**Modelling of Carotenoids Content in Red Clover Sprouts Using Light of Di**ff**erent Wavelength and Pulsed Electric Field**

**Ilona Gał ˛azka-Czarnecka 1,\*, Ewa Korzeniewska 2,\*, Andrzej Czarnecki 1, Paweł Kiełbasa <sup>3</sup> and Tomasz Drózd ˙ ˙ z <sup>3</sup>**


Received: 4 May 2020; Accepted: 11 June 2020; Published: 16 June 2020

**Abstract:** The paper presents the results of influence the light of different wavelengths and pulsed electric fields on the content of carotenoids. Seeds germination was carried out in a climatic chamber with phytotron system. The experiment was carried out under seven growing conditions differing in light-emitting diode (LED) wavelengths and using pulsed electric fields (PEFs) with different strength applied before sowing. Cultivation of the sprouts was carried out for seven days at relative humidity 80% and 20 ± 1 ◦C. Different light wavelengths were used during cultivation: white light (380–780 nm), UVA (340 nm), blue (440 nm), and red (630 nm). In addition, the pulsed electric field (PEF) with three values of strength equal to 1, 2.5 and 5 kV/cm, respectively, was applied to three series of sprouts before sowing. Sprouts treated with the PEF were grown under white light (380–780 nm). The light exposure time for all experimental series of sprouts was 12/12 h (12 h light, 12 h dark for seven days). Lutein is the dominant carotenoid in germinating red clover seeds, the content of which varies from 743 mg/kg in sprouts grown in red light, 862 mg/kg in sprouts grown in UVA, to 888 mg/kg in sprouts grown in blue light. Blue light in the cultivation of red clover sprouts had the most beneficial effect on the increase of carotenoids content and amounted to 42% in β-carotene, 19% in lutein, and 14% in zeaxanthin. It confirms that modelling the content of carotenoids is possible using UVA and blue light (440 nm) during seed cultivation. An increase in the content of β-carotene and lutein in red clover sprouts was obtained in comparison to the test with white light without PEF pre-treatment, respectively by 8.5% and 6%. At the same time a 3.3% decrease in the content of zeaxanthin was observed. Therefore, it can be concluded that PEF pre-treatment may increase mainly the content of β-carotene.

**Keywords:** carotenoids; traditional food; light-emitting diodes; pulsed electric field; lutein; zeaxanthin; β-carotene

#### **1. Introduction**

Carotenoids play a key role in human health and should therefore be a regular part of the daily diet. It is important that they are delivered from various sources. Several carotenoids, including β- carotene, β-cryptoxanthin, and α-carotene are classified as provitamin A. Carotenoids other than provitamin A are also important in the human diet, because their high intake is correlated with a

lower risk of developing chronic degenerative diseases including age-related, with special attention to age-related macular degeneration (AMD), cardiovascular diseases and some types of cancer [1–3].

Carotenoids are pigments soluble in lipids. Carotenoids are isoprenoid metabolites synthesized by all photosynthetic organisms (including algae, plants and even cyanobacteria) and some non-photosynthetic organisms such as archaea, fungi, bacteria or animals. Carotenoids are in over 1100 naturally occurring compounds that give colour to many edible parts of plants and flowers from yellow, through to orange and red. In addition, carotenoids can be cleaved to produce compounds with roles as growth regulators, such as abscisic acid (ABA) and strigolactones, as well as other bioactive molecules.

Carotenoids are found in many plant products. They are components of supplements and are also an additive to feed (e.g., lutein, zeaxanthin, β-cryptoxanthin, α-xanthine) in order to obtain the right colour of, for example, farmed fish eggs [3,4].

The literature on the subject has shown that, except for plants stained orange or red, what is associated with the colour of these compounds (e.g., papaya, carrots, peppers, also green plants such as sprouts) is a very good source of carotenoids [3]. In addition, sprouts belong to low-processed food. Their cultivation is fast (several days), easy and relatively cheap. However, there are no reports on the content of carotenoids in red clover sprouts. Commonly, carotenoids are associated with products that are from yellow to red, but these compounds are also commonly found in products containing chlorophylls. Carotenoids in chloroplasts help to absorb an excess of energy and dissipate it in the form of heat. In photosynthesis, carotenoids help to absorb light, but also play an important role in getting rid of solar energy excess. When a leaf is exposed to the full sun, it receives a large portion of energy. If this energy is not properly managed, it can destroy elements essential for photosynthesis. The seed germs are an interesting product containing carotenoids, obtained during just a few days of their cultivation. The germ is called an embryo that has pierced through the seed coat and developed a system (root and cotyledon) for self-feeding.

The European Union (EU) Commission Regulation 208/2013 from 11 March 2013 defines sprouts as a product obtained as a result of germination of seeds and their development in water or other carriers, which can be collected before the formation of proper leaves and intended for consumption as a whole, including seeds [5]. Sprouted seeds are one of the most nutritious and tastiest types of food in the world. Most sprouts can be eaten fresh. They are an addition or the base of salads, they are also suitable for many snacks and dishes, such as soups, dressings, dips and cocktails. Sprouts can enrich the nutritional value of many dishes, such as pizza, casseroles, croquettes, burgers, meat dishes, etc. Currently, the use of germinated seeds is unlimited and original desserts, smoothies and even sweets are prepared.

Sprouts in some especially Asian cuisines have been consumed for a long time and in Europe and the US they occupy an important place in vegetarian diets. Sprouted seeds, due to their composition, can be called a health bomb, because they contain nutrients and many compounds that have a positive effect on human health; they are primarily antioxidant compounds, including plant dyes such as carotenoids. The advantages of germinated seeds are primarily their nutritional and taste qualities, as well as their growing availability and diversity on the market. Seed cultivation can be carried out throughout the year using various solutions. Modern crops can be grown in phytotron (climatic) chambers with an automatic irrigation and temperature regulating system and additional lighting. In addition, assimilation lighting is one of the most important factors of plants grown in such conditions. Unlike photosynthesis, photoreaction is a more qualitative reaction and it depends on the wavelength. Thanks to this it is possible to stimulate photophysiological processes in the plant, affecting the content of bioactive compounds and obtaining their more often favourable composition.

To produce young edible seedlings (sprouts), it is possible to use the seeds of plants belonging, among others, to the *Fabaceae* family (bean beetles). The red clover (*Trifolium pratense L*) belongs to this family. Sprouted red clover seeds have strong antioxidant properties, mainly due to the high content of bioactive compounds. Studies have shown that they also contain a favourable qualitative

and quantitative composition of phytoestrogenic compounds (isoflavones such as daidzein, genistein, formononetin and biochanin A), which have a beneficial effect on the human body. Sprouted red clover seeds can be included in the daily diet. In addition, light-emitting diodes (LEDs) open the possibility of their use for plant growth in a closed environment [6]. More advantageous and more efficient light sources are based on light-emitting diodes (LEDs) due to their advantages, such as light emission in a narrow spectrum band, high efficiency compared to traditional lamps, low voltage operation, photosynthetic regulation, photosynthetic photon flux density (PPFD) and low heat emission [7].

Plant growth, including germ and the profile of biologically active compounds, depends on the genotype, type of exposure (monochrome, combined or white light), its intensity and time [8]. The content of vitamins and microelements increases, and anti-nutritional components, such as trypsin inhibitors, are removed during germination as a result of intensive metabolism. It makes the germs safe for human consumption. The enzymes are activated in seed during germination, including amylolytic, proteolytic and lipolytic ones. Their activity favourably changes the composition of germinated seeds. Starch, proteins and fats are broken down, becoming a source of energy and substrates for the synthesis of new substances. Literature data [9] indicate the relationship between the content of antioxidant compounds in sprouts and their growth conditions (i.e., seed location, temperature and humidity). Seed cultivation can be carried out using many germination methods, not only differentiated by the method of moisturizing, temperature, and access to light, but also by the substrate [6,10,11]. Studies on the processes occurring in germinating seeds also explore the importance of light as an intermediary in the regulation of enzyme activity [12]. Seed viability can be expressed as the ability to germinate, which leads to the formation of a plant capable of reproduction. Sufficiently long viability affects the vigour of seeds, which expresses their ability to produce healthy and well-growing seedlings and plants. The cultivation of seeds both in terms of their germination efficiency and the content of bioactive compounds can be stimulated in various ways. An interesting factor is, for example, the usage of the pulsed electric field (PEF) on seeds before cultivation. The usage of the pulsed electric field on seeds is a phenomenon described in various ways in the literature. It has been shown that the effect of the PEF on seeds before germination has a positive effect on germination efficiency and seed growth rate [13,14]. Research was also conducted on the use of PEF [15–20], magnetic field [21] and UV light [22] in food processing and preservation. In biological material that has been subjected to an electric field, as a result of the impact of electrical impulses on cell membranes, a significant increase in their conductivity is observed, mainly through the formation of free spaces on the surface, the so-called pores. Their presence allows the free flow of various components through the cell membrane. Thanks to this phenomenon, it becomes possible to transfer ions, molecules and even more complex compounds (i.e., drugs, nucleic acids, monoclonal antibodies, oligonucleotides or plasmids) into the cell [23].

It is believed that the PEF can potentially be used to control and optimize the process of sprout growth and modify its composition, in particular nutritional values and bioactive ingredients [24]. PEF can also effectively stimulate germ growth and positively affect metabolism and nutrient content [24,25].

The effect of PEF on germination depends on the type of plant and the strength of the used field, while on some species such as marigold tomato or radish no significant effect was observed. In the case of lentil, a 50% increase in germination rate was observed. The effect of changes in germination rate induced by PEF is probably associated with changes in the metabolism of amino acids occurring in seeds [25] while PEF induces electroporation causing increased membrane permeability. Electroporation is a reversible process, however, when too high values of the PEF process parameters are used, irreversible changes in the structure of the cell membrane can occur [26]. Depending on the duration and the number of pulses and the strength of the electric field, the cell membrane may even be destroyed (i.e., irreversible electroporation) [25].

Red clover *Trifolium pratense* L. is an interesting plant. Throughout the world, it is most often known as a feed plant because it is used for fodder. There are many publications on bioactive compounds and their variability resulting from the cultivation of a mature plant. Many pharmaceutical preparations and dietary supplements [27,28] are also obtained on the basis of this plant. There is little research on carotenoids in red clover, and no such data were found in the available literature. Research and modelling of carotenoids content in red clover sprouts are innovative.

It should be noted that it has been confirmed that red clover sprouts are a rich source of bioactive compounds, including isoflavones, compounds with similar effects to oestrogen and can supplement the daily diet [6].

Another factor more and more often considered in the scientific literature, regarding the plants' cultivation, including sprouts, is the effect of light of different wavelengths on growth factors, as well as the content of biologically active compounds, including carotenoids [29]. Light, its intensity and wavelength have a significant impact on germination and plant development. Analysing the reports of various authors, the impact of light of varying wavelengths on sprouts of plants of different species is different. There is no information on the effect of light of varying wavelengths on the content of carotenoids in red clover sprouts. Therefore, the purpose of this work is to attempt to model the content of carotenoids in red clover sprouts using the effects of the PEF on their cultivation and the light of different wavelengths during their growth.

#### **2. Materials and Methods**

The experiment was carried out for seven days under different growing conditions. In the experiment, different wavelengths of light and pulsed electric fields (PEF) were used. The light-emitting diodes (LED) were used as the source of light. In the conducted research the light of wavelengths, ranging from 340–780 nm, was applied to the tested material. The strength of the pulsed electric field was chosen from the ranges 1 kV/cm, 2.5 kV/cm and 5 kV/cm. During the experiment, favourable conditions for seed development were created. The red clover seeds were put in a phytotron chamber where the light of different wavelengths was applied. Another group of seeds was treated with PEF before sowing and then they were cultivated in white light conditions. Cultivation of seeds was carried out to obtain the edible sprouts. The collection time was determined based on the quality features and content of bioactive compounds in red clover sprouts, including ascorbic acid and flavonoids (phytoestrogens). These compounds are present in the largest quantities between the 6th and 8th day of cultivation [6,11]. Thus, the germination in this experiment took place after 7 days of cultivation (after 168 h).

In the experiment, the sprouts were grown without soil and without supporting substrates containing mineral substances. Therefore, further development of the plant would be inhibited as a result of depletion of spare substances stored in seeds. Since whole sprouts of red clover are intended for consumption, the division into cotyledon and hypocotyl was not included in the study. It was found that on the day of harvesting red clover sprouts were firm, had green leaves with a cucumber-pea smell and were properly shaped, but depending on the cultivation conditions, the obtained weight (biomass) varied.

#### *2.1. Pulsed Electric Field*

A laboratory stand was built to conduct the experiment. It consisted of a high-voltage pulse generator with the voltage output in the range of 0 to 30 kV. The generated signal was the rectangular shape. The control system was used to obtain the proper value of the electric field strength. It allowed setting the number of pulses and the time interval between them. The process was conducted in the chamber in which the discharge occurred. There were two flat electrodes, between which a cylindrical, Teflon cell with seeds was placed.

The following parameters of the electroporation process were selected: pulse repeatability 10 s; the number of impulses 20; the electric field strengths 1 kV/cm, 2.5 kV/cm and 5 kV/cm. Higher field strength values were not used due to the destruction of the tested material. During the process, the constant value of temperature 20 ◦C was ensured. The temperature was measured with a thermocouple. The PEF was applied to red clover seeds before germination.

#### *2.2. Raw Material*

The seeds of red clover (*Trifolium pratense L*.), Rosette variety, suitable for germination supplied by FN Granum (Wodzierady, Poland) were chosen as the tested material. The plants were divided into 49 experimental series. Each series consisted of 5 g seeds. In each series there were three containers.

#### *2.3. Sprouts Cultivation*

Red clover (*Trifolium pratense* L.) seeds were grown using modified conditions. Seeds germination was carried out in the climatic chamber with a phytotron system (modified KBWF 720 Binder, Tuttlingen, Germany). Cultivation of the sprouts was carried out for 7 days at relative humidity 80% and 20 ± 1 ◦C. The LEDs which emitted the light with different wavelengths were used as the light sources in the process of sprouts growing. The different light wavelengths were used during cultivation: white light (380–780 nm), UVA (340 nm), blue (440 nm), red (630 nm). A photosynthetic photon flux density (PPFD) of 150 <sup>±</sup> <sup>5</sup> <sup>μ</sup>mol m-2 s-1 was maintained.

Since it is not possible to use sunlight in the phytotron chamber, white light (cool white) was used for all experimental samples to maintain constant growing conditions. Such lighting was also used by other authors in the cultivation of plants, including sprouts [30]. It would be possible to use sunlight in the experiment, but the sunlight would be variable in time and the environmental conditions would vary (e.g., in sprout machine).

In addition, the pulsed electric field (PEF) with three values of strength, 1, 2.5 and 5 kV/cm, respectively were applied on three series of sprouts before sowing. Seeds after PEF treatment were stored for 14 days at room temperature (T = 20 ◦ C) and then grown.

Sprouts treated with PEF were grown under white light (380–780 nm).

During cultivation, lighting was used repeatedly for 12 h and after that period the plants were stored 12 h in the dark. Sprouts were cultivated in portions of 5 g in containers (polypropylene) with a perforated bottom set on a tray. Sprouting was conducted in triplicate for each treatment (3 containers for each experiment). On the first day, the seeds were soaked by the addition of 15 mL of water, and in the subsequent days, the examined sprouts were irrigated to maintain high moisture in the culture. During cultivation, sprout weight growth was analysed every 24 h. Red clover sprouts were weighed immediately after harvesting on an analytical balance (RADWAG, PS 06.R2, Radom, Poland). The obtained germs of the sprouts are the average of three samples of 5 g seeds each. From the time the seeds were sown, the samples were weighed every 24 h.

The whole sprouts were examined. Sprouts were harvested manually every 24 h from the day of sowing. Analyses from the average sample from each container (3 containers for each treatment) were performed in one test samples taken from each container. After each harvest, the weight of the harvested sprouts was determined. The content of carotenoids was determined after 7 days of red clover sprouts growing.

These samples after harvesting were frozen immediately in liquid nitrogen and stored at −80 ◦C until further analysis.

#### *2.4. Germination Energy*

For each cultivation variant, four hundred seeds from the International Seed Testing Association (ISTA) (red clover and red clover after PEF treatment) were collected, grown in separate moulds made of polipropylen (PP) plastic, dedicated to conducting the germination process. During the study, the percentage of healthy, correctly germinated seeds was determined. During the cultivation, seed viability indexes were determined for energy of red clover seeds.

Germination energy (GE) was determined as the percentage of seeds that germinated during the first 4 days (96 h) [31].

#### *2.5. Sample Preparation and Determination of Carotenoids*

The samples of sprouts were placed in liquid nitrogen using an analytical mill (A 11 basic, IKA Works GmbH & Co. KG, Staufen, Germany)

The method proposed by Kimura and Rodriguez-Amaya [32] with some modifications was used for the extraction of carotenoids. First, 1 g of grounded and homogenized sprouts was placed in a plastic tube. Extraction was performed twice, first with 5 mL of acetone and next with 5 mL of ethanol (100%). Each extraction step was performed by vortex mixing for 2 min followed by centrifugation at 15,000 RCF (relative centrifugal force) for 10 min at 4 ◦C (MPW-360R, MPW Med. Instruments, Warsaw, Poland). The supernatants were evaporated to dryness under a stream of N2 at 35 ◦C. The residue was redissolved in the mobile phase, centrifuged and diluted with the mobile phase to a suitable concentration.

The carotenoid extracts (injection volume 20 μL) were analysed on an HPLC system (Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany) comprising of degasser of mobile phase Manager 5000, Pump 1000, autosampler model 3935 (maintained at 6 ◦C). A Gemini column maintained at 40 ◦C was used (5u C18110A, 150 × 4,60 mm 5 μm, Phenomenex, Torrance, CA, USA). The HPLC system was coupled with a photodiode array detector (PDA) model 2800. Quantification was performed at a detection wavelength of 445 nm. A gradient system (from A:B 75:25 to 0:100 in 13 min, maintaining this proportion until the end of the turn) was applied with methanol:water (90:10 *v*/*v*) as eluent A and acetonitrile:2-propanol (63:37 *v*/*v*) as eluent B. The flow rate was 1 mL/min. The carotenoids were identified based on the retention time of the standards and quantified with the help of peak areas against the standard calibration curves. Standards of carotenoids were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

#### *2.6. Statistical Analysis*

Statistical analysis was based on the determination of the average values of three measurements and their standard deviation. The data were analysed using a one-way analysis of variance (ANOVA) with the Tukey's range test at the significance level *p* < 0.05. All data were tested for normality using the Shapiro–Wilk test. To test for homogeneity of variance, Levene's test was used. The calculations were performed using the software STATISTICA for Windows (version 10, Statsoft, Krakow, Poland).

#### **3. Results and Discussion**

In the experiment, a research hypothesis was put forward that it is possible to modify the content of carotenoids in red clover sprouts (*Trifolium pratense* L) by applying PEF to seeds before sowing and using light of varying wavelengths during cultivation. Until now, these factors have not been combined in such a way. Thus, this is an innovative approach to this issue. The highest sprout mass was obtained after applying red light to the seeds (38.9 g) and in the case of seeds cultivated in white light after PEF (5 kV/cm) pre-treatment (35.5 g). The lowest final mass was obtained in the case of UVA irradiation.

The seeds cultivation in white light after PEF pre-treatment with the field strengths of 1, 2.5 and 5 kV/cm increased the yield by 0.38%, 3.37% and 6.12%. Table 1 presents the final mass of the 100 g samples after 7 days of cultivation (on the day of harvest) in the case of different conditions: in UVA, blue, red and white light, without and after PEF pre-treatment with the field strengths of 1, 2.5 and 5 kV/cm. (Table 1). Statistically significant differences between the experimental series are presented in Table 2.


**Table 1.** Germination energy given per 100 pieces of seeds and the final mass of the samples after their cultivation in the different growth conditions, recalculated to the initial weight of 100 g sprouts. Values are presented as mean ± SD.

\* The performed analysis of variance does not allow for the rejection of the hypothesis of equality of means (F = 1.82, *p* = 0.143). Kruskal–Wallis test was conducted to examine the differences on germination energy according to the treatment taken. No significant differences (Chi-squared = 10.91, *p* = 0.09) were found among all the seven growing conditions. \*\* Statistically significant differences are presented in the Table 2.

**Table 2.** *p*-Values of Tukey's post hoc tests after one-way ANOVA for final mass of sprouts with treatments as fixed factors (F = 11.50, *p* < 0.05). Statistically significant differences are bolded.


Other authors also show a differentiated effect on the growth and yield of different plants depending on the wavelength of the used light. The results of the research indicate that the usage of red LED light in the cultivation of seeds and plants can cause an increase in biomass [33,34]. The red light is the most effective photosynthetically active radiation [35] referring to absorption. Research shows that blue light can also be used as a factor to increase biomass [36].

In the case of crops of different varieties of lettuce sprouts, it was found that the fresh weight increased in the case of using red or blue light in the growing process compared to crops under white light. The sprouts were irradiated for 12 h and left in the dark for 12 h. The best effect of the growth of germ mass was obtained when the sprouts were exposed to 3 h of blue light, and in one case it was observed that it increased by 23% [8]. On the other hand, in other studies [25] where wheat seeds before cultivation were treated with 50 pulses of 6 kV/cm PEF, a significant increase in fresh weight was noted, which is positively correlated with PEF energy.

A statistically significant effect of PEF (used at the initial stage of seed preparation for cultivation) on the mass of obtained red clover sprouts was observed. The exposure of seeds to the pulsed electric field can activate processes at an early stage of photomorphogenesis and thus positively affect its development. That line of research requires continuing experiments to confirm this observation. It was not the primary purpose of the presented studies.

In the case of wheatgrass (*Triticum aestivum* L.) the usage of 0.5 kV/cm PEF did not affect the growth of sprouts compared to the control ones. Increasing the PEF strength to 1.4 kV/cm had a positive effect on sprout growth, however, the 2 kV/cm PEF treatment of the tested seeds had an adverse effect and a lower growth was obtained than compared to the control [24].

Figure 1 shows the mass of sprouts depending on the day of cultivation. The initial mass of each sample was 5 g. The obtained mass in the harvest day depended on the pre-treatment and cultivation conditions.

**Figure 1.** Mass of red clover sprouts during 7 days of sprout growth depending on irradiation and pulsed electric field (PEF) treatment prior to sowing. Values are presented as mean ± SD (*n* = 3).

The germination energy of red clover seeds (Table 1) in the case of PEF 5 kV/cm (in the process of seed pre-treatment before sowing) was the highest but did not differ statistically in comparison to other treatments. Moreover, all analysed groups had no significant differences in germination energy. The use of monochromatic LED sources and white light with the addition of 1 and 2.5 kV/cm PEF did not affect germination energy compared to white light crops (no statistically significant differences were found).

A statistically significant increase in germination energy was found after 4 days using a field of energy 240 J (12 kV) for parsley and 960 J (12 kV, the number of pulses was changed) for parsley, winter wheat, winter barley, lettuce, tomato and garden rocket [37].

In other studies, germination energy measured after 24 h was positively correlated with the increase in intensity (energy of pulse electric field) and in the case of 6 kV/cm PEF with 50 pulses it was 92% compared with 84% for non-treated sprouts [25].

For *Haloxsylon ammodendron* seeds, it has been observed that germination energy increases from 72.9% to 90.3% and 98.0% in the cases of using the strengths of 10 and 20 kV/cm, respectively [38].

It was observed that red clover sprouts after 7 days of cultivation contain β-carotene, lutein and zeaxanthin. However, their content varies depending on the factors used (PEF, different lengths of light). In this experiment, monochromatic light sources were used in the process of red clover sprouts growing. Moreover, the chosen wavelengths had a documented effect on photosynthesis and photomorphogenesis [36,39–41].

Green light was not chosen to process the experiment because it was documented in studies that a green monochromatic light source did not affect sprout development. Only slight enrichment of the spectrum with other light wavelengths was contemplated to improve photomorphogenesis [39,42].

In this study, it was observed that the content of carotenoids depends on the conditions of the used light (Figure 2). The dominant carotenoid in germinating red clover seeds is lutein, the content of which varies from 743 mg/kg in sprouts grown in red light, to 862 mg/kg in sprouts grown in UVA, and 888 mg/kg in sprouts grown in blue light. Lutein is a carotenoid with widely documented health-promoting properties. Obtaining new sources of lutein is desirable. At the same time, it was observed that the use of UVA or blue light during cultivation had a positive effect on the formation of other carotenoids, including β-carotene and zeaxanthin. UVA and blue light are the most preferred for obtaining carotenoid dyes. Under these cultivation conditions, the highest content of the total value of tested carotenoids (β-carotene, lutein and zeaxanthin) in red clover sprouts was obtained, respectively 1750 mg/kg (kg of dry weight) in the case of UVA irradiation and 1892 mg/kg using blue irradiation. Comparing these results to sprouts grown in white light, the increase in the content of these dyes in sprouts irradiated with blue light is statistically significant and equal to 42% in β-carotene, 19% in lutein and 14% in zeaxanthin (Figure 2, Tables 3–5). The content of lutein in sprouts from crops cultivated under red light was slightly lower than under white light, but the difference is not statistically significant (Table 4).

**Figure 2.** Influence of different light and PEF pre-treatment on carotenoids. The height of each bar and the error bars indicate the means and standard errors, respectively, from three independent measurements.




**Table 4.** *p*-Values of Tukey's post hoc tests after one-way ANOVA for lutein with treatments as fixed factors (F = 21.10, *p* < 0.05). Statistically significant differences are bolded.

**Table 5.** *p*-Values of Tukey's post hoc tests after one-way ANOVA for zeaxanthin with treatments as fixed factors (F= 11.07, *p* < 0.05). Statistically significant differences are bolded.


Carotenoid content was studied in mature red clover plants for fodder grown in the field. Research shows that the main carotenoid is lutein, its content was 136 mg/kg DM (dry matter), and the total β-carotene content was 29 mg/kg DM [43]. In other studies, the composition of the carotenoids in red clover is different, and the content of lutein, β-carotene and zeaxanthin is much higher, respectively 237.7, 100.3 and 91.7 mg/kg DM. It should be noted that in recent studies, clover after harvesting has been subjected to a consolidation process [44].

Other authors report that the content of β-carotene in red clover is about 200 mg/kg DM, and the content of this carotenoid in *Trifolium repens* varies from 300 to 730 mg/kg DM depending on the harvesting place [45].

In the experiment where the subject was Alsike clover, also belonging to the genus *Trifolium*, the content of lutein and β-carotene was much higher and equalled 208.9–243 mg/kg and 35.4–123.4 mg/kg fresh weight, respectively [46].

The effect of various types of LED lighting on the content of carotenoids in alfalfa sprouts (also belonging to *Fabaceae* family) was investigated [47]. The combination of the red and blue LEDs used during cultivation increased lutein content from 82.6 to 108.2, and β-carotene from 26.6 to 44.6 mg/kg fresh mass of sprouts [47]. However, the research did not cover the whole sprout, only cotyledon. Increasing the intensity of blue light during the cultivation of beetroot (*Beta vulgaris* L.) and parsley (*Petroselinum crispum* Mill.) caused an increase in the content of carotenoids as compared to irradiation with red light. It has been shown that increasing the proportion of blue light intensity in these microgreens results in an increase in lutein content from 103.8 to 118.5 mg/kg of fresh beet mass, from 122.9 to 190.7 mg/kg of fresh parsley, and β-carotene, respectively from 0.09 to 0.87 and 0.54 to 0.86 mg/kg, and zeaxanthin from 1.39 to 3.20 and up to 0.84–14.4 mg/kg, respectively [35].

Lefsrud in his research observed an increase in the content of lutein and β-carotene in kale when illuminated with both blue and red light. The maximum accumulation of lutein and β-carotene in fresh kale mass was found at 640 nm and 440 nm, respectively, calculated on a fresh mass basis. However, when converted to dry mass, the maximum lutein content in kale also occurred at 440 nm. [1].

There are no reports in the literature on the content of carotenoids in red clover sprouts, as well as on the impact of PEFs on the content of carotenoids. Therefore, our research can only be compared with the unique research carried out by Ahmed et al. [25] who studied the effect of PEF on the content of plant dyes in wheat germ. A 35% increase in the content of carotenoids was observed in the PEF treatment with strength of 6 kV/cm and 50 impulses compared to untreated sprouts [25].

In our experiment, no such significant effect of PEF treatment of red clover seeds on the content of carotenoids was found.

Modelling the content of carotenoids in sprouts which are grown in different lighting conditions, especially in ultraviolet radiation with wavelengths up to 400 nm and blue light, forces the plant to adapt to the changes to avoid their harmful effects. Many compounds (e.g., flavonoids as well as carotenoids) can fulfil the role of protection against the harmful effects of UV light [6,48]. Carotenoids are the auxiliary photoreceptors of chlorophyll and absorb light mainly in the blue region. Cryptochromes are the receptors of blue light in the 390–480 nm range. They stimulate leaf expansion. In the blue and UVA spectrum, phototropins, responsible for plant phototropism, bending of shoots towards the light, opening of stomata and leaf expansion are also photoreceptors in the blue and UVA spectrum [49,50].

The increase of carotenoids content is plants' response to stress is associated with high irradiance. High exposure does not always lead to the growth of carotenoids, it can sometimes cause photodegradation of pigment particles [1,51,52]

In the presented experiments, we observed that monochromatic blue light increases the content of carotenoids. The positive effect of blue light on the content of these dyes was also observed by other authors [52,53] Opposite observations were made by Tuan et al. [29]. Research on tartary buckwheat sprouts has shown that blue light causes a decrease in the content of carotenoids compared to white light. However, it should be noticed that in the recalled work, the full spectrum range is not given in the description of the characteristics of the white light source (only 380 nm is mentioned (i.e., UV wavelength)).

The effect of pulsed electric field on seeds (before cultivation) had an impact on the increase of carotenoid content, including β-carotene, lutein and zeaxanthin. It was observed that the application of 5 kV/cm had the most favourable effect on the increase in the content of carotenoids in red clover sprouts compared with the reference test which grew seeds in white light. An increase in the content of β-carotene and lutein in red clover sprouts was obtained in comparison to the test with white light and without PEF pre-treatment by 8.5% and 6%, respectively, and a decrease in the content of zeaxanthin by 3.3% was also found. Therefore, PEF pre-treatment may increase mainly the content of β-carotene. At the same time, it should not be assumed that the use of a higher value of electric field strength (above 5 kV/cm) will work more favourably. The use of higher field strength can damage or destroy the seed epidermis and damage its internal structure, which means that the seed will not be able to germinate. The results of this experiment indicate that the PEF pre-treatment on seeds before their cultivation can be one of the factors that can model the carotenoids content in germinated seeds. The obtained results should be considered as an introduction to further research.

Photosynthetically active radiation includes a wide range of light wavelengths that do not participate in the process of photosynthesis but can stimulate the vegetative growth of plants and modify the chemical composition of the leaves [54].

#### **4. Conclusions**

The dominant carotenoid in germinating red clover seeds is lutein, whose content varies from 743 mg/kg in sprouts grown in red light, 862 mg/kg in sprouts grown in UVA, to 888 mg/kg in sprouts grown in blue light. UVA and blue light are the most preferred for obtaining carotenoid dyes. The highest content of the total value of tested carotenoids (β-carotene, lutein and zeaxanthin) in red clover sprouts was obtained, respectively at 1750 mg/kg (kg of dry weight) in the case of UVA irradiation and 1892 mg/kg using blue irradiation.

Modelling the content of carotenoids in red clover sprouts is possible because by using UVA and blue light (440 nm) during seed cultivation, a significant increase in β-carotene, lutein and zeaxanthin is obtained. An interesting result of the presented research is also the use of PEF pre-treatment on the seeds before the cultivation process. It is a factor that, although to a lesser extent than blue light and UVA, also causes an increase of the carotenoids in sprouts. It is possible that PEF pre-treatment of seeds will also have a beneficial effect on other seeds intended for germination. It requires further testing and confirmation. At the same time, the use of red light resulted in a greater mass of red clover sprouts, because red light has a beneficial effect on photomorphogenesis.

Consumption of 100 g fresh red clover sprouts which were grown for 7 days in UVA, blue, red or white light provided 14.0, 15.1, 12.1 and 12.2 mg total sum of tested carotenoids, respectively. Although the PEF treatment does not significantly affect the carotenoids' content, in the case of PEF strength 5 kV/cm the intake of carotenoids increased by 4% compared to cultivation only in white light and equals 12.7 mg/100 g.

It is worth noting that red clover sprouts may also be a new source of carotenoids including lutein and zeaxanthin. The recommended dietary allowances (RDA) of lutein and zeaxanthin in the human diet have not been established. For reducing the risk of AMD, the efficacious intake level for lutein may be ~ 6 mg per day [55]. Therefore, the consumption of approx. 40 g of red clover sprouts may cover the daily requirement.

Cultivation of seeds for sprouts in closed conditions (in a phytotron chamber) with properly selected blue or UVA lighting can stimulate an increase in the content of bioactive compounds, including carotenoids, and at the same time ensure the proper development of leaves. Changing the spectral composition of radiation during cultivation can be used to produce plants intended for several days of germination and consumption. It is possible to obtain a significantly improved product in terms of nutritional value in a short time using the tested conditions.

**Author Contributions:** Conceptualization, I.G.-C. and E.K.; methodology, I.G.-C., E.K. and P.K.; validation, I.G.-C., E.K., A.C., P.K. and T.D.; formal analysis, I.G.-C. and E.K.; investigation, I.G.-C., E.K., A.C., P.K. and T.D.; resources, I.G.-C. and P.K. and T.D.; data curation, I.G.-C., P.K. and T.D.; writing—original draft preparation, I.G.-C., E.K. and A.C.; writing—review and editing, I.G.-C., E.K. and A.C.; visualization, A.C.; supervision, I.G.-C.; project administration, I.G.-C. and E.K.; funding acquisition, I.G.-C.; P.K. and T.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Bunch Microclimate A**ff**ects Carotenoids Evolution in cv. Nebbiolo (***V. vinifera* **L.)**

**Andriani Asproudi 1, Maurizio Petrozziello 1,\*, Silvia Cavalletto 2, Alessandra Ferrandino 2, Elena Mania <sup>2</sup> and Silvia Guidoni <sup>2</sup>**


Received: 30 April 2020; Accepted: 29 May 2020; Published: 31 May 2020

**Abstract:** This study investigates the impact of bunch microclimate on the evolution of some relevant carotenoids in Nebbiolo grapes. Four bunch-zone microclimates, defined by different vineyard aspect and vine vigor, were characterized by radiation and temperature indices. Berry samples were collected from green phase up to harvest, during two consecutive seasons and carotenoid determination was assessed by High-Performance Liquid Chromatography (HPLC). High carotenoid concentrations were highlighted in Nebbiolo. Lutein and neoxanthin contents (μg berry<sup>−</sup>1) varied similarly in both seasons achieving a concentration peak after veraison especially in the cooler plots while a variety effect on the lutein seasonal trend was presumed. Conversely, β-carotene content remained generally constant during ripening, with the exception of the south plots showing dissimilar evolution between the seasons. Furthermore, higher temperature in the less vigorous and south facing vineyards led to lower amounts of carotenoids, both during ripening and at harvest. Bunch zone temperature and light condition may affect both synthesis and degradation of grape carotenoids determining their amount and profile at harvest. These findings add further knowledge about the influence of climate changes on grape aroma precursors, and are useful to adapt cultural strategies and preserve grape quality consequently.

**Keywords:** vineyard aspect; vineyard topography; vine vigor; heat accumulation; temperature; photosynthetically active radiation; lutein; neoxanthin; β-carotene

#### **1. Introduction**

Plant carotenoids are essential for photosynthesis and photoprotection due to their multiple functions as potent free radical quenchers, singlet oxygen scavengers and lipid antioxidants. They are present in the photosynthetic tissues as part of photosystem II [1]. Carotenoids also give rise to the formation of numerous biologically active cleavage products such as aroma compounds, vitamins, phytohormones, and apocarotenoid pigments [2].

Grape carotenoids were identified as precursors of certain key odorants in wine, namely C13-norisoprenoids, which are low threshold aroma compounds characterized by floral and fruity pleasant notes strongly linked to increases in wine quality, especially for non-floral varieties [3]. The formation of norisoprenoids is thought to occur from the biodegradation of the parent carotenoid, followed by enzymatic conversion to the aroma precursor (e.g., a glycosylated or other polar intermediate), and finally by the acid-catalyzed conversion to the aroma compound [4], which may be then subjected to further acid reaction during wine aging [5]. A family of region-specific carotenoid cleavage dioxygenase (CCD) enzymes is implicated in the initial biodegradation and oxidative cleavage

of carotenoids to form plant apocarotenoids, e.g., C13-norisoprenoids [6–8]. The expression of a CCD capable of producing C13-norisoprenoids from lutein and zeaxanthin (*VvCCD1*) was reported to increase at veraison [6], while the reported increase in expression of a CCD4 gene (*VvCCD4*) after veraison is suggestive of a possible role of this enzyme on norisoprenoid formation during the late stage of berry ripening [8]. Carotenoids could also be precursors of norisoprenoids during fermentation and wine aging [9–11].

Lutein and β-carotene represent nearly 85% of the total carotenoids in grapes and they are mostly involved in degradation reactions in grapes, juice, and wine. The carotenoids directly involved in the aroma of wine are β-carotene, generating β-ionone, and neoxanthin generating β-damascenone. Lutein and violaxanthin also undergo breakdown reactions that may produce norisoprenoid compounds in wines [3,12]. Lutein, for instance, is reported to be an important precursor of 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) [13] while the formation of megastigmane-3,9-diol and of 3-oxo-α-ionol glucosides from the ε-cycle of lutein has also been illustrated [14]. Moreover, the involvement of lutein epoxide (Lx) cycle, an additional slower and reversible mechanism of photoprotection that supplements the violaxanthin cycle (violaxanthin and zeaxanthin), was recently demonstrated [8,15]. Authors observed a higher accumulation of lutein 5,6-epoxide in shaded berries [15] that is de-epoxidized to lutein following normal light aspect [8]. Because of the activation of the xanthophyll and Lx cycles at the end of the ripening period [14], processes of bioconversion between different carotenoids, also induced by modified light conditions, may take place, and influence the formation of norisoprenoids [6,14,16].

Generally, carotenoids are thought to be mostly synthesized between berry set and veraison. For this reason, the aromatic profile of the wine also depends on the carotenoid composition of immature berries while the end of veraison (and not the beginning as thought in the past) appears to be a key moment for the changes in the ratio carotenoid norisoprenoid [17]. Several variables may promote the norisoprenoid final content in grapes: some favor carotenoid synthesis during the herbaceous phase of berry growth, some others stimulates their degradation to norisoprenoids occurring from veraison onwards [16,18]. Therefore, carotenoid evolution during ripening can be considered as an indicator of grape aromatic potential [2,19,20].

As reported in many studies, the concentration of carotenoids in ripe grapes depends on cultivars [21], ripening stage, climate region [22], altitude [23], soil water retention capacity [21], and degree of bunch exposure to sunlight [15]. The highest carotenoid concentration occurred in the hot regions, likely due to the higher amount and intensity of the received solar radiation [22,24]. In warm climates, the level of β-carotene at harvest resulted as three–six-fold higher than that of lutein [18,19,25]. In particular, light is the main factor responsible for the changes in the biosynthesis of carotenoids [4] promoting both their accumulation before veraison and causing their degradation during ripening [26]. The degree of the bunch exposure to sunlight appeared to influence the ratio epoxyxanthophylls: non-epoxyxanthophylls whereas high UV-B levels favored carotenoid degradation [23,27], actually, higher rates of degradation emerged during the hotter period of grape ripening [28].

Furthermore, vineyard topographic features, such as slope gradient and aspect or altitude, along with the heterogeneity of vineyard vigor and different vine management may generate a great variability in microclimatic conditions (light, temperature, and humidity) within vineyards, canopy and bunch zone, likely influencing grape ripening and quality. The impact of the vineyard microclimatic characteristics on Nebbiolo grape development, ripening and anthocyanin accumulation, as well as on the evolution of grape norisoprenoid precursors has already been investigated [29–31]. Until now, only one study regarding the evolution of the carotenoid compounds in Nebbiolo grapes has been carried out [28].

Many studies explored the impact of artificial regulation of the bunch exposure to sunlight, by leaf removal or other canopy manipulation, on grape metabolic composition [15,22,23,32]. Nevertheless, integrated studies focusing on the impact of vineyard aspect and natural vine vigor on bunch microclimate and grape aroma precursors are lacking.

Therefore, the aim of this study was to complete a previous research by assessing the concentration and seasonal accumulation pattern of the most relevant carotenoids in Nebbiolo grapes as well as to study the link between bunch microclimate and carotenoid evolution during ripening.

#### **2. Materials and Methods**

#### *2.1. Vineyard Site and Treatments*

This study is complementary of a previous research on Nebbiolo grapes [31]. The experimental vineyard, site and treatments are widely described in the cited article. Briefly, the study was performed during 2012 and 2013 in two commercial vineyards located in North-West Italy (44◦36 04" N, 8◦00 34" E; 428 m above sea level). Four vineyards differing in terms of slope aspect (South and West) and natural vine vigor (two level of vigor in each vineyard: V+ and V−) were identified by assessing the Normalized Difference Vegetation Index of the parcel, as previously described [33]. In 2012, the vineyards compared were SouthV+, SouthV−, WestV+; in 2013, the WestV− vineyard was included. In each vineyard, three replicates of 50 vines were used for berry sampling.

#### *2.2. Microclimate Assessment*

The thermopluviometric characterization of the two seasons was assessed by bioclimatic indices calculated by the observation of an agrometeorological station belonging to the Regione Piemonte network. In order to characterize the four microenvironments in terms of radiation and thermal conditions, Photosynthetically Active Radiation (PAR) and air temperature inside the bunch zone were measured at intervals of 20 each, from pea size stage to harvest as described in the previous research and according to an established protocol [31,33]. Then, the integral of daily amount of PAR (SPAR) and maximum daily temperatures (ST) and other cumulative thermal indices, such as Normalized Sum of the Degrees Celsius (SD) and Number of Hours (NH), were calculated over four periods corresponding to different phases of berry growth. The daily values of SPAR [MJ m−<sup>2</sup> d−1] were obtained by the cumulative sum of the hourly mean values of PAR irradiance [J m−<sup>2</sup> s−1] multiplied by 3600 s. NHs were the number of hours over the considered period in which the mean hourly maximum temperature (Tmax, [◦C]) met three established ranges: Tmax ≥ 15 ◦C and Tmax < 25 ◦C (NH15 - 25); Tmax ≥25 ◦C and Tmax < 35 ◦C (NH25 - 35); Tmax ≥ 35 ◦C (NH ≥ 35). The same thresholds were used to calculate the SD indices by adding together the mean hourly maximum temperature (Tmax, [◦C]) that were simultaneously higher than the minimum value of the threshold and lower than the maximum one, thus, SD15 - 25, SD25 - 35 and SD ≥ 35 respectively were assessed. To eliminate the influence of the period (from early summer to early autumn) in which the data were recorded, and of the different length of each period, the values of all variables were normalized by Equation (1):

$$\text{Normalized value} = \text{(VALUE} - \text{MEAN}) \text{(MAX} - \text{MIN)} \tag{1}$$

where VALUE was the value of the variable in a specific vineyard in a specific period; MEAN, MAX, and MIN, were, respectively, the mean, the maximum, and the minimum values of the variable calculated over all the vineyards in the considered period.

#### *2.3. Berry Sampling*

Four subsequent samplings of 400 berries were carried out randomly on each vineyard replicate, from about BBCH code75 (five–six weeks after bloom) until harvest [31]. In more detail:, in 2012, samplings were conducted on 12 July (about 24 days before veraison: dbV), 31 July (about 5 dbV), 27 August (about 22 days post veraison: dpV), 5 October (about 60 dpV); in 2013, 22 July (about 25 dbV), 21 August (about 5 dpV), 9 September (about 24 dpV), 15 October (about 60 dpV). For carotenoid analysis, three replicates of 50 g of berries were analyzed as described below.

#### *2.4. Extraction and Determination of Carotenoids*

#### 2.4.1. Extraction from Grape Material

The procedure of carotenoid extraction was adapted from the method of Oliveira and others [21], as optimized by Crupi and collaborators [34]. Approximately 50 g of fresh berries, without seeds, added of 100 mg Na2S2O5, were homogenized for 5 min in presence of magnesium carbonate basic. The homogenate was spiked with 200 μL of 183.2 mg/L of β-apo-8-carotenal (Fluka, Porto, Portugal, ref. 10,810) as internal standard, and diluted with 40 mL of water (Milli-Q, Millipore). Extraction was first carried out with 40 mL of ether/hexane (1:1, *v*/*v*), agitating for 30 min, then repeated twice with further 20 mL of ether/hexane. The upper layer was separated each time. The final extract was concentrated to dryness at 20 ◦C (Laborota 4001, Heidolph instruments) and resuspended in 1 mL of acetone/hexane (1:1, *v*/*v*) for High Performance-Liquid Chromatography (HPLC)/DAD determination. Each sample was injected in duplicate. Sample handling, homogenization, and extraction were carried out on ice under dim yellow light to minimize light-induced isomerization and oxidation of carotenoids.

#### 2.4.2. High Performance-Liquid Chromatography (HPLC) Determinations

An Agilent Model 1200 quaternary solvent system equipped with quaternary pump solvent delivery and a UV-visible photodiode array detector was used. The absorption spectra were recorded at 447 nm and the sample injection volume was of 20 μL. The reversed stationary phase employed was a Lichrospher 100 RP C18 (5 μm) LichroCART (250 × 4 mm i.d.). Mobile phase was performed with solvent A: acetone/water (70:30 *v*/*v*), solvent B: acetone 100% (Sigma pure-grade), flow rate = 1 mL min<sup>−</sup>1. The analytical gradient was: 0–20 min (from 100% to 0% of A), then from 20 to 30 min isocratic with 100% of B [25].

#### 2.4.3. Identification and Quantification

The most relevant carotenoids were identified by comparison of UV-visible spectra with those of commercially available standards, β-carotene (Sigma 95%, synthetic,) (C-9750), lutein (Sigma 70%, from alfalfa) neoxanthin (0234.1) from CaroteNature GmbH (Erlenauweg 17, 3110 Münsingen, Switzerland), matching also different information such as position of absorption maxima (λmax) and the degree of vibration fine structure (% III/II) (Table 1) [34].


**Table 1.** High Performance-Liquid Chromatography (HPLC)/DAD characteristics of carotenoids found in Nebbiolo grapes.

β-Carotene 11.82 (428); 452; 478 25 a % III/II is the ratio of the height of the longest-wavelength absorption peak, designated III, and that of the middle absorption peak, designated as II, taking the minimum between the 2 peaks as baseline.

Quantification of individual compounds was done by calibration curves using the respective standards for lutein and β-carotene with R<sup>2</sup> = 0.9997 and R<sup>2</sup> = 0.9991, respectively, whereas neoxanthin was expressed as lutein equivalent because of the unavailability of a fresh neoxanthin standard. The results were expressed in terms of concentration (mg kg−<sup>1</sup> of berries) and content (μg berry−1) to avoid an overestimation of the changes that may be the result of an altered berry surface area to volume ratio.

#### *2.5. Statistical Analyses*

Data were statistically analyzed by ANOVA (SPSS 15.0 for Windows, Chicago, USA and SAS 9.4 SAS Institute, Cary, USA), Tukey's test was used to assessed the differences as regards microclimatic variables and carotenoids both among treatments for each sampling date and between sampling dates for each treatment; the general effect of factors such as vineyard microenvironments (by treatment) and seasons (by year) and their possible interaction (year\*treatment) were assessed too. Moreover, a 3-way-ANOVA, as regards carotenoid data of South treatments, was assessed, in order to evidence the vigor effect and interactions between year, sampling date and vigor level.

A preliminary ANOVA on normalized microclimatic variables (SD, NH, ST:SPAR) assessed the differences among sampling periods and the opportunity that these latter could be used as replicates for the comparison of vineyard microclimates and seasons. No differences among the periods emerged for none of the microclimatic variables; thus, the periods were used as replicates when microenvironments and seasons were compared. A Hierarchical Clustering Analysis (HCA) was also carried out using the method of centroide distance to evaluate the clustering of the vineyards based on the microclimatic conditions.

With the aim of identifying a model able to describe the impact of microclimate on berry composition, several Principal Component Analysis (PCA) were also performed on both microclimate (NH, SD, ST:SPAR) and carotenoids related variables including concentration (mg kg−1), content (μg berry−1), proportion (%) of lutein, β-carotene and neoxantin, lutein:β-carotene ratio, and sum of carotenoids (lutein+β-carotene+neoxanthin) as mg kg−<sup>1</sup> of berries and μg berry<sup>−</sup>1. The results reported here, refer to the data set of variables that explained the higher amount of the model variance. HCA and PCA were performed by SAS 9.4 (SAS Institute, Cary, USA).

#### **3. Results**

#### *3.1. Seasonal Meteorological Trends*

The two seasons presented some peculiarities from the meteorological point of view. In terms of annual values, the mean minimum and maximum temperatures were higher in 2012 than in 2013, as well as the average of the maximum monthly temperature that in 2012 exceeded the 2013 value by 2.7 ◦C. In the summer of 2012, the maximum temperatures exceeded 30 ◦C for 78 days, whereas in 2013 for 66. The hot condition of 2012 was also certified by the cumulative amount of Growing Degree Days (GDD) which exceeded the value of the 2013 by approximately 10%. Moreover, 2012 was drier than 2013, both in terms of rainfall amount and number of rainy days (> 1 mm). The differences between the years were also reflected on the growing period (Table 2). The warmest condition of 2012 affected the timing of the phenological phases that occurred earlier in 2012 than in 2013 [31]. In 2013, in fact, a delay of around 10 days for bud burst, a couple of weeks for bloom, a week for veraison and 10 days for commercial harvest was observed in comparison to 2012.

#### *3.2. Bunch Microclimate*

The thermal and radiative normalized indices calculated for each sampling date, were able to separate the two vineyard aspects when a cluster analysis was carried out, whereas clear separations between the levels of vigor, between seasons and among sampling dates were not evident (Figure 1 HCA). In more detail, ANOVA analysis, showed that differences among the four environments emerged for singular variables in both years (Table 3). When negative, the normalized indices indicated a negative difference compared to the average value calculated for all vineyards, and vice versa when positive. The higher the absolute values of the index, the higher were the differences. In 2012, the year with higher temperatures and lower rainfall SD15 - 25, NH15 - 25, and ST:SPAR were negative in the vineyards facing south, therefore, lower than the average calculated on all the vineyards, and they were significantly different from those of the vineyards facing west. In addition the vigor of the plants affected NH15 - 25 and ST:SPAR indices of the south facing vineyards showing both a lower value in

the less vigorous condition (V−). No differences among vineyards emerged for the indices referring to the other temperature ranges.

**Table 2.** Meteorological characterization of the two seasons (the values are calculated both for the entire year and for the grapevine vegetative period: April–October). Data were registered by Serralunga Boscareto's meteorological station (Agrometeorological Network, Regione Piemonte).


<sup>1</sup> Days with precipitation over 1 mm.

**Figure 1.** Dendrogram of hierarchical clustering analysis of the four microenvironment (SV−, SV+, WV−, and WV+) obtained by analyzing the meteo-climatic indices reported in Table 2. (1, 2, 3, and 4, as the first digit in the treatment acronym, correspond to the phenological period; 2, 3, as the second digit, correspond to the season 2012 and 2013, respectively; W and S represent the West and South vineyard aspect, respectively; V+ and V− indicate a higher or lower plot vigor, respectively).


**Table 3.** Normalized Sum of the Degrees Celsius (SDs), number of hours (NHs) related to the temperature ranges (15– 25; 25– 35; ≥35) and ST:SPAR; ST=summation of the maximum daily temperatures; SPAR=summation of the daily integrals of PAR.

For the same year, means fallowed by different letters are significant different for *p* < 0.05. ns—not significant; \* *p* < 0.05, \*\*\* *p* < 0.001 indicate the significance of differences among treatments.

In 2013, the coolest and wettest year, the differences emerged in the first year were confirmed. Furthermore, SD ≥ 35 and NH ≥ 35, were higher in the vineyards facing south and, in particular, in the less vigorous ones. On the contrary, in the vineyards facing west, the influence of vigor did not emerge. Therefore, differences among vineyards were evident for the indices relating to the lowest and highest temperature ranges whereas for the intermediate range (25 - 35) both SD and NH were similar in all the vineyards. For none of the indices, differences between the years emerged while the year \* vineyard interaction was significant only for SD ≥ 35 and NH15 - 25.

#### *3.3. Evolution of Carotenoids in Nebbiolo Grapes*

The compounds that mostly contributed to the total amount of carotenoids in Nebbiolo grapes were lutein and β-carotene. The evolution of each compound during ripening, both as μg berry−<sup>1</sup> (Figure 2a, Figure 3a, and Figure 4a) and as mg kg−<sup>1</sup> of fresh berries (Figure 2b, Figure 3b, and Figure 4b), is shown for both years 2012 and 2013 (Table 4).

### • Lutein

In 2012, at the first sampling date, at 24 days before veraison (dbV) similar amounts of lutein were detected in green berries from both west and south exposed vineyards (Figure 2a). Lutein concentration (mg kg−<sup>1</sup> of berries) and content (μg berry<sup>−</sup>1) remained then constant until the beginning of veraison (5 dpV, second sampling) (Figure 2a,b and Table 4). In 2013, at the first sampling (25 dbV), the lutein concentration (mg kg<sup>−</sup>1) was significantly higher in SouthV+ when compared to the other treatments and to the previous year. Afterwards, at the second sampling (5 dpV), lutein content decreased significantly only in south-exposed vineyards (Figure 2a,b, Table 4).


**Table 4.** Seasonal changes of the carotenoid concentration and of the ratio Lutein: β-carotene in Nebbiolo grapes during 2012 and 2013.

For the same line and year means followed by different letters indicate significant differences among sampling dates for 2012 and 2013 respectively. \*, \*\*, \*\*\* indicate, respectively, significant differences for *p* < 0.05, *p* < 0.01, and *p* < 0.001. nd—not detectable, ns—not significant, sign §—statistical differences by date, by T—Treatment; by Y—year; T\*Y—interactions between Treatment and Year; 1—Lutein+β-carotene+neoxanthin.

Concurrently to the increase of the berry weight and sugar concentration [31], a significant increase of lutein content per berry was noticed in both years reaching a peak at about 4 weeks after veraison, with the exception of SouthV− in 2013. This increase of lutein content was proportional to the vine vigor, thus more important for the most vigorous vines. After veraison, in SouthV− a minor increase was observed in 2012 and a constant trend in 2013, thus, at the time of the peak, significant differences were noticed between WestV+ and SouthV− in both seasons. At the final stage of ripening, a significant lutein degradation was observed for all treatment in 2012; significant differences between SouthV− (1.66 mg kg−<sup>1</sup> of berries or 2.78 μg berry−1) and WestV+ parcels (3.75 mg kg−<sup>1</sup> of berries or 6.78 μg

berry<sup>−</sup>1) were found at harvest (Figure 2a,b and Table 4). The concentration decline observed in 2013 was less remarkable for all vineyards compared to 2012, resulting significant only for the most vigorous plots, WestV+ (mg kg−<sup>1</sup> of berries) and SouthV+ (μg berry<sup>−</sup>1). The content of lutein in the less vigorous SouthV<sup>−</sup> at harvest was significantly lower (4.4 <sup>μ</sup>g berry−<sup>1</sup> or 2.52 mg kg−<sup>1</sup> of berries) than in the other vineyards. Thus, the warmest vineyard had the lowest lutein concentration at harvest in both years. Lower amounts of lutein were detected at harvest in 2012 for all treatments, but significantly lower only for SouthV−, when compared to 2013 (average at harvest, 2.69 mg kg−<sup>1</sup> and 4.73 <sup>μ</sup>g berry−<sup>1</sup> in 2012; 3.61 mg kg−<sup>1</sup> and 6.7 μg berry−<sup>1</sup> in 2013). The interaction year\*treatment was not significant regardless of the unit (Table 4).

**Figure 2.** Seasonal changes of lutein as μg berry−<sup>1</sup> (**a**) and mg kg<sup>−</sup>1of berries, (**b**) in Nebbiolo grapes in 2012 (on the left) and 2013 (on the right), as a function of vine vigor and vineyard aspect. Averages ± standard error (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01 indicate the significance of differences among treatments.

• β-Carotene

In 2012, the β-carotene concentrations (mg kg−<sup>1</sup> of berries) decreased during the season with no significant differences among treatments at any sampling point except at harvest, when significantly higher amounts were noticed in the WestV+ (4.07 mg kg−<sup>1</sup> or 7.33 <sup>μ</sup>g berry−1) than in the SouthV<sup>−</sup> grapes (2.54 mg kg−<sup>1</sup> or 4.24 μg berry<sup>−</sup>1). In 2013, the β-carotene decline between the first and second sampling was significant for south-exposed vineyards while the post veraison increase of β-carotene content in 2013, was significant only for the SouthV+ grapes (Figure 3a,b, Table 4). β-carotene content (μg berry−1) and concentration (mg kg−<sup>1</sup> of berries) were significantly lower in SouthV<sup>−</sup> than in

WestV+ both at the third sampling and at harvest (3.2 mg kg−<sup>1</sup> or 5.6 <sup>μ</sup>g berry−<sup>1</sup> for SouthV<sup>−</sup> and 4.2 mg kg−<sup>1</sup> or 7.9 μg berry−<sup>1</sup> for WestV+). Moreover, differences between the two years at harvest, were not significant when comparing the values as mg kg−<sup>1</sup> (averagely 3.41 versus 3.60, in 2012 and 2013, respectively) but they were significant when comparing the values as μg berry−<sup>1</sup> (5.94 versus 6.7, in 2012 and 2013, respectively). The interaction year\*treatment was not significant regardless the unit (Table 4).

(**b**)

**Figure 3.** Seasonal changes of β-carotene as μg berry−<sup>1</sup> (**a**) and mg kg−<sup>1</sup> of berries (**b**) in Nebbiolo grapes in 2012 (on the left) and 2013 (on the right), as a function of vine vigor and vineyard aspect. Averages ± standard error (n = 3). \* *p* < 0.05, \*\* *p* < 0.01 \*\*\* *p* < 0.001 indicate the significance of differences among treatments. b.

### • Neoxanthin

In the green berries, undetectable amounts (2012) or traces (2013) of neoxanthin were observed for all treatments. The important increase after veraison, observed for the most vigorous plots was more remarkable in 2012 than in 2013, and was then followed by a significant degradation of this compound until harvest. In 2012, as for lutein and β-carotene, significant differences were observed at harvest between SouthV<sup>−</sup> (0.11 mg kg−<sup>1</sup> of berries or 0.18 <sup>μ</sup>g berry−1) and WestV+ grapes (0.25 mg kg−<sup>1</sup> of berries or 0.46 μg berry−1). Similarly to lutein, a peak of concentration was achieved 4 weeks after veraison but, differently from lutein and β-carotene, was higher in 2012 (averagely 0.49 μg kg−<sup>1</sup> and 0.81 μg berry<sup>−</sup>1) than in 2013 (averagely 0.30 μg kg−<sup>1</sup> and 0.49 μg berry<sup>−</sup>1) (Figure 4).

**Figure 4.** Seasonal changes of neoxanthine as μg berry−<sup>1</sup> (**a**) and mg kg−<sup>1</sup> of berries (**b**) in Nebbiolo grapes in 2012 (on the left) and 2013 (on the right), as a function of vine vigor and vineyard aspect. Averages ± standard error (n = 3). \* *p* < 0.05, \*\* *p* < 0.01 indicate the significance of differences among treatments.

In both years, this peak was followed by a significant degradation of neoxanthin until harvest period in SouthV+, WestV−, and WestV+ (Figure 3a). Moreover, in both years, a high degradation rate was highlighted especially for the most vigorous SouthV+ that influenced neoxanthin amount at harvest time. In SouthV−, instead and similarly to lutein, neoxanthin levels followed a more flattened evolution showing a minor peak mostly in 2013. In both years after veraison, SouthV− attained the lowest values compared to the other treatments especially when μg berry−<sup>1</sup> were considered. The vine vigor (V−, V+) did not affect the neoxanthin content at harvest. Differences between the years were not significant; the interaction year\*treatment was significant when values were expressed as μg berry−<sup>1</sup> (Figure 4, Table 4).

### • Lutein: β-Carotene Ratio and Sum of Carotenoids

The ratio lutein: β-carotene increased between green phase (first sampling) and complete veraison (third sampling), whereas a decrease was evident during the weeks preceding commercial harvest. In 2012, the ratio was in favor of β-carotene whereas in 2013 the ratio was often in favor of lutein. Comparing both years, the ratio varied at harvest from 0.65 to 0.79 in the warm and more exposed plots (SouthV−) and from 0.93 to 1.13 in the cooler and more vigorous WestV+. In average, the difference between years was significant as well the interaction years\*treatments (Table 4). In general, the sum of the concentration (mg kg−1) of the three considered carotenoids, decreased from the first to the last sampling, whereas, when expressed as μg berry<sup>−</sup>1, a peak was observed at the third sampling in both years. However, at harvest in 2012 and at all sampling dates in 2013, the sum of the carotenoids (concentration and content) was lower in SouthV−, than in WestV+.

Summarizing, the season significantly influenced all the variables except the neoxanthine and β-carotene concentration. Regardless the season, the treatment significantly influenced the content of all compounds. Nevertheless, the interaction year\*treatment was significant only for neoxantine (μg berry<sup>−</sup>1) and lutein: β-carotene ratio. Focusing on the results of the 3-way ANOVA, carried out on the south facing vineyards, significant differences between the two levels of vine vigor emerged for all compounds, but neither the interaction year\*vigor nor year\*vigor\*sampling date were significant, regardless the unit of measurement of carotenoids (Supplementary Table S1).

Performing a PCA on a data matrix including microclimatic indices and berry carotenoids an effective distribution of the vineyards along the first component (PC1), and of the sampling dates along the second component (PC2), emerged (Figure 5).

**Figure 5.** Score plot of the observations in the Cartesian coordinate system identified by Principal Component Analysis: analysis including variables related to carotenoids and microclimatic indexes (the variables included in the analysis are reported in Table 4). 1, 2, 3, and 4 refer to the data sampling; S—South, W—West, V+—high vigor, V−—low vigor. Symbols in black color—2012; symbols in grey color—2013; •—SouthV−, -—SouthV+, —WestV−, and —WestV+.

Prin1 and Prin2 explained, respectively, 39% and 27% of the total variance. The case distribution along Prin1 was well represented by a linear combination of the variables NH15 - 25, ST:SPAR and sum of carotenoids (positively correlated with Prin1). The case distribution along Prin2 was well represented by a linear combination of the variables Neoxanthine (%) and lutein: β-carotene ratio (positively correlated with Prin2) (Table 5).

**Table 5.** Principal Component Analysis (PCA): Percentage of variance explained by the three principal components (Prin1, Prin2, and Prin3), eigenvalues and loadings indicating the correlation between the variables and the three principal components.


#### **4. Discussion**

The two seasons of the study were different from a weather point of view. In fact, 2012 was generally warmer and drier than 2013 (Table 1). The conditions of 2013 enhanced vine vigor avoided summer water stress and delayed the timing of all phenological phases. Nevertheless, dry and warm condition from mid-September to mid-October prolonged vine metabolic activity, and allowed it to reach an optimal level of berry ripening.

The thermal and radiative microclimate indices allowed separating south facing vineyards from the west facing ones, but neither the sampling dates, nor the vigor levels (Figure 1). In general, the southern vineyards were the hottest even if, as evidenced by the significant interaction year\*treatment for SD ≥ 35 and NH15 - 25, the differences between the south and west vineyards were amplified in the cooler and wetter season (2013) whereas they were mitigated in the warmer season (2012). ST:SPAR was smaller in the southern than in the western vineyards in both years. This index is an expression of two synergistic effects. Firstly, in hilly conditions such as those of the study, the southern aspect intercepted higher amount of solar radiation (higher values of SPAR). Secondly, the west side of the hill registered the maximum daily temperature (thus, the high value of ST) when, in the afternoon, the Sun's rays were perpendicular to the slope. This index showed a good potential in characterizing the microclimates and it contributed to their separation when inserted into the dataset analyzed by PCA.

The PCA analysis conducted on both microclimatic indices and carotenoid compounds, effectively separated the west facing vineyards from the south facing ones and, in the latter case, also the two levels of vigor (Figure 5). The vineyards separation along Prin1 was determined by the sum of the carotenoids and by both the microclimatic indices NH15 - 25 and ST:SPAR, whose values were greater in the west facing plots. All these variables were positively correlated with Prin1 (Table 5), thus, the overall amount of the carotenoids was favored by cooler conditions. The differences between the vintages and between the levels of vigor were evident in the warmer vineyards where the intercepted radiation was greater (South) but not in the fresher ones (West) whose conditions were evidently less favorable for the degradation of carotenoids (Figure 5). This observation led to think that the negative effect of higher temperatures prevails over the positive effect of a higher interception of radiation on grape carotenoid concentration at harvest. In the first part of this study [31], higher amount of C13 norisoprenoids was found in the sunniest year and in the more sunlit vineyards. The decrease in the concentration of those compounds in the warmer SouthV− plot in the last ripening stage was likely attributable to the effect of high temperatures. This decrease was more evident for some compounds and less for others, thus microclimate had an effect on both concentration and profile of C13-norisoprenoids in Nebbiolo grapes [31] confirming, at least in part, results already issued [14].

The higher carotenoid content found in Nebbiolo grapes when compared to other varieties [35–38], agrees with the levels found so far for cv. Nebbiolo [28]. A peak of lutein concentration, that was proportional to the vine vigor, was noticed after veraison in both seasons. A similar peak was reported for Touriga Franca [21], Nebbiolo, and Barbera [28] and, more recently, for Merlot [36]. Therefore, it is possible that cultivar differences exist in the timing of lutein synthesis and degradation thus, a probable delay in VvCCD1 expression can be hypothesize for these varieties [8]. In the present study, the lutein peak after veraison was more evident in the most vigorous plants. This result may be attributable to the high vigor which often reflects a greater synthesis of chlorophyll so, probably, of carotenoids and/or to the lower degradation of this compound under the cooler conditions of these plants. Furthermore, a higher content of lutein-5,6-epoxide (not quantified in this study) in shaded berries prior the veraison can be presumed; the accumulation of this compound has been shown to be a plant early response to shade conditions [8,15]. Therefore, a higher transformation of this compound in lutein may occur after veraison. In both years, the peak of lutein after veraison was less evident in SouthV−; high temperatures and a higher exposure to sunlight probably promoted the carotenoid degradation in post veraison, as proposed in literature [39]. Lutein is also reported to be more efficient than violaxanthin in preventing ROS formation, thus, it could be further used by grapevines for photoprotection under stress conditions [40]. This could also explain the lower lutein concentration and its higher rate of degradation under more stressing condition, such as in south facing vineyard and in the warmer periods of the season, where high radiation and temperature (>35 ◦C) were achieved for many hours a day in both years [31]. As regards the other treatments, the west-exposed and the more vigorous plots maintained the highest amounts at harvest. Our findings agree with previous research reporting that grapes grown in shaded conditions [21,41] had higher carotenoid levels. On the contrary, under high UV-B levels, lower concentrations of total carotenoids were found [27].

In 2012, constant levels for β-carotene were observed during ripening in all experimental plots when results were expressed in μg berry−1, while a significantly lower amount was registered in SouthV− in both years at harvest. In 2013, instead, higher contents of β-carotene than in 2012 were highlighted in green berries (three weeks before veraison) and for the south facing vineyards a notable decrease was registered thereafter during veraison. The decline of β-carotene during that period was already observed for Nebbiolo and Barbera grapes and was attributed to the high temperature [28]. According to other studies, instead, β-carotene content in grapes, shows an increase from preveraison to veraison, and a decline thereafter until harvest [23,25,36]. In our study, the condition of west facing vineyards, less sunlit and lower temperature than in south ones, likely led to a lower degradation of this compound allowing the maintenance of a constant content (μg berry<sup>−</sup>1) in both years. Other research reported also a greater impact of microclimatic variations on lutein more than on β-carotene [42]. Our results showed a different impact of microclimate onβ-carotene if compared to the two xanthophylls since high temperatures of south vineyards in the second year led to a higher degradation of β-carotene during veraison more than at the final stages of ripening.

The warmer conditions registered 4 weeks after veraison in the second year of the study, probably penalized also the concentration (μg kg−1) and content (μg berry−1) of the neoxanthin that resulted particularly lower at the supposed peak moment, in the south-facing plots. The behavior of this compound was similar to lutein.

The lutein: β-carotene ratio is an indicator of flux to the a- and b-branches, respectively, of the carotenoid metabolic pathway [15]. In literature is reported an influence of the variety on this ratio; in some cultivars indeed (i.e., Syrah, Sauvignon, Pinot Noir, and Merlot), the lutein level was almost twice than that of β-carotene. In Chardonnay and Carignan, the concentration of the two carotenoids was very similar while a higher level of β-carotene was found in Grenache, Gamay, and Sauvignon blanc [15,25,26,38]. In addition, the growing region and topographic features of the site [34,38], as well as cultural practices, such as leaf removal [15], may also affect this ratio. The lutein: β-carotene ratio in

Nebbiolo grapes, varied during berry development also depending on season and vineyard aspect (Table 4). Lutein prevailed on β-carotene after veraison. However, this did not happen, neither in the early stages nor at harvest, nevertheless, in both years, the ratio in the later phases was lower in the warmest nor more exposed plot in accordance with previous research [15,19].

Recent research on the effect of bunch zone leaf removal on Sauvignon blanc [15] concerned mostly the light effect since irrelevant temperature differences within the bunches were registered. Under these conditions, the concentration of the major carotenoids decreased during berry development, following the behavior of chlorophylls, whereas specific xanthophylls, such as lutein, resulted more abundant during the early stages of berry development in berries more exposed to sunlight. In addition, as already reported, the intensity of solar radiation exerts a great role on the degradation of grape carotenoids [26]. Nevertheless, another study highlighted that the carotenoid concentration from veraison to harvest was positively correlated with temperature but less correlated with both rainfall and radiation [19]. According to our study, individual carotenoids respond in a different way depending on the microclimatic conditions of each specific period during ripening. Nevertheless, differently from the previous studies, a major synthesis of lutein occurred after veraison in the vigorous plots where both radiation and temperature were lower whereas in the warmer and more exposed plots (SouthV−), lutein accumulation and final content were penalized (Figure 2). β-carotene content was lower in the warmer period and in the more sunlit south plots with respect to the cooler and more shaded west plots, as well. Actually, the differences emerged between the growing seasons and between vineyard aspects highlighted the influence of the temperature on the rate of synthesis and/or degradation of β-carotene, (Figure 3, Table 4). In any case, the positive effect of radiation did not clearly emerge in our study since the highest peaks were recorded in the less sunlit vineyards (Figure 2).

According to the literature, the amount of berry carotenoids at harvest seemed to be more dependent on the condition of the earlier developmental stage that impact on their synthesis, rather than on the condition of the final phases that impacts their degradation [26]. As a result of the current study, the lower temperature of the early phases of 2013, favored the amounts of carotenoids in the green berries, even in conditions of lower radiation [31]. The particularly warmer condition of the period after veraison in the less vigorous south plots, promoted a higher degradation even with different rate depending on the compound. Nevertheless, a higher total content (μg berry<sup>−</sup>1) of carotenoids was averagely measured at harvest in 2013 than in 2012, confirming the importance of the accumulation phase on the final content at harvest [26].

#### **5. Conclusions**

This study confirms most results of previous studies and illustrates the effect of vineyard aspect and bunch microclimate on both synthesis and degradation of the most relevant grape carotenoids during berry development in Nebbiolo grapes. Normalized microclimate indices appeared useful to characterize growing seasons and the microenvironments and to explain the compositional differences between the examined environments.

Lutein and neoxanthin responded in a similar way to environment variability having a similar peak after veraison in both years, whereas a variety effect can be presumed as regards lutein trend during ripening. A different trend was observed for β-carotene content depending on season and vineyard aspect. Generally, the warmer conditions of the most sunlit south facing vineyards led to low amounts of all compounds at harvest. On the other hand, less warm conditions, like those of west-exposed vineyards or more vigorous vines, likely favored the synthesis and/or induced a lower degradation of carotenoid compounds. Nevertheless, carotenoids seemed to respond to microclimate variability differently depending on the compound. In addition, the amount of radiation accumulated in specific periods, and mainly the prolonged high temperatures during the last stages of grape ripening, determined the evolution of carotenoids during season and their profile and quantity at harvest. Furthermore, a repeatability of these results can be expected since the relationships observed among the environments were maintained in both years and despite the overall differences between them from a meteorological point of view.

Our results highlight also that in earlier vintages, driven by the climate warming, the grapes ripen in a warmer period when high temperature determine a higher degradation influencing considerably both berry carotenoid profile and concentration. Nowadays, winegrowers are called to face such warmer climatic conditions, therefore, our study add further knowledge to target vineyard cultural strategies in order to modulate aroma potential of grapes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/10/11/3846/s1, Table S1: Supplementary Table S1. Results of the 3-way ANOVA, carried out on the carotenoid content (μg berry-1) and concentration (mg kg-1) of the South facing vineyard considering the two levels of vigour (V+ and V-), the two seasons and the four sampling dates as factors of variability.

**Author Contributions:** Conceptualization, S.G.; Formal analysis, A.A., S.C., E.M. and S.G.; Funding acquisition, S.G.; Investigation, A.A. and E.M.; Methodology, A.A., S.C. and S.G.; Resources, M.P.; Supervision, S.G.; Validation, M.P. and S.C.; Writing–original draft, A.A.; Writing–review & editing, A.A., M.P., S.C., A.F. and S.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding

**Acknowledgments:** The authors would like to thank Fondazione Dalmasso and the Azienda Agricola G.D. VAJRA in Barolo, for supporting this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

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