*Review* **Emerging Non-Thermal Technologies for the Extraction of Grape Anthocyanins**

**Antonio Morata \* , Carlos Escott , Iris Loira , Carmen López , Felipe Palomero and Carmen González**

enotecUPM, Chemistry and Food Technology Department, Technical University of Madrid UPM, 28040 Madrid, Spain; carlos.escott@gmail.com (C.E.); iris.loira@upm.es (I.L.); carmen.lopez@upm.es (C.L.); felipe.palomero@upm.es (F.P.); carmen.gchamorro@upm.es (C.G.)

**\*** Correspondence: antonio.morata@upm.es

**Abstract:** Anthocyanins are flavonoid pigments broadly distributed in plants with great potential to be used as food colorants due to their range of colors, innocuous nature, and positive impact on human health. However, these molecules are unstable and affected by pH changes, oxidation and high temperatures, making it very important to extract them using gentle non-thermal technologies. The use of emerging non-thermal techniques such as High Hydrostatic Pressure (HHP), Ultra High Pressure Homogenization (UHPH), Pulsed Electric Fields (PEFs), Ultrasound (US), irradiation, and Pulsed Light (PL) is currently increasing for many applications in food technology. This article reviews their application, features, advantages and drawbacks in the extraction of anthocyanins from grapes. It shows how extraction can be significantly increased with many of these techniques, while decreasing extraction times and maintaining antioxidant capacity.

**Keywords:** non-thermal technologies; grapes; wine; anthocyanins; HHP; UHPH; PEFs; US; irradiation

#### **1. Introduction**

Anthocyanins are flavonoid pigments responsible for the color of many fruits, flowers and vegetable tissues. Extensive details on their properties and features can be found in the literature [1–9]. They have been extensively studied for their potential applications as natural colorants [10–12] as they are innocuous and safe molecules, but also for their positive impact on health due to their antioxidant properties [13,14] and their effect on the gut microbiome [15]. Anthocyanin color depends on the substitution pattern in the B-ring and the acylation patterns, both of which affect the electron density and the observed color, ranging in grapes from red orange (brownish red) to bluish red (purple), with typical ranges from 518 nm of maximum absorption for cyanidin to 528 nm for malvidin [16]. Acylation normally increases the maximum absorption (e.g., malvidin 528 nm to coumaroyl malvidin 535 nm) (Table 1). The color of anthocyanins is also affected by low pH, which increases the color intensity by the hyperchromic effect, shifting the equilibria to increase the amount of pyrilium cation. Additionally, anthocyanins can also undergo SO<sup>2</sup> bleaching and co-pigmentation processes that produce bluish red pigments by bathochromic shifts in the maximum wavelength of absorbance [16].

In most grape varieties, anthocyanins are located in the exocarp (skins) (Figure 1A,B), which are the layers of cells in the outer surface of the berry; only a few varieties also have anthocyanins in the pulp [16]. The skins have a thicker cell wall than the pulp to protect the berry mechanically and against rot and pests.

**Citation:** Morata, A.; Escott, C.; Loira, I.; López, C.; Palomero, F.; González, C. Emerging Non-Thermal Technologies for the Extraction of Grape Anthocyanins. *Antioxidants* **2021**, *10*, 1863. https://doi.org/ 10.3390/antiox10121863

Academic Editors: Agustín G. Asuero and Noelia Tena

Received: 2 November 2021 Accepted: 22 November 2021 Published: 23 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).



Molecular structure

<sup>1</sup> Obtained experimentally with HPLC-DAD-ESI/MS; <sup>2</sup> From [17].

**Figure 1.** (**A**) Red grape skin (exocarp) *Vitis vinifera* L. Tempranillo variety by 60 µm optical camera built-in part of the AFM. (**B**) 3D Topography of the same skin by atomic force microscopy.

The structure and shape of the cells in the berries are flat cells in the skin and large polyhedral cells in the pulp (Figure 2A). Anthocyanins are located in the cells of the skin,

inside the vacuole (Figure 2B). To extract the anthocyanins and to keep enough color (not only in red winemaking, but also in red juice production), it is necessary to disaggregate the cell wall polysaccharides, mainly the pectins (Figure 2C). In conventional winemaking, depolymerization of the cell wall and separation of polysaccharide fibers is achieved during maceration by means of soaking, fermentation temperature and mechanical treatments (i.e., punch downs, pump overs, délestage) [18].

**Figure 2.** (**A**) Red grape section with flat colored cells in the skins and polyhedral cells in the pulp. (**B**) Skin cells shape and structure. (**C**) Cell wall fiber components.

In addition, cryomacerations (cold soak) by heat exchanger cooling or dry ice can be used to preferentially extract anthocyanins and aroma compounds in the absence of fermentation [19]. This is advantageous because it reduces the extraction of tannins whose solubility is lower in the absence of alcohol and better reduces astringency in young wines and juices. Another powerful technology to quickly degrade cell wall pectins and promote the extraction of anthocyanins, tannins and aroma compounds is the use of pectolytic enzymes [20], especially endo-polygalacturonases that break the pectin sequence depolymerizing the cell wall and releasing the pigments in the juice.

Currently emerging non-thermal technologies are increasingly being used to improve the extraction of bioactive compounds, such as anthocyanins, from food products, but also to decrease or eliminate spoilage or pathogenic microorganisms and sometimes even to inactivate oxidative enzymes [21,22]. Among them, continuous and discontinuous high-pressure technologies (i.e., High Hydrostatic Pressure (HHP) and Ultra High Pressure Homogenization (UHPH) [23–25], Pulsed Electric Fields (PEFs) [26–29], Ultrasound (US) [30–34] and β-irradiation [35–37].

This review is focused on the features of emerging non-thermal technologies that make them suitable for the extraction of anthocyanins from grape skins while protecting their natural coloring and antioxidant properties.

#### **2. Use of High-Pressure Technologies to Extract Anthocyanins**

The use of high-pressure technologies is growing exponentially in the food industry. A PubMed search using the keywords high, pressure and food yields 40,077 research articles in the period 1970–2021 with 36,920 since 2000. Although several technologies can be found, research with continuous (Ultra)-High Pressure Homogenization processes (UHPH and HPH) and discontinuous High Hydrostatic Pressure (HHP) technologies stand out. All

of them share gentle food processing as they are non-thermal treatments with low impact on food quality, sensory constituents and nutraceutical components [25,38–41]. HHP and UHPH technologies are industrially implemented and several brands compete in the market. In batch technologies, the leading companies are Hiperbaric (https://www.hiperbaric. com/es/ (accessed on 2 November 2021)) and Avure (https://www.jbtc.com/es/northamerica/foodtech/products-and-solutions/brands/avure-technologies (accessed on 2 November 2021)). In UHPH (continuous processing), the most effective technology is the one developed by Ypsicon (https://www.ypsicon.com/ (accessed on 2 November 2021)). These technologies have specific characteristics that will be described separately below.

#### *2.1. High Hydrostatic Pressure (HHP)*

HHP involves the application of high pressures to the food by means of a fluid (hydrostatic), which is usually water. The fluid is pumped into a high-strength steel vessel containing the food product, where pressures above 100 MPa, commonly in the range of 400–600 MPa, are reached during processing [42]. HHP treatments consist of pressurizing the food product in this pressure range for 2–10 min. The main effect of this is the destruction of the cell walls and membranes of microorganisms, but plant and animal tissue cells are also similarly affected. HHP can be considered a non-thermal technology because, even when compression produces adiabatic heating, this is quite moderate and ranges between 2 and 3 ◦C/100 MPa. This slight increase in temperature can be controlled by cooling the vessel or lowering the temperature of the food at the inlet. Moreover, HHP processing is not able to affect covalent bonds, so pigments, aroma and flavors are usually protected [24,38,42].

The effect of HHP on plant tissues is to damage the integrity of the cell walls, resulting in small pores or fissures that can facilitate the extraction of metabolites from the cell wall. HHP has been used to enhance the extraction of anthocyanins from grapes [23,24] and grape pomace [26]. The extraction of anthocyanins by HHP has been increased compared to controls in the range of 23–82% (Table 2). The extraction of phenolic compounds (tannins) is also increased in grapes, with a total polyphenol index of +26% [24], and the antioxidant activity of the extracts is higher than in controls [23,26]. HHP is a powerful technology to extract anthocyanins from plant tissues, and specifically from grape skin, preserving or enhancing the antioxidant capacity of the extracts, working at low temperatures (<30 ◦C, at 550 MPa for 10 min [24]), even under refrigeration. Furthermore, anthocyanin extraction has been reported to be selective depending on the acylation pattern and the methoxilation ratio on the B ring [23]. In grapes, anthocyanin migration from the skin to the pulp and seeds is observed after HHP treatment (Figure 3), which is evidence of cell wall poration and anthocyanin migration into the berry under the effect of pressure [24]. HHP causes the intensification of mass transfer phenomena, thus affecting cell permeability and molecule diffusion [43]. However, the external shape and structure of the berry are completely preserved (Figure 3). It is noticeable that similar extractions can be achieved in the range of 200–550 MPa, so it is possible to use milder HHP conditions, making the process cheaper while working at lower temperatures by adiabatic compression. Lower temperatures help to protect the anthocyanins during extraction and later in the ongoing process [44], probably reducing the risk of oxidation that can occur under thermal conditions.

The processing of grapes by HHP can be done with whole grapes (Figure 3), but it is also possible to process the separated skins that can be obtained from by-products such as pomace. Additionally, the stability of anthocyanins can be improved by using additives such as ethanol or other preservatives. HHP processing of berries also helps to sanitize them by easily removing yeasts and highly reducing bacterial loads [21,24]. This helps to obtain healthier anthocyanin extracts with reduced microbial loads, which facilitates the implantation of starters if these extracts are subsequently used in fermented foods [45]. Additionally, gentle extraction together with inactivation of microorganisms and higher antioxidant activity can reduce the use of antioxidants such as sulfites in the extracts, and also in the subsequent use of these extracts in food products [21,46].

**Figure 3.** External shape and appearance of control and pressurized grapes (200 MPa, 10 min), and details of the internal structure showing colored pulp and seeds in HHP-processed grapes.

**Table 2.** Emerging non-thermal technologies, processing conditions, and effects on the extraction of anthocyanin.



#### **Table 2.** *Cont.*

#### *2.2. Ultra High Pressure Homogenization (UHPH)*

UHPH consists of the continuous pressurization of a fluid to 200 MPa or more, through a special valve, and its subsequent release at low pressure (usually atmospheric pressure) [25,41,60,61]. Typical processing conditions are the use of 300 MPa with valve residence times less than 0.2 s. The process can be heat-assisted by using upstream heat exchangers, which greatly increases efficiency. The short processing time, even when high temperatures are used in the valve, produces a very gentle treatment with high nutritional and sensory quality [25]. The preservation of antioxidant activity [62], the control of oxidative enzymes such as polyphenol oxidases (PPOs) [62–64], the preservation of delicate aromatic molecules such as terpenes [64], and the absence of thermal markers have been observed in the processing of grape juices by UHPH [64].

The UHPH process and the passing through the valve produce high impact forces and intense shear stresses, and the result is a significant nanofragmentation of plant tissues with removal of microorganisms, including spores depending on the temperature in the valve, inactivation of enzymes and nanofragmentation of colloidal particles. The consequence is increased extraction by cell disruption and improved bioaccessibility [65]. The mechanical effect is highly dependent on the valve design, and the antimicrobial effect with mild impact on the residence time and the design of the heat exchangers upstream and downstream of the valve [25] (Figure 4). One of the most effective designs is the one developed by Ypsicon [66].

There is a size requirement concerning the maximum size of colloidal particles in the grape juice before pressurization due to the cross-sections in the fluidic components of the pump and valve. Particles in the fluid should be less than 500 µm to avoid clogging (Figure 4) [25]. After the treatment, the particles are fragmented in the range of 100–500 nm [25,64]. When grape juice, which has many colloidal constituents with a poly-

hedral appearance (Figure 5A), is processed by UHPH, a finer structure can be observed (Figure 5B) without large fragments [47].

**Figure 4.** Scheme of the structure and components of a UHPH-Ypsicon valve. Intense impact and shear stresses together with the help of heating produce: pasteurization/sterilization, nanofragmentation, enzyme inactivation, nano-coating and nano-encapsulation.

**Figure 5.** (**A**) Atomic Force Microscopy (AFM) topography of the surface of a dried red grape juice showing polyhedral granules, which are the colloidal particles of the juice (i.e., plant cell fragments and fibers). (**B**) The same dried red grape juice by AFM after UHPH treatment, with smaller granules and a flatter surface (no large polyhedral granules).

#### **3. Pulsed Electric Fields (PEFs) in the Extraction of Anthocyanins**

Like the previous ones (HHP and UHPH), PEFs have become a global technology with numerous applications in food processing, preservation and stabilization [67–71]. PEF is based on the use of high intensity electric fields (3–40 kV/cm) for a very short time (milli-micro seconds). Food is processed by PEF when it passes through two electrodes. The Electric Field Strength (E) is the voltage (kV) divided by the distance between the electrodes (cm), i.e., E = V/d. PEF systems are currently available on an industrial scale for food processing in the range of 50–10,000 L/h for fluids and 1–70 tonnes/h for solids such as French fries. The effect of PEFs is the poration of cells at the nanoscale, which affects the selective permeability [72]. These pores are difficult to observe by electronic microscopy. However, the pores produce various effects depending on size and number,

tending to increase cell permeability, thus facilitating the extraction of cell compounds (e.g., anthocyanins and many others), the entry of compounds and the temporal or definitive inactivation of cells depending on the intensity [73]. Pulses can be applied in several modalities. The main parameters are the pulse shape (i.e., squared, exponential, sinusoidal), the polarity (i.e., monopolar or bipolar), the number of pulses and the pulse duration (Figure 6). The intensity and effectiveness of the treatments depend on the above parameters with squared bipolar pulses being more effective and the number of pulses making the process more powerful. Even when the pulse duration also improves the efficacy, it should be kept at a low value because it affects the temperature of the food by ohmic heating.

**Figure 6.** Types of pulses. (**A**) Squared monopolar. (**B**) Squared bipolar. (**C**) Exponentially decaying. (**D**) Sinusoidal.

Plant cells need lower intensities than microorganisms, especially bacteria, depending on size and shape. To induce permeabilization in plant cells (size 40–200 µm), E must be 1–2 kV/cm, while in microorganisms (size 1–10 µm), 12–20 kV/cm are required [74]. Therefore, to extract bioactive molecules from vegetal tissues, less than 5 kV/cm is necessary, however, for microbial inactivation, E should normally be higher than 10 kV/cm. When plant cells are pored (i.e., grape skins), the consequence is an increased extraction of biomolecules such as anthocyanins, tannins and aroma compounds (Figure 7).

**Figure 7.** Electroporation and cell permeabilization.

At pilot and industrial scale, several works have demonstrated the efficiency of PEFs to increase the extraction of anthocyanins, and other phenols at low temperature while preserving their antioxidant capacity (Table 1). Currently grapes or by-products (grape pomace) can be processed continuously at a flow rate of several hundreds to a few tonnes of kg per hour (118 kg/h, [51]; 500 kg/h [53], 1900 kg/h [52]). Usually, the crushed gape is pumped by a progressive cavity pump [51] or a peristaltic pump [52] and later processed in a collinear chamber by applying exponentially decay pulses or, more frequently, squared pulses of an electric field strength ranging from 2 to 10 kV/cm [26,28,48,49,52,53]. Anthocyanin extraction increases in the range of 17–100% (Table 1) depending on processing conditions and post-maceration time. The temperature is increased by only 2–15 ◦C [55], therefore it is easy to work at room temperature or under refrigerated conditions. In addition to improved anthocyanin and phenol extraction, PEFs can be used for gentle nonthermal pasteurization of the must, thus improving the implantation of non-*Saccharomyces* starters [55] and potentially reducing the use of SO2. The effect of PEFs on the extraction

of phenolic compounds from seeds has also been reported and should be considered in winemaking processes [75,76].

#### **4. Ultrasounds (USs) in the Extraction of Anthocyanins**

Ultrasounds (USs) are mechanic waves with a frequency above 20 kHz, which is not perceptible to the human ear (typically in the range 20 Hz–20 kHz) [77]. It is a key technology for obtaining bioactive compounds (e.g., anthocyanins), like the others described above, as it can be considered a sustainable 'green' extraction method [78] as it does not use organic solvents and is gentle to heat-sensitive molecules [79]. The compression and rarefaction of the products produced by the US waves produce the successive reduction in size and expansion of the bubbles formed by cavitation (Figure 8). When these bubbles collapse, large amounts of energy are released, reaching localized temperatures of 5000 ◦K and pressures of 200 MPa [80]. These phenomena are responsible for the depolymerization of biostructures [80] and facilitate the extraction of molecules from plant tissues. Depolymerization can occur by bubble collapse, cavitation or degradation of the polymer by impact with radicals formed during sonication [81]. Depolymerization of cell wall polysaccharides accelerates the release of anthocyanins from the skin cells in grapes (Figure 9). The extraction of anthocyanins in water within a few minutes and the increase in temperature due to the cavitation effect can be observed. High power ultrasounds with the best extraction potential are considered to be in the range of 20–25 kHz [78].

**Figure 8.** Implosion of bubbles and cavitation produced by alternative compression-rarefaction effects generated by US waves [22].

Figure 9 shows the application of USs on grape berries by means of a sonotrode and reveals, after a few minutes, how the anthocyanins are extracted to the surrounding media (water) due to the depolymerization of the cell walls of the grape skins. Additionally, the heating effect produced by cavitation can be observed, which in this case is about 5 ◦C in the center of the flask according to infrared thermography.

There are several systems for applying USs to plant tissues with the aim of favoring the extraction of compounds: Ultrasound baths, sonotrodes, sonoplates. However, on an industrial scale, the most effective system is the use of continuous tubular exchangers on the external surface of which sonoplates are distributed to apply US waves during the flow of the mash or liquid through the exchanger. For a better distribution of the sonoplates on the exchange surface, the section is usually hexagonal instead of circular (Figure 10).

**Figure 9.** Use of USs in the extraction of grape anthocyanins and effect on temperature measured with an infrared camera. Left: before ultrasonication, right: after US treatment.

**Figure 10.** Cavitation cells arranged in a hexagonal tubular exchanger with the sonoplates for applying US waves [22].

This technology has been used to process Tempranillo grapes, achieving the same anthocyanin content in a final wine after only continuous US treatment and 72 h of skin maceration as in the control wine [82]. In discontinuous treatment at the laboratory scale, the USs have been shown to increase the extraction of anthocyanins and phenols by more than 50% compared to controls [56]. US can also be applied continuously after the application of pectolytic enzymes at industrial level, increasing color intensity by 18% and total polyphenols by 21% in wines [57]. The use of US-assisted extraction can be improved by optimizing other physicochemical parameters (temperature, ethanol and time), thus reaching a maximum of 6.26 mg/mL under the best conditions of 45.14 ◦C, 52.3% ethanol

and 24.5 min [83]. USs can be used to improve extraction and/or reduce extraction time in grapes [56,57,82], and by-products such as pomace [33] and lees [58]. USs have been applied to *Vitis vinifera* L. varieties Cabernet Franc [84], Tempranillo [82], Tannat [56], and Monastrell [57]. The influence of the US frequency has also been analyzed, considering the values of 12.5, 25, and 37.5 kHz, as well as from 12.5 to 25 kHz, the extraction of anthocyanins increased by 18% in grape pomace, however, the higher the frequency, the lower the extraction [59].

#### **5. Effect of E-Beam Irradiation in the Extraction of Anthocyanins**

Electron beam (e-beam) irradiation or β-irradiation involves the use of accelerated electrons at high energy, typically 10 MeV [21,37,85], to process foods and eliminate microorganisms, allowing pasteurization (1–5 kGy) or sterilization (>10 kGy) depending on the dose [21,37,85]. The irradiation dose is measured in Grays (Gy) or kGrays (kGy). One Gy is defined as the absorption of 1 Joule of energy per kg of irradiated mass. Irradiation is widely used to preserve food in more than 55 countries and is considered a safe technology approved by WHO, FAO and IAEA [86]. This technique is cheap on a large scale, environmentally friendly and time efficient [87]. e-Beam irradiation is a complex technology that requires expensive irradiation accelerators and large-scale facilities. e-Beam irradiation can be applied on an industrial scale in a continuous process [21]. The radiation dose can be monitored and verified by placing radiochromic dosimeters on the treated food (Figure 11). This can be used to verify the real dose received by the food at every width. e-Beam irradiation can be considered a gentle non-thermal technology with temperature increments of less than 5 ◦C at doses up to 10 kGy [21]. In addition, e-beam irradiation has been proposed as an alternative to sulphites in wine preservation [88] and has demonstrated its ability to delay browning in plant foods [89]. However, some negative effects have been observed such as loss of aroma [37] and reduction in vitamin C content [85], due to free radical-mediated oxidation [90].

**Figure 11.** Red grapes in plastic bags after e-beam irradiation. The white arrows indicate the location of radiochromic dosimeters.

Even when the external appearance of the grapes after irradiation remains unchanged (Figure 12A), the release of some juice in the bags can be observed, especially at high doses (10 kGy). This leakage of juice from the grapes shows the weakening of the plant tissues due to irradiation. The main effect of e-beam irradiation on plant tissues is the fragmentation of fibrillar polymers such as pectins and other polysaccharides, promoting the release and extraction of cell components, including anthocyanins. It has been reported

that the molecular weight of pectins can be reduced by 90% using doses of 3–10 kGy [91]. The effect on grapes is the increased extraction of phenols and anthocyanins [37], which can be observed in the more intense color of the running juice, especially when grapes are processed at 10 kGy (Figure 12B). Up to 1 kGy, the anthocyanin extraction is low and not too high compared to the controls, however at 10 kGy, the anthoyanins extracted in the running juice were 125 mg/L compared with 72 mg/L on average in the controls (+71%) [37]. Better antioxidant and sensory properties and higher phenol content have also been observed in grapes processed up to 2 kGy [92]. With blueberries, the use of e-beam irradiation at doses below 3 kGy has demonstrated to be a gentle processing that does not affect monomeric anthocyanin and antioxidant activity [93]. Protection of anthocyanins, color, phenols and antioxidant activity has also been observed in strawberries processed at 1 kGy [94].

**Figure 12.** Effect of e-beam irradiation on the external appearance of grapes (**A**). Running juice from grapes processed by e-beam irradiation at various doses (**B**).

#### **6. Pulsed Light**

Pulsed light (PL) treatment involves the application of high-intensity, low-duration pulses of radiation in the 200 nm (UV) to 2500 nm (IR) range [95,96]. The intensity can be higher than 10<sup>6</sup> fold that of sunlight at sea level in the summer midday and the duration ranges from micro to milliseconds. Detailed parameters for pulsed light processing conditions have recently been revised [97]. The temperature rise after standard PL treatments is usually less than 5 ◦C, so it can be considered a mild non-thermal technology that can be used in delicate foods [96].

The ability to extract anthocyanins and phenols from plant tissues is lower than some of the previous techniques and the literature reports inconclusive results. Non-significant differences have been found in the anthocyanin content of wines made from PL-processed grapes compared to controls [98]. Temperature increases in grape skins of 2–3 ◦C after pulsed light treatments have also been reported without severe damage to the skin surface observed by AFM scanning [99]. Furthermore, PL processing of strawberries at 4–8 J/cm<sup>2</sup> does not affect the quality and antioxidant capacity [100].

#### **7. Conclusions**

The use of emerging non-thermal technologies is a successful tool for the extraction of anthocyanins from grapes, increasing the yield, accelerating the process and preserving the antioxidant capacity. Many of these techniques can be applied in continuous flow (UHPH, PEF, US, Irradiation and PL), which is suitable for industrial processing. Most of

these techniques can work at room temperature or even using refrigerated crushed grapes, although temperature is always a synergistic parameter. These technologies can be used for the extraction of anthocyanins from grapes, and also from by-products such as pomace, generating high-value pigments from them. Additionally, emerging technologies can be used to improve the winemaking process by increasing the extraction of anthocyanins and phenolic compounds in maceration and controlling oxidations.

**Author Contributions:** Conceptualization, A.M.; methodology, A.M. and C.G.; writing—original draft preparation, A.M., C.E., I.L., C.L., F.P.; writing—review and editing, A.M., C.E., I.L., C.L., F.P. and C.G.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Ministerio de Ciencia, Innovación y Universidades project: RTI2018-096626-B-I00.

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

#### **References**


## *Review* **Metabolomics of Chlorophylls and Carotenoids: Analytical Methods and Metabolome-Based Studies**

**María Roca and Antonio Pérez-Gálvez \***

Food Phytochemistry Department, Instituto de la Grasa (CSIC), Building 46, 41013 Sevilla, Spain; mroca@ig.csic.es

**\*** Correspondence: aperez@ig.csic.es; Tel.: +34-954611550

**Abstract:** Chlorophylls and carotenoids are two families of antioxidants present in daily ingested foods, whose recognition as added-value ingredients runs in parallel with the increasing number of demonstrated functional properties. Both groups include a complex and vast number of compounds, and extraction and analysis methods evolved recently to a modern protocol. New methodologies are more potent, precise, and accurate, but their application requires a better understanding of the technical and biological context. Therefore, the present review compiles the basic knowledge and recent advances of the metabolomics of chlorophylls and carotenoids, including the interrelation with the primary metabolism. The study includes material preparation and extraction protocols, the instrumental techniques for the acquisition of spectroscopic and spectrometric properties, the workflows and software tools for data pre-processing and analysis, and the application of mass spectrometry to pigment metabolomics. In addition, the review encompasses a critical description of studies where metabolomics analyses of chlorophylls and carotenoids were developed as an approach to analyzing the effects of biotic and abiotic stressors on living organisms.

**Keywords:** antioxidants; carotenoids; chlorophylls; extraction methods; novel analytical technologies; metabolomics; mass spectrometry; metabolism; pathways; pigments

## **Citation:** Roca, M.; Pérez-Gálvez, A. Metabolomics of Chlorophylls and Carotenoids: Analytical Methods and Metabolome-Based Studies. *Antioxidants* **2021**, *10*, 1622. https:// doi.org/10.3390/antiox10101622

Academic Editor: Agustín G. Asuero

Received: 4 August 2021 Accepted: 12 October 2021 Published: 15 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

## **1. Introduction**

Metabolomics is an essential approach that allows for the acquisition of knowledge regarding the actual composition of complex mixtures of extracts from tissues of plant or animal origin. The development of metabolomics is only feasible with a holistic methodology, applying a multifaceted and interdependent sequence of experiments, techniques, and computational tools [1]. Accordingly, the successful application of metabolomics depends on the successful selection or development of extraction protocols; the arrangement of the suitable analytical platform for analyses; the implementation of software for data gathering, handling, and analysis of results, where an expert-curated learning attitude is fundamental; and, finally, the application of statistics to extract the information within a biological context [2]. Nevertheless, the importance of metabolomics lies in the information regarding the physiology of an organism, tissue, cell, etc. Indeed, metabolomics is a source that reflects a biochemical state or activity.

This review is focused on the metabolomics of chlorophylls and carotenoids, which was named "pigmentomic", as a tool for exploring their antioxidant features within the secondary plant metabolism. The antioxidant properties of both families of pigments have been deeply investigated and recently reviewed [3]. To gain an idea of the present impact of this topic, we performed a reference search in the Web of Science (ISI Web of Knowledge) databases, introducing "metabolomic\*" and "chlorophyll\*" as topics, and a total of 380 results were obtained (Figure 1). Moreover, when the topics "metabolomic\*" and "carotenoid\*" are selected, 499 results arise. However, the interesting point of both surveys is their time evolution, as half of the manuscripts were published in 2019 or thereafter, a clear signal of the exponential growth rate of metabolomics studies focusing

on chlorophylls and carotenoids. This review includes metabolomics and metabonomics studies, as the difference between both terms is author dependent, and each term was defined as a subset of the other. It can be assumed that in metabolomics (stated by Fiehn and collaborators in 2001) [4], studies are necessary to identify and quantify all endogenous metabolites, while the metabonomics assessment (created by Nicholson et al. in 1999) [5] aims to identify a metabolite fingerprint. In a broad sense, a metabolomics strategy utilizes a mixture of separation techniques, such as HPLC or GC-MS, while in metabonomics studies, the use of NMR spectroscopy is more frequent. after, a clear signal of the exponential growth rate of metabolomics studies focusing on chlorophylls and carotenoids. This review includes metabolomics and metabonomics studies, as the difference between both terms is author dependent, and each term was defined as a subset of the other. It can be assumed that in metabolomics (stated by Fiehn and collaborators in 2001) [4], studies are necessary to identify and quantify all endogenous metabolites, while the metabonomics assessment (created by Nicholson et al. in 1999) [5] aims to identify a metabolite fingerprint. In a broad sense, a metabolomics strategy utilizes a mixture of separation techniques, such as HPLC or GC-MS, while in metabonomics studies, the use of NMR spectroscopy is more frequent.

surveys is their time evolution, as half of the manuscripts were published in 2019 or there-

*Antioxidants* **2021**, *10*, x FOR PEER REVIEW 2 of 24

**Figure 1.** Number of publications since 2011 in the Web of Science (ISI Web of Knowledge) databases, introducing "metabolomic\*" and "chlorophyll\*" (green series) and "metabolomic\*" and "carotenoid\*" (orange series) as topics. The year 2021 does not cover the whole year and takes into account publications from January to September only. **Figure 1.** Number of publications since 2011 in the Web of Science (ISI Web of Knowledge) databases, introducing "metabolomic\*" and "chlorophyll\*" (green series) and "metabolomic\*" and "carotenoid\*" (orange series) as topics. The year 2021 does not cover the whole year and takes into account publications from January to September only.

We summarize the current understanding of how metabolomics describes fluctuations in chlorophylls and carotenoids, which perform essential functions and actions in photosynthetic organisms and animals that incorporate them through diet. Their involvement in plant biochemistry as key network components suggests that chlorophylls and carotenoids are key compounds involved in significant metabolic pathways. The review starts with a general description of the application of techniques for sample preparation and the acquisition of extracts suitable for analysis; a picture of the analytical platform and technologies applied for the identification and quantification of the pigment profile; and the workflow for data analysis, including software tools and the application of metabolite databases and statistics. Next, we compile the works where the metabolomics of chlorophylls and/or carotenoids has proved extremely valuable in the recognition or tuning of metabolic pathways correlated with responses to different abiotic and biotic factors; physiologic and biologic studies; and even applications to animal health. In this sense, the aim of the present review is to strengthen the potentiality of the metabolomics studies of chlorophylls and carotenoids. Moving on from an analytical determination, metabolomics is a powerful tool for comprehensive research, with multiple and diverse applications, as will be shown in this review. We summarize the current understanding of how metabolomics describes fluctuations in chlorophylls and carotenoids, which perform essential functions and actions in photosynthetic organisms and animals that incorporate them through diet. Their involvement in plant biochemistry as key network components suggests that chlorophylls and carotenoids are key compounds involved in significant metabolic pathways. The review starts with a general description of the application of techniques for sample preparation and the acquisition of extracts suitable for analysis; a picture of the analytical platform and technologies applied for the identification and quantification of the pigment profile; and the workflow for data analysis, including software tools and the application of metabolite databases and statistics. Next, we compile the works where the metabolomics of chlorophylls and/or carotenoids has proved extremely valuable in the recognition or tuning of metabolic pathways correlated with responses to different abiotic and biotic factors; physiologic and biologic studies; and even applications to animal health. In this sense, the aim of the present review is to strengthen the potentiality of the metabolomics studies of chlorophylls and carotenoids. Moving on from an analytical determination, metabolomics is a powerful tool for comprehensive research, with multiple and diverse applications, as will be shown in this review.

#### **2. Biochemistry of Chlorophylls and Carotenoids**

#### *2.1. Chlorophylls*

Chlorophylls comprise a homogeneous group of more than 100 different structures with a unique configuration in nature. Their primary function is associated with photosynthesis, being functionals during the charge separation in the reaction centers or transferring

energy in the harvesting complex. Unfortunately, this essential role has masked, other actions of chlorophylls in nature, including the interrelation with the general metabolism, and has led to the underestimation of their physiologic functions. ferring energy in the harvesting complex. Unfortunately, this essential role has masked, other actions of chlorophylls in nature, including the interrelation with the general metabolism, and has led to the underestimation of their physiologic functions.

Chlorophylls comprise a homogeneous group of more than 100 different structures with a unique configuration in nature. Their primary function is associated with photosynthesis, being functionals during the charge separation in the reaction centers or trans-

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**2. Biochemistry of Chlorophylls and Carotenoids**

*2.1. Chlorophylls*

Chlorophylls are tetrapyrroles with an additional fifth isocyclic ring (Figure 2). They are coordinated generally with a central atom of magnesium, although this can be substituted by hydrogen or other divalent cations. In parallel, the propionic acid at C17<sup>3</sup> is esterified with a phytyl chain (C20H39), but different chlorophyll structures arise from esterification with multiple alcohols, and they can even occur in a non-esterified form (as pheophorbides). Chemically, depending on the degree of unsaturation of the macrocycle, chlorophylls could be classified as chlorin type (chlorophyll *a* and *b* among others), porphyrin type (chlorophyll *c*), or bacteriochlorin type (as certain bacteriochlorophylls), which are responsible for a complex array of different chlorophyll metabolites. Moreover, during natural (senescence or ripening) metabolism or food processing or storage, chlorophylls can be oxidized to form new chlorophylls. Among the most common are 13<sup>2</sup> -hydroxychlorophylls, which are formed if the hydroxyl group is introduced at C13<sup>2</sup> . In addition, C15<sup>1</sup> -hydroxylactone-chlorophylls are formed if a lactone group is formed at C15<sup>1</sup> , and pyroderivatives are formed if the carboxymethoxy group at C13<sup>2</sup> is lost. Chlorophylls are tetrapyrroles with an additional fifth isocyclic ring (Figure 2). They are coordinated generally with a central atom of magnesium, although this can be substituted by hydrogen or other divalent cations. In parallel, the propionic acid at C17<sup>3</sup> is esterified with a phytyl chain (C20H39), but different chlorophyll structures arise from esterification with multiple alcohols, and they can even occur in a non-esterified form (as pheophorbides). Chemically, depending on the degree of unsaturation of the macrocycle, chlorophylls could be classified as chlorin type (chlorophyll *a* and *b* among others), porphyrin type (chlorophyll *c*), or bacteriochlorin type (as certain bacteriochlorophylls), which are responsible for a complex array of different chlorophyll metabolites. Moreover, during natural (senescence or ripening) metabolism or food processing or storage, chlorophylls can be oxidized to form new chlorophylls. Among the most common are 13<sup>2</sup> hydroxy-chlorophylls, which are formed if the hydroxyl group is introduced at C13<sup>2</sup> . In addition, C15<sup>1</sup> -hydroxylactone-chlorophylls are formed if a lactone group is formed at C15<sup>1</sup> , and pyroderivatives are formed if the carboxymethoxy group at C13<sup>2</sup> is lost.

**Figure 2.** Main chlorophyll structures present in organisms or food due to natural metabolism or during processing or storage. **Figure 2.** Main chlorophyll structures present in organisms or food due to natural metabolism or during processing or storage.

Biochemically, the chlorophyll metabolism is divided among synthesis, the chlorophyll cycle, and degradation, and it is independently regulated. Few interesting reviews have detailed the complete set of biochemical reactions, enzymes, and genes implicated Biochemically, the chlorophyll metabolism is divided among synthesis, the chlorophyll cycle, and degradation, and it is independently regulated. Few interesting reviews have detailed the complete set of biochemical reactions, enzymes, and genes implicated in their metabolism [6–9], and, consequently, we only delineate the main reactions for a general outlook (Figure 3) in this review. Chlorophyll synthesis is initiated from the amino acid metabolism, specifically from aminolevulinic or glutamic acids, depending on the researcher. Different condensation, reductions, and decarboxylations generate protoporphyrin IX, which is the first colored chlorophyll metabolite. This point of the route is a

hotspot, as it is where the branch toward heme metabolism occurs if Fe-chelatase inserts Fe in the tetrapyrrole, or, similarly, where the branch toward the chlorophyll metabolism occurs if Mg-chelatase catalyzes the reaction. Following Mg-protoporphyrin IX and after several reactions, protochlorophyllide *a* is formed. This compound is an interesting metabolite because the subsequent reaction is light dependent in angiosperms and thus responsible for the etiolated plants in dark conditions. After several reactions, chlorophylls *a* and *b* are synthesized, with the functional capacity of interconversion through a plastic chlorophyll cycle [9]. Such flexibility in the chlorophyll metabolism is based on the capacity to modify the relative amounts of chlorophyll *a* and chlorophyll *b* depending on the light intensity, modifying the proportion of antenna complexes and, consequently, the photosynthetic apparatus. While chlorophyll synthesis is completely developed in the chloroplast, the catabolic reactions start in the green organelle but run through the cytosol, finishing in the vacuole (Figure 3). Chlorophyll *a* is degraded to pheophorbide *a* in two reactions, liberating the magnesium atom and de-esterifying the phytol chain. Recently, it was demonstrated that phytol yielded from chlorophyll catabolism is essential for tocopherol synthesis [10]. Next, the macrocycle is oxygenolytically opened to form the first linear chlorophyll catabolite, the so-called phyllobilins due to their resemblance to the heme-derived bilins. At present, more than 40 different phyllobilins have been described [11] with unknown functions, although an antioxidant role has been assigned to them. After reduction, a fluorescent chlorophyll catabolite (FCC) is produced and exported from the chloroplast to the cytosol. FCCs could be modified in the cytosol and imported into the vacuole, where the acidic pH promotes isomerization to non-fluorescent chlorophyll catabolites (NCCs). Although a phyllobilin database for *Arabidopsis thaliana* [12] is already available, a complete database containing all phyllobilins identified at present in multiple species is necessary. hotspot, as it is where the branch toward heme metabolism occurs if Fe-chelatase inserts Fe in the tetrapyrrole, or, similarly, where the branch toward the chlorophyll metabolism occurs if Mg-chelatase catalyzes the reaction. Following Mg-protoporphyrin IX and after several reactions, protochlorophyllide *a* is formed. This compound is an interesting metabolite because the subsequent reaction is light dependent in angiosperms and thus responsible for the etiolated plants in dark conditions. After several reactions, chlorophylls *a* and *b* are synthesized, with the functional capacity of interconversion through a plastic chlorophyll cycle [9]. Such flexibility in the chlorophyll metabolism is based on the capacity to modify the relative amounts of chlorophyll *a* and chlorophyll *b* depending on the light intensity, modifying the proportion of antenna complexes and, consequently, the photosynthetic apparatus. While chlorophyll synthesis is completely developed in the chloroplast, the catabolic reactions start in the green organelle but run through the cytosol, finishing in the vacuole (Figure 3). Chlorophyll *a* is degraded to pheophorbide *a* in two reactions, liberating the magnesium atom and de-esterifying the phytol chain. Recently, it was demonstrated that phytol yielded from chlorophyll catabolism is essential for tocopherol synthesis [10]. Next, the macrocycle is oxygenolytically opened to form the first linear chlorophyll catabolite, the so-called phyllobilins due to their resemblance to the hemederived bilins. At present, more than 40 different phyllobilins have been described [11] with unknown functions, although an antioxidant role has been assigned to them. After reduction, a fluorescent chlorophyll catabolite (FCC) is produced and exported from the chloroplast to the cytosol. FCCs could be modified in the cytosol and imported into the vacuole, where the acidic pH promotes isomerization to non-fluorescent chlorophyll catabolites (NCCs). Although a phyllobilin database for *Arabidopsis thaliana* [12] is already available, a complete database containing all phyllobilins identified at present in multiple species is necessary.

in their metabolism [6–9], and, consequently, we only delineate the main reactions for a general outlook (Figure 3) in this review. Chlorophyll synthesis is initiated from the amino acid metabolism, specifically from aminolevulinic or glutamic acids, depending on the researcher. Different condensation, reductions, and decarboxylations generate protoporphyrin IX, which is the first colored chlorophyll metabolite. This point of the route is a

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**Figure 3.** Brief description of the biosynthesis and catabolism of chlorophylls and routes related to other phytochemicals.

As previously stated, in addition to their key role in photosynthesis, chlorophyll compounds are implicated in different physiological actions and biochemical reactions. The photodynamic properties of several chlorophyll metabolites allow them to be implicated in the ROS response [13] and, consequently, as shown below, on different mechanisms, such as defense, stress, and cell death. However, multiple pieces of evidence demonstrate the antioxidant properties of chlorophylls [3]. Another example of the superficial valuation of chlorophylls is the simple determination of chlorophylls as a simple symptom of senescence. If we bear in mind the fact that the physical presence of chlorophylls *a* and *b* is necessary for the assembly of the photosynthetic apparatus, it can be understood that organisms named stay-greens (with a deficiency in senescence) have, in many cases, been identified as mutants in chlorophyll degradation genes.

#### *2.2. Carotenoids*

Carotenoids are a family of naturally occurring yellow, red, and orange pigments chemically derived from isoprenoids that group together ca. 1200 compounds [14]. Carotenoids are lipophilic compounds synthesized in plastids. In chloroplasts, carotenoids have an essential role in photosynthesis, assisting in harvesting light energy by transferring it to the chlorophylls and protecting the photosynthetic apparatus by quenching triplet excited states of chlorophyll molecules, singlet oxygen, and carboxy radicals [15]. Additionally, they are precursors to phytohormones and other signaling compounds [16,17]. These functions in photosynthesis, photoprotection, and key metabolic pathways make carotenoids essential metabolites. However, the biosynthesis of secondary taxon-specific carotenoids also occurs in chromoplasts, and it is linked with other roles and actions, such as antioxidant activity not being related to photosynthesis and carotenoids serving as intermediates in plant-animal interactions by furnishing flowers and fruits with fragrances and colors [18,19]. Carotenoids with specific structural arrangements are precursors for vitamin A, which has a direct impact on the function of these pigments in human nutrition [20]. Their action as antioxidants and other not yet fully understood activities in mammals have prompted evidence for their role in human health [13]. Furthermore, there is a commercial demand for carotenoids for the food, pharmacy, and cosmetics industries [21]. Altogether, this explains the enormous interest in carotenoid biosynthesis and the possibility of manipulating and engineering the carotenoid biosynthetic pathway to answer fundamental research questions and identify practical applications [22].

Carotenoid biosynthesis (Figure 4) starts with a series of isoprene condensations to yield phytoene, a substrate that undergoes desaturation and isomerization steps (yielding a group of intermediates) to form lycopene. These initial steps configure the basic structure that characterizes plant carotenoids: the typical C40 skeleton with a central polyene system that condenses the physicochemical properties of these pigments and conditions and the subsequent enzymatic processes that continue the route [23,24]. From this point, cyclization and subsequent oxygenation of the cyclic intermediates emerge as the breakthrough to the origin of a considerable diversity of carotenoid structures [25]. Different combinations of cyclic arrangements (type β and type ε) at one or both ends of the polyene system generate the first branch in the route, while the introduction of hydroxyl, keto, epoxide, etc. functions produces the classification of carotenoids in carotenes (pure hydrocarbons) and xanthophylls (oxygenated products of carotenes).

At this point, the main issue to consider in metabolome-based studies of carotenoids is the site of carotenoid biosynthesis and accumulation and the structural features that characterize the precursors, products, and catabolites of this family of natural pigments. First, carotenoids are synthesized in plastids and chloro- and chromo-plasts, meaning that compartmentation approaches can be successfully used to focus metabolomics studies and specifically analyze how the pathway operates in this separate location a priori without unexpected competition. Second, the structure of the carotenoids is the premise of solving the analytical challenge of their identification and quantification, while it includes the possibility of expanding the analysis to both parent compounds and metabolic products.

Carotenoids present a common basic skeleton (Figure 4), the polyene chain, and a combination of cyclic/linear arrangements at the ends of the skeleton with the introduction of oxygen functions at specific carbon atoms expands the number of structural blends [26]. Moreover, these structural features seem to correspond exclusively to this family of natural pigments. However, the correct identification is only feasible through the acquisition of several layers of information from different technologies (UV-visible spectrophotometry, mass spectrometry, nuclear magnetic resonance, and circular dichroism) combined with a variety of derivatization processes and a comparison with reference standards. The presence of geometric isomers, a frequent feature of carotenoids, complicates the identification task and requires the introduction of secure workflow models and a combination of analytical techniques for successful classification [27–30]. *Antioxidants* **2021**, *10*, x FOR PEER REVIEW 6 of 24

**Figure 4.** Scheme of the carotenoid biosynthesis route including catabolism to abscisic acid, volatile compounds, and strigolactones. MEP: methylerythritol phosphate; GGPP: geranylgeranyl pyrophosphate; PSY: phytoene synthase; PDS: phytoene desaturase; Z-ISO: ζ-carotene isomerase; ZDS: **Figure 4.** Scheme of the carotenoid biosynthesis route including catabolism to abscisic acid, volatile compounds, and strigolactones. MEP: methylerythritol phosphate; GGPP: geranylgeranyl pyrophosphate; PSY: phytoene synthase; PDS: phytoene desaturase; Z-ISO: ζ-carotene isomerase; ZDS: ζ-carotene desaturase; CRTISO: carotene isomerase.

#### ζ-carotene desaturase; CRTISO: carotene isomerase. **3. The Praxis of Metabolomics: Essential Steps and Challenges for the Experimental Design**

At this point, the main issue to consider in metabolome-based studies of carotenoids is the site of carotenoid biosynthesis and accumulation and the structural features that characterize the precursors, products, and catabolites of this family of natural pigments. First, carotenoids are synthesized in plastids and chloro- and chromo-plasts, meaning that compartmentation approaches can be successfully used to focus metabolomics studies and specifically analyze how the pathway operates in this separate location a priori without unexpected competition. Second, the structure of the carotenoids is the premise of solving the analytical challenge of their identification and quantification, while it includes the possibility of expanding the analysis to both parent compounds and metabolic prod-When working with chlorophylls and carotenoids, as with other phytochemicals, different metabolomic approaches can be developed (Figure 5). If the goal of a study can be solved with observations and the quantification of a rather limited number of metabolites, which are chosen based on previous literature reports or self-experience, targeted metabolomics is performed. If we encounter a study without a previous hypothesis, then we aim to obtain a global picture of the metabolome, measuring as many metabolites as possible, which means that untargeted metabolomics are suitable. Using this strategy, when samples are classified based on their metabolite profile, without identification of the individual peaks, fingerprinting is carried out. On the contrary, when as many compounds as possible are identified and subsequently quantified, metabolite profiling is carried out.

combination of analytical techniques for successful classification [27–30].

ucts. Carotenoids present a common basic skeleton (Figure 4), the polyene chain, and a

[26]. Moreover, these structural features seem to correspond exclusively to this family of natural pigments. However, the correct identification is only feasible through the acquisition of several layers of information from different technologies (UV-visible spectrophotometry, mass spectrometry, nuclear magnetic resonance, and circular dichroism) combined with a variety of derivatization processes and a comparison with reference standards. The presence of geometric isomers, a frequent feature of carotenoids, complicates the identification task and requires the introduction of secure workflow models and a **Design**

**3. The Praxis of Metabolomics: Essential Steps and Challenges for the Experimental** 

When working with chlorophylls and carotenoids, as with other phytochemicals, different metabolomic approaches can be developed (Figure 5). If the goal of a study can be solved with observations and the quantification of a rather limited number of metabolites, which are chosen based on previous literature reports or self-experience, targeted metabolomics is performed. If we encounter a study without a previous hypothesis, then we aim to obtain a global picture of the metabolome, measuring as many metabolites as possible, which means that untargeted metabolomics are suitable. Using this strategy, when

vidual peaks, fingerprinting is carried out. On the contrary, when as many compounds as possible are identified and subsequently quantified, metabolite profiling is carried out.

**Figure 5.** An outlook of the planning of a metabolomics study including the selection of the workflow (hypothesis-driven or hypothesis generation), examples of instrumental techniques, and data **Figure 5.** An outlook of the planning of a metabolomics study including the selection of the workflow (hypothesis-driven or hypothesis generation), examples of instrumental techniques, and data preprocessing and interpretation.

#### preprocessing and interpretation. *3.1. Material Preparation and Extraction Protocols*

*3.1. Material Preparation and Extraction Protocols* In metabolomics studies, frequent potential sources of bias are as follows: an unclear selection of the development stage at harvesting time, a lack of references to provide guidance on the light period and harvest duration, and a lack of a record of environmental variables and growth conditions [31]. This is crucial when working with chlorophylls and carotenoids, because the type of sample handling and applied treatments are critical to avoiding alterations to the metabolites. Additionally, it should be kept in mind that fluxes and accumulation rates are different depending on the class of metabolites (chlorophylls or carotenoids) or the metabolic process (for example, metabolites involved in the photo-In metabolomics studies, frequent potential sources of bias are as follows: an unclear selection of the development stage at harvesting time, a lack of references to provide guidance on the light period and harvest duration, and a lack of a record of environmental variables and growth conditions [31]. This is crucial when working with chlorophylls and carotenoids, because the type of sample handling and applied treatments are critical to avoiding alterations to the metabolites. Additionally, it should be kept in mind that fluxes and accumulation rates are different depending on the class of metabolites (chlorophylls or carotenoids) or the metabolic process (for example, metabolites involved in the photosynthesis, antioxidant activity, catabolism of degradation products during ripening, and tissue senescence) that is focused on [32].

synthesis, antioxidant activity, catabolism of degradation products during ripening, and tissue senescence) that is focused on [32]. If the analysis of metabolites does not need the pre-processing of the tissue, then the direct flash freezing of the sample in liquid nitrogen stops metabolic conversions, and the frozen sample is homogenized into a fine powder to enhance and standardize metabolite extraction. A significant research effort has been made to refine the protocols for specific chlorophyll and carotenoid extraction, minimizing the sources of errors and increasing the reliability of the data [33]. The experimental design of most protocols aims to reduce the processing time while increasing the efficiency of the extraction. In addition, factors such as economic viability and sustainability have been introduced in the experimental If the analysis of metabolites does not need the pre-processing of the tissue, then the direct flash freezing of the sample in liquid nitrogen stops metabolic conversions, and the frozen sample is homogenized into a fine powder to enhance and standardize metabolite extraction. A significant research effort has been made to refine the protocols for specific chlorophyll and carotenoid extraction, minimizing the sources of errors and increasing the reliability of the data [33]. The experimental design of most protocols aims to reduce the processing time while increasing the efficiency of the extraction. In addition, factors such as economic viability and sustainability have been introduced in the experimental design of those protocols. Therefore, different "green extraction techniques" can be applied, such as supercritical fluid extraction, microwave-assisted extraction, ultrasound-assisted extraction, pulsed electric field extraction, and extraction assisted by enzymes. These techniques have been applied mainly for carotenoid extractions, although several assays were developed for chlorophyll extractions [34]. Supercritical fluid extraction presents several advantages, such as its high purity of the extraction, simplicity, safety, and moderate temperatures [35,36]. On the contrary, it is essential to optimize the temperature and pressure conditions for a specific sample. Better results seem to be obtained when taking advantage of microwave irradiation and when applying microwave-assisted extraction [37,38]. The direct generation of heat within the matrix increases the recovery of the pigments. The studies using ultrasoundassisted extraction showed a significant reduction in the extraction time and an increase in the pigment extraction yields [39]. Pulsed electric field extraction was also used to improve

pigments extractions [40,41], but its effectivity depends greatly on the intensity, amplitude, duration, number, and frequency of repetitions. However, besides the excellent results obtained with these protocols, the application of ionic solvents could be considered the most modern extraction methodology at present. An ionic solvent can be defined as compounds completely composed of ions with a melting point below 100 ◦C. However, additional steps of purification are required when similar structures and/or polarities are present. Therefore, an additional improvement is the set-up of the liquid-liquid extraction process using aqueous solutions of tensioactive ionic liquids and vegetable oil as an alternative to the conventional extraction processes [42], with excellent results for chlorophylls and carotenoids. However, these modern extraction protocols require a considerable amount of time to be generalized, while solvent extraction techniques are the universal protocol applied to obtain chlorophylls and/or carotenoid extracts. The high recovery and stability of the extracted compounds should be poised, and several different solvents are suitable to achieve this aim (methanol, ethanol, acetone, and mixtures at different ratios of water vs. organic solvent(s) at an acidic pH), with the help of sonication and vortex mixing. The selection of the solvent should be made considering the wide range of the polarity of compounds if untargeted metabolomics are pursued, while some solvent mixtures could be tested to extract those compounds of interest selectively for targeted metabolomics. In this case, the appearance of sample matrix effects in the subsequent instrumental analysis diminishes, while interference due to the matrix during analysis and quantification could be a serious issue in the case of metabolite profiling, which requires an almost complete extraction of metabolites.

#### *3.2. Technologies: Instrumental Techniques for the Acquisition of Spectroscopic and Spectrometric Data*

Once the extract is ready for analysis, the instrumental technique performs the acquisition of a set of data, whose complexity is related to the selected strategy for the metabolomic study (Figure 5). Fingerprinting is typically performed with <sup>1</sup>H-NMR, ignoring the problem of making individual assignments of peaks [43]. Here, the main issue is to work with signals that are typically evident as multiple peaks, hindering the analysis of data. To overcome this problem, the acquisition of <sup>13</sup>C-NMR spectra with modern probes and systems purposely created to increase the sensitivity has been noted [44]. With these approaches, the aim is to find a group of marker compounds, which are inferred from shifts of different nuclei that characterize skeletons, aromatic rings, heteroatoms, and typical structural arrangements but are not fully identified. Subsequently, statistical analysis is conducted to classify the samples and draw conclusions based on discrimination, aggrupation, or differentiation of selected variables [45,46]. Technical improvements were made in the last two decades to make definitively the combination of NMR spectroscopy with LC a successful arrangement [47,48] if metabolite profiling or targeted metabolomics is the strategy of the metabolomic study. Metabolite profiling and targeted metabolomics make use of GC, while LC can be applied to targeted and untargeted metabolomics. These techniques of analysis are coupled with one or several detection systems to achieve both the compound separation and detection, collecting spectroscopic and/or mass spectrometric data on individual components of the extract.

GC coupled with mass spectrometry (MS) is a robust chromatographic instrumental technique (Figure 5) with a high compound separation efficiency (peak widths of 2–5 s) that yields reproducible retention times. This feature allows the quick building of spectral libraries of reference analytes that boost the identification of a compound profile in the extract, with a high level of certainty in identification [49]. However, GC is only able for the analysis of thermally stable and volatile compounds (directly from the extract, or once the extract is derivatized to produce volatile products), such as carotenoid degradation products (Figure 4) or phytol (arising from chlorophyll degradation, Figure 3). Additionally, the availability of standards of carotenoid and chlorophyll volatile metabolites is still very limited, so the great advantages of the reproducibility of GC retention times and direct matching with mass spectral libraries are fully usable in metabolomic studies of primary

metabolites, which have commercially available standard compounds [50–52], but not in pigmentomic studies. Despite these limitations, GC-MS is a suitable technique for the identification of the links between carotenoids, their putative signaling molecules (aroma profile), and the antioxidant potential during fruit ripening, as shown in melon [53], red pepper [54], citrus, and tomato [55], or during the processing of black tea [56] and *Mentha* species [57]. However, the applications remain scarce in the case of chlorophylls [58].

LC emerged from the principles of classic chromatography and the instrumental advances designed for GC, typically used for chlorophylls and carotenoids (Figure 5). The number of possible combinations for mobile phase composition, the increasing amount of packing materials for column building, and the high speed of the cumulative working pressure have definitively improved the efficiency and resolution of this technique, which could be easily combined with a wide range of detection systems in a single workflow [59–61]. Liquid chromatography in the classic high-pressure arrangement or the modern ultraperformance technology is typically coupled with different detectors based on optical detection (UV-visible, diode array, fluorescence, evaporative light-scattering, and differential refractive index detectors) applied to carotenoids and chlorophylls in foods [62,63] and biological samples [64,65], or in electrical detection (conductivity, electrochemical, and Corona-charged aerosol detectors), as was shown for the measurement of carotenoid bioavailability [66] and antioxidant potential [67] and in vitamin A equivalence studies [68] in humans. However, while the application of electrochemical detectors for chlorophyll analysis is rather limited [69], the holistic strategy that features metabolomics requires the application of further instrumental techniques to obtain as much information as possible from a single run, so the above-noted detection systems have begun to be combined with infrared, Raman, and NMR spectroscopies. This is the case of the metabolite profiling of microalgae species [70] and vegetable purees [71]. Soft-ionization techniques (electrospray ionization, ESI; atmospheric pressure chemical ionization, APCI) that yield protonated (positive mode) or de-protonated (negative mode) molecular ions are appropriate for the analysis of the most relevant groups of plant secondary metabolites [29,72,73], which are mainly separated with a reversed-phase column providing an efficient retention time and separation index, with a particular emphasis on the detection of isomeric compounds. Usually, APCI is used for carotenoids [74–76] and non-polar chlorophylls (chlorophylls and pheophytins) [77–79], and ESI is used in the analysis of polar chlorophylls (pheophorbide and chlorophyllide) and phyllobilins [80,81]. However, different configurations of both the ion source and mass analyzer have been implemented, including ion mobility [82] and MALDI [83,84].

To increase the reliability of data in the case of metabolite profiling/targeted metabolomics, where (tentative) identification of pigments is the aim, the acquisition of MS in a highresolution mode, in combination with tandem MS, is almost a pre-requisite. This combination of working conditions and online experiments allows the analyst to obtain different pieces of information that are conveniently arranged in pairs of independent and orthogonal data of physicochemical properties, facilitating the implementation of workflow protocols for the characterization of chlorophyll and carotenoid metabolic profiles tailored to the selected strategy implemented in the study (targeted metabolomics, fingerprinting, or metabolite profiling) [85].

#### *3.3. Application of Different Approaches to Pigment Metabolomics*

Table 1 contains some representative work dealing with mass spectrometry in the analysis of chlorophylls and carotenoids that we examine in detail in this section. These studies may serve as the starting point to follow current strategies that successfully enhance the analysis of these plant pigments (Figure 5). The aims of these studies were diverse, so the difficulties and bottle-neck issues that were faced boosted the application of methodological approaches and solutions. Hegeman et al. [86] applied the stable isotope-assisted assignment of elemental composition to constrain the number of potential positive hits for a mass peaking procedure in the identification of chlorophyll derivatives. Similarly, Giavalisco et al. [87] provided a comprehensive multi-isotope labeling-based strategy in combination with a fractionated metabolite extraction protocol to perform unambiguous qualitative and quantitative metabolomics using *A. thaliana* leaf and root extracts.

The characteristic isotopic pattern of the copper chlorophyll derivatives is selected as a fast and specific procedure to characterize precisely the presence of metallo-chlorophyll complexes applied to improve the green coloration of food products [88]. A novel strategy that combines UPLC coupled with traveling wave ion mobility (TWIN) and UV-visible detection is proposed to improve the characterization of chlorophylls and carotenoids analyzed in complex biological matrices [82]. A workflow strategy to perform targeted metabolomics of chlorophyll catabolites is applied to data analysis obtained by HPLC/ESIhr-QTOF-MS from leaf and fruit senescent tissues [81,89]. Automated data analysis using multivariate curve resolution algorithms to study multi-component systems that follow additive bilinear models (pure spectrum and related time profile) is also an appropriate strategy for the analysis of pigment metabolites. With this method, Wehrens et al. [90,91] examined the metabolite profiles (carotenoids, tocopherols, and chlorophylls) of grapes (*Vitis vinifera*) and cassava (*Manihot escullenta*). Watanabe et al. [92] described a combination of analytic tools that can be used to obtain comprehensive metabolite profiles in the *A. thaliana* plant model. Another interesting approach in the metabolomic studies of chlorophylls is the determination of phytol, a direct metabolite produced by chlorophyll degradation [93] that is analyzed by GC-MS. The incorporation of Bayesian approaches to cluster accessions of *Brassica rapa* of different morphotypes and origins allows for the acquisition of association mapping between different markers and metabolites, including chlorophylls and carotenoids [94]. Authors correct for kinship and population structure with the main aim of reducing the rate of false-positive associations. The implementation of alternative separation procedures, such as supercritical fluid extraction/chromatography coupled with MS, which reduce the extraction time and analysis run time, is an increasingly applied option to achieve a reduction in inter-sample variability and the setting of batchtype applications [95,96].



235

#### **4. Metabolome-Based Studies of Chlorophylls and Carotenoids 4. Metabolome-Based Studies of Chlorophylls and Carotenoids** During the initial development of metabolomics, compounds such as amino acids,

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During the initial development of metabolomics, compounds such as amino acids, organic acids, and carbohydrates were the focus of the studies. However, "pigmentomic analysis" is increasing exponentially, as researchers noticed the metabolic significance of chlorophylls and carotenoids in photosynthetic organisms. Indeed, they are valuable compounds for cells, with physiologic and economic implications. Next, we describe the main applicability areas where the metabolomics of chlorophyll and carotenoids contributes to deciphering a metabolic response. In some cases, the studies integrate metabolite and physiological data with transcriptional information to confirm both molecular and metabolic modifications. Figure 6 presents different pathways that might emerge during a metabolomics study related to chlorophylls and carotenoids. organic acids, and carbohydrates were the focus of the studies. However, "pigmentomic analysis" is increasing exponentially, as researchers noticed the metabolic significance of chlorophylls and carotenoids in photosynthetic organisms. Indeed, they are valuable compounds for cells, with physiologic and economic implications. Next, we describe the main applicability areas where the metabolomics of chlorophyll and carotenoids contributes to deciphering a metabolic response. In some cases, the studies integrate metabolite and physiological data with transcriptional information to confirm both molecular and metabolic modifications. Figure 6 presents different pathways that might emerge during a metabolomics study related to chlorophylls and carotenoids.

**Figure 6.** Brief description of some plant pathways and reaction processes that could be related to the biosynthesis and catabolism of chlorophyll and carotenoid pigments. **Figure 6.** Brief description of some plant pathways and reaction processes that could be related to the biosynthesis and catabolism of chlorophyll and carotenoid pigments.

#### *4.1. Application in Abiotic Factors Studies 4.1. Application in Abiotic Factors Studies*

Biological tolerance is a complex process that includes not only physio-biochemical modifications but also molecular changes. Such metabolic adjustments are required to respond to environmental signals. Consequently, metabolite profiling brings an opportunity to understand the fundamentals of tolerance by searching for modified or different signatures associated with tolerance ability. In this sense, the adaptations of the chlorophyll and carotenoid metabolism of cells exposed to different stresses have been investigated regarding cesium [105], nitric oxide [106], cadmium [107,108], graphene oxide [109], iron [110], nitrogen depletion [111–113], and extreme irradiation environments [114,115]. Another research field where the metabolomics of chlorophylls and carotenoids finds successful applications is the study of nanotoxicology, which aims to determine the toxicity of metals and micro- and nano-particles to environmental organisms and how the latter respond to the former. In this regard, it was studied how copper oxide nanoparticles, CuO microparticles, and copper ions perturb the metabolism of aquatics organisms [116] and even the effect of ZnO nanoparticles on the cultivation of terrestrial plants [117]. The most striking advance made in these studies is that the experimental design based in metabolic flux measurements might point out specific responses, which include chlorophyll break-Biological tolerance is a complex process that includes not only physio-biochemical modifications but also molecular changes. Such metabolic adjustments are required to respond to environmental signals. Consequently, metabolite profiling brings an opportunity to understand the fundamentals of tolerance by searching for modified or different signatures associated with tolerance ability. In this sense, the adaptations of the chlorophyll and carotenoid metabolism of cells exposed to different stresses have been investigated regarding cesium [105], nitric oxide [106], cadmium [107,108], graphene oxide [109], iron [110], nitrogen depletion [111–113], and extreme irradiation environments [114,115]. Another research field where the metabolomics of chlorophylls and carotenoids finds successful applications is the study of nanotoxicology, which aims to determine the toxicity of metals and micro- and nano-particles to environmental organisms and how the latter respond to the former. In this regard, it was studied how copper oxide nanoparticles, CuO microparticles, and copper ions perturb the metabolism of aquatics organisms [116] and even the effect of ZnO nanoparticles on the cultivation of terrestrial plants [117]. The most striking advance made in these studies is that the experimental design based in metabolic flux measurements might point out specific responses, which include chlorophyll breakdown and the tuning of carotenoids' metabolism. These responses reveal metabolomic-based strategies to allow acclimation of the organisms to the factor under study.

down and the tuning of carotenoids' metabolism. These responses reveal metabolomic-

based strategies to allow acclimation of the organisms to the factor under study.

As chlorophylls and carotenoids are photosynthetic pigments, there are numerous metabolomics studies regarding the influence of light in the physiology of organisms. In this sense, the metabolomics approach has been applied to investigate the effect of light and dark cycles on the lipid metabolome [118], lineage-specific pathways [119], irradiationinduced stress [120], and photo-regulatory processes [121]. Moreover, metabolomics studies were developed to investigate the influence of LED light on the modulation of the fruit metabolome [122] or the porphyrin and chlorophyll metabolism itself [123]. These studies make use of state-of-the-art comprehensive omics analysis, together with a holistic effective treatment of data, although the translation of the results to the productive field requires further testing.

Another line of research is drought stress, considered to be one of the most important limiting environmental factors for agriculture and responsible for great losses of global food production. Once cells detect water stress, a cascade of signals activates multiple biochemical pathways (Figure 6): hormone induction, gene expression regulation, reactive oxygen species scavenging, carbohydrate and energy metabolism, nitrogen assimilation and amino acid metabolism, fatty acid metabolism, etc. Consequently, high-throughput "omics" techniques are essential to gain a holistic panoramic view of the plant response. In general, transcriptome and metabolite profiling reveals that plants respond to drought by modulating several secondary metabolic pathways and particularly by modifying the production of carotenoids or chlorophylls [124–128], including the extreme example of the adaptation to desiccation, as exhibited by resurrection plants [129].

Regarding salinity, plants have developed several mechanisms to adapt to this stress caused by osmoregulation, such as vacuolar H<sup>+</sup> -ATPases, which are key in cytosol detoxification, as they create an electrochemical H<sup>+</sup> gradient across the membranes [130]. Transcriptome and metabolome analyses revealed the crucial biological pathways involved in the fast-adaptive response to salt stress, including carotenoid biosynthesis and the metabolism of porphyrin and chlorophyll [131–134]. An additional multi-omics analysis was used to unveil thermal adaptation strategies of extremophile bacteria [135] and plants [136,137], where the lipid or carotenoid metabolism seems to be implicated. The main effort that requires multi-omics analysis is to select complementary signals in the experimental design, so that the studied signals allow for a deeper understanding of the molecular adaptation of the organism to stress.

A completely different research area where pigment metabolomics was applied is the study of the environmental metabolome, which elucidates the relationship between living organisms and their ecosystem. Through the characterization of the metabolites obtained from the environment, paleometabolites (diagenetic products of chlorophylls and carotenoids derived from photosynthetic algae and bacteria) can be identified [138]. In addition, this technique can be used to determine the toxic effects of organophosphates on the species in freshwater ecosystems [139]. The above compilation is an example of the increasing research areas where chlorophyll and carotenoid metabolomics are involved. This growing trend is broadening our horizons in new, diverse disciplines with a variety of research focuses, such as the determination of metabolic turnover [140], the effects of biostimulants on the metabolome [141,142], and sustainable soil control [143]. All of them are examples of research areas where the metabolomics of chlorophylls and carotenoids has revealed as a successful approach to gather essential information.

#### *4.2. Application in Biotic Factor Studies*

The interrelation between organisms is a subject that has been scarcely studied, surely due to its complexity. However, it is in this subject where metabolomics could successfully contribute to advances in knowledge because of the inherent capacity of this approach of studying several physiological pathways, responses (Figure 6), and behaviors at the same time. Therefore, through metabolomic studies of chlorophylls and carotenoids, significant advances have been achieved with regard to the interplay between biotic stressors; the effect of single- vs. multiple-pest infestations on the biochemistry of plants [144]; the

fluctuation of the leaf metabolome in response to arbuscular mycorrhizal fungi [145]; the microbial networks established during the assemblage of symbiotic microbials, such as in lichens [146]; the ecological interactions that occur at algal surfaces within microbial communities [147]; and the study of the evolutionary origin of symbiosis [148]. In parallel, metabonomic studies have also investigated the interplay between biotic and abiotic stresses, such as the effect of selenium treatments on the oxidative stress response of plants when infected [149] by the enhanced content of chlorophylls and carotenoids and related enzymatic activities. It should be highlighted that this application is very complex, such as the response to interrelation changes at the organ level, while triggering different biosynthetic pathways to down- or up-regulate them.

#### *4.3. Application in Physiologic and Molecular Biology Studies*

Metabolomics is a powerful tool that can be used not only to analyze the response of photosynthetic organisms to external abiotic or biotic stressors but also to conduct an in-depth investigation of their physiology in the widest meaning of the term. This promising research line with economic consequences is, nevertheless, a complicated area of study, taking into account the multiple variables that are accounted for. Examples of the potentiality of this implementation include the study of the mechanisms controlled by the circadian clock [98] and the effects of the auto-tetra-polyploidy on the balance between the primary and secondary metabolisms [150]. However, the main area of applicability is the behavioral patterns in the accumulation of metabolites (chlorophylls, carotenoids, etc.) paired with specific ripening stages, harvesting periods, cultivars, traceability, and plant tissue functions. Sometimes, such correlations are successfully established despite the genetic background or in a timeline fashion [101,151–156]. Additionally, it is possible to distinguish different genetic backgrounds with chemotaxonomic purposes [157], establishing species- and lineage-specific metabolites in marine microalgae [158], and differentiate chemotypes of selected accessions [159]. The metabolomics of chlorophylls and carotenoids could also be used to analyze the effects of postharvest treatments on the metabolism of edible plants [160–164] or for the identification of fast and unequivocal biochemical markers in breeding programs [165,166].

Another field of application is the utilization of metabolomics as a tool to investigate the biochemical pathways implied in the biosynthesis and degradation of these pigments (Figures 3 and 4), identifying pathway cascades [167,168] and revealing the effects of specific genes [169–173]. As a further step, the metabolomics study of pigments could be used as a platform for the development of strategies to engineer fluxes in complex biosynthetic networks [174–176]. A subset of pigment-targeted metabolomics is synthetic biology, which combines known molecular components and genes for the implementation of different molecular pathways displaying novel functions and dynamic behavior that do not occur naturally [177]. A workflow that combines gene expression and quantitative metabolomics with mathematical modeling to identify strategies in order to increase production yields of nutritionally significant pigments has even been proposed [178]. This overall approach, although highly informative and practical, could become difficult to apply as a routine method. Lastly, metabolomics is a common and useful approach for identification purposes [57,85,179,180] and the detection of food processing [71,181,182].

#### *4.4. Application in Human Health (Health Status, Cancer, Hypertension, and Digestive Efficiency) Studies*

In addition to all of these applicability areas, pigment metabolomics has also been applied to the investigation of human health. This is possible thanks to the fact that the concept of health status has moved from just "a state" to "the ability to adapt", which was denoted as phenotypic flexibility. In this context, metabolomics and proteomics were adopted to correlate micronutrients with the characteristics of metabolic parameters and, ultimately, to health-related processes [183]. A poorly scientifically explored research area is the potential bioactivity of metabolites yielded via the catabolism of chlorophylls and carotenoids. The wide array of catabolic products (Figures 3 and 4), including phyllobilins (bilin-type

catabolites of chlorophylls) [184], volatile and non-volatile apocarotenoids arising from the asymmetric cleavage of carotenoids [185], and the carotenoid-derived hormones abscisic acid and strigolactones [186], deserve attention, because they perform antioxidant activities in their natural environment. Additionally, metabolomics was also proposed as a noninvasive and reliable screening technology as an alternative for cancer detection. Currently, diagnostic procedures are costly and invasive, and novel methodologies that could reduce such features of evaluation tests for patients are urgently required [187,188]. Alternative strategies to address the study of cancer are the identification of new compounds against the proliferation of selected cancer cells [189] and the review of the validity of established biomarkers of dietary intake and the identification of novel ones [190]. These studies are still in the hypothesis testing stage and although they embrace a great potential, the focus should be to establish the complex map of cancer-related activities before pointing out a direct link, either positive or negative, between carotenoids and chlorophyll metabolites and cancer effects.

#### **5. Conclusions**

Chlorophylls and carotenoids, known antioxidants, are often evaluated in metabolomics studies with regard to the matter under scrutiny (abiotic/biotic stress) and not as a marker of the metabolic status of the organism. Moreover, the evaluation of these plant pigments is performed with instrumental techniques that yield a global profile count rather than via an in-depth description of both the qualitative and quantitative aspects of the pigment catabolites. This review suggests that the assessment of processes for both primary and secondary metabolisms should consider chlorophylls and carotenoids as key contributors to metabolic study and not simply as "signaling" compounds to determine easily whether something is going wrong or well. Thus, the recently increasing number of published papers, summarized in this manuscript, addressing photosynthetic pigments and metabolomics is generating strong expectations for significant advances in our knowledge of metabolomics as a central piece of functional genomics. Indeed, the study of chlorophyll and carotenoid metabolites requires the development of a wide range of protocols, technical applications, and methodologies. This fact reflects the key role of photosynthetic pigments in the plant metabolism, chemotaxonomy, food technology, and animal health.

**Author Contributions:** Conceptualization, M.R. and A.P.-G.; investigation, M.R. and A.P.-G.; writingoriginal draft preparation, M.R. and A.P.-G.; writing-review and editing, M.R. and A.P.-G.; funding acquisition, M.R. and A.P.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is part of the I+D+i projects RTI2018-095415-B-I00 and AGL2017-87884-R funded by MCIN/ AEI /10.13039/501100011033, and the European Regional Development Fund, FEDER "Una manera de hacer Europa".

**Acknowledgments:** This article is dedicated to Juan Garrido-Fernández, scientist, teacher, friend, on his retirement as *Ad Honorem Scientist* at the Spanish National Research Council (CSIC).

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

#### **References**


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