**2. The species** *Cynara cardunculus* **L.**

#### **2.1. Origin and population structure**

**1. Introduction**

74 Technological Approaches for Novel Applications in Dairy Processing

Greece and Portugal [13–17].

occurs with the autumn rains, and the cycle starts over [16].

and the seed yield was 603 kg/ha after the second growing season [16].

is on average 11% palmitic, 4% stearic, 25% oleic and 60% linoleic fatty acids.

*Cynara cardunculus* L. is a herbaceous perennial diploid plant. It is the most widespread species of the genus *Cynara* and belongs to the Asteraceae family. Recent revisions about *Cynara* genus classification have sparked discussion over whether wild cardoon (*C. cardunculus* var. *sylvestris*), cultivated cardoon (*C. cardunculus* var. *altilis*) and globe artichoke (*C. cardunculus* var. *scolymus* L.) should be classified as different species, or as subspecies [1]. Studies about morphology and phytogeography of the *Cynara* genus supports that the referred plants belongs to a single species and should consequently be classified as subspecies [2–8]. Regardless the subspecies, cardoon is part of Mediterranean flora, distributed throughout the Mediterranean basin, Macaronesia (Madeira and Canary Islands), North Africa, Cyprus and Turkey [2, 9–11], is also a colonizer in Mexico, California, Argentina, Chile, Peru, Australia, China and West Africa [11–13]. Regarding artichoke, its production is worldwide disseminated, with great economic impact, especially in Italy, Spain, France and Turkey [12]. The production of cultivated cardoon seems to be more restricted to South Europe, namely in Spain, Italy, France,

Wild cardoon grows spontaneously in marginal areas of field crops, pastures and along paths in dry areas and in soils of various characteristics. The plant, either cultivated or wild, can persist for a number of years, over 10 years, re-sprouting annually from its large perennial taproot [18, 19]. New seedlings usually germinate after the autumn rains, then first cotyledons emerge, growing slowly through in a rosette arrangement. Cardoon plants hold in the rosette stage during winter and early spring, when stalks start to elongate. As the flower stems develop, the lower rosette leaves begin to die off. Plants usually flower in the early summer, followed by the dieback of their aerial growth. Seedlings do not generally flower in their first year, as their energy is absorbed on the development of its deep taproot. New growth

Both plant agronomic characteristics and human selection of certain phenotypes, over the years, can possibly explain the use specificity, of each subspecies, for different purposes. *C. cardunculus* L. species lignocellulosic fraction shows great potential as solid biofuel [14], as well as for biogas production [20, 21] and bioethanol [22, 23]. *C. cardunculus* L. biomass productions can reach 14–20 t DW/ha per year, while according the studies of Pesce et al. [24], the wild cardoon accession was less productive in terms of biomass accumulation (11.8 t DW/ ha per year) compared to cultivated forms (*Altilis* 41′ and 'Bianco avorio') producing, respectively, 19.1 t and 16.8 t DW/ha per year. Stalks and capitula of cultivated cardoon may also be used to produce pulp fibers [14, 25, 26]. In Southern Portugal, a region characterized by very hot and dry summers, a large scale cultivation of the cardoon *C. cardunculus* L. for biomass production was installed in a total of 77.4 ha. The field biomass yield was estimated at 7.5 t/ha,

*C. cardunculus* seed's oil fatty acid composition has also revealed a great potential for biodiesel production [14, 27]. *C. cardunculus* seed yield, and see oil composition is quite similar to sunflower oil. *Cynara* crop seed yield has been estimated at 1.36 t/ha per year [28], and a maximum seed oil content of 32.47% has been described by Curt et al. [29], while oil composition The taxonomy on the gender *Cynara* has evolved over time, and it seems to exist some confusion about the type of plants that fall under the designation of *C. cardunculus* L. species; even for the use in cheese manufacture, the descriptions of plants have been a little variable. In fact, the definition of the species that comprises the genus *Cynara* is somewhat complex, because some of them take different names, appearing eventually placed in other genera, with which there are affinities. For example, the number of species considered in the genus review of Wiklund [2] is eight, whereas the previous treatment of the same genus, in 1838, considered only three.

Illustrating the designations complexity or classification, Bailey and Bailey [46] refer to 10 Mediterranean species, 7 of which are referred to by Tutin et al. [47] as European: *C. scolymus*, *C. tournefortii*, *C. humilis*, *C. cardunculus*, *C. cornigera*, *C. alba* and *C. algarbiensis*. However, the species *C. humilis* is often identified as belonging to the genus *Bourgea* (*Bourgea humilis* L.) [2, 48] and *C. alba* corresponds, according to Valdes et al. [48], to the species *C. baetica*, also referred as belonging to the genus *Cirsium*. From this, one species, *Cirsium vulgare*, was used by Wiklund [2] as a test for its study of the genus *Cynara*. *C. scolymus* is referred to as spontaneously unknown by Tutin et al. [47] and Franco [49]. Moreover, Foury [3] distinguish only three species: (i) *C. cardunculus*, whose distribution coincides with that found by Wiklund [2] for *C. cardunculus* spp. *flavescens* (the NW Mediterranean region, whereas *C. cardunculus* spp. *Cardunculus* generally occurs in regions with coastal influence, Macaronesia, Portugal, Central and NE Mediterranean distribution); (ii) *C. sibthorpiana* (Greece, Cyprus and Crete), designated by Tutin et al. [47] by *C. cornigera* and (iii) *C. syriaca*, in Palestine. Valdés et al. [48] distinguish *C. cardunculus* L., *C. humilis* L., *C. algarbiensis* and *C. baetica* in Andaluzia. Franco [49], in Portugal flora, refers to *C. scolymus*, *C. cardunculus*, *C. humilis*, *C. algarbiensis* and *C. tournefortii*, the last one being excluded of *Cynara* genus by Wiklund [2].

The agronomic plant characteristics, combined with human selection over the years, possibly explain the specificity of the use of each subspecies for each different purpose. Different parts of the plant, such as leaves and inflorescences, with high relevant biochemical profiles, are

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77

Throughout history, cardoon use had curious applications including torment weapon, confectionery, medicine, besides its role as a coagulant in cheesemaking. According to Barreira [57], the reference of cardoon in the Bible is associated with "torment" or "suffering", as, for example, (i) "*But Jehoash king of Israel replied to Amaziah king of Judah: A thistle in Lebanon sent a message to a cedar in Lebanon, 'Give your daughter to my son in marriage.' Then a wild beast in Lebanon came along and trampled the thistle underfoot"* (2 Chronicles 25:18); (ii) "*It will produce thorns and thistles for you, and you will eat the plants of the field"* (Genesis 3:18); (iii) "*But land that produces thorns and thistles is worthless and is in danger of being cursed. In the end it will be burned"* (Hebrews 6:8). Columella [58], in the treatise "*De Re rustica"* (1st century BC), mentioned some interesting references about the use of cardoon as a milk coagulant, for example, "*It will be necessary too not to neglect the task of cheese-making, especially in distant parts of the country, where it is not convenient to take milk to the market in pails. Further, if the cheese is made of a thin consistency, it must be sold as quickly as possible while it is still fresh and retains its moisture; if, however, it is of a rich and thick consistency, it bears being kept for a longer period. Cheese should be made of pure milk which is as fresh as possible, for if it is left to stand or mixed with water, it quickly turns sour. It should usually be curdled with rennet obtained from a lamb or a kid, though it can also be coagulated with the flower of the wild thistle or the seeds of the safflower, and equally well with the liquid which flows from a fig-tree* 

The only treatise on the ancient gastronomy that is known nowadays is "*Artis magiricoe libri X*", also known as "*De Re coquinaria*" written by Apicio in the first century AD [4]. In this treatise some recipes using cardoon can be found in Chapter XIX of Book III [5], as, *for example,* "*CARDUI ("cardoons"): thistles are eaten with a salty dressing, olive oil, and hard-boiled eggs"* (adapted)*; "ALITER CARDUI ("other cardoons"): rind, mint, coriander, finely chopped fennel, pepper, levis and salt water and olive oil"* (adapted)*; "ALITER CARDUOS ELIXOS ("other cooked cardoons"): served with pepper, cumin, sauce and olive oil"* (adapted). In addition, in Spanish manuscripts of fifteenth century [59], it is possible to find a recipe of candied *C. cardunculus* describing that the ideal months for *C. cardunculus* picking are April/May, when the stalks are more tender and sweet. The process of confection begins with stalk cutting, then rinsed in cold clear water for 1 or 2 days, followed by cooking in clear water and ending in a process of soaking in a syrup of sugar and water. Later, in the seventeenth century, the use of *C. cardunculus* is also mentioned in the elaboration of dishes along with butter, onion, pepper, nutmeg, ginger, eggs, among other ingredients, like milk clot (e.g., "*Almojavanas de quajada*"). The first cookbook printed in Portugal, known as *"Art of Cooking divided in two parts"* (from the original *"Arte de Cozinha dividida em duas partes"*) brought up some references of recipes of the banquets to be served during April, with dishes using cardoon such as "*Cardoon curdled with eggs*", "*Bundle of cardoon garnished with the same cardoon*" and "*Italian style cardoon garnished with cream*". Also in

used as food providing the selection for larger, tender and non-spiny plants [12, 56].

**2.2. Historical and etymological archives**

*if you make an incision in the bark while it is still green*.".

The recent revisions on *Cynara* genus classification have sparked discussion over whether the wild cardoon (*C. cardunculus* var. *sylvestris*), cultivated cardoon (*C. cardunculus* var. *altilis*) and globe artichoke (*C. cardunculus* var. *scolymus* L.) should be classified as different species, or as subspecies [50]. According to the results of Gatto et al. [12], the origin of these forms may have new scenarios: (i) the globe artichoke was domesticated a long time ago from wild material in Sicily/ northern Africa; (ii) the leafy cardoon was derived from western Mediterranean (Portugal, Spain) wild cardoon and (iii) the eastern wild cardoon from Italy, Greece, Tunisia and Malta represents the only original wild form, that gave origin to both globe artichoke and cultivated cardoon. Furthermore, the leafy cardoon might have returned to wild forms, giving rise to the so-called western wild cardoon (Spain, Portugal). So, plant types considered before as different species or varieties, as *C. scolymus* L., cultivated for artichoke, and *C. cardunculus* L. var. *altilis* DC, known for its juicy young leaves [4], or *C. cardunculus* L. subsp. *cardunculus* and *C. cardunculus* L. subsp. *flavescens* [2], with differences on size, leaves and flowers of plants, and on spinyness of bracts, are considered now as coming from the perennial wild, *C. cardunculus* L. var. *sylvestris* (Lam.).

All these different forms of plants belong actually to a single species, *C. cardunculus* L. [4]. Studies about morphology and phytogeography of the *Cynara* genus support that the referred plants belong to a single species and should consequently be classified as subspecies [2–8]. The variability of characteristics within the species is a conclusion drawn by different authors [2, 3] and could be observed before in Portugal by Morbey [51], who collected plants of *C. cardunculus* prior to settle of experimental fields. However, despite the richness of wild cardoon germplasm, the identification and characterization of its genetic resources are scarcely investigated. In Portugal, *C. cardunculus* L. has an increasingly limited distribution, becoming restricted to certain areas where it has benefited of some care, even being sometimes cultivated [52].

Molecular data provided evidence that the western wild cardoon, the *C. cardunculus* L. subsp. *flavescens*, distributed in Spain and Portugal and characterized by more robust plants, is genetically closer related to cultivated cardoon, while the eastern wild cardoon, the *C. cardunculus L.* subsp. *cardunculus*, might be the progenitor of the globe artichoke [1, 7, 50], confirming the conclusions of recent revisions of *Cynara* genus. The two crops have possibly been derived from human pressure selection for either large non-spiny heads on one side, or non-spiny large stalked tender leaves on the other side [1, 5]. In the last decades, several molecular markers (RAPD, AFLP, SSR and ISSR) have been used for *C. cardunculus* population characterization. Among genetic markers available, the simple sequence repeats (SSR) are highly informative since they are codominant and generally highly polymorphic [53]. Recent studies have been conducted in Tunisia by Ben Ammar et al. [54] and Khaldi et al. [55], which revealed a large variability among wild cardoon populations.

The agronomic plant characteristics, combined with human selection over the years, possibly explain the specificity of the use of each subspecies for each different purpose. Different parts of the plant, such as leaves and inflorescences, with high relevant biochemical profiles, are used as food providing the selection for larger, tender and non-spiny plants [12, 56].

#### **2.2. Historical and etymological archives**

for *C. cardunculus* spp. *flavescens* (the NW Mediterranean region, whereas *C. cardunculus* spp. *Cardunculus* generally occurs in regions with coastal influence, Macaronesia, Portugal, Central and NE Mediterranean distribution); (ii) *C. sibthorpiana* (Greece, Cyprus and Crete), designated by Tutin et al. [47] by *C. cornigera* and (iii) *C. syriaca*, in Palestine. Valdés et al. [48] distinguish *C. cardunculus* L., *C. humilis* L., *C. algarbiensis* and *C. baetica* in Andaluzia. Franco [49], in Portugal flora, refers to *C. scolymus*, *C. cardunculus*, *C. humilis*, *C. algarbiensis* and *C. tournefortii*,

The recent revisions on *Cynara* genus classification have sparked discussion over whether the wild cardoon (*C. cardunculus* var. *sylvestris*), cultivated cardoon (*C. cardunculus* var. *altilis*) and globe artichoke (*C. cardunculus* var. *scolymus* L.) should be classified as different species, or as subspecies [50]. According to the results of Gatto et al. [12], the origin of these forms may have new scenarios: (i) the globe artichoke was domesticated a long time ago from wild material in Sicily/ northern Africa; (ii) the leafy cardoon was derived from western Mediterranean (Portugal, Spain) wild cardoon and (iii) the eastern wild cardoon from Italy, Greece, Tunisia and Malta represents the only original wild form, that gave origin to both globe artichoke and cultivated cardoon. Furthermore, the leafy cardoon might have returned to wild forms, giving rise to the so-called western wild cardoon (Spain, Portugal). So, plant types considered before as different species or varieties, as *C. scolymus* L., cultivated for artichoke, and *C. cardunculus* L. var. *altilis* DC, known for its juicy young leaves [4], or *C. cardunculus* L. subsp. *cardunculus* and *C. cardunculus* L. subsp. *flavescens* [2], with differences on size, leaves and flowers of plants, and on spinyness of bracts, are considered now as coming from the perennial wild, *C. cardunculus* L. var. *sylvestris* (Lam.).

All these different forms of plants belong actually to a single species, *C. cardunculus* L. [4]. Studies about morphology and phytogeography of the *Cynara* genus support that the referred plants belong to a single species and should consequently be classified as subspecies [2–8]. The variability of characteristics within the species is a conclusion drawn by different authors [2, 3] and could be observed before in Portugal by Morbey [51], who collected plants of *C. cardunculus* prior to settle of experimental fields. However, despite the richness of wild cardoon germplasm, the identification and characterization of its genetic resources are scarcely investigated. In Portugal, *C. cardunculus* L. has an increasingly limited distribution, becoming restricted to certain areas where it has benefited of some care, even being sometimes cultivated [52].

Molecular data provided evidence that the western wild cardoon, the *C. cardunculus* L. subsp. *flavescens*, distributed in Spain and Portugal and characterized by more robust plants, is genetically closer related to cultivated cardoon, while the eastern wild cardoon, the *C. cardunculus L.* subsp. *cardunculus*, might be the progenitor of the globe artichoke [1, 7, 50], confirming the conclusions of recent revisions of *Cynara* genus. The two crops have possibly been derived from human pressure selection for either large non-spiny heads on one side, or non-spiny large stalked tender leaves on the other side [1, 5]. In the last decades, several molecular markers (RAPD, AFLP, SSR and ISSR) have been used for *C. cardunculus* population characterization. Among genetic markers available, the simple sequence repeats (SSR) are highly informative since they are codominant and generally highly polymorphic [53]. Recent studies have been conducted in Tunisia by Ben Ammar et al. [54] and Khaldi et al. [55],

which revealed a large variability among wild cardoon populations.

the last one being excluded of *Cynara* genus by Wiklund [2].

76 Technological Approaches for Novel Applications in Dairy Processing

Throughout history, cardoon use had curious applications including torment weapon, confectionery, medicine, besides its role as a coagulant in cheesemaking. According to Barreira [57], the reference of cardoon in the Bible is associated with "torment" or "suffering", as, for example, (i) "*But Jehoash king of Israel replied to Amaziah king of Judah: A thistle in Lebanon sent a message to a cedar in Lebanon, 'Give your daughter to my son in marriage.' Then a wild beast in Lebanon came along and trampled the thistle underfoot"* (2 Chronicles 25:18); (ii) "*It will produce thorns and thistles for you, and you will eat the plants of the field"* (Genesis 3:18); (iii) "*But land that produces thorns and thistles is worthless and is in danger of being cursed. In the end it will be burned"* (Hebrews 6:8).

Columella [58], in the treatise "*De Re rustica"* (1st century BC), mentioned some interesting references about the use of cardoon as a milk coagulant, for example, "*It will be necessary too not to neglect the task of cheese-making, especially in distant parts of the country, where it is not convenient to take milk to the market in pails. Further, if the cheese is made of a thin consistency, it must be sold as quickly as possible while it is still fresh and retains its moisture; if, however, it is of a rich and thick consistency, it bears being kept for a longer period. Cheese should be made of pure milk which is as fresh as possible, for if it is left to stand or mixed with water, it quickly turns sour. It should usually be curdled with rennet obtained from a lamb or a kid, though it can also be coagulated with the flower of the wild thistle or the seeds of the safflower, and equally well with the liquid which flows from a fig-tree if you make an incision in the bark while it is still green*.".

The only treatise on the ancient gastronomy that is known nowadays is "*Artis magiricoe libri X*", also known as "*De Re coquinaria*" written by Apicio in the first century AD [4]. In this treatise some recipes using cardoon can be found in Chapter XIX of Book III [5], as, *for example,* "*CARDUI ("cardoons"): thistles are eaten with a salty dressing, olive oil, and hard-boiled eggs"* (adapted)*; "ALITER CARDUI ("other cardoons"): rind, mint, coriander, finely chopped fennel, pepper, levis and salt water and olive oil"* (adapted)*; "ALITER CARDUOS ELIXOS ("other cooked cardoons"): served with pepper, cumin, sauce and olive oil"* (adapted). In addition, in Spanish manuscripts of fifteenth century [59], it is possible to find a recipe of candied *C. cardunculus* describing that the ideal months for *C. cardunculus* picking are April/May, when the stalks are more tender and sweet. The process of confection begins with stalk cutting, then rinsed in cold clear water for 1 or 2 days, followed by cooking in clear water and ending in a process of soaking in a syrup of sugar and water. Later, in the seventeenth century, the use of *C. cardunculus* is also mentioned in the elaboration of dishes along with butter, onion, pepper, nutmeg, ginger, eggs, among other ingredients, like milk clot (e.g., "*Almojavanas de quajada*"). The first cookbook printed in Portugal, known as *"Art of Cooking divided in two parts"* (from the original *"Arte de Cozinha dividida em duas partes"*) brought up some references of recipes of the banquets to be served during April, with dishes using cardoon such as "*Cardoon curdled with eggs*", "*Bundle of cardoon garnished with the same cardoon*" and "*Italian style cardoon garnished with cream*". Also in medicine, several references on the use of cardoon can be found in treatises, such as "*Pedacio Dioscorides anazarbeo, Acerca de la materia medicinal y de los venenos mortiferos traduzido de lengua griega en la vulgar castellana & illustrado con claras y substantiales annotationes, y con las figuras de innumeras plantas exquisitas y raras*". This work consists of the translation from Greek to Castilian of the treatise *De Materia Medica*, by Pedanius Dioscorides (first century BC) with the inclusion of personal annotations. In Chapter XIII is included the description of the benefits of cardoon on the stomach, liver, bladder and kidneys, but also in the prevention of the bad odors from the human body.

be followed. The seed germination occurs when the soils water content and the environmental temperature are favorable, normally in the autumn or spring of Mediterranean climates. After this initial growth cycle, the aerial biomass production is usually lower due to the larger development of the root system, but can increase in the next growth cycles, depending on the environmental conditions. In the case of a cold autumn (early frosts), spring sowing may constitute a better option. It is advisable to accomplish the spring sowing as soon as the period of frosts is over. Usually, plants will reach summertime in the rosette stage; after, and due to the high temperatures, some leaves dry up. Later, when the environmental conditions are milder, *Cynara* spp. resumes its vegetative growth, and the size of the leaf rosette becomes bigger.

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After, the growing process of the plant can be considered finished in the next summer and *Cynara* spp. can be harvested [14, 26]. Harvest process should be performed after the conclusion of the plants growing cycle,but before seed dispersal. The aerial biomass of *Cynara* spp. should be dry (less than 15% moisture) and seeds must be ripe. The research and development of proper machinery for harvesting *Cynara* spp. plantations is under process aiming for

*C. cardunculus* adult plant can reach a height of 3 m, spread over an area of 1.5 m in diameter [16], and can contain 15 inflorescences, 7 in average [16]. These inflorescences do not open all

The flower harvest is performed through a cut, and should be done when the inflorescences are mature and open. To obtain high-quality flower with a minimum of impurities, like straw, the pistils should be collected as high as possible on the plant. It is empirically and generally accepted that the more blue-purple the collected material is, the more value it has for traditional cheesemaking [16], concerning at least the milk clotting activity. The harvest of the flower is usually done between the end of June and the beginning of July [51, 63], depending on the year and on the varieties; it is usually performed manually, with a bucket with two hooks where the inflorescence fits. The scissors used by the pickers are long and very sharp, being able to collect all of the flower at once. There are some recent developments regarding mechanical harvesting attempts, but so far, there is no specific device commercially available. The traditional preservation process for cardoon flower is at room temperature (25–30°C) with air dehydration for about 30–60 days [67]. The purple parts of the cardoon flower (styles and stigmas) are collected along the flowering season and placed to dry at room temperature, protected from sunlight, and with regular turnings of the material to prevent unwanted fermentations and fungi growth [51, 64]. The drying process can decrease the coagulant activity [65]. In spite the fact that average flower milk clotting activity (MCA)/g of dried, and non-dried flower was similar, the authors refer to losses of milk clotting activity varying from 20 to 50% when expressed on dry basis or nitrogen (N) total basis. The traditional drying process, used to preserve the flower throughout the cheese production season, tends to standardize the flower composition and coagulant activity and although carried out at low temperature leads to high losses of flower enzymatic activity as measured by coagulant activity. The evaluation of the amount of these losses, together with the decline in clotting activity during conservation,

harvesting the whole biomass in one operation.

**2.4. Harvest and conservation of** *Cynara cardunculus* **L. flower**

at the same time because they have different maturation states.

As in a previous treatise [58], several ways of preparing and consuming cardoon are reported, especially using young and tender plants, like the preparation using wine, salt and pepper. Andres Laguna [60] differentiates the "cardoon" from the "artichoke" referring to the latter on as "lush" for which reason should be placed in the list of foods recommended to the bride and groom, however can be used as milk coagulant. A manuscript about the cheesemaking process in the Alentejo region, reports different ways of preparing *C. cardunculus* aqueous extracts [61]. The author also points out that the consumption of cardoon can lead to food craving, and mentions a method for growing cardoon: "*The truth is that planting them* [the thistles]*, covering all them with earth, makes them more white, more tender and tastier, like they were from a different species*" [60]. This description is in line where Priest Isidoro de Barreira [57], who, referring to the biblical meaning of the cardoon, describes: "(…) *that* [torment] *which he suffers* [thistle] *before being suitable for eating*: (…) *when they bind him, and cover him with earth, in which will be mortified to lose its bitterness*." Cardoon crops are present in paintings from the beginning of the seventeenth century by Caravaggio and Juan Sanchez Cótan [50]. Later during the seventeenth century, de Cabreira [62] included the use of "holy thistle" in the treatment of sores in his compendium of medicines for surgery.

#### **2.3. The natural growth cycle of** *Cynara cardunculus* **L.**

*C. cardunculus*, as other Mediterranean species, is a plant fully adapted to the local climate conditions, where raining is low, irregular and mainly concentrated in autumn/winter. On the other hand, the hot dry summers are not favorable for plant growth, especially if no irrigation is used. The first stage of *C. cardunculus* growing cycle initiates after seeds germinate, usually in the first weeks of autumn. In this stage, two fresh cotyledons arise from the ground, followed by several leaves and, later, a leaf rosette. This rosette usually grows in a slow but steady manner and, the *Cynara* spp. may take all wintertime to early spring at this rosette stage. By late spring, *Cynara* spp. develops a leaf-branched floral scape including several heads. After full blossom and flower fertilization, the fruits ripen and finally the aerial biomass dries up in the summer. When the weather conditions become milder the perennating buds on the basal plant part sprout and a new development cycle starts. This succession of annual growth cycles may last several years [14].

The cultivation as an industrial crop for industrial application of *Cynara* spp. resembles most of the stages of the natural growth reported before, that is, as a perennial field crop in dry farming. As a perennial crop, and with a very deep plant root system, a basal dressing before sowing is recommended. After subsoiling and plowing are recommended, a thorough harrowing should be followed. The seed germination occurs when the soils water content and the environmental temperature are favorable, normally in the autumn or spring of Mediterranean climates. After this initial growth cycle, the aerial biomass production is usually lower due to the larger development of the root system, but can increase in the next growth cycles, depending on the environmental conditions. In the case of a cold autumn (early frosts), spring sowing may constitute a better option. It is advisable to accomplish the spring sowing as soon as the period of frosts is over. Usually, plants will reach summertime in the rosette stage; after, and due to the high temperatures, some leaves dry up. Later, when the environmental conditions are milder, *Cynara* spp. resumes its vegetative growth, and the size of the leaf rosette becomes bigger.

After, the growing process of the plant can be considered finished in the next summer and *Cynara* spp. can be harvested [14, 26]. Harvest process should be performed after the conclusion of the plants growing cycle,but before seed dispersal. The aerial biomass of *Cynara* spp. should be dry (less than 15% moisture) and seeds must be ripe. The research and development of proper machinery for harvesting *Cynara* spp. plantations is under process aiming for harvesting the whole biomass in one operation.

#### **2.4. Harvest and conservation of** *Cynara cardunculus* **L. flower**

medicine, several references on the use of cardoon can be found in treatises, such as "*Pedacio Dioscorides anazarbeo, Acerca de la materia medicinal y de los venenos mortiferos traduzido de lengua griega en la vulgar castellana & illustrado con claras y substantiales annotationes, y con las figuras de innumeras plantas exquisitas y raras*". This work consists of the translation from Greek to Castilian of the treatise *De Materia Medica*, by Pedanius Dioscorides (first century BC) with the inclusion of personal annotations. In Chapter XIII is included the description of the benefits of cardoon on the stomach, liver, bladder and kidneys, but also in the prevention of the bad

As in a previous treatise [58], several ways of preparing and consuming cardoon are reported, especially using young and tender plants, like the preparation using wine, salt and pepper. Andres Laguna [60] differentiates the "cardoon" from the "artichoke" referring to the latter on as "lush" for which reason should be placed in the list of foods recommended to the bride and groom, however can be used as milk coagulant. A manuscript about the cheesemaking process in the Alentejo region, reports different ways of preparing *C. cardunculus* aqueous extracts [61]. The author also points out that the consumption of cardoon can lead to food craving, and mentions a method for growing cardoon: "*The truth is that planting them* [the thistles]*, covering all them with earth, makes them more white, more tender and tastier, like they were from a different species*" [60]. This description is in line where Priest Isidoro de Barreira [57], who, referring to the biblical meaning of the cardoon, describes: "(…) *that* [torment] *which he suffers* [thistle] *before being suitable for eating*: (…) *when they bind him, and cover him with earth, in which will be mortified to lose its bitterness*." Cardoon crops are present in paintings from the beginning of the seventeenth century by Caravaggio and Juan Sanchez Cótan [50]. Later during the seventeenth century, de Cabreira [62] included the use of "holy thistle" in the treat-

*C. cardunculus*, as other Mediterranean species, is a plant fully adapted to the local climate conditions, where raining is low, irregular and mainly concentrated in autumn/winter. On the other hand, the hot dry summers are not favorable for plant growth, especially if no irrigation is used. The first stage of *C. cardunculus* growing cycle initiates after seeds germinate, usually in the first weeks of autumn. In this stage, two fresh cotyledons arise from the ground, followed by several leaves and, later, a leaf rosette. This rosette usually grows in a slow but steady manner and, the *Cynara* spp. may take all wintertime to early spring at this rosette stage. By late spring, *Cynara* spp. develops a leaf-branched floral scape including several heads. After full blossom and flower fertilization, the fruits ripen and finally the aerial biomass dries up in the summer. When the weather conditions become milder the perennating buds on the basal plant part sprout and a new development cycle starts. This succession of

The cultivation as an industrial crop for industrial application of *Cynara* spp. resembles most of the stages of the natural growth reported before, that is, as a perennial field crop in dry farming. As a perennial crop, and with a very deep plant root system, a basal dressing before sowing is recommended. After subsoiling and plowing are recommended, a thorough harrowing should

odors from the human body.

78 Technological Approaches for Novel Applications in Dairy Processing

ment of sores in his compendium of medicines for surgery.

**2.3. The natural growth cycle of** *Cynara cardunculus* **L.**

annual growth cycles may last several years [14].

*C. cardunculus* adult plant can reach a height of 3 m, spread over an area of 1.5 m in diameter [16], and can contain 15 inflorescences, 7 in average [16]. These inflorescences do not open all at the same time because they have different maturation states.

The flower harvest is performed through a cut, and should be done when the inflorescences are mature and open. To obtain high-quality flower with a minimum of impurities, like straw, the pistils should be collected as high as possible on the plant. It is empirically and generally accepted that the more blue-purple the collected material is, the more value it has for traditional cheesemaking [16], concerning at least the milk clotting activity. The harvest of the flower is usually done between the end of June and the beginning of July [51, 63], depending on the year and on the varieties; it is usually performed manually, with a bucket with two hooks where the inflorescence fits. The scissors used by the pickers are long and very sharp, being able to collect all of the flower at once. There are some recent developments regarding mechanical harvesting attempts, but so far, there is no specific device commercially available.

The traditional preservation process for cardoon flower is at room temperature (25–30°C) with air dehydration for about 30–60 days [67]. The purple parts of the cardoon flower (styles and stigmas) are collected along the flowering season and placed to dry at room temperature, protected from sunlight, and with regular turnings of the material to prevent unwanted fermentations and fungi growth [51, 64]. The drying process can decrease the coagulant activity [65]. In spite the fact that average flower milk clotting activity (MCA)/g of dried, and non-dried flower was similar, the authors refer to losses of milk clotting activity varying from 20 to 50% when expressed on dry basis or nitrogen (N) total basis. The traditional drying process, used to preserve the flower throughout the cheese production season, tends to standardize the flower composition and coagulant activity and although carried out at low temperature leads to high losses of flower enzymatic activity as measured by coagulant activity. The evaluation of the amount of these losses, together with the decline in clotting activity during conservation, was estimated to be about 75% of the potential enzymatic coagulant available in the flower expressed per unit of dry matter [66]. Reducing exposure time to traditional drying conditions may limit such losses, and it is possible to use dehydration at higher temperatures while shortening the drying time, with MCA losses still lower than those with traditional drying.

of these cheeses benefit from protected designation of origin (PDO) status in which *C. cardunculus* flower aqueous extracts have been successfully employed and legally required (**Table 1**).

*Cynara cardunculus:* Use in Cheesemaking and Pharmaceutical Applications

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With the exception of some standardized formulas recently commercially available, the general preparation of cardoon flower extracts for cheesemaking use, remains generally as described in very old references. Coagulating enzymes are extracted from dry flower (styles and stigmas) on a day-to-day basis [83]. The required amount of cardoon flower is placed in bottled water infusion during a variable period of time, being carefully macerated and ground with water, usually in a mortar. The mixture is then filtered, obtaining a purplish or brown

This traditional use is based on the observation of previous clotting times, and when appropriate, compensation for any loss of enzyme activity coagulant is made by correcting the amount of flower applied per liter of milk as a correction given the empirical use of the coagulant. Control of extracts coagulant activity is performed by controlling the amount of flower necessary to the milk batch volume, frequently equivalent to 0.2–0.6 g flower/L milk [84].

So, the cardoon extract is an aqueous extract of edible parts of flowers from plants, prepared with a variable proportion of bottled water, sometimes with some salt addition during maceration for flower proteinases extraction, therefore usually not standardized. With the maceration in a mortar, with a 5% sodium chloride solution and sand added as an abrasive

**Cheese Country Type of milk Coagulant (reference in law)** Mestiço de Tololosa Portugal Ewe and goat Animal Rennet or *Cynara cardunculus*

cow

cow

**Table 1.** PDO cheeses made with aqueous extract of *Cynara cardunculus* L. in Portugal and Spain [82].

Ewe, goat and cow

*Cynara cardunculus* or *Cynara scolymus*

≤50% animal rennet

rennet

≥50% *Cynara cardunculus* or *Cynara scolymus*

*Cynara cardunculus* or *Cynara scolymus* or animal

de Nisa Portugal Ewe *Cynara cardunculus* de Castelo Branco Portugal Ewe *Cynara cardunculus* de Évora Portugal Ewe *Cynara cardunculus* Serpa Portugal Ewe *Cynara cardunculus* de Azeitão Portugal Ewe *Cynara cardunculus* Serra da Estrela Portugal Ewe *Cynara cardunculus* La Serena Spain Ewe *Cynara cardunculus* Torta del Casar Spain Ewe *Cynara cardunculus*

Flor de Guía Spain Ewe, goat and

Guía Spain Ewe, goat and

Media Flor de Guía Spain/Gran Canaria Island

**3.1.** *Cynara cardunculus* **L. flower aqueous extracts**

liquid which is added to the milk [71, 80].

Martins [66] also studied the effect of cardoon flower drying under different conditions (25–30°C for 7 days, 50°C for 5 days, and 100°C for 5 h). The author concluded that when compared to traditional drying process (25–30°C for 30 days), MCA average was significantly higher drying only for 7 days (MCA in dry matter about 35% higher). MCA of dried flower at 55°C for 5 days in a dry basis, was about 17% higher, while drying at 100°C for 5 h caused a loss for flower MCA of about 5% in a dry basis. The moisture content of the dried flower at 25–30°C for 7 days, about 6% (w/w), was similar to that of traditionally dried flower, showing average water activity (aw) of 0.585. This means that by controlling the moisture content throughout, the drying period can be decreased, and thus the MCA can be significantly preserved if adequate storage conditions are initially respected. Although there are always losses related to flower storage, the dried flower remains well preserved until next harvest period under conditions of reduced relative humidity and at room temperature. After 300 days of dry flower storage at 25°C, Martins [66] found MCA losses of about 35% of the original MCA, near the MCA losses after storage at 4°C for 150 days.
