J. ISSAAS Vol. 18, No.1: 70-75 (2012) COMPARATIVE STUDY ON SAMPLE PREPARATION METHODS FOR THE HPLC QUANTIFICATION OF ITURIN FROM CULTURE SUPERNATANT OF AN ANTAGONISTIC BACILLUS STRAIN Kenji Yokota, Maiko Yatsuda, Eitaro Miwa, and Kyoko Higuchi Department of Applied Biology and Chemistry, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan Corresponding author: yokota@nodai.ac.jp (Received: November 11, 2011; Accepted: March 12, 2012) ABSTRACT Iturin is one of the antimicrobial cyclic lipopeptides produced by antagonistic strains of Bacillus spp. This study evaluated sample preparation methods for high-performance liquid chromatography (HPLC) quantification of iturin from culture supernatant of an iturin-producing Bacillus strain. The acid precipitation and methanol extraction (APME) method showed a lower efficiency than the butanol extraction and methanol substitution (BEMS) method. Direct application of culture supernatant to HPLC analysis showed the lowest efficiency. The BEMS method was the most effective method for quantifying the amount of iturin from culture supernatant based on the percent recovery and the time required to perform the analysis. Key words: lipopeptides, biosurfactant, antifungal, biological control INTRODUCTION The antagonistic strains of Bacillus spp. are recognized as biological control agents against several kinds of plant diseases, and are expected to use in countries of Southeast Asia region. Bacillus spp. show highly tolerance against high temperature, dryness and storage, because of its endospore forming. Furthermore, in the Southeast Asia region, it has been thought that there are many of biological resources which are effective Bacillus strains as biological control agents. Iturin is a circular lipopeptide produced by antagonistic strains of Bacillus spp. (Hiradate et al., 2002). Iturin consists of a heptapeptide circularized with a -amino fatty acid, which has homologous variants of different lengths, and the fatty acid chains have differing isometry (Fig. 1) (Hiradate et al., 2002). Lipopeptides with antifungal activity have been identified in a broad range of host strains (Phae and Shoda, 1991), and many Bacillus spp. that produce iturin have been employed as biological control agents against several plant diseases; e.g., mulberry anthracnose caused by Colletotrichum dematium (Yoshida et al., 2001). A dose-dependent effect on disease suppression when iturin was applied to soil against tomato damping-off caused by Rhizoctonia solani (Mizumoto et al. 2007). Therefore, the evaluation of iturin production by antagonistic Bacillus strains is one of the critical factors in screening for effective strains to act as antagonists. Several chemical properties of iturin have been reported so far. Besson et al. (1976), reported that iturin in the culture supernatant of B. subtilis is precipitated by acidification to ph 3.0 by using HCl and can be extracted from the precipitates using organic solvents. 70
Comparative study on sample preparation methods for the HPLC quantification of iturin.. Two major sample preparations for high-performance liquid chromatography (HPLC) -based quantification of cyclic lipopeptides have previously been reported (Phae and Shoda. 1991; Yazgan et al., 2001). The acid precipitation and methanol extraction (APME) method was developed by Phae and Shoda (1991) and widely used for the quantification of iturin (Hsieh et al., 2008; Grover et al., 2010). The other method, the 1-butanol extraction and methanol substitution (BEMS) method was developed by Yazgan et al. (2001) and widely used for quantification of antimicrobial lipopeptide in culture supernatants ( Romero et al. 2007). This present study evaluated these sample preparation methods for the quantification of iturin in culture supernatant. Strain and culture conditions MATERIALS AND METHODS Bacillus subtilis ATCC 21556 was used as an iturin-producing bacterium. No.3S medium containing 1% Polypepton S (Nihon Pharmaceutical, Tokyo Japan), 1% glucose, 0.1% K 2 HPO 4, and 0.05% MgSO 4 7H 2 O was used as the culture medium. (Tsuge et al., 2001). A culture shaken for 2 days at 25 C was used for the quantification of iturin. Partial purification of iturin from solid-state culture Partially purified iturin was obtained from a solid-state culture of B. subtilis ATCC 21556 for the recovery test. Soybean curd residue (Soyaflour, The Nisshin OilliO Group, Ltd., Tokyo, Japan) was used as a solid-state medium. One hundred grams of solid-state medium (60% water content) was placed in a 500-mL plastic case and autoclaved at 121 C for 30 min. A B. subtilis ATCC 21556 culture shaken for 2 days was inoculated onto the solid-state medium at an initial concentration of 10 7 cells g -1 fresh weight (FW) a nd incubated for 5 days at 25 C. The solid-state culture (100g) was extracted using 300 ml of 1-butanol and shaking for 10 min at room temperature. The butanol fraction was collected by centrifugation at 5000 g for 5 min at 20 C, washed with distilled water, and dried using a rotary evaporator. The residue was dissolved in 200 ml of methanol, and 10 g of ODS resin (Wakosil, Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to the methanol extraction. Distilled water was added to the ODS mixture (up to 30% of methanol concentration). The ODS resin was washed using 30% methanol and applied to the column. Iturin was eluted from the column using 100% methanol. Preparation procedures Culture supernatants were collected by centrifugation at 8000 g for 5 min at 4 C and divided for further preparation. 71
J. ISSAAS Vol. 18, No.1: 70-75 (2012) APME This method was developed by Phae and Shoda (1991). Forty milliliters of culture supernatant was acidified to a ph of 2.0 using 12 M HCl and then incubated for 18 h at 4 C. Precipitates were collected by centrifugation at 8000 g for 10 min at 4 C and then extracted with 4 ml of methanol for 30 min by shaking. The methanol extract was collected by centrifugation at 8000 g for 10 min at 4 C. BEMS This method was according to Yazgan et al. (2001) with some modifications. Four milliliters of culture supernatant was extracted using 1 ml of 1-butanol by vortex mixing for 20 s. After centrifugation at 10000 g for 2 min at 20 C, the organic (upper) layer was collected by a pipette. The remaining aqueous layer was extracted twice using 300 L of 1-butanol. The organic fractions were combined and evaporated by centrifugal evaporator (VC -36S, Tietech Co., Ltd., Nagoya, Japan), and the dried sample was dissolved using 300 L of methanol. Direct method The direct method was carried out by direct application of a culture supernatant for HPLC analysis. All samples were filtered using a 0.45- m PTFE membrane (ADVANTEC, Advantec MFS, Inc., Dublin, CA USA) prior to HPLC analysis. The precision of the APME and BEMS methods was defined as percent column volume (CV) based on 10 replications of each method. Recovery test The recovery test of iturin from culture supernatant was carried out by adding partially purified iturin to a culture supernatant of B. subtilis ATCC 21556. Ten milliliters of partially purified iturin methanol solution (1.8 mg ml -1 ) was added to 600 ml of culture supernatant and then divided for further preparation. HPLC analysis HPLC analysis was performed using a Shimadzu LC-10 HPLC system at 205 nm with an LC-10Av UV-VIS spectrophotometer (Shimadzu, Kyoto, Japan); the column was a Mightysil RP-18 GP containing ODS (5 μm, 250 mm 4.6 mm ID; Kanto Chemical Co., Tokyo, Japan). The column conditions for iturin analysis were 65% methanol (mobile phase), 1 ml/min, 45 C. Injection volume was 10 L. Data collection was performed using a Chromato-PRO data integrator (Run Time Co., Kanagawa, Japan). Iturin standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). The lower limit of detection was 0.57 g ml -1 by HPLC analysis for each iturin homologs (data not shown). RESULTS AND DISCUSSION The antimicrobial lipopeptide iturin has homologous variants of fatty acid moieties; 7 homologs (A2 to A8) have been identified so far (Fig. 1). This study detected 5 homologs of iturin, A2 to A6, from an iturin standard purchased from Sigma-Aldrich. A chromatograph of iturin homologs A2 to A6 in the culture supernatant of B. subtilis ATCC21556 is shown in Fig. 2. Each peak of iturin was collected and confirmed by MALDI-TOF-MS (Shimadzu, Kyoto, Japan). 72
Comparative study on sample preparation methods for the HPLC quantification of iturin.. Fig. 2. Chromatogram of iturin homolog extract using the BEMS method and culture supernatant of Bacillus subtilis ATCC 21556. Iturin concentrations in the culture supernatant of B. subtilis ATCC21556 prepared using the APME, BEMS, and direct methods are shown in Table 1. With the BEMS method, each homolog of iturin was detected at concentrations greater than 3 times those found using the APME method. The BEMS method also had lower CV values for each homolog compared to the APME method. The ratio of each homolog (iturin A2 to A5) was approximately the same between the APME and BEMS methods. The direct method had the lowest efficiency for iturin quantification. Percent recovery values of each iturin homolog are shown in Table 2. The BEMS method had the highest percent recovery among the 3 preparation methods. For the BEMS method, all homologs were almost completely recovered. In contrast, the APME method recovered less than 15% of the total iturin. However, the recovery of iturin A2 was much lower than A6 recovery by APME in this study. This suggests that the acid precipitation step in the APME method might cause the low efficiency of recovery. Bie et al. (2004) repor ted a related study on sample preparation method for HPLC quantification of iturin. They screened the main factors affecting extraction of iturin from the precipitates which were collected by centrifugation followed by acidified of the culture supernatant in APME method of this study. They observed that methanol or ethanol showed higher efficiency than propanol or 1-butanol, which was used as the organic solvent for extraction of iturin. The reason for the low efficiency associated with the APME method remains unclear. For the BEMS method, we modified the ratio of culture supernatant and 1-butanol for extraction from the original Yazgan s method ( Yazgan et al., 2001). In the original method, they extracted by one-fourth volume of 1-butanol against culture supernatant for 3-times; whereas this study used one-fourth volume of 1-butanol once, and further extraction by 3/40-times volume of 1-butanol twice. There was no significantly difference in the recoveries of iturin from culture supernatant between the original and modified method (data not shown). The direct method showed the lowest percent recovery for all iturin homologs. No iturin homologs were recovered other than iturin A2. Thimon et al. (1992) reported that the critical micelle concentration of iturin in 0.1 M NaHCO 3 solution is 43 M at 25 C. The concentration of 73
J. ISSAAS Vol. 18, No.1: 70-75 (2012) total iturin in the culture supernatant of B. subtilis ATCC21556 determined by the BEMS method was approximately 700 M. Therefore, it is likely that the low recovery efficiency for the direct method was caused by the formation of micelles of iturin in the culture supernatant. Table 1. Effect of sample preparations for iturin quantification. Preparation Iturin (mg ml -1 ) a A2 A3 A4 A5 A6 Total A2 A3 A4 A5 A6 Total CV b APME c 14.2 25.5 82.8 67.5 5.4 195.4 0.088 0.086 0.0820.107 0.117 0.089 BEMS d 46.3 101.5 296.1 251.2 37.8 732.8 0.064 0.071 0.0700.070 0.065 0.069 Direct 1.2 0.2 0.6 n.d e n.d 2.0 n.t f n.t n.t n.t n.t n.t Table 2. Percent recovery of iturin homologs by preparation method Preparation Recovery of iturin (%) a A2 A3 A4 A5 A6 Total APME b 3.7 20.5 14.2 11.9 58.1 14.1 BEMS c 97.0 98.5 99.2 100.6 98.6 99.6 Direct 11.5-2.0-0.3 0 0 0.5 a Mean value of 10 replications b Acid precipitation and methanol extraction c Butanol extraction and methanol substitution CONCLUSIONS In this study, we evaluated the sample preparation methods for antifungal lipopeptide iturin in culture supernatant for HPLC analysis. Based on percent recovery and method precision, we conclude that the BEMS method is the most effective sample preparation method for iturin quantification in the culture supernatant of antagonistic Bacillus strains. ACKNOWLEDGEMENTS We thank Mr. Shin Kanuma of the Nisshin OilliO Group, Ltd. for providing soybean curd residue. This study was supported by Takano Life Science Research Foundation and the Academic Frontier Research Project at the Tokyo University of Agriculture. REFERENCES Besson, F., Peypoux, F., Michel, G., and Delcambe, L. 1976. Characterization of Iturin A in antibiotics from various strains of Bacillus-subtilis. J. Antibiot. 29:1043-1049 Bie, X.M., Lu, Z.X., Lu, F.X., and Zeng, X.X. 2005. Screening the main factors affecting extraction of the antimicrobial substance from Bacillus sp fmbj using the Plackett-Burman method. 74
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