Impaginato 25 Adv. Hort. Sci., 2023 37(1): 25­32 DOI: 10.36253/ahsc­13857 Phytonutritional and aromatic profiles of Tulbaghia simmleri Beauv. edible flowers during cold storage I. Marchioni 1, B. Najar 1, 2 (*), A. Copetta 3, B. Ferri 2, B. Ruffoni 3, L. Pistelli 2, L. Pistelli 1, 4 1 Dipartimento di Scienze Agrarie, Alimentari e Agro‐ambientali (DISAAA‐a), Università di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy. 2 Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano, 12, 56126 Pisa, Italy. 3 CREA Centro di Ricerca Orticoltura e Florovivaismo, Corso Inglesi, 508, 18038 Sanremo (IM), Italy. 4 Centro Interdipartimentale di Ricerca Nutraceutica e Alimentazione per la Salute (Nutrafood), Università di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy. Key words: Antioxidant activity, enzyme activity, low temperature, postharvest, secondary metabolites, sweet wild garlic, volatile organic com­ pounds. Abstract: Edible flowers are appreciated due to their aesthetic features, nutri­ ti o n a l v a l u e a n d a n ti o x i d a n t p r o p e r ti e s . T u l b a g h i a s i m m l e r i B e a u v . (Amaryllidaceae family) flowers are characterized by a pleasant garlic taste and are consumed both as fresh and dried products. The aim of this work was to assess the effect of chilling temperature (+4°C) on the visual quality, nutritional content, and aroma profile of T. simmleri flowers after two (T2) and six (T6) days of storage. Colorimetric analysis highlighted a reduction in petal bright­ ness at T6 and hence their darkening, due to a significant increase in a* coordi­ nate and the decrease in the b* one. Total polyphenols and flavonoids content remained unchanged until the end of the experiment, while total anthocyanins increased at T2. Flowers antioxidant activity (DPPH assay) decreased progres­ sively during cold storage, while catalase (CAT) and ascorbate peroxidase (APX) activities increased. The aroma profile was analyzed by HS­SPME associated with GC­MS, underlining that fresh flowers were dominated by high content in monoterpenes (around 80%), with 1,8­cineol as main compound (53.1%). Cold storage reduced this class of volatiles while sesquiterpenes and non­terpenes increased; between them, benzyl benzoate reached 12%. 1. Introduction Edible flowers (EFs) are traditionally consumed since ancient times (Mlcek and Rop, 2011). Some of them are commonly recognised as veg­ etables (e.g. artichokes, broccoli, capers), while others are still considered (*) Corresponding author: basmanajar@hotmail.fr Citation: MARCHIONI I., NAJAR B., COPETTA A., FERRI B., RUFFONI B., PISTELLI L., PISTELLI L., 2023 ­ Phytonutritional and aromatic profiles of Tulbaghia simmleri Beauv. edible flowers during cold storage. ­ Adv. Hort. Sci., 37(1): 25­32. Copyright: © 2023 Marchioni I., Najar B., Copetta A., Ferri B., Ruffoni B., Pistelli L., Pistelli L. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 14 October 2022 Accepted for publication 13 December 2022 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-13857 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(1): 25­32 26 “unusual food” (reviewed in Pires et al., 2019). EFs straights rely on their colours, shapes, flavours, tastes, and nutrients (e.g. carbohydrates, proteins, vitamins, phytochemical compounds with antioxidant and healthy properties) (Fernades et al., 2017). Their market is constantly expanding, and new species with attractive sensorial features and good storage attitude are required. Tulbaghia (common name: wild garlic) is a genus of monocotyledonous plants (Amaryllidaceae family) indigenous to South Africa (Lyantagaye, 2011). Herbaceous perennial bulbs, corms or rhizomes char­ acterize its species. Tulbaghia spp. flowers, held in umbels in groups of ten or more, are strongly fra­ grant and characterised by tubular shape (Zschocke and Van Staden, 2000). A raised crown­like structure or a fleshy ring at the centre of the flower tube are distinctive features of this genus (Vosa, 2000). The colours are different, mainly white, pink or mauve. Flowers and rhizomes produce cysteine­derived sul­ phur compounds (e.g. marasmicin), which confer to this organs a pleasant alliaceous smell, especially when bruised or during senescence (Aremu and Van Staden 2013; Kubec et al., 2013). The peculiar aroma and the pungent garlicky taste of flowers make sever­ al Tulbaghia spp. interesting for the food industry (Kubec et al., 2013). T. simmleri Beauv. is mainly known as ornamental plant, which flowers consist of six tepals and a cen­ tral crown of six lobes, fused for more than a third of their length to form a tube. The lobes have pointed tips, giving the crown a fringed edge (Vosa, 2000). In the southern hemisphere, its period of blooming ranges between April to October, even though, with particular climate conditions, it could be extended until early spring (Zschocke and Van Staden, 2000). In the northern hemisphere, however, its period of blooming ranges between October to April. Several bioactive compounds characterize this plant, since it is used to treat fever, colds, headaches, asthma, and tuberculosis in South African traditional medicine (Zschocke and Van Staden, 2000). T. simmleri has been severely neglected when compared to the most common T. violacea, for which several culinary uses are known, also concerning flowers (Aremu and Van Staden, 2013; Rivas­García et al., 2022). Further investigation on T. simmleri worth to be performed, since this species produce deep mauve, long lasting edible flowers, which period of bloom does not over­ lap the one of T. violacea (not available in autumn and winter). This will ensure the availability of EFs with garlic taste for most of the year. Moreover, Takaidza et al. (2018) highlighted good total polyphe­ nolic and flavonoid content, and hence good antioxi­ dant activity, in T. simmleri plants, in comparison with other seven Tulbaghia species, T. violacea included. Postharvest technologies are common methods to extend EFs shelf­life, as it is generally rather short (2­ 10 days) (Fernandes et al., 2019, 2020). Flowers are high value products, which must be picked with care, p a c k a g e d p r o p e r l y t o p r o t e c t t h e m f r o m a n y mechanical damage, and stored at proper tempera­ ture until consumption (Fernandes et al., 2020). Improperly handled/stored edible flowers suffer tis­ sue browning, flower wilt, dehydration, petal discol­ oration, and abscission. The senescence process is a s s o c i a t e d w i t h p h y s i o l o g i c a l c h a n g e s a n d catabolism, which are linked to accelerated respira­ tory levels, weight reduction, and/or plant hormone response (Kou et al., 2012; Landi et al., 2018). To address these concerns, fresh edible flowers are often stored under low temperatures, generally at chilling ones (4­5°C) (Fernandes et al., 2020). Since different EFs species showed different behaviour at cold storage (Landi et al., 2018; Marchioni et al., 2020 a, 2020 b), postharvest studies should be per­ formed for each flower, in order to elucidate their physiological response to low temperature and hence their shelf­life. The aim of this work was to evaluate the phytonu­ tritional and aromatic profile of T. simmleri EFs s t o r e d a t 4 ° C f o r 0 , 2 a n d 6 p o s t h a r v e s t d a y s . Spectrophotometric and chromatographic analyses were performed in order to highlight any changes in polyphenolic content (flavonoids and anthocyanins included), antioxidant activity, and volatile organic compounds (VOCs) during cold storage. 2. Materials and Methods Plant material and postharvest conditions Tulbaghia simmleri plants were provided by the Chambre d’Agriculture des Alpes­Maritimes (CREAM, Nice, France) and were grown at Research Centre for Vegetable and Ornamental Crops (CREA, Sanremo, Imperia, Italy, GPS: 43.816887, 7.758900). Details on plant cultivation is reported in Najar et al. (2019). Full open flowers were picked in April, weighed and cold stored as described in Marchioni et al. (2020 b), for two (T2) and six (T6) postharvest days. Fresh flowers Marchioni et al. ‐ Changes of T. simmleri edible flowers during cold storage 27 were considered as control (T0). Weight loss and colour determination Flowers weigh was measured (Ohaus® analytical Standard Series™ Model AS60S, Ohaus Corporation, Florham Park, N.J. USA) before cold storage (T0) and at the end of each experimental point (T2 and T6) to calculate their weight loss (formula reported in Fernandes et al., 2018). Once flowers had been weighed, their colour was evaluated with a spec­ trophotometer SP60 series (X­Rite Incorporated, Michigan, USA). L* (lightness), a* (redness) and b* (yellowness) colour coordinates (CIELAB scale, CIE 1976) were measured in different point of at least ten flowers, in order to best describe their colour variations. Biochemical analyses Biochemical analyses were performed using frozen samples. Total phenolic, flavonoid and antho­ cyanins content were determined as reported by Marchioni et al. (2020 b). Data were reported as mg gallic acid equivalents (GAEq)/g fresh weight (FW) (polyphenols), mg catechin equivalents (CEq)/g FW (flavonoids), and mg malvin chloride equivalents (MEq)/g FW (anthocyanins). Radical scavenging activ­ ity (DPPH assay) of each sample was determined as described by Brand­Williams et al. (1995). Data was expressed in IC50, which represent the concentration of the sample able to inhibit by 50% the radical DPPH. All absorbance were read in a UV­1800 spec­ trophotometer (Shimadzu Corp., Kyoto, Japan). Enzymatic activities Frozen flowers (200 mg) were pulverized and homogenized in 2 mL of extraction buffer, consisting of 50 mM sodium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2% (w/v) insoluble polyvinylpolypyrrolidone (PVPP), as reported by Pistelli et al. (2017). Samples were centrifuged at maximum speed for 30 min at 4°C and the supernatant was used for enzyme activi­ ties. The soluble protein content was determined according to Bradford (1976) using bovine serum albumin as standard. Catalase (CAT, EC 1.11.1.6) activity was measured by monitoring the decomposition of hydrogen perox­ ide (H2O2), recording the decline in absorbance per minute at 240 nm (Zhang and Kirkham, 1996). The reaction started by adding 20 μL of extract to 980 μl of 8.8 mM H2O2 solution in 50 mM sodium phosphate buffer. One unit of CAT is determined as the amount of enzyme required to detoxify 1 μmole of H₂O₂ (ε= 394 M­1 cm­1) per minute. Data were expressed as unit of CAT per mg of soluble proteins (μmol min­1 mg­1). Ascorbate peroxidase (APX, EC 1.11.1.11) activity w a s d e t e r m i n e d b y f o l l o w i n g t h e d e c r e a s e i n absorbance at 290 nm (ε = 2.7 mM­1 x cm­1) due to enzymatic ascorbate oxidation (Nakano and Asada, 1981). The reaction started by the addition of 50 mM H 2O 2 solution to the reaction mixture (20 μl of extract, 0.15 mM disodium EDTA and 0.37 mM ascor­ bic acid in 50 mM sodium phosphate buffer). A unit of APX is defined as the amount needed to oxidize 1 μ m o l e o f a s c o r b i c a c i d p e r m i n u t e . D a t a w e r e expressed as unit of APX per mg of soluble proteins (μmol min­1 mg­1). Spontaneous emission analysis The spontaneous emission analysis was per­ formed as reported in our previous work (Marchioni et al., 2020 b). Briefly, and after the chosen storage time had elapsed (0, 2 and 6 days at 4°C), 1g of T. simmleri was properly weighted to be sealed in a 25 mL glass flask and kept at laboratory temperature (around 21°C) for 15 min (equilibration time). Once the time expired, the 100 μm polydimethylsiloxane PDMS fiber (Supelco, Bellefonte, PA, USA), was exposed to the flask headspace for 10 min, to be than transferred into the GC­MS instrument. Statistical analysis The normal distribution of the residuals and the homogeneity of variance was determined and then data were statistically analyzed by one­way analysis of variance (ANOVA) (Past3, version 3.15), using Tukey Honestly Significant Difference (HSD) with a cut­off significance of p<0.05 (letters). 3. Results and Discussion Weight loss and chromatic changes during cold stor‐ age The visual quality of T. simmleri flowers has been almost entirely maintained up to the sixth days of cold storage (T6) (Fig. 1, Table 1). The main changes observed during postharvest treatment were the decrease in flowers fresh weight, brightness (L*) and bluish parameter (b*), along with the increase in the reddish parameter (a*) (Table 1). Taken together, these variations resulted in a slight darkening of the petals at the end of the experiment, without any evi­ Adv. Hort. Sci., 2023 37(1): 25­32 28 dent loss of flower firmness. The decrease in fresh weight is due to the loss of cell turgor, which is correlated to flower shape. Significant water loss can determine decreased floral diameter, as well as petals curling and crumpling (Kou et al., 2012; Ahmad and Thair, 2016; Marchioni et al., 2020 b). Nevertheless, the weight loss in T. simmleri flowers was very limited (around 7%), show­ ing, therefore, a good aptitude to cold storage. Moreover, the latter was observed to reduce the brightness of seven different EFs (Landi et al., 2018), as well as T. simmleri flowers (Table 1). This decrease in L* values is indicative of tissue darkening, com­ monly associated with the oxidation of phenolics and their polymerization into dark brown pigments, as a result of the activities of polyphenol oxidase (PPO), peroxidase and phenylalanine ammonia lyase (PAL) (Landi et al., 2018; Hu and Shen, 2021). The same process could also be responsible for the changes in the color coordinates a* and b*, which turn towards darker hues (Table 1). Antioxidant compound and enzyme activities Polyphenols are considered as the most important and widest natural compounds with antioxidant activity (Cavaiuolo et al., 2013). Thanks to their bioactive potential, these molecules can help to pre­ vent chronic degenerative diseases, cardiovascular disorders, and different types of cancer (Pires et al., 2019; Skrajda­Brdak et al., 2020). Postharvest treat­ ment should maintain unaltered flowers polyphenols concentration to guarantee health benefit until flow­ ers consumption. Our results satisfied this statement, because no changes were observed up to T6 for p o l y p h e n o l a n d fl a v o n o i d s a m o u n t s ( T a b l e 2 ) . Indeed, a short increase in the total anthocyanins content was quantified already after 2 days (T2) that could be correlated to the interchange between bluish and reddish parameters (Table 1). Despite this positive trend, it should be noted that T. simmleri fresh flowers are characterized by low amount of phenolic compound than other well­known and cur­ rently consumed EFs (Li et al., 2014; Chen et al., 2018). Moreover, higher quantities of polyphenols and flavonoids were also reported in other species of the same genus, such as T. cominsii and T. violacea, probably connected to the use of different extraction methods (Landi et al., 2018; Rivas­García et al., 2022). Nevertheless, maintaining the levels of pheno­ lic compounds in T. simmleri flowers could indicate that this species did not show substantial signs of decay up to the end of the experiment. As regards total anthocyanins content, their increase was previ­ Table 1 ­ Weight loss and chromatic changes of T. simmleri flowers at 0 (T0), 2 (T2), and 6 (T6) postharvest days (storage at 4°C) Data are reported as mean ± standard error (weight loss, n = 4; L*, a*, b*, n = 15). Different letters indicate statistically signifi­ cant differences (p<0.05; Tukey’s HSD test). Table 2 ­ Antioxidant compounds, radical scavenger activity (DPPH assay), catalase (CAT) and ascorbate peroxidase (APX) activities of T. simmleri flowers at 0 (T0), 2 (T2), and 6 (T6) postharvest days (storage at 4°C) Data are reported as mean ± standard error (n = 6). Different letters indicate statistically significant differences (P<0.05; Tukey’s HSD test). Parameters Days 0 2 6 Weight loss (%) 0 c 3.21 ± 0.04 b 7.34 ± 0.69 a L* 56.01± 1.25 a 55.54 ± 1.19 a 49.45 ± 0.68 b a* 22.71 ± 0.64 c 25.78 ± 0.65 b 27.84 ± 0.46 a b* ­14.16 ± 1.08 a ­19.73 ± 0.69 b ­20.02 ± 0.51 b Parameters Days 0 2 6 Total polyphenols (mg GAEq/g FW) 1.22 ± 0.01 a 1.30 ± 0.04 a 1.32 ± 0.03 a Total flavonoids (mg CEq/g FW 0.30 ± 0.01 a 0.32 ± 0.01 a 0.29 ± 0.01 a Total anthocyanins (mg MEq/g FW) 0.21 ± 0.02 b 0.29 ± 0.01 a 0.24 ± 0.02 a DPPH assay (IC50 mg/ml) 4.20 ± 0.28 a 3.46 ± 0.19 a 5.22 ± 0.09 b CAT activity (µmol min­1 mg­1) 12.68 ± 0.41 b 9.05 ± 0.24 c 21.25 ± 0.27 a APX activity (µmol min­1 mg­1) 0.66 ± 0.04 b 0.64 ± 0.04 b 0.83 ± 0.04 a Fig. 1 ­ Visual appearance of T. simmleri flowers after different times of cold storage (4°C): freshly picked flowers (A); after 2 days of cold storage (T2) (B); and after 6 days of cold storage (T6) (C). Bar scale: 1 cm. Marchioni et al. ‐ Changes of T. simmleri edible flowers during cold storage 29 ously observed also in other EFs stored at low tem­ perature, but the regulatory mechanisms in flowers are still under debate (Shvarts et al., 1997; Landi et al., 2015; Marchioni et al., 2020 b). Senescence and flowers exposure to low tempera­ tures are tightly associated with a rise in reactive oxy­ gen species (ROS) level in the cells, whose production is accompanied by the activation of several enzymes involved in ROS scavenging (Cavaiuolo et al., 2013; Darras, 2020). Polyphenolic compounds also take part to this process, as demonstrated by the reduc­ tion of flowers antioxidant activity observed at T6 (Table 2). In this work, the attention was paid to the ROS scavenging enzymes that use hydrogen peroxide (H2O2) as substrate, namely catalase (CAT) and ascor­ bate peroxidase (APX). T. simmleri flowers showed that CAT activity is higher than the one of APX (Table 2), suggesting a greater involvement of CAT in H2O2 inactivation. Moreover, both the enzymes increased their activity at T6 (Table 2). To the best of our knowledge, very few papers investigated ROS scav­ enging enzymes activity in EFs stored at chilling tem­ perature as single postharvest treatment. In fact, Chrysargyris et al. (2018, 2019) combined the conser­ vation at 5°C with preharvest salinity treatment and modified atmosphere packaging to observe the stor­ age aptitude of Tagetes patula and Petunia × hybrida flowers. Nevertheless, in agreement with our results, APX activity was lower than the one of CAT in T. pat‐ ula flowers, after both 7 and 14 postharvest days (Chrysargyris et al., 2018). CAT activity was also investigated by Rizzo et al. (2019), highlighting differ­ e n t t r e n d d e p e n d i n g o n t h e s p e c i e s a n d t h e polypropylene (PP) film used. In the control thesis (comparable with our experiment), CAT activity increases significantly after 6 days of cold storage only in half out of the four studied flowers (Malva sylvestris and Papaver rhoeas), similarly to what we observed for T. simmleri. Aroma profile Monoterpenes were the main class of compounds, regardless the storage time and their percentage, that represented at least 50% of the identified fraction ( T a b l e 3 ) . I n t e r e s ti n g t o n o t e i s t h e d r a s ti c a l l y decrease in oxygenated hydrocarbons content which was of 77% (passing from 0­ to 2­day conservation) and 60% (passing from 0­ to 6­day conservation) respectively. On the contrary, this decrease was some­ how compensated by the increase in the monoter­ pene hydrocarbons after 2­day storage (an increase of about 2­folds) and by non­terpene compounds after 6­day storage (an increase of about 2.5­folds). In detail of composition, the fresh flower (T0) was rich in linalool and 1,8­cineol and these compounds almost completely disappear after 2 days of storage. A decrease of linalool content was observed also in papaya “Golden” fruit stored at low temperature (Gomes et al., 2016). Interestingly is also the increase of limonene content, about 5­folds, from T0 and T2 (3.01% vs 14.78%, respectively), the same compound conserved the latter percentage even at T6. Worthy to note, the presence of benzyl­benzoate in the flow­ ers is only noticeable after 2­ and 6­days of refrigera­ tion, and its quantity is tripled during this time. This work reported for the first time the chemical composition of spontaneous emission of the studied species. Also noteworthy is the absence of sulfur compounds. Almost similar behavior has been seen in T. violacea, where such compounds were present in a negligible amount, which were around 1.2% in leaves and do not exceed 4% in roots detected using the same analysis technique (HS­SPME) (Staffa et al., 2020). Rhizomes’ essential oil (EO) of a South African species of T. violacea was also reported to be rich in 2,4­dithiapentne, which represent more than the half of the identified fraction (Soyingbe et al., 2013). Hydrocarbons were the major compounds in the hexane extract of T. violacea calli from Cairo (Egypt) (55.0%), while the flowers were rich in oxygenated compounds (74.6%) (Eid and Metwally, 2017). On the contrary, the EO from the same species studied by the same team but published two year before under­ line the prevalence of sulfur compounds in both leaves and flowers and represented 79.7% and 57.5%, respectively (Eid, 2015). 4. Conclusions Cold storage can reduce some biochemical reac­ tions, although stress conditions increase the reactive s p e c i e s o f o x y g e n ( R O S ) i n s i d e p l a n t ti s s u e s . Tulbaghia simmleri flowers maintain almost unal­ tered their visual quality, and their content in antioxi­ d a n t c o m p o u n d s , u p t o 6 p o s t h a r v e s t d a y s . Moreover, cells counteract ROS production increas­ ing CAT and APX activity. The aroma profiles changed during the cold treatment, even if monoterpenes remained the most represented class of volatile com­ pounds. Looking at the main characteristics of the flowers we can conclude that T. simmleri showed a good aptitude to chilling temperature, suggesting the need to test longer period of storage. 30 Adv. Hort. Sci., 2023 37(1): 25­32 Table 3 ­ Aroma profile of T. simmleri flowers detected by headspace solid phase microextraction (HS­SPME) at 0 (T0), 2 (T2), and 6 (T6) postharvest days N° Class Component L.R.I Days 0 2 6 1 nt (E)­3­hexen­1­ol 866 2.37 ± 0.10 2 mh α­Thujene 932 0.20 ± 0.00 tr 3 mh α­Pinene 939 0.19 ± 0.02 3.36 ± 0.83 1.78 ± 0.12 4 mh Camphene 953 0.38 ± 0.08 0.19 ± 0.01 5 nt Benzaldehyde 961 0.93 ± 0.18 6 mh Sabinene 976 0.53 ± 0.11 0.26 ± 0.00 7 nt 1­octen­3­ol 978 4.89 ± 0.16 8 mh β­Pinene 980 1.18 ± 0.27 0.59 ± 0.02 9 nt 3­Octanone 988 2.90 ± 0.16 10 om 2,3­dehydro­1,8­cineole 991 0.96 ± 0.08 11 mh Myrcene 992 1.48 ± 0.61 12 nt 3­Octanol 993 2.09 ± 0.13 0.80 ± 0.21 13 mh δ­3­Carene 1011 0.44 ± 0.00 14 mh α­Terpinene 1018 0.18 ± 0.04 1.03 ± 0.37 0.84 ± 0.08 15 mh p­Cymene 1026 0.12 ± 0.01 7.26 ± 2.92 4.72 ± 0.70 16 mh Limonene 1031 3.10 ± 0.08 14.74 ± 6.42 14.80 ± 1.13 17 om 1,8­Cineole 1033 53.10 ± 0.08 10.38 ± 0.25 18 om (Z)­β­ocimene 1033 0.20 ± 0.06 0.14 ± 0.03 19 mh (E)­β­ocimene 1040 0.62 ± 0.00 0.38 ± 0.08 20 nt Phenyl acetaldeyde 1043 1.30 ± 0.04 21 mh γ­Terpinene 1062 0.63 ± 0.08 5.74 ± 0.12 3.82 ± 0.02 22 om cis­Sabinene hydrato 1068 0.82 ± 0.16 23 mh Terpinolene 1088 0.32 ± 0.15 1.23 ± 0.39 1.05 ± 0.02 24 mh Linalool 1098 15.51 ± 0.10 1.32 ± 0.68 0.92 ± 0.04 25 nt Phenyl ethyl alcohol 1110 1.31 ± 0.03 26 om trans­Limonene oxide 1139 1.24 ± 0.06 27 om trans­Pinocarveol 1140 0.83 ± 0.10 28 om Camphor 1143 1.89 ± 0.44 1.95 ± 0.14 29 om Menthone 1154 0.46 ± 0.03 0.51 ± 0.01 30 om Isomenthone 1164 0.40 ± 0.18 0.22 ± 0.01 31 om Borneol 1165 0.76 ± 0.06 0.59 ± 0.02 32 om δ­Terpineol 1167 tr 33 om trans­linalool oxide 1172 0.47 ± 0.05 0.32 ± 0.02 34 om neo­Menthol 1174 0.76 ± 0.14 35 om cis­Pinocamphone 0.71 ± 0.06 36 om 4­Terpineol 1177 0.24 ± 0.08 1.57 ± 0.40 1.19 ± 0.09 37 om α­Terpineol 1189 5.27 ± 0.08 0.79 ± 0.15 0.26 ± 0.04 38 nt Decanal 1204 0.64 ± 0.06 39 om Verbenone 1205 0.30 ± 0.05 40 om Lilac alcohol B 1210 1.08 ± 0.11 41 nt Methyl 4­nonenoate 0.36 ± 0.14 42 om trans­Carveol 1217 0.32 ± 0.08 43 om Methyl carvacrol 1244 0.68 ± 0.30 0.68 ± 0.12 44 om Linalyl acetate 1257 2.14 ± 0.33 1.95 ± 0.35 45 om Isobornyl acetate 1285 2.11 ± 0.49 1.98 ± 0.35 46 om Myrtenyl acetate 1325 1.43 ± 0.16 0.30 ± 0.00 47 om Methyl perillate 0.15 ± 0.07 48 sh α­Cubebene 1351 0.23 ± 0.00 49 sh α­Longipinene 1352 1.98 ± 0.35 50 sh α­Copaene 1376 0.63 ± 0.11 0.27 ± 0.07 51 sh β­Caryophyllene 1418 0.99 ± 0.06 2.48 ± 0.49 2.89 ± 0.21 52 sh α­Guaiene 1439 0.35 ± 0.04 53 sh Aromandrene 1442 0.12 ± 0.00 0.17 ± 0.02 54 ac­12 (E)­geranyl acetone 1453 tr Data are reported as mean ± standard deviation (SD) (n=2). ... to be continued Marchioni et al. ‐ Changes of T. simmleri edible flowers during cold storage 31 Acknowledgements T h i s w o r k w a s s u p p o r t e d b y a g r a n t f r o m European Union in the frame of INTERREG ALCOTRA V­A France­Italy ANTEA Project n.1139 ­ Attività inno­ vative per lo sviluppo della filiera del fiore edule/ Fleurs comestibles: innovations pour le development d’une filière transfrontalière, and INTERREG ALCO­ T R A V ­ A F r a n c e ­ I t a l y A N T E S P r o j e c t n . 8 3 3 6 ­ Capitalizzazione di progetti Antea e Essica. 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