digestibility of the diet components, metabolic turnover, tissue and compound specificity, growth rate, and animal age. δ¹⁵N signatures of sheep and cattle products have been related mainly to diet signatures, which are quite variable among farms and between years. Although few data exist, a minor influence in δ¹⁵N signatures of animal products was attributed to N losses at the farm level, whereas stocking rate showed divergent findings. Correlations between mode of production and δ²H and δ¹⁸O have not been established, and only in one case of an animal product was δ³⁴S a satisfactory marker for mode of production. While many data exist on diet–tissue isotopic discrimination values among domesticated animals, there is a paucity of data that allow a direct and statistically verifiable comparison of the differences in the isotopic signatures of organically and conventionally grown animal products. The few comparisons are confined to beef, milk, and egg yolk, with no data for swine or lamb products. δ¹³C appears to be the most promising isotopic marker to differentiate organic and conventional production systems when maize (C4) is present in the conventional animal diet. However, δ¹³C may be unsuitable under tropical conditions, where C4 grasses are abundant, and where grass-based husbandry is predominant in both conventional and organic systems. Presently, there is no universal analytical method that can be applied to differentiate organic and conventional animal products.     

Passage of stable isotope-labeled grass silage fiber and fiber-bound protein through the gastrointestinal tract of dairy cows

Fractional passage rates are required to predict nutrient absorption in ruminants but data on nutrient-specific passage kinetics are largely lacking. With the use of the stable isotope ratio (δ) as an internal marker, we assessed passage kinetics of fiber and fiber-bound nitrogen (N) of intrinsically labeled grass silage from fecal and omasal excretion patterns of δ¹³C and δ¹⁵N. In a 6 × 6 Latin square, lactating dairy cows received grass silages [455 g/kg of total diet dry matter (DM) ] in a 2 × 3 factorial arrangement from ryegrass swards fertilized at low (45 kg of N/ha) or high (90 kg of N/ha) levels of N and harvested at 3 maturity stages. Feed intake (16.7 ± 0.48 kg of DM/d; mean ± standard error of the mean) and milk yield (26.7 ± 0.92 kg/d) increased at the high level of N fertilization and at decreasing maturity. Nutrient digestibility decreased with increasing plant maturity, particularly at the high level of N fertilization, essentially reflecting dietary treatment effects on the nutritional composition of the grass silage. Fractional rumen passage rates (K₁) were highest and total mean retention time in the gastrointestinal tract (TMRT) was lowest when based on the external marker chromium mordanted fiber (Cr-NDF; 0.047/h and 38.0 h, respectively). Fecal δ¹³C in the acid detergent fiber fraction (¹³CADF) provided the lowest K₁ (0.023/h) and the highest TMRT (61.1 h) and highest peak concentration time (PCT; 24.3 h) among markers. In comparison, fecal fiber-bound N (¹⁵NADF) had a considerably higher K₁ (0.032/h) and lower TMRT (46.4 h) than ¹³CADF. Total N (measured with ¹⁵NDM) had a comparable K₁ (0.034/h) to that of ¹⁵NADF but provided the highest fractional passage rates from the proximal colon-cecum (K₂; 0.37/h) and lowest PCT (17.4 h) among markers. A literature review indicated unclear effects of grass silage maturity on K₁ and unknown effects of N fertilization on K₁. Our study indicated no effect of advancing maturity on fecal K₁ and a trend for K₁ to increase with the high level of N fertilization. Parameter K₂ increased, whereas PCT and TMRT generally decreased with the high level of N fertilization. Omasal digesta sampling largely confirmed results based on fecal sampling. Results indicate that the use of δ¹³C and δ¹⁵N can describe fiber-specific passage kinetics of forage.     

Geographical origin of polished rice based on multiple element and stable isotope analyses

We determined carbon and nitrogen contents (C and N contents) and stable carbon, nitrogen, and oxygen isotopic compositions (δ¹³C, δ¹⁵N, and δ¹⁸O) of polished rice in order to develop a simple method to discriminate its geographical origin. As a first attempt, we examined a single cultivar, Koshihikari rice, from 14 different cultivation areas including Australia (n = 1), Japan (n = 12), and USA (n = 1). For all rice samples, C and N contents and the isotopic compositions are consistent with those of general plant materials, being 37.2–40.0% (C content), 0.8–1.4% (N content), −27.1 to −25.4% (δ¹³C), +0.4 to +9.0% (δ¹⁵N), and +18.8 to +22.9% (δ¹⁸O). However, its cultivated area is clearly distinguished by a pentagonal radar plot based on the elemental and isotopic compositions. Thus, the comparison of C and N contents and δ¹³C, δ¹⁵N, and δ¹⁸O values would potentially be useful for rapid and routine discrimination of geographical origin of the polished rice.     

Seasonal variation in the C,N and S stable isotope composition of retail organic and conventional Irish beef

The objective of this study was to investigate seasonal variation in the C, N and S stable isotope composition of retail organic and conventional Irish beef. A total of 242 beef samples (127 organic, 115 conventional) was collected in a one-year survey and analysed by isotope ratio mass spectrometry. The δ¹³C time series in conventional beef was significantly non-random, with a pronounced seasonal positive shift of >2‰ between December and June, whilst δ¹³C in organic beef was less variable and significantly more negative. In conventional beef, δ¹⁵N was remarkably invariant (remaining close to 7‰) throughout the year, while organic beef was more variable and also significantly lower in δ¹⁵N. The S isotope composition (δ³⁴S) exhibited a complex seasonal pattern in both types of beef. These results show that seasonal patterns can occur in the isotopic composition of beef, probably reflecting seasonality in animal feeding practices modulated by tissue turnover rates. Such seasonal variation needs to be considered in the isotopic authentication of beef and other animal-derived products.     

Seasonal influence on the prevalence of thermotolerant Campylobacter in retail broiler meat in Denmark

In Denmark, the incidence of human campylobacteriosis cases, as well as the Campylobacter prevalence in broiler flocks, is strongly influenced by season with a summer peak in July-August. Therefore, it was considered that the prevalence of Campylobacter in broiler meat sold at retail in Denmark might also be influenced by season. A retrospective survey analysis was performed on 2001-2007 national surveillance data of the prevalence of thermotolerant Campylobacter in all conventional broiler flocks at slaughter, and in randomly sampled broiler meat at retail. There was a significant effect of season on the occurrence of Campylobacter in meat at retail; the largest effect was found for domestic chilled meat. Thus, the Campylobacter prevalence in Danish broiler flocks, which fluctuated with season, was found to be a strong predictor for the occurrence of Campylobacter in fresh, chilled, Danish broiler meat. However, besides flock prevalence, there was also a direct effect of season on the occurrence of Campylobacter in Danish broiler meat at retail.     

Comparison of polyphenolic composition and antioxidant activity of wild Italian blueberries and some cultivated varieties

Four varieties of cultivated blueberries (Vaccinium corymbosum) and a wild crop (Vaccinium miyrtillus) originating form the Modena region in Italy (Mirtillo nero dell’Appennino Modenese) and protected by the mark of origin, were examined in order to determine their antioxidant activity as related to their phenolic composition. The antioxidant activity was measured as radical scavenging activity, ferric reducing activity, and by an amperometric method; the total phenolics and total anthocyanins were determined by colorimetric methods; individual anthocyanins were evaluated by HPLC. Results showed that total phenolics and total anthocyanin concentrations were, respectively two fold and three fold higher in the wild fruits, which also had a higher anthocyanin-to-total phenolic ratio. Determination of individual anthocyanins put in evidence some differences between the cultivated and wild varieties, in particular the absence of acylated anthocyanins in wild blueberries. The antioxidant activity was much higher in wild blueberries than in the cultivated ones, and it was more related to the total phenolic rather that to the anthocyanin concentration.     

Stable carbon isotopes and the study of prehistoric human diet

Mass spectrometric analysis of the stable carbon isotope composition (13C/12C or delta 13C) of bone collagen from human remains recovered at archaeological sites provides a direct chemical method for investigating dietary patterns of prehistoric human populations. This methodology is based on the facts that (1) different food items within the human diet have distinct delta 13C values, and (2) the delta 13C value of human bone collagen is determined by the delta 13C value of the diet. Studies of the development of subsistence patterns based on corn agriculture, one of the most significant developments in North American prehistory, can benefit from the use of stable carbon isotope techniques because corn has a high delta 13C value relative to other components of the human diet. Measurements of delta 13C of bone collagen from prehistoric human skeletal remains from southeastern Missouri and northeastern Arkansas indicate that intensive corn agriculture began in this region around A.D. 1000, that the incorporation of corn into the human diet was a rapid phenomenon, and that 35 to 77% of the human diet from A.D. 1000 to A.D. 1600 consisted of corn. Results from an isochronous population in southeastern South Dakota (A.D. 1400) suggest that 78 to 90% of the diet of this group consisted of corn, with no difference between males and females. Coupled with more traditional archaeological methods, stable carbon isotope analysis of bone collagen can significantly enhance reconstruction of dietary patterns of prehistoric humans.     

Principles and Limitations of Stable Isotopes in Differentiating Organic and Conventional Foodstuffs: 1. Plant Products

Among the lighter elements having two or more stable isotopes (H, C, N, O, S), δ¹⁵N appears to be the most promising isotopic marker to differentiate plant products from conventional and organic farms. Organic plant products vary within a range of δ¹⁵N values of +0.3 to +14.6%, while conventional plant products range from negative to positive values, i.e. ?4.0 to +8.7%. The main factors affecting δ¹⁵N signatures of plants are N fertilizers, biological N₂ fixation, plant organs and plant age. Correlations between mode of production and δ¹³C (except greenhouse tomatoes warmed with natural gas) or δ³⁴S signatures have not been established, and δ²H and δ¹⁸O are unsuitable markers due to the overriding effect of climate on the isotopic composition of plant-available water. Because there is potential overlap between the δ¹⁵N signatures of organic and conventionally produced plant products, δ¹⁵N has seldom been used successfully as the sole criterion for differentiation, but when combined with complementary analytical techniques and appropriate statistical tools, the probability of a correct identification increases. The use of organic fertilizers by conventional farmers or the marketing of organic produce as conventional due to market pressures are additional factors confounding correct identification. The robustness of using δ¹⁵N to differentiate mode of production will depend on the establishment of databases that have been verified for individual plant products.     

Chemical composition, in vitro cytotoxic and antioxidant activities of the essential oil and major constituents of Cymbopogon jawarancusa (Kashmir)

The chemical composition of the hydrodistillate of aerial parts of Cymbopogon jawarancusa, a natural grass considered as major forage for animal nutrition, used in food because of the presence of sufficient concentration of minerals like calcium and potassium was analysed by capillary GC–FID, GC–MS and ¹³C NMR. Seventeen constituents representing 97.8% of the total oil with piperitone (58.6%) and elemol (18.6%) as major constituents were identified. In vitro cytotoxicity of the oil and its constituents on human cancer cell lines THP-1 (leukemia), A-549 (lung), HEP-2 (liver) and IGR-OV-1 (ovary) was evaluated by Sulphorhodamine-B assay. The oil was found to be more potent than its components against cancer cell lines tested with IC₅₀ of 6.5 μg/ml (THP-1), 6.3 μg/ml (A-549), 7.2 μg/ml (HEP-2) and 34.4 μg/ml (IGR-OV-1). Antioxidant activity of oil and its constituents was evaluated by DPPH assay. In conclusion, the results demonstrate potent cytotoxic and antioxidant effects of oil, and its components like piperitone, α-pinene, β-caryophyllene and β-elemene.     

Seasonal variations on total fatty acid composition of fillets of zander (Sander lucioperca) in Beysehir Lake (Turkey)

Seasonal variations on total fatty acid compositions of zander, Sander lucioperca in Beysehir Lake, were determined by using GC. Polyunsaturated fatty acids (PUFA) were found to be higher than saturated (SFA) and monounsaturated fatty acids (MUFA) in all seasons. Palmitic acid was the major SFA (57.0–64.0% of total SFA) in all seasons. Oleic acid was identified as the major MUFA (45.0–58.0% of total MUFAs). Docosahexaenoic acid (DHA), linoleic acid (LA), eicosapentaenoic acid (EPA), and arachidonic acid (AA) were the most abundant PUFA. Relating to the total fatty acid composition of zander, the percentages of DHA, LA, AA, and EPA ranged between 17.1–23.3%, 5.40–15.4%, 6.72–9.94% and 4.22–5.93% of total lipid, respectively. The percentages of total ω3 fatty acid were higher than those of total ω6 fatty acid in the fatty acid composition of zander with ω3/ω6 ratios of 1.49, 1.45, 1.22, 0.72 in spring, autumn, winter, and summer, respectively. It was shown that the fatty acid composition and ω3/ω6 fatty acids ratio in the muscle of zander were significantly influenced by spawning and season.     

基于碳、氮稳定同位素技术的羊肉产地溯源可行性研究

选择内蒙古12个旗县,共计141只羊的背最长肌、股二头肌、臂三头肌、和腹下肌四个部位肌肉,共564份样品,运用元素分析仪-同位素比质谱仪联用(EA-IRMS)法测定样品δ13C和δ15N值,确定不同部位、不同产地羊肉的同位素组成,以及用其组成特征进行羊肉产地溯源的可行性分析。结果显示,四个部位的δ13C、δ15N值差异性不显著(p>0.05);用δ13C和δ15N值进行二维投射和层聚类分析,可以完全的将呼伦贝尔和锡林郭勒放牧羊肉与西部区四子王旗、和林县和达拉特旗舍饲羊肉区分。结果表明,通过稳定同位素δ13C和δ15N值可以区分部分旗县羊肉样品,为通过该方法实现肉制品的产地溯源提供了技术支撑和理论依据。     

The combination of stable isotope abundance ratios of H,C, N and S with Sr/Sr for geographical origin assignment of orange juices

Within the EU-project “Pure Juice” established stable isotope methods (δ²H, δ¹³C, δ¹⁵N) have been applied and improved in order to determine and verify the geographical origin of orange juices. In addition, new approaches employing analyses of δ³⁴S and ⁸⁷Sr/⁸⁶Sr have been developed and tested. Approximately 150 authentic orange juice samples from several regions in North- and South-America, Africa and Europe have been analysed. A discrimination of orange producing regions, based on the results which ultimately depend on geographical, climatic and lithological differences was successfully performed. Furthermore, we demonstrate that blending of single strength juice by adding concentrate can be revealed by comparing ⁸⁷Sr/⁸⁶Sr of soluble and insoluble components of the juices. We conclude that regional assignment of orange juice samples is most successful when single parameters are combined in a “multi-element approach”.     

Polyphenolic composition and content in the ripe berries of wild Vitis species

To explore wild genetic resources for improving fruit and processing quality of cultivated grape cultivars, the polyphenolic composition and content in the ripe berries of 147 grape accessions from 16 Vitis species for two consecutive years were characterised. A total of 48 polyphenolic compounds, including 28 anthocyanins, 6 flavonols, 6 flavanols, 6 hydroxycinnamic derivatives, and 2 hydroxybenzoic acids, were identified via HPLC-MS and quantified by HPLC-DAD. These wild grape species had unique presence of abundant di-glucoside derivatives of anthocyanins. In addition, anthocyanins in most wild species were predominantly nonacylated. The mean contents for anthocyanins, flavanols, flavonols, hydroxycinnamic derivatives, and hydroxybenzoic acids were 9.610, 0.769, 0.093, 0.441 and 0.027 mg g⁻¹ FW, respectively. They were about 2 to 10 folds higher than their respective counterparts in the most widely cultivated grape species Vitis vinifera. As expected, most of these groups of compounds were correlated negatively with berry weight, but positively with the content of total soluble solids.     

Comparative chemical composition,free radical-scavenging and cytotoxic properties of essential oils of six Stachys species from different regions of the Mediterranean Area

The chemical composition of essential oils of six Stachys species, S. cretica L. ssp. vacillans Rech. fil., S. germanica L., S. hydrophila Boiss., S. nivea Labill., S. palustris L. and S. spinosa L., obtained by hydrodistillation, was studied by GC and GC–MS. All the oils have in common a great percentage of fatty acids and esters (24.2–58.5%) and a high amount of sesquiterpenes (16–35.9%), with the exception of the oil from S. palustris, which consisted mainly of carbonylic compounds (25.4%). The antioxidant activity by DPPH test and the antiproliferative activity on a series of human cancer cell lines (C32, amelanotic melanoma and ACHN, renal cell adenocarcinoma) were investigated for all the oils. S. palustris,S. cretica and S. hydrophila showed the highest antiradical effect, with IC₅₀ values of 0.482, 0.652 and 0.664 mg/ml, respectively. The most antiproliferative essential oil against C32 cell line was the oil of S. germanica with a 77% of inhibition at a concentration of 100 μg/ml. S. germanica, S. palustris and S. spinosa showed the most antiproliferative activity on ACHN cell line, at a concentration of 100 μg/ml,with 81%, 77% and 73% inhibition, respectively.     

The prevalence, antimicrobial resistance and PFGE profiles of Laribacter hongkongensis in retail freshwater fish and edible frogs of southern China

Laribacter hongkongensis is a novel emerging pathogen associated with human gastroenteritis. We aimed to investigate the prevalence, antimicrobial resistance and genotypic relationship of 199 L. hongkongensis isolates from 690 intestinal samples of fish and frogs. These samples were collected from retail markets in the city of Guangzhou in southern China from October 2008 to September 2009. L. hongkongensis was detected in from 80 (16.3%) out of 490 freshwater fish, and this number included 76 (32.3%) out of 235 grass carp and 4 (14.8%) out of 27 bighead carp. A higher isolation rate of 59.5% (119 out of 200) was observed in edible frogs. The isolation rate was highest in the spring in comparison with other seasons. Notably, 63.8% of the isolates were resistant to at least one class of antimicrobial agents. Analysis by pulsed-field gel electrophoresis (PFGE) revealed that the isolates could be grouped into three clusters. Isolates from fish intestines were grouped into two clusters: cluster I and II. Isolates of frog-origin and several fish-origin isolates were grouped into cluster III. Two patient-derived strains could be classed into cluster III. Extensive genetic heterogeneity among the isolates was observed. The results indicate that L. hongkongensis isolates exhibits host tropism, extensive resistance to widely used antimicrobials and diverse biological evolution in an aquatic environment. The frog is more likely than the freshwater fish to be the potential source for human infection with L. hongkongensis.     

Determination of the geographical origin of Slovenian black locust,lime and chestnut honey

The geographical origin of three Slovenian unifloral honey types (black locust, lime and chestnut) was investigated by analysis of some physico-chemical parameters, the elemental content using total reflection X-ray fluorescence spectrometry (TXRF) and the stable carbon and nitrogen isotope ratios using isotope ratio mass spectrometry (IRMS). The results were interpreted by chemometric methods. A total of 122 samples of Slovenian black locust, lime and chestnut honeys were collected from domestic beekeepers all over Slovenia for three years. Slovenia is a small country by area, but paedologically and climatically diverse, therefore offering interesting possibilities for studying geographical influences. The combination of the investigated parameters offers the possibility of distinguishing among samples of specific honey types from the four different Slovenian natural-geographical macroregions, namely the Alpine, Dinaric, Pannonnian and Mediterranean regions. Lime honey samples were 100% correctly classified, while the success rates for black locust and chestnut honeys were slightly lower at 98.2% and 94.6%, respectively.     

Selective concentration of EPA and DHA using Thermomyces lanuginosus lipase is due to fatty acid selectivity and not regioselectivity

The selectivity of anchovy oil hydrolysis was optimised for Thermomyces lanuginosus lipase, so that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were concentrated and partially separated from each other. Enzyme concentration and pH control were important factors for effective hydrolysis. Monitoring percent hydrolysis using capillary chromatography with flame ionisation detector (Iatroscan) and fatty acid selectivity using gas chromatography (GC) indicated that during hydrolysis DHA primarily remained on the glycerol backbone, while EPA was progressively removed. ¹³C nuclear magnetic resonance (NMR) data showed that selectivity of hydrolysis was primarily due to fatty acid selectivity and not regioselectivity, with hydrolysis from both sn-1,3 and sn-2 sites being equally favoured.     

Chemical composition and insecticidal properties of some aromatic herbs essential oils from Algeria

The biological effects on Sitophilus granarius were evaluated for three aromatic herbs essential oils: Foeniculum vulgare, Rosmarius officinalis and Lippia citriodora. Stored grain pests were currently controlled by chemical pesticides. This control method leads to pollution of the environment and intoxication of consumers. Essential oils of aromatic plants are more considered as good control alternative tools. Filter papers treated with 5, 50 and 500 μl of test oil were placed in the bottom cover of 1 l plastic bottle. The insects, 50 adults per bottle, were exposed for 1–5 days. Cumulative mortalities were determined 24 and 120 h after treatment. All treatments were replicated five times. The components of the essential oils were identified through GC and GC–MS. The identity of the constituents was confirmed and their relative proportions determined. The results indicate that these natural products may find potential application as useful, environmentally safe insect control and crop protectant agents.     

Classification of geographical origins and prediction of δ13C and δ15N values of lamb meat by near infrared reflectance spectroscopy

Near infrared spectroscopy (NIRS) combined with chemometric analysis was investigated for its potential to classify the geographical origin and predict δ(13)C and δ(15)N values of lamb meat samples (n=99) from three pastoral regions and two agricultural regions of China. Principal component analysis (PCA), discriminant partial least squares analysis (D-PLS), linear discriminant analysis (LDA) and partial least squares regression (PLSR) were used for data analysis. D-PLS and LDA correctly classified 100% of the both pastoral and agricultural region samples, and gave a total correct classification of 88.9% and 75% to the five individual region samples, respectively. The best PLSR calibration models for predicting δ(13)C and δ(15)N of lamb meat were obtained with the determination coefficient (R(2)) 0.76 and 0.87, respectively. These results show that NIRS combined with chemometrics can be used as a rapid and effective method to discriminate the geographical origin and estimate the δ(13)C and δ(15)N of lamb meat.