NOVEL TECHNOLOGIES

In recent years, the use of emerging and innovative technologies for the extraction of plant secondary metabolites have shown significant advancements [36]. Several pretreatment methods, such as, physical, biological, and chemical treatments are capable to improve cell structure and to increase the permeability of membrane during the extraction of plant secondaiy metabolites.

Extraction with different physical methods, e.g., HPA [33, 53], NPCA [65, 98, 111], high pressure homogenization-assisted (HPHA) [7, 34, 46]. microwave-assisted (MA) process [27, 41, 47, 121, 122, 124], UA [1, 85, 101,116, 119], MEFA[40,63,88, 102], pulsed electric field-assisted (PEFA) [30,66,94,99,113] and HVEDA [66,99] have received significant attention. Beside these physical methods, extraction process with different enzymes are used to break for the cell structure and to release the secondary metabolites from cell-matrix [24, 39, 59, 64, 92]. In a similar mamier, extraction with chemical pretreatment has been used to increase the inner or outer membrane permeabilization and to decrease the overall internal resistance for releasing plant secondary metabolites from matrix [57, 91].

Emerging technologies are considered as a hallmark in the platform of ‘process intensification.’ Novel extraction teclmiques are applicable for the extraction of different classes of plant secondary metabolites, with specific requirements in terms of low-temperature processing [36]and lower use of organic solvents [59]. These techniques have been used to enhance the mass transfer and increase of extraction yield and reduce the energy consumption during the extraction of plant secondaiy metabolites [36]. Modifications of the extraction methods are still under development according to the choice of researcher for a specific purpose [9].

EXTRACTION WITH ENZYMATIC TREATMENT

In enzymatic extraction, hydrolytic enzymes with highly specified catalytic activity are employed in the disruption of plant matrix before the extraction stage. It provides better quantity of yield compare to primitive extraction process [24, 39, 59, 64, 92]. In this context, commonly used hydrolytic enzymes are proteases, cellulases, lipases, and amylases. In Figure 9.2, the enzymatic extraction process for the extraction of plant-based secondaiy metabolites is represented.

The study has reported that significant improvement in the release of phenolic compounds from the hydro-distilled residual leaves after enzymatic pretreatment [18]. The enzymatic extraction has also been effective to increase the extraction efficiency of polyphenols from citrus peel [64], blackcurrant [61], ginger [76] and carotenoids from marigold flower [14].

EXTRACTION ASSISTED WITH PULSED ELECTRIC FIELD (PEFA)

PEFA extraction has significant potential to improve cell permeability of membrane through a phenomenon of irreversible electroporation. In this method, the application of moderate electric field strength with 0.1 to 40 kV/cm for microseconds increases the pore size in cell membranes and disrupts the cellular membrane, which increase the permeabilization of plant metabolites from matrix [37, 68]. Figure 9.3 shows PEFA extraction process for the extraction of plant secondary metabolites.

EA extraction process for the recovery of secondary metabolites from plants. Source

FIGURE 9.2 EA extraction process for the recovery of secondary metabolites from plants. Source: Self-developed with concepts from Refs. [24, 92].

PEFA extraction process for the recovery of secondary metabolites from plants. Source

FIGURE 9.3 PEFA extraction process for the recovery of secondary metabolites from plants. Source: Self-developed with concepts from Refs. [37, 68].

According to a recent study, the PEFA extraction process has been effectively utilized in winemaking process to increase the polyphenol content, antioxidant activity and color intensity in red wine [30]. Likewise, the PEFA extraction process was satisfactorily applied in brown rice to extract antioxidant compounds, such as, phenolic acids, polyphenols, y-oryzanol, and saturated and unsaturated fatty acids [94].

EXTRACTION USING HIGH VOLTAGE ELECTRICAL DISCHARGE (HVEDA)

FIVEDA extraction is a non-thermal treatment to disrupt cells in liquid samples and it subsequently increases the recovery of valuable components from the plant matrix. During this process, energy is released directly into the medium through the submerged electrodes. It is an extremely destructive treatment, where electric shock converts to mechanical energy and disintegrates cell-walls and cell-membranes [30, 66, 94, 99,113]. Figure 9.4 shows the HVEDA extraction process for the extraction of secondary metabolites from plants.

This method has been satisfactorily employed to boost the recovery of polyphenol compounds from white grape pomace [66]. Moreover, it has been used to enhance the extraction efficiency of polyphenols and proteins with more effective energy inputs than the ultrasound and PEFA methods [99].

HVEDA extraction process for the recovery of secondary metabolites from plant matrix

FIGURE 9.4 HVEDA extraction process for the recovery of secondary metabolites from plant matrix.

Source: Self-developed with concepts from Refs. [99].

HIGH-PRESSURE-ASSISTED EXTRACTION (HPA)

Although HPA is considered for the inactivation of pathogens in the food industry, yet this process has received attention for rupturing plant cell-walls for efficient extraction of plant secondary metabolites. The HPA extraction process involves compression, holding of the sample at the specific pressure and release of pressure. During the application of HPA, the sample should be dried prior to size reduction, because the presence of moisture may reduce the extraction efficiency. In this process, generally, pressure between 100-600 MPa is applied [53]. Figure 9.5 presents HPA extraction process for the extraction of secondaiy metabolites from plant matrix.

It has been reported that high hydrostatic pressure has a positive effect on the recovery of polyphenols and anthocyanins from various red fruit-based products. Furthermore, HPA treatment at moderate temperature increases the extraction efficiency of colored pigments and polyphenol content from fruits [33].

NEGATIVE PRESSURE CAVITATION-ASSISTED EXTRACTION (NPCA)

The NPCA extraction process is eco-friendly and efficient technology for the recovery of plant metabolites, where cavitation is formed by negative pressure with continuous introduction into the liquid-solid system to increase the turbulence, mass transfer and collision between the solvent and plant tissue. During this process, millions of tiny vapor bubbles are formed and collapsed.

HPA extraction process for the recovery of plant secondary metabolites. Source

FIGURE 9.5 HPA extraction process for the recovery of plant secondary metabolites. Source: Self-developed with concepts from Refs. [33, 53].

This collapse causes the release of high energy, which raises the local temperature and pressure at reaction sites. It increases the reaction rate in the system. Through continuous addition of water to the system, turbulence increases the mass transfer and collision between the matrix and solvent. This facilitates the permeabilization of target secondary metabolites from the plant matrix [50, 65, 111]. This method is effective in improving the mixing of substrate and enzyme. The NPCA extraction method has also been used to extract alkaloids, polyphenols, polysaccharides, and flavonoids from different plant parts [67]. Figure 9.6 shows the NPCA extraction process to extract secondary metabolites from the plant tissue.

The research study has demonstrated the effectiveness of NPCA extraction in combination with other methods, such as: microwave, homogenization, enzyme, ionic liquid solvents and deep eutectic solvents [98].

NPCA extraction process for the recovery of secondary metabolites from plants. Source

FIGURE 9.6 NPCA extraction process for the recovery of secondary metabolites from plants. Source: Self-developed with concepts from Ref. [50].

EXTRACTION USING HIGH-PRESSURE HOMOGENIZATION (HPH)

High-pressure homogenization is a non-thermal process, where the cell membrane is disrupted using high-intensity fluid-mechanical stresses. In this process, the flow of the process fluid with high pressures (between 50-400 MPa) through homogenization is used. Samples are forced to pass through a narrow gap in the homogenizing valve, and high shear and turbulence create acceleration, compression, and pressure drop. Moreover, it results in the deformation and subsequent disruption of cells and macromolecules in the fluid [7, 34, 50]. Figure 9.7 shows the HPHA extraction for recovery of plant secondary metabolites is represented.

HPHA extraction process for the recovery of plant secondary metabolites. Source

FIGURE 9.7 HPHA extraction process for the recovery of plant secondary metabolites. Source: Self-developed with concepts from Ref. [89].

Recently, researchers have found that HPHA extraction has potential for the recovery of secondary metabolites from the plant. High-pressure hornog- enizer or ball mill was the most efficient for the recovery of carotenoids from microalgae (Haemotococcus pluvialis, Chromochloris zofingiensis, and Chlorella sorokiniana) [46,110]. It has been found that HPHA extraction has an effect on changing the microstructure and bioaccessibility of carotenoids from tomato pulp. In the same way, HPHA extraction decreased the particle size of tomato because of matrix disruption. It also increased the consistency of the product [48].

MICROWA VE-ASSISTED EXTRACTION (MVA)

Microwaves are electromagnetic waves, which are used at 2.45 GHz and interact with polar molecules in the sample to generate heat [122]. During MA extraction, evaporation of the moisture from the cells enhances the porosity of the cell-matrix. It successively improves the penetration of a solvent into the matrix [47]. The elevated temperature can also improve solubility and yield of extraction. Compared to conventional extraction methods, MVA extraction has advantages, such as, higher recovery yield, less solvent consumption and shorter extraction tune [122].

The change can be observed with light microscopy and scanning electron microscopy. The application of microwave during extraction provides a greater extent of cell disruption of the plant materials, which increase the rate of extraction [38]. However, microwave irradiation can change some target components due to chemical reactions [41, 121, 124]. Thus, MA extraction may improve extraction yield by modifying the structures of the target compounds [102]. In Figure 9.8, the MA extraction process for the extraction of plant secondaiy metabolites is represented.

MA extraction process for the recovery of plant secondary metabolites. Source

FIGURE 9.8 MA extraction process for the recovery of plant secondary metabolites. Source: Self-developed with concepts from Refs. [122,124].

ULTRASOUND-ASSISTED EXTRACTION (UA)

Ultrasound can improve the recovery process of plant metabolites through the interaction of acoustic waves with solvent to plant tissue. Due to the application of waves, dissolved gas creates bubbles to generate heat [8,119]. This process induces significant cavitation phenomena, which is responsible for cell-wall disruption [42, 85]. UA extraction can enhance the extraction yield and aqueous extraction processes without using solvents, therefore, it provides the opportunity to use green solvents. Ultrasound treatment improves the extraction performance and extraction of heat-sensitive plant secondary metabolites [119]. UA extraction is an efficient technique to reduce the loss of plant secondary metabolites during extraction by decreasing extraction tune [101, 116]. In Figure 9.9, the UA extraction process for the extraction of plant secondaiy metabolites is presented.

UA extraction of polyphenols from the bark [42], amino acids from grape [21] and isoflavones from soybean [85] have been reported.

MODERATE ELECTRIC FIELD-ASSISTED EXTRACTION (MEFA)

MEFA extraction uses an alternating current electric field usually from 1-1000 V cnr1 through two highly conductive electrodes to the biological material placed between them. It is a green method. The term moderate electric field can be defined as a process where electric field strength includes thermal treatment (ohmic heating) or excludes the heating process (electro-permeabilization) [105]. Unlike HVEDA, MEFA extraction involves direct use of the electric current in the form of alternating current at considerably lower strength than the pulsed electric field [40]. The use of an alternating current reduces the effect of electrolysis and the formation of undesired compounds around the electrodes. It can be operated at high temperature due to ohmic heating or at low temperature to minimize thermal effects. The method can reduce extraction time and energy with high quality and yield. The heating rate highly depends on the nature of biological materials [102].

UA extraction process for the recovery of plant secondary metabolites. Source

FIGURE 9.9 UA extraction process for the recovery of plant secondary metabolites. Source: Self-developed with concepts from Ref. [85].

In the MEFA extraction process, quality, and yield of the products is affected by electric field strength, process tune, frequency, and conductivity of the extraction medium and biological material. The types of electrode materials in the process also play a significant role in the performance of

MEFA extraction. The most commonly used electrode materials are stainless steel, titanium, platinum, and platinized titanium [40].

The MEFA extraction enhances permeabilization because of triggering of trans-membrane potential difference across the cell membrane (non-thermal effect) and high temperature [63]. It can be applied without solvent (by direct pressing of electrodes) or by using a conductive extraction medium. Salt solutions have also been used as extraction medium (solvent) during the MEFA extraction process. For example, the use of potassium chloride [88] or sodium chloride solution (0.05%) [63] has been reported in literature. However, in MEFA extraction process, energy consumption is reduced and the extraction yield is enhanced by enhancing the mass transfer. Process optimization is important to enhance the quality and quantity of the specifically extracted biomolecule and to minimize the electrochemical reactions, which may occur during the extraction process [40, 102]. In Figure 9.10, MEFA extraction process for recoveiy of plant secondary metabolites is presented.

MEFA extraction process for the recovery of plant secondary metabolites. Source

FIGURE 9.10 MEFA extraction process for the recovery of plant secondary metabolites. Source: Self-developed with concepts from Refs. [102,105].

The extraction of oils using distilled water as a medium has been used during the extraction of phenolic compounds [63]. MEFA extraction has also been successfully used to extract plant metabolites from potato tissue using different temperatures and treatment times. The total energy consumption in the conventional extraction process was about 300 kJ kg'1, which was significantly reduced to 28 kJ kg'1 during MEFA extraction of phenolic compounds from colored potato [88].

In Table 9.4, some examples of the extraction of plant secondaiy metabolites from different biological materials by MEFA process are presented.

Research studies were conducted using emerging technologies to increase the recoveiy efficiency of secondaiy metabolites from the plant matrix. In Table 9.5, some examples for the extraction of plant secondaiy metabolites with emerging technology are represented.

TABLE 9.4 Examples of Extraction of Plant Secondary Metabolites from Different Biological Materials by the MEFA Process

Sample Matrix

Extracted Compounds

References

Beetroot

Betanin

[63]

Colored potato

Phenolic compounds

[88]

Gac (Momordica cochinchinensis)

p-carotene and lycopene

[1]

Green microalgae (Chlorella vulgaris)

Carotenoids and chlorophyll

[35]

Microalga (Heterochlorella luteoviiidis)

Carotenoids

[49]

Tomato

Phenolic compounds

[43]

White Bran

Phenolic compounds

[4]

TABLE 9.5 Recovery of Plant Secondary Metabolites from Plant Matrix with Emerging Technologies

Plant Material

Extraction Method

Bioactive Components

References

Red grape skins Green tea leaves

HPA

Anthocyanin Caffeine

[28] [54]

Marigold flowers Bay leaves

EA

Carotenoids Essential oil

[14] [18]

Brown rice

PEFA

y-oryzanol, polyphenols, and phenolic acids, saturated, and unsaturated fatty acids

[4]

Pigeon pea leaves

NPCA

Flavonoids

[67]

Microalgae

Tomatoes

HPHA

Carotenoid Carotenoid

[110] [87]

Tea Tobacco leaves

MA

Phenols Solanesol

[108] [124]

Grapes Spruce wood bark

UA

Amino acids Polyphenols

[21] [42]

Pigeon pea leaves Radix Astragali (Astragalus)

NPCA

Flavonoids Polysaccharides

[66] [50]

Olive kernel

HVEDA

Polyphenols and proteins

[99]

SEPARATION AND PURIFICATION OF SECONDARY METABOLITES

Efficient separation and purification is another important issue for the recovery of plant metabolites. Plant secondary metabolites are often mixed with a wide range of compounds with similar polarities and structures. Due to this fact, the separation of plant secondary metabolites is considered a great challenge. A number of different separation and purification methods, such as membrane-based separation process [13] and chromatographic process [12, 93] are commonly used to obtain pure plant secondary metabolites [12, 13]. Apart from that, non-chromatographic techniques (such as distillation, evaporation, freeze-drying, adsorption, etc.), can be used for separation and purification of secondary metabolites from plant matrix.

9.3.2.10.1 Membrane Separation

Membranes are selective barriers, which are used for the separation of desired molecules. The membrane separation process is a pressure-driven process, where particles with larger sizes than the membrane pores are retained and smaller molecules move through the membrane pore. Based on the pore sizes, membranes can be ordered as microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) [13]. In the industrial process, both cross-flow and dead-end membrane modules are used for purification and fractionation of plant secondary metabolites. In Figure 9.11, different types of membranes and their principle of separation process are presented.

The UF membrane has been used to concentrate polyphenols from grape seeds [66]; and to recover phenolic compounds from extracts of black tea [112]. Furthermore, the membrane separation process was successfully employed to concentrate bioactive components from kola-nut extract [80]. Although the membrane separation process is important to concentrate and recover plant metabolites, yet its performance is sometimes poor due to concentration polarization on the membrane surface and fouling of membrane [16].

9.3.2.10.2 Chromatographic Purification Techniques

Chromatography-related separation is used for the separation of a biomolecule from a mixture of different chemical compounds with unique polarity and molecular weight [6]. Different types of chromatographic processes (such as: adsorption chromatography, partition chromatography, ion exchange- chromatography, size-exclusion chromatography, thin-layer chromatography, paper chromatography, gas chromatography and high-performance liquid chromatography) are used to isolate secondary metabolites from plant tissues [12, 17]. In Figure 9.12, the classifications of chromatographic techniques are represented.

The separation efficiency of target metabolites is highly dependent on their adsorption affinity to the stationary phase. Subsequently, different spectroscopic techniques (such as: infrared nuclear magnetic resonance spectroscopy, ultraviolet (UV)-visible spectroscopy and tandem mass spectroscopy) are used to identify the separated and purified biological compounds [90].

(A) Different types of membranes depending on their molecular weight

FIGURE 9.11 (A) Different types of membranes depending on their molecular weight

cut-off (pore size), (B) Principle of membrane separation process.

Source: Self-developed with concepts from Ref. [13].

Classifications of chromatographic techniques. Source

FIGURE 9.12 Classifications of chromatographic techniques. Source: Self-developed with concepts from Refs. [12,17].

The separation of secondaiy metabolites from the herbal matrix is not an easy task due to the presence of a wide variety of phytochemicals. This problem can be overcome by changing polarity of mobile phase. Therefore, in chromatographic process, gradient elution mode is frequently used instead of isocratic elution mode for separation of bioactive compounds. As plant metabolites have highly variable properties (polarity, chemical structure, glycosidic linkages and spectral characteristics), no single technique is appropriate for universal application of separation of plant secondaiy metabolites [73].

 
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