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Sosa, R.A.;  Nevárez-Moorillón, G.V.;  Ochoa-Velasco, C.E.;  Navarro-Cruz, A.;  Hernández-Carranza, P.;  Cid-Pérez, T.S. Saffron Authentication and By-Products. Encyclopedia. Available online: (accessed on 11 December 2023).
Sosa RA,  Nevárez-Moorillón GV,  Ochoa-Velasco CE,  Navarro-Cruz A,  Hernández-Carranza P,  Cid-Pérez TS. Saffron Authentication and By-Products. Encyclopedia. Available at: Accessed December 11, 2023.
Sosa, Raul Avila, Guadalupe Virginia Nevárez-Moorillón, Carlos Enrique Ochoa-Velasco, Addí Navarro-Cruz, Paola Hernández-Carranza, Teresa Soledad Cid-Pérez. "Saffron Authentication and By-Products" Encyclopedia, (accessed December 11, 2023).
Sosa, R.A.,  Nevárez-Moorillón, G.V.,  Ochoa-Velasco, C.E.,  Navarro-Cruz, A.,  Hernández-Carranza, P., & Cid-Pérez, T.S.(2022, November 11). Saffron Authentication and By-Products. In Encyclopedia.
Sosa, Raul Avila, et al. "Saffron Authentication and By-Products." Encyclopedia. Web. 11 November, 2022.
Saffron Authentication and By-Products

Plants and vegetables are major sources of food bioactives. Spices and herbs are plant materials that provide a wide range of biologically active compounds. In addition to being used as sources of aroma, flavor, and color and as preservatives, spices and herbs have been used for medicinal purposes and health and wellness for centuries. Aromatic spices can be added to food in their natural state as a powder or extract. In the food industry, it is not only the active parts of vegetables or plants that are important since there are several uses for their waste or by-products as ingredients in different food formulations. Saffron is the commercial name for the dried red stigmas of the Crocus sativus L. flower. It owes its sensory and functional properties mainly to the presence of its carotenoid derivatives, synthesized throughout flowering and also during the whole production process. 

saffron adulteration plants

1. Saffron Authentication

Due to its high market price, saffron is the most adulterated spice in history, which is most frequently carried out by adding adulterants such as pulverized stigmas [1][2] since diverse plants with similar color and morphology to saffron function as adulterants when mixed [3]. Saffron adulteration can be classified into five common practices, as follows: (1) Adulteration using material from other plants such as calendula, arnica, gardenia, beet, pomegranate, turmeric, achiote, and safflower [2][4][5][6] or with other plant parts of C. sativus besides the stigmas; (2) Increasing saffron mass by moistening with honey, corn silk, sugar, fat, inorganic compounds, vegetable oils, or glycerin [6][7]; (3) Using natural or artificial food-grade colorants such as tartrazine, ponceau-4R, quinoline, methyl orange, sunset yellow, Sudan II, and Allura red [8][9]; and other less-used adulteration methods including (4) The addition of exogenous components mixed with food flavorings (erythrosine) and extracted spent saffron (recolored or old), and (5) Geographic origin tagging fraud [4][10][11][12].
The chemical composition of food is an indicator of quality, origin, authenticity, and/or adulteration. The chemical profile, also known as spectral fingerprinting or chemo typing, is considered a characteristic pattern [13]. In food, variations in a profile are related to alterations in production systems, the geographical origins of raw materials, storage conditions, or adulterant practices [14]. It should be emphasized that it is important to identify the adulterant and quantify the adulteration level [15]. Furthermore, the ISO/TS 3662 spectrophotometric technique does not differentiate between genuine and adulterated saffron [16][17]. Saffron authentication is based on a pharmacognostic analysis (microscopic examination of histomorphological features). It is time-consuming and requires the availability of trained and experienced personnel [2][18].
Regulatory systems evaluate saffron using sensory inspections (macroscopic and microscopic examinations) as well as conduct quantitative determinations of specific chemical compounds [19]. Authentication is based on detecting known chemical compounds obtained with instrumental signals [20]. However, these kits yield many characteristics or compounds, making it necessary to establish the chemical markers of authenticity [21]. Spectral fingerprinting can also detect and quantify adulterations using statistical data [20]. Chemometrics uses mathematical and statistical methods to create a correlation between the sample properties and chemical data obtained from analytical instruments [22]; this area is based on optimizing the experimental design and extracting useful information from large and complex data sets [14]. Therefore, analytical chemometric coupling could notably decrease the number of characteristics/compounds/signals and generate the markers responsible for different authenticity issues (adulteration detection, variety or geographical origin, discrimination, organoleptic profile, maturation, and production method). In addition, the identified markers would help to establish databases containing complete and standardized information on the chemical profiles [21].
The following research summary is based on determining chemical compounds as authentication markers (of genuine saffron or adulterants used) using different analytical techniques to determine the spectral fingerprints and/or even using chemometrics to obtain the amount of the adulterant or even the detection limits of the adulterant. Saffron adulteration determination by the inclusion of tepals and/or stamens was carried out by Senizza et al. [16]. They determined 232 compounds using UHPLC-QTO-MS. Among them, 77 chemicals were present in trace quantities including the presence of flavonoids: 11 flavanols (tepals had a high content) and 7 anthocyanins (pigments of flowers, fruits, and other plant organs), which increased in the adulterated samples. On the other hand, lignans (12 compounds) were found in low amounts in the authentic samples. Zeaxanthin and picrocrocin, which decreased in the adulterated samples, suggested a possible “dilution effect” when adding adulterants. Moras et al. [5] determined, through UHPLC-DAD-MS, the presence of iridoids as a marker for saffron adulteration, yielding positive test results when gardenia extract was added.
Investigations using analytical techniques and chemometrics to quantify the adulterant and the minimum detection to detect fraud have been presented. A method for deducing saffron authenticity using LC-MS with derivatives of kaempferol and geniposide was developed by Guijarro-Díez et al. [11]. They detected a minimum quantifiable value of adulteration (0.2%) regardless of the adulterant (linear regression lineal and ANOVA), the specific method, and saffron quality control. Sabatino et al. [23] used HPLC-PDA-ESI-MS to identify unusual concentrations of adulterants in saffron (10–67% safflower, calendula, and turmeric). Their results showed that the ISO did not detect the addition of 10% of adulterants. Moreover, marker molecules such as picrocrocin, trans-5-nG, trans-4-GG, trans-4-ng, cis-3-Gg, cis-4-GG, and cis-2-gg were not found in the adulterated spices. They determined the addition of 5% of safflower or calendula and 2% addition of turmeric in the analyzed samples.
Saffron stigma adulteration with up to 20% of plant derivatives (saffron stamens, calendula, safflower, turmeric, buddleja, and gardenia) was determined by Petrakis and Polissiou [15] using a DRIFTS method and chemometric techniques. PLS-DA was applied to perform saffron authentication based on infrared fingerprints (4000–600 cm). Identification was carried out with data from the 2000–600 cm−1 region to develop the mathematical models and detection limits ranging from 1.0 to 3.1% (p/p). Another (NIR) spectroscopy investigation combined with multivariate data analysis was performed by Shawky et al. [24]. They performed saffron stigma authentication with other plants (safflower, pomegranate peel, calendula flower, paprika, turmeric, hibiscus, saffron stamens, and re-extracted saffron stigma), modeling them with data at the spectral region (9000–4000 cm−1). The use of PLS-DA allowed them to differentiate between authentic, adulterated, and mixed adulterant samples, with a detection limit of up to 10 mg/g of the adulterant. In addition, they quantified other added adulterants.
Saffron stigma authentication using artificial intelligence (simulating senses: sight, smell) was reported by Heidarbeigi et al. [25]. They determined plant adulterants (safflower and dyed corn using beetroot as a colorant, in addition to their mixtures) through signals obtained by the e-nose (managing to differentiate adulterated and unadulterated saffron). They also applied PCA and artificial neural networks (ANN) to determine fraud in saffron stigmas, determining adulteration levels higher than 10%. Kiani et al. [26] used CVS (camera, lighting system, and software) and an e-nose in combination with multivariate methods (PCA, HCA, and SVMs) to detect saffron stigma adulterants (colored safflower and saffron style) based on color and aroma profiles. The test demonstrated the ability to identify the adulterated samples and this was achieved using ANN-MLP models, concluding that neural networks allowed color (89%) and aroma-intensity (100%) prediction. CVS was used by Minaei et al. [27] to characterize saffron color by sample image analysis. The use of PCA to group color characteristics and the use of PLS, MLR, and MLP neural networks (color characteristics used: R, Y, I, and Cr) related color and dye force (ISO 3632), with a correlation coefficient of 0.89 and a success rate of 96.67%.
Another interesting application is the use of an e-nose (non-conventional technique), compared to IR-MS and GC-MS (conventional techniques) to discriminate among saffron samples with different origins, ages, and types of drying. The e-nose, in conjunction with PLS-DA, was able to discriminate between samples of saffron with different origins; this unconventional methodology was proposed to detect adulterates [28]. Recently, molecular techniques for detecting fraud by adulterations have gained interest. Safflower adulteration stamens as saffron adulterants were also studied by Babaei et al. [17], using a multiplex PCR technique. Khilare et al. [6] described three methods to achieve saffron authentication (microscopic examination, ISO3632 standard, and DNA barcode). They evaluated 36 saffron samples and showed that the ISO only determines the color and aroma, while the microscopic method indicates color purity and uniformity (possible adulterants).
Finally, DNA codes (gene code used: rbcL) have allowed researchers to authenticate saffron’s origin and quality. Torelli et al. [2] used SCAR to detect adulteration or contamination. SCAR markers can represent a rapid, reliable, and inexpensive method for saffron authentication. Other rapid techniques for determining saffron adulteration were proposed by Zhao et al. [29] via DNA extraction. They used a recombinase polymerase amplification (RPA-LFD), which allowed them to perform the rapid visual detection of the saffron and adulterated samples. Finally, when saffron was immersed in water, it expanded immediately; when a diphenylamine and sulfuric acid solution was added, the saffron was colored with a blue tone and quickly became reddish brown. Saffron phenylethanol varies according to the spice preparation and is related to the stamen pollen [4]. Table 1 shows a summary of the various research works and techniques for the determination of the different types of adulterants. As regards the adulteration of saffron by its origin or PDO products, saffron has a high value on the market so some saffron producers falsify the product’s origin [30][31]. In Europe, a PDO label carries a regional valuation that identifies the products produced, processed, and prepared in a specific geographic area [32]. There are five brands recognized with this label: “Krokos Kozanis” (Greece), “Azafrán de la Mancha” (Spain), “Zafferano dell ‘Aquila”, “Zafferano di San Gimignano”, and “Zafferano di Sardegna” (Italy) [30]. There have been a considerable number of studies on origin adulteration [10][28][31][32][33][34][35][36]. La Mancha in Spain and Kashmir in India are two regions where saffron maintains higher prices [35]. Therefore, labeling saffron samples with a PDO implies that the product is of high quality [31]. Moreover, Senizza et al. [16] determined the chemical markers capable of discriminating PDO saffron samples from non-PDO. Chemical fingerprints were obtained using UHPLC-ESI-QTOF-MS and multivariate statistics, obtaining the flavonoids belonging to the flavonols and flavones (pelargonidin 3-O-6-succinyl-glucoside, isoxanthohumol, nobiletin, jaceosidin, 6-hydroxyluteolin, 3-methoxysinenset, 7-dimethylquercetin, quercetin 6-O-malonylglycitin), phenolic acids (protocatechuic aldehyde, 4-hydroxybenzaldehyde, vanillin, 2/3/4-hydroxybenzoic acids, benzoic acid, sinapine, p-coumaroyl malic acid, p-coumaric acid, cinnamoyl glucose, 4-hydroxyphenylacetic acid), lignans, and other polyphenols.
A suitable method is the use of NMR in conjunction with multivariate statistical analysis. Principal component analysis allowed the discrimination between the samples of Italian PDO and commercial saffron, despite the year of harvest, date of purchase, and storage time [33]. Bosmali et al. [30] proposed a molecular approach for the authentication of the “Krokos Kozanis” brand using specific ISSR (inter-simple sequence repeat) markers to evaluate the variability within the C. sativus L. species (differences in bands produced by other Crocus species). The species-specific markers such as HRM analysis were developed in conjunction with the DNA barcode regions.

2. Saffron By-Products

The preparation of saffron is expensive due to the intense harvesting work and postharvest processes (dehydration and storage) required [42]. It is known that in order to produce 1 kg of stigma, around 1000 kg of flowers are treated by weight, which represents 220,000–260,000 flowers [43][44]. Therefore, saffron cultivation is not highly profitable in terms of biomass, which increases the interest in minimizing losses and ensuring efficient waste management [45]. Several reports have focused on the stigma, which is the plant’s biologically active part [46]; its bioactivity is attributed to the composition, containing the main chemical components and their synergy with other compounds [47].
However, the by-products are also important since their use could increase the C. sativus flower’s economic value, considering that other parts of the plant contain compounds with sensorial properties or biological activity [44][45]. C. sativus tepals are the main by-product of saffron production [48] but the flowers have low safranal content so they cannot be consumed or sold as saffron on the spice market [43]; only the leaves are used as forage [49]. Using HPLC-DAD, Serrano-Díaz et al. [50] determined kaempferol 3-Osophoroside and delphinidin 3,5-di-O-glucoside as the main components of the aqueous by-products of saffron flowers. Tepal and stamen biomarkers were determined by Mottaghipisheh et al. [51] using HPLC-DAD; they reported crocin, crocetin, picrocrocin, safranal, kaempferol-3-O-sophoroside, kaempferol-3-O-glucoside, and quercetin-3-O-soforoside. Tepal’s main component was kaempferol-3-O-sophoroside with crocin, crocetin, and picrocrocin; safranal was not detected in any of the analyzed samples. Table 2 shows the principal agro-industrial by-products of saffron that have been investigated and their possible uses. Lahmass et al. (2017) determined that the corms, leaves, and spasms of C. sativus may possess anti-aging or anticancer properties.
These investigations generate interest in valorizing the various parts of saffron flowers and improving small-scale farmers’ incomes. These results could contribute to the development of innovative products from saffron flowers and more effective biological waste management and exploitation [57]. It is important to emphasize knowledge of the components’ depth (majority or minority) within each potentially valuable plant part of the saffron plant, which could help in determining the most suitable application [58].


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