1362 Alexis Kotland1,2 Imane Hadef2 Jean-Hugues Renault2 Mahmoud Hamzaoui2 Agathe Martinez2 Nicolas Borie2 Arnaud Guilleret1 Romain Reynaud1 Jane Hubert2 1 Soliance, Pomacle, France 2 Universite ´ de Reims
Champagne-Ardenne, Reims, France Received November 8, 2012 Revised December 21, 2012 Accepted January 28, 2013
J. Sep. Sci. 2013, 36, 1362–1369
Research Article
Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts Sophorolipids represent an important class of natural surfactants with a variety of environmental, cosmetic, and pharmaceutical applications. Despite their promising physicochemical and biological properties, the use of sophorolipids is hampered by the lack of information regarding their individual structure-activity relationships. The major difficulty in isolating pure sophorolipids arises from the high complexity of crude fermentation media composition and from their strong structural similarities. In this work, a centrifugal partition chromatography method was developed in an original gradient elution mode for the separation of sophorolipids produced by the yeast Candida bombicola. Experiments were realized by using three sets of solvent systems composed of n-heptane, ethyl acetate, nbutanol, methanol, and water in different proportions. The separation was performed at 5 mL/min in the ascending mode by increasing progressively the polarity of the organic mobile phase. In these conditions, more than 80% of the sophorolipids present in the initial crude fermentation extract were eluted successively from the most hydrophobic lactone forms to the most hydrophilic acid forms. The structures of the isolated sophorolipids were further elucidated by HPLC and NMR analyses. Keywords: Candida bombicola / Centrifugal partition chromatography / Sophorolipids DOI 10.1002/jssc.201201033
1 Introduction Sophorolipids represent an important class of microbial glycolipids, which are increasingly used as natural surfactants in a variety of environmental, cosmetic, or pharmaceutical applications. Sophorolipids are synthesized by nonpathogenic yeasts such as Candida bombicola, Candida apicola, or Thodotorula bogoriensis when grown on carbohydrates and lipid substrates [1]. They are released in the extracellular medium as a mixture of structurally related molecules composed of one sophorose unit linked -glycosidically to a hydroxy fatty acid chain with 16–18 carbon atoms and one or more unsaturations. Sophorose can be acetylated at the 6 and/or 6 position and the hydroxy fatty acid can be linked to the sophorose unit at the terminal or subterminal position. The carboxylic end of the fatty acid chain is either free (acid forms) or esterified to one hydroxyl group of the sophorose unit (lactone forms) (Fig. 1).
Correspondence: Dr. Jane Hubert, UMR CNRS 7312, Universite´ ˆ 18, Moulin de la Housse, BP de Reims Champagne-Ardenne, Bat. 1039, 51687 Reims, Cedex 2, France E-mail:
[email protected] Fax: +33-326913166
Abbreviation: CPC, centrifugal partition chromatography
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Sophorolipids possess strong surface-active properties and are used in cosmetic formulations due to their excellent skin compatibility. Biological investigations have also demonstrated that sophorolipids possess an antimicrobial activity against plant pathogenic fungi [2], a capacity to inhibit the human immunodeficiency virus [3] and more recently a potential for liver cancer treatment [4]. The physicochemical and biological properties of sophorolipids are strongly dependant on their structure, particularly on the distribution between the lactone and acid forms released during the fermentation process [5, 6]. It was also demonstrated that the surfactant properties and biodegradability of sophorolipids were both influenced by the fatty acid chain length [6, 7]. Acetyl groups can also reduce the hydrophilicity of sophorolipids and enhance their antiviral and cytokine stimulating effects [3]. Nevertheless, the exact structure-activity relationships within each class of sophorolipids remain unclear and the introduction of sophorolipid mixtures in cosmetic or pharmaceutical products is hampered by the lack of information regarding the properties of individual species and their toxicity toward human systems. In this context, efficient separation and purification tools are required to examine each compound individually. The major difficulty in isolating individual sophorolipid structures arises from their strong structural similarities within each class. Often the molecular species vary only in the
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2 Materials and methods 2.1 Chemicals n-heptane, chloroform (CHCl3 ), ethyl acetate (EtOAc), acetonitrile (CH3 CN), n-butanol (n-BuOH), methanol (MeOH), and TFA were purchased from Carlo Erba Reactifs SDS (Val de Reuil, France). CH3 CN and TFA were of analytical HPLC grade. Deuterated methanol (MeOD) and DMSO-d6 were purchased from Eurisotop (Saint-Aubin, France). The sophorolipids used in this work were synthesized by the yeast C. bombicola (industrial strain) through an industrial fermentation process (Soliance, Pomacle, France).
2.2 Apparatus
Figure 1. Structural characteristics of sophorolipids.
number of acetyl groups, in the length of the fatty acid chain or degree of unsaturation. Separation of the polar sophorolipid acid structures from the less polar lactone forms can be easily achieved by selective crystallization in buffer solutions or in organic solvents [8]. However, very few studies have described the purification of individual sophorolipids within each group [9–12]. The purpose of the present work was to explore a new strategy for the separation of individual sophorolipids from a crude extract of C. bombicola yeasts by using centrifugal partition chromatography (CPC). CPC refers to a particular type of solid support free liquid–liquid chromatographic system where analytes are separated according to their partition coefficients between two immiscible liquid phases [13, 14]. This technique is very attractive in terms of selectivity, sample loading capacity, and scaling-up ability [15, 16]. It also enables the introduction of crude samples into the column without extensive pretreatments and therefore constitutes an interesting alternative for the fractionation and purification of individual sophorolipids [17]. The CPC separation method was developed in an original gradient elution mode using three solvent systems composed of n-heptane, ethyl acetate, n-butanol, methanol, and water. The polarity of the mobile phase was progressively increased in order to transfer selectively the different sophorolipid species from the stationary phase to the mobile phase. In order to obtain highly pure sophorolipids, preparative HPLC (prep-HPLC) was further combined to CPC as an orthogonal technique. As a result, 11 sophorolipid structures were confirmed by NMR analyses among which three structures are described for the first time. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
R apparatus (Armen CPC was performed on an ASCPC250 Instrument, Vannes, France) equipped with a 250-mL column containing 21 stacked disks engraved circumferentially with 504 twin cells. The total volume of the cells was 214 mL and the volume of interconnecting ducts was 36 mL. The rotation speed can be adjusted from 500 to 3000 rpm, creating a maximum centrifugal force field of 600 × g. The solvents were pumped by an Armen Pump Light version equipped with a double piston and delivering flow rates up to 50 mL/min with low residual pulsation. The maximum pressure was 150 bar. The samples were introduced into the CPC column by using an Armen injector equipped with a 20-mL sample loop. The system was coupled to an ultraviolet detector (Armen) monitored at 207 nm. Fractions (10 mL) were collected by a LS-5600 collector (Armen). Data were acquired using the Armen Glider CPC control software V2.9.
2.3 Preparation of the crude fermentation extract A C. bombicola fermentation extract rich in sophorolipids was obtained industrially after removal of the cell debris by decantation of the whole fermentation media. The resulting fermentation extract (51.2% dry weight) was then adjusted to pH ≈ 9 in order to avoid crystallization and maintain the liquid nature of the final product.
2.4 CPC separation procedure In an attempt to optimize our gradient elution method, three biphasic solvent systems were prepared independently by mixing n-heptane, EtOAc, n-BuOH, MeOH, and H2 O in the proportions 37.5:10:2.5:12.5:37.5 v/v for system 1, 20:27.5:2.5:12.5:37.5 v/v for system 2, and 0:0:50:7.5:42.5 v/v for system 3, respectively (Table 1). The use of these five solvents was necessary to progressively increase the polarity of the mobile phase during the gradient from system 1 (n-heptane-rich upper phase) to system 2 (EtOAc-rich upper phase) and system 3 (n-BuOH-rich upper phase) while www.jss-journal.com
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Table 1. Solvent systems and gradient elution method optimized for the CPC separation of sophorolipids
Solvent systems n-heptane Ethyl n-butanol Methanol Water (%, v/v) acetate (%, v/v) (%, v/v) (%, v/v) (%, v/v) System 1 System 2 System 3
37.5 20 0
10 27.5 0
2.5 2.5 50
12.5 12.5 7.5
37.5 37.5 42.5
Gradient elution method Time (min)
Up 1 (%, v/v)
Up 2 (%, v/v)
Up 3 (%, v/v)
0 10 70 80 140 230
100 100 25 0 0 0
0 0 75 100 0 0
0 0 0 0 100 100
Up 1, upper phase of system 1; Up 2, upper phase of system 2; Up 3, upper phase of system 3.
ensuring the biphasic character of the global system (with MeOH and water as major constituents in the lower phase). The sample solution was prepared by directly dissolving the crude fermentation extract (400 mg corresponding to 205 mg dry weight) in 10 mL of the upper phase of system 1 mixed to the lower phase of system 2 (25:75, v/v). The sophorolipid separation was performed by using a gradient elution procedure. The CPC column was filled with the lower aqueous phase of system 2 at 500 rpm. The rotation speed was then adjusted to 1200 rpm. The upper organic phase of system 1 was used as the initial mobile phase in the ascending mode. After loading the sample solution into the column through the 10-mL sample loop, the flow rate was progressively increased from 0 to 5 mL/min in 5 min. The gradient elution was performed at 5 mL/min by pumping from 100% of the upper phase of system 1 to 75% of the upper phase of system 2 in 60 min, to 100% of the upper phase of system 2 in 10 min, to 100% of the upper phase of system 3 in 60 min, and finally maintained at this composition for 90 min as described in Table 1. Experiments were conducted at room temperature. 2.5
1
H NMR characterization of the stationary phase composition during the gradient CPC separation process
In order to evaluate how the evolution of the mobile phase gradient can affect the stationary phase composition during the CPC separation procedure, an aliquot (200 L) of the aqueous stationary phase was taken at its initial state (fresh aqueous phase of system 2 = stat initial), at t = 0 (corresponding to the aqueous phase of system 2 equilibrated with the organic phase of system 1), at t = 80 min (corresponding to C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the previous stationary phase at t = 0 equilibrated with the organic phase of system 2) and at t = 140 min (corresponding to the previous stationary phase at t = 80 min equilibrated with the organic phase of system 3). This aliquot was mixed with 300 L of DMSO-d6 and analyzed by 1 H NMR at 298 K on a Bruker Avance AVIII-600 MHz spectrometer (Bruker, Germany). Spectra were acquired with 1 scan (no dummy scan) over a spectral width of 12 ppm using the standard zg pulse program and employing a relaxation delay of 20 s. The 1 H NMR signals of n-heptane at 1.2 ppm (10H), of ethyl acetate at 1.9 ppm (3H), of methanol at 3.2 ppm (3H), of water at 3.8 ppm (2H), and of n-BuOH at 3.4 ppm (2H) were integrated on each spectra and the volume percentage (V% ) of each solvent was calculated as V% = (A/n)×(MW/d), with A = signal area, n = number of proton(s), MW = molecular weight of the solvent, and d = density of the solvent at 298 K. 2.6 TLC and HPLC analyses of the collected fractions The composition of each collected fraction (10 mL) was checked by TLC on Merck 60 F254 silica gel plates (Merck), developed with CHCl3 /MeOH/H2 O (80:20:2, v/v), sprayed by a sulfuric acid solution (50% in water) and revealed after heating for 1 min at 200⬚C. Fractions were combined according to their TLC composition similarities to yield nine pools, noted from F1 to F9. Analytical HPLC analyses were performed on a Summit HPLC system equipped with an Ultimate 3000 pump, an ASI-100 autosampler, and an UVD340S diode array detector monitored at 207 nm (Dionex, Sunnyvale, CA, USA). After in vacuo solvent removal, samples (about 5 mg) were dissolved in 1 mL CH3 CN/H2 O (1:1, v/v) and acidified with 1% TFA (pH ≈ 1–2). The chromatographic separation was performed on a Luna RP C18 column (250 × 4.6 mm, 5 m, Phenomenex, Le Peck, France) maintained at 40⬚C. The injection volume was 10 L. The mobile phases were composed of acetonitrile (solvent A) and distilled water (solvent B). Solvent A was maintained at 20% for 2 min, then increased to 100% in 38 min and remained for 10 min. Then solvent A recycled back to the initial conditions of 20% in 1 min and remained for 9 min. 2.7 Preparative HPLC Each pool from F2 to F8 was further purified on the same HPLC system, but the purifications were performed on a semipreparative Luna RP C18 column (250 × 10 mm, 5 m, Phenomenex, Le Peck, France) maintained at 40⬚C. Chromatograms were recorded and integrated by using the Chromeleon version 6.0.1 software (Dionex). The injection volume was 200 L. The mobile phases were composed of acetonitrile (solvent A), distilled water (solvent B), and distilled water acidified with TFA at 0.025% (solvent C). The flow rate was set at 4 mL/min. An isocratic elution was performed at 70:30 (solvent A/solvent B, v/v) for the less polar pools (from F2 to F4), and at 50:50 (solvent A/solvent C, v/v) for the more polar pools (from F5 to F8). www.jss-journal.com
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Figure 2. HPLC chromatogram of the crude extracellular extract obtained from the fermentation of Candida bombicola yeasts and after NaOH treatment (pH 9). More than 20 compounds were detected among which 13 were identified in the present study. 1 = free fatty acid C18:1, 2 = free fatty acid C18:2, 3 = SLl I, 4 = SLl III, 5 = SLl II, 6 = SLl IV, 7 = SLl V, 8 = SLl VI, 9 = SLo I, 10 = SLo II, 11 = SLo III, 12 = SLo IV, and 13 = SLo V.
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tained by microbial fermentation of the yeast C. bombicola. In order to satisfy industrial constraints, the crude extract was stabilized at pH 9 with sodium hydroxyde to ensure the liquid state and physico-chemical properties of the final product. This treatment of course has led to some modifications of the sophorolipid structures naturally produced by C. bombicola, particularly at the level of molecular sites sensitive to basic conditions such as acetyl or lactonic functions. As a result and surprisingly, instead of obtaining only nonacetylated and open acid forms, a wide diversity of sophorolipid structures was detected. Our objective was therefore to characterize these resulting individual sophorolipid species. More than 20 compounds were initially observed by HPLC in the initial crude fermentation extract (Fig. 2). No predominant compound was observed but many structures were distributed within a large polarity range.
2.8 NMR identification of sophorolipids An aliquot (from 5 to 10 mg) of each sophorolipid recovered as pure compound was dissolved in 600 L MeOD and analyzed by NMR spectroscopy at 298 K on a Bruker Avance AVIII600 spectrometer. 1 H and 13 C NMR spectra were acquired at 600.15 and 150.91 MHz, respectively. Additional HSQC, HMBC, and COSY 2D-NMR experiments were performed using standard Bruker microprograms.
3 Results and discussion 3.1 General context of the study In the present study, a CPC method was developed in the gradient elution mode for the fractionation and purification of different sophorolipids present in an industrial extract ob-
3.2 Separation of sophorolipids by CPC Gradient elution in CPC is an efficient approach that enables the fractionation of complex crude mixtures in less time than classical isocratic elution procedures [18–21]. However, its applications in solid support free liquid–liquid chromatography remain scarce, mainly because the modifications of the stationary phase composition during the mobile phase gradient are poorly understood and thus poorly controlled. The CPC separation procedure developed in the present work was performed in a gradient elution mode by using three sets of biphasic solvent systems composed of n-heptane, ethyl acetate, n-butanol, methanol, and water. As given in Table 1, the organic phase of the less polar solvent system 1 was used as the initial mobile phase, the organic phase of the most polar solvent system 3 was used as final mobile phase while
Figure 3. TLC profiles of the fractions collected during the CPC separation procedure.
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Table 2. 1 H NMR characterization of the stationary phase composition during the CPC separation procedure
Solvent
n-heptane MeOH BuOH EtOAc H2 O
Stationary phase composition (%, v/v) Stat initial
Stat t = 0 min
Stat t = 80 min
Stat t = 140 min
< 0.1 31.6 0.2 8.1 60.1
< 0.1 30.4 1.3 4.7 63.6
< 0.1 29.5 1.8 9.0 59.7
< 0.1 22.8 14.1 2.6 60.5
Stat, stationary phase.
the aqueous phase of the intermediary system 2 was used as stationary phase inside the CPC column. These systems enabled a good solubility of the crude sample and a selective elution of the different sophorolipid classes according to their hydrophobicity and chemical structures. After loading 200 mg of the crude sophorolipid extract, the initial retention of the stationary phase inside the CPC column was 75%. As illustrated in Fig. 3 by TLC analyses, the initial mobile phase rich in n-heptane enabled the successive elution of free fatty acids and of the most hydrophobic sophorolipid structures (lactonic and acetylated structures) while keeping the more hydrophilic compounds in the aqueous stationary phase. Fractions containing free fatty acids according to their TLC and HPLC profiles were combined (pool F1) and directly analyzed by NMR spectroscopy, confirming the presence of oleic acid (C18:1) as major fatty acid chain and linoleic acid
(C18:2) as minor one (spectra not shown). The following fractions eluting from 23 to 100 min were also combined according to their composition similarities to yield the pools F2, F3, and F4, which only contained sophorolipids under their lactonic forms. During this period, the mobile phase was progressively enriched in ethyl acetate (Table 1). This increasing amount of ethyl acetate enabled to increase the mobile phase polarity and thus also to start with the elution of the open acidic sophorolipid structures at a retention time of about 100–120 min (pool F5). Finally, increasing further the polarity of the mobile phase with n-butanol allowed the complete elution of the open acid forms of sophorolipids from 120 to 230 min (pools F6, F7, and F8, see Fig. 3).
3.3 Investigation of the stationary phase composition modifications during the CPC separation process At the end of the experiment, the final stationary phase retention was only 30%. Considering the strong surfactant character of sophorolipids, this important loss of stationary phase is not surprising. In addition, working in the gradient elution mode implies necessarily continuous modifications of the mobile phase composition. Inside the CPC column, these modifications may have resulted in some perturbations in the biphasic solvent system hydrodynamic and/or thermodynamic equilibrium, leading to a slight but continuous loss of stationary phase during the gradient process. This carryover was more significant between 80 and 140 min when increasing progressively the mobile phase of system 3 rich in
Table 3. 1 H and 13 C NMR chemical shifts of Sophorose ␣ and Sophorose  recovered in the fraction pool F9. NMR analyses were performed in MeOD
Sophorose ␣
Sophorose 
1 H (ppm)
13 C (ppm)
1 H (ppm)
13 C (ppm)
4.5 5.4a) 3.5a) 3.6 3.4 3.3 3.9 3.8 3.3 3.4 3.7–3.9 3.7–3.9
104.5 92.1 81.6 76.5 76.3 73.9 72.2 71.4 70.0 69.9 61.2 61.2
4.7 4.6a) 3.4a) 3.3 3.3 3.3 3.3 3.3 3.4 3.3 3.7–3.8 3.9–3.7
103.6 95.3 82.9 76.8 76.7 76.5 76.5 74.1 70.1 69.9 61.3 61.2
a)The coupling constant JH1’-H2’ is 3.66 Hz for sophorose ␣ and 7.74 Hz for sophorose .
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J. Sep. Sci. 2013, 36, 1362–1369 Table 4.
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C NMR chemical shifts of identified sophorolipids
Position
SLl I
SLl II
SLl III
SLl IV
SLl V
SLl VI
SLo I
SLo II
SLo III
SLo IV
SLo V
C1’ sucre C2’ sucre C3’ sucre C4’ sucre C5’ sucre C6’ sucre C1" sucre C2" sucre C3" sucre C4" sucre C5" sucre C6" sucre COO (C1) C=C C=C C=C C=C C2 C3 CH2 fatty acid
102.4 82.0 75.8 70.5 73.3 63.6 103.9 75.2 74.0 69.8 72.7 62.1 173.4 130.0 129.7 – – 34.4 24.7 30.1 30.0 29.7 29.0 28.3 28.2 28.1 27.2 26.9 25.3 37.5 79.4 21.1 171.6 170.5 20.9 20.8
102.5 81.0 75.5 70.3 73.4 63.4 103.3 74.2 74.1 69.7 72.8 62.1 173.3 130.4 129.5 128.7 128.1 34.4 24.7 25.3–37.3
102.1 79.0 77.2 70.1 73.5 63.4 102.2 72.6 77.2 68.9 73.6 63.4 173.8 129.8 129.4 – – 34.0 24.1 29.8 29.7 29.5 28.4 27.6 27.3 27.3 27.1 26.2 24.1 36.9 78.1 21.0 171.3 171.3 19.5 19.3
101.8 79.2 77.4 70.0 76.3 61.4 102.3 72.6 77.2 68.9 73.6 63.4 173.8 129.7 129.4 – – 34.0 24.2 29.8 29.6 29.5 28.4 27.6 27.4 27.3 27.1 26.2 24.6 36.3 77.6 20.9 – 171.3 – 19.2
102.1 82.3 76.4 70.0 76.2 61.3 103.9 75.3 73.5 70.3 72.0 62.4 173.0 130.0 129.3 – – 33.4 25.0 30.2 30.1 29.7 28.8 28.7 28.1 27.9 27.7 26.9 26.6 37.2 78.3 20.2 – 170.8 – 19.4
100.7 83.5 76.6 70.0 76.2 61.3 105.1 75.0 76.2 69.6 74.2 62.9 173.9 129.9 129.3 – – 33.6 24.3 29.6 29.4 29.2 28.6 28.2 27.8 27.4 26.8 25.6 25.1 36.1 76.2 20.3 – – – –
101.2 82.4 76.4 70.1 73.5 63.5 104.3 74.7 76.1 70.0 74.2 63.4 176.4 129.5 129.4 – – 33.6 24.7 29.5 29.4 29.4 29.0 28.9 28.9 28.8 26.8 26.7 24.9 36.5 77.2 20.5 171.3 171.3 19.3 19.5
101.0 82.5 76.6 70.0 76.3 61.3 104.3 74.7 76.1 70.0 74.2 63.6 176.3 129.5 129.4 – – 33.6 24.7 29.5 29.4 29.4 29.0 28.9 28.8 28.8 26,8 26.7 24.8 36.3 76.7 20.4 – 171.3 – 19.5
101.5 80.2 76.7 70.1 73.6 63.4 103.2 74.5 76.4 70.4 77.0 61.7 176.4 129.5 129.4 – – 33.6 24.7 29.5 29.4 29.2 29.0 28.9 28.8 28.8 26,8 26.7 24.9 36.6 78.1 20.5 171.2 – 19.3 –
101.4 80.5 76.9 70.1 76.4 61.7 103.3 74.5 76.4 70.4 77.0 61.4 176.9 129.5 129.4 – – 34.0 24.9 29.5 29.4 29.4 29.0 28.9 28.9 28.8 26.8 26.7 24.9 36.4 77.5 20.5 – – – –
101.6 81.4 76.5 70.0 76.3 61.3 103.5 74.5 76.4 70.1 76.9 61.3 176.3 129.5 129.4 – – 25.5–34.2
C16 C17 C18 C=O (Ac’) C=O (Ac") OCOCH3’ OCOCH3"
37.3 79.6 21.3 171.8 170.6 20.9 20.8
69.7 – – – –
SLl x, Sophorolipid lactone form x; SLo x, Sophorolipid open form x; Ac, acetyl function.
n-butanol. In order to investigate these perturbations in more details and to see if the mobile phase gradient has strongly affected the initial state of the stationary phase, the exact compositions of both phases were determined at different times of the process (t0 min , t80 min , and t140 min ) by 1 H NMR spectroscopy. As shown in Table 2, the amount of n-heptane in the stationary phase was negligible and the amount of water was stable around 60% over the whole CPC experiment. By contrast, the amounts of methanol, n-butanol, and ethyl acetate in the stationary phase were significantly different from the beginning to the end of the process. The amount of methanol for instance decreased from 31.6 to 22.8%, that of ethyl acetate from 8.1 to 2.6% while the amount of n-butanol increased from 0.2 to 14.1%. These data therefore suggest that the hydrodynamic perturbations occurring during the CPC separation procedure may have resulted not much from the tensioactive properties of sophorolipids, but above all C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
from the modifications of the stationary phase composition during the mobile phase gradient. This hypothesis was confirmed by performing the same gradient elution experiment without injection. The loss of stationary phase observed during this blank experiment was similar to that observed after loading the crude sophorolipid extract (data not shown). The loss of stationary phase was observed from 96 to 166 min, corresponding to the increase of the n-butanol concentration in the mobile phase. In order to recover the most hydrophilic compounds still retained inside the CPC column at this end of the experiment, the stationary phase was finally extruded with n-heptane in the descending mode, yielding the fraction pool F9. This pool was directly analyzed by NMR, indicating the only presence of sophorose ␣ as major compound and of sophorose  as minor one. The 1 H and 13 C NMR chemical shifts of both compounds are given in Table 3. www.jss-journal.com
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Table 5. 1 H NMR chemical shifts of identified sophorolipids
Position
SLA I
SLA II
SLA III
SLA IV
SLA V
SLA VI
SLD I
SLD II
SLD III
SLD IV
SLD V
H1’ sucre H2’ sucre H3’ sucre H4’ sucre H5’ sucre H6’ sucre H1"’ sucre H2" sucre H3" sucre H4" sucre H5" sucre H6" sucre HC=CH HC=CH HC=CH HC=CH H2 H3 CH2 fatty acid chain H16 H17 H18 OCOCH3’ OCOCH3"
4.5 3.7 3.7 3.4 3.4 4.3/4.4 4.6 3.5 3.7 5.0 3.7 4.2/4.4 5.4 5.4 – – 2.4 1.6 1.2–2.1 1.5 3.8 1.2 2.1 2.1
4.5 3.6 3.7 3.5 3.5 4.3/4.4 4.6 3.6 3.7 5.0 3.7 4.2 5.4 5.4 5.4 5.4 2.4 1.7 1.2–2.8 1.6 3.8 1.3 2.1 2.1
4.4 3.5 3.6 3.3 3.5 4.2/4.4 4.9 3.4 5.0 3.5 3.6 4.3/4.4 5.4 5.4 – – 2.4 1.4 1.2–2.1 1.4/1.5 3.7 1.2 2.1 2.1
4.4 3.5 3.6 3.3 3.3 3.7/3.9 4.9 3.4 5.0 3.5 3.6 4.2/4.4 5.4 5.4 – – 2.4 1.7 1.2–2.1 1.4/1.5 3.8 1.2 – 2.1
4.5 3.4 3.6 3.3 3.3 3.6 4.7 3.3 3.6 4.9 3.7 4.1 5.4 5.4 – – 2.4 1.5 1.3–2.2 1.5/1.6 3.7 1.3 – 2.1
4.5 3.3 3.6 3.3 3.3 3.7/3.9 4.5 3.3 3.4 3.4 3.4 4.2/4.4 5.4 5.4 – – 2.4 1.6 1.3–2.1 1.7 3.8 1.2 – –
4.5 3.4 3.6 3.3 3.5 4.2/4.4 4.6 3.3 3.4 3.3 3.5 4.2/4.4 5.4 5.4 – – 2.3 1.6 1.3–2.1 1.5/1.6 3.8 1.2 2.1 2.1
4.5 3.4 3.8 3.3 3.3 3.7/3.9 4.6 3.3 3.4 3.3 3.5 4.2/4.4 5.4 5.4 – – 2.3 1.6 1.2–2.1 1.4/1.6 3.6 1.2 – 2.1
4.5 3.5 3.6 3.3 3.5 4.2/4.4 4.7 3.3 3.4 3.3 3.3 3.7/3.9 5.4 5.4 – – 2.3 1.6 1.3–2.2 1.5/1.6 3.8 1.3 2.1 –
4.5 3.5 3.6 3.3 3.3 3.7/3.9 4.7 3.3 3.4 3.3 3.3 3.7/3.9 5.4 5.4 – – 2.3 1.6 1.3–2.1 1.5/1.6 3.9 1.3 – –
4.4 3.5 3.6 3.4 3.3 3.7/3.9 4.6 3.3 3.4 3.3 3.3 3.7/3.9 5.4 5.4 – –
3.4 Sophorolipid identification In order to obtain highly pure sophorolipids, the fraction pools from F2 to F8 were further subjected to semipreparative HPLC. In total, 11 compounds were recovered, dried and their structures were characterized by analytical HPLC and NMR analyses. HPLC retention times were determined by adding an aliquot of each isolated sophorolipid to the initial crude extract in separate HPLC runs. As a result, each peak of the crude extract chromatogram and corresponding to an identified structure was annotated (Fig. 2). It should be noted that the HPLC retention times of the sophorolipids analyzed on the reverse-phase C18 chromatographic column were totally inverted as compared to the elution order of the same structures during the CPC separation process. For instance, on the HPLC chromatogram of Fig. 2, the less polar lactone forms were only eluted from 30 to 40 min after the open acid forms, while the same lactone forms eluted at the beginning of the CPC process (from F2 to F4) before the open acid forms. These data are absolutely logical since both techniques (HPLC and CPC) are working in a partition mode. 1D and 2D NMR experiments enabled the validation of sophorolipid structures either under lactone (noted SLl) or open acid forms (SLo). The 13 C and 1 H NMR chemical shifts of these structures are presented in Tables 4 and 5, respectively. In agreement with previously reported NMR signal assignments [22, 23], SLl I was identified as a diacetylated sophorolipid with a lactonic bond at the C4 sugar position and linked to an oleic (C18:1) chain at the subter C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1.1–2.4
3.6/3.9 – –
minal C17 position (Fig. 1). SLl II was identical to SLI I except the fatty acid part that was a linolenic (C18:2) chain. SLl III, SLl IV, SLl V, and SLl VI were all identified as lactonic sophorolipids with the sugar part linked to an oleic chain at the subterminal position. The differences between these four structures arise from the acetylation degree and from the position of the lactone bond (Fig. 1). The lactone bond located at the C4" sugar position (SLl I, SLl II, SLl V) is usual among sophorolipid structures [5]. However, to our knowledge this is the first time that sophorolipids having a lactone bond at the C3 (SLl III and SLl IV) or C6 (SLl VI) sugar positions, are presented. These new structures were confirmed by additional 2D NMR experiments (hsqc, hmbc, and cosy, data not shown). Recent data dealing with the microbial synthesis of sophorolipids have reported that the lactonization step occurs between the fatty acid carboxyl group and the C4 position of the sophorose unit [24]. In some rare cases lactonization could also occur at the C6 or C6 position, but up until now, no specific enzyme mediating this lactonization step either at the C4", C6 , C6 , or C3 has been identified. SLo I was identified as a diacetylated open acid form linked to a C18:1 fatty acid chain in the subterminal position, SLo II and SLo II were identical to SLo I but were monoacetylated at the C6 and C6 position, respectively. SLo IV was identified as an unacetylated open acid form linked to a C18:1 fatty acid chain in the subterminal position and SLo V was identified as an unacetylated open acid form linked to a C18:1 fatty acid chain in the terminal position. www.jss-journal.com
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4 Concluding remarks
[7] Shin, J. D., Lee, J., Kim, Y. B., Han, I. S., Kim, E. K., Bioresource Technol. 2010, 101, 3170–3174.
A centrifugal partition chromatography method developed in the gradient elution mode was investigated for the purification of individual sophorolipid species from a crude fermentation extract of C. bombicola yeasts. The results demonstrate that, even starting from a highly complex extract containing more than 20 target compounds and exhibiting tensioactive properties, CPC enables the removal of free fatty acids and residual sophorose in a single run while fractionating the different sophorolipid species in a wide hydrophobicity range. The combination of CPC to semipreparative HPLC as orthogonal technique has resulted in the recovery of highly pure sophorolipids that were individually identified. This purification strategy can be interesting for further biological evaluation of individual glycolipid structures as well as for a better understanding of their structure-activity relationships.
[8] Hu, Y. M., Ju, L. K., J. Biotechnol. 2001, 87, 263–272.
The authors thank the society “Soliance” for financial support, workforce, and discussions. The authors have declared no conflict of interest.
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