SG

Ra

Cb

D

a

ARRA

KSFEA

1

itofeafcoisehcpeo

y

0h

Industrial Crops and Products 51 (2013) 163– 170

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h om epage: www.elsev ier .com/ locate / indcrop

eparation of total saponins from the pericarp of Sapindus mukorossiaerten. by foam fractionation

ui Lia, Zhao Liang Wua,∗, Yan Ji Wangb,∗∗, Ling Ling Lia

School of Chemical Engineering and Technology, Hebei University of Technology, No. 8 Guangrong Road, Dingzi Gu, Hongqiao District, , Tianjin 300130,hinaKey Lab of Green Chemical Technology & High Efficient Energy Saving, Hebei University of Technology, No. 8 Guangrong Road, Dingzi Gu, Hongqiaoistrict, Tianjin 300130, China

r t i c l e i n f o

rticle history:eceived 31 May 2013eceived in revised form 30 July 2013ccepted 30 August 2013

eywords:

a b s t r a c t

Sapindus mukorossi Gaerten., as a rich source of saponins, is an important agricultural economic tree intropical and subtropical regions. Its fruit pericarp has a high content of triterpenoid saponins of highsurface activity and important biological activities. Thus the current work adopted a two-stage foamfractionation technology to separate the saponins from the pericarp. A spiral internal component andelevated temperature were utilized to improve enrichment ratio. Using this technology, the enrichment

apindus saponinsoam fractionationnrichment rationtimicrobial activity

ratio of the sapindus saponins reached 133.4 with a recovery of over 36.4% and the separated saponinshad a high purity of 90.3%. The product was analyzed by FTIR and HPLC–MS to determine its ingredients,including plentiful triterpenoid saponins and bits of sesquiterpene glucosides. The subsequent bioactivityanalysis made sure that the product had moderate but long-term antimicrobial activity. Therefore thecurrent work had industrial implication in producing high-purity saponins for food, cosmetics and evenpharmaceutical fields.

. Introduction

Sapindus mukorossi Gaerten., a handsome deciduous tree of fam-ly Sapindaceae, is an important economic agricultural plant inropical and subtropical regions of Asia. Its fruit is a rich sourcef natural products of medicinal importance and it is also exploitedor widespread use in many other fields (Verma et al., 2013; Reddyt al., 2013; Dobhal et al., 2007). The fruit pericarp is widely useds a natural detergent and its extract is commercially utilized as aoam stabilizing and emulsifying agent in cleansers, shampoos andosmetics. The reason is that the pericarp is abundant in saponinsf high surface activity (Yang et al., 2010). The major saponinsn the pericarp are triterpenoid-type and their structures are pre-ented in Table 1 (Sharma et al., 2013; Huang et al., 2003; Nakayamat al., 1986). Previous work has found that the sapindus saponinsave molluscicidal effects against pomacea canaliculata, moderateytotoxicity against human tumor cells, hemolysis effect and high

reservative efficacy (Verma et al., 2013; Saha et al., 2010; Chent al., 2010). Thus they will also have a bright prospect in the fieldsf insecticides, medicine and food.

∗ Corresponding author. Tel.: +86 222656 4304; fax: +86 222656 4304.∗∗ Corresponding author. Tel.: +86 222656 4061; fax: +86 222656 4061.

E-mail addresses: zhaoliangwu hebut@163.com (Z.L. Wu),jwang@hebut.edu.cn (Y.J. Wang).

926-6690/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rittp://dx.doi.org/10.1016/j.indcrop.2013.08.079

Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

The reported separation technology of saponins consists ofcrude leaching and further purification. In the leaching process,the commonly utilized solvents include water, methanol, ethanol,etc. Roy et al. (1997) found that the leaching percentage of thesapindus saponins using water was higher than that of using theorganic solvents. These organic solvents are more expensive thanwater and unfriendly to the environment. Moreover their recov-ery needs complicated procedures and this will increase the cost interms of large-scale saponin production. So water will be selectedin the current work as the leaching solvent. Techniques for purify-ing saponins from the crude leaching liquor include ultrafiltration,column chromatography, foam fractionation and high performanceliquid chromatography (HPLC) (Yan et al., 2011; Saha et al., 2010;Nie et al., 2009; Wei et al., 2008). Among these techniques, foamfractionation is more desirable because of its low cost, low energyconsumption and free pollution (Khalesi et al., 2013; Vivek andRamkrishna, 2013). Separation of saponins from the crude leachingliquor using this technique has great potential to be industrialized.

The crude leaching liquor from the pericarp of S. mukorossiGaerten. has complex ingredients such as saponins, saccharides,proteins and other unknown ones. Among them, saponins havethe highest content and the highest surface activity, so it is fea-

sible for foam fractionation to separate the saponins. However,the product will not be satisfactorily pure if only single stage offoam fractionation is adopted. Yan et al. (2011) developed a two-stage foam fractionation technology for separating tea saponins

ghts reserved.

164 R. Li et al. / Industrial Crops and Products 51 (2013) 163– 170

Table 1Triterpenoid saponins from the pericarp of S. mukorossi Gaerten.

Formula/molecular weight Sapogenin R1 R2

C58H94O26/1206 Ara (2→1) Rha (3→1) Ara Glc (2→1) GlcC59H92O25/1200 Rha (3→1) Ara-2-O-Ac Glc (2→1) Glc-2-O-AcC53H86O22/1074 Ara (2→1) Rha Glc (2→1) Glc

C50H78O18/966Ara (2→1) Rha (3→1) Ara-2,4-O-di-Ac HAra (2→1) Rha (3→1) Ara-3,4-O-di-Ac HAra (2→1) Rha (3→1) Xyl-3,4-O-di-Ac H

C48H76O17/924Ara (2→1) Rha (3→1) Ara-3-O-Ac HAra (2→1) Rha (3→1) Ara-4-O-Ac H

C46H74O15/866 Ara (2→1) Rha (3→1) Ara HC H O /750 Ara (2→1) Rha H

fiteffw

itEttBsc(rfaDd(reGhc

41 66 12

rom their crude leaching liquor. In practice, the saponin purityn their work is considered not to be greatly improved because ofhe relatively high initial saponin concentration (2.08 g/L) and lownrichment ratio (3.47). Then a different type of two-stage foamractionation where the foamate of the first stage is served as theeeding solution of the second stage will be adopted in the currentork to improve the saponin purity.

The saponin purity is closely related to its enrichment ratio. Its generally acknowledged that enrichment ratio is determined bywo essential processes: interfacial adsorption and foam drainage.nhancement of interfacial adsorption is obtainable by increasinghe bubble residence time in the liquid phase, adjusting the solu-ion properties and adding a reflux device (Martin et al., 2010;hattacharjee et al., 1997; Weijenberg et al., 1978). However theurface excess will not greatly increase when the surfactant con-entration increases to or over its critical micelle concentrationCMC). In this case, foam drainage will play a more importantole in improving enrichment ratio. Effective attempts to enhanceoam drainage include design of a new column and insertion ofn internal component into a conventional column (Li et al., 2011;ickinson et al., 2010). In the present work, a novel componentesigned by Yang et al. (2011), the spiral internal componentSIC) shown in Fig. 1, will be adopted to increase enrichmentatio. Elevating temperature is also an effective method to increase

nrichment ratio (Jiang et al., 2011; Kumpabooth et al., 1999;rieves and Dhattacharyya, 1964.). Accordingly it will be adoptederein to achieve a high enrichment ratio at a high saponin con-entration.

Fig. 1. Schematic diagram of the foam fractionation column with SIC.

In this work, a two-stage foam fractionation technology with SICand different temperatures will be adopted to obtain high-puritysapindus saponins. For simplifying the technology, the naturalpH was selected in all the foam fractionation experiments. Thesaponins will be measured by a modified vanillin-perchloric acidcolorimetry. Then the saponins obtained by this technology willbe analyzed by Fourier transform infrared spectrometer (FTIR)and HPLC-mass spectrometer (MS) to determine their ingredients.Finally the antimicrobial activity of the saponins will be analyzed.All the attempts are aimed at providing a cost-effective method forobtaining the sapindus saponins of high quality.

2. Materials and methods

2.1. Plant material

The pericarp powder of S. mukorossi Gaerten. was purchasedfrom Fujian Yuanhua Forestry Biological Technology Co., Ltd., Chinaand its saponin content was about 42.5 mg/g.

2.2. Preparation and preprocessing of the leaching liquor

The pericarp powder was triply leached with tap water of 55 ◦Cat a ratio of 100 g/1000 mL, and each leaching time was 3 h. Theleaching percentage of the sapindus saponins reached 75.1% andthey had a low purity of 21.1% in the crude leaching liquor. Aflocculant (1% (wt%) chitosan–5% (v/v) acetic acid solution) wasadded into the liquor at a ratio of 2% (v/v) to improve the total-saponin purity. The flocculating time was 8 h and the precipitatewas removed by centrifugation at 5000 rpm. Finally the prepro-cessed leaching liquor of 40.7% in the total-saponin purity wasobtained and stored at 4 ◦C for use.

2.3. Separation of the total saponins from their leaching liquorusing foam fractionation

Fig. 1 presents a schematic diagram of the foam fractionationcolumn. The foam fractionation column consisted of a trans-parent plexiglass tube which had 50 mm in inner diameter and1200 mm in height. A spiral internal component (SIC) of 50 mmin thread pitch was installed into it. The column was tightly twinedby a silicone tube connected to a 501 ultrathermostat (Shang-hai Experimental Instrument Factory Co. Ltd., China) to controlthe temperature inside the column. The temperature was mon-itored by a thermometer attached to the top of the column. Allthe foam fractionation experiments were carried out in batchmodel.

A two-stage foam fractionation technology was adopted topurify the sapindus saponins from their leaching liquor and thefoam fractionation column in Fig. 1 was used in both the two stages.Its schematic diagram is presented in Fig. 2 and the dotted lines

R. Li et al. / Industrial Crops and Products 51 (2013) 163– 170 165

o-sta

irfop

a(

E

R

�

wsots

2

2

t1pDoIU

Fig. 2. Schematic diagram of the tw

ndicate a modification for this technology which would be elabo-ated in the following. The key point of this technology was that theoamate of the first stage would serve as the feed solution of the sec-nd stage which would be further enriched. In this case the highlyurified saponins would be obtainable under suitable conditions.

The parameters for evaluating the foam fractionation perform-nces were defined as enrichment ratio (E), recovery percentageR) and total-saponin purity (�f).

= Cf

C0(1)

= Vf Cf

V0C0× 100% (2)

f = mfss

mf× 100% (3)

here C0 and Cf are the total-saponin concentrations in the feedolution and the foamate, respectively; V0 and Vf are the volumesf the feed solution and the foamate, respectively; mfss and mf arehe masses of the total saponins and the total solid content in theampled foamate, respectively.

.4. Analytical methods

.4.1. Measurement of the total-saponin concentrationThe concentration of the sapindus saponins was measured by

he vanillin-perchloric acid colorimetry (Yan et al., 2011; Hiai et al.,976). The standard sapindus saponins cannot be purchased atresent, so oleanolic acid (purity ≥ 98%, Xinan Xiaocao Botanic

evelopment Co. Ltd., China), structurally similar to the sapogeninf the sapindus saponins, was used as the standard reference.ts maximum absorption wavelength was 550 nm determined by3010-UV spectrophotometer (Hitachi Corp., Japan).

ge foam fractionation technology.

The measurement procedures are as follows. (1) A suitable vol-ume (v) of the oleanolic acid (or the sapindus saponins) solutionwas sampled into a 50 mL triangular flask and dried; (2) 0.4 mL 5%(wt%) vanillin–glacial acetic acid solution and 1.4 mL perchloric acidwere added into the flask; (3) The flask was shaken and heated inwater bath of 70 ◦C for 15 min, and then placed in ice bath; (4) 10 mLglacial acetic acid was added into the flask after 3 min ice bath andfinally the absorbance was measured. The standard equation wasA = 0.00394m + 0.0268, R = 0.99934, where A is the absorbance, m isthe sample mass ranging from 50 �g to 200 �g and R is the linearcorrelation. Thus the sample concentration in a solution (C) wasequal to m/v and it was an oleanolic acid equivalent.

2.4.2. Ingredient analysisFTIR spectra were measured on a Vector-22 spectrophotome-

ter (Brucker, Germany) using a KBr matrix. HPLC–MS (LCQ DecaXP MAX, Thermo Fisher, USA) and HPLC (G1311A, Agilent Tech-nologies) were used to analyze the ingredients of the foamfractionation product. HPLC purification of the product was madeon a SymmetryTM C18 column (250 mm × 4.6 mm, 5 �m, Shimadzu,Japan) with acetonitrile/deionized water (0–30 min, 2:3; ∼30 min,3:2) at a flow rate of 0.2 mL/min and 40 ◦C. The total saponins weremeasured by a UV-detector at the maximal absorption wavelengthof 210 nm. Then each ingredient was ionized in the electrosprayionization (ESI) and operated in positive mode for MS analysis. TheMS scanning range was m/z = 700–1500.

2.4.3. Antibacterial activity analysisAntibacterial activity of the foam fractionation product was

determined by a modified agar diffusion assay. Micrococcusflavus NCIB 8166 (Microbial Culture Collection Center by Chinese

Academy of Sciences) was used as the indicator strain and nisin(1000 IU/mg, Tianjin Kangyi Bioengineering Co., Ltd., China), a high-efficiency antibacterial polypeptide, was used as a reference. Themedium and the experimental procedures had been specified by

166 R. Li et al. / Industrial Crops and P

Table 2Effects of SIC on enrichment ratio and recovery percentage at initial total-saponinconcentrations of 0.10 g/L and 2.00 g/L.

0.10 g/L 2.00 g/L

E R E R

WhwDt

a

3

3t

3

pegfct2tw0p

ee2(qat(

With SIC 27.5 ± 1.3 16.5 ± 0.7% 1.53 ± 0.5 80.8 ± 3.8%Without SIC 12.3 ± 0.5 36.9 ± 1.5% 1.21 ± 0.4 90.1 ± 4.1%

u et al. (2009). The differences from their work were that only oneole was bored into each agar plate and both nisin and the productere dissolved in distilled water with a concentration of 0.3 g/L.iameter of each inhibition zone was measured as an equivalent of

he antibacterial activity.All the experiments were triply repeated and all the data were

nalyzed using SPSS software.

. Results and discussion

.1. Two-stage foam fractionation technology for separating theotal saponins

.1.1. Improvement of enrichment ratio with SICYang et al. (2011) and Li et al. (2012) demonstrated that SIC

layed an important role in improving enrichment ratio, so itsffects on foam fractionation of the total saponins were investi-ated at two initial concentrations of 0.100 g/L and 2.00 g/L. Theoam fractionation column without SIC was used as a contrastedolumn and the experiments were carried out under the condi-ions of liquid loading volume 500 mL, volumetric gas velocity00 mL/min, room temperature (25 ◦C) and natural pH. Note thathe natural pH value of the saponin-containing solution changedith the total-saponin concentration and herein pH = 7.0 ± 0.1 at

.100 g/L and pH = 6.3 ± 0.2 at 2.00 g/L. The gas distributor had aore diameter of 400 �m and the results are presented in Table 2.

Table 2 presents that SIC effectively improved the total-saponinnrichment ratio but decreased the recovery percentage, and thenrichment ratio increased by 123.6% at 0.100 g/L and 26.4% at.00 g/L with SIC. The results consisted with those of Yang et al.2011). The SIC allowed the liquid draining from the rising foam to

uickly return the bulk liquid, so it could enhance foam drainagend improve enrichment ratio. When viscosity increased with theotal-saponin concentration, the velocity for the interstitial liquidliquid between bubbles) flowing onto the SIC walls decreased. As

Fig. 3. Effects of the initial total-saponi

roducts 51 (2013) 163– 170

a result, the ability of SIC to enhance foam drainage was weakened.Therefore the enrichment ratio at 2.00 g/L was less effectivelyimproved by SIC. In a word, SIC would be used in the follow-ing two-stage foam fractionation to improve the total-saponinenrichment ratio.

3.1.2. Optimization of the initial concentration for the first stagefoam fractionation

Effects of the initial total-saponin concentration were investi-gated to select a suitable initial concentration for the two-stagefoam fractionation technology aimed at obtaining high-puritysaponins. The initial total-saponin concentration ranged from0.10 g/L to 2.00 g/L, corresponding to the pH values from 7.0 ± 0.1to 6.3 ± 0.2. The other conditions were the same as those describedin the above section and the results are presented in Fig. 3.

From Fig. 3, the enrichment ratio decreased from 27.5 to 1.53while the recovery percentage increased from 16.5% to 80.8%with increasing the initial total-saponin concentration. This trendconsisted with the work of Yang et al. (2011). It was explained bythat the liquid holdup in the foam increased because the increasein the initial concentration increased the solution viscosity andfoam stability. Fig. 3 also illustrated that the total-saponin puritydecreased from 82.8% to 42.3% while their foamate concentra-tion decreased from 2.75 g/L to 2.49 g/L and then increased to3.06 g/L. The major surface-active substances in the feeding solu-tion were saponins, so their purity had the same increasing trendas their enrichment ratio. The high foamate concentration could beattributed to a great enrichment ratio or a high initial concentration,but the low enrichment ratio made the latter meaningless. All theabove considered, 0.10 g/L was determined as the suitable initialconcentration where the highest enrichment ratio was obtainable.

3.1.3. Optimization of the first stage foam fractionation usingorthogonal experiments

With initial total-saponin concentration and pH fixed at 0.10 g/Land 7.0 ± 0.1 (natural pH), liquid loading volume, volumetric gasvelocity and pore diameter of the gas distributor (or bubblesize) were optimized to obtain satisfactory foam fractionationperformances. Effects of the three factors on foam fractionationwere well known by researchers (Liu et al., 2013; Rujirawanich

et al., 2013; Stevenson, 2007), so this work just optimized themusing orthogonal experiments on the foundation of preliminaryexperiments not presented herein. The levels of the three factorsand the orthogonal results are presented in Table 3 and Table 4,

n concentration on E, R, �f and Cf .

R. Li et al. / Industrial Crops and P

Table 3Factors and levels of the orthogonal experiments for the first stage foamfractionation.

Factor Level

1 2 3

A, liquid loading volume (mL) 500 700 900B, volumetric gas velocity (mL/min) 100 200 300C, pore diameter of the gas distributor (�m) 100 200 400

Table 4The results of the orthogonal experiments for the first stage foam fractionation.

No. A B C E R/%

1 500 100 100 23.4 ± 1.0 10.7 ± 0.52 500 200 200 18.3 ± 0.7 25.6 ± 1.23 500 300 400 13.4 ± 0.6 34.9 ± 1.74 700 100 200 21.5 ± 1.0 19.6 ± 1.05 700 200 400 19.4 ± 0.9 35.7 ± 2.06 700 300 100 11.1 ± 0.6 46.8 ± 2.27 900 100 400 29.5 ± 1.4 9.40 ± 0.98 900 200 100 17.7 ± 0.6 40.4 ± 2.09 900 300 200 10.8 ± 0.5 50.4 ± 2.5M1E 18.37 24.80 17.40M2E 17.33 18.47 16.87M3E 19.33 11.77 20.77M1R 23.73 13.23 32.63M2R 34.03 33.90 31.87

reomat(

rgt1gpa3e

M3R 33.40 44.03 26.67KE 2.00 13.03 3.90KR 10.03 30.8 5.96

espectively. In Table 4, MiE (MiR) referred to the mean of all thenrichment ratios (recovery percentage) obtained at the ith levelf any one factor and KE (KR) referred to the difference between theaximum and the minimum among all the values of MiE (MiR) of

ny one factor. Furthermore KE (KR) of any one factor representedhe degree of the effect that this factor had on enrichment ratiorecovery percentage).

According to the KE and KR values, both enrichment ratio andecovery percentage were most greatly influenced by volumetricas velocity. In terms of enrichment ratio its optimal condi-ions were liquid loading volume 900 mL, volumetric gas velocity00 mL/min and pore diameter 400 �m. Under these conditions areat enrichment ratio of 29.5 was obtained with a low recovery

ercentage of 9.40%. The optimal conditions for recovery percent-ge were liquid loading volume 900 mL, volumetric gas velocity00 mL/min and pore diameter 200 ± 10 �m, where a high recov-ry percentage of 50.4% and a relatively low enrichment ratio of

Fig. 4. Effects of temperature on E, R

roducts 51 (2013) 163– 170 167

10.8 were obtained. However both the results were not suitable ifused as those of the first stage because of the relatively low recov-ery percentage or enrichment ratio. From Table 2, an enrichmentratio of 17.7 and a recovery percentage of 40.4% at liquid load-ing volume 900 mL, volumetric gas velocity 200 mL/min and porediameter 100 �m. Both enrichment ratio and recovery percentagewere relatively high, so these conditions were considered to besuitable for the first stage foam fractionation.

3.1.4. Optimization of the second stage foam fractionationThe second-stage feed solution (pH = 6.5 ± 0.2), i.e. the first-

stage foamate, had a high total-saponin concentration of 1.77 g/Land at this concentration, a great enrichment ratio would not bereadily obtained at room temperature. Previous work (Yan et al.,2011; Jiang et al., 2011) demonstrated that the elevated tem-perature could effectively enhance foam drainage via decreasingthe solution viscosity, and thus enrichment ratio was effectivelyimproved even if at high initial concentrations. Therefore, in thesection, only the optimization of temperature was specified. Theother conditions for the second stage foam fractionation, volumet-ric gas velocity, liquid loading volume and pore diameter of the gasdistributor, were optimized and fixed at 70 mL/min, 500 mL and400 �m, respectively, based on preliminary efforts not presentedin this article. The results are presented in Fig. 4.

Fig. 4 illustrates that the enrichment ratio of the total saponinsincreased from 1.53 to 7.73 while their recovery percentageincreased from 33.4% to 40.1% and then decreased to 25.4% withelevating temperature from 25 ◦C to 75 ◦C. The elevated tem-perature could intensify molecular movement and decrease thesolution viscosity. The decreased viscosity weakened the resistancefor surface-active molecules adsorbing onto bubble surfaces. As aresult, the saponin molecules were more quickly adsorbed ontobubble surfaces. This explained the reason why the foamabilityof the feed solution, expressed as foaming height measured byRoss–Miles method (Careya and Stubenraucha, 2009), increasedwith elevating temperature from 25 ◦C to 65 ◦C. The increase infoamability increased the amount of the saponins adsorbed ontobubble surfaces. However, with increasing temperature over 45 ◦C,the more intense molecular movement slowed down the increasein the adsorption of the saponin molecules, so the increase in foam-ability of the feed solution decreased. In terms of foam drainage,with increasing temperature over 45 ◦C, its enhancement was

more effective due to the continually decreased viscosity and thecontinually intensified bubble coalescence. Therefore the total-saponin recovery decreased with temperature increasing over45 ◦C while the enrichment ratio still greatly increased. When

, foaming height and viscosity.

168 R. Li et al. / Industrial Crops and Products 51 (2013) 163– 170

action

tsmsfsws3

3t

putitatotipb

Fig. 5. FTIR spectra of the foam fr

emperature increased over 65 ◦C, the saponin molecules could nottably adsorb at bubble surfaces owing to their overquick thermalovement. Thus the foamability and the amount of the adsorbed

aponins decreased. Combined with the more effectively enhancedoam drainage, this resulted in fast decrease in the recovery andlight increase in the enrichment ratio. All above considered, 65 ◦Cas selected herein as the optimal temperature where the total-

aponin enrichment ratio and recovery percentage were 7.54 and2.6%, respectively.

.1.5. Modification for the two-stage foam fractionationechnology

Using the two-stage foam fractionation technology, the recoveryercentage of the total saponins was as low as 13.2% if the resid-al solutions of both the two stages were discharged. Therefore theechnology was modified by reusing the residual solutions and it isllustrated in Fig. 2 by the dotted lines. The residual solutions of bothhe two stages had the total-saponin concentrations of 0.061 g/Lnd 1.25 g/L and their pH values were 7.0 ± 0.1 and 6.7 ± 0.2, respec-ively, so they were mixed at a ratio of 29.5/1 (v/v) to form a solutionf pH = 6.9 ± 0.2 serving as the first-stage feed solution. Moreover

he excess residual solution of the first stage was used as the leach-ng solvent to separate the sapindus saponins from the pericarpowder. Thus the simple modification would effectively improveoth the recovery of the total saponins and the usage of water.

Fig. 6. ESI-total ion chromatogram of the foam

ation product and oleanolic acid.

According to the experimental results, the total recovery of thesapindus saponins reached more than 36.4% with a great enrich-ment ratio of 133.4 and a high purity of 90.3% using the modifiedtwo-stage foam fractionation technology.

3.2. Product analysis

3.2.1. FTIR analysis of the productThe foamate from the second stage foam fractionation was dried

to obtain milky amorphous powder while the product of Huanget al. (2003) was white, so our product did not have 100% in purity.The FTIR spectrum of our product was measured using oleanolicacid as a reference and both the spectra are presented in Fig. 5.

It is found in Fig. 5 that both the product and oleanolicacid had OH (∼3436.94 cm−1, oleanolic acid; ∼3424.65 cm−1, theproduct) and OH of the product had stronger absorption thanthat of oleanolic acid. Oleanolic acid and the product had bothCH2 and CH3 (∼2943.06 cm−1, ∼2871.74 cm−1, ∼1463.21 cm−1,∼1384.76 cm−1, oleanolic acid; ∼2926.96 cm−1, ∼2855.17 cm−1,∼1462.73 cm−1, ∼1377.69 cm−1, the product), but the peaks at∼2926.96 cm−1 and ∼2855.17 cm−1 indicated more CH2 than CH3

in the product. In addition there was no absorption at ∼720 cm−1

and this meant that the product had no (CH2)n≥4. The rela-tively strong absorption at ∼1695.85 cm−1 (oleanolic acid) and∼1729.24 cm−1 and 1697.20 cm−1 (the product) was attributed to

fractionation product in positive model.

R. Li et al. / Industrial Crops and P

Fw

C∼e∼wa

sabct

3

sCCCictttd(crsa

3

iTCpwnmmdta

ig. 7. Variation of antibacterial activity of nisin and the foam fractionation productith time.

O. Moreover the strong absorption in the product spectrum at1256.83 cm−1, ∼1136.7 cm−1 and ∼1050.53 cm−1 presented thexistence of C O C and carboxylic ester. The weak absorption at1637.21 cm−1 (oleanolic acid) and ∼1632.73 cm−1 (the product)as resulted from C C, and this could be confirmed by the weak

bsorption at 970–800 cm−1.The similarity of the oleanolic acid spectrum to the product

pectrum confirmed the existence of sapogenin and the strongerbsorption of OH, CH2 and C O C in the product spectrum coulde attributed to many glycosides in the product. Therefore it isoncluded that the product of the two-stage foam fractionationechnology was rich in the sapindus saponins.

.2.2. HPLC–MS analysis of the productReferring to Table 1, it is found in Fig. 6 that the triterpenoid

aponins in the product concluded C58H94O26 (m/z: 1229 [M+Na]+),46H74O16 (m/z: 905 [M+Na]+), C41H66O12 (m/z: 773 [M+Na]+),48H76O17 (m/z: 947 [M+Na]+) and C50H78O18 (m/z: 989 [M+Na]+).48H76O17 and C50H78O18 had been reported to have two and three

somerides (Huang et al., 2003), respectively, but they were notompletely separated under the present conditions. In additionhere were other unknown compounds in the product, of whichhe molecular masses were 877, 1114, 1146, 1169 and 1183, andhey were conjectured to be sesquiterpene glucosides on the foun-ation of their molecular masses and relatively low surface activityKasai et al., 1986). The subsequent HPLC analysis under the aboveonditions presented that the area percentage of the total saponinseached 87.5%, corresponding to a high purity. Thus the sapindusaponins were effectively purified by the two-stage foam fraction-tion technology.

.2.3. Antimicrobial activity analysis of the productIt had been reported that the sapindus saponins had growth

nhibition against gram-positive bacteria (Yang et al., 2010).he results in Fig. 7 consisted with those of the previous work.ompared to nisin, antimicrobial activity of the foam fractionationroduct was lower and this meant that the dosage of the productould be more if it had the same antibacterial effect as nisin. Iteeded to be further investigated in the future work whether theore dosage of the product had negative effects. Nisin could be

ore readily inactive than the saponins, so its antibacterial activity

ecreased faster with time than that of the product. Moreoverhe sapindus saponins had wider and easier source than nisin,nd the two-stage foam fractionation technology was indeed a

roducts 51 (2013) 163– 170 169

cost-effective way for obtaining the high-quality saponins. There-fore the current work had significant and industrial implication inproviding a cheap antibacterial product for the food, cosmetic andeven pharmaceutical fields.

4. Conclusions

The SIC and the elevated temperature greatly improved thetotal-saponin enrichment ratio but largely decreased the recov-ery percentage in single stage foam fractionation. The modifiedtwo-stage foam fractionation technology successfully purified andrecovered the sapindus saponins from their leaching liquor. Usingthis technology, a great enrichment ratio of 133.4 and a highpurity of 90.3% were obtained with a recovery of more than 36.4%.The major components in the highly purified product were thetriterpenoid saponins including C58H94O26, C46H74O16, C41H66O12,C48H76O17 and C50H78O18. The product had moderate but long-term antimicrobial activity against gram-positive bacteria, thushaving a promising prospect as a natural preservative. In termsof practical significance, this present work provided a green andcost-effective technology for purifying and recovering naturalsurface-active products.

Acknowledgement

This work was financially supported by the Natural ScienceFoundation of China (21236001) and the Natural Science Founda-tion of Hebei, China (B2011202056).

References

Bhattacharjee, S., Kumar, R., Gandhi, K.S., 1997. Prediction of separation factor infoam separation of proteins. Chem. Eng. Sci. 52, 4625–4636.

Careya, E., Stubenraucha, C., 2009. Properties of aqueous foams stabilized by dode-cyltrimethylammonium bromide. J. Colloid Interface Sci. 333, 619–627.

Chen, H.Y., Kuo, P.L., Chen, Y.H., Huang, J.C., Ho, M.L., Lin, R.J., Chang, J.S., Wang,H.M., 2010. Tyrosinase inhibition, free radical scavenging, antimicroorganismand anticancer proliferation activities of Sapindus mukorossi extracts. J. TaiwanInst. Chem. Eng. 41, 129–135.

Dickinson, J.E., Laskovski, D., Stevenson, P., Galvin, K.P., 2010. Enhanced foamdrainage using parallel inclined channels in a single-stage foam fractionationcolumn. Chem. Eng. Sci 65, 2481–2490.

Dobhal, U., Bisht, N.S., Bhandari, S.L., 2007. Traditional values of Sapindus mukorossiGaerten. vern. Ritha: a review. Plant Arch. 7, 485–486.

Grieves, R.B., Dhattacharyya, D., 1964. The effect of temperature upon foam frac-tionation. J. Am. Oil Chem. Soc. 42, 174–176.

Hiai, S., Oura, H., Nakajima, T., 1976. Color reaction of some sapogenins and saponinswith vanillin and sulfuric acid. Planta Med. 29, 116–122.

Huang, Y.C., Liao, S.H., Chang, F.R., Kuo, Y.H., Wu, Y.H., 2003. Molluscicidal saponinsfrom Sapindus mukorossi, inhibitory agents of golden apple snails, pomaceacanaliculata. J. Agric. Food Chem. 51, 4916–4919.

Jiang, C.S., Wu, Z.L., Li, R., Liu, Q., 2011. Technology of protein separation from wheywastewater by two-stage foam separation. Biochem. Eng. J. 55, 43–48.

Kasai, R., Fujino, H., Kuzuki, T., Wong, W.H., Goto, C., Yata, N., Tanaka, O., Yasuhara,F., Yamaguchi, S., 1986. Acyclic sesquiterpene oligoglycosides from pericarps ofSapindus mukorossi. Phytochemistry 25, 871–876.

Khalesi, M., Venken, T., Deckers, S., Winterburn, J., Shokribousjein, Z., Gebruers, K.,Verachtert, H., Delcour, J.A., Martin, P., Derdelinckx, G., 2013. A novel method forhydrophobin extraction using CO2 foam fractionation system. Ind. Crops Prod.43, 372–377.

Kumpabooth, K., Scamehorn, J.F., Wsuwan, S., Harwell, J.H., 1999. Surfactant recov-ery from water using foam fractionation: effect of temperature and added salt.Sep. Sci. Technol. 34, 157–172.

Li, R., Wu, Z., Wang, L., Yang, Q., Zhu, H., 2012. Enhancing foam drainage by spiralinternal components of different thread pitches and inclined angles and theirapplications to enrichment of SDS. Sep. Purif. Technol. 98, 109–117.

Li, X.L., Evans, G.M., Stevenson, P., 2011. Process intensification of foam fractionationby successive contraction and expansion. Chem. Eng. Res. Des. 89, 2298–2308.

Liu, Z.M., Wu, Z.L., Li, R., Fan, X.J., 2013. Two-stage foam separation technology forrecovering potato protein from potato processing wastewater using the columnwith the spiral internal component. J. Food Eng. 114, 192–198.

Martin, P.J., Dutton, H.M., Winterburn, J.B., Baker, S., Russell, A.B., 2010. Foam frac-tionation with reflux. Chem. Eng. Sci. 65, 3825–3835.

Nakayama, K., Fujino, H., Kasai, R., Tanaka, O., Zhou, J., 1986. Saponins of pericarps ofChinese Sapindus delavayi (Pyi-shiaulzu), a source of natural surfactants. Chem.Pharm. Bull. 34, 2209–2213.

1 and P

N

R

R

R

S

S

SV

70 R. Li et al. / Industrial Crops

ie, W., Luo, J.G., Wang, X.B., Wan, X., Kong, L.Y., 2009. An insight into enrichment andseparation of oleanane-type triterpenoid saponins by various chromatographicmaterials. Sep. Purif. Technol. 65, 243–247.

eddy, V., Torati, R.S., Oh, S., Kim, C., 2013. Biosynthesis of gold nanoparticles assistedby Sapindus mukorossi Gaerten. fruit pericarp and their catalytic application forthe reduction of p-nitroaniline. Ind. Eng. Chem. Res. 52, 556–564.

oy, D., Kommalapati, R.R., Mandava, S.S., Valsaraj, K.T., Constant, W.D., 1997. Soilwashing potential of a natural surfactant. Environ. Sci. Technol. 31, 670–675.

ujirawanich, V., Triroj, M., Pornsunthorntawee, O., Chavadej, J., Chavadej, S., 2013.Recovery of surfactant from an aqueous solution using continuous multistagefoam fractionation: mixed surfactant system. Sep. Sci. Technol. 48, 757–765.

aha, S., Walia, S., Kumar, J., Dhingra, S., Parmar, B.S., 2010. Screening for feedingdeterrent and insect growth regulatory activity of triterpenic saponins fromDiploknema butyracea and Sapindus mukorossi. J. Agric. Food Chem. 58, 434–440.

harma, A., Sati, S.C., Sati, O.P., Sati, M.D., Kothiyal, S.K., 2013. Triterpenoid saponinsfrom the pericarps of Sapindus mukorossi. J. Chem., 1–5, Article ID 613190.

tevenson, P., 2007. Hydrodynamic theory of rising foam. Miner. Eng. 20, 282–289.erma, N., Amresh, G., Sahu, P.K., Mishra, N., Singh, A.P., Rao, C.V., 2013. Antihy-

perglycemic activity, antihyperlipidemic activity, haematological effects andhistopathological analysis of Sapindus mukorossi Gaerten. fruits in streptozo-tocin induced diabetic rats. Asian Pac. J. Trop. Med. 5, 518–522.

roducts 51 (2013) 163– 170

Vivek, R., Ramkrishna, S., 2013. An inexpensive strategy for facilitated recovery ofmetals and fermentation products by foam fractionation process. Colloids Surf.B 104, 99–106.

Wei, F.Y., Xie, H., Yu, J.H., Wu, Y., 2008. Separation and purification ofsapindus-saponin by ultrafiltration. Membr. Sci. Technol. (in Chinese) 28,85–88.

Weijenberg, D.C., Mulder, J.J., Drinkenburg, A.A.H., Stemerding, S., 1978. The recoveryof protein from potato juice waste water by foam separation. Ind. Eng. Chem.Process Des. Dev. 17, 209–213.

Wu, Z.L., Wang, L., Jing, Y.J., Li, X.L., Zhao, Y.L., 2009. Variable volume fed-batch fer-mentation for nisin production by Lactococcus lactis subsp. lactis W28. Appl.Biochem. Biotechnol. 152, 372–382.

Yan, J., Wu, Z.L., Zhao, Y.L., Jiang, C.S., 2011. Separation of teasaponin by two-stage foam fractionation. Sep. Purif. Technol. 80,300–305.

Yang, C.H., Huang, Y.C., Chen, Y.F., Chang, M.H., 2010. Foam properties, detergent

abilities and long-term preservative efficacy of the saponins from Sapindusmukorossi. J. Food Drug Anal. 18, 155–160.

Yang, Q.W., Wu, Z.L., Zhao, Y.L., Wang, Y., Li, R., 2011. Enhancing foam drainage usingfoam fractionation column with spiral internal for separation of sodium dodecylsulfate. J. Hazard. Mater. 3, 1900–1904.