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Crossflow microfiltration for the cold stabilization of roselle (Hibiscus sabdariffa L.)extract

Mady Cisse a, Fabrice Vaillant b,c, Doudjo Soro d, Max Reynes c, Manuel Dornier c,⇑a Ecole Supérieure Polytechnique, Université Cheikh Anta Diop, B.P. 5085, Dakar Fann, Senegalb Centro de Investigación de Tecnología de Alimentos, Universidad de Costa Rica, Codigo Postal 2060, San José, Costa Ricac CIRAD, Montpellier SupAgro, UMR Qualisud, 73 rue J.F. Breton, TA B-95/16, F-34398 Montpellier Cedex 5, Franced Laboratoire des Procédés Industriels, de Synthèse et de l’Environnement (LAPISEN), Institut National Polytechnique Félix Houphouët-Boigny (INP-HB), BP 1313Yamoussoukro, Cote d’Ivoire

a r t i c l e i n f o

Article history:Received 14 December 2010Received in revised form 25 March 2011Accepted 3 April 2011Available online 8 April 2011

Keywords:MicrofiltrationSterilizationOptimizationVolumetric reduction ratio

a b s t r a c t

In this study, roselle extract was cold sterilized by crossflow microfiltration. At laboratory scale the opti-mal operating conditions at volumetric reduction ratio (VRR) 1 cannot be generalized when VRR isgreater than 1. Indeed, at VRR 1 the optimal transmembrane pressure was 2.5 bar with a permeate fluxof 185 L h�1 m�2, while at VRR 20 optimal pressure was 3.7 bar with a permeate flux of 95 L h�1 m�2. Asimple model that allowed to represent the effect of VRR and transmembrane pressure on the permeateflux was adjusted with the experiments done at laboratory scale. This model used to predict the permeateflux using a semi-industrial pilot scale equipment gave a good match between the experimental and cal-culated performances. Permeate was collected aseptically and stored at 4, 20 and 37 �C for 90 days. Aroselle extract storage at 4 and 20 �C was appropriate for consumption after a 3 month shelf life.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Hibiscus sabdariffa L. (Malvaceae) known as bissap in Senegal isan annual crop. Commonly called roselle or red sorrel, karhade, H.sabdariffa is also widely grown for its red calyx in Central, East andWest Africa, as well in southeast Asia and Central and southAmerica (Cisse et al., 2009b). In these countries, in most cases, it iscultivated with the purpose of using the calyx of their flowers toproduce a soft drink highly appreciated all over the world for theparticular sensation of freshness conveyed and its ruby color dueto its high content in anthocyanin (Wong et al., 2002; Cisse et al.,2009b; Juliani et al., 2009). In addition these extracts are also usedin folk medicine for their potential health effects (Chen et al., 2003;Chang et al., 2005, 2006). Indeed, beneficial effects for controllinghuman diseases have also been reported in the literature includingthe reduction of risks of coronary heart disease, cancer, and stroke(Wrolstad 2004a,b; Liu et al., 2006; Lin et al., 2007a,b; Lee et al.,2009). These positive physiological effects could be related to thepresence of anthocyanins with a high antioxidant activity (Prenestiet al., 2007; Juliani et al., 2009). However, the main problem of

anthocyanins is their instability in the presence of endogenous en-zymes (e.g., peroxidase, polyphenoloxidase, and b-glucosidase),oxygen, ascorbic acid, light, metallic ions (Starr 1972; Mazza andBrouillard 1987; Francis 2000; Dyrby et al., 2001; Malien-Aubertet al., 2001; Tiwari et al., 2008) and mostly heat (Kirca et al.,2006, 2007; Cisse et al., 2009c; Koca and Ustun 2009). Indeed Cisseet al. (2011) show that using high temperature during extractionand stabilization leads to a rapid decrease in red color of the prod-uct during storage.

Cold process membrane technologies represent an alternativeto high-temperature treatments. Crossflow microfiltration (CMF)has been applied successfully to some highly thermosensitivejuices, resulting in microbiologically stabilized clarified juice thatpreserves the major part of the fruit’s original aroma (Camposet al., 2002; Carneiro et al., 2002; Matta et al., 2004; Vaillantet al., 2005). The nutritional quality is also maintained. The appli-cation of CMF to roselle extract has not been reported yet. In theaqueous extract of roselle, the absence of compounds such ashemicelluloses, cellulose, tannins, proteins, starch and low contentof pectins may limit fouling during CMF.

The research described in this paper was carried out to studythe use of crossflow microfiltration for the stabilization of roselleextract in the first step. In the second part, studying the microfil-tration of roselle extract in accordance with the volumetric reduc-tion ratio (VRR) we aimed to develop a strategy that would allow afruit juice industry to produce efficiently stabilized roselle juice. A

0260-8774/$ - see front matter � 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2011.04.001

Abbreviations: AIS, alcohol-insoluble solids; CMF, crossflow microfiltration;MW, molecular weight; ORAC, oxygen radical absorbance capacity; TSS, totalsoluble solids; VRR, volumetric reduction ratio.⇑ Corresponding author. Tel.: +33 467617186.

E-mail address: dornier@cirad.fr (M. Dornier).

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simple predictive model for permeation flux decline is also used inthis work.

2. Equipment and methods

2.1. Preparation of extracts

The roselle extract was prepared with dried calyxes from aSenegal Thai variety (Cisse et al., 2009a) mixing them with deminer-alized water using a mass ratio of calyx to water of 1:15. After 3 h,the extract was then filtered successively through a stainless steelsieve (1 mm) and a bag filter system made in polyester with 5 lmof micron rating (GAF, Belgium). The dried calyxes were obtainedafter sundrying during seven days on mats directly on the floor.

2.2. Microfiltration

Two pieces of equipment were used during this study: a semi-industrial pilot scale and a laboratory scale equipments. The semi-industrial microfiltration unit IMECA (Clermont L’Hérault, France)featured a tubular ceramic membrane (Membralox� 1P19-40, PallExekia, Bazet, France), with the following attributes: 19 channelswith an internal diameter d = 4 � 10�3 m, length L = 1.02 m, aver-age pore size diameter of 0.2 lm, and a total effective filtrationarea of 0.22 m2. Temperature was controlled, using cold water cir-culating through a tubular heat exchanger. The equipment fea-tured a closed loop that was fed by a positive-displacementpump. The pump itself was regulated by a geared engine thathad an integrated frequency converter to ensure constant trans-membrane pressure (DP). This pressure was manually controlledfor the entire duration of the trials. A centrifugal pump whichwas included in the close circuit, gave a crossflow velocity ofU = 4.0 ms�1. The TIA laboratory scale equipment (Bollene, France)was featured by four identical mono-tubular membranes T1-70/250 (d = 7 � 10�3 m, L = 0.25 m) with an effective filtration areaof 0.0055 m2 each membrane and 0.2 lm pore size. The processconditions were crossflow velocity between 2 and 6 m s�1, temper-ature of 25–35 �C and applied transmembrane pressure from 1 to3.75 bar.

The trials were carried out following two classical modes: withand without concentration i.e., with or without permeate extrac-tion (recycling the permeate). According to the concentrationmode followed, without retentate extraction, the volumetricreduction ratio at time t (VRRt) was defined as the ratio betweenthe total amount of juice fed into the close circuit and the final vol-ume of retentate which remained constant throughout trials andcorresponded to the total volume of the circuit. Then, the VRRt

was calculated according to Eq. (1).

VRRt ¼ 1þ Vp

V cð1Þ

with Vp; Vc, respectively, the volume of permeate extracted at timet and the volume of the circuit. With this mode, all trials were donewith continuous feed of extract and constant collection of permeatewithout retentate extraction. The feed tank contained 60 and 3 L forthe IMECA and TIA equipment respectively. Permeation fluxes (Jp)

were determined every 10 min by measuring the volume of perme-ate collected for 1 min. At the end of the experiments, the flux resis-tances were calculated by the resistance in series model (Eq. (2)).

Rt ¼ Rm þ Rf ¼DP

l � Jpð2Þ

in which Jp is the permeate flux (m3 s�1 m�2), DP is the appliedtransmembrane pressure (Pa), l is the viscosity of the permeate(Pa s), Rt the total resistance (m�1), Rm is the intrinsic membraneresistance measured with water and Rf the fouling layer and polar-ization resistance.

2.3. Stability during storage

During the trials, using the semi-industrial pilot scale equip-ment, microfiltered extract was collected in sterilised glass bottlesusing a closed system. The bottles were stored avoiding light expo-sure in a stove (Memmert, Schwabach, Germany) at 37 �C, in a po-sitive ventilated enclosure (LIEBHERR, Germany) at 20 �C and in arefrigerator (Hoelzle & Chelius HC, Germany) at 4 �C. Samples wereanalyzed every 15 days during 30 days and every 30 days during2 months.

2.4. Physical and chemical analysis

All extracts were analyzed for pH, titratable acidity, and densityusing standard methods (AOAC, 1990). The total soluble solids(TSS) content was measured with an Abbe refractometer (Atago, Ja-pan), Vitamin C (ascorbic acid and dehydroascorbic acid) was as-sessed by high-performance liquid chromatography (HPLC)(Dhuique-Mayer et al., 2007) using an Agilent 1100 system (Massy,France). Sucrose, fructose, and glucose were determined accordingto Cataldi et al. (2000).

The total anthocyanin content was assessed by the pH differen-tial method (Lee et al., 2005). All absorbance readings weredone against distilled water, which was used as control. Spectro-photometric measurements were carried out using Shimadzuspectrophotometers (UV-1605 and UV-2450, Kyoto, Japan). Con-centrations were expressed as delphinidin 3-xylosylglucosideequivalents for roselle (MW = 577 g mol�1). The molar extinctioncoefficient at pH 1 and 510 nm, used for calculation, was26,000 L mol�1 cm (Cisse et al., 2009c). All of the reagents usedwere of analytical grade and were purchased from Sigma (L’isled’Abeau, France).

Alcohol-insoluble solids (AIS) such as pectins were determinedby dispersing a previously weighed homogenous aliquot of reten-tate into boiling ethanol (80 vol%) (Vaillant et al., 1999). Oxygenradical absorbance capacity (ORAC) assay was done in accordancewith Ou et al. (2001), using a microplate spectrofluorimeter with96-well plates made of black polypropylene (Gancel et al., 2008).The ORAC value was expressed as Trolox equivalents (mmol L�1).Total flora, yeasts, and molds, fecal and total coliforms were as-sessed using standard methods (Vanderzant and Splittstoesser1992).

Nomenclature

C0 initial concentration (g kg�1 or g L�1)Ct concentration at time t (g kg�1 or g L�1)Jp permeate flux (L h�1 m�2 or m3 s�1 m�2)R resistance (m�1)V volume of permeate or circuit (L or m3)

DP transmembrane pressure (bar or Pa)l dynamic viscosity (Pa s)gp yield of production of permeatet1/2 half-lives (d)

M. Cisse et al. / Journal of Food Engineering 106 (2011) 20–27 21

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2.5. Permeate flux modeling

Based on the thin-film theory, permeation flux ðJpÞ can be ex-pressed (Vaillant et al., 2008) by the general equation:

Jp ¼ klnVRR1VRRt

� �ð3Þ

where, VRR1 and VRRt are the volumetric reduction ratio at time1and t, k is a global mass transfer coefficient. After linearization theEq. (3) becomes:

Jp ¼ a� blnVRRt ð4Þ

with a ¼ klnðVRR1Þ ¼ JpðVRRt ¼ 1Þ and b ¼ k. The coefficient a andb were fitted using the least square method (Sigma Plot V.10).

2.6. Sensorial test

Sensorial tests used the triangular test method with a panel of20 regular consumers of sorrel drink. Two descriptors were chosen:color and smell. First, to evaluate the effect of the microfiltrationprocess on sensorial quality, the clarified extract obtained aftermicrofiltration was compared with the original extract. Secondly,to evaluate the effect of the storage temperature on sensory qual-ity, extract at 4, 20 and 37 �C were compared with each other andwith the original extract.

3. Results and discussion

3.1. Optimization of the process at laboratory scale

Two sets of tests were carried out. A first one at VRR constantequal to 1 was done followed by a second one with increasedVRR up to 20.5. For all the tests, the temperature was set at 35 �Caccording to the thermal degradation of anthocyanins (Cisseet al., 2009c). The crossflow velocity was fixed at 6 m s�1. Thus,only the effects of transmembrane pressure on permeate flux hasbeen studied.

Fig. 1 shows some examples of permeate flux at VRR 1 as func-tion of processing time where we observed a typical behavior of acrossflow microfiltration process curve. Up to 40 min, an acute de-cay of the permeate flux was noted, followed by a period whereflux stabilization occured. The sharp initial flux decline was dueto the formation of a fouling layer mainly caused by the alcohol-insoluble solids (Table 1) in our case and the concentration polar-ization. Similar results were observed during microfiltration of

many juices, drinks, aqueous solutions, water (Campos et al.,2002; Vaillant et al., 2008; Castaing et al., 2010; Ebrahimi et al.,2010). As shown in Fig. 2 (VRR = 1), by increasing the transmem-brane pressures (DP), the permeate flux increases significantly upto DP values of 2.5 bar. Between 1.2 and 2.5 bar the permeate fluxincreases by 35% against 5% when the transmembrane pressureranges from 2.5 to 3.7 bar. Permeate flux ranges between 136and 193 L h�1 m�2. At the same time, the total resistance (Rt) in-creases with the transmembrane pressure (Fig. 3). It’s clear thatthe intrinsic resistance of the membrane (Rm) was insignificantcompared to the resistance due to fouling (Rf) which represents93% of total resistance (Rt). The same distribution was reportedduring the microfiltration of different juice or model solutions(Ushikubo et al., 2006, 2007; Buetehorn et al., 2010; Mirsaeedghaziet al., 2010; Yao et al., 2010).

Permeate flux varies slightly in the range of the transmembranepressures tested. It is unlikely that the increasing of the foulingresistance is linked to an increase in concentration polarization.It would therefore be related to the increase of the hydraulic resis-tance of the external fouling that may be compressible.

These initial trials show that crossflow microfiltration of roselleextract at VRR 1 can be carried out at transmembrane pressure of2.5 bar with higher permeate flux around 185 L h�1 m�2.

Fig. 1. Permeate flux during the crossflow microfiltration of roselle extract usingthe laboratory scale equipment at VRR 1 and different transmembrane pressures(T = 35 ± 2 �C, U = 6 m s�1), (error bars represent standard deviation for n = 3).

Table 1Characteristics of roselle extracts – original, and microfiltered using the semi-industrial pilot scale equipment; numbers in parentheses indicate the standarddeviation of three replicates (T = 35 ± 2 �C, U = 4 m s�1, 1 < VRR < 4, DP = 1.50 bar).

Parameters Roselle extractoriginal

Roselle extractmicrofiltered

pH 2.25 (0.05) 2.30Total soluble solids (TSS)

(g kg�1)45 (2) 45 (2)

Titratable acidity (g malicacid kg�1)

185 (9) 183 (7)

Vitamin C (g kg TSS�1) 10 (0.6) 9.5 (0.2)Sucrose (g kg TSS�1) 3.1 (0.2) 3.0 (0.1)Fructose (g kg TSS�1) 17.5 (1.1) 17.0 (1.0)Glucose (g kg TSS�1) 23.4 (1.3) 22.8 (1.5)Anthocyanins (mg L�1) 254 (10) 250 (12)ORAC (lmol Trolox g�1) 166 (1.3) 151 (1.1)Alcohol-insoluble solids

(g kg TSS�1)21 (1.2) 2.5 (0.2)

Total flora (CFU mL�1) 2.103 <30Yeast and molds (CFU mL�1) 1.102 <30Fecal coliforms (CFU mL�1) 0 0Total coliforms (CFU mL�1) 0 0

Fig. 2. Permeate flux versus transmembrane pressure during the crossflowmicrofiltration of roselle extract using the laboratory scale equipment at differentvolumetric reduction ratios (VRR) (T = 35 ± 2 �C, U = 6 m s�1).

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To illustrate the influence of the VRR on permeate flux, cross-flow microfiltration was carried out with a roselle extract havingthe similar characteristics than the extract used at VRR 1 (Table 1).In that case the laboratory scale equipment of microfiltration wasconstantly fed with roselle extract to keep the hold-up volumeconstant (3 L) while permeate was recovered. After 17 L of roselleextract, feeding was stopped. As shown in Fig. 4, by increasingthe VRR, the permeate flux decreased whatever the applied trans-membrane pressure. However, for a high VRR value, permeate fluxremains proportional to the transmembrane pressure. Using Fig. 4we can predict permeate flux as a function of transmembrane pres-sure and volumetric reduction ratio, during crossflow microfiltra-tion of roselle extract. For example, at 2.1 bar of transmembranepressure, the permeate flux is at least of 50 L h�1 m�2. For indus-trial purposes, the VRR is directly related to the yield of the oper-ation (Eq. (5)) and is an essential indicator of the microfiltrationviability. For each product we can determine the critical valuesof VRR and permeate flux. In our case at VRR 20, the maximum per-meate flux is 95 L h�1 m�2 to a transmembrane pressure of 3.7 barwith a yield of 95%. This pressure is significantly different from thatfound during microfiltration at VRR 1. With a VRR of 20 permeateflux is 1.5 times lower at 2.5 bar of transmembrane pressure thanat 3.7 bar. Moreover, the time required to reach VRR 20 remainsunchanged between 2.5 and 3.7 bar of transmembrane pressure.

It’s of 370 min. With 1.5 and 2.0 bar, the time required to reachVRR 20 is, respectively 480 and 390 min.

gp ¼ 100� 1� 1VRR

� �ð5Þ

where gp is the yield of production of permeate (%) and VRR the vol-umetric reduction ratio.

As in trials at VRR 1, the total resistance (Rt) increases withtransmembrane pressure during the tests at increasing VRR. Fur-thermore, it logically increased with the volumetric reduction ratio(Fig. 5) that is correlated to the increase of the fouling power of theproduct when it is concentrated.

According to these results, the parameter optimization of thecrossflow microfiltration at VRR 1 does not seem to be the bestway to choose the value of parameters for industrial purpose.The hypothesis considering that the values of optimum parametersof crossflow microfiltration are both the same at constant andincreasing VRR, is clearly not validated in our case.

3.2. Permeate flux modeling

In order to predict permeate flux during crossflow microfiltra-tion, the model based on the thin-film theory is used accordingto Eq. (4). As shown in Fig. 6 experimental data matched the modelwell.

To compare the accuracy of the model in predicting experimen-tal permeate flux values it was carried out at two transmembranepressures 1.5 and 3.0 bar. Experimental permeate flux were thencompared with calculated values (Table 2). In all cases, the modelgave similar results, with estimates being close to the experimentalvalues. The model accuracy was thus verified, and the approachfollowed in this study was validated. Using the thin-film theorywith linear regression coefficient between 0.994 and 0.980, Fig. 7gave k and VRR1 values according to the transmembrane pressure.Up to 2.5 bar of transmembrane pressure, value of k is almost con-stant while VRR1 increases with pressure. From 2.5 to 3.7 bar, kdecreases. It therefore seems that the fouling particles induce lim-iting permeation flux, independent on pressure when of the gel orcake layer reach equilibrium. The gel or cake layer built up from ro-selle extract appears squeezable and is subject to compression oreven flocculation.

3.3. Validation at a semi-industrial scale

Based on previous results, further trials of crossflow microfiltra-tion were carried out using semi-industrial pilot scale equipment.

Fig. 3. Hydraulic resistances versus transmembrane pressure during crossflowmicrofiltration of roselle extract using the laboratory scale equipment (T = 35 ± 2 �C,U = 6 m s�1) (error bars represent standard deviation for n = 3).

Fig. 4. Permeate flux versus volumetric reduction ratio (VRR) during the crossflowmicrofiltration of roselle extract using the laboratory scale equipment for selectedvalues of transmembrane pressure (T = 35 ± 2 �C, U = 6 m s�1), (error bars representstandard deviation for n = 3).

Fig. 5. Total résistance versus transmembrane pressure at different volumetricreduction ratio (VRR) during crossflow microfiltration of roselle extract using thelaboratory scale equipment (T = 35 ± 2 �C, U = 6 m s�1) (error bars represent stan-dard deviation for n = 3).

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So, the model established with the laboratory scale equipment wasused to predict the permeate flux as a function of the volumetricreduction ratio. Fig. 8 shows the variation of permeate flux duringa microfiltration trial of the roselle extract in order to produce acommercial sterile extract. The behavior of the permeate flux couldbe separated into two steps. First there was a decay of the perme-ate flux followed by a period when the decrease was less impor-tant, tending to stabilization. Similar behavior of permeate fluxhas been reported for different juices (Carneiro et al., 2002;Ushikubo et al., 2007). By increasing the VRR, the permeate flux de-

creased to below 95 L h�1 m�2 when the VRR reach 3. Interestingly,a VRR value of 3 was seen when the permeate flux remained stable.These results are consistent with those obtained with the labora-tory scale equipment. According to these results, the costs of pro-ducing commercial sterile roselle extract would be competitiveand would have a higher production yield. Calculated permeateflux using the model were close to those obtained experimentallybut greater than 9–15%. This difference could be related to thehydrodynamic conditions that were different in both installations(tube diameter, mono or multichannel configuration).

Therefore, model developing with the laboratory scale equip-ment can be used to validly predict the permeate flux duringsemi-industrial trials at transmembrane pressure ranging between1.2 and 3.7 bar and at increasing VRR at least up to 3.5.

3.4. Cold sterilization of roselle extract by crossflow microfiltration

Table 1 presents the results of physical–chemical and micro-biological analysis of the original extract and the extract aftercrossflow microfiltration. Except vitamin C, alcohol-insoluble solids(AIS) such as pectins and total flora, all parameters have very closevalues. The vitamin C content of the clarified extract is reducedonly by 5%. This loss may have been due to oxygen exposure andcould have been reduced by implementing a degassing treatmentbefore microfiltration. The antioxidant capacity decreased alsoslightly from 166 to 151 lmol Trolox g�1. As the content of antho-cyanin remains constant; the slight reduction of antioxidant capac-ity may be due to the vitamin C loss. The pectin content wasreduced by 90% after CFM. Microbiological analyses of the perme-ate extract showed that this product does not contain any yeast ormold, fecal coliforms nor total coliforms. The total flora was under30 CFU mL�1. The microorganisms were retained by the mem-brane. These parameters were maintained during the storageperiod of 90 days, at temperature conditions of either 4, 20 and37 �C. The low pH also contributes to the microbiological stabilityof the product. In fact during the storage time pH and titratableacidity of microfiltered extract did not change with storagetemperature at all. However anthocyanins and vitamin C contentdecrease regularly (Fig. 9). After 90 days at 37 �C losses of 68% and52% were respectively observed for vitamin C and anthocyanins.While at 4 and 20 �C losses in anthocyanins are, respectively of15% and 30 % after 90 days of storage.

Anthocyanin and vitamin C contents decreased during storageaccording to time and temperature for all the storage tempera-tures. Linear regression confirmed that the degradation of thesenutritional compounds in the roselle extract during storage can

Fig. 6. Permeate flux versus logarithm of volumetric reduction ratio (VRR) duringthe crossflow microfiltration of roselle extract using the laboratory scale equipmentat different transmembrane pressures (T = 35 ± 2 �C, U = 6 m s�1) (error bars repre-sent standard deviation for n = 3).

Table 2Comparison between experimental values of permeate flux (L h�1 m�2) obtainedusing the semi-industrial pilot scale equipment, and those calculated using the model(Eq. (3)) (T = 35 ± 2 �C, U = 6 m.s�1, 1 < VRR < 21, 1 < DP < 4 bar).

VRR 1.5 bar 3.0 bar

Experimental Model Experimental Model

1 162 161 188 1895 95 92 138 13110 66 62 106 10715 46 44 90 9320 38 32 79 82

Fig. 7. Model parameters values (Eq. (3)) versus transmembrane pressure forcrossflow microfiltration of roselle extract using the laboratory scale equipment(T = 35 ± 2 �C, U = 6 m s�1) (error bars represent standard deviation for n = 3).

Fig. 8. Calculated and experimental permeate flux versus volumetric reductionratio (VRR) during crossflow microfiltration of roselle extract with the semi-industrial pilot (DP = 1.50 bar, U = 4 m s�1, T = 35 ± 2 �C).

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be fitted by a first-order reaction model. So, the first-order reactionrate constants (k), half-lives (t1/2) and decimal reduction time (D)were calculated by the following equations and their values are gi-ven in Table 3.

lnCt

C0

� �¼ �k � t ð6Þ

t12¼ � lnð0:5Þ � k�1 ð7Þ

Whatever the storage temperature is, it has a strong influenceon the degradation of anthocyanins and vitamin C. In fact, storageat 37 �C resulted in a much faster degradation compared withrefrigerated storage at 4 �C. For example, with vitamin C, reactionrate k ranges from 5.787 to 13.888 � 10�8 s�1 to respective storagetemperatures of 4 and 37 �C . The t1/2 values of roselle anthocya-nins were 198 days at 4 �C, 115 days at 20 �C and only 77 days at37 �C. Vitamin C appears to be more susceptible to degradationthan the anthocyanins during storage. Indeed at 20 �C, the half-lifeof vitamin C was of 87 days against 115 days for anthocyanins withreaction rate k of 9.259 and 4.630 10�8 s�1, respectively. Isother-mal kinetic parameters determined according to (Cisse et al.,2009c) using the two Arrhenius and Ball models are presented inTable 4. All models were observed to properly match the tempera-ture dependence of k and D (0.990 < R2 < 0.999). The activationenergies were 18.9 and 20.4 kJ mol�1, respectively, for vitamin Cand anthocyanin while value of D0 were, respectively, of 4.36 and6.20 � 107 s. These results confirm the greater sensitivity to thedegradation of vitamin C compared to anthocyanin during storageat temperatures between 4 and 37 �C. Compared to other work(Cisse et al., 2011) anthocyanins of H. sabdariffa appears to havegreater stability after microfiltration. In fact, extracts obtained at30 �C or 100 �C and then stabilized by pasteurization at 90 �C for5 min shows values of D0 two times smaller and a z factor valueof 67 �C against 83 �C for the anthocyanin in microfiltered roselleextract.

However, these differences, particularly in terms of color, werenot verified by visual observation according to sensorial tests usingthe triangular test method with a panel of 20 regular consumers ofsorrel drink. Indeed, according to the sensorial tests, no significantdifferences (at 95% confidence level) could be noticed between theextract at 4 �C and the extract at 20 �C. Compared to a sample pre-pared in situ (original extract) and have not undergone the micro-filtration treatment, the two extracts at 4 and 20 �C show nosignificant differences according to color and smell with the origi-nal extract in accordance with 95% of panelists. The crossflowmicrofiltration process used had no significant effect on the senso-rial quality of the extract. On the other hand, all the tasters agreedthat the extract stored at 37 �C has a significant difference in colorcompared to other extracts. The comparison between the visibleabsorption spectra of the extracts corroborated the results of sen-sorial evaluation of the color. Between 400 and 700 nm, spectra ofthe microfiltrated extracts stored 90 days at 4 or 20 �C were veryclose to the spectra of the initial extract (Fig. 10). On the contrary,the spectra of the extract microfiltered and stored at 37 �C wascompletely different. At this temperature, storage strongly affectedthe color of the extract.

These results show the potential of crossflow microfiltration tosterilize roselle extract and to keep at an acceptable level for nutri-tional and sensory qualities of the product. Nevertheless, the stor-age temperature must not exceed 20 �C for a good preservation ofthe color during 3 months.

Fig. 9. Evolution of the contents of anthocyanin (1) and vitamin C (2) of themicrofiltered extract during the storage at different temperatures.

Table 3Effect of temperature on k, t1/2, D values for anthocyanin and vitamin C degradation inmicrofiltered roselle extract obtained using the semi-industrial pilot scale equipment(T = 35 ± 2 �C, U = 4 m s�1, 1 < VRR < 4, DP = 1.50 bar), numbers in parentheses indi-cate the standard deviation of three replicates.

Compounds Temperature (�C) k � 10�8 (s�1) t1/2 (day) D (days)

Anthocyanin 4 1.157 (0.002) 198 65820 4.630 (0.004) 115 38437 9.260 (0.005) 77 256

Vitamin C 4 5.787 (0.002) 139 46120 9.259 (0.003) 87 28837 13.888 (0.008) 58 192

Table 4Kinetic parameters for the anthocyanin and vitamin C degradation during storage based on Arrhenius and Ball models (Cisse et al., 2009c), numbers in parentheses indicate thestandard deviation of three replicates.

Compounds Arrhenius model Ball model

k1 � 10�4 (s�1) Ea (kJ mol�1) R2 D0 � 107 (s) z (�C) R2

Anthocyanin 2.97 (0.02) 20.4 (1.2) 0.995 6.20 (0.25) 83 (2) 0.990Vitamin C 2.18 (0.002) 18.9 (1.3) 0.999 4.36 (0.12) 91 (1) 0.996

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4. Conclusions

Crossflow microfiltration has a great potential to stabilize ro-selle extract. The stabilized extract presents physico-chemical,nutritional and sensorial properties that are comparable with thefresh roselle extract. The cold pasteurized product is stable whenstored at 4 and 20 �C during 90 days. Roselle extract remained agood source of anthocyanins with microbiological safety require-ments. Also, an acceptable level for nutritional and sensory quali-ties of the product was preserved. Crossflow microfiltration ofroselle extract makes it possible to reach a high average perme-ation permeate flux of about 95 L h�1 m�2 at VRR = 20 and givinga global yield of 95%. Transmembrane pressure presents a high ef-fect in permeate flux and total resistance that is composed by theresistance due to fouling at 93%. Optimizing transmembrane pres-sure at VRR = 1 and at higher VRR did not give the same optimumvalues. So, it seems necessary to do the optimization of variousparameters of microfiltration at increasing VRR. Especially inindustry, the VRR is directly related to the yield of the operationand is an essential indicator of the microfiltration viability. Thepredictive model for permeation flux developed gives good corre-lation between the experimental flux of permeate and those calcu-lated with the model.

Acknowledgements

The authors wish to thank the Agence Universitaire de la Fran-cophonie (AUF) and CIRAD for its financial support.

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