A Buyer's Guide to DNA and RNA Prep Kits

ACTIVE MOTIF http://www.activemotif.com

For more information, please visit phenol purification technology.

Active Motif's Nautilus™ product line includes kits to isolate plasmid and bacterial artificial chromosome DNA, as well as genomic DNA from blood, cell cultures, bacterial cultures, and tissues. The company also offers the Fenozol™ kit to isolate total RNA, and the mTRAP™ kit for messenger RNA.

AGENCOURT BIOSCIENCES http://www.agencourt.com

Agencourt caters to high-throughput labs with its high-performance Solid Phase Reversible Immobilization (SPRI) product line. The key to SPRI: paramagnetic beads that bind plasmid DNA, eliminating the need for centrifugation or vacuum filtration steps. Options include the SprintPrep™ kit, for high-copy-number nucleic acids, and CosMCPrep™, for both high- and low-copy-number plasmids, bacterial artificial chromosomes, and so on.

AMBION http://www.ambion.com

Ambion bills itself as "The RNA Company," and its product line reflects that fact. In addition to a variety of isolation kits for total and messenger RNA, the company offers such accessories as a plant RNA isolation aid and RNAlater®-ICE, which preserves RNA in frozen tissue samples as they thaw.

AMERSHAM BIOSCIENCES http://www.amershambiosciences.com

Amersham Biosciences offers purification kits for genomic and plasmid DNA, and for total and messenger RNA. The Quick Prep™ Micro mRNA purification kit can squeeze mRNA out of a single eukaryotic cell, while the Nucleon™ DNA extraction kits can isolate genomic DNA from blood or cultured cells, plants, or from paraffin-embedded or hard tissue samples.

APPLIED BIOSYSTEMS http://www.appliedbiosystems.com

Best known for its instrumentation, Applied Biosystems produces genomic DNA isolation kits, too. The company's NucPrep™ chemistry enables the purification of DNA from plant and animal tissues, while BloodPrep™ processes whole blood or cultured cells. Both are optimized for use with Applied Biosystems' hardware.

ARCTURUS http://www.arctur.com

As a manufacturer of laser-capture microdissection instruments, Arcturus specializes in microgenomics, the molecular analysis of microscopic tissue samples. The company offers its PicoPure™ DNA and RNA isolation kits, which can isolate nucleic acids from a single cell.

BAY GENE http://www.baygene.com

Industry newcomer Bay Gene, founded in 2002, offers kits for purification of plasmid and genomic DNA, as well as total RNA. On the genomic DNA front, the company has kits to harvest nucleic acids from tissues, plants, blood, cultured cells, and bacteria. And, Bay Gene provides three levels of plasmid prep purity: rapid, ultra-pure, and endotoxin-free.

BECKMAN COULTER http://www.beckmancoulter.com

Beckman Coulter offers a series of high-throughput purification systems under Promega's Wizard® brand name. While the Wizard SV 96 system uses a series of transfer steps to effect purification, the Wizard MagneSil™ system uses paramagnetic beads, obviating the need for transfer, centrifugation, and vacuum filtration steps.

BD BIOSCIENCES CLONTECH http://www.clontech.com

Clontech offers a variety of products that vary by sample source and intended application. Sequencing-grade plasmid? Try the NucleoSpin Plus plasmid kit. But for transfections, the company recommends its NucleoBond product line. Other kits are available, including options for tissue and cell cultures, plants, and blood, and for purification of total and messenger RNA.

BIO-RAD LABORATORIES http://www.bio-rad.com

Bio-Rad offers a wide range of products for DNA and RNA isolation. The company's product line includes the AquaPure™ kits, for purification of genomic DNA and total RNA, and the Aurum™ system, which includes kits for plasmid and RNA purification. Aurum employs a binary system of stacked microtiter plates that minimizes sample handling, combines lysate filtration and plasmid binding in a single step, and works with both manual and automated methods.

DYNAL BIOTECH http://www.dynalbiotech.com

Dynal's product line features Dynabeads®, whose superparamagnetic properties ease sample purification. Offerings include kits to isolate mRNA from lysates, whole blood, or total RNA, plus kits to purify genomic DNA.

EDGE BIOSYSTEMS http://www.edgebio.com

Edge's product portfolio includes products for DNA purification. In addition to a bacterial genomic DNA isolation kit, the company offers a series of high-throughput plasmid miniprep kits. Designed for use in special "growth block" microtiter plates, the Plasmid 96 miniprep kit uses alkaline lysis chemistry to effect purification.

EPICENTRE http://www.epicentre.com

Epicentre's product line includes MasterPure™ isolation kits for DNA and RNA from yeast, blood, plant leaves, tissues, and buccal swab samples. The SoilMaster™ kit is available for extracting PCR-ready DNA from soil.

EPPENDORF http://www.eppendorf.com

Eppendorf brands many of its nucleic acid isolation kits using the adjective perfect; the plasmid miniprep kits are called Perfect-prep®, and the RNA isolation kits, Perfect RNA®. Eppendorf also offers other superlatives: The Fastplasmid™ plasmid isolation kits can isolate plasmid DNA in just nine minutes.

GENTRA SYSTEMS http://www.gentra.com

Gentra Systems' line of DNA and RNA purification kits includes PUREGENE® and PURESCRIPT® kits for purification of DNA and RNA respectively. Also offered, the company's GENERATION® DNA purification systems and new VERSAGENE® RNA purification kit. VERSAGENE isolates total RNA from cultured cells or tissues at room temperature without organic solvents.

GENOVISION http://www.genovision.com

GenoVision, a Qiagen company that focuses on histocompatibility antigen typing, offers the GenoM™-6 instrument for automated nucleic acid isolation and purification. The system employs GenoPrep™ cartridges preloaded with all the reagents necessary to isolate DNA from blood.

INVITROGEN http://www.invitrogen.com

Invitrogen's product portfolio includes kits for isolation of total and messenger RNA, as well as plasmid preparation. On the RNA front, the company offers everything from TRIzol® reagent to the FastTrack® 2.0 mRNA isolation kit. For DNA, Invitrogen offers the S.N.A.P.™ miniprep kit for purification of sequencing-quality nucleic acids, PureLink™ for transfection-quality DNA, and more.

MACCONNELL RESEARCH http://www.macconnell.com

MacConnell Research's motto is "Making manual mini preps a thing of the past," and the company offers two systems to make that possible. The Mini-Prep-24 and MiniPrep-96 robots both purify plasmid DNA using special sample cassettes. The device lyses the cells, resolves the plasmid DNA through a pre-cast agarose gel onto a membrane, washes the membrane, and finally purifies the DNA by electroelution. The two machines differ in the number of preps performed per hour.

MARLIGEN BIOSCIENCES http://www.marligen.com

Marligen sells the products previously offered by Life Technologies under the CONCERT™ brand. The company also offers kits to purify total RNA, as well as plasmid prep systems based on either silica membrane or anion exchange chemistries (the latter produces higher-quality nucleic acids).

MILLIPORE http://www.millipore.com

Millipore's Montage™ Plasmid Miniprep96 kit and BAC96 Miniprep kit are designed for use with automated liquid handlers. The kits require no centrifugation or precipitation steps; instead, the protocol employs a series of filtration steps, aided by a vacuum manifold, to effect purification.

MO BIO LABORATORIES http://www.mobio.com

Mo Bio caters to all manner of nucleic acid isolation needs. In addition to a collection of Ultra-Clean™ plasmid prep kits, the company offers products for isolating DNA from soil, blood, tissues, plants, yeast, and water, as well as a kit for forensic labs. Mo Bio also offers a range of RNA isolation kits.

MOLECULAR RESEARCH CENTER http://www.mrcgene.com

MRC offers reagents for DNA and RNA isolation, based on reagents named DNAzol® and TRI Reagent®, respectively. The former is a guanidine-detergent formulation; the latter is a monophasic mixture of phenol and guanidine thiocyanate. DNAzol kits are available for cells, tissues, plants, whole blood, and bacteria; TRI Reagent kits exist for tissues, cells, and blood. According to MRC, the TRI reagent can be used to purify DNA, RNA, and protein simultaneously from a single sample.

NOVAGEN http://www.novagen.com

Novagen, part of EMD Biosciences, offers kits to purify plasmid DNA, genomic DNA (from bacteria and blood), and mRNA. The company's Straight A's™ mRNA isolation kits and Magprep™ genomic isolation kits employ magnetic beads, while its Mobius™ and Ultra-Mobius™ plasmid prep kits use alkaline lysis chemistry.

PROMEGA http://www.promega.com

Promega's portfolio includes products to isolate plasmid DNA, genomic DNA, and total and messenger RNA. Among the company's offerings, the PolyA-Tract® system 1000 uses magnetic beads to purify mRNA directly from cells or lysates. The company also offers Wizard® plasmid purification systems, such as those licensed to Beckman Coulter.

QBIOGENE http://www.qbiogene.com

Qbiogene, which acquired BIO 101 several years ago, offers a variety of plasmid prep kits that incorporate its subsidiary's GLASSMILK matrix, including the RapidPURE™ and RPM® kits. In addition, the company offers genomic DNA isolation kits such as the GNOME® and FLORACLEAN™ systems, as well as kits for isolation of total RNA.

QIAGEN http://www.qiagen.com

Qiagen supplies a wide array of kits for the isolation of plasmid and genomic DNA, as well as both total and messenger RNA. When it comes to plasmid preps, the company caters to every throughput and scale needed. Options range from the QIAfilter plasmid Giga kit (10 mg of DNA) to the micro R.E.A.L. Prep 384 plasmid kit (1.5 μg of DNA per well). The company offers three distinct levels of purity, from standard to ultrapure.

ROCHE APPLIED SCIENCE http://www.roche-applied-science.com

Roche Applied Science offers kits to isolate DNA and RNA. In the genomic DNA category, the company has kits to purify nucleic acids from mammalian tissues, cultured cells, bacteria, yeast, plant tissues, and blood. Its MagNA Pure line uses magnetic beads to ease purification steps. Roche also provides kits to isolate plasmid DNA and total and messenger RNA.

SIGMA-ALDRICH http://www.sigmaaldrich.com

Chemical giant Sigma-Aldrich offers a range of systems for genomic and plasmid DNA, as well as total and messenger RNA. Most of the kits are sold under the GenElute™ brand name, but the company also provides Extract-N-Amp™ systems for PCR-ready nucleic acid extraction.

STRATAGENE http://www.stratagene.com

Stratagene's product portfolio includes kits for plasmid DNA and total RNA isolation. The StrataPrep® plasmid miniprep and StrataPrep EF plasmid midiprep kits use alkaline lysis and a nucleic acid-binding matrix to effect purification. The Absolutely RNA® purification kits isolate total RNA.

ZYMO RESEARCH http://www.zymoresearch.com

Zymo offers a range of kits for DNA and RNA isolation. Examples include the YeaStar Genomic DNA isolation kit, for use with yeast, and the Pinpoint Slide DNA isolation system, for use in collecting DNA from a specific region of a microscopic slide. The company offers a similar kit, the Pinpoint Slide RNA isolation system, for RNA recovery from a microscopic slide, as well as a variety of plasmid miniprep systems.

Extraction plays an important role for isolation and purification of many bioactive components from food material. In order to obtain the extract from the food sample, steps like size reduction, extraction, filtration, concentration and drying should be noted (Azmir.J et al. 2013; Živković et al. 2018). Figure  illustrates the general flow diagram for the isolation method of polyphenols from foods. Various extraction techniques have been analyzed to estimate the polyphenol recovery from foods ranging from traditional methods to modern methods. The most extensively used techniques for extraction include Soxhlet extraction, maceration, ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, high-voltage electric discharge, pulse electric field extraction and enzyme-assisted extraction (Pasrija and Anandharamakrishnan 2015; Luo et al. 2018). Apart from these technologies, membrane separation and encapsulation methods have also shown their potential for better extraction of polyphenols (de Santana Magalhães et al. 2019; El-Messery et al. 2019).

The solvent is recovered, and the extract is obtained using filtration process

Solvent in the flask is heated, it vaporizes into the thimble containing sample and condenses back into the flask. When the liquid reaches the top, the contents get emptied and the extraction continues

Apart from the advantages and simplicity in design, maceration technique poses few disadvantages. There may be batch-to-batch variations leading to error. Additionally, with the upcoming technology and fast-growing world, time could play a key role in this extraction. A huge amount of time is required for reaching equilibrium. Table shows the comparison between Soxhlet extraction and maceration technique (Ozel and Kaymaz 2004 ; Azwanida 2015 ; Shukla et al. 2016 ) as they have extensively used for extraction studies.

The speed of agitation and time are the two most important factors to be considered in this technique. The speed of the magnetic stirrer may lead to a vortex formation, which leads to a turbulence when the speed of the stirrer is varied. Due to these parameters, an increase in mass transfer rate may also be possible. Thus, the speed of the stirrer should be maintained between 180 and 240 rpm. If the speed is increased, there is high variations in equilibrium concentration and hence the diffusion coefficient (Shewale and Rathod 2018 ). Thus, the full study is undergone until all the compounds are extracted and process reaches equilibrium (Amita Pandey and Tripathi 2014 ).

Maceration is one of the go-to methods for determination of polyphenolic compounds (Ćujić et al. 2016 ). This is due to its simplicity, least experimental set-up, low cost and environmentally friendly characteristics.

On the other hand, maceration is considered a more suitable extraction technique as it uses lower temperatures, lesser time duration and gave higher yield of polyphenolic content. For instance, a study was done for determining the phenolic and flavonoid content of Syzygium cumini. L seed kernel. Soxhlet extraction gave a total phenolic content (TPC) of 30.05 mg GAE/g at 100 °C in 6 h, while batch extraction gave a phenolic content of 79.87 mg GAE/g at 50 °C in 105 min (Mahindrakar and Rathod 2020 ). Another investigation on extraction of curcumin was analyzed using process intensification methods. It was concluded that batch extraction gave higher yield (7.89 mg/g) at lower temperatures (30 °C) as compared to Soxhlet, which took almost the same yield at increased temperatures (Shirsath et al. 2017 ). Thus, it can be concluded that maceration is a more promising and affordable technique.

Due to its increased simplicity, the Soxhlet extraction method is still considered. However, there are some drawbacks to this technique. It uses large amounts of samples (10–30 g), long extraction times (18–24 h depending on the sample), large amounts of solvent usage (300–500 mL per sample) and excessive loss of heat energy (Hawthorne et al. 2000 ).

Soxhlet extraction has always been the most extensively used process for extraction purposes involving concentration of analyte leading to separation of bioactive constituents from natural products. Sample preparation is the most critical and can be done using variety of techniques (Luque de Castro and Priego-Capote 2010 ). Leaching is considered as one of the traditionally and practically used methods for solid pretreatment. When it comes to environmentally friendly methods, Soxhlet extraction is one of the relevant techniques. In conventional Soxhlet, sample is added inside a thimble and fresh solvent is added in the round-bottomed flask. The fresh solvent is passed through the thimble during the extraction process and then used as recovery. When the liquid reaches the top, the siphon drops the solvent back into the round-bottomed flask through the thimble holder. This operation is repeated until the process reaches saturation. The extraction of polyphenols is carried out for about 24–50 h, and more than half of the solvent is used for extraction studies (Sen et al. 2017 ).

Heat reflux extraction is a much preferred technique as compared to percolation and decoction as it requires less extraction time and solvent (Zhang et al. 2018b ). The technology involves heating the matrix for a particular time, leading to a complex chemical reaction. As the process uses reflux extractor as the main reactor, better mass transfer and contact efficiency are achieved between solvent and treated matrix (Tian et al. 2016 ). The vapor trickles down to the flask, thus controlling the temperature of the reaction. The technology has been preferred due to its simplicity and easy operation (Zhang et al. 2018a ). The technology has found application in extracting many natural, phytochemical compounds and essential oils (Gao and Liu 2005 ; Aliboudhar and Tigrine-Kordjani 2014 ).

Decoction involves boiling the crude aqueous extract to a certain volume for a specific time to get the heat-stable materials. The liquid settles down and is cooled, strained or filtered. The technique can be used for extracting water-soluble constituents. However, it should be noted that this procedure holds inefficient for heat and light-sensitive compounds. Additionally, mass transfer and kinetic effects are needed to be considered. Keeping negatives aside, this technique has found applications in various aromatic (Fuleky and Czinkota 1993 ) and medicinal plants (Rijo et al. 2014 ; Hashemi et al. 2019 ).

Percolation is a traditional procedure used for separation of active compounds from fluid extract. It consists of a narrow percolator (generally cone-shaped). The food sample is mixed thoroughly with water, and the solution is added from the top through the column into a closed container. The mixture seeps down with time (24–48 h depending on the sample), thus obtaining the pure extract. The enriched wet extract is concentrated in evaporators to get the desired concentration. The technique has been used for isolating a variety of polyphenolics from food matrices (Hansen and Møller 1975 ; Rathore et al. 2012 ).

As polyphenolic compounds are usually obtained inside different foods, beverages and plant matrices in small quantities, extraction methods become necessary. Pretreatment techniques like drying, crushing and grinding may be required depending on the samples. After extraction, isolation and purification are done to obtain the active compounds (Chuo et al. 2020 ). Conventional extraction methods mainly include percolation, decoction, heat reflux extraction, Soxhlet extraction and maceration. These are varied depending on the composition and characteristics of food samples. Although conventional methods are easy to use, they pose negative effects like high extraction time, huge energy consumption and solvent wastage (Zwingelstein et al. 2020 ).

Non-conventional technologies

Various studies have shown the potential of conventional extraction methods like Soxhlet extraction and maceration technique with promising results. However, such methods require huge usage of solvent, time and energy. The commercially used techniques for the extraction include ultrasound-assisted extraction, microwave-assisted extraction and supercritical fluid extraction (Luo et al. 2018). These techniques have shown a promising potential for improvements in polyphenolic content by 32–36% with about 17.6-fold lower energy consumption as compared to thermal treatments (Maza et al. 2019). Table shows the unique characteristics of the above technologies (Azwanida 2015; Llompart et al. 2019; Fayaz et al. 2020; Wen et al. 2020). Emerging techniques like high-voltage electric discharge, pulse electric field extraction and enzyme-assisted extraction have also gained interest and have been investigated with different food and plant matrices. These techniques show high quality of extract with the least consumption of raw material and energy. Figure  shows the emerging technologies required for the isolation of polyphenols. Figure  a shows ultrasound-assisted extraction systems (probe and bath sonicator) illustrating the cavitation phenomenon due to application of frequency on food matrix. Figure  b gives a schematic of the heat exchange occurring between the food material and environment on application of microwave energy. Figure  c depicts the mechanism of supercritical fluid extraction with CO2 as critical solvent.

Table 2

CriteriaUltrasound-assisted extraction (UAE)Microwave-assisted extraction (MAE)Supercritical fluid extraction (SFE)TerminologyInvolves the application of ultrasound energy on the food material leading to an increase in surface area between solvent and sample owing to increased yields

Application of microwave energy for the separation of materials using a solvent.

The microwave radiation interacts with the sample causing heat transfer by conduction

Involves separation of one food (a bioactive compound) component from the sample using supercritical fluids as an extracting solvent. CO2 is the most extensively used supercritical solventUnique characteristics

Alterations in the physical and chemical properties of the sample.

Enhancement of mass transfer of solvent into the plant material

Improved sample recoveries with high efficiencies observedEasy alterations can be done by changing temperature, pressure or adding a solvent with least effect on final sampleOptimum extraction parameters20–2000 kHz, 10–30 min400–600 W, 10–30 min, with appropriate temperature depending on samples31 °C, 7380 kPaSelectivityHighMethod prefers to interact with polar molecules and solvents with high di-electric constantHighBenefits and ease of useSimple set-up, low solvent usage and least extraction timeLeast extraction time and solvent usage as compared to conventionalNo cost of buying solvent and least sample usageDisadvantagesCavitation issues may arise and should be taken care ofThermal degradation of compounds may occur and proper conditions should be maintainedCan only be used for extracting bioactive compounds with high yieldsCostLow cost technology with least energy usageLow set-up costHigh initial equipment costScalabilityUsed for small and large-scale polyphenolic extractionsHigh power consumption if working on a large scaleHigh generally used for optimization studiesOpen in a separate window

Ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) deals with the application of high-intensity ultrasonic waves into the treated food sample. The technology is known for its simplicity and is comparatively cheaper as compared to other conventional extraction techniques (Dai and Mumper 2010). The introduction of high-frequency waves leads to a disturbance in solute–solvent mixture, resulting in breakage of cell walls and solvent diffusion (Cares et al. 2010). Criteria such as swelling rate, disruption and particle size post-treatment need to be considered for obtaining a higher efficiency (Xu et al. 2007). An increase in intensity during the process leads to intramolecular forces breaking the particle–particle bond. This leads to bond breakage and excessive penetration of solvent into the compounds resulting in cavitation.

The reason why ultrasound extraction holds an edge with respect to technology is due to shorter residence time between particles to solvent, the usage of small amounts of material, the least amount of solvents needed for use (100 ml minimum) and increased yields in overall polyphenolic extraction (Chmelová et al. 2020; Oroian et al. 2020). It holds useful in the isolation of bioactive elements within a very small period of time. One disadvantage is the requirement of a surplus amount of constant ultrasound energy for extraction of phenolic content (Savic and Savic Gajic 2020).

Microwave-assisted extraction

Microwave-assisted extraction (MAE) is another advanced technique used for isolation of polyphenolic compounds. The introduction of electric and magnetic field leads to heat transfer and conduction, resulting in a dipole moment between solvent and sample. The rotation also leads to successive collisions because of which thermal energy is produced in a closed environment. The phenomenon takes place very fast as the heating takes place in a closed medium. In this process, it heats the whole sample thoroughly by convection (Wen et al. 2020). Microwave extraction has smaller extraction time, minimal solvent requirement, increased extract purity, cost-effective, and better phenolic extraction yield in comparison with traditional methods. However, a tremendous amount of heat and energy loss occurs while conducting the process (Périno-Issartier et al. 2011).

Supercritical fluid extraction

In recent years, supercritical fluid extraction (SFE) has gained a lot of interest to extract bio-actives from plants at atmospheric temperatures preventing thermal denaturation. Supercritical fluid extraction is considered an efficient technique for separation studies due to its design and simplicity of construction. Transport phenomena studies allow better understanding of flow behavior and boundary conditions making it a faster extraction method than conventional techniques. Important criterions such as temperature, pressure, sample volume, solvent additions, flow rate controlling are to be strictly considered. Many solvents were tried due to the special conditions desired for carrying out the extraction procedures. Solvents like hexane (Lee et al. 2019), pentane (Lanças et al. 1994), toluene (Pripakhaylo et al. 2019), nitrous oxide and sulfur hexafluoride (Sakaki et al. 1990) were considered to be the most suitable for studies. However, carbon dioxide (CO2) has been the most extensively used solvent due to its ease of solvent removal and least cost (Pasrija and Anandharamakrishnan 2015). It is considered as a promising solvent because of its supercritical existence. Additionally, the gas is non-corrosive, inexpensive, colorless and odorless making it one of the ideal choices for isolation and purification in food industry (Sánchez-Vicente et al. 2009; Campalani et al. 2020).

However, there may also be few disadvantages when considering the following extraction studies. The phase equilibrium plays an essential role during the designing of a highly sensitive process with too many operating conditions to be followed. A large amount of pressure and environmental conditions also need to be met for the initiation of separation studies (Chaves et al. 2020).

Keeping the negatives aside, supercritical fluid extraction has shown positive signs as compared to other conventional techniques for extraction of polyphenolic compounds.

High-voltage electrical discharge

High-voltage electric discharge (HVED) technique is a sustainable technology involving the application of high voltages into an aqueous solution. It can be a good alternative as compared to thermal and conventional treatments. Figure  a shows the mechanism of high-voltage electric discharge treatment. The two submersed electrodes having high voltage release energy through a plasma channel into the mixture causing the disturbance (Roselló-Soto et al. 2015). The principle of high-voltage electric discharge can be described in two phases: the first phase (prebreakdown phase) and the second phase (breakdown phase). The first phase involves the introduction of light shock waves to the food sample, thus forming small bubbles. Adjusting the electric field intensity leads to the initiation of breakdown phase, resulting in release of active ingredients from the sample. However, the modification of electric field should be performed carefully as transition from prebreakdown phase to breakdown phase (electrohydraulic phase) could lead to several effects such as strong shock waves, structural damage to the sample, plasma bubbles inside the sample and strong liquid turbulence (Li et al. 2019). Energy usage forms an important for better recovery of active compounds and determining the efficiency of the system. The energy of the treatment (Rajha et al. 2014; Barba et al. 2015) can be shown in Eq. (1) and Eq. (2):

E=∑i=1nWHVEDm

1

WHVED=∫0tVIdt

2

where E is the specific energy (kJ/kg), m the product mass, WHVED is pulse energy (kJ/pulse), V is the voltage (V) and I is the current applied (A).

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In recent years, the technology has been extensively used for extracting polyphenols from various foods like grapes (Brianceau et al. 2016), pomegranate peel (Xi et al. 2017), peanut shells (Yan et al. 2018) and papaya (Lončarić et al. 2020). The technology thus poses as a sustainable method for the extraction of polyphenols.

Pulse electric field extraction

Pulse electric field extraction (PEF) is a green technology used for extraction of phytochemicals from many food materials in the absence of heat. The method involves the usage of electric pulses of moderate intensity leading to rupturing of cell membranes. The sample is placed between two electrodes, and electric field is varied depending on the sample. The release of compounds is due to the intermittent pulses of electricity produced during the process. (Puértolas et al. 2010). Figure  b shows the mechanism of pulse electric field extraction.

Electric field strength (E = V/d), pulse duration and number of pulses play an integral role in determining the efficiency of the whole sample. The intensities range from low (< 100–200 V/cm) to high (> 1500 V/cm). However, it is noted that short pulses are most effective for extraction of polyphenols in pulse electric field.

The energy consumption for this treatment (EPEF) (El Darra et al. 2013) can be calculated as per Eq. (3):

EPEF=∫0tVIdtm

3

where V is the electric field voltage (V), I the current (A), t is time (s) and m is the mass of the food sample (g).

In recent years, the technique has been in demand for analyzing many bioactive compounds in fruits and vegetables like strawberry (Stübler et al. 2019), orange (Luengo et al. 2013), red beet (Loginova et al. 2011), grapes (Delsart et al. 2012; Brianceau et al. 2015), tea (Liu et al. 2019) and onion (Liu et al. 2018).

Enzyme-assisted extraction

Enzyme-assisted extraction (EAE) is a sustainable technology dealing with introduction of enzymes into a mixture enhancing overall efficiency. Figure  shows the mechanism of enzyme-assisted extraction. The basic mechanism involves disruption of cell wall of food material by hydrolyzing it using an enzyme as a catalyst under optimum extraction conditions for release of bioactive components (Nadar et al. 2018). Addition of an enzyme softens the cell wall of the sample giving it easier access to the solvent medium. Since bioactive compounds, polyphenols and other phytochemicals exist inside the cells and are difficult to extract, this technique helps to release such compounds. The enzymes mainly used for extraction are cellulose (Yuliarti et al. 2015; Wikiera et al. 2016), protease (Oliveira et al. 2020) and pectinase (Marić et al. 2018; Domínguez-Rodríguez et al. 2021). Table shows the estimation of total phenolic content extracted from food samples using different enzymes. Particle size and enzyme ratios play an integral role in moderating the yield of polyphenols.

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Table 3

Food sampleEnzyme usedConditionsTotal phenolic content (in terms of gallic acid equivalent (GAE))ReferenceChokeberry pomaceViscozyme L and CeluStar XLEnzyme to solute ratio: 6% v/w, 40 °C, pH: 3.5, 7 h

With enzyme: 15 mg GAE/g

Without enzyme: 11.6 mg GAE/g

Contact us to discuss your requirements of Hydrogen Peroxide Plant Licensor. Our experienced sales team can help you identify the options that best suit your needs.

Kitrytė et al. (2017)Grape pomaceCellulase, tannaseAcetate buffer, pH: 5, 45 °C, 2 h0.74–0.76 mg GAE/gMeini et al. (2019)Guava leavesCellulase, xylanase

Dried powdered guava leaf (5 g) with water pH: 5, 12 h

Enzyme dosage: 0.5 g

With cellulase enzyme: 27.2

No significant influence on xylanase enzyme

Wang et al. (2017)Citrus peelViscozyme LCitrus peel (0.5 g), acetate buffer, pH: 4.8, 60 °C, 0.8% concentration of enzyme usage, 1 h

With enzyme: 1590

Without enzyme: 1169.23

Nishad et al. (2019)Open in a separate window

The technology is an environmentally friendly method majorly using water as solvent medium. Additionally, the extraction is carried out at low temperatures and requires low energy, thus preventing polyphenols from degradation. However, not many enzymes have investigated specifically for isolation in polyphenols from foods. The technology has extensively been used for extraction of polyphenols from vegetables and beverages like cabbage (Huynh et al. 2014) and wine (De Camargo et al. 2016).

Table shows the estimation of polyphenolic content using conventional and non-conventional technologies as discussed above for different foods. The method selection depends on factors like raw material, concentration, bioactivity, target molecule, process yields, cost, energy consumption and impact on the environment (Maroun and Chacar 2018).

Table 4

Food sampleTechniqueConditionsTotal phenolic content
(mg gallic acid equivalent (GAE)/g)ConclusionsReferenceCantaloupe melon peel and seedsEthanol extraction using water

200 mg powder of peels and seeds.

Solvent usage: 10 ml, 6 h, 50 °C

Peel: 25.48

Seed: 1.50

Improvement in radical stability between hydrogen and phenoxyl radicals.

Melon extracts could be used in food and cosmetic products

Vella et al. (2019)Citrus peelsMaceration0.3 g sample, 50 ml ethanol–water (20:80 v/v), 15 min, 90 °C280–673

Highest polyphenol content was found in the flavanone hesperidin.

Evaluated citrus peel by-products could be transformed into value-added products

Gómez-Mejía et al. (2019)Mango peelMaceration and ultrasound-assisted extraction (UAE)

Maceration: 5 g peel powder, 40 °C, 5000 rpm, 10 min

Ultrasound-assisted extraction: Frequency: 35 kHz@Temperature: 35, 45, 55 °C@Solvent analyzed: Methanol and ethanol

Maceration: 18.66

UAE: 67.58

UAE proved as a better extraction technique. Mango peel has an adequate amount of phenolics, making it a suitable ingredient for preparation of functional foodsSafdar et al. (2017)Grape seedsMaceration, ultrasound-assisted extraction and Soxhlet extraction

Soxhlet extraction

Seed sample: 25 g, 50 °C, 300 ml n-hexane solvent usage for 6 h

UAE with maceration:

Seed sample: 25 g, 20 kHz, 150 W, 30 min at 30 °C-50 °C.

Maceration time: 12 h

105.20Better oil recovery observed when grape seeds were subjected to UAE as compared to traditional SoxhletDa Porto et al. (2013)AppleUltrasound-assisted extraction (UAE) vs ultrasound-assisted extraction with hydrostatic treatment25 kHz, 70% amplitude, 20 °C, 60 min748High extraction efficiency and inactivation of enzymes observed during ultrasound extraction combined with hydrostatic treatmentAbid et al. (2014)Cantaloupe melonUltrasound-assisted extraction376 W/cm2, 10 min–Juice homogeneity improvements during treatmentFonteles et al. (2012)Rosemary and thyme extractsConventional vs ultrasound-assisted extraction

Conventional: 1200 rpm

Ultrasound-assisted extraction: 28.7 W/cm2 400 W, 40 °C

Thyme: 158

Rosemary: 15.4

Ultrasound stimulated activity of Bifidobacterium.

Polyphenolic compounds noticed in both thyme and rosemary.@Growth rate improvement of salmonella enterica in thyme.

Carotenoids improvements from thyme and rosemary

Munekata et al. (2020)Citrus peelsMaceration and ultrasound-assisted extractionAcetone (1:3), 30 min, 37 °C21.99Ultrasound-assisted extraction turned out to be an ideal technique in terms of yield, total phenols, flavonoids and antioxidant activitySaini et al. (2019)Green teaMicrowave-assisted extraction (MAE)

120,360,600 W

1,3,5 min

116.58Optimum conditions were 350.65 W power, 5 min irradiation timeTaşkın and Aksoylu Özbek (2020)Sea buckthorn bushConventional extraction vs microwave-assisted extraction

Microwave extraction (MAE): 400 W, 20–100 °C, 15 min

Conventional: 8000 rpm, 5 min

Microwave-assisted extraction (MAE): 1147

Conventional: 741

MAE turned out to be more preferred extraction method as compared to conventional for determining polyphenolic content antioxidant activity of the bush foodPérino-Issartier et al. (2011)Carrot juiceMicrowave-assisted extraction165 W, 9.39 min extraction time, 8.06:1 g/g oil to waste ratio215Enriched flaxseed oil used in carrot juice was in good quality, high in phenolic content and antioxidant activity (70% inhibition)Elik et al. (2020)PropolisMaceration, microwave-assisted extraction, ultrasound-assisted extraction

Maceration: 24 h, 250 rpm, room temperature

ultrasound extraction: 20 kHz, 15 min

microwave extraction: 140 W, 1 min

185–504

Ultrasound-assisted extraction proved as a much efficient technique for isolation of polyphenolics extraction efficiency and flavonol content.

Sample preparation of propolis played a critical role in obtaining the results

Oroian et al. (2019)Carob barkMicrowave-assisted extraction80 °C, 35% ethanol, 29.5 min33.6High amount of gallic acid was seen. Microwave extraction was found to be a suitable technique for revalorization of agro-food wasteQuiles-Carrillo et al. (2019)Lamiaceae herbsSupercritical fluid extraction (SFE)35 MPa, 100 °C–

Highest amount phenolic diterpenes detected in thyme and sage.

Improved antioxidant activity as compared to commercial antioxidants

Babovic et al. (2010)Spearmint leavesSupercritical fluid extraction and solvent extractionSupercritical extraction: 100–300 bar, 40–60 °C, 60–90 min–

Greater flavonoid content with higher yields.

Best yield was achieved at 200 bar, 60 °C and 60 min.

Solvent extraction: 257.67 mg/g

Supercritical fluid extraction: 60.57 mg/g

Bimakr et al. (2011)Apple seedsSupercritical fluid extraction and Soxhlet extraction24 MPa, 40 °C, 1 L/h of CO2, 140 min

Supercritical fluid extraction: 2.96 μg

Soxhlet: 1.56 μg

SFE gave higher oxidative stability than Soxhlet extraction.

The final product post-supercritical extractionwas rich in linoleic acid (63.76 g/ 100 g of oil)

Ferrentino et al. (2020)Strawberry leavesSupercritical fluid extraction308 K, 20 MPa, ethanol: CO2: 1:101709.1 μgSolvent density, solubility of organic compounds and vapor pressure played an important role influencing phenolic content and antioxidant activitySato et al. (2019)Sesame cakeHigh-voltage electric discharge (HVED)Energy input: 83 kJ/kg, 10% ethanol, 0.5 Hz, pulse duration: 10 μs54.3–440.3Technique showed least usage of organic solvents with higher diffusion giving increased efficiencySarkis et al. (2015)Orange peelsHigh-voltage electric discharge and enzyme-assisted extractionEnergy input: 222 kJ/kg, 80 min700Intense extraction of biomolecules with high polyphenols and reducing sugar yields from defatted orange peels was found during the combination of high-voltage electric discharge and enzyme-assisted extractionEl Kantar et al. (2018b)Grape seedsHigh-voltage electric dischargeNumber of discharges: 300, electric field strength: 40 kV/cm, electrode diameter: 25 mm8300Peleg’s model showed the best extraction kinetics (R2 > 0.995)Liu et al. (2011)Pomegranate peelsHigh-voltage electric dischargeVoltage: 40 kV, electrode diameter: 35 mm, electric field strength: 10 kV/cm, Time taken: 7 min46High-voltage electric discharge improved the recovery of polyphenols by 3 for ultrasound extraction and by 1.3 times for pulse electric field extractionRajha et al. (2019)Grape fruit peelsHigh-voltage electric dischargeEnergy: 7.27 kJ/kg to 218 kJ/kg, 20% aqueous glycerol86

Addition of glycerol reduced pretreatments by 6 times.

Same diffusivity of polyphenols was obtained in water from high-voltage electric discharge at 218 kJ/kg and in aqueous glycerol at 36 kJ/kg

El Kantar et al. (2019)OnionPulse electric field extraction (PEF)2.5 kV/cm, 90 pulses, 45 °C102.86Technique proved an environmentally friendly method with greater extraction yields and least sample consumption as compared to Soxhlet extractionLiu et al. (2018)TeaPulse electric field extraction1.25 kV/cm, 100 pulses, energy: 22 kJ/kg, 2 h398

77% of total polyphenols were extracted on application of electric field.

Significant increase in extraction recovery implied improvement in cell membrane permeability post-treatment

Liu et al. (2019)Lemon residuesPulse electric field extraction

0,3.5, 7 kV/cm,

0, 2.5, 5 bars pressure,

extraction time: 45 min

292Huge variations in phenolic content after pressing the sample and increasing the field strengthPeiró et al. (2019)Orange, pomelo, lemonPulse electric field extraction3 kV/cm and 10 kV/cm–Efficiency increased post-pressing by: 25% for orange, 37% for pomelo and 59% for lemonEl Kantar et al. (2018a)Open in a separate window

The emerging novel food processing technology for isolation of polyphenols has a promising potential to produce safe products with high quality. These techniques could minimize adverse losses taking place during conventional processing of bioactive compounds from fruits, vegetables and other products. Advanced technologies like membrane separation and encapsulation have also paved their way at a commercial level. These are discussed below.

Membrane separation

The use of membrane separation technology has been gaining lot of interest for separating as well as concentrating phenolic compounds and purifying them. The technology offers far better replacement as compared to traditional technologies like Soxhlet extraction as they possess low operating costs, easy scaleup and give higher product quality (Castro-Muñoz et al. 2019). In recent years, pressure-driven membrane process mainly ultrafiltration, microfiltration and nanofiltration have been largely studied in the agro-food sector. Figure  shows the membrane process technology used for isolating polyphenolic compounds from a food sample. Studies done for polyphenol recovery from artichoke (Conidi et al. 2014), wine lees (Giacobbo et al. 2017) and other natural compounds (Cañadas et al. 2020) have shown variation in improved recoveries with greater efficiencies. The processing of juices and liquid foods has also yielded useful health benefits and given higher recoveries when treated with membrane technology (Avram et al. 2017). Table shows membrane separation as a technology for isolation of polyphenolic compounds from different foods.

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Table 5

Food sampleTechniqueConditionsPhenolic content–(mg gallic acid equivalent (GAE)/g)ConclusionsReferencesPistachio hullMembrane separation with ultrasound-assisted extraction (UAE)

2 stage membrane process:

1 kDa cellulose membrane, 4 bar pressure, 250 rpm

120.31

Highest amount of phenolic compound and antioxidant activity in the retentate part.

34 compounds were found. Most abundant were gallic acid, galloylshikimic acid, pyrogallol and quercetin

Seifzadeh et al. (2019)Red wine leesMicrowave-assisted extraction (MAE) and membrane separation

3 stage membrane process

MAE: 356 Wh, 0.5–3 min, 1:10 wine: solvent used

Membrane area: 13.85 cm2, 68.9 bar

Pore size: 0.15 μm

933–1939Usage of membrane separation technology gave more importance to membrane material used and pore sizes. Aliphatic polyamide membrane gave the highest retention toward polyphenolic compounds as compared to polyvinylidene fluoride and cross-linked membranesArboleda Meija et al. (2019)Pomegranate juiceMembrane separation using polyvinylidene fluoride and polysulfone membraneAbsorbance: 765 nm

Polyvinylidene fluoride membrane: 1934.3

Polysulfone membrane: 1888.1

Lower retention of polyvinylidene fluoride membranes as compared to polysulfone membranesGaliano et al. (2016)Roselle extractUltrafiltration and nanofiltration membranesMembrane area: 0.0155 m2, thermal bath temperature (35 °C)

Ultrafiltration: 29.1

Nanofiltration:28.4

Nanofiltration membranes gave higher (95%) permeate fluxes and retention values for total soluble solids, acidity and bioactive components.

No damages in quality of the extract

Cissé et al. (2012)Lyciumbarbarum. L extractsMembrane separation with aqueous extractionPolyethersulphone membrane (0.3–0.4 kDa)1870.773–80% retention values of total polyphenols were observedConidi et al. (2020)Open in a separate window

Membrane-assisted solvent extraction has shown to be an efficient alternatives compared to traditional extraction techniques in terms of toxicity and quality of extract. This is mainly because of the least solvent usage (approximately 800 µL) for extraction. The analyte passes through the membrane to the acceptor phase according to the partition coefficient in the sample-solvent mixture (Vincelet et al. 2010). Additionally, the entire extraction is carried out in a vial on a flat membrane separating the aqueous phase with organic phase, thus not requiring any space to perform (Barbara Hauser 2002). Nonpolar solvents are generally preferable to inhibit loss of solvent through membrane. The technology with direct coupling to large-volume injection and gas chromatography detection has shown to be a quick and economic procedure for estimation of bioactive compounds from different foods and wastewaters (Schellin and Popp 2005; Rodil et al. 2007; Antónia Nunes et al. 2019).

However, one of the major challenges faced during separation and isolation of compounds is membrane fouling. Membrane fouling is the most influential factor that restricts the performance of membranes in its long-term operations. Factors such as origin of foulants, pore size and materials used play a vital role during characterization and control aspects (Chang et al. 2019). There have been several studies done to reduce the blockage of membranes and fabrication improvements (Dickhout et al. 2019; Li et al. 2020; Xu et al. 2020). Due to this reason, many membranes are used more as a purification process than as an extraction technology. The general method to evaluate membrane fouling is to compare the water flux through original and used membranes keeping same parameters. It can be determined using the formula (Seifzadeh et al. 2019) as given by Eq. (4):

Fw=ΔPμwR

4

where Fw is pure water flux (L/m2 h), ΔP is feed pressure (Pa), μw is water viscosity (Pa s) and R is membrane resistance.

Thus, it can be concluded that although the technology has progressed through the years, advancements still need to be carried out for better scaleup purposes.

Encapsulation techniques

Encapsulation technology has been considered as a highly advanced method for enabling modification of physical properties or isolation of food materials. In recent years, various researchers have recommended carrier agents and phenolic compounds, which can be used for producing micro- or nanocapsules (Lohith Kumar and Sarkar 2018; Saini et al. 2020). The encapsulation technique gives polyphenols and other micronutrients protection from the environment (Ezhilarasi et al. 2013). Certain factors need to be considered for determining the performance of encapsulation technology. Figure  a shows the experimental procedure involved in encapsulation. Figure  b illustrates the step-by-step mechanism for extraction of polyphenols using encapsulation technique using suitable wall material. Selection of technique and wall material holds a key factor to encapsulate phenolic compounds. The carrier or wall material is important as it controls the release action and protects the efficiency. Several techniques and wall materials have been evaluated for encapsulation of polyphenolic compounds as discussed in Table .

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Table 6

Encapsulating techniqueProcess technologyPolyphenols extractedWall materialFood sampleInferencesReferenceLiposomes

Flexible system which can entrap both oil and water functional compounds.

Generally used for entrapping aqueous solution within a lipid membrane

Catechin, epicatechin, quercetin, vanillinChitosanCoca hull via drinking yoghurtReduction in phenolic degradation by protecting them from interacting with other proteins in yoghurtAltin et al. (2018)Electro-spinning

Rapid technique involving the application of electric field to stretch the ultra-thin filaments using a syringe needle.

Generally carried out at room temperature to stop the degradation of polyphenol compounds

Phenolic acids and anthocyaninsGelatinSour cherryEight times better protection of glucoside molecules as compared to non-encapsulated sour cherry concentrateIsik et al. (2018)Electro-spraying

Single-step process where the solution is subjected to electric field and is broken into droplets due to high electric potential

The technology is a slight modification of electrospinning process

CurcuminWater-soluble proteinTurmeric

Elimination of interactions between curcumin and muscle proteins

Reduction of antioxidant activity observed

Gómez-Estaca et al. (2015)Spray drying

Technology involves dispersion of phenolic compounds into the carrier material follower by atomization in a hot chamber.

The solid particles formed from liquid droplets offer increased stability and solubility

Polyphenolic compoundsSodium alginateOlive leafProtection and controlled release of oleuropein under gastric conditions observedGonzález et al. (2019)Freeze-dryingProcess involves pressure reduction with removal of water from frozen food materials.@Involves a phase change from solid to gaseous phase. Generally used for encapsulating water-soluble bioactive compoundsFlavonoidsWhey proteins, pectinYellow onionResults showed that the encapsulated polyphenols can be used as a functional food ingredient and had improvement on consumer’s healthMilea et al. (2019)EmulsificationEncapsulation technique involves dispersion of two or more immiscible liquids where one liquid gets dispersed in the form of droplets.@Technique offers better stability and controlled release of polyphenolic compoundsResveratrol moleculesChitosanNutraceuticalsSlowing down of diffusion rate and release kinetics were studied using encapsulation techniquesSanna et al. (2015)Open in a separate window

However, there remains a huge gap in finding a universally applicable technique for polyphenol encapsulation due to their complex structure. Another challenge lies in keeping up to the consumer standards in terms of nutritional value, product quality, safety and cost. In the last few years, several other innovative green technologies have also been analyzed for improving the characteristics and yield of polyphenols in foods. Table gives an overview of such green technologies, which are in their early stages and have a potential for scaleup.

Table 7

Technology involvedProcess methodologyFood analyzedReferencesCloud point extraction

One-step procedure involving the extraction of polyphenolic and bioactive compounds using nonionic surfactants.

The surfactants tend to separate out from the main solution yielding a cloud formation when heated.

Simple and rapid process with reduced extraction time, less toxic and yields negligible environmental pollution as compared to conventional techniques

Olive oilKiai et al. (2018)Ultrasound-assisted extraction (UAE) using glycerol-based natural eutectic mixtures

Technology involving mixing of two solid materials with high melting points which do not interact to form a new chemical compound.

Hydrogen bonding interactions and phase behaviors play a key role in studying this process

Agri-food wastesMouratoglou et al. (2016)Infrared irradiation technology

One of clean energy sources for improved extraction of natural products and bioactive compounds using a ceramic infrared emitter.

Entire extraction requires low energy, easy to use, economical and has a great potential for scaleup

Pomegranate, olive, apricot pomaceAbi-Khattar et al. (2019); Rajha et al. (2019)Rapid solid–liquid dynamic extraction

An innovative solid–liquid cyclic pressurization process involving the rapid extraction of polyphenols from their organic or inorganic solvent mixtures. The technique uses liquid pressure and takes place at room temperature (or slightly lower) in order to avoid thermal stress on phenolic compounds.

The technique is environmentally friendly and requires less energy as compared to conventional extraction process

WineGallo et al. (2019)Vacuum-based solvent-free microwave extraction

Green extraction method which does not require solvent usage.

The food matrix is exposed to microwave radiation leading to expansion of cells resulting in extraction of solutes. The application of a vacuum condition allows the boiling point of solvent (water) to become lower than ambient pressure. Thus, the water can continuously boil at a reduced pressure and temperature allowing much efficient mixing preventing polyphenols from degradation

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Medicinal herb (C. nutans)Othman et al. (2020)Open in a separate window