Stella A. Ordoudi

Screening method for the detection of artificial colours in saffron using derivative UV-Vis spectrometry after precipitation of crocetin

A screening method for the detection of artificial colours (naphthol yellow, tartrazine, quinoline yellow, Sunset yellow, Allura red, amaranth, azorubine, Ponceau 4R and Red 2G) in saffron is described. The method involves removal of crocins by precipitation of crocetin (pH 0.1, 90°C) before adsorption of the artificial colours on polyamide SPE cartridges (pH 2). After washing with methanol, acetone and methanol, elution was done with a methanol:ammonia solution (95:5 v/v), and detection was performed by derivative spectrometry. Sample pretreatment changes the UV-Vis saffron extract profile in such a way that second derivative spectra can be used to identify the presence of added colours. Erythrosine, which was found to be pH dependent, could not be detected under the above conditions. The lowest detectable amount for each colour was strongly dependent on chemical structure. The recovery of carminic acid was very low possibly due to irreversible retention on the polyamide. This procedure can replace the current ISO TLC method (2003) and be used alternatively or in combination with HPLC procedures adopted in the same standard.

On the quality control of traded saffron by means of transmission Fourier-transform mid-infrared (FT-MIR) spectroscopy and chemometrics

The present study aimed to extend application of the FT-MIR technique to the quality control of traded saffron that suffers various types of fraud or mislabelling. Spectroscopic data were obtained for samples stored for different periods in the dark. Samples with the highest quality according to ISO 3632 specifications produced a typical spectrum profile (reference set). Principal Component Analysis (PCA) of spectroscopic data for this set along with HPLC-DAD analysis of major apocarotenoids assisted identification of FT-IR bands that carry information about desirable sensory properties that weaken during storage. The band at 1028cm(-1), associated with the presence of glucose moieties, along with intensities in the region 1175-1157cm(-1), linked with breakage of glycosidic bonds, were the most useful for diagnostic monitoring of storage effects on the evaluation and test set samples. FT-IR was found to be a promising, sensitive and rapid tool in the fight against saffron fraud.

Kinetics of individual crocetin ester degradation in aqueous extracts of saffron (Crocus sativus L.) upon thermal treatment in the dark.

Kinetics of individual crocetin ester degradation in aqueous extracts of saffron upon thermal treatment in the dark has been studied. Special attention has been paid to the comparison between saffron extracts and aqueous solutions of a crocetin ester rich fraction, with a lower stability of the latter observed. The degradation reaction was the same for all crocetin esters whether they were in saffron extracts or whether they were purified, although it was affected by external factors that modified their kinetic and thermodynamic parameters, making some of them less stable than others.

Saffron Quality: Effect of Agricultural Practices, Processing and Storage

Saffron, the most expensive spice worldwide, is comprised of the dried stigmas of the plant Crocus sativus Linnaeus of the Iridaceae family, a sterile triploid not found in the wild. According to the definition given by FAO (Food and Agricultural Organisation) it forms ‘a loosely matted mass of dark, reddish-brown flattened threads, amongst which a few narrower yellow ones can be distinguished. The upper, enlarged part of the flattened threads is the stigma of the flower, the lower narrower portion is the style’ (FAO, 1986). Saffron is mainly used as a spice that imparts colour to food but its medicinal and dyeing properties are also well known and appreciated. C. cartwrightianus, a possible progenitor of C. sativus, as well as more than 80 other species belonging to genus Crocus originate from the eastern Mediterranean basin from where the cultivation of the plant was spread to other parts of the ‘Old World’. Many of the Crocus species occur in the Aegean islands and Crete (Greece) that may be considered as ‘the birthplace’ of the cultivated plant, highly appreciated in the early civilizations of those areas for its exceptional properties. Famous fresco fragments exhibited today in the archaeological museums in Heraklion (Crete), Santorini and that in Athens offer evidence for the ritual significance and the use of Crocus plant in the every day life of the prehistorical natives. In addition, written information on pottery tablets give unequivocal evidence for the participation of the plant material in the economy of the Cretan kings of Knossos (1500–1450 B.C). The small amounts of the final product, the dried stigmas, reported on those records, equivalent to a few grams up to half a kilo, indicate that it commanded continually through the centuries an exceptional high commercial price. The ancient Greek name ‘krokos’ survived in the current language of this small country to characterise both the plant and the spice whereas the word saffron of Arabic (or old French?) roots (that means ‘yellow’) that may dates even back to the Assyrian empire (2300 B.C) and comes from the name of a town called Azupirano (Saffron town) (Basker and Negbi, 1983) is used in many languages (‘safran’ in French and German, ‘saufuran’ in Japanese, ‘azafran’ in Spanish, ‘zafora’ in Greek). The Hebrew word found in Bible is ‘karkom’ and the Chinese names are ‘fan-hong-hua’ (foreign red flower) or ‘zang-hong-hua’ (Tibet red flower).

On the Traceability of Commercial Saffron Samples Using 1H-NMR and FT-IR Metabolomics

In previous works on authentic samples of saffron of known history (harvest and processing year, storage conditions, and length of time) some biomarkers were proposed using both FT-IR and NMR metabolomics regarding the shelf life of the product. This work addresses the difficulties to trace back the “age” of commercial saffron samples of unknown history, sets a limit value above which these products can be considered substandard, and offers a useful tool to combat saffron mislabeling and fraud with low-quality saffron material. Investigations of authentic and commercial saffron samples of different origin and harvest year, which had been stored under controlled conditions for different lengths of time, allowed a clear-cut clustering of samples in two groups according to the storage period irrespectively of the provenience. In this respect, the four-year cut off point proposed in our previous work assisted to trace back the “age” of unknown samples and to check for possible mislabeling practices.

Ion-pair assisted extraction followed by 1H NMR determination of biogenic amines in food and biological matrices

A selective method for the extraction and determination of six biogenic amines (BAs) by NMR is presented. Briefly, BAs are extracted into an organic solvent via the use of an ion pairing agent, followed by a back extraction in D2O in order to acquire the (1)H NMR spectra. The method is studied with respect to the critical experimental parameters and is successfully applied to selected food substrates (dark chocolate, banana, gouda cheese) and biological samples (urine and blood plasma) signifying its potential as an alternative tool for BAs determination. Accurate and precise results are consistently achieved with all matrixes studied. The calculated limits of detection and limits of quantitation were found to be in the ranges 0.05-0.13μg/mL and 0.14-0.38μg/mL, respectively, for biological samples while for food samples they were in the ranges 2.25-6.25μg/g and 6.75-18.7μg/g, respectively.

Insight of Saffron Proteome by Gel-Electrophoresis

Saffron is a spice comprised of the dried stigmas and styles of Crocus sativus L. flowers and, since it is very expensive, it is frequently adulterated. So far, proteomic tools have never been applied to characterize the proteome of saffron or identify possible cases of fraud. In this study, 1D-Gel Electrophoresis was carried out to characterize the protein profile of (i) fresh stigmas and styles of the plant; (ii) dried stigmas and styles from different geographical origins (Spanish, Italian, Greek and Iranian) that had been stored for various periods of time after their processing; and (iii) two common plant adulterants, dried petals of Carthamus tinctorius L. and dried fruits of Gardenia jasminoides Ellis. A selective protein extraction protocol was applied to avoid interference from colored saffron metabolites, such as crocins, during electrophoretic analyses of saffron. We succeeded in separating and assigning the molecular weights to more than 20 proteins. In spite of the unavailability of the genome of saffron, we were able to identify five proteins by Peptide Mass Fingerprinting: phosphoenolpyruvate carboxylase 3, heat shock cognate 70 KDa protein, crocetin glucosyltransferase 2, α-1,4-glucan-protein synthase and glyceraldehydes-3-phosphate dehydrogenase-2. Our findings indicate that (i) few bands are present in all saffron samples independently of origin and storage time, with amounts that significantly vary among samples and (ii) aging during saffron storage is associated with a reduction in the number of detectable bands, suggesting that proteases are still active. The protein pattern of saffron was quite distinct from those of two common adulterants, such as the dried petals of Carthamus tinctorius and the dried fruits of Gardenia jasminoides indicating that proteomic analyses could be exploited for detecting possible frauds.

Consideration of fluorescence properties for the direct determination of erythrosine in saffron in the presence of other synthetic dyes

Direct selective detection of erythrosine in saffron in the presence of other synthetic dyes considers its fluorescence at 532 nm excitation/548 nm emission. Saffron pre-treatment was according to the ISO 3632-2 trade standard test methods. On account of calculated quantum yield values, none of the yellow dyes is expected to interfere. Among red ones, reservations about allura red AC, azorubine and red 2G were not verified by experimentation, signifying excellent method specificity. Detection and quantification limits (0.56 and 1.70 nM) were of the same magnitude as those reported in the literature after chromatographic separation of erythrosine. The percentage recovery from spiked saffron samples ranging from 63 to 141 was acceptable for residue levels in foods. The matrix effect from crocins (saffron pigments) was evidenced only at a lower spiking level (0.02  mg kg−1). The minimum required performance limit (MRPL) was 0.04 mg kg−1, indicating that the method is appropriate for determining traces of erythrosine in saffron. The approach offers improved sensitivity (by three orders of magnitude) and specificity than the direct spectrophotometric detection of certain synthetic dyes in saffron and deserves attention by the ISO Technical Committee for ‘Herbs, culinary spices and condiments’.

Enhanced Bioaccessibility of Crocetin Sugar Esters from Saffron in Infusions Rich in Natural Phenolic Antioxidants

The present study aims to examine whether and to what extent the bioaccessibility of the major saffron apocarotenoids, namely crocetin sugar esters (CRTSEs), is affected by the presence of strong water-soluble antioxidants, ingredients of the herbs found in commercial tea blends with saffron. An in vitro digestion model was applied to infusions from these products to investigate the possible changes. All of the studied infusions were rich in total phenols (9.9–22.5 mg caffeic acid equivalents/100 mg dry infusion) and presented strong DPPH radical scavenging activity regardless of the composition of the corresponding herbal blends. RP-HPLC-DAD and LC-MS analysis enabled the grouping of the infusions into hydroxycinnamic acid-rich and in flavan-3-ol-rich ones. CRTSEs in herbal tea infusions were found to be significantly more bioaccessible (66.3%–88.6%) than those in the reference saffron infusion (60.9%). The positive role of strong phenolic antioxidants (caffeic acid, rosmarinic acid) on the stability of CRTSEs was also evidenced in model binary mixtures. On the contrary, cinnamic acid, exerting no antioxidant activity, did not have such an effect. Our findings suggest that strong radical scavengers may protect the crocetin sugar esters from oxidation during digestion when present in excess.

A stepwise approach for the detection of carminic acid in saffron with regard to religious food certification

The stepwise approach takes advantage of simple, versatile, low-cost screening tools that can be applied to several posts of the saffron trade chain to specifically detect adulteration with carminic acid (CA). This natural dye is of insect origin and should not be present in Kosher and Halal foods such as saffron. For gross adulteration levels (>25.0%, w/w) reaction with diphenylamine-sulfuric acid was found adequate to indicate the presence of extraneous matter but not its identity. FT-IR analysis of the dry material combined with chemometrics served to rapidly sort out samples containing >10.0% CA without any sample pretreatment except grinding. Aqueous extracts prepared according to ISO 3632-2 were then examined by tristimulus colorimetry and derivative UV-Vis spectrometry to detect adulteration down to the level of 2.0% (w/w). Determination of CA down to 0.2%, w/w was achieved by RP-HPLC-DAD using aqueous acetonitrile elution solvent (pH=2.8).