José Luis Ríos
1,* and Isabel Andújar
2Introduction
Crocus sativus L., a plant from the iris family (Iridaceae), is mainly grown in the Mediterranean and south- western Asia (Gresta et al. 2008). Commercial saffron consists of the dried red stigma of the fl ower with a small portion of the yellowish style attached (Fig. 4.1). While saffron is principally used in cooking and baking, it is also used to add fl avor and color to both alcoholic and nonalcoholic beverages. Other less extended applications include its use as a dye in the textile industry and as an excipient in pharmaceutical preparations (Ríos et al. 1996). Saffron is recognized as the most expensive, and therefore the most interesting and attractive, spice in the world, prized for its coloring, bitterness, and aromatic power of its dried stigmas (Gresta et al. 2008).
Various authors have published interesting reviews on saffron, covering all aspects of this spice throughout history, including the use of the dried red stigmas and saffron’s cultivation, processing, chemistry and standardization, with a fi nal overview and outlook for the future (Sampathu et al. 1984, Ríos et al.
1996). More recently, Gresta et al. (2008) conducted an extensive review of various agricultural aspects of saffron such as its origins and distribution, genetic traits, botanical characteristics, culture adaptation, management techniques, and qualitative characteristics, including the latest instrumental methods for quality assessment. Other reviews include Kumar et al.’s (2009) compilation of the most recent agronomic advances in saffron’s commercial fl ower and corm production: Carmona et al.’s (2007) review of saffron’s aromatic characteristics, Winterhalter and Straubinger’s (2000) paper on saffron’s chemical composition and the most recent fi ndings on saffron aroma formation, Maggi et al.’s (2010) analysis of changes in the spice’s volatile profi le depending on its storage time, and Carmona et al.’s (2005) study on the infl uence of different drying and aging conditions on the spice’s constituents.
Commercial saffron for use in cooking, that is, the dried stigmas of the fl ower itself, should contain no more than 12% water and 7% mineral matter (Ríos et al. 1996). The main components responsible for its fl avor and aroma are the essential oil, bitter principles, and dye material. The dye material consists
1 Departament de Farmacologia, Universitat de Valencia, 46100 Burjassot (Spain).
Email: riosjl@uv.es
2 COMAV, Universidad Politécnica de Valencia, 46022 Valencia (Spain).
Email: isanpe@upvnet.upv.es
*Corresponding author
mainly of both water-soluble and fat-soluble carotenoids (8%), with the latter containing a remarkable amount of lycopene, α-carotene, β-carotene, and zeaxanthin. The relevant water soluble components are carotenoid glycosides such as crocin and the free aglycone trans-crocetin, although other minor water- soluble compounds are also present, such as fl avonoids (kaempferol, quercetin, and naringenin derivatives), cianidins (delphinidin and petunidin glucosides), and anthraquinones (emodin derivatives), which are the principal phenolic compounds (Ríos et al. 1996, D’Auria et al. 2004, Gresta et al. 2008, Padmavati et al.
2011).
Among the bitter principles, picrocrocin is the most important. It can be hydrolyzed to glucose and the volatile aglycone, safranal (Fig. 4.2). The volatile fraction or essential oil (0.3–1.5%) is a colorless liquid, which gives the spice its characteristic, intense odor. It is highly unstable due to its high capacity for oxygen absorption and browning. It is slightly levorotatory, with a density between 0.9514 and 0.9998.
The essential oil is made up of monoterpenes, mainly aldehydes (70%), with safranal as the most abundant component (47% of the volatile fraction). All the aldehydes have a similar structure, with 2,6,6-trimethyl- cyclohexene-1-carboxaldehyde being the most prevalent. For a complete chemical overview, see the reviews from Ríos et al. (1996), D’Auria et al. (2004), Gresta et al. (2008) and Padmavati et al. (2011).
Botanical aspects
Saffron is a stemless perennial geophyte herb, which fl owers in autumn. It is a sterile triploid derived from saffron, a triploid species (x = 8; 2n = 3x = 24) that is self- and out-sterile and mostly male-sterile and therefore unable to produce seed, so it is propagated principally through corms. It is derived from a wild ancestor, probably through fertilization of either a diploid unreduced egg cell by a haploid sperm cell or a haploid egg by two haploid sperm cells (Gresta et al. 2008, Kumar et al. 2009). Some authors consider C. cartwrightianus Herb. cv. albus to be a possible ancestor of C. sativus since ultrastructural observations have revealed that the pollen of both taxa show numerous pollen germination anomalies which are different from those of other closely related species such as C. thomasii Ten. or C. hadriaticus Herb. (Siracusa et al. 2013). Moreover, fl owering in C. cartwrightianus shares close similarities to that in C. sativus (Gresta et al. 2008). Morphologically, it has a tuberous-bulb formation referred to as a corm, which is covered
Figure 4.1. Saffron fl ower and stigmas.
by several reticulated fi brous tunics. Leaves are erect, narrow, grass-like, and dark green in color. The fl owers (one to 12) have six violet petals that are connate at the base in a long and narrow tube, with a pistil composed of an inferior ovary from which a slender style divides into three dark red branches, called stigmas (Gresta et al. 2008). The dry stigmas constitute the valuable spice.
Saffron grows best in friable, loose, low-density, well-watered, and well-drained clay calcareous soils (Kumar et al. 2009). The biological cycle of saffron starts with the fi rst autumn rains with the emission of the aerial parts, leaves, and fl owers, and fi nishes with the replacement the corms. The fl owering season is from mid-October to the end of November, depending on climatic conditions (Gresta et al. 2008). Traditional cultivation methods generally respect this biological cycle. As for the soil, it should be completely cleared of weeds, plowed at a depth of 25–30 cm, and left to rest, either for a few weeks or for the entire winter.
Disinfestation of soil before planting avoids fungal infection and sowing by hand is recommended.
Climate and soil, planting time, seed/corm rate, planting depth, corm size/weight, crop density, nutrient management, weed management, growth regulators, harvest, and post-harvest management all infl uence saffron quality and yield (Kumar et al. 2009).
However, with the application of new mechanical techniques in its cultivation and harvest, some relevant modifi cations can be made to the biological cycle of saffron. For example, the induction of hysteranthy, or fl owering prior to leaf appearance, may be of great interest as it facilitates the mechanization of the fl ower harvest without damaging the leaves. This physiological phenomenon can be induced by controlling the temperature during corm storage (Gresta et al. 2008).
Figure 4.2. Chemical composition of saffron. Major components.
Use and applications as dye, perfume and in food
While the essential oil of saffron is not directly used in food science, it has great potential as a condiment in food: both solid foods and beverages can be improved with the use of this special spice. Saffron not only improves the smell, fl avor, and taste of meals, but it can also improve the health of those who consume it daily.
The use of saffron as a dye stems from the presence of water-soluble carotenoids, principally α-crocetin, which is mostly responsible for the yellow color saffron gives to food. It was extensively used in ancient cultures (Assyria, Egypt, Greece, and Rome) as a dye for wool, silk, and hair, among other things. However, its use has decreased with the introduction of cheaper synthetic dyes. Today, saffron is used as a coloring agent mostly in the food and beverage industry. Fresh saffron is odorless, but as it dries, its characteristic aroma appears. This odor is due to safranal, which is released after the enzymatic or thermal hydrolysis of its corresponding glycoside, picrocrocin, present in the fresh stigmas.
Saffron is an extensively used spice in many typical Mediterranean and Asian dishes and is highly appreciated for its bitter taste and the luminous yellow-orange color it gives to foods. There are many reasons for using saffron in cooking, including its colorant properties, pleasing fl avor, delicate aroma, and bitter taste. It is used as a condiment in both meat and fi sh dishes, in soups or cakes, and in sauces or liquor. Saffron is also used in rice dishes, creams, cheese, chicken, mayonnaise, bouillabaisse, and other traditional foods, as well as an additive in alcoholic and nonalcoholic beverages worldwide (Sampathu et al.
1984). The recommended fi nal concentrations in which saffron should be used to avoid an overwhelming taste or smell, which would change the fi nal properties of the food and beverages containing it are given in Table 4.1.
Because the consumption of saffron has stabilized during the past few years, alternative applications have been created to open up new markets. The use of saffron for beverage production is one of these new alternatives. For example, in Spain saffron is used to fl avor soft drinks and other beverages, whereas in Italy is added to liquors and in Greece is used in the preparation of alcoholic distillates. The concentration of saffron in non-alcoholic beverages should generally be around 1.3 ppm, whereas in alcoholic beverages the concentration is higher, about 200 ppm (Table 4.1). In the USA, tinctures of saffron are used for fl avoring liquors, such as ‘Boonekamp’, a special bitter brandy, or ‘Strega’, in which saffron gives color, taste, and fl avor to this highly alcoholic liquor, otherwise known as ‘saffron gin’.
Aside from its organoleptic properties and its potential as a medicinal drug, saffron contains a series of highly interesting nutrients and minerals. For example, the principal elements present in saffron are carbohydrates, vitamin C, and manganese (Table 4.2). Of the total content in carbohydrates, about 20%
are reducing sugars (glucose, fructose, gentiobiose, xylose, and rhamnose), but pentosans, gums, and dextrins are also present (10%). The lipid fraction is around 3–8%, with the presence of fatty acids such
Table 4.1. Final concentration of saffron in ppm in the fi nal product.
Saffron powder Saffron extract
Nonalcoholic beverages 1.3 1.3–7.5
Alcoholic beverages 200 −
Baked goods 10 1.9–14.0
Meats 260 −
Ice cream, ices, … − 1.3–9.0
Candy − 6.3
Condiments − 50
(Sampathu et al. 1984)
as palmitic, stearic, oleic, linoleic, and linolenic acids, as well as the phytosterols sitosterol, campesterol, and stigmasterol. The mineral content is 1.0–1.5%, with a high proportion of magnesium and potassium salts. The presence of proteins, amino acids, and nitrogen compounds is over 11–13% (Ríos et al. 1996, Padmavati et al. 2011). The spice also contains vitamins, especially ribofl avin and thiamine (Table 4.2).
Varieties and quality of saffron
There are different qualities of saffron and various denominations depending on its origin. According to both national and international specifi cations, saffron can be classifi ed in different ways. The International Organization for Standardization (ISO) has created a classifi cation system for saffron based on the minimal requirements for each quality level (ISO 3632). Thus, ISO 3632 establishes four categories (I–IV) of quality in which different points, such as color (due to crocin), fl avor (due to picrocrocin), and aroma (due to safranal), are evaluated. In addition, other parameters such as fl oral waste and foreign matter are also taken into account (Table 4.3).
Category IV has the poorest quality, with a maximum fl oral waste mass fraction of 10, double that stipulated for category III. Moreover, each country establishes its own rules and categories. In Spain, for example, the standards and categories established by the government are Coupe, Mancha, Rio, Standard, Table 4.2. Nutritional values per 100 g of saffron. Source United States Department of Agriculture, Agricultural (USDA) National Nutrient Database. Percentages are expressed as the recommended daily allowance (RDA).
Principle Nutrient Value Percentage of RDA
Energy 310 Kcal 15.5%
Carbohydrates 65.37 g 50%
Protein 11.43 g 21%
Total lipid (fat) 5.85 g 29%
Cholesterol 0 mg 0%
Dietary fi ber 3.9 g 10%
Vitamins Nutrient Value Percentage of RDA
Folates 93 μg 23%
Niacin 1.46 mg 9%
Pyridoxin 1.01 mg 77%
Ribofl avin 0.267 mg 20%
Vitamin A 530 UI 18%
Vitamin C 80.8 mg 135%
Electrolytes Nutrient Value Percentage of RDA
Sodium 148 mg 10%
Potassium 1724 mg 37%
Minerals Nutrient Value Percentage of RDA
Calcium 111 mg 11%
Copper 0.33 mg 37%
Iron 11.10 mg 139%
Magnesium 264 mg 66%
Manganese 28.41 mg 1235%
Phosphorus 252 mg 36%
Selenium 5.6 μg 10%
Zinc 1.09 mg 10%
and Sierra, in which the coloring strengths, expressed as direct reading of the absorbance of crocin at about 440 nm, are 190, 180, 150, 145, and 110, respectively. In other countries with a different legal codex, there are relevant differences in quality. For example, the Indian Pharmaceutical Codex establishes the maximum values of water and volatile matter at 103ºC to be 14 and 8 for saffron in fi laments and in powder, respectively (Sampathu et al. 1984), whereas in the international codex (ISO 3632), these values are 12 and 10, respectively (Table 4.4).
Table 4.3. Classifi cation of saffron in categories by physical criteria in fi lament form (ISO/TS 3632-2: 2003) and crocin content (absorbance at 440 nm).
Characteristics Categories
I II III IV
Floral waste, mass fraction, % max 0.5 3.0 5.0 10
Foreign matter, mass fraction, % max 0.1 0.5 1.0 1.0
Crocin content (absorbance at 440 nm) > 190 150–190 110–150 80–110
Table 4.4. Quality requirements for saffron in function of its categories according to the ISO/TS 3632-1: 2003’s specifi cations.
Categories
I II III
Water and volatile matter at 103ºC maximum (% w/w)
Filament 12 12 12
Powder 10 10 10
Total ash on the dry basis maximum (%) 8 8 8
Ash insoluble in HCl on the dry basis (% w/w) max 1.0 1.0 1.5
Extract soluble in cold water on the dry basis (% w/w) min 70 55 40
Coloring strength, on dry basis, min
257 nm, min (maximum absorbance of picrocrocine) 70 55 40
330 nm, min (maximum absorbance of safranal) 20 20 20
330 nm, max (maximum absorbance of safranal) 50 50 50
440 nm, min (maximum absorbance of crocines) 190 150 100
Artifi cial water-soluble acid colorants 0 0 0
Saffron is the most expensive spice in the world and for this reason, its adulteration is common. This is easier in the case of powder, but it also occurs when presented as fi laments. There are different kinds of adulterants, most of them of plant origin. They usually include styles, stamen, and strips of the corolla of the saffron fl ower itself or stigmas from other Crocus species such as C. vernus or C. speciosus. Mixing new saffron with condensed or older saffron or addition of other parts of the saffron fl ower are also common adulteration techniques. Sometimes, saffron weight can be increased by the addition of substances such as water to increase the humidity percentage. Other techniques include soaking in syrup, honey, glycerin, or olive oil; adding mineral salts such as barium sulfate, calcium carbonate, potassium hydroxide, potassium nitrate, monopotassium tartrate, and sodium borate; or the addition of organic compounds such as lactose, starch, and glucose.
In other cases, the additives used come from other species, for example, fl orets of saffl ower (Carthamus tinctorius), maize (Zea mays), and calendula (Calendula offi cinalis), sometimes colored with methyl orange. Other adulterants are obtained from arnica (Arnica montana), pomegranate (Punica granatum), common golden thistle (Scolymus hispanicus), sliced poppy (Papaver rhoeas) fl owers, Cape saffron (Cassine peragua), red sandalwood (Santalum paniculatum), madder (Rubia tinctorum), or the outer skin
of onions (Allium cepa). In these cases, the uncolored material is cut into similarly sized pieces and dyed with eosin. Other natural colorants used as adulterants are carnation perianths (Dianthus caryophyllus), turmeric (Curcuma longa), annatto (Bixa orellana), ground red pepper (Capsicum frutescens), and small roots from allium porrum (Allium ampeloprasum var. porrum). Sometimes even salted and dried meat fi bers or colored gelatin fi bers are added.
Adulteration is easier in the case of saffron powder, in which different colorants are used. These include Martins yellow, tropeolin, fuchsine, picric acid, tartrazine, erythrosine, azorubine, cochineal red A, orange yellow, naphthol yellow, rocelline yellow, and methyl orange, among others. There are different methods for detecting the presence of contaminants or adulterants in saffron; these differ depending on the presentation (styles or powder). For example, the ISO/TS 3632, 2003 recommends the use of High- Performance Liquid Chromatography (HPLC) as it is the most sensitive method for detecting adulterants.
However, for non-specialized laboratories or in the home, other methods exist.
An examination of the macro- and micromorphology of saffron stigmas could be a fi rst step in the detection of adulteration and an analysis of general quality. For example, a stigma length over 30 mm with styles measuring 23 to 24 mm, hard brilliant color, and a strong aroma are all characteristic of the highest quality (high select) whereas poor quality saffron tends to have stigmas that are either broken or
< 20 mm long (Table 4.5).
Table 4.5. Spanish quality of saffron. Morphologic and organoleptic characteristics.
Stigma length Style length Organoleptic characteristics
Very select 30 mm 23–24 mm Hard brilliant color, strong aroma
Select 30 mm 23 mm Brilliant dark red color, thick thread
Superior 28 mm 22 mm Dark red color, whole strong threads
Medium 25 mm 21 mm Good odor, color and appearance
Ordinary 20–24 mm 20–24 mm Pleasant odor
Slack < 20 mm 23–24 mm Broken stigma, dark color
Unfortunately, the quality grades for saffron differ from country to country. With this in mind, one good way to assess product purity is through microscopic examination to identify several characteristic traits. Presence of the epidermis of the style, a vascular bundle with a rounded section, upper epidermis, and parenchyma, together with the characteristic papilla and smooth grains of pollen, are all easily identifi able, thus facilitating evaluation. In addition, pollen should not contain more than three germinal pores (Fig. 4.3).
Another simple way to detect contamination or fraud is Thin-Layer Chromatography (TLC), which is cheaper and easier to carry out than HPLC. The standard TLC analysis uses silica gel as the stationary phase and different mobile phases, such as butanol/acetic acid/water 4:1:1 or ethyl acetate/isopropanol/
water 65:25:10. In the latter system, crocin and crocetin appear as yellow spots (Rf 0.15–0.25) in daylight, but show fl uorescence-quenching in UV254 (as does picrocrocine: Rf 0.55), becoming dark violet-blue when anisaldehyde-sulfuric acid is used as a reagent. The lack of these characteristic spots helps detect different contaminants or adulterants, such as curcuma, saffl ower, or calendula. The use of high-performance thin-layer chromatography (HPTLC) has also been proposed, with silica gel F254 plates and chloroform/
methanol/acetic acid 10:1:0.13 as the mobile phase.
Yet another simple method to evaluate product quality is the spectrophotometric assay, taking into account the characteristics outlined in the ISO/TS 3632 standard. The aqueous extract of saffron presents three characteristic absorption maximums at 443, 330, and 257 nm, corresponding to crocine and its derivatives, safranal and picrocrocine, respectively. When the spice has been adulterated with foreign substances, alterations appear in the spectrum. For example, the minimum extinction values for water extracts at 440 nm (corresponding to the maximum absorbance of crocines) give the values of 190, 150, 100, and 80 for categories I, II, III and IV, respectively (see Table 4.4).
Analysis with HPLC is the best method for detecting all kinds of contaminants and adulterants.
Different systems can be used for a good separation. For example, in reverse phase HPLC, the suggested
mobile phases are either methanol/water (60:40) at a fl ow rate of 1 mL/minute and detection at 440 nm, or a gradient with methanol 5 until 95% (lineal 3.3% per minute) and detection at 254 nm for picrocrocin and 438 nm for crocin. Another alternative is the use of acetonitrile instead of methanol, albeit with the same gradient (over 30 minutes, 1.0 mL/minute) or water/acetonitrile gradient from water 10%, linear gradient to 40% (45 minutes), isocratic (5 minutes), and back to initial conditions in the last 12 minutes (Lechtenberg et al. 2008). Some more recent chromatographic techniques have been used to analyze saffron, including a non-destructive method based on a supercritical carbon dioxide extraction combined with HPLC and Gas Chromatography (GC) for determining the safranal content (Lozano et al. 2000);
an LC-ESI-MS system for the determination of crocetin esters, picrocrocin and its related compounds (Carmona et al. 2006); and a micellar electrokinetic chromatographic (MEKC) method for quantifi cation of the main metabolites (Gonda et al. 2012).
Pharmacological properties of saffron and its components
The medicinal uses and pharmacological properties of saffron have been extensively studied and reviewed.
For example, Adbulla ev (2002) and Fernández (2006) reviewed the anticancer and chemopreventive properties of saffron, while Ríos et al. (1996), Schmidt et al. (2007), Srivastava et al. (2010), Bathaie and Mousavi (2010), Mousavi and Bathaie (2011), and Hosseinzadeh and Nassiri-Asl (2013) published all the known information on the pharmacological properties of this spice and its components. In addition, Ulbricht et al. (2011) carried out an evidence-based systematic review, which included written and statistical analysis of scientifi c literature, expert opinion, and pharmacological and toxicological data.
Still, other researchers have studied the different activities of compounds isolated from saffron.
Thus, Mehdizadeh et al. (2013) looked into the cardioprotective effects of safranal while Rezaee and Figure 4.3. Typical microscopic elements from saffron. (A) Pollen grain and rest of parenchyma and epidermis ×100;
(B) Pollen grain ×400; (C) Papilla ×100; (D) Papilla ×400.