*2.4. Spectrophotometric Analyses*

The quantification of carotenoid content was performed by spectrophotometric analysis (spectrophotometer UV-1800, Shimadzu, Kyoto, Japan) using specific calibration curves for lycopene (Supelco, purity ≥90% Darmstadt, Germany) and β-carotene (Sigma Aldrich, purity ≥97%, Darmstadt, Germany) constructed by dissolving the related standards at different concentrations in dichloromethane solutions. Dichloromethane (λ cut-off ≤ 233 nm) (Sigma Aldrich, purity ≥99.9%, Darmstadt, Germany) was chosen because it can dissolve both carotenoids; moreover, preliminary tests were performed to check the spectra between 250 and 600 nm of lycopene and β-carotene solutions dissolved in dichloromethane and in the other solvents (*n*-hexane/acetone, ethyl acetate/ethyl lactate and menthol/lactic acid). The resulting peaks were very similar for each solution. Absorbance peaks were identified for lycopene at 477 nm and 481 nm, and for β-carotene at 461 nm. Both wavelengths were considered for lycopene (477 nm for the extractions using the green solvent solutions according to the literature [58] and 481 nm for the extracts obtained with the conventional one according to the absorbance peak obtained from the traditional extract). The calibration curve of lycopene at 481 nm has the equation y = 0.2481x (R<sup>2</sup> = 0.9957), the one at 477 nm y = 0.2393x (R<sup>2</sup> = 0.9829), and the curve of β-carotene y = 0.0042x + 0.0265 (R<sup>2</sup> = 0.9928).

The absorbance of each extract was determined using a UV-spectrophotometer at the abovementioned wavelengths against the respective blank (the different solvent mixtures) and linear regression equations (R<sup>2</sup> > 0.99) were obtained for the three calibration curves and used to determine the lycopene and β-carotene content (μg/g) in each extract; the absorbance of the solvent (blank) concentrations was expressed as μg carotenoids per g of by-product weighted. A regular check was carried out for the accuracy and reproducibility of the absorbance and wavelength scales as well as for stray light.

Color of the extracts was also determined. A quartz cuvette was filled with the extract, and the transmittance was read on a Jasco dual beam spectrophotometer model V-550 UV-Vis (Jasco, Tokyo, Japan). Results were expressed using the CIEL\*a\*b\* scale.

### *2.5. Data Processing and Statistical Analysis*

Data processing and calculation were carried out with Microsoft® spreadsheet program 2016 (Microsoft Corp., Redmond, WA, USA). Analysis of variance (Analysis of variance (one-way ANOVA, Tukey's HSD, *p* < 0.05) and PCA were performed with XLSTAT (Addinsoft Corp., Paris, France).

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

### *3.1. Water Content and Water Activity (aw) in the Raw and Treated By-Products*

The analyses of moisture level and water activity are relevant to assess how each treatment described in Section 2.3 can preserve the by-product by drying it out. As seen in Table 1, the initial moisture content was 63% while the water activity was 0.99. The freeze-dried by-product had the lowest level of moisture content and a w, followed by the non–heat-dried by-product obtained with the prototype. This highlights the satisfactory action of the air-drying prototype, since it allows the level of a w to decrease below 0.7, while the lowest level of a w was reached with the freeze-drier. According to the literature, at an a w lower than 0.75, bacterial growth is inhibited [59]. Finally, the heat-dried byproduct had a w values comparable to those obtained with the prototype, thus supporting possible inhibition of bacterial activity despite the higher moisture content observed for this treatment compared to the two other drying methods; these results are similar to those found in the literature for comparable products [60].

**Table 1.** Mean ( ±SD) moisture content (%) and mean a w for each tomato by-product, both raw and treated, as described in Section 2.3. By-product: raw (P), freeze-dried (L), non-thermal air-dried (E), and heat-dried (S).


### *3.2. Lycopene and β-Carotene Content in the Extracts*

The carotenoid content in extracts strongly differ in relation to both the solvent used and the drying method (Table 2).

Concerning the solvent mixture, the one with the highest extraction potential was the traditional one, acetone:*n*-hexane, which had some practical issues related to evaporation of the solvent during the heating/ultrasound-assisted extraction. This could be useful in a closed system, with possible solvent recovery and extract concentration, but in an open system, it is not sustainable. The use of the DES mixture, composed of ethyl acetate:ethyl lactate, showed promising results in terms of amount of lycopene extracted. In fact, the DES eutectic mixture (samples with code "G") was the second-best option for extraction of carotenoids from the raw by-product, and in particular lycopene, compared to the ultrasound-assisted extraction conducted with the use of traditional solvents (code "T" in Table 2).

In fact, from the raw by-product treated with the greener solvent solution, it is possible to obtain extracts with 27.44 μg/g of lycopene (sample G), which is significantly lower than the 34.11 μg/g obtained with acetone:*n*-hexane (sample T). The use of DES allows practical issues to be overcome related to the high volatility of the traditional organic solution. A similar result was seen for β-carotene with 1510.19 μg/g (sample G) obtained with ethyl acetate:ethyl lactate compared with 2117.64 μg/g from the traditional extraction (sample T). **Table 2.** Carotenoid content in the extract obtained with the three different solvent solutions in combination with the three drying methods. The samples were named using the combination of codes, identifying the different solvent solution, acetone:*n*-hexane (1:1 % *v*/*v*) (T), ethyl acetate:ethyl lactate (30:70 %*v*/*v*) (G), and DL-menthol:lactic acid (8:1 mol/mol) (ML), and the three drying techniques, freeze-drying (L), non-thermal air-drying (E), and heat drying (S) (e.g., MLL: extract obtained from the use of DL-menthol:lactic acid from the freeze-dried by-product).


Different lowercase letters indicate significant differences (one-way ANOVA, Tukey's HSD, *p* < 0.05) among the extracts obtained by the raw and differently treated by-products using the same solvent mixtures. Different capital letters indicate significant differences (one-way ANOVA, Tukey's HSD, *p* < 0.05) among the extracts, obtained by the raw and the treated by-product with the same drying technology, or non-treatment, using different solvent mixtures. \* ND: not detected.

On the other hand, the DL-menthol:lactic acid solution showed difficulties in the interaction between the solvents in the mixture and the by-product due to its viscosity, thus lowering the extraction potential, with 12.32 μg/g and 492.46 μg/g, respectively, for lycopene and β-carotene (sample ML).

Moreover, on the dried by-product, both the drying method and the choice of the extraction mixture had a strong influence on the lycopene and β-carotene content in the final extracts (Table 2). In particular, concerning the drying methods, the prototype (E) showed good potential while preserving the carotenoid content, as shown in Table 3. The use of non-thermal air-drying was superior to freeze-drying in terms of extractions with the DES mixture for both lycopene and β-carotene. The nature of the prototype allows the by-product to be pulverized, and this appears extremely useful, rendering the subsequent interaction between solvent and smashed by-product more effective. Indeed, by reducing the particle size of the raw material, it is possible to enhance the extraction of lycopene and β-carotene; in fact, according to the literature, the extraction potential is inversely proportional to the particles dimension [61]. Indeed, the concentration of lycopene in the extracts obtained from the three different solvent mixtures of the by-product dried with the use of the prototype are comparable: (i) 81.20 μg/g with acetone:*n*-hexane (sample "TE"), (ii) 75.86 μg/g with ethyl acetate:ethyl lactate (sample "GE"), and (iii) 82.86 μg/g with menthol:lactic acid (sample MLE) (Table 2). These results confirm the high potential of the unconventional non-thermal drying procedure. In fact, it allows the by-product to be uniformly pulverized, thus enhancing the extraction potential, thanks to the synergic action of the flux of air and the rotation of the blade rotor, while preserving the most important biological compounds. For this reason, with non-thermal air-drying, the greener solvents performed at their best in terms of extractive capacity.


**Table 3.** CIE coordinates of the color space regarding the different samples of tomato by-product extracts. L\* indicated the lightness, a\* the red/green coordinate, and b\* the yellow/blue. Higher values of a\* are redder, while higher values of b are more yellow rather than blue.

Different lowercase letters indicate significant differences (one-way ANOVA, Tukey's HSD, *p* < 0.05) among the extracts obtained by the raw and the differently treated by-products using the same solvent mixtures. Different capital letters indicate significant differences among the extracts, obtained by the raw and the treated by-product with the same drying technology using different solvent mixtures. \* ND: not detected.

The lycopene concentration of the by-product obviously has a decisive influence on the yield. In fact, Silva and colleagues [58], adopting the same extractive conditions, reported a higher lycopene content (1334.8 μg/g of dried material). Different storage conditions of the by-product in the factory before sampling and diverse tomato variety appear to be responsible for this variability. To verify if it is convenient to extract carotenoids and lycopene from a by-product of the tomato canning industry, the starting point is an evaluation of the quality and quantity of these components in the by-product itself. In addition, the lycopene content in extracts is lower than that of β-carotene; this is due to the previously mentioned storage and processing conditions that can lead to the degradation of lycopene, since its degradation rate is higher than that of β-carotene [62].

Concerning heat drying with the use of the oven (samples coded with "S"), the tests performed show that the lycopene and β-carotene content in the extracts were significantly lower than those found when applying the other drying treatments (Table 2). The main cause may be temperature, which is responsible for the degradation of carotenoids, and, in particular, the total decay of these compounds occurs at 100–145 ◦C according to the literature, even if a proportion is also affected by the increase in temperature, up to 80 ◦C for 2 h, used in the oven [57]. It is clear that heat treatments should be avoided when tomato pomace is used, independently of the solvent used for the subsequent extraction, since the carotenoid yield is dramatically reduced (Table 2).

### *3.3. Color and Principal Component Analysis*

The treatment to dry out the by-product and the choice of solvent mixture also showed a significant impact on the color of the extracts, in terms of L\*a\*b\* (Table 3).

Based on some considerations, in terms of quality, explained in Section 3.2, the different extracts are clearly discriminated in a Principal Component Analysis (PCA) biplot (Figure 1), which takes into account, besides the determination of lycopene and β-carotene contents, the results of color determination.

**Figure 1.** PCA biplot built with the results of the color analysis and the lycopene and β-carotene content for all the samples under consideration.

As shown in Figure 1, the lycopene and β-carotene contents were negatively correlated with the L\* (r = −0.807 and r = −0.905, respectively, *p* ≤ 0.05). On the other hand, b\* was positively correlated with the β-carotene content (r = 0.651, *p* ≤ 0.05). The variable b\*, if positive, is considered as a yellow index [63]. Extracts obtained from freeze-dried and non-thermal air-dried tomato pomace showed a higher level of carotenoids vs. fresh and heat dried (see Table 2) and higher percentages of the yellow and red components (see Table 3). The differences in color among the samples, also related to the content of lycopene and β-carotene, should be taken into consideration for the formulation of different cosmetic, food, and pharmaceutical products, such as the type of the solvent used for the extraction (e.g., food grade), its residual amount in a completely dried extract, or other possible restrictions through laws.
