Study of the oligopeptide fraction in Grana Padano and Parmigiano-Reggiano cheeses by liquid chromatography electrospray ionization mass spectrometry

The oligopeptide fractions of Grana Padano and Parmigiano Reggiano cheese samples, at various stages of ageing (6, 12, 18 and 24 months), were analysed by means of high-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (ESI-MS). The main oligopeptides (< 5000 Da) present in the samples were extracted by using an originally developed method which allowed good enrichment of the oligopeptide fractions and identified according to their molecular weights. The casein sequences compatible with the found molecular weights were determined and the exact oligopeptide sequences were identified by using the fragmentation peaks originated by in-source collisionally-induced dissociation (CID). The ESI-MS method reported here allowed the sequence identification of oligopeptides very quickly with a single liquid chromatography-mass spectrometry run. Oligopeptides were also semi-quantified by comparison with a suitable internal standard (the dipeptide phenylalanyl-phenylalanine, Phe-Phe). This methodology demonstrated that, as far as the main oligopeptides are concerned, Grana Padano and Parmigiano Reggiano presented very similar composition. Anyway, the evolution of the fractions during ageing time was characteristically distinct among the two cheeses: in Grana Padano cheeses most of the oligopeptides reached a maximum at 12 months of ageing and then decreased, whereas in Parmigiano Reggiano cheeses the oligopeptide amounts were usually lower and had a less regular trend. The reason for this different behaviour may be ascribed to the different production techniques.     

Ripening and geographical characterization of Parmigiano Reggiano cheese by 1H NMR spectroscopy

Samples of Italian Parmigiano Reggiano cheese of different ripening stages (14, 24 and 30 months) were analyzed by (1)H NMR spectroscopy and the water-soluble metabolites content compared with samples of "Grana type" cheese from east Europe countries. Different multivariate statistical protocols were examined to build up a stable model for both ripening and geographical discrimination among the samples. The discriminant approach revealed a larger metabolite content increasing with ripening process; in particular younger samples were characterized by a higher content in leucine and isoleucine, while 30 months samples by a larger extent of threonine. The use of a classification approach resulted in a better discrimination of geographical samples. Foreign "Grana type" samples resulted well differentiated with respect to all Italian samples of Parmigiano Reggiano cheese. Foreign samples were characterized by a large amount of leucine and isoleucine, compounds that typified young Italian samples (14 months), and also by lactate, butanoate and acetate, thus suggesting short ripening for foreign samples. Parmigiano Reggiano samples were instead characterized by a large amount of all other compounds, in particular by threonine, which typified old samples (30 months), and other amino acids.     

delta(13)C- and delta(18)O-values of glycerol of food fats

The average values of carbon and oxygen isotopic contents (delta(13)C and delta(18)O) of 36 glycerol samples from fats have been determined. The examined samples arise from many fats of animal and plant origin, as well as from the three Italian hard cheeses Parmigiano-Reggiano, Grana Padano and Trentingrana. The total (13)C content allows one to distinguish between glycerol from plants with the C-4 carbon fixation pathway (maize, mean delta(13)C = -14.4 per thousand) and that from plants with the C-3 pathway (mean delta(13)C = -30.7 per thousand). The delta(13)C-values of glycerols of animal origin seem to depend on the diet of the animal, as suggested by the mean values -29.6, -29.0 and -25.1 per thousand, respectively, observed for Parmigiano-Reggiano, Trentingrana and Grana Padano. Additionally, the mean total (18)O content of glycerol samples of vegetable origin is approximately 23.8 per thousand, while that from animal fat is 15.1 per thousand. However, the delta(18)O mean values relative to Parmigiano-Reggiano, Grana Padano and Trentingrana are 11.8, 16.0 and 13.8 per thousand, respectively. The combination of the (13)C and (18)O measurements relative to the fat glycerol of the three cheeses might be considered a potential criterion of authentication.     

H,C, N and S stable isotopes and mineral profiles to objectively guarantee the authenticity of grated hard cheeses

In compliance with the European law (EC No. 510/2006), geographical indications and designations of origin for agricultural products and foodstuffs must be protected against mislabelling. This is particularly important for PDO hard cheeses, as Parmigiano Reggiano, that can cost up to the double of the no-PDO competitors.     

Does natural weathering change the stable isotope composition (2H, 13C, 15N, 18O and 34S) of cattle hair?

Stable isotope analysis of hair has found applications in many fields of science because it provides a temporally resolved, fairly stable isotopic archive of mammalian individuals. We investigated whether this hair archive is modified by natural weathering while attached to a living animal. We analyzed the tail switch hairs of one suckler cow, sampled seven times over a period of four annual summer pasture–winter stall feeding cycles. We compared relative isotope ratios (δ²H, δ¹³C, δ¹⁵N, δ¹⁸O and δ³⁴S) of sections of hair that grew simultaneously but were exposed to natural weathering conditions over different periods of time. Natural wear caused a loss of mass of approx. 0.13% day^–1, with no apparent effect of environmental conditions. Changes in δ²H, δ¹³C, δ¹⁵N and δ¹⁸O were below the detection limit, indicating that hair is a reliable archive for the isotopes of these elements. In contrast, δ³⁴S values increased during the grazing period by about 1 ‰, with exposure to UV radiation appearing to have a major influence on this result. The δ³⁴S values decreased during the subsequent stall period, probably due to abrasion. Seasonal variation in δ³⁴S may indicate alternating environments that differ in their weathering conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

A new method for the determination of the 34S/32S ratio of water-soluble sulphur in soil

A new method for the determination of the ³⁴S/³²S ratio of water-extractable sulphate in soil is described. Soils are extracted directly with deionized water, which is evaporated down. The remaining residue is then rehydrated and transferred to tin cups containing an adsorbent and mixed with an oxygen donor (V₂O₅). Samples are then analysed using a continuous flow isotope ratio mass spectrometer. The new method requires around 10?g soil per determination, compared to much larger amounts (up to kilograms) of soil required for the previous methods. Sample preparation and subsequent analysis is quick and efficient. The method is demonstrated using a number of soils collected from around the world to provide a range of determined δ³⁴S values. The δ³⁴S values of water-extractable sulphur were broadly similar to those of the soil total sulphur.     

Sulphur isotopes in animal hair track distance to sea

Stable sulphur isotope ratios (³⁴S/³²S) in animal tissues have been suggested as a tracer of coastal residency of terrestrial animals, but data are lacking that quantify the inland range of the sulphur coastal signal and the effects of seasonality. Here, we present δ³⁴S measurements of sheep wool collected seasonally on eight farms across Ireland and wool samples collected opportunistically along the west and east coasts. We observed large (>10‰) δ³⁴S differences across the island and we show that wool δ³⁴S values were negatively correlated with distance to the west coast. We propose that this is due to the predominantly (south‐)westerly airflow, possibly combined with the influence of anthropogenic sulphur deposited from the east. While essentially all the sulphur contained in west‐coast wool is of marine origin, relatively high δ³⁴S values were still measured >100 km inland, suggesting that marine sulphur can be carried over long distances. Seasonal variations are small at the individual level for sedentary grazing animals. We conclude that sulphur isotopes ratios measured in archival keratinous tissues can be used to describe regional δ³⁴S isoscapes primarily defined by distance to coasts and thus provide a tool to detect short‐term movements of domestic, feral and wild animals within such isoscapes. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of acidification on elemental and isotopic compositions of sediment organic matter and macro‐invertebrate muscle tissues in food web research

Stable isotope techniques in food web studies often focus on organic carbon in food sources which are subsequently assimilated in the tissue of consumer organisms through diet. The presence of non‐dietary carbonates in bulk samples can affect their δ¹³C values, altering how their results are interpreted. Acidification of samples is a common practice to eliminate any inorganic carbon present prior to analysis. We examined the effects of pre‐analysis acidification on two size fractions of sediment organic matter (SOM) from marine and freshwater wetlands and pure muscle tissue of a common freshwater invertebrate (Cherax destructor). The elemental content and isotopic ratios of carbon and nitrogen were compared between paired samples of acidified and control treatments. Our results showed that acidification does not affect the elemental or isotopic values of freshwater SOM. In the marine environment acidification depleted the δ¹³C and δ¹⁵N values of the fine fraction of saltmarsh and δ¹⁵N values of mangrove fine SOM. Whilst acidification did not change the elemental content of invertebrate muscle tissue, the δ¹³C and δ¹⁵N values were affected. We recommend to researchers considering using acidification techniques on material prepared for stable isotope analysis that a formal assessment of the effect of acidification on their particular sample type should be undertaken. Further detailed investigation to understand the impact of acidification on elemental and isotopic values of organic matter and muscular tissues is required. Copyright © 2010 John Wiley & Sons, Ltd.     

Is high‐resolution magic angle spinning NMR a practical speciation tool for cheese samples? Parmigiano Reggiano as a case study

High-resolution magic angle spinning (HRMAS) NMR is probably the most apt NMR method to analyze complex materials involving a solid phase, e.g. foodstuffs. We present here an HRMAS analysis of grated cheese (Parmigiano Reggiano). A full NMR characterization of this cheese allows the identification of the presence of fatty acids (saturated and unsaturated), amino acids and other small organic molecules. Since the presence and relative concentration of these molecules have previously been shown to correlate with organoleptic, origin and age characterization, HRMAS NMR of cheese is likely to provide a good complimentary tool for the analysis of this food material.     

Measurement of the δ34S value in methionine by double spike multi‐collector thermal ionization mass spectrometry using Carius tube digestion

Methionine is an essential amino acid and is the primary source of sulfur for humans. Using the double spike (³³S‐³⁶S) multi‐collector thermal ionization mass spectrometry (MC‐TIMS) technique, three sample bottles of a methionine material obtained from the Institute for Reference Materials and Measurements have been measured for δ³⁴S and sulfur concentration. The mean δ³⁴S value, relative to Vienna Canyon Diablo Troilite (VCDT), determined was 10.34 ± 0.11‰ (n = 9) with the uncertainty reported as expanded uncertainties (U). These δ³⁴S measurements include a correction for blank which has been previously ignored in studies of sulfur isotopic composition. The sulfur concentrations for the three bottles range from 56 to 88 µg/g. The isotope composition and concentration results demonstrate the high accuracy and precision of the DS‐MC‐TIMS technique for measuring sulfur in methionine. Published in 2010 by John Wiley & Sons, Ltd.     

Differentiation among dairy products by combination of fast field cycling NMR relaxometry data and chemometrics

A set of commercial milk and Sicilian cheeses was analysed by a combination of fast field cycling (FFC) nuclear magnetic resonance (NMR) relaxometry and chemometrics. The NMR dispersion (NMRD) curves were successfully analysed with a mathematical model applied on Parmigiano–Reggiano (PR) cheese. Regression parameters were led back to the molecular components of cheeses (water trapped in casein micelles, proteins and fats) and milk samples (water belonging to hydration shells around dispersed colloidal particles of different sizes and bulk water). The application of chemometric analysis on relaxometric data enabled differentiating milk from cheeses and revealing differences within the two sample groups of either cheeses or milk samples. Marked differences among cheeses were evidenced by statistical analysis of the sole quadrupolar peaks parameters, suggesting that these contain information on the nature of the milk used during cheese production. Hence, combination of FFC NMR and chemometrics represents a powerful tool to investigate alterations in dairy products.     

A novel approach to measure isotope ratios via multi-collector—inductively coupled plasma—mass spectrometry based on sample mixing with a non-enriched standard

In this work, a novel approach to measure isotope ratios via multi-collector—inductively coupled plasma—mass spectrometry (MC-ICP-MS) for low amounts of target element is proposed. The methodology is based on mixing of the sample (target element isolate) with a non-enriched in-house standard, previously characterized for its isotopic composition. This methodology has been applied to isotopic analysis of Cu and of Fe in whole blood samples. For this purpose, different mixtures of sample + in-house standard were prepared and adjusted to a final concentration of 500 μg/L of the target elements for isotopic analysis. δ⁶⁵Cu, δ⁵⁶Fe, and δ⁵⁷Fe varied linearly as a function of the amount of in-house standard (or of sample) present in the mixture. The isotopic composition of the sample was calculated considering the isotope ratios measured for (i) the mixture and (ii) the in-house standard and (iii) the relative concentrations of target element contributed by the sample and the standard to the mixture, respectively. For validation purposes, the isotopic analysis of whole blood Cu was carried out using both the conventional (using 2 mL of whole blood) and the newly developed approach (using 500 μL of whole blood). The δ⁶⁵Cu values obtained using mixtures containing 40 % (200 μg/L) of Cu from the blood samples and 60 % (300 μg/L) of Cu from the in-house standard were in good agreement with the δ⁶⁵Cu value obtained using the conventional approach (bias ≤0.15?‰).     

Concentration effects on laser-based δ18 O and δ2 H measurements and implications for the calibration of vapour measurements with liquid standards

Recently available isotope ratio infrared spectroscopy can directly measure the isotopic composition of atmospheric water vapour (δ¹⁸O, δ²H), overcoming one of the main limitations of isotope ratio mass spectrometry (IRMS) methods. Calibrating these gas‐phase instruments requires the vapourisation of liquid standards since primary standards in principle are liquids. Here we test the viability of calibrating a wavelength‐scanned cavity ring‐down spectroscopy (CRDS) instrument with vapourised liquid standards. We also quantify the dependency of the measured isotope values on the water concentration for a range of isotopic compositions. In both liquid and vapour samples, we found an increase in δ¹⁸O and δ²H with water vapour concentration. For δ¹⁸O, the slope of this increase was similar for liquid and vapour, with a slight positive relationship with sample δ‐value. For δ²H, we found diverging patterns for liquid and vapour samples, with no dependence on δ‐value for vapour, but a decreasing slope for liquid samples. We also quantified tubing memory effects to step changes in isotopic composition, avoiding concurrent changes in the water vapour concentration. Dekabon tubing exhibited much stronger, concentration‐dependent, memory effects for δ²H than stainless steel or perfluoroalkoxy (PFA) tubing. Direct vapour measurements with CRDS in a controlled experimental chamber agreed well with results obtained from vapour simultaneously collected in cold traps analysed by CRDS and IRMS. We conclude that vapour measurements can be calibrated reliably with liquid standards. We demonstrate how to take the concentration dependencies of the δ‐values into account. Copyright © 2010 John Wiley & Sons, Ltd.     

A survey of isotopic composition (2H, 3H, 18O) of groundwater from Vojvodina

Isotopes of hydrogen (³H, ²H) and oxygen (¹⁸O) are perfect candidates for groundwater tracers. A survey of isotopic composition of 34 groundwater samples and one Lake from Vojvodina region (Serbia) is presented here. Tritium activity concentration and stable isotope composition (δ²H, δ¹⁸O), as well as deuterium excess, were determined. The groundwater samples lie on the groundwater regression line. Minor deviations and a few lower deuterium excess values indicate waters recharged in a different climate regime and subjected to evaporation, respectively. According to the obtained results, most of the analyzed groundwater can be characterized as modern waters, recharged mostly from precipitation.

     

δ18O values of Sus scrofa blood water and bone phosphate; a marked discrepancy between domestic and wild specimens

δ¹⁸O analyses of water in the blood of domestic and wild pigs indicated that large isotopic differences exist between domestic and wild specimens of the same species (Sus scrofa) living in the same area. Similar isotopic differences are found between the δ¹⁸O(PO₄^3–) values of bones from the two groups of animals. When δ¹⁸O values obtained from recent wild boar bones are introduced in the equation of the isotopic scale determined for domestic pigs, totally unreliable δ¹⁸O values of local meteoric water are obtained. The δ¹⁸O(PO₄^3–) values measured in three groups of modern wild boar specimens allow the calculation of a first approximate equation which is quite different from that of domestic pigs. This isotopic scale should be accurately re‐calibrated for wild animals. Copyright © 2011 John Wiley & Sons, Ltd.     

Geographical traceability of wheat and its products using multielement light stable isotopes coupled with chemometrics

The present study was aimed to investigate the variation of stable isotopic ratios of carbon, nitrogen, hydrogen, and oxygen in wheat kernel along with different processed fractions from three geographical origins across 5 years using isotope ratio mass spectrometry (IRMS). Multiway ANOVA revealed significant differences among region, harvest year, processing, and their interactions for all isotopes. The region contributed the major variability in the δ¹³C ‰, δ²H ‰, δ¹⁵N ‰, and δ¹⁸O‰ values of wheat. Variation of δ¹³C ‰, δ¹⁵N ‰, and δ¹⁸O ‰ between wheat whole kernel and its products (break, reduction, noodles, and cooked noodles) were ?0.7‰, and no significant difference was observed, suggesting the reliability of these isotope fingerprints in geographical traceability of wheat‐processed fractions and foods. A significant influence of wheat processing was observed for δ²H values. By applying linear discriminant analysis (LDA) to the whole dataset, the generated model correctly classified over 91% of the samples according to the geographical origin. The application of these parameters will assist in the development of an analytical control procedure that can be utilized to control the mislabeling regarding geographical origin of wheat kernel and its products.     

Simultaneous δ2H and δ18O analyses of water inclusions in halite with off‐axis integrated cavity output spectroscopy

Stable isotope compositions of ancient halite fluid inclusions have been recognized to be valuable tools for reconstructing past environments. Nevertheless, in order to better understand the genesis of halite deposits, it could be of great interest to combine both δ²H and δ¹⁸O measurements of the water trapped as inclusions in the defects of the mineral lattice. We developed a method combining off‐axis integrated cavity output spectroscopy (OA‐ICOS) connected on line with a modified elemental analyzer (EA‐OA‐ICOS) to perform those measurements. The first step was to test the method with synthetic halite crystals precipitated in the laboratory from isotopically calibrated waters. Water isotopic signatures have been measured with conventional techniques, equilibration for δ¹⁸O and chromium reduction for δ²H. Then, we modified and optimized a conventional EA to connect it online with an OA‐ICOS instrument for H₂O measurements. The technique is first evaluated for calibrated free water samples. The technique is also evaluated for salt matrix effect, accuracy, and linearity for both isotopic signatures. Then, the technique is used to measure simultaneously δ²H and δ¹⁸O values of halite water inclusions precipitated from the evaporation experiments. Data generated with this new technique appeared to be comparable with those inferred from prior off‐line technique studies. The advantages offered by the OA‐ICOS technique are the simultaneous acquisition of both isotopic ratios and the substantial reduction of data acquisition time and sample aliquot size. Natural halite samples have been analyzed with this method. Natural halite samples as old as Precambrian have also been analyzed with this method.     

Micro‐scale (1.5 µm) sulphur isotope analysis of contemporary and early Archean pyrite

We present a method for in situ sulphur (S) isotopic analysis of significantly small areas (1.5 µm in diameter) in pyrite using secondary ion mass spectrometry (NanoSIMS) to interpret microbial sulphur metabolism in the early earth. We evaluated the precision and accuracy of S isotopic ratios obtained by this method using hydrothermal pyrite samples with homogeneous S isotopic ratios. The internal precision of the δ³⁴S value was 1.5‰ at the level of 1 sigma of standard error (named 1SE) for a single spot, while the external reproducibility was estimated to be 1.6‰ at the level of 1 sigma of standard deviation (named 1SD, n = 25). For each separate sample, the average δ³⁴S value was comparable with that measured by a conventional method, and the accuracy was better than 2.3‰. Consequently, the in situ method is sufficiently accurate and precise to detect the S isotopic variations of small sample of the pyrite (less than 20 µm) that occurs ubiquitously in ancient sedimentary rocks. This method was applied to measure the S isotopic distribution of pyrite within black chert fragments in early Archean sandstone. The pyrite had isotopic zoning with a ³⁴S‐depleted core and ³⁴S‐enriched rim, suggesting isotopic evolution of the source H₂S from ?15 to ?5‰. Production of H₂S by microbial sulphate reduction (MSR) in a closed system provides a possible explanation for both the ³⁴S‐depleted initial H₂S and the progressive increase in the δ³⁴S_H2S value. Although more extensive data are necessary to strengthen the explanation for the origin of the MSR, the results show that the S isotopic distribution within pyrite crystals may be a key tracer for MSR activity in the early earth. Copyright © 2010 John Wiley & Sons, Ltd.     

The effects of α-cellulose extraction and blue-stain fungus on retrospective studies of carbon and oxygen isotope variation in live and dead trees

Tree‐ring carbon and oxygen isotope ratios from live and recently dead trees may reveal important mechanisms of tree mortality. However, wood decay in dead trees may alter the δ¹³C and δ¹⁸O values of whole wood obscuring the isotopic signal associated with factors leading up to and including physiological death. We examined whole sapwood and α‐cellulose from live and dead specimens of ponderosa pine (Pinus ponderosa), one‐seed juniper (Juniperous monosperma), piñon pine (Pinus edulis) and white fir (Abies concolor), including those with fungal growth and beetle frass in the wood, to determine if α‐cellulose extraction is necessary for the accurate interpretation of isotopic compositions in the dead trees. We found that the offset between the δ¹³C or δ¹⁸O values of α‐cellulose and whole wood was the same for both live and dead trees across a large range of inter‐annual and regional climate differences. The method of α‐cellulose extraction, whether Leavitt‐Danzer or Standard Brendel modified for small samples, imparts significant differences in the δ¹³C (up to 0.4‰) and δ¹⁸O (up to 1.2‰) of α‐cellulose, as reported by other studies. There was no effect of beetle frass or blue‐stain fungus (Ophiostoma) on the δ¹³C and δ¹⁸O of whole wood or α‐cellulose. The relationships between whole wood and α‐cellulose δ¹³C for ponderosa, piñon and juniper yielded slopes of ~1, while the relationship between δ¹⁸O of whole wood and α‐cellulose was less clear. We conclude that there are few analytical or sampling obstacles to retrospective studies of isotopic patterns of tree mortality in forests of the western United States. Published in 2011 by John Wiley & Sons, Ltd.