**Contents**


### **About the Editor**

**Pasquale Crupi** is currently Researcher at the Interdisciplinary Department of Medicine—University of Bari "Aldo Moro". His research interests are mainly focused on metabolomic analyses of food products; in particular, metabolic fingerprinting and metabolite profiling analyses by high resolution and high-throughput technologies (GC–MS, HPLC–MSn, HPLC–MS/MS, NMR) of primary and secondary metabolites (sugars, amino acids, organic acids, polyphenols, carotenoids, aromatic compounds, vitamins, etc.) present in fruit and vegetables (such as grape, cherry, carob, artichoke, olive etc.) and biotransformed products (such as juice, extra virgin olive oil, and wine). He is coauthor of more than 60 publications in international peer-reviewed journals.

## **Preface to "Carotenoids in Fresh and Processed Food: Between Biosynthesis and Degradation"**

Nowadays, consumers look at food not only as a source of energy and nutrition but also an affordable way to promote health and prevent future diseases.

In this context, studying the qualitative and quantitative profiles of natural compounds such as carotenoids, which have antioxidant and anti-inflammatory properties, in food is very relevant. In the last decades, many studies have demonstrated the importance of a diet rich in carotenoids in lowering the onset of certain diseases, such as for numerous types of cancer, cardiovascular diseases, age-related macular degeneration, etc.

Regardless, new insights in this research field are still necessary. Therefore, this *Applied Sciences* Special Issue has collected relevant contributions on interesting aspects related to the composition pattern, biosynthesis and degradation, and overall chemical properties of carotenoids in fresh and processed food, which can improve knowledge in the sector of food science and food chemistry. In this sense, it is worth pointing out that the gathered manuscripts represent valuable advancements in the study of carotenoids.

Of course, naturally thank all authors contributing to this Special Issue in *Applied Sciences* for their scientific input and experimental efforts throughout the project.

> **Pasquale Crupi** *Editor*

### *Editorial* **Carotenoids in Fresh and Processed Food: Between Biosynthesis and Degradation**

**Pasquale Crupi**

Dipartimento Interdisciplinare di Medicina, Università degli Studi Aldo Moro Bari, P.zza Giulio Cesare, 70124 Bari, Italy; pasquale.crupi@uniba.it; Tel.: +39-347-125-2849

#### **1. Introduction**

Currently, there is a general trend in food science to link food and health in line with consumers' concern about what is in their food and how what they eat can promote wellbeing. Thus, food is considered today not only a source of energy but also an affordable way to prevent future diseases. In this context, studying carotenoids content in food is very relevant. Indeed, epidemiological studies have demonstrated that the consumption of diets rich in carotenoids is associated with a lower incidence of cancer, cardiovascular diseases, and age-related macular degeneration, mainly due to their antioxidant and provitamin A activity [1]. Although many works have been conducted concerning the presence and properties of carotenoids in food [2], some challenges must be still faced in this research field: The role of carotenoids as antioxidants and its mechanism of action need to be investigated further; detailed qualitative and quantitative composition of carotenoids in underutilized fruits and vegetables is required in order to contribute significant information to select nutrient rich plants for food formulation; how emerging packaging and processing techniques (i.e., high electric field pulse, high-pressure CO2, etc.) can preserve the content of carotenoids in processed food products needs to be understood; the complete understanding of carotenoid biosynthesis, regulation, and roles of various carotenoid derivatives for edible plants and animals is still not well established; and detailed studies for identifying the pre- and post-harvesting favorable factors (i.e., elicitors, cooking methods, etc.), which improve the bioavailability and bioaccessibility of carotenoids from different foods, are necessary.

The Special Issue "Carotenoids in Fresh and Processed Food: Between Biosynthesis and Degradation" was aimed to invite worldwide scholars (particularly experts in the sector of food science and food chemistry) to submit their most interesting communications, reviews, and original articles that can improve the knowledge in the field of carotenoids in food.

Potential topics included, but were not restricted to, carotenoids and apocarotenoids chemistry and biosynthesis, structural isomerization and degradation, content in vegetable and non-vegetable foods, and bioavailability and bioaccesibility methods of analysis.

#### **2. Carotenoids in Fresh and Processed Food: Between Biosynthesis and Degradation**

The aim of this Special Issue was to group the most recent and relevant research in relation to the aforementioned topics regarding carotenoids in food into a single document. Subsequently, the possibility of publishing a book with the contributions of all authors has been assessed. There were six papers submitted to this Special Issue, and five of them were accepted. In the following paragraphs, a summary of these papers with their most relevant findings is presented.

The first paper [3] deals with the protection of β-Carotene from photodegradation. The authors of this work showed how β-Carotene degrades rapidly in a 2% oil-in-water emulsion, made from food-grade soy oil with 7.4 mg β-carotene/mL oil, during storage and when exposed to light. However, the addition of clove oil (2.0, 4.0, or 8.0 μL/mL of

**Citation:** Crupi, P. Carotenoids in Fresh and Processed Food: Between Biosynthesis and Degradation. *Appl. Sci.* **2022**, *12*, 1689. https://doi.org/ 10.3390/app12031689

Received: 22 January 2022 Accepted: 3 February 2022 Published: 7 February 2022

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

**Copyright:** © 2022 by the author. 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/).

emulsion) prevented the photodegradation of β-carotene, regardless of the ratio between clove oil and β-carotene in the concentration range studied. Since plant phenols have been demonstrated to efficiently regenerate carotenoids from their initial photooxidation products, the authors concluded that the observed regeneration of β-carotene was due to eugenol, the main plant phenol of clove oil to occur in the oil-water interface. Therefore, clove oil in low concentrations may find use as a natural protectant of provitamin A in enriched foods during retail display.

The second paper [4] presented how two typical plant hormones, namely salicylic acid (SA) and methyl jasmonate (MeJA), were able to regulate the accumulation of flavonoids (i.e., eriocitrin, narirutin, and poncirin) and carotenoids (i.e., β-cryptoxanthin) in the juice sacs of Satsuma mandarin in vitro. The results showed that SA treatment was effective in enhancing the contents of eriocitrin, narirutin, poncirin, and β-cryptoxanthin, whilst MeJA treatment inhibited these compounds accumulation in the juice sacs (*p* < 0.05). Moreover, gene expression analysis confirmed that the changes of flavonoid and carotenoid contents were highly regulated at the transcriptional level. In particular, a transcriptional factor CitWRKY70 was identified in the microarray analysis, which was induced by the SA treatment while being suppressed by the MeJA treatment. Since the change in the expression of CitWRKY70 was consistent with that of flavonoid and carotenoid biosynthetic key genes, this finding indicated that CitWRKY70 might be involved in the regulation of the investigated compounds content in the juice sacs of citrus fruit in response to SA and MeJA treatments.

In the third article, Gał ˛azka-Czarnecka et al. [5] studied the influence of light at different wavelengths (white light at 380–780 nm, UVA at 340 nm, blue light at 440 nm, and red light at 630 nm) and pulsed electric field (PEF) at different strength (1, 2.5 and 5 kV/cm) on the content of carotenoids (i.e., lutein, zeaxanthin, and β-carotene) in red clover sprouts. The experiment was carried out in a climatic chamber with phytotron system under seven growing conditions differing in light-emitting diode (LED) wavelengths and PEF strength applied before sowing. Lutein was found as the dominant carotenoid in germinating red clover seeds, with content varying from 743 mg/kg in sprouts grown in red light to 888 mg/kg in sprouts grown in blue light. Blue light treatment during the red clover sprouts growing had the most beneficial effect in enhancing carotenoids content up to 42% in β-carotene, 19% in lutein, and 14% in zeaxanthin. An increase of β-carotene (8.5%) and lutein (6%) amount was also obtained with white light without PEF pre-treatment; conversely zeaxanthin decreased by about 3.3%. Therefore, the authors concluded that PEF pre-treatment may increase mainly the content of β-carotene in red clover sprouts.

The presence of carotenoids in grape berries is well documented [6]; the grape variety and viticulture practices, but also climate conditions and geographic origin, can influence their qualitative and quantitative profile as well as their degradation during grape ripening from véraison to harvest [7]. The last two works, belonging to this SI, treated about effective practices for conditioning carotenoids degradation in grapes. In particular, Asproudi et al. [8] investigated the impact of bunch microclimate on the evolution of some relevant carotenoids (i.e., neoxanthin, luteinin, and β-carotene) in Nebbiolo grapes, collected from green phase up to harvest, during two consecutive seasons. Overall, higher temperature in the less vigorous and south facing vineyards led to lower amounts of carotenoids, both during ripening and at harvest. Lutein and neoxanthin contents (μg/berry) varied similarly in both seasons and achieved a maximum after veraison, especially in the cooler plots. Therefore, a variety effect on the lutein seasonal trend was hypothesized. Conversely, b-carotene content remained generally constant during ripening, with the exception of the south plots showing dissimilar evolution between the seasons. This observation allowed the authors to conclude that bunch zone temperature and light condition may affect both synthesis and degradation of grape carotenoids determining their amount and profile at harvest.

Crupi et al. [9] aimed to study the effect of the foliar application of yeast extracts (YE) to Negro Amaro and Primitivo grapevines on the carotenoid content during grape ripening

and the difference between the resulting véraison and maturity (ΔC). The results showed that β-carotene and (allE)-lutein were the most abundant carotenoids in all samples, ranging from 60% to 70% of total compounds. Their levels, as well as those of violaxanthin, (9-Z) neoxanthin, and 5,6-epoxylutein, decreased during ripening. This was especially observed in treated grapes, with ΔC values from 2.6 to 4.2-fold higher than in untreated grapes. Thereby, the YE treatment has proved to be effective in improving the C13-norisoprenoid aroma potentiality of Negro Amaro and Primitivo, which are fundamental cultivars in the context of Italian wine production.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


### *Communication* **Clove Oil Protects** *β***-Carotene in Oil-in-Water Emulsion against Photodegradation**

**Yi-Ming Zhou 1, Hui-Ting Chang 1, Jian-Ping Zhang 1,\* and Leif H. Skibsted 2,\***


**Abstract:** *β*-Carotene degrades rapidly in a 2% oil-in-water emulsion, made from food-grade soy oil with 7.4 mg *β*-carotene/mL oil, during storage and when exposed to light. Added clove oil (2.0, 4.0, or 8.0 μL/mL of emulsion) protects against the photodegradation of *β*-carotene, regardless of the ratio between clove oil and *β*-carotene in the concentration range studied, suggesting that the regeneration of *β*-carotene is caused by eugenol, the principal plant phenol of clove oil to occur in the oil-water interface. Therefore, clove oil in low concentrations may find use as a natural protectant of provitamin A in enriched foods during retail display.

**Keywords:** eugenol; photoprotection; provitamin A

#### **1. Introduction**

While *β*-Carotene (*β*-Car) is an important provitamin A, it is sensitive to light and degrades rapidly in plant oils or in plant oil emulsions during storage under ambient conditions [1–4]. Vitamin A deficiency is a major challenge worldwide, especially for children's nutrition, and urgently calls for practical solutions [5,6].

Recently, it was found that plant phenols regenerate *β*-Car and other carotenoids (Car) from their initial photooxidation product, the carotenoid radical cations (Car•+), through electron transfer from the reducing phenol group *ϕ*-OH [7]:

$$\text{Car}^{\bullet+} + \text{ $\varphi$ -OH} \rightarrow \text{Car} + \text{ $\varphi$ -O}^{\bullet} + \text{H}^{+} \tag{1}$$

The regeneration of *β*-Car corresponding to the reaction of Equation (1) was surprisingly found to be the most efficient for moderately reducing plant phenols, such as eugenol, while strongly reducing plant phenols, like tea catechins, showed no regeneration of *β*-Car, but displayed enhanced photobleaching [7–9].

Eugenol and isoeugenol, the main constituents of clove oil [10], are moderately reducing plant phenols that have been found to regenerate *β*-Car efficiently from the radical cation formed by photolysis of *β*-Car. This reduction occurs in alkaline chloroform/methanol as an electron-withdrawing solvent [11]. The ordering of the anions of the plant phenols according to the rate of regeneration of carotenoids could further be accounted for by the Marcus theory of electron transfer [12]. According to this theory, the maximal rate of electron transfer corresponds to a driving force matching the reorganization energy in the transition state for electron transfer. Notably, for a larger driving force, the rate of electron transfer enters the so-called inverted region with a higher activation barrier, and accordingly, lower rates are seen for quercetin and tea catechins [7,12].

The more practical aspects of the Marcus theory for electron transfer have not yet been exploited in relation to food preservation. However, the protection of *β*-Car, as a provitamin A in an oil-in-water emulsion in a functional food, could provide a proof of

5

**Citation:** Zhou, Y.-M.; Chang, H.-T.; Zhang, J.-P.; Skibsted, L.H. Clove Oil Protects *β*-Carotene in Oil-in-Water Emulsion against Photodegradation. *Appl. Sci.* **2021**, *11*, 2667. https:// doi.org/10.3390/app11062667

Academic Editor: Pasquale Crupi

Received: 19 February 2021 Accepted: 13 March 2021 Published: 17 March 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/).

concept, where the use of Marcus theory could be moved from model systems involving chlorinated solvents of high pH into a real food system. Accordingly, clove oil, with high content of moderately reducing plant phenols and its worldwide use in food and beverages, was combined with *β*-Car in an oil-in-water emulsion and stored under illumination in ambient conditions with the objective of protecting provitamin A against degradation during retail display. The present study aimed to explore whether the Marcus theory for electron transfer could be used to design optimal protection of a light-sensitive vitamin.

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

#### *2.1. Materials*

All-*trans*-*β*-carotene (*β*-Car) was from Sigma-Aldrich (St. Louis, MO, USA). Clove oil, containing 85% eugenol and isoeugenol, was from O'plants (Shanghai, China). Soybean oil was from Yihai Kerry Food Co., Ltd. (Beijing, China). Whey protein isolates (WPI) were from HIRMAR (Los Angeles, CA, USA). Lecithin (95%) was from Arbor Star Biological Technology Co., Ltd. (Beijing, China).

#### *2.2. Preparation of Emulsion*

*β*-Car (40 mg, accurately weighted) and lecithin (2400 mg) were mixed in 5.4 mL soybean oil and stirred at 1000 rpm for 3 h in the dark to fully dissolve the mixture in the oil phase. The water phase contained 12 g WPI in 280 mL deionized water with the pH of the aqueous solution adjusted to 7.0. This solution was adjusted by dropwise addition of dilute HCl and NaOH, while pH was monitored electrochemically. The emulsion was prepared by mixing the oil phase with the aqueous solution and homogenizing the mixture at 13,000 rpm for 5 min using an FA25 homogenizer (Shanghai, China). Subsequently, 10 mL of emulsion samples were added to glass jars before adding the clove oil (0.02 mL, 0.04 mL, or 0.08 mL) to different samples, which were homogenized at 13,000 rpm for 2 min. In this study, 10 mL emulsion without clove oil served as the control sample. The emulsion had a fat content of approximately 2%, which is comparable to milk and other nutritive beverages. The final concentration of eugenol and isoeugenol from clove oil was 1.7, 3.4, or 6.8 μL/mL emulsion. All samples were stored under light (spectral distribution in the 300−800 nm range, 22,000 Lx warm white similar to light used for illumination during retail display) at 25 ◦C. Control emulsion samples were stored in the dark at 25 ◦C. The main experiment, as described in Figures 1 and 2 as well as in Table 1, was in storage for three weeks. The standard deviation of each color measurement was less than 1%.

**Figure 1.** Appearance of oil-in-water with *β*-Car during storage under ambient conditions and on exposure to light in glass jars with and without addition of 4 μL/mL emulsion of clove oil.

**Figure 2.** Red bleaching (relative redness *a*/*a*0) of oil-in-water emulsion with *β*-Car with or without the addition of clove oil during storage in the dark or exposed to light of 22,000 Lx at 25 ◦C.

**Table 1.** The redness parameter *a* of *β*-Car and *β*-Car-clove oil on light exposure compared to dark storage for different days. The redness parameter of 0 day is defined as *a*0.


#### *2.3. UV-Visible Absorption Spectroscopy*

UV-visible absorption spectra were measured on a Cary50 spectrophotometer (Varian Inc., Palo Alto, CA, USA), using 1.0 cm quartz cells. According to Lambert-Beer's law, the soybean oil acted as a mixed low-polarity solvent for the concentration of *β*-Car as it relates to the absorbance:

$$
\mathcal{L} = \frac{A\_{\lambda}}{\varepsilon\_{\lambda} \cdot b} \tag{2}
$$

Equation (2) was used for quantification in this study [13]. In Equation (2), *c* is the molar concentration (mol·L<sup>−</sup>1) of *<sup>β</sup>*-Car. *<sup>A</sup>*<sup>λ</sup> is the measured absorbance, and *<sup>ε</sup>*<sup>λ</sup> is the molar extinction coefficient of *<sup>β</sup>*-Car in pure soybean oil (1.43 × 105 <sup>L</sup>·mol−1·cm<sup>−</sup>1) at the maximal absorption wavelength (λ) of 462 nm. *b* is the optical pathlength of the cuvette (1 cm).

#### *2.4. Color Measurement*

The LAB Hunter values of the emulsion samples were measured multiple times by a PR-780 Spectrophotometer (Photo Research, Los Angeles, CA, USA) during storage for up to 21 days. In the main experiment, photographs of the samples were taken regularly during storage using a digital camera. The light source for color measurement was a tungsten lamp (40 W), and a standard, white tile served as a background.

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

*β*-Car is lipophilic (log*P* = 12.2) and dissolves in the soy oil of the oil-in-water emulsion. Clove oil consists mainly of eugenol with log*P* = 2.49 [14,15] which distributes between the oil and the aqueous phase. As evident from Figure 1, the emulsion appeared homogeneously red. The concentration of *β*-Car in the emulsion oil phase was 7.4 mg/mL soy oil, while the phenols from clove oil were distributed between the two phases. Soy oil was selected for the oil phase of the emulsion as it is edible, with good nutritive value, and is available worldwide. Lecithin with 8.4 mg/mL emulsion was also added because it is commonly used as an emulsifier in foods.

When stored in the dark, the color remained constant, as depicted in Figure 2. In Table 1, *a*, i.e., the redness parameter of the LAB color system, is shown for 21 days of storage, while *a*<sup>0</sup> is the redness parameter of day 0. The presence of clove oil did not affect the color during dark storage at any of the three concentrations. This finding was in agreement with the robustness toward uncatalyzed degradation of *β*-Car which was previously observed [16].

Upon exposure to light, the redness faded, as was evident from visual inspection; see Figure 1. Carotenoids are generally sensitive to radiation, including light and *γ*irradiation [17]. The redness parameter *a* also showed a significant decrease during storage when exposed to light (Table 1 and Figure 2). The presence of clove oil clearly provided protection, as bleaching was reduced to approximately half of that in the emulsion without clove oil. Notably, the protection of color, and accordingly, of *β*-Car, was not dependent on either the amount of clove oil added or the concentration of the plant phenols in the concentration range studied (clove oil between 2.0 μL/mL and 8.0 μL/mL), due to the saturation of plant phenols at the emulsion interface. The decrease of the redness parameter *a* could be described by a mono-exponential model function for each of the three independent experiments for which the rate constant was 0.089 days<sup>−</sup>1, and was not dependent on the clove oil concentration. This type of protection was similar to that of plant phenols toward the carotenoids involved in the visual function [18].

The light source used for the storage experiment had an intensity of 22,000 Lx, was mainly in the visible region, and had a minor UV-component. The glass of the jars further served as a UV-filter. As seen from the absorption spectra of Figure 3, the light was absorbed by *β*-Car rather than by the clove oil phenols. Excitation of *β*-Car to the singlet or triplet states generated radical cations, leading to bleaching:

$$\text{Car} + \text{h}\nu \to \, ^1\text{Car} \ast \tag{3}$$

$$^1\text{Car}\* \to ^3\text{Car}\*\tag{4}$$

$$\text{ $^3$ Car\*/} ^1 \text{Car\*} \text{--} \text{ $^\*$ Ar\*} + \text{e}^- \text{ (solv.)} \tag{5}$$

$$\text{Car}^{\bullet,+} \rightarrow \text{Degradation products} \tag{6}$$

**Figure 3.** Absorption spectra of *β*-Car in methanol normalized at 450 nm and of clove oil in methanol normalized at 283 nm.

According to Equations (1) and (6), the regeneration of the carotenoid from radical cations will compete with its degradation. As seen in Figure 2, the bleaching was independent of the concentration of eugenol in the concentration range studied.

The regeneration rate previously found for homogeneous solution [7]:

$$\frac{d[\text{Car}]}{dt} = k\_2[\text{Car}^+] \left[\text{ $\varphi$ -OH}\right] \tag{7}$$

seems for the present conditions independent of the total phenol concentration in the emulsion. This apparent zero-order dependence on the plant phenol for the emulsion probably indicated: (i) a rapid electron transfer, and (ii) an apparent similar excess of phenol available for reduction under all conditions investigated. These observations pointed toward a mechanism occurring in the emulsion interface that was saturated with the plant phenols. The rate expression of Equation (7) was based on a series of more systematic kinetic studies in homogenous solutions [7–9,11]. The observed kinetics for the photodegradation of *β*-Car in the oil-in-water emulsion can be accommodated within this theory, including the partition of eugenol between the homogeneous aqueous phase and the heterogeneous oil phase. The distribution between water and oil may be adjusted as eugenol is consumed.

In the oil-in-water emulsion, the protection of *β*-Car by clove oil is an important finding, since regeneration occurs at neutral pH as compared to the conditions of high pH used in model studies [7–9,11]. The phenols of clove oil and not only their anions are sufficiently reducing for the donation of an electron, and have matching reduction potential according to Marcus' theory to reduce the carotenoid radical cation [12]. Isoeugenol and especially eugenol may be unique in this respect; nevertheless, other plant oils and plant phenols with similar, moderate reduction potentials are now being investigated for their ability to protect carotenoids against light degradation in food.

Designing functional foods with better shelf life is encouraging, as there is a serious problem with vitamin A deficiency worldwide [19]. Moreover, the use of plant oils will provide such products with a natural image of sustainability. The practical application of these findings still needs further development, but in light of the simple procedures required, the perspective seems encouraging, especially for developing countries.

#### **4. Conclusions**

Our results show that clove oil protects *β*-carotene in an oil-in-water emulsion from photodegradation due to the content of moderately reducing plant phenols. It serves as a proof of concept for the use of the Marcus theory for electron transfer as a strategy for the protection of vitamin A and provitamin A compounds, thus addressing a global problem.

**Author Contributions:** Conceptualization of the study and wrote the manuscript, J.-P.Z. and L.H.S.; designed experiments, performed experiments and analysis of data, Y.-M.Z. and H.-T.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been supported by the Natural Science Foundation of China (Grant Nos. 21673288, 21573284).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data and analyses from the current study are available from the corresponding authors upon reasonable request.

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

#### **References**


#### *Article*
