Phytonutritional and aromatic proﬁles of Tulbaghia simmleri Beauv. edible ﬂowers during cold storage

: Edible ﬂowers are appreciated due to their aesthetic features, nutri­ tional value and antioxidant properties. Tulbaghia simmleri Beauv. (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%.


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 "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(Fernandes et al., , 2020)).Flowers are high value products, which must be picked with care, packaged properly to protect them from any 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 associated with physiological changes and 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., 2020Marchioni et al., a, 2020 b) 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 stored at 4°C for 0, 2 and 6 postharvest days.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.

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 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 IC 50 , 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 (H 2 O 2 ), 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 H 2 O 2 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 M 1 cm 1 ) per minute.Data were expressed as unit of CAT per mg of soluble proteins (μmol min 1 mg 1 ).
Ascorbate peroxidase (APX, EC 1.11.1.11)activity was determined by following the decrease in absorbance at 290 nm (ε = 2.7 mM 1 x cm 1 ) due to enzymatic ascorbate oxidation (Nakano and Asada, 1981).The reaction started by the addition of 50 mM H 2 O 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 μmole of ascorbic acid per minute.Data were expressed as unit of APX per mg of soluble proteins (μmol min 1 mg 1 ).

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).

Weight loss and chromatic changes during cold storage
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 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 polyphenol and flavonoids amounts (Table 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  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 (H 2 O 2 ) 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 H 2 O 2 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. (2018Chrysargyris et al. ( , 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. patula flowers, after both 7 and 14 postharvest days (Chrysargyris et al., 2018).CAT activity was also investigated by Rizzo et al. (2019), highlighting differ ent trend depending on the species and the 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 (Table 3).Interesting to note is the drastically 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).

Conclusions
Cold storage can reduce some biochemical reac tions, although stress conditions increase the reactive species of oxygen (ROS) inside plant tissues.Tulbaghia simmleri flowers maintain almost unal tered their visual quality, and their content in antioxi dant compounds, up to 6 postharvest days.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.