Isolation and characterization of Alstroemeria hookeri ssp. ...

RESEARCH ARTICLE

Isolation and characterization of Alstroemeria hookeri ssp.hookeri starch in comparison with potato starch

Zormy Correa1, Alejandro Zuniga1, Carolina Garfias2 and Luis Arturo Bello-Perez3

1Centro de Investigación de Polímeros Avanzados (CIPA), Beltrán Mathieu 224, piso 2, Concepción, Chile2Departamento de Ingeniería de Materiales, Facultad de Ingeniería, Universidad de Concepción, Barrio Universitario S/N,Concepción, Región del Bío-Bío, Chile

3 Instituto Politécnico Nacional, CEPROBI, Carretera Yautepec-Jojutla, Yautepec, Morelos, México C.P.

Starch was isolated from a non-conventional source, Alstroemeria tubers and its physicochemicalproperties were evaluated. Commercial potato starch was used for comparison. The chemicalcompositions of the starches were similar, except in ash, protein, and lipids contents, whereAlstroemeria showed lower values compared to potato. Starch recovery ratio for Alstroemeria was86.4%. AM content and degree of crystallinity were similar. Granule size distribution forAlstroemeria was narrower compared to potato. The swelling properties of Alstroemeria werehigher than for potato, increasing as temperature increased. Alstroemeria exhibited higher peakviscosity, breakdown and final viscosity; and lower gelatinization and pasting temperaturecompared to potato. AP MWand branch chain-length distribution were studied. Alstroemeria hadhigher molar mass (Mw), gyration radii (Rz), and density (r) compared to potato. Potato starch hada higher proportion of intermediate/short chains (DP 13-24) and Alstroemeria starch had a higherproportion of long chains (DP � 37).

Received: November 17, 2012Revised: March 19, 2013

Accepted: March 22, 2013

Keywords:Alstroemeria starch / Chemical analysis / Isolation / Physical properties

1 Introduction

Starch is one of the most promising materials forbiodegradable plastics due to its low cost, availability, andability to impart a broad range of functional properties to foodand nonfood applications [1]. Most commercially availablestarches are isolated from grains such as corn, rice, wheat andfrom tubers such as potato and tapioca. These conventionalsources of starch have been researched extensively [2–5].Recently, tubers as chayote [6] and taro [7] were used for starchisolation, and physicochemical and functional propertieswere evaluated.

Alstroemeria, commonly called the Peruvian Lily or Lily ofthe Incas is a South American genus of about 50 species offlowering plants. Alstroemeria hookeri ssp. hookeri is one ofthe Alstroemeria species. In Chile, it is found in the Mauleand Bío–Bío regions and grows from sandy loam soils todeserts [8].

First descriptions of Alstroemeria were done by Linneoin 1762 [8] and by Reiche in 1910 [9]. Both describedAlstroemeria as perennial plant with cylindrical fleshy tubers,having high starch content with nutritious and medicinalproperties.

Although the origin of Alstroemeria can be found in SouthAmerica, in the middle of the sixties it was for the first timegrown inWestern Europe. In 1990 there were 84 ha (hectare).of cut flower Alstroemeria grown in Holland (AlstroemeriaCultivation Guide, 2006, http://www.roskamhorticultura.com). The standard production in Holland under idealcircumstances is 200–300 stem per year per m2 with a cropplanting density of 3.2 plants per gross m2. The crops areplanted from December to June being the life time ofAlstroemeria plant of 3–5 years. It is a wild plant with goodadaptation and of easy cultivar, which do not compete with the

Correspondence: Dr. Zormy Correa, Centro de Investigación dePolímeros Avanzados (CIPA), Beltrán Mathieu 224, piso 2,Concepción, ChileE-mail: [email protected]: þ56-41-3168649

Abbreviations: DSC, differential scanning calorimetry; Mw,molecular mass; PT, pasting temperature; RVA, rapid viscoanalyzer; Rz, gyration radii; SP, swelling power; WAI, water-absorption index; WSI, water-solubility index

DOI 10.1002/star.201200265Starch/Stärke 2013, 65, 991–998 991

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com

http://www.roskamhorticultura.com/

http://www.roskamhorticultura.com/

human food sources as potato, wheat, rice or corn. As anadvantage compared to potato, Alstroemeria does not need tobe peeled off, which reduces the processing cost (Directivasde Cultivo, Alstroemeria, http://www.horticom.com/pd/images/pdf/73178.pdf).

In the field of ornamental cultivars, Alstroemeria hasattained international renown but unfortunately, there is nopublished data on tuber production and the integral use ofthis resource. Only data related to flowering is found in theliterature.

The aim of this work was the isolation and thephysicochemical characterization of starch from Alstroemeriatubers and its comparison to potato starch.

2 Materials and methods

2.1 Materials

Alstroemeria hookeri spp. hookeri wild tubers were collected12 km northwest from Concepción city (Bío–Bío region). It isa low altitude area (<1.5 m), between Bío–Bío river and SanVicente Bay. Commercial potato starch produced by EmslandGroup (Germany) was used for comparison.

2.2 Starch extraction

Extraction of starch was done according to the techniquedescribed by Bello-Pérez et al. [10] with some modifications.The tubers were washed and blended in an aqueous solution(tubers:water ratio 1:1) for 10 min at maximum speed. Theobtained homogenate was sieved through ASTM 60(250 mm) and ASTM 100 (150 mm) meshes, in order toextract starch completely. The procedure was repeatedthree times. The starch milk obtained after sieving wasallowed to settle overnight and subsequently, decanted.Finally, the upper non-white layer was removed and thewhite layer (starch) was collected and oven-drying at 40°Cfor 24 h.

2.3 Chemical analysis

Moisture content was taken as weight loss of 2 g sample afteroven-drying the starch powders at 130°C for 2 h. Ashand protein were determined according to AOAC methods923.03 and 920.87, respectively [11]. The starch contentwas determined by using Megazyme total starch assay kit(Megazyme International, Ireland, UK). Lipids content wasobtained by the Bligh and Dyer method [12]. Determinationswere conducted in triplicate.

The AM content of Alstroemeria and potato starcheswas tested using the enzymatic amylose/amylopectin assaykit (Megazyme International) [13]. Absorbance wasmeasured at 510 nm by using a UV mini-1240 spectropho-

tometer (Shimadzu Scientific Instruments, Japan). TheAM content of the isolated starch was determined intriplicate.

2.4 Physical properties

2.4.1 Morphology of starch granules

The morphology of the granules in Alstroemeria and potatostarches was studied with a scanning electron microscopemodel JSM 6380 (JEOL, Japan). The starch powders werecovered with gold using an etching unit model (Edwards S150Sputter Coater, MA, USA). Acceleration voltage used was15 kV.

2.4.2 Granule size distribution

Granule size determination was carried out at roomtemperature using a Malvern Mastersizer (Malvern Instru-ments, Worcestershire, UK) particle size analyzer based onthe principle of laser light-scattering. Software was basedon Standard Operating Procedures (SOP), capable ofmeasuring sizes from 0.02 to 2000 mm. The volumedistribution was obtained according to the Mie scatteringtheory [14]. Size distribution was calculated in duplicateand expressed as (d0.1), (d0.5), and (d0.9), the diameterof the granules for which 10, 50, and 90% of the starchvolume was made up.

2.4.3 XRD

The XRD pattern of both native powder starches wererecorded on an X-ray D5000 Siemens diffractometer(Siemens, Germany). The anode generator was operated at40 kV and 30 mA, with the Cu Ka radiation (l ¼ 1.5406 A

�).

The scanning rate of the diffraction angle 2-u (2–80°) was0.02°/10 s. Software analysis used for crystallinity determi-nation was Diffrac Plus, V 15.2009, TOPAS V4.0. Duplicatemeasurements were performed.

2.4.4 Swelling properties

Water-absorption index (WAI), water-solubility index (WSI),and swelling power (SP) were determined in triplicate,according to Anderson et al. [15]. Starch slurries were stirredand heated in a thermostated water bath from 50 to 80°C, andthen cooled to room temperature before centrifuging at4900 rpm for 30 min.

2.4.5 Gelatinization properties

The gelatinization properties were measured using differen-tial scanning calorimetry (DSC) (Mettler Toledo, DSC1Stare System, Switzerland). The starch sample (4.27 mg of

992 Z. Correa et al. Starch/Stärke 2013, 65, 991–998


http://www.horticom.com/pd/images/pdf/73178.pdf

http://www.horticom.com/pd/images/pdf/73178.pdf

Alstroemeria, 3.96 mg of potato) was weighed directly in analuminum pan of 40 mL capacity and moistened with 8 mL ofdeionized water by a microsyringe. Pans were hermeticallysealed and allowed to stand for 1 h at room temperaturebefore DSC measurements. Indium was used for calibration.Sample pans were heated from 25 to 130°C at a rate of 10°C/min. Onset temperature (To), peak temperature (Tp), andconclusion temperature (Tc) were assessed in triplicate.

2.4.6 Pasting properties

Pasting properties of starches weremeasured on a rapid viscoanalyzer (RVA-Super 3, Newport Scientific, Warriewood,NSW, Australia), using the RVA General Pasting Method 1,Version 5. Starch sample (3.5 g) was weighed directly in thealuminum RVA sample canister, and distiller water (25.0 g)was added to a total constant sample weight of 28.5 g (14%moisture basis). Temperature and time were set as follows:the sample was held at 50°C for 1 min before heating from 50to 95°C at 12°C/min, held at this temperature for 2.5 min,cooled to 50°C at 12°C/min and held at this temperature for2 min. The total test time was 13 min. The initial paddlespeed was 960 rpm and then decreased to 160 rpm for theremainder of the experiment. Pasting parameters werecalculated from the pasting curve, using the Thermoclinesoftware. All measurements were replicated three times.

2.4.7 Amylopectin molecular weight and branchchain-length distributions

Starch granule solubilization was performed followed by theprocedure described by Bello-Pérez et al. [16]. Briefly, 20 mgof starch were added into a Teflon cup containing 10 mL ofdeionized water. The cup was introduced into a polycarbonatevessel (Parr Instrument Co., Moline, IL, USA) and centeredinside a microwave oven at maximum power during 1 min(1100 Watt). Cooling step was carried out by immersion in anice bath for 30 min. The solution was filtered through a 5 mmnylon membrane (Millipore, Bedford, MA, USA) andimmediately injected onto the HPSEC column.

The HPSEC analysis combined with multi-angle laser-light scattering (MALLS) (Dawn 8þ Heleos, Wyatt Technolo-gy Corporation, CA, USA) and RI (Agilent 1100, modelG1362A, Agilent Technologies, UK) detectors were usedfor AP structural study. A Shodex OH PAK SB-807 HQanalytical column (Showa Denko K.K., Tokyo, Japan) wasused to obtain the AP MW distribution. The mobile phasewas distilled-deionized water with 0.02% sodium azide,flow rate was 0.3 mL/min. Astra software (version 5.3.4.14,Wyatt Technology Corporation, CA, USA) was used for dataanalyses.

Starch was debranched using isoamylase (Megazyme,Wicklow, Ireland), and the branch chain-length distributionwas determined using an ICS-5000 high-performance anion-

exchange chromatograph (HPAEC) equipped with a pulsedamperometric detector (PAD, Dionex, Sunnyvale, CA, USA),according to the procedure described by Jane and Chen [17],and Wong and Jane [18]. A CarboPac PA 200 anion-exchangeanalytical column (250 � 3 mm2) was used for sampleseparation. Themobile phase used for separation consisted ofeluent A (deionized water), eluent B (200 mMNaOH), eluentC (100 mM NaOH), and eluent D (150 mM NaOH and500 mM NaAc) with a flow rate of 0.5 mL/min. Duplicatemeasurements were performed.

2.5 Statistical analysis

Statgraphics Centurion XV software was used to evaluate theone-way and two-way analysis of variance (ANOVA) with 95%confidence interval to establish differences in mean values ofevaluated properties. Statistically significant differences wereevaluated based on p<0.05.

3 Results and discussion

3.1 Chemical analysis

The percentage of starch for the Alstroemeria tubers (based onsolids content) was 40.87%. The starch recovery ratio afterisolation was 86.40% with moisture content of 8.39%. Thisvalue of starch content (40.87%) is low compared to potato,70.01% [6].

Table 1 shows the main chemical composition of bothstarches. A higher moisture percentage was obtained forAlstroemeria starch (8.39%) compared to potato starch(6.17%). The moisture content of both starches was belowthe range of starch minimum deterioration [19].

As aforementioned, starch recovery ratio was 86.40%.This value was greater compared to that of potato (76.54%)evaluated in this work.

Other components, ash, protein, and lipids, were presentin starch as minor constituents (Table 1). Ash content washigher for potato (0.38%) than for Alstroemeria (0.28%). It canbe seen that Alstroemeria starch has fewer impurities and

Table 1. Chemical composition of starch from Alstroemeria andpotatoa)

Characteristics (%) Alstroemeria Potato

Moisture 8.39 � 0.25 6.17 � 0.07Starch 86.40 � 0.96 76.54 � 0.64Ash 0.28 � 0.02 0.38 � 0.03Protein 0.07 � 0.01 0.11 � 0.01Lipids 0.16 � 0.01 0.26 � 0.04AM content 29.25 � 1.21 29.75 � 1.95

a)Data were reported as means � SD (n ¼ 3).

Starch/Stärke 2013, 65, 991–998 993


they will not affect its physicochemical and functionalproperties.

Protein content for Alstroemeria (0.07%) was lower thanfor potato (0.11%) starch. For the lipids content, this washigher for potato starch (0.26%) compared to Alstroemeria(0.16%). Low protein content is important in starches that areused in glucose and fructose syrups due to that low amount ofMaillard reaction products can be produced [20–21]. For bothstarches there were significant statistical differences (p<0.05)for starch content (p ¼ 0.0001), ash (p ¼ 0.0055), moisture(p ¼ 0.0001), and protein (p ¼ 0.0029).

AM content calculation using the Megazyme kit showednot significant differences (p ¼ 0.72). AM content ofAlstroemeria starch can be considered as “normal” starch.

3.2 Morphology of starch granules

In Fig. 1, the starch morphology can be seen. The scanningelectron micrographs (SEM) of Alstroemeria and potato starchgranules are shown in Fig. 1a and Fig. 1b, respectively.

Alstroemeria starch presents composite granules, due tofusion of two or more starch granules (see arrows in Fig. 1a).Diverse shapes of starch granules were observed aselongated, round, and irregular, but all present irregularitiesin the surface. The majority of Alstroemeria starch granuleshad a size up to 25 mm. Potato starch showed two maingranules populations, with average size between 5–15 and25–40 mm. The granules presented round and oval shapes.The surface of the granules was smooth and no brokengranules were observed. Similar shape and size for potatostarch granules were reported [3, 6, 22, 23].

3.3 Granule size distribution

Figure 2 shows a representative curve of the granule sizedistribution of the starches. For Alstroemeria, diametersvalues were between 14.16 and 79.62 mm. For the granulesize distribution of potato starch, the predominant peak wascentered on particle size between 11.25 and 112.47 mm. FromFig. 2, it can be seen that granule size distribution forAlstroemeria was narrower than for potato starch. Somesmaller particles were observed in two shoulders for thepotato starch at sizes below 8.93 mm that was observed bySEM. However, it represents only 5% of the total volumeeluted.

From the obtained data, potato starch granules exhibitedthe largest granule size at 50% (d0.5) and 90% (d0.9) diametercompared to those ofAlstroemeria starch. It can also be seen inFig. 2, where the mean peak volume for Alstroemeria waslocated at lower particle size values.

Granule size distribution of starch depends on techniqueused for isolation and is an important characteristicthat can influence its functional properties and applica-tions [24–27].

Figure 1. SEMmicrographs of granular starches for (a)Alstroemeriaand (b) potato. Figure 2. Alstroemeria and potato granule size distribution.



3.4 XRD

In Fig. 3XRDpatterns of the starches can be seen. The strongestdiffraction peak was at 17° 2-u (main peak) and a few smallpeaks at around 2-u values of �14°, 19°, and 22° were observedfor both starches, indicating similar crystalline structures.

The studied Alstroemeria and potato starches can beclassified as B-type due to the peak at 2-u ¼ 5°, and that wasbigger for potato starch [28, 29]. The “d” spacing, angleintensity, and peak intensity were found to be similar for bothstarches (not shown).

The degree of crystallinity of the starches was almostsame. Potato degree of crystallinity was 27.9 � 3.3% andAlstroemeria degree of crystallinity was 27.0 � 0.2%.

3.5 Swelling properties

WAI, WSI, and SP of both starches presented a similarbehavior as can be seen in Table 2. The process ofgelatinization depends mainly on water content and tempera-ture conditions [19, 30].

The swelling properties values increased when tempera-ture increased. Alstroemeria starch at 50°C has lower WAI andSP values than potato, but when temperature increased to 60°C, Alstroemeria presented higher values. Diversity of SP canbe attributed to variation in the structure (AM and AP) of thestarches [4, 28, 31, 32].

At 70°C, it was not possible to determine experimentallytheWAI,WSI, and SP of potato starch, because a firm gel wasformed and there was not residue after centrifugation.Similar pattern was observed for Alstroemeria at 80°C.

A two-way ANOVA analysis was done taking intoaccount the starch type and temperature. For all theswelling properties, the p value was 0.00 having significantdifferences.

3.6 Gelatinization properties

Alstroemeria gelatinization properties were slightly differ-ent, and lower than that of potato starch. For Alstroemeria,the gelatinization properties were: To ¼ 58.67 � 0.6°C,Tp ¼ 62.88 � 0.3°C, and Tc ¼ 66.85 � 1.0°C. For potato,the gelatinization properties were: To ¼ 63.89 � 1.7°C,Tp ¼ 69.87 � 2.1°C, and Tc ¼ 76.32 � 4.2°C.

Gelatinization is associated with starch granuleswelling and loss of molecular order [33]. The range ofgelatinization (R) was 8.18 for Alstroemeria and 12.43for potato. A wide melting range might imply a largeamount of crystals of varying stability, whereas a narrowrange could suggest crystals of a more homogeneousquality and similar stability [34]. More structural studiesare necessary.

For the peak temperature (Tp), the p value was 0.0049having significant differences.

3.7 Pasting properties

The pasting properties of the starches measured using a RVAare shown in Table 3. The pasting properties depends ongranule size, AM content, branch chain-length distributionof AP, degree of interactions between starch and other

Figure 3. XRD diffractograms of Alstroemeria and potato starches.

Table 2. Swelling properties of Alstroemeria and potato starchesa)

Temperature (°C)

Alstroemeria Potato

WAIb) WSIc) SPd) WAIb) WSIc) SPd)

50 3.77 � 0.26 1.28 � 1.12 3.59 � 0.53 3.82 � 0.67 0.39 � 0.15 3.83 � 0.6760 8.11 � 0.42 1.71 � 0.15 8.17 � 0.41 5.45 � 0.59 0.92 � 0.13 5.47 � 0.5970 14.05 � 0.25 6.18 � 0.58 14.69 � 0.34 –

e)–e)

–e)

a)Data were reported as means � SD (n ¼ 3).b)WAI: water-absorption index (g/g).c)WSI: water-solubility index (g/g).d) SP: swelling power (g/g).e) At 70°C a firm gel was formed for potato starch.

Starch/Stärke 2013, 65, 991–998 995


components present in the starch suspension duringheating [35–37].

Pasting temperature (PT) of 71.63°C for potato washigher than that Alstroemeria starch 65.30°C. The higherpotato PT suggested that the bonding force between starchchains was stronger in this starch than in Alstroemeriastarch, retarding the swelling of the granules during theheating [30].

As aforementioned, Alstroemeria had lower PT and inTable 2, it can be seen that it showed higher SP at 60°C,indicating their ability to swell and increased viscosity atlower temperature compared to potato starch. Also, gelatini-zation temperature forAlstroemeria (Tp ¼ 62.88 � 0.3°C) waslower compared to potato (Tp ¼ 69.87 � 2.1°C).

As shown by the results of granule size distribution(Fig. 2), small granules of Alstroemeria starch had a muchgreater surface area than large granules like in potato, whichmay result in higher water absorption [38].

Potato starch showed lower breakdown value, indicatingstability of swollen starch granules at shear stress. Breakdownvalue is related to peak viscosity and to swelling of the starchgranules [4, 39]. This was the case ofAlstroemeria, which had ahigher peak viscosity (9420 cP) and breakdown (4513 cP)than potato with a peak viscosity of 7349 cP and breakdown of3495 cP.

During the setback, the temperature cooled down, theviscosity increased, and starch retrogradation or reassociationof AM molecules took place, which resulted in a gel ornetwork formation [2, 40]. Re-association was slightly lower inAlstroemeria starch than in potato as indicated by its setbackvalue.

Significant statistical differences (p<0.05) were found forPT (p ¼ 0.0000) and peak viscosity (p ¼ 0.0000).

3.8 Amylopectin molecular mass (Mw) and branchchain-length distributions

Table 4 shows the results for molar mass (Mw), gyration radii(Rz), and molecular density (r) of the starches. Alstroemeriahad higher Mw, Rz, and r compared to potato. Reportedvalues for potato were Mw ¼ 1.7 � 108; Rz ¼ 356 nm;and r ¼ 3.8 g/mol/nm3 [41], comparable with the valuesfound for the studied starches.

In Table 5 the chain length distribution of AP is shown. Itcan be seen that potato starch had the higher proportion ofintermediate/short chains (DP 13-24) and Alstroemeria starchhad the higher proportion of long chains (DP � 37). Yooet al. [42] studied the effect of chain length distribution onswelling and pasting behavior of six native tuber and rootstarches. They found that high-Mw AP molecules increasedswelling (see Table 2) as can be seen forAlstroemeria. Han andHamaker [43], and Vandeputte et al. [44], mentioned that ahigher percentage of long chains (DP � 37) preservedgelatinized granule structure due to entanglements withother APmolecules and thenmaintain granule integrity. Alsolong chains (DP � 37) increases radii of gyration of AP andwould help to maintain viscosity. Alstroemeria had high peakviscosity and breakdown of the paste, after gelatinizedgranules are disrupted. Also, long chains of AP affect peakviscosity which promotes higher swelling [45]. Intermediate/short chains would decrease paste breakdown for potato. It isimportant to note that AM content of both starches wassimilar. Potato had fewer long chains (DP � 37) compared toAlstroemeria. Therefore its Mw was smaller (1.4 � 0.02 � 108)and also its density was lower (1.8 g/mol/nm3) [5].

Table 3. Pasting properties of the starches measured by RVAa)

Viscosity (cP)

Sample Pasting temperature (°C) Peak time (min) Peak Breakdown Setback Final

Alstroemeria 65.30 � 0.05 3.13 � 0.01 9420 � 42 4513 � 98 486 � 141 5393 � 129Potato 71.63 � 0.55 3.80 � 0.07 7349 � 96 3495 � 87 539 � 61 4392 � 49

a)Data were reported as means � SD (n ¼ 3).

Table 4. Molar mass (Mw), gyration radii (Rz), and moleculardensity (r) of starchesa)

Sample Mw (�108) Rz (nm) r (g/mol/nm3)

Potato 1.4 � 0.02 296.5 � 6.5 1.8Alstroemeria 1.7 � 0.01 352.2 � 5.3 3.1

a)Data reported are the averages of duplicate analyses.

Table 5. Chain-length distribution of starch APa)

Sample

Distribution (%)

DP � 12 DP 13-24 DP 25-36 DP � 37

MaximundetectableDP

Potato 23.3 � 0.3 37.9 � 0.2 16.0 � 0.3 22.8 � 0.6 70Alstroemeria 19.6 � 0.2 30.3 � 0.4 17.1 � 0.5 32.8 � 0.4 71

a)Data reported are the averages of duplicate analyses.



4 Conclusions

A new source of starch was isolated using a simple procedure.Although morphology, AM content, and crystallinity weresimilar, Alstroemeria starch had higher starch recovery ratio,purity (lower levels of lipids, protein and ash), higher swellingproperties, and narrower granule size distribution, comparedto potato. Alstroemeria had lower gelatinization temperature.Also, it exhibited higher peak and final viscosity. Structuralstudies showed that Alstroemeria had higher molar mass(Mw), gyration radii (Rz), density (r), and higher proportion oflong amylopectin branch chains (DP � 37), compared topotato. Potato starch had higher proportion of intermediate/short chains (DP 13-24). Therefore, Alstroemeria tuber couldbe an alternative for starch isolation with specific physico-chemical and functional characteristics in applicationsrequiring purity, higher water retention, and solubility. Thissuggests a possible use of this starch as thickening agentwhere high viscosity was needed.

Wewant to express our gratitude to the Centro de Investigaciónde Polímeros Avanzados CIPA-CONICYT Regional R08C1002and CIPA-CONICYT GORE BÍO BÍO R08C1002 projectsfor the financial support. Also, the authors wish to thankDr. Roberto Rodríguez, from Universidad de Concepción Granotec,Universidad de Chile, BioMar Chile and CEPROBI-IPN for theirassistance in the experimental measurements.

The authors have declared no conflict of interest.

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