Pressurized Liquid Extraction of Polyphenols and Anthocyanins from Saffron Processing Waste with Aqueous Organic Acid Solutions: Comparison with Stirred-Tank and Ultrasound-Assisted Techniques

Abstract

Fοllow up with our previous study on the extraction of saffron processing waste polyphenols using deep eutectic solvents, the objective of this examination was a comparative evaluation of pressurized liquid extraction (PLE), stirred-tank extraction (STE) and stirred-tank extraction with ultrasonication pretreatment (STE/UP) with respect to the recovery of pigments and antioxidant polyphenols from saffron processing waste. Aqueous solutions of citric and lactic acids at two different concentrations were used as green solvents. The extracts obtained under the specified conditions were analyzed for total pigment and total polyphenol yields as well as for their ferric-reducing power and antiradical activity. Furthermore, each produced extract was analyzed with liquid chromatography–mass spectrometry to profile its analytical polyphenolic composition. In all cases, PLE provided inferior results compared to the two other techniques, producing extracts with lower polyphenolic concentration and weaker antioxidant properties. On the other hand, no specific pattern was detected concerning the effect of ultrasonication, acid type and acid concentration. Hierarchical cluster analysis indicated that stirred-tank extraction with 1% (w/v) lactic acid and ultrasonication pretreatment might be the highest-performing combination, providing extracts with increased polyphenol and pigment concentration; however, it also enhanced antioxidant activity. It was also concluded that the significantly shorter extraction time when using PLE might be an important element in further optimizing the process, buttressing the use of this technique for the establishment of innovative and sustainable-by-design extraction methodologies.

1. Introduction

As the world’s population and industrial activity are rapidly expanding, resource depletion and environmental aggravation are challenges which need to be imminently addressed [1]. The agricultural and food industries are responsible for a large share of waste and byproducts generated as a result of farming practices and the harvesting and processing of raw materials. These side-streams are particularly rich in organic substances, and their uncontrolled dumping results in environmental pollution with detrimental consequences to the neighboring eco-systems and public health [2]. On the other hand, agri-food waste represents a vast pool of materials which can be used for the production of bio-based chemicals, bio-fuels, and high value-added substances [3]. Thus, in the framework of the circular economy, the rational utilization of agri-food waste biomass within a biorefinery concept may contribute to a fully sustainable agri-food sector.

Plant processing by-products consist mainly of peels, seeds, stems, flowers, and disfigured and undersized or damaged tissues. These residues are rich in a spectrum of bioactive compounds including polyphenols, carotenoids, oils, pectins, etc., and their promotion as cheap and abundant bioresources is state-of-the-art for the commercialization of commodities in the food, pharmaceutical, and cosmetics industry [4]. Thus, proper management of these byproducts should target their re-introduction to the production line as raw materials for the obtaining of novel products with health-related properties and added value through sustainable technologies of extraction [3]. The principles pertaining to the Green Chemistry concept are mainly focused on reducing waste and promoting efficient use of energy and resources. Concerning extraction processes, these principles may include (but are not limited to) the use of alternative solvents such as water or other bio-based solvents, reducing energy consumption by implementing innovative technologies, reducing unit operations for safer and more robust processes, and targeted generation of extracts with increased or improved bioactivity [2].

Pressurized liquid extraction (PLE) is also known as pressurized solvent extraction and accelerated solvent extraction. When water is used as the solvent, this technique is characterized as sub-critical water extraction, pressurized hot water extraction (PHWE), or superheated water extraction. It is based on liquid solvents used at a relatively high temperature and pressure, which enables improved extraction performance compared to techniques carried out at near-atmospheric temperature and pressure by boosting the solubility of the target molecules and increasing mass transfer [5]. PLE is considered a green extraction process and is performed mainly with non-toxic, non-volatile and reusable solvents such as water; it has been applied to the extraction of several plant matrices for the effective recovery of bioactive metabolites such as polyphenols, terpenes, oil, etc. [6,7].

In addition to replacing conventional extraction processes with more efficient green processes, pretreatment of samples prior to extraction with ultrasonication has also been shown to improve extraction performance. This effect has been demonstrated with the extraction of polyphenols from elderberry flowers [8], red grape pomace [9], and wheat bran [10]. Extraction enhancement by ultrasonication occurs through mechanisms involving mostly cavitation phenomena, which result in the generation of cavitation bubbles. These bubbles implode on the surface of the solid matrix, generating micro-jetting and other effects such as erosion, particle breakdown, surface peeling, micro-mixing and macro-turbulence [11].

Saffron (Crocus sativus, Iridaceae) is a herb acknowledged since the ancient time for its medicinal properties and culinary uses [12]. The stigma is the most valuable tissue of the plant; these are manually collected and dried to produce a highly appreciated spice. After separation, the remaining parts of the flower, composed of the tepals (undifferentiated petals and sepals), are rejected as processing waste. However, the evidence accumulated by recent studies has shown that saffron tepals may contain several bioactive substances, including flavonol glycosides and anthocyanins. Some of these phytochemicals have been reported to exhibit multiple beneficial bioactivities [13,14], and on this ground several extraction processes have been proposed with the aim of producing polyphenol-enriched extracts from saffron processing waste (SPW) [15,16].

Nevertheless, to the best of the authors’ knowledge there is only one extant examination of PLE extraction of SPW, which focused only on total polyphenols [17]. Moreover, aqueous organic acid solutions such as those tested on other plant materials [18,19] have never been used for the extraction of SPW polyphenols. This being the case, the study reported herein describes an investigation of the extraction of flavonols and anthocyanins from SPW employing PLE. This technique was compared to conventional batch stirred-tank extraction both with and without ultrasonication pretreatment. The differences in the extraction yield among the techniques tested were revealed by determining the analytical metabolite profile of each obtained extract.

2. Materials and Methods

2.1. Chemicals

For chromatographic determination, the solvents used were of HPLC grade. Pelargonin chloride (pelargonidin 3,5-di-O-glucoside) was from Extrasynthese (Genay, France). Kaempferol 3-O-glucoside, rutin (quercetin 3-O-rutinoside) (>94%), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenylpicrylhydrazyl (DPPH), and Folin-Ciocalteu reagent were from Sigma-Aldrich (St. Louis, MO, USA). Citric acid anhydrous and iron chloride hexahydrate were from Merck (Darmstadt, Germany). Ascorbic acid (99.5%), L-lactic acid, and sodium carbonate anhydrous (99%) were from Penta (Praha, Czechia). Gallic acid hydrate was from Panreac (Barcelona, Spain).

2.2. Plant Material

Saffron (Crocus sativus L.) processing waste (SPW) consisting of saffron tepals was collected from a processing plant in Kozani (West Macedonia, Greece) immediately after manual processing of the saffron flowers. The material was transferred to the laboratory within 24 h and freeze-dried in a Biobase BK-FD10P freeze-drier (Shandong, China) for 24 h. The dried tissue was then ground in a domestic blender to give a powder with an average particle diameter of 0.637 mm and stored in air-tight tubes at −18 °C until used.

2.3. Pressurized-Liquid Extraction (PLE)

The equipment used was a PLE system (Fluid Management Systems, Inc., Watertown, MA, USA). An analytical description of the device is given in Figure 1. For the purposes of this study, static extraction was performed using a 100-mL stainless steel cell. An amount of SPW (2 g) was loaded onto the extraction cell and capped with two filtration end fittings. The PLE system then automatically pressurized and heated the sample while pumping solvent into the chamber for a predetermined resident time and solvent flow. The specific settings used were: filling (with solvent) time, 1.3 min; pressurization, 0.5 min; preheating (at 120 °C) time, 5 min; extraction (at 120 °C) time, 10 min; cooling (at T < 50 °C), 7 min; depressurization time, 0.02 min; solvent flush, 1.3 min; nitrogen flush, 1 min. Under these conditions, the liquid-to-solid ratio was 40 mL g⁻¹.

During heating, a temperature rise (overshooting) was observed which in no case exceeded 4 °C, while the temperature set was restored within 1–2 min. After the completion of the extraction, the extract was drained and transferred to a suitable vial for further processing or analysis. The choice of the extraction temperature and time was based on previous data [20,21,22], as well as preliminary experimentation. The solvents used were distilled water (pH = 5.61), 1% (w/v) citric acid (pH = 2.88), 5% (w/v) citric acid (pH = 2.42), 1% (w/v) lactic acid (pH = 2.71), and 5% (w/v) lactic acid (pH = 2.48).

2.4. Stirred-Tank Extraction in Batch Mode

An exact mass of 0.250 g of SPW was mixed with 10 mL of solvent (liquid-to-solid ratio 40 mL g⁻¹) in a 25-mL Duran glass vial and stirred continuously at 500 rpm for 180 min at 80 °C into an oil bath heated by a thermostat-equipped hotplate (Witeg, Wertheim, Germany). After the extraction, all samples were centrifuged at 10,000× g for 10 min.

2.5. Batch Stirred-Tank Extraction with Ultrasonication Pretreatment

Prior to batch stirred-tank extraction samples were subjected to ultrasonication in pulse mode for 15 min in an Elma S 100 (H) heated ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany) at a frequency of 37 Hz and nominal power of 550 W. During this resident time, increases in the initial temperature (31 °C) in no case exceeded 6 °C. The actual ultrasonication power (P) dissipated into the system, as well as the acoustic energy density (AED), were determined using the following equations:

$$P=mC\mathrm{p}\frac{dT}{dt}$$

(1)

$$\mathrm{AED}=\frac{P}{V}$$

(2)

where m corresponds to the mass of the coupling liquid (water) contained in the ultrasonication bath (in g), C_p corresponds to the specific heat capacity of water (4.2 J g⁻¹ K⁻¹), and $\frac{dT}{dt}$ represents the temperature rise per s, which was calculated by fitting temperature change (dT) as measured by a thermocouple as a function of time [23]. P and AED were determined to be 159.6 W and 39.9 W L⁻¹, respectively.

2.6. Determinations

Determination of total polyphenols was performed with Folin-Ciocalteu reagent and results were given as mg gallic acid equivalents (GAE) per g of dry mass (dm). The antiradical activity (A_AR) was estimated with a DPPH radical probe using a stoichiometric assay, and results were given as μmol DPPH per g dm. Ferric-reducing power (P_R) was determined using the TPTZ chromophore probe; results were expressed as μmol ascorbic acid equivalents (AAE) per g dm. The analytical protocols for all these methodologies have been previously reported in detail [24]. Total pigments were likewise determined with a protocol reported elsewhere [25].

2.7. Liquid Chromatography–Diode Array-Mass Spectrometry (LC-DAD-MS)

A published methodology was employed [24] using a Finnigan AQA mass spectrometer coupled to a Finnigan (San Jose, CA, USA) MAT Spectra System P4000 pump and a UV6000LP diode array detector. Chromatography was performed on a Fortis RP-18 column, 150 mm × 2.1 mm, 3 μm, at 40 °C, with a 10-μL injection loop. Μass spectra were acquired at 20 and 70 eV with electrospray ionization (ESI) in positive ion mode. Mass acquisition settings were as follows: temperature 250 °C, probe source voltage 25 V, detector voltage 450 V, and capillary voltage 4 kV. Elution was carried out with (A) 2% acetic acid and (B) methanol at a flow rate of 0.3 mL min⁻¹, as follows: 0–30 min, 0–100% methanol; 30–40 min, 100% methanol.

2.8. High-Performance Liquid Chromatography (HPLC)

A Shimadzu CBM-20A liquid chromatograph (Shimadzu Europa GmbH, Duisburg, Germany) along with a Shimadzu SPD-M20A diode-array detector was used, interfacing with Shimadzu LC solution software. Analyses were run on a Phenomenex Luna C18(2) column (100 Å, 5 μm, 4.6 × 250 mm) (Phenomenex, Inc., Torrance, CA, USA) at 40 °C. Information concerning the elution program, calibration standards and calibration curves used for the quantification have been previously reported in detail [24].

2.9. Statistical Analyses

At least two individual extractions were accomplished for each treatment, and all determinations were performed in triplicate. The values reported are the average ± standard deviation (SD). Linear regressions were established using SigmaPlot™ 12.5 (Systat Software Inc., San Jose, CA, USA), and distribution analyses with JMP™ Pro 13 (SAS, Cary, NC, USA). All statistical analyses were performed with at least at a 95% significance level.

3. Results and Discussion

3.1. Yield in Total Polyphenols and Total Pigments

Figure 2A illustrates the yield in total polyphenols (Y_TP) achieved with the various solvents tested through deployment of pressurized liquid extraction (PLE), stirred-tank extraction (STE) and stirred-tank extraction with ultrasonication pretreatment (STE/UP). The highest Y_TP (38.24 mg GAE g⁻¹ dm) was recorded for the STE/UP performed with 1% (w/v) lactic acid (LA). Extractions carried out with 5% (w/v) citric acid (CA) and 5% LA under the same conditions had comparable performance, since there was no statistical difference amongst the Y_TP obtained (p < 0.05). The same held true for the extractions carried out with water, 1% CA, 5% CA and 5% LA in stirred-tank mode without ultrasonication pretreatment. These findings show that there was no clear effect of either the type of the acid or its concentration on extraction efficiency. Furthermore, ultrasonication pretreatment offered virtually no advantage over simple stirred-tank extraction. On the other hand, all samples generated with PLE displayed lower Y_TP, which reached statistical significance (p < 0.05) irrespective of the acid used or its concentration.

The results on the yield in total pigments (Y_TPm) provided a different image (Figure 2B). The extractions carried out with 1% CA, 1% LA and 5% LA in stirred-tank mode, and those with 1% CA and 1% LA in stirred-tank mode with ultrasonication pretreatment exhibited the highest and most statistically significant Y_TPm (p < 0.05). On the contrary, PLE performed with any of the extraction media tested showed significantly lower Y_TPm (p < 0.05). In this case as well, a distinction between CA and LA or between STE and STE/UP was not apparent. Taking into consideration both Y_TP and Y_TPm, the samples prepared with 1% CA-STE, 5% CA-STE, 5% LA-STE, and 1% LA STE/UP were those with the richest composition.

3.2. Effect on the Antioxidant Properties

The results on the determination of reducing power (P_R) and antiradical activity (A_AR) are depicted in Figure 3. Extracts produced with water, 5% CA and 5% LA in stirred-tank mode had significantly higher A_AR (p < 0.05). The same was observed for the extract generated with 5% CA in stirred-tank mode after ultrasonication pretreatment. By contrast, all extracts produced with PLE showed lower A_AR (p < 0.05) (Figure 3A). The pattern concerning P_R was essentially similar, with the samples prepared with 1% CA-STE, 5% CA-STE, 5% LA-STE and 5% CA STE/UP showing the highest values. On the other hand, all PLE samples as well as 1% LA-STE/UP had significantly lower P_R (Figure 3B). Considering both A_AR and P_R, the samples prepared with 5% CA-STE, 5% LA-STE and 5% CA-STE/UP were the most efficacious in terms of expressing antioxidant activity.

3.3. Effect on the Flavonol and Anthocyanin Profile

The extract generated from each assay was subjected to HPLC analysis in order to portray the analytical polyphenolic composition and detect differences attributed to different extraction modes. Chromatograms were traced at both 360 and 520 nm; in all cases, seven principal compounds could be tentatively identified and quantified: four flavonol glycosides and three anthocyanin pigments (Figure 4). The tentative identification of these substances was based on mass spectral data, as previously described [24]; the quantitative data concerning flavonol and anthocyanin composition are given in

Table 1. Analytical flavonol composition of the extracts produced in this study; values reported are means ± standard deviation.

Extraction Mode	Yield (mg g−1 dm)
	KSG	QS	KS	KG	Total Flavonols
Water-PLE	3.16 ± 0.04 c	1.69 ± 0.02 b	20.87 ± 0.06 c	1.41 ± 0.01 c	27.13 c
1% CA-PLE	2.45 ± 0.00 b	1.28 ± 0.00 b	15.79 ± 0.02 b	1.19 ± 0.01 b	20.70 b
5% CA-PLE	1.49 ± 0.00 b	1.14 ± 0.00 b	9.59 ± 0.02 b	1.09 ± 0.01 b	13.31 b
1% LA-PLE	2.49 ± 0.03 b	2.05 ± 0.01 c	16.99 ± 0.09 b	1.14 ± 0.01 b	22.67 b
5% LA-PLE	2.40 ± 0.01 b	1.91 ± 0.00 b	16.29 ± 0.04 b	1.30 ± 0.01 b	21.90 b
Water-STE	3.81 ± 0.01 a	2.93 ± 0.03 a	30.33 ± 0.07 a	1.70 ± 0.01 a	38.77 a
1% CA-STE	3.21 ± 0.02 c	2.85 ± 0.01 a	22.32 ± 0.13 c	1.45 ± 0.00 c	29.83 c
5% CA-STE	2.84 ± 0.00 c	2.28 ± 0.02 c	18.98 ± 0.10 c	1.55 ± 0.01 c	25.64 c
1% LA-STE	3.48 ± 0.02 a	2.83 ± 0.01 a	24.59 ± 0.14 c	1.55 ± 0.02 c	32.45 c
5% LA-STE	3.48 ± 0.02 a	2.79 ± 0.02 a	23.91 ± 0.07 c	1.63 ± 0.04 a	31.81 c
Water-STE/UP	3.23 ± 0.02 c	2.74 ± 0.04 a	31.69 ± 0.24 a	1.72 ± 0.03 a	39.38 a
1% CA-STE/UP	3.22 ± 0.01 c	2.05 ± 0.00 c	22.04 ± 0.10 c	1.46 ± 0.01 c	28.78 c
5% CA-STE/UP	3.03 ± 0.05 c	1.97 ± 0.01 c	20.28 ± 0.07 c	1.67 ± 0.01 a	26.95 c
1% LA-STE/UP	4.20 ± 0.02 a	3.13 ± 0.02 a	34.12 ± 0.24 a	1.87 ± 0.01 a	43.32 a
5% LA-STE/UP	3.65 ± 0.03 a	2.61 ± 0.01 c	25.85 ± 0.10 c	1.77 ± 0.01 a	33.88 a

Values with different superscripted letters within columns are statistically different (p < 0.05).

and

Table 2. Anthocyanin pigment composition of the extracts produced in this investigation; values reported are means ± standard deviation.

Extraction Mode	Yield (mg g−1 dm)
	DDG	PDG	DG	Total Anthocyanins
Water-PLE	0.57 ± 0.12 a	0.74 ± 0.02 b	0.42 ± 0.01 b	1.72 b
1% CA-PLE	0.35 ± 0.03 a	0.58 ± 0.06 b	0.72 ± 0.09 b	1.64 b
5% CA-PLE	0.19 ± 0.01 b	0.48 ± 0.02 b	1.02 ± 0.07 c	1.69 b
1% LA-PLE	0.39 ± 0.07 a	0.79 ± 0.05 b	0.82 ± 0.01 c	2.00 c
5% LA-PLE	0.23 ± 0.02 c	0.76 ± 0.04 b	1.01 ± 0.01 c	2.00 c
Water-STE	0.33 ± 0.02 c	1.17 ± 0.02 a	0.38 ± 0.04 b	1.89 b
1% CA-STE	0.28 ± 0.09 c	1.17 ± 0.03 a	1.42 ± 0.02 a	2.87 a
5% CA-STE	0.12 ± 0.01 b	0.90 ± 0.06 c	1.70 ± 0.01 a	2.72 a
1% LA-STE	0.25 ± 0.02 c	1.16 ± 0.02 a	1.17 ± 0.05 c	2.58 c
5% LA-STE	0.12 ± 0.04 b	0.99 ± 0.01 c	1.50 ± 0.01 a	2.60 a
Water-STE/UP	0.15 ± 0.09 b	1.18 ± 0.05 a	0.32 ± 0.00 b	1.65 b
1% CA-STE/UP	0.29 ± 0.06 c	0.91 ± 0.07 c	1.06 ± 0.04 c	2.26 c
5% CA-STE/UP	0.19 ± 0.01 b	0.91 ± 0.01 c	1.38 ± 0.01 a	2.48 c
1% LA-STE/UP	0.49 ± 0.00 a	1.36 ± 0.05 a	1.41 ± 0.03 a	3.25 a
5% LA-STE/UP	0.21 ± 0.03 c	1.10 ± 0.04 a	1.81 ± 0.05 a	3.11 a

Values with different superscripted letters within columns are statistically different (p < 0.05).

, respectively.

The most abundant flavonol glycoside was kaempferol 3-O-sophoroside (K_S), followed by kaempferol 3-O-sophoroside 7-O-glucoside (K_SG), quercetin 3-O-sophoroside (Q_S) and kaempferol 3-O-glucoside (K_G). This finding was in accordance with recent findings on SPW extraction with a deep eutectic solvent [24]. The pattern of flavonol composition in the samples produced with PLE was identical to those seen in samples generated with STE and STE/UP, which showing that there was no selectivity towards any SPW flavonol. The extraction with 1% LA-STE/UP was proven to be the most efficient for all flavonols, affording significantly higher yields (p < 0.05). On the contrary, extractions with 1% CA-PLE, 5% CA-PLE and 5% LA-PLE were the least efficient in this regard. Considering the total flavonol yield, the extractions with Water-STE, Water-STE/UP, 1% LA-STE/UP and 5% LA-STE/UP were of equivalent performance.

These findings suggest that ultrasonication pretreatment enabled the extraction of increased flavonol amounts. On the other hand, the use of citric acid appeared to disfavor flavonol recovery.

With respect to anthocyanins, extraction with 1% LA-STE/UP was once again the most efficacious, while 1% CA-STE, 5% CA-STE, 5% LA-STE and 5% LA-STE/UP were of comparable efficiency.

However, unlike flavonols, PLE afforded significantly higher levels of delphinidin 3,5-di-O-glucoside (D_DG) compared to both STE and STE/UP. This outcome indicates that D_DG recovery might be favored with PLE. On average, the most abundant anthocyanin was delphinidin 3-O-glucoside (D_G), followed by petunidin 3,5-di-O-glucoside (P_DG) and delphinidin 3,5-di-O-glucoside (D_DG). These findings contrast with a recent investigation in which it was demonstrated that D_DG was the predominant anthocyanin [24,26].

Taking into account the total anthocyanin yield, it was evident that STE and STE/UP were of higher efficiency compared to PLE; however, the distinction between STE and STE/UP was unclear. By jointly considering the yield in total flavonols and total anthocyanins, the highest-performing system was 1% LA-STE/UP. This was corroborated by the data on Y_TP, Y_TPm (Figure 2), A_AR and P_R (Figure 3). To confirm these observations, a hierarchical cluster analysis was performed including the yield in all individual polyphenols as well as A_AR and P_R. As can be seen in Figure 5, 1% LA-STE/UP was clustered separately, which could be considered sound evidence of its supremacy over all other extracts. Apart from Water-PLE, all other PLE samples were grouped together, which clearly points out their similarity in extraction yield and antioxidant properties.

This outcome suggests that under the PLE conditions employed, the addition of citric acid or lactic acid does not foster extraction efficiency or antioxidant activity. Likewise, 5% CA-STE, 5% LA-STE, 5% CA-STE/UP and 5% LA-STE/UP were on the same cluster, an indication of their comparable efficiency. Thus, it can be supported that extraction with aqueous solution containing 5% of either citric or lactic acid showed no significant differences, while ultrasonication pretreatment offered no detectable statistically significant advantage. It is also to be noted that the categorization of Water-STE and Water-STE/UP in the same cluster provides additional evidence that ultrasonication pretreatment of SPW might not always provide a significant benefit in terms of increasing polyphenol extraction yield and enhancing antioxidant activity.

Such an outcome apparently contradicts recent examinations in which ultrasonication pretreatment significantly boosted polyphenol extraction using various means, including β-cyclodextrin [27], deep eutectic solvents [8], and hydroethanolic solutions [9]. However, negative effects have also been reported [28]. Therefore, it would be reasonable to presume that different plant matrices may behave in a different manner as a response to ultrasonication prior to performing stirred-tank extraction. Furthermore, the role of extraction media should also be taken into account. Early investigations highlighted the importance of the type and concentration of acid on the aqueous extraction of anthocyanins from red grape pomace [29]. In the same line, a more recent study has suggested that aqueous solutions of acetic acid are more efficient for flavanol extraction from red grape pomace compared to citric acid solution [30]. In that study, a significant role in the extraction performance was also attributed to acid concentration. Such an approach was more thoroughly carried out by deploying response surface methodology, where it was demonstrated that acidification with lactic acid provided a more effective means of recovering flavonoids from red grape pomace compared to acetic, tartaric and citric acids [31].

The efficacy of PLE compared to both conventional STE and STE implemented after ultrasonication pretreatment was lower, as judged by the Y_TP, A_AR, P_R, as well as the yield of major polyphenols. These results contrasted with previous studies where PLE outperformed both conventional and emerging techniques applied for anthocyanin extraction [32,33] and other polyphenols [22]. However, other investigations showed that these differences might be marginal [34]. Nevertheless, it should be emphasized that the yields attained using PLE cannot be overlooked considering the significantly shorter required extraction time. Furthermore, although PLE was carried out at 120 °C, considerably higher than the 80 °C used for STE and STE/UP, no alteration in the polyphenolic profile was observed. This may indicate that SPW polyphenols are rather stable under these conditions. Such evidence could be a key element in the future optimization of PLE methodology, which is anticipated to shed more light on the potential of PLE for obtaining high extraction yields and extracts with improved antioxidant properties from SPW. Generally, PLE processes have the advantage of providing important enhancements compared to conventional extraction procedures, including higher extraction yields and recoveries, faster extraction, and lower solvent volumes. The use of a high temperature results in an increase in the rate of mass transfer, enhancement of the solubility of the target compounds, and a decrease in solvent viscosity [7]. Moreover, the use of alternative solvents such as deep eutectic solvents should also be considered. The use of such a solvent composed of lactic acid and glycine has been demonstrated to significantly enhance the performance of polyphenol extraction from SPW compared to conventional solvents [24]. Thus, a combination of deep eutectic solvents with PLE might provide a highly effective means of polyphenol and pigment extraction from SPW.

4. Conclusions

Follow up with a previous study of ours on the use of deep eutectic solvents, in this study, pressurized-liquid extraction was compared to conventional stirred-tank extraction and stirred-tank extraction integrated by ultrasonication pretreatment, in order to obtain evidence regarding their suitability for antioxidant polyphenol and pigment extraction from saffron processing wastes (floral residues). The solvents used were green aqueous solutions of citric and lactic acids. The outcome of the investigation evidenced that stirred-tank extraction and stirred-tank extraction including ultrasonication pretreatment outperformed pressurized-liquid extraction under the conditions employed. This appraisal was based on the yields of total pigments and total polyphenols, as well as the antioxidant properties of the obtained extracts. Determination of the analytical polyphenolic composition also showed that the extracts generated with either stirred-tank extraction or stirred-tank extraction with ultrasonication pretreatment were richer in the major substances identified. On the other hand, the significantly shorter extraction time used for pressurized-liquid extraction should not be overlooked. It is suggested that optimization of the pressurized-liquid extraction process based on the data reported in this study might establish a green and efficient methodology for the recovery of polyphenols and pigments from saffron processing waste. Such an approach would contribute to more effective promotion of this precious residue.