In the last two decades, compounds with antioxidant capacities have attracted increasing interest [1
], in particular polyphenols, carotenoids and vitamins (mainly E and C). This specific attention derives from the ability of these compounds to scavenge free radicals and reactive oxygen species which are known to be involved in the development of cardiovascular diseases and several cancers [2
]. Since one of the main sources of antioxidants are fruits and vegetables, these foodstuffs have gained great interest and widespread usage in the nutritional strategies applied to prevent these pathologies [2
In this context, tomato (Lycopersicon esculentum
and pepper (Capsicum
spp.), both belonging to the Solanaceae
family, are considered to be important sources of natural carotenoids and phenols [3
Among vegetables, tomato, which is consumed either as raw fruit or as a processed product, is the second most important vegetable crop in the world and one of the main components of the Mediterranean diet [5
]. Furthermore, the industrial processing of tomato leads to by-products, namely tomato seeds and peels, representing 10–40% of total processed tomatoes [6
]. The management of tomato by-products is considered an important problem faced by tomato processing companies, as they cannot be discharged into the environment due to their high polluting potential [4
]. The bioactive compounds present in industrial tomatoes and their processing by-products include tocopherols, polyphenols, carotenoids (mainly lycopene), some terpenes, and sterols [7
]. Thus, tomato wastes are a cheap resource to be recovered and recycled within the food chain, and a sustainable strategy able to address the current challenges of the industrialized world is required [8
Besides tomato, red pepper (Capsicum
spp.) is also an important vegetable consumed worldwide. Due to their circulatory stimulant functions, chili peppers (Capsicum annuum
L.) are of ethnopharmacological importance and are also widely used as fresh fruits and savoury food additives due to their colour, pungency, and aroma [9
]. Furthermore, the presence of many bioactive components such as vitamin C, phenolics and carotenoids [11
] makes peppers extremely attractive for the phytochemical manufacturing industry as well. In particular, among carotenoids, which increase in concentration greatly during pepper maturation [14
], the most representative are α and β-carotene, β-cryptoxanthin, capsanthin, lutein, and zeaxanthin [16
]. The phenolic fraction includes mainly phenolic acids (cinnamic acid derivatives and hydroxy-substituted benzoic acids) and flavonoids (e.g., quercetin and luteolin) [10
In this context, the recovery of bioactive compounds (mainly carotenoids and polyphenols) from tomato wastes and peppers requires the use of mild extraction technologies, able to preserve the nutritional and pharmacological properties of these molecules, but also their antioxidant power [17
]. Conventional Solvent Extraction (CSE), such as organic solvent extraction, has been widely used to extract carotenoids and/or phenols from plant material. Traditionally, CSE used n-hexane, propanol, methanol, tetrahydrofuran or ethyl acetate to extract carotenoids. This method usually requires long extraction times, large amounts of organic solvents and high temperatures, which can lead to extensive degradation of thermo-sensible molecules, as well as leave trace amounts of potentially toxic solvents in the extract [17
]. Moreover, the sustainability of the extraction process and the purification of the bioactive compounds is of the utmost importance [19
Nowadays, supercritical fluid extraction (SFE) employing Sc-CO2
is an established industrial process for the production of high added-value products. In 2014 there were more than 150 SFE industrial plants with a total extraction volume of more than 500 L in the world [19
]. Many of these production plants are generally devoted to the SFE process involving a preliminary Sc-CO2
extraction of natural products, leading to the recovery of high-value products which provide interesting options for their use in the nutraceutical and functional food industry [20
In the last decades, the search for bioactive compounds or “target molecules” from natural sources or their by-products has become the most important application of SFE technology [21
As reported in the literature [21
], the bioactivities from natural compounds extracted by SFE from 2010 to 2015 were mainly the antioxidant (41%), antitumor (18%), and antibacterial (10%) ones. As widely reported [25
] SFE is a green technology that shows immediate advantages over traditional extraction techniques: (i) it is a flexible process due to the possibility of continuous modulation of the solvent power/selectivity of the supercritical fluid (SF); (ii) it allows the removal of polluting organic solvents as well as that of the expensive extract post-processing used for solvent elimination, thus ensuring a safe separation process both for human health and the environment.
Based on the work of Melo et al. [26
] and references within, it is possible to affirm that the last decades have seen great advances; among them full characterization and quantification of supercritical extracts, assessment of kinetic and equilibrium aspects, and phenomenological modelling and optimization of operating conditions. In particular, many authors have studied the dependence of the solubility of different carotenoids in supercritical CO2
with temperature and pressure [26
]. Most of the solubility data of these works were correlated using the semi-empirical Chrastil’s model [28
], which provides a proportional relationship between the solubility and density of CO2
]. Furthermore, the majority of the SFE studies for the recovery of carotenoids have focused on tomato products and industrial tomato by-products, as they constitute a good source of lycopene, and, to a lower extent, of β-carotene [4
Nonetheless, according to Melo et al. [26
], solute-matrix interactions can be better understood and correctly taken into account by reliable predictive models. In such a context, to better analyze the experimental results and to optimize the working parameters (temperature, pressure, etc.), this extractive technology could greatly benefit from mathematical models that are not only suitable and reliable, but also easy to use.
Different phenomena as phase equilibrium, mass transfer, and flow of Sc-CO2
through packed beds, are differently involved in the kinetic models reported in the literature [29
]. In many cases, the extraction of the first fraction of extracts is essentially limited by its solubility, whereas the extraction rate of the remaining fraction is limited by internal diffusion through the matrix [30
As reported in previous papers, a simplified mathematical model was introduced to describe the time evolution of SFE of the lipid fraction from oilseeds [31
] and microalgae [34
]. Using Chrastil’s equation [28
], it was also possible to correlate the maximum extraction rate with both working pressures and temperatures.
On this basis, a simplified method to estimate the time evolution of carotenoid extraction by Sc-CO2
from two different matrices (chili peppers and tomato by-products) can be developed in order to highlight the “matrix effect” on the SFE of carotenoids. This new method could potentially simplify the identification of the best working conditions to promote SFE of carotenoids from these two matrices as a function of temperature, pressure, flow-rate of Sc-CO2
, and amount of matrix, and to reduce the load of the related experimental activity [29
Furthermore, in a previous paper [31
] we reported on the use of a pilot scale SFE apparatus aimed at studying a two-sequential step procedure to intensify the extraction of oil and phenolic compounds from sunflower seeds.
In this context, this research had two main objectives: (i) to verify the effectiveness of a two-step SFE process (a preliminary Sc-CO2 extraction of carotenoids followed by the recovery of polyphenols with ethanol coupled with Sc-CO2) to obtain bioactive extracts from two different matrices (chili pepper and tomato by-products); (ii) to test the validity of the mathematical model proposed to describe the kinetics of SFE of carotenoids, the knowledge of which is required to establish the role played in the extraction process by the characteristics of the matrix.
2. Materials and Methods
As reported in a previous paper [36
], SFE were performed using a commercial pilot plant apparatus (Sitec, Maur, Switzerland) which allows the recovery and the subsequent recycling of the solvent, with a minimal loss of CO2
. A supplementary pump provides the addition of a co-solvent to the CO2
stream, when desired.
In order to obtain tomato peels as by-products, fresh fruits of L. esculentum L. were washed, cut, and parenchyma, seeds and percolation juice were removed. Then, both tomato peels and fresh fruits of C. annuum L. were lyophilized and ground to a particle size of 0.37 mm. All the samples were then stored under inert atmosphere (N2) and protected from light until use.
SFE of the carotenoidic fraction was performed using 280 g of lyophilized material for each run, with working pressures (P) of 40 and 70 MPa and temperatures (T) of 40 °C and 60 °C. The extraction time was 180 min, while the flow rate of Sc-CO2
was 10 kg·h−1
. Extraction yields were determined gravimetrically, while the carotenoid concentration in extracts and lyophilized fruits was determined spectrophotometrically (Cary 300 UV-Vis, Agilent Technologies, Santa Clara, CA, USA) [38
]. In particular, samples were first solubilized in hexane:acetone:ethanol 2:1:1 (by volume), shaken for 30 min, then distilled water was added and the samples were left to separate as a function of their polarity; the content of carotenoids was then obtained by measuring the specific absorbance and expressed as β-carotene (λ = 479 nm) for pepper and as lycopene (λ = 472 nm) for tomato. Extraction of carotenoids by percolation with n-hexane for 180 min was also performed using a Soxhlet apparatus (SER 148-3, Velp Scientifica, Usmate, Italy).
The recovery of the phenolic fraction was carried out using the samples resulting from carotenoid SFE and left in the extractor.
As reported in the literature [16
], pure Sc-CO2
is a poor solvent for these polar compounds and water is not suitable as co-solvent because, in the operating conditions adopted, its very reduced presence in the homogeneous phase is unable to significantly modify the polarity of the final Sc-CO2
/water mixture; thus, ethanol (EtOH) was used, both coupled to Sc-CO2
) and in pure form. Extractions were carried out using a pressure (P) of 30 MPa and 50 MPa and a temperature (T) of 50 °C and 80 °C. Because of the different EtOH/CO2
ratios used, variable solvent flow rates were employed, whereas the extraction time was fixed to a maximum of 180 min. Extraction yields were determined gravimetrically, while the polyphenolic concentration in both extracts and lyophilized fruits was determined spectrophotometrically according to the Folin-Ciocalteau method [39
], and expressed as chlorogenic acid (λ = 765 nm). Extraction of polyphenols by percolation with pure EtOH for 180 min was also performed using a Soxhlet apparatus.
All the reagents utilized were provided by Sigma Aldrich s.r.l. Milano, Italy, while the CO2 was supplied by SOL s.p.a, Monza, Italy.
As widely reported in the literature [26
], SFE is based on the solvating properties of SF. In particular, the extraction by SF depends on a tuneable nature of SF like temperature, pressure and some extrinsic features like the characteristics of the sample matrix, interactions with targeted analysts, and many environmental factors [43
In this context, on the basis of the results obtained, it was possible to introduce a simplified kinetic model that was able to describe the time evolution of extraction of bioactive compounds (mainly carotenoids and phenols) from different substrates. Moreover, the utilization of this simplified kinetic model together with the Chrastil’s equation allowed the prediction of the time evolution of SFE as a function of the main working conditions adopted.
The high values assumed by the square of the correlation coefficient seemed to confirm the suitability of the hypotheses introduced and gave a measure of the validity of the kinetic model proposed.
In particular, while both C. annuum L. and L. esculentum L. were confirmed as good sources of bioactive antioxidant compounds, when the same operating conditions (T, P, pre-treatments carried out on the substrate) were used, the extraction process of both carotenoids and phenols from C. annuum L. was significantly faster. The obtained results allowed us to tentatively describe the role played by the matrix in SFE of bioactive compounds.