Sustainability: Obtaining Natural Dyes from Waste Matrices Using the Prickly Pear Peels of Opuntia ficus-indica (L.) Miller

In this work, the effect of dyes extracted through the Naviglio method (an eco-innovative solid-liquid extraction technique) was tested, which proved interesting for such an extraction. The dyes extracted from Opuntia ficus-indica (L.) Miller waste were treated to maximize the extraction of the coloring molecules. The extraction method was compared with conventional methods under the same conditions. The results showed that the extracts obtained with the different techniques, in the same extraction conditions (solvent, pH, temperature, time), were richer in the pigments for the extract obtained with the Naviglio method. The stability of the dyes was tested before the staining. A plant-based fiber, cotton, as well as an animal-based fiber, wool, were chosen for the staining tests. For the two types of fiber, two etching methods were used: one with rock alum, a method widely used to fix a color and one with lemon juice, a method used for a long time by rural populations who practiced the coloring of hides and tissues. In addition, the qualitative content of the pigments was also verified with an HPLC profile of the Opuntia ficus-indica (L.) Miller extracts. Ultimately, the results suggest that the Opuntia ficus-indica (L.) Miller fruit can be of great interest as a natural source of dyes and be used for tinctures.


Introduction
The agricultural and food industries are particularly sensitive to the disposal and/or reuse of waste, especially due to the increasing amounts of waste in their production processes. In Italy only, in 2012, with increasing data up until 2018, it was estimated that 3,037,092 tons of vegetable waste were produced and that 96% of it was derived from the food, beverage and tobacco industries [1,2]. transported to the laboratory within a maximum of 3 h after harvest. Subsequently, the pears were washed thoroughly with distilled water to remove impurities and thorns. All the samples were left frozen at −24 °C until they were analyzed. The samples were prepared manually; the peel was separated from the pulp and the seeds together with the mucilage were separated.

Preparation of Prickly Pear Peel Extracts
The sample to solvent ratio was kept constant (plant material:solvent = 1:10, w/v) for the two methods of extraction and for all experiments.
The plant materials were extracted by two different extraction methods; moreover, two different pure solvents and a mix of them were used: ethanol (EtOH), bidistilled water (H2O) and a solution of EtOH and H2O in a ratio of 8:2 (v/v). The latter ratio was selected on the basis of previous extraction studies found in the literature, which testified that this EtOH and H2O ratio was the most promising for extracting the various components present within the matrix [9,10,[17][18][19][20][21][22][23].
Extractions were conducted at room temperature for 2 h.

Apparatus
The Extractor Naviglio ® represents an eco-innovative technological solution in solid-liquid extraction. This extraction was achieved through the discovery of a new principle of solid-liquid extraction, called "Principio di Naviglio" (Naviglio's Principle) [24]. The Extractor Naviglio ® is based on a suction effect, with a pressure of approximately 9-10 bar for a specific time (the defined cycle) and is followed by an immediate decompression at atmospheric pressure. The rapid release of the liquid extract from the inside of a solid matrix, because of the pressure gradient, mechanically transports the extractable compounds contained in the solid matrix outwards (Naviglio's Principle). An extractive cycle includes a static phase followed by a dynamic phase. During the static phase, the system is subjected to pressure for a time which is determined by the composition of the solid matrix from which molecules have to be extracted; in this phase the liquid penetrates into the solid and it permeates all the empty spaces passing through the cellular membranes of vegetables in a manner more efficacious than maceration. When the static phase is finished, the dynamic phase starts immediately. It accomplishes two objectives; the first is the generation of a negative pressure gradient between the inside and the outside of the solid matrix, which represents the dragging effect of the extraction (Naviglio's Principle), while the second objective is to stir the liquid in the entire system again to prevent the formation of a super saturation zone of extracted substances near the solid surface.
All absorbance measurements were carried out using a Merck Pharo 300 spectrophotometer (Merck Millipore GmbH, Milano, Italy). A 1.0 cm long optical path glass cell was used in all measurements.
A Metrohm 827 pH meter (Metrohm Italiana S.r.l., Origgio (VA), Italy), a two-channel laboratory pH measuring instrument for measuring pH/mV and temperature, was used for the pH measurement.
Specific gravities were measured with a Brix & Gravity Aichose Refractometer (Qingdao Xindacheng Plastic Machinery Co. ltd, Jiaozhou City, Qingdao, China) with automatic temperature compensation (ATC) that features a 0%-32% Brix Scale and a Specific Gravity Scale from 1.000-1.130.

Maceration Extraction (ME)
Ten grams of prickly pear peel were extracted by macerations with 100 mL of EtOH, H2O and their mixture in a ratio of 8:2 (v/v) under continuous stirring and in the dark, for 24 h. After maceration, the extracts were filtered through paper to remove any floating matter.

Extractor Naviglio ® Extraction (NE)
The extraction was then repeated on another sample (with the pulp and peel of prickly pears) using the Extractor Naviglio ® (ATLAS FILTRI ITALIA, Limena, Padova, Italy). A quantity of 60 g of the sample was placed in a 50 µm filtering membrane bag and placed into the 500 mL chamber of the Extractor Naviglio ® . Then, the chamber was filled with the solvent. The extractions were carried out with solutions of EtOH, H2O and their mixture in a ratio of 8:2 (v/v). Each extraction cycle consisted of a 2 min static phase, followed by a 2 min dynamic phase; the total number of cycles was 360 for a total time of 24 h. After filtration, the clear solution extract obtained was stored in a dark container.

Soluble Solid Contents and pH
The soluble solid content (°Bx) and pH were measured in all extracts by means of a refractometer and Metrohm pH meter.

Liquid-Liquid Extraction
The extracts obtained with the different techniques were analyzed by HPLC. After the evaluation of the chromatograms, the separation of the pigments and the bioactive components was carried out, including a liquid-liquid separation of the separating funnel. Solvents with increasing polarity were used, such as n-hexane, chloroform and butanol. The liquid-liquid separation led to obtaining four fractions in different solvents which were desolvated with a rotary evaporator. The remaining fraction in double distilled water was concentrated and stored at 4 °C in sealed dark glass bottles. These samples were used as dyes for the coloration of animal and vegetable type textile fibers, wool and cotton, respectively.
The fractions that were obtained through the liquid-liquid separation, carried out with solvents with increasing polarity, allowed for the extraction of several compounds of well known biological interest and that could have nutraceutical uses: compounds such as fatty acids, small molecules like polyphenols and amino acids were found.

Chemical Characterization of the Extracted Dyes
The extracts of Opuntia ficus-indica (L.) Miller were chemically characterized with spectroscopic and chromatographic techniques. The dye extracts were subjected to UV-VIS spectrophotometry (Merck Pharo 300) after appropriately diluting the solution containing the extracted dye with water (0.5 mg/mL) [25]. UV-VIS spectra were acquired in a visible region of 350-650 nm. For the quantitative internal standard, a calibration curve was prepared using final concentrations of 0.015, 0.032, 0.065, 0.130, 0.25, 0.5 and 1.0 mg/mL in triplicate. Quantitative determination was made taking into account the maximum absorbance of the absorption signal, at the wavelength of the analyzed colored compound reported and expressed in milligrams per gram of the prickly pear peels: the estimated amount of coloring compounds was 5•10 −3 g/gpeel. The R 2 interval was between 0.9849 and 0.9990 and the relative standard deviation (RSD) slope values between the measured samples were less than 5%.
The HPLC separation of the active compounds was carried out by using a C-18 column (Dr. Maisch GmbH, 250 mm × 4.6 mm, particle size 5 µm) set for a gradient elution. Eluent A was water (0.1% trifluoroacetic acid) and eluent B was acetonitrile (0.1% trifluoroacetic acid). The gradient program was 15% B for 5 min, 15%-20% B over 5 min, 20%-25% B over 10 min, 25%-35% B over 10 min and 35%-50% for 10 min. The flow rate was 1.0 mL•min −1 and the injection volume was 20 µL at 25 °C. The identification of these compounds was carried out by comparing the retention time in the extracts of the injected samples to those of the standard HPLC compounds. The qualitative determinations were made taking into account the absorbance signal of each peak relative to the analytical standards (betalain, quercetin, luteolin, kaempferol and isorhamnetin) of the compounds found at the same wavelength. Chromatograms were observed at a 340-540 nm wavelength.

Dyeing Fiber with Opuntia ficus-indica (L.) Miller Dye Extract
Cotton and wool were the fibers selected for the study. They were previously leached in an aqueous solution containing 0.5 g/L of sodium carbonate and 2 g/L of non-ionic detergent at 50 °C for 60 min. Subsequently, threads of fiber material, between 20 cm (wool fibers) and 35 cm (cotton fibers) were prepared for dyeing. The dyes obtained from Opuntia ficus-indica (L.) Miller were fixed to the fibers with the use of mordants. The mordants used were rock alum (potassium alum sulphate) and lemon juice; both methods were reported by popular tradition. The fibers were dyed with the extract dye of Opuntia ficus-indica (L.) Miller without the use of mordants in obscured balloons, sealed and placed under agitation with a magnetic stir bar.
The etching solutions were prepared by placing 245 mg of rock alum in 500 mL of deionized and double distilled water (solution 1) and placing 100 mL of lemon juice in 500 mL of deionized and double distilled water (solution 2). The solutions were placed under magnetic stirring and heated for 60 min at 80 °C.
Two processes were used: pre-mordant, a process in which the sample is first mordanted and subsequently dyed and a process in which the fibers were dipped directly into the coloring solution.
The fiber samples were weighed (1 g) and immersed in the solutions. For the pre-mordant method, the tissues were first immersed in the aqueous solutions of each of the aforementioned mordants at 80 °C for 60 min. The samples were then removed and without drainage were immersed in an aquatic extract of Opuntia ficus-indica (L.) Miller (10 mL) at 80 °C for 24 h with constant stirring (Figures 1, 2 and 3). Subsequently, the drained and dried fabrics were washed twice (2 min) at 40 °C with distilled water.
For direct coloring without mordants, the fibers were immersed in a bath containing the coloring extract of Opuntia ficus-indica (L.) Miller at room temperature (25 °C) and under stirring for 24 h. After dyeing, the surplus of dye was removed from the colored materials by rinsing twice with water at 40 °C.
Samples of the non-colored fibers were used as controls. The experiments were performed by triplicates.

Colorimetric and Fastness Properties of Fiber Dyed with Opuntia ficus-indica (L.) Miller Dye Extract
The tissues dyed with the Opuntia ficus-indica (L.) Miller extracts ( Figure 4) discussed in this study (see Section 2.10) were subjected to reflectance color measurements using a FRU WR-10 QC colorimeter from ShenZhen Wave Optoeletronics Technology with a viewing area of 4 mm in diameter. The following measurement conditions were selected: light source D65, which represented a daylight phase, with a correlated color temperature (CCT) between 5000-6500 K and a standard CIE (International Commission on Illumination, from its French name, "Commission internationale de l'Éclairage") 10° observer. The measurements were made using the method reported in the work of Chan-Bacab et al., 2015 [26], that reported the right method to apply to the analysis of fiber coloring using CIE LAB (also known as CIE L*a*b*, expresses the color through three values L, a and b as explained below) standards [27].
Therefore, the color measurements were analyzed considering the CIE L* a* b* or CIELAB color system (CIE 1986) [27], widely endorsed by both the scientific community and industry [28][29][30][31]. The CIELAB color system organized the perception of the three-dimensional color space (which appears to be approximately uniform in color) represented in rectangular coordinates: L*, a*, b*. The L* axis is associated with the lightness of the color going from value 100, corresponding to white, to value 0, corresponding to black, while the axes a* and b* are associated with the changes of red-green (where when a* is positive it corresponds to red and when a* is negative it corresponds to green) and yellowblue (where when b* is positive it corresponds to yellow and when b* is negative it corresponds to blue). These reading parameters allow to easily calculate, through the use of tabulated equations, the attributes of the perceived coloring: intensity and tonality. These attributes are calculated as follows: The compared samples were the colored samples with the non-colored ones and the colored samples with and without the use of mordants. The samples compared were the colored samples with the non-colored samples and the colored samples with and without the use of stain. Between ten and fifteen measurements were made, carried out in random positions of the textile fibers. On the basis of these repeated measurements, the minimum number of measurements necessary to quantify the color of the colored textile material under examination was established, taking into account the cumulative averages of the coordinates of the CIELAB color system as also defined according to work by Prieto et al., 2010 [32]. The

Results and Discussions
With a view to the circular economy, the dyes obtained from natural sources and specifically from waste matrices deriving from the agro-food industry are increasingly at the center of research for their ecological value and non-toxicity. For example, in 2018, the global market for dyes from natural sources was estimated to be around 2.3 billion for natural food dyes alone, for their application in beverages. This shows the economic importance of these biomolecules that lies in modern bioeconomies.
In this context, the Opuntia ficus-indica (L.) Miller and in particular its waste identified in its peel, was selected for the extraction of the dyes [12].
It is possible to identify within the literature of the studies carried out on the use of dyes extracted from other vegetable waste matrices, such as the study on pomegranate peel (Punica granatum L.) [35], the use of different dye extraction and fixing methods.
Different parameters of the extracts obtained with the different extraction techniques were measured [36]. The values of pH, specific gravities and dry residue were collected for all extracts at 24 h. The values are shown in Table 1. An important result (in degrees Brix) was obtained in the evaluation of the soluble solid, that within the extracts, the specific gravity was very low in relation to the other components present [37].
The extracts were primarily analyzed by UV-VIS spectrophotometry to evaluate the extraction of the pigments present within the extracts obtained with different extraction techniques and different solvents. The spectra obtained showed the presence of coloring molecules belonging to the betalain family. By comparing the spectra obtained for the extracts obtained through conventional maceration and the Naviglio method (data not shown for brevity), they showed that the extracts obtained under the same conditions (solvent, time, pH, ratio sample:solvent) with the Naviglio method had a higher content of coloring molecules compared to those obtained by maceration. Thus, it was concluded that the use of the Naviglio method allowed to obtain richer extracts in less time, as reported in the literature for other applications [38][39][40]. Figure 5 shows the spectra obtained from the analysis of the extracts of Opuntia ficus-indica (L.) Miller with the Naviglio method, which shows a better content of coloring molecules, even if only slightly, for the extract obtained with water. The extracts were dried with a rotavapor and dissolved in bidistilled and deionized water to study their stability according to some parameters.
The storage stability of the coloring extracts was studied. Storage stability was studied at different pH values (3 < pH < 7) and at 4°C and 25 °C. The pigment extracts showed a very high storage stability, which was at its maximum at pH 5 and 4 °C ( Figure 6). A t1/2 of 20 days was found for red-violet peel extract at 25 °C and pH 5. The visible absorption spectra of the extracts of the red-violet peels from the Opuntia ficus-indica (L.) Miller with the Extractor Naviglio ® in water solvent, stored at 4 °C, are shown in Figure 6. Initially, only one peak at 535 nm could be detected, due to the presence of pigment molecules. These compounds were progressively degraded, while absorbance at 535 nm decreased at the same time. At pH 7, a small shoulder was observed with a maximum absorbance close to 410 nm, as a result of the degradation process. At pH 5, a new shoulder in the region between 455-465 nm could be detected, which was more intense in the samples incubated for longer times (more than 22 h). At pH 3, no shoulders were observed, while a peak in the 455-465 nm region was clearly depicted, especially in samples incubated from more than 6 h. The action of air and light, as well known, over time play an important role in the degradation of the pigment molecules of natural extracts [41].
Commercial betanin diluted with dextrin (which contains a mixture of betanin and isobetanin) purchased from Sigma-Aldrich represents one of the main betalainic sources commercially used for coloring purposes in North America and Europe.
The comparison between the UV-VIS spectra obtained from the Opuntia ficus-indica (L.) Miller extracts and the commercial betanin solution allowed to identify its presence, as also reported in the literature [42].
A series of chromatographic analyses was performed to define the presence of the specific molecular components of coloring pigments. The thin layer chromatography (TLC) profiles of the Opuntia ficus-indica (L.) Miller extracts (data not shown) revealed and confirmed the presence of natural dyes in the extract and the data were consistent with those obtained in the absorption spectra ( Figure 5), in accordance with the literature [43]. In all the extracts obtained, the presence of betacyanins was detected, which may be responsible for the signals observed in the absorption spectra, which are responsible for the metabolites responsible for staining. The presence of betacyanins such as betanin and isobetanin (Figure 7) was expected as shown in the chromatograms obtained by HPLC (Figure 8). The chromatogram, obtained at 340 nm, showed the presence of other compounds of the flavonoids family. The selected wavelength of 340 nm was a compromise to show the presence of the extracted compounds. Other analyses were performed by HPLC (data not shown) at wavelengths more suitable to only show the presence of the coloring compounds. Under the same conditions it was established that for the sample containing commercial betanin there are only two more important peaks, associated precisely with betanin and isobetanin; it was calculated that betanin has a shorter retention time than isobetanin (ΔtR = 0.4 min) in the HPLC analysis, as reported

A.
B. C.
in this work. This difference is explained by the fact that isobetanin has a stronger interaction with the non-polar-stationary phase of the chromatographic column, as also shown in the literature [44,45].  Cotton and wool fibers colored with the extracts of Opuntia ficus-indica (L.) Miller were subjected to colorimetric measurement and the color measurement parameters were recorded, L*, a*, b*, C*ab and hab, according to the CIELAB color system.
It was verified that the minimum number of measurements required to characterize these parameters in each sample of colored textile material was in line according to what has already been shown in the literature [46], in which this minimum parameter was defined by the fixed section of the inverted exponential decay graph, obtained by CIELAB color coordinates (L*, a* and b*) with respect to the number of measurements. Eight measurements were made to characterize an area between 4.00 and 7.50 cm 2 approximately, with an opening diameter of 4 mm.
The color of each tissue sample stained with the Opuntia ficus-indica (L.) Miller extract was characterized by making the eight measurements in random positions on three replicates of each type of sample, following the conditions described in Section 2.11. The values of the CIELAB color coordinates (L*, a*, b*, C*ab and hab) for the colored fibers treated in this study are shown in Table 2.  The data were obtained from the average of 8 measurements with the relative standard deviation that is shown in brackets, performed at random points on three replicates. Undyed   The L* parameter (color lightness) varied between 49.39 and 40.10 CIELAB units for colored cotton fiber samples and between 38.73 and 50.14 for colored wool fiber samples, having 99.11 and 92.46 CIELAB units, as the control L* value for the cotton and wool fibers, respectively (identified as non-colored fabric, U), which indicates a clearly perceptible change in the color of the fibers due to dyeing, or ΔL* > 40 CIELAB units, indicating a significant color difference. The value of a* (associated with the change from greenness (−) to redness (+)) varies in a data range from −0.54 (U) to 2.59 (DWM) and 25.82 (DPA), for cotton fiber and in a data range from −1.79 (U) to 7.49 (DWM) and 27.47 (DPA), for wool fiber: this numerical identification means that the fabric was given a reddish color. The variation in b* (associated with the variation in the range from blueness (−) to yellowness (+)) occurs in a data range from 1.75 (U) to 5.18 (DWM) and 17.40 (DPA), for cotton fiber and in a data range from 8.37 (U) to 12.04 (DPA) and 13.74 (DPL), for wool fiber: an increase ranging, for cotton fibers, between 3-15 units and 4-5 units for wool fibers. The C*ab (color saturation) parameter varies in a data range from 1.86 (U) to 5.88 (DWM) and 31.14 (DPA), for cotton fiber and in a data range from 8.56 (U) to 15.48 (DWM) and 30.74 (DPL), for wool fiber; in both cases there was an increase in the value between 4-30 CIELAB units, which indicates a more marked color saturation.
The hue angle, hab, for fibers colored with and without mordants, fell within the range 25.53° (DPL) and 65.45° (DWM), therefore, they were in the area of the red, red-pink hue.
The extraction of the coloring pigment molecules was compared with the maceration and the Naviglio extraction methods. The Naviglio method proved, as expected, to be a more effective extraction method that acts in a shorter time.
The use of two mordants, rock alum and lemon juice, was compared, together with the natural extract from the skins of Opuntia ficus-indica (L.) Miller. Rock alum is widely used by industries that apply dyes to fabrics and fibers, while the use of lemon juice has been traced within testimonies provided by rural populations who in the past practiced the coloring of skins and fabrics with natural methods.
It was therefore observed that the color components isolated from Opuntia ficus-indica (L.) Miller led to a specific color of the type of fruit used. The two mordants used influenced the depth of the shades, as the literature suggests [47][48][49]. The mordants allowed for the fixing of the dye obtained from the extracts of Opuntia ficus-indica (L.) Miller to the vegetable cotton fibers and the animal wool fibers. This was certainly due to the formation of a chemical bridge between the dye and the fibers [50,51].
Stain-free dyed fabrics (DWM) had lighter shades, i.e., higher L* values, with the exception of the lemon juice stained wool fiber (DPL).
The hab value represents the main attribute of color perception, therefore these results led us to conclude that the use of mordants had no significant alteration effects on the color values. As can be seen in Table 3, the differences in the absorption of the dye, between the cotton and the wool, is higher for cotton, which seems to be a good support for the dye obtained from Opuntia ficus-indica (L.) Miller.
An evaluation of the resistance of the coloring to washing and exposure to light was performed [52,53]. The washing and light fastness values with a brief description of the fastness evaluation are shown in Table 4. The dye of the extract of Opuntia ficus-indica (L.) Miller itself, without the use of stains (DWM), produced properties of washing fastness equivalent but not superior (6-7) to stained fabrics. Its light fastness (7) was lower than that of most samples.
Considering these results, we can indicate that the fastness properties were not significantly influenced by the use of mordants and therefore, the use of the natural pigment without mordants can be a good solution and alternative to the coloring treatment.

Conclusions
This work highlights the possibility of using waste matrices such as those of prickly pear peels which within the agro-food chain, represent an important cost for companies in terms of disposal. Therefore, using such scraps or more 'secondary raw materials' to obtain natural dyes, but not only, can represent a step towards reducing the important costs for companies and increasingly directing them towards a circular economy. The use of the Naviglio extractor, an eco-innovative equipment, would allow various companies working in the agro-food sector to obtain molecules with a high intrinsic value from the 'secondary raw materials' (waste of by-products) to be used in various sectors, such as that of natural dyes.
The compounds contained within the fractions obtained from the liquid-liquid extractions could have an important role. Their recovery, also linked to the extractive method used, is of fundamental interest in the qualitative and quantitative evaluation that can be done. These bioactive compounds are a valid choice for use in the nutraceutical field and they could be an important step for innovation within the agro-food chain and an added value for companies willing to innovate their systems and productions; ultimately, this process can also be considered environmentally friendly.
In recent years, natural dyes have become an interesting alternative to synthetic dyes, all thanks to their best properties. They are incredibly more healthy for humans and the environment. For this reason, they fall into the category of important molecules with a view to a circular and zero-impact economy, since they are obtained from processing waste. With this study, we want to open a line of analysis on other endemic and non-endemic plants, present within the Campania landscape. The fibers dyed with the Opuntia ficus-indica (L.) Miller extract show a good receptivity to absorb the color both with the aid of the mordants and without.
In fibers dyed with the Opuntia ficus-indica (L.) Miller extract, the mordant influenced the depth of the shades, although it did not have significant effects on the color values. The fastness properties were not significantly affected by the use of mordants. All dyed fibers had a similar color, with the hue angle values indicating a pinkish color.
The use of a natural mordant such as lemon juice, widely used in popular tradition, shows excellent etching and solidity characteristics for the color extracted from Opuntia ficus-indica (L.) Miller.