Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx
INNFOO-01286; No of Pages 8
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Innovative Food Science and Emerging Technologies
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Influence of the treatmentmedium temperature on lutein extraction assisted by pulsedelectric fields from Chlorella vulgaris
Elisa Luengo, Juan Manuel Martínez, Andrea Bordetas, Ignacio Álvarez, Javier Raso ⁎Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, c/Miguel Servet, 177, 50013, Spain
⁎ Corresponding author at: Tecnología de los AlimeUniversidad de Zaragoza, c/Miguel Servet, 177, 5001976762675; fax: +34 976761590.
Influence of the of temperature of biomass (10–40 °C) treated by pulsed electric fields (PEFs) at different inten-sities (10–25 kV/cm) on electroporation of the microalgae Chlorella vulgaris and on the extraction of lutein wasinvestigated. The occurrence of reversible and irreversible electroporation increased with electric field strengthand medium treatment temperature. On the other hand, increasing temperature increased the sensitivity ofC. vulgaris cells to irreversible electroporation. Response surface methodology was used to identify optimal PEFtreatment conditions for enhancing lutein extraction yield (LEY) from fresh C. vulgaris biomass. Consideringthe cultivation temperature of C. vulgaris (25–30 °C) and the low increase in the LEY when the PEF treatmentswere applied at temperatures above 30 °C, a treatment of 25 kV/cm–100 μs at 25–30 °C increased the LEY around3.5–4.2-fold in comparison with the control, resulting in the most suitable treatment conditions formaximizing the lutein extraction at the lowest energy cost.Industrial relevance: In recent years, industrial interest in microalgae as source of bioproducts such as naturaladditives or active ingredients for food and cosmetic formulations has arisen. However, there are still severalobstacles to fully take advantage, such as the ability to successfully extract these compounds from the cellbiomass. Electroporation of microalgae by PEF-technology could be an alternative to conventional cell disruptiontechniques. Therefore, identifying critical factors affecting the enhancement of bioproduct extraction frommicroalgae is necessary to establish PEF as a true option.
In recent years, industrial interest in microalgae as source ofbioproducts such as natural additives or active ingredients for foodand cosmetic formulations has arisen (Becker, 2007; Schwenzfeier,Wierenga, & Gruppen, 2011). Chlorella vulgaris is a unicellularmicroalgathat not only contains a high amount of green photosynthetic pigmentssuch as chlorophylls a and b but also contains lutein and other primarycarotenoids such as α and ß-carotenes (Gonzalez & Bashan, 2000;Kitada et al., 2009).
Lutein is a xanthophyllic compound used as food colorant by theEuropean Union (E-161 b). This xanthophyll has also a potential rolein preventing retinal degeneration, some types of cancer, and cardiovas-cular diseases due to its antioxidant capabilities (Arnal et al., 2009;Carpentier, Knaus, & Suh, 2009). Lutein is located in the microalgalchloroplast and thermal degradation of lutein starts to be significant attemperatures above 60 °C. Currently, the commercial source of lutein
ntos, Facultad de Veterinaria,3 Zaragoza, Spain. Tel.: +34
fluence of the treatment medce and Emerging Technologies
is marigold flowers (Tagetes erecta L.) (Hojnik, Skerget, & Knez, 2008;Sowbhagya, Sampathu, & Krishnamurthy, 2004). Recently, microalgaehave been proposed as a potential source of this compound becausesome microalgal species have a higher lutein content than marigoldflowers, and they have shown yield productivities hundreds of timeshigher than marigold crops on a per-square-meter basis (Del Campo,Garcia-Gonzalez, &Guerrero, 2007; Fernandez-Sevilla, Acien Fernandez,& Molina Grima, 2010). In addition, compared to higher plants,microalgae can be cultivated in bioreactors on a large scale and thusthey are a continuous and reliable source of the product withoutdepending on environmental conditions, once it can be cultivatedindoors.
Obtaining high-value bioactive extracts from microalgae requiresculturing the microalgae, recovering the biomass, and purifying themetabolite from the biomass. The ability to successfully and efficientlyextract the compounds from the cell biomass without causing signifi-cant degradation is one of the main goals to taking full advantage ofmicroalgae as a valuable source of bioproducts. Although some productsof interest are excreted bymicroalgae to the growthmedium, generallyproducts produced bymicroalgae are localized in the intracellular spaceeither accumulated in vesicles or in cytoplasm. The presence in mostmicroalgal species of a cell wall surrounding the cells but especially ofan intact cytoplasmicmembranewhich acts as a semipermeable barrier
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greatly influences extraction of these compounds. Traditionally, extrac-tion of microalgal products is conducted from dry biomass with organicor aqueous solvents, depending on the polarity of the compound to beextracted (Ambrozova et al., 2014; Ceron et al., 2008). Dryingmicroalgalbiomass requires a significant amount of energy and may cause loss ofvaluable food compounds through oxidation. It is preferable to usemoist biomass in the product recovery scheme to reduce energy costsand prevent degradation of compounds.
The pulsed electric fields (PEF) are a technology causing cellmembrane permeabilization. PEF is based on the fact that when acell membrane is exposed to a sufficiently intense electric field ofshort duration (milliseconds to microseconds), it undergoes electri-cal breakdown which renders it permeable to molecules otherwiseunable to cross it. This external electric field needs to be above acritical value to induce electroporation. Depending on the processingparameters applied, the membrane can either become transiently orpermanently permeable, making electroporation either reversible orirreversible.While in reversible electroporation, pores created by theelectric field are able to reseal after the treatment application, inirreversible electroporation the pores in the cytoplasmic membranestay permanent. Application of PEF for improving extraction ofcompounds of interest from microalgae requires irreversiblerather than reversible electroporation. Several studies have demon-strated the potential of PEF to enhance extraction of compoundssuch as lipids and carotenoids from fresh microalgal biomass(Flisar, Meglic, Morelj, Golob, & Miklavcic, 2014; Goettel, Eing,Gusbeth, Straessner, & Frey, 2013; Grimi et al., 2014; Luengo,Condon-Abanto, Alvarez, & Raso, 2014; Zbinden et al., 2013). How-ever, practical application of technology requires conducting furtherresearch to understand the influence of main processing parametersto optimize processing conditions for obtaining maximummicroalgal electroporation with lower energy requirements. Besideselectric field strength and treatment time that are the characteristicprocessing parameters of PEF technology, processing temperaturehas been demonstrated to be a key parameter affecting cellmembrane electroporation. When PEF has been investigated as anonthermal method for microbial inactivation, several studies havedemonstrated that application of PEF at higher temperaturesdecreases the critical electric field required to kill microorganismsand causes a greater level of microbial inactivation at temperaturesthat are not lethal (Saldana, Alvarez, Condon, & Raso, 2014;Timmermans et al., 2014). On the other hand, it has been also re-ported that higher temperatures enhance electroporation of plantcell tissues (Lebovka, Praporscic, Ghnimi, & Vorobiev, 2005). How-ever, the effect of the temperature on the PEF-induced electropora-tion of microalgae and on the subsequent improvement in theextraction of compounds of interest from microalgae has not beeninvestigated.
The aim of this investigation was to assess the influence of tempera-ture of applied PEF on reversible or irreversible electroporation of themicroalgae C. vulgaris and on the extraction enhancement efficiency oflutein.
2. Material and methods
2.1. Cell culture
C. vulgaris (BNA 10–007, National Bank of Algae, Canary Islands,Spain), were grown in BG-11 medium contained the following compo-nents: 15 g L−1 NaNO3; 4.0 g L−1 K2HPO4; 7.5 g L−1 MgSO4·7H2O;3.6 g L−1 CaCl2·2H2O; 0.6 g L−1 Citric acid; 6 g L−1 ammonium ferriccitrate green; 0.1 g L−1 EDTA·Na2; 2.0 g L−1 Na2CO3; and trace metalsolution (H3BO3 2.86 g L−1; MnCl2·4H2O 1.81 g L−1; ZnSO4·7H2O0.22 g L−1; Na2MoO4·2H2O 0.39 g L−1; CuSO4·5H2O 0.08 g L−1;Co(NO3)2·6H2O 0.05 g L−1). For a solid medium, 15 g of technical agar
Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
was added to 1 L of the medium. Medium BG 11 (liquid and solid)was autoclaved at 121 °C for 20 min.
Cells were cultured photoautotrophically in a 1 L Roux flask bubbledwith air (6 mL/s), at 30 °C, in light:dark cycles (12:12) with white fluo-rescent lamps (15 μmol m−2 s−1). Cultures were initially inoculatedwith 1 × 106 cells/mL. Cell density was determined by a microscope(microscope L-Kc, Nikon, Tokyo, Japan) in a cell chamber Thoma(ServiQuimia, Constantí, Spain). Cells between the 10th and 15th days(stationary phase) were subjected to experiments.
For dry weight determination, 1 mL of culture was dried untilachieving constant weight (GeneVac Ltd, UK).
2.2. PEF treatments
The PEF equipment used in this investigation was previouslyintroduced by Saldaña et al. (2010). Microorganisms were treatedin a tempered batch parallel-electrode treatment chamber at differ-ent temperatures (10.0, 25.0, 40.0 ± 1.0 °C) with a distance betweenelectrodes of 0.25 cm and an area of 1.76 cm2. The temperature of thetreatment medium was measured with a thermocouple before andafter the PEF treatment and the temperature variations were alwayslower than 2 °C. The energy per pulse (W) was calculated using thefollowing equation:
W ¼Zi
0
σ � E tð Þ2dt ð1Þ
where k (S/m) is the electrical conductivity of the treatmentmedium; E (V/m) is the electric field strength; and t(s) is theduration of the pulse. The total energy (kJ) applied (W) was calculat-ed bymultiplying the energy per pulse (W′) by the number of pulses.The total specific energy (kJ/kg) applied (W) was determined bydividing the total energy by the mass of treated medium.
Before treatment, microorganisms were centrifuged at 3000 ×gfor 10 min at 25 °C and resuspended in a citrate–phosphateMcIlvaine buffer (1 mS/cm; pH 7). The microbial suspension(0.44 mL) at a concentration of 109 CFU/mL was placed into thetreatment chamber by means of a 1 mL sterile syringe (TERUMO,Leuven, Belgium). C. vulgaris cells were subjected up to 50 square3 μs waveform pulses at 0.5 Hz of 2.5, 3.75, 5 and 6.25 kV applied be-tween the electrodes separated by a gap of 0.25 cm. These voltagesresulted in electric field strengths of 10, 15, 20, and 25 kV/cm respec-tively that corresponded with specific energies per pulse of 0.30,0.66, 1.2, and 1.86 kJ/L of culture (concentration 109 cells/mL).Total specific energy ranged from 1.5 to 93 kJ/L of culture. The cur-rent intensities were 19, 33, 43 and 55 A when the voltages appliedwere 2.5, 3.75, 5 and 6.25 kV respectively.
2.3. Counting of viable cells
PEF-treated and control cell suspensions were serially diluted inMcIlvaine buffer (1 mS/cm; pH 7) sterile solution. From selecteddilutions, 20 μl were streak plated into solidmedia. Plates were incubat-ed at 30 °C for 7 days with the same light regime used for the liquidculture, and the number of CFU per milliliter was counted to determinethe % dead cells after treatment. Longer incubation times did notincrease the microalgal counts.
2.4. Staining cells with propidium iodide
Two alternative staining protocols were followed under the sameexperimental conditions. Cells were either stained with propidium io-dine (PI) (Sigma-Aldrich,) before PEF-treatment or once the treatmenthad finished. PI was used to investigate electroporation of microalgae
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because its molecular weight (668.4) is similar to the molecular weightof lutein (568.8).
2.4.1. Staining cells before PEF treatmentsBefore PEF treatments, microorganismswere centrifuged at 3000 ×g
for 10 min at 25 °C and resuspended in a citrate–phosphate McIlvainebuffer (1 mS/cm; pH 7) to a final concentration of approximately109 cells/mL. Next, PI was added to cell suspensions to a final concentra-tion of 0.8 mM. Once the PEF treatment was finished, cell suspensionswere incubated for 10 min. Previous experiments had shown thatlonger incubation times did not influence fluorescence measurements.Next, cell suspensions were centrifuged and washed two times untilno extracellular PI remained in the buffer, and the dye trappedinside the cells was measured. Fluorescence was measured with aspectrofluorophotometer (mod. Genios, Tecan, Austria) using a535 nm excitation filter (523–547 nm) and a 625 nm emission filter(608–642 nm).
Fluorescence data for cell suspensions were expressed as a percent-age of permeabilized cells based on the fluorescence value obtained forcells permeabilized after a PEF treatment (150 μs at 25 kV/cm) in acitrate–phosphate buffer of pH 7.0, sufficient to inactivate more than99% of cells. Under these conditions, permeabilization of the cellpopulation was checked using a fluorescence microscope (Nikon,Mod. L-Kc, Nippon Kogaku KK, Japan).
The degree of permeabilization evaluated following this protocolcorresponds to the sum of the irreversible and reversible cellpermeabilization.
2.4.2. Staining cells after PEF treatmentsPI was added to a final concentration of 0.8mM.With the purpose of
standardizing the staining protocol PI was added 5 min after the PEF-treatment. No differences in the PI uptake were observed when PI wasadded within 1 h after the treatment. Cell suspensions were incubatedfor 10 min, centrifuged, and washed two times until no extracellularPI remained in the buffer, and fluorescence was measured. In this case,the degree of permeabilization corresponds only to irreversiblepermeabilization.
Fluorescence measurements were based on mean values obtainedfrom two different microalgal suspensions.
2.5. Pigment extraction
100 μl of non-treated or PEF-treated suspension was added to 1 mLof 96% ethanol and vortexed. The mixture was macerated in the darkat room temperature for 20 min and centrifuged at 6000 ×g for 90 s.
2.6. HPLC analysis of carotenoids
HPLC/DAD analyses were performed on a Varian ProStar high-performance liquid chromatograph (Varian Inc., Walnut Creek, CA,USA) equipped with a ProStar 240 ternary pump, a ProStar 410autosampler, and a ProStar 335 photodiode array detector. The systemwas controlledwith a Star chromatographyworkstation v.6.41 (Varian).A reversed-phase columnMicrosorb-MV100-5 C18 (25× 0.46 cm; 5 μmparticle size) with a precolumn (5 × 0.46 cm; 5 μm particle size) of thesamematerialwas used. The temperature of the columnand precolumnwas maintained at 30 °C.
Pigments were eluted isocratically using acetonitrile:water:methanol(65%:2%:23) as a mobile phase for 25 min. The flow rate throughthe column was 1.5 mL/min, sample injection 30 μl, and absorbancedetection wavelength 443 nm. Prior to injection, all samples werefiltered through a 0.2 μm sterile syringe filter of cellulose acetate(VWR, West Chester, PA, USA).
Luteinwas identified by comparing its retention time and visible ab-sorption spectra with those of its standard. A calibration curve of luteinwas injected to determine its concentration in the extract.
Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
2.7. Experimental design
Response surface methodology (RSM) was used to evaluate the ef-fect of the treatment parameters, electric field strength, treatmenttime, and temperature on the lutein extraction yield (LEY) fromC. vulgaris.
The obtained data after the cells were treated with the conditionsdescribed in Section 2.2 were modeled with the following second-order polynomial equation:
Y ¼ β0 þXki¼1
βiXi þXki¼1
βiiXi2 þ
XkiN j
βi jXiXj ð2Þ
where Y is the response variable to be modeled, Xi and Xj are indepen-dent factors, β0 is the intercept, βi is the linear coefficient, βii is the qua-dratic coefficient, βij is the cross-product coefficient, and k is the totalnumber of independent factors. A backward regression procedure wasused to determine the parameters of the models. This procedure sys-tematically removed the effects that were not significantly associated(p N 0.05) with the response until a model with a significant effectwas obtained.
The CCD and the corresponding analysis of the datawere carried outby using the software package Design-Expert 6.0.6 (Stat-Ease Inc., Min-neapolis, MN, USA).
2.8. Statistical analysis
Experiments were performed in triplicate and the presented resultsare means ± standard deviation. One-way analysis of variance(ANOVA) using Tukey's test was performed to evaluate the significanceof differences between the mean values. The differences were consid-ered significant at p b 0.05. GraphPad PRISM (GraphPad Software, SanDiego, California, USA) was used to perform the statistical analysis.
3. Results and discussion
3.1. Effect of PEF on the permeabilization of C. vulgaris cells
Fig. 1 shows the influence of PEF treatments of 75 μs (25 pulses of3 μs) at several electric field strengths and different temperatures (10,25 and 40 °C) on the electroporation of the cytoplasmatic membraneof C. vulgaris evaluated by the uptake PI added before (A) and after (B)the PEF treatment.
Independently of the staining protocol, the uptake of PI increasedwith the electric field strength and the treatment temperature. WhenPI was added before the application of the PEF-treatment to detect theoccurrence of reversible and irreversible permeabilization, the maxi-mum percentage of PI uptake was observed even at the lower electricfield investigated (10 kV/cm) when cells were treated at 25 and 40 °C.However, at 10 °C, 15 kV/cm or higher electric field strengths were re-quired to obtain the same degree of permeabilization. On the otherhand, to obtain the maximum irreversible electroporation (addition ofPI after the treatment) (Fig. 1B), it was necessary to apply treatmentsof at least 15 kV/cm at 25 and 40 °C. At the lower temperature investi-gated (10 °C), the maximum degree of electroporation obtained at 25and 40 °C was not achieved even at the highest electric field applied(25 kV/cm).
The difference between the % of PI uptakewhen PIwas added beforeand after the PEF treatment reveals the existence of cells reversiblyelectroporated. At 25 and 40 °C, reversible electroporation was onlyobserved at the lowest electric field assayed (10 kV/cm). While at15 kV/cm or higher, the % of PI uptake with the two staining protocolsassayed was similar, at 10 °C, the % of PI uptake was always lowerwhen PI was added after the PEF treatment. For example, at 10 kV/cm,75% of PI uptake was detected when the dye was added before the
ium temperature on lutein extraction assisted by pulsed electric fields(2015), http://dx.doi.org/10.1016/j.ifset.2015.02.012
Fig. 1. Influence of electric field strength at different treatment temperatures on the PI uptake when PI was added before (A) and after (B) the PEF treatment. Treatment time: 75 μs (25pulses of 3 μs). 10 °C (●), 25 °C (○), 40 °C (■).
0 20 40 60 80 1000
20
40
60
80
100
R2:0.796
% death cells
% P
I up
take
Fig. 2. Relationship between the percentages of cell permeabilization assessed by PIstaining after PEF against the percentage of dead cells. To show the degree to whicheach treatment causes membrane permeabilization, a theoretical straight line withslope = 1 and intercept = 0, is included. 10 °C (●), 25 °C (○), 40 °C (■).
4 E. Luengo et al. / Innovative Food Science and Emerging Technologies xxx (2015) xxx–xxx
treatment, but no uptakewas observedwhen added after the treatment,meaning that the cells of C. vulgaris electroporated during the treatmentwere able to reseal their membranes after the treatment. According tothese results, increasing the treatment temperature decreases the criti-cal electric field to induce irreversible electroporation in the cell mem-branes of C. vulgaris. While at 10 °C permanent electroporationrequired electric field strengths higher than 10 kV/cm, at 25 and 40 °C,treatments at 10 kV were sufficient to render cells irreversiblyelectroporated.
Results shown in Fig. 1 indicate that in the range of temperaturesinvestigated, the uptake of PI by the population of C. vulgaris cells wasnot influenced by the temperature at electric field strengths above15 kV/cm when PI was added before the treatment. In this range ofelectric field strength, while the electroporation of the cells ofC. vulgaris was irreversible for most of the population at 25 and 40 °C,at 10 °C, a proportion of the cells were reversibly electroporated duringthe treatment. So these cells were able to reseal pores after thetreatment, and its membrane became impermeable to PI when addedafter the treatment. Theoretical models proposed to explain electropo-ration indicate that the electric field induces a potential across thecytoplasmic membrane, causing a structural reorganization of the lipidbilayer that leads to the formation of aqueous pores (Joshi, Hu,Schoenbach, & Hjalmarson, 2002; Saulis & Venslauskas, 1993). Depend-ing on the parameters of the electric field pulses, electroporation can beeither reversible or irreversible. According to results shown in Fig. 1when PEF was applied at 10 °C, the increase of the permeability of thecytoplasmic membrane during the treatment was similar to theincrease observed at higher temperatures. However, while at 25 and40 °C the electroporation was irreversible, at 10 °C, the membrane of aproportion of the cells returned to its natural state after the treatment.Reversibility or irreversibility of electroporation has been correlatedwith the size and the number of pores in the lipid bilayer by electropo-ration. Phase transitions of the membrane phospholipids from gel toliquid-crystalline phase are temperature-related, rendering thephospholipid bilayer less ordered and packed at higher temperatures(Reigada, 2014; Stanley, 1991). The higher organization of the lipidbilayer at lower temperatures could cause external electric fields of agiven intensity inducing a smaller number of pores or small size poresthan at higher temperatures, facilitating that the membranes return toits natural state afterward.
Application of PEF for improving extraction of compounds of interestfrom microalgae requires permanent electroporation, because theestimated lifetimes of the pores after the field is removed when tran-sient electroporation occurs are estimated to be in the range from
Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
milliseconds up to few minutes (Saulis, 2010). These times result tooshort to enhance the extraction of the compounds localized in theintracellular space of microalgae, because generally they are located invacuoles, chloroplasts, or vesicles. Generally, in the literature it isassumed that only irreversible electroporation is directly correlatedwith cell death (Unal, Yousef, & Dunne, 2002; Wouters, Bos, &Ueckert, 2001). The relationship between the percentage of PI uptakewhen the PI was added after the PEF treatment and the percentage ofdead cells is estimated by plate counting after the treatment is shownin Fig. 2. The figure shows all the inactivation data obtained at differenttemperatures by applying the treatment conditions to the C. vulgarispopulation described in theMaterial andmethods section. A theoreticalstraight line with slope 1 and intercept 0 that would represent a perfectagreement between% of PI uptake (permeabilization) and cell death hasbeen included in Fig. 2. Fig. 2 shows that when the percentage ofirreversible permeabilized cells was lower than 80%, the percentage ofdead cells was higher than the number of irreversible permeabilizedcells independent of the temperature of PEF application. Thereforethese results indicate that a number of cells inactivated during thetreatment were able to return the cytoplasmic membrane to its initial
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state after the treatment. As it is expected that the cytoplasmicmembrane will remain unpermeabilized in these cells, determinationof the number of cells inactivated by PEF could not be a good indicatorfor quantification of the level of C. vulgaris cells that have been irrevers-ibly electroporated in a process that aims to improve extraction ofintracellular compounds by PEF.
3.2. HPLC profile of pigments extracted from C. vulgaris
A suitable procedure for improving extraction of biocompoundsfrom microalgae should improve the extraction yield without causingsignificant degradation in the compounds of interest. The extracts ob-tained from C. vulgaris using ethanol as a solvent after the applicationof PEF treatments of different intensities at different temperatureswere analyzed by reverse-phase HPLC. Fig. 3 compares chromatogramprofiles detected at 443 nm for extracts obtained in an extraction fromuntreated and pretreated by PEF (40 °C–25 kV/cm–50 pulses of 3 μs)C. vulgaris cells. Similar chromatogram profiles were obtained for theextracts obtained in different experimental conditions investigated. Ac-cording to Fig. 3, the application of a PEF treatment in the most intenseconditions increased the amount of compounds extracted but did notaffect the extraction of a selected compound. As reported by other au-thors (Updike & Schwartz, 2003), PEF treatment did not cause pigmentdegradation. Similar results have been observedwhen the application ofPEF has been investigated to improve the extraction of other com-pounds of interest from the cells of plant tissues (Lopez, Puertolas,Hernandez-Orte, Alvarez, & Raso, 2009; Puertolas, Cregenzan, Luengo,Alvarez, & Raso, 2013).
3.3. Improvement of the extraction of lutein from C. vulgaris by PEF treat-ments at different temperatures
The lutein extraction yield (LEY) resulting from the extractionwith ethanol from the control (untreated C. vulgaris cells) and thePEF-treated C. vulgaris cells at the treatment conditions describedin Section 2.2 is shown in Table 1. Values of LEY varied from 163 to753 μg/g dw of C. vulgaris culture. These values are within the
Fig. 3. HPLC profile at 443 nm for extracts obtained in an extraction from pretreated b
Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
range of the values reported in the literature by other authors thathave investigated the accumulation of this pigment in thesemicroalgae (Jeon et al., 2014; Kitada et al., 2009). Results shown inTable 1 indicate the potential of PEF for improving the extraction oflutein from a fresh biomass of C. vulgaris. As compared with the un-treated biomass, the concentration of lutein was around 4.5-foldhigher when the fresh biomass was previously electroporated at40 °C by a PEF of 25 kV/cm for 75 μs. Treatments of the same intensityat 10 and 25 °C increased the LEY 3.2- and 2.3-fold, respectively. Thehigh ethanol concentration used for extraction could cause the dena-turation of the cytoplasmatic membrane facilitating the lutein re-lease. However, our results indicate that this denaturation is notvery effective in improving mass transfer through the cytoplasmaticmembrane because a previous electroporation increased significant-ly the extraction of lutein.
The improvement in the extraction of lutein as a consequence ofthe prior permeabilization of the cytoplasmic membrane ofC. vulgaris by PEF depended on the PEF treatment conditions. Whencells were treated at 10 °C, no statistically significant increase inLEY was observed at the lowest electric field strengths applied (10and 15 kV/cm) even after the application of the longest treatmentduration (150 μs). At this temperature, these results agree with thedata obtained in the % of PI uptake when the dye was added afterthe treatment to detect irreversible electroporation, since the % ofPI uptake was lower than 5%. However, at 15 kV/cm, 40% of PI uptakedetected when the cells were treated for 150 μs did not correspondwith the lack of lutein extraction improvement observed. These re-sults could indicate that the number or the size of the pores pro-duced by these PEF treatments allows PI uptake, but they were notbig enough to improve lutein extraction. At 25 and 40 °C, the LEY in-creased significantly at electric fields of 15 kV/cm or higher even atthe shortest treatment time applied (15 μs). However, at thesehigher temperatures, although a PEF treatment of 20 kV/cm for75 μs was enough to obtain 100% of PI uptake, to obtain the maxi-mum LEY at each temperature, it was necessary to apply more in-tense electric field strengths (25 kV/cm). According to theseresults, although some positive correlation (R = 0.8, data not
y PEF (40 °C–25 kV/cm–50 pulses of 3 μs) (A) and untreated (B) C. vulgaris cells.
ium temperature on lutein extraction assisted by pulsed electric fields(2015), http://dx.doi.org/10.1016/j.ifset.2015.02.012
Table 2Coefficients, F-values and p-values of the ANOVA analysis for the quadratic model devel-oped to describe the influence of the temperature (T), treatment time (t) and electric fieldstrength (E) on the lutein extraction yield from C. vulgaris cells.
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Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
shown) was observed between the PI uptake and the lutein extrac-tion in the cells of C. vulgaris treated by PEF at different tempera-tures, data on irreversible electroporation evaluated by the uptakeof PI are not suitable to define the PEF treatment conditions to obtainthe highest LEY.
3.4. Mathematical modeling
Toquantify the influence of PEF processingparameters (electricfieldstrength, treatment time and temperature) on the LEY from C. vulgarisfresh biomass, a multiple regression analysis was conducted fitting theexperimental data presented in Table 1 for Eq. (2). Ethanol was usedas solvent for untreated and PEF treated cells of C. vulgaris. Backwardregression procedure did not eliminate any equation term because allthe terms of Eq. (2) were statistically significant (p b 0.05). Coefficientsof Eq. (2) and the statistics used to test the adequacy of the model areshown in Table 2. The p-value of the models was less than 0.05, whichindicates that the model is significant and therefore the terms in themodels have a significant effect on the responses. The determinationcoefficient was 0.90, which means that only 10% of the total responsevariation remained unexplained by the model developed. The adjustedR2 values that correct the R2 according to the number of responses andterms in the model were close to the R2. The value of the RMSE param-eter shows that model produced predictions close to the observed data.A bias factor (Bf) of 1 means that themodel was a good predictor of LEYand the accuracy factors (Af) show that, on average, the predictionsdiffer from the observations by 18%. The F-values for the model param-eters are useful indicators of the significance of the effects of the vari-ables and their interactions. According to the F-values shown inTable 2, changes in electric field strength and the temperature had themost significant effect on the LEY. However, since the square terms ofthe electric field strength and temperature were also significant, thismeans that its effect on LEY is nonlinear. The negative effect of thesquare term of temperature and time indicates an optimum value fortemperature and time; above this value, the temperature or time in-crease did not substantially increase the LEY.
3.5. Influence of electric field strength, treatment time and temperature onthe improvement of the extraction of lutein from C. vulgaris
Graphical representationswere obtained using the regressionmodel(Table 2) considering the responses within the range of experimentalconditions assayed (Fig. 4A and B). The effect of treatment time (num-ber of pulsesmultiplied by the pulsewidth) and temperature of applica-tion of the PEF treatment at 25 kV/cm to a fresh biomass of C. vulgaris onthe subsequent extraction of lutein is shown in Fig. 4A. At any temper-ature, the LEY increased with the treatment time until around 100 μs.Beyond this time, further increases of the treatment time did not appre-ciably increase the lutein extraction. Increasing the treatment tempera-ture led to a reduction of the treatment time required to obtain a givenLEY and, as a consequence, it resulted in a decrease in the total specificenergy required. For example, a lutein extraction of 400 μg/g dw ofC. vulgaris culture required a treatment of 100 μs that corresponded toan energy input of 71 kJ/L of culture when the treatment temperaturewas 10 °C. However, the same LEY was obtained with a treatment of15 μs at 25 °C that corresponded to an energy input of 31 kJ/L of culture.
These results support other studies in which it has been observedthat the increase of the temperature of application of PEF treatmentscauses a reduction of the specific energy input required to obtain agiven reduction in the viability of a microbial population with the aimof food pasteurization (Saldana, Puertolas, Monfort, Raso, & Alvarez,2011).
The influence of the PEF electric field strength to the fresh biomass ofC. vulgaris at different temperatures on the subsequent extraction oflutein is shown in Fig. 4B. Treatment time selected to plot Fig. 4B was100 μs because according to Fig. 4A, it was the shortest treatment
ium temperature on lutein extraction assisted by pulsed electric fields(2015), http://dx.doi.org/10.1016/j.ifset.2015.02.012
Fig. 4. Influence of treatment time (A) and electric field strength (B) on the lutein extraction yield obtained from C. vulgaris cells PEF-treated at several temperatures. 10 °C (—), 20 °C (- -),30 °C (—), 40 °C (- -).
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duration that permitted obtaining the highest LEY at the temperaturesinvestigated. It is observed that at any temperature, the LEY increasedby augmenting the intensity of the electric field strength applied tothe fresh microalgal biomass. However, increases in the range of 10 to20 kV/cmwere less effective than in the range of 20 to 25 kV/cm. For ex-ample, at 20 °C, while an increase of 5 kV/cm units of electric field from10 to 15 kV/cm increased the extraction of lutein to 39 μg/g dw ofC. vulgaris, from 20 to 25 kV/cm the observed increases were 112 μg/gdw of C. vulgaris. This behavior could be due to the fact that lutein is lo-cated inside the chloroplast and needs to cross not only the cytoplasmicmembrane but also the chloroplast membrane to be extracted from thefresh biomass of C. vulgaris. At higher electric field strengths, the addi-tion of the cytoplasmic membrane to the PEF treatment could alsoelectroporate the membrane of the chloroplast, facilitating lutein ex-traction. Electric field strength threshold is inversely related to cellsize (Kotnik, Kramar, Pucihar, Miklavcic, & Tarek, 2012). Since chloro-plast size is smaller than cell size, a higher electric field strength couldbe necessary to electroporate it. The electric fields (100 kV/cm) usedby different authors for permeabilization of cell organelles with pulsesof a duration of nanoseconds confirm that higher electric fields are re-quired to affect the integrity of the chloroplast (Esser, Smith,Gowrishankar, Vasilkoski, & Weaver, 2010).
The electroporation of the chloroplastmembrane could be also influ-enced by the temperature of application of PEF, since the degree of per-meabilization is larger at higher temperatures. According to the resultsshown in Fig. 1B, independently of the temperature of application ofthe PEF treatment, the complete population of C. vulgaris biomass waspermeabilized after a treatment at 25 kV/cm for 100 μs. However,Fig. 4B shows that an increase of the temperature of PEF treatment ap-plication at this intensity from 10 to 40 °C increased the LEY from 400to 700 μg/g dw of C. vulgaris. The higher electroporation of the chloro-plast membrane at 40 than at 10 °C could explain the higher extractionof lutein observed at higher temperatures. Since the chloroplast mem-brane is a lipid bilayer like the cytoplasmic membrane, the higher orga-nization of the lipid bilayer at lower temperatures could also be thecause because an external electric field of a given intensity induced asmaller degree of electroporation at lower temperatures. Independentof the reason that an increase of the temperature of application of thePEF treatment causes an improvement in the amount of extracted lu-tein, Fig. 4B shows that enhancement in the extraction was dependenton the range of temperatures inwhich the increase is performed. For ex-ample, at 25 kV/cm, increasing the temperature of 10 °C to 20 °C in-creased the LEY by 35%, but an increase lower than 10% was observedby raising the treatment temperature from 30 to 40 °C. From a practicalpoint of view, the smaller increases in LEY were observed when thetreatment temperature is above 30 °C and the energy cost required to
Please cite this article as: Luengo, E., et al., Influence of the treatment medfrom Chlorella vulgaris, Innovative Food Science and Emerging Technologies
increase the temperature of the biomass advises against applying thetreatment at temperatures higher that 30 °C to improve the extractionof lutein from C. vulgaris biomass.
4. Conclusions
In this investigation it has been demonstrated that in addition to theelectric field strength and treatment time, treatment temperature is acritical parameter that influences electroporation of C. vulgaris and sub-sequent extraction of lutein from a fresh biomass of this microalgaetreated by PEF. Evaluation of the irreversible electroporation of cells ofC. vulgaris detected by the increase of the fluorescence of the populationon thedye PIwas added after the PEF treatment or quantification of cellsof C. vulgaris inactivated by the PEF treatment was not a suitable proce-dure for defining optimal PEF treatment conditions at different temper-atures to achieve the maximum extraction of lutein from a freshbiomass of C. vulgaris.
Commercial viability of the products derived frommicroalgae is sig-nificantly dependent on the cost of these extraction processes, and gen-erally the downstream recovery of the products can be substantiallymore expensive than the cultivation of the microalgae. Indeed, tomake production of extracts from microalgae economically feasible, itis necessary to optimize the biomass processing formetabolite recovery.
Considering that the cultivation temperature of C. vulgaris is be-tween 25 and 30 °C, the low increase in the LEYwhen the PEF treatmentis applied at temperatures above 30 °C and the energy cost derived fromincreasing the temperature of the culture to be treated by PEF, from apractical point of view, the most appropriate approach for improvingextraction of lutein by PEF at the lowest energy cost would be to locatethe PEF treatment chamber in the outflow tube of the photobioreactorswhere the microalgae are cultivated.
Acknowledgments
This research was supported by the Government of Aragón (Grupode Investigación Consolidado A20), and the European Social Fund. E.L.gratefully acknowledges the financial support for her doctoral studiesfrom the Department of Science, Technology and University of theAragon Government.
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