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Applied and Environmental Microbiology, January 2004, p. 631-634, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.631-634.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Bacilysin, Chlorotetaine, and Iturin A Produced by Bacillus sp. Strain CS93 Isolated from Pozol, a Mexican Fermented Maize Dough

Trevor G. Phister, Daniel J. O'Sullivan, and Larry L. McKay^*

Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108

Received 21 April 2003/ Accepted 10 October 2003

	ABSTRACT

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Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry. The three compounds were iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.

	INTRODUCTION

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Pozol is a fermented maize dough that is consumed by the indigenous Mayan peoples of southeastern Mexico (2, 13). Present-day ethnic groups with Mayan ancestry use pozol as a source of nutrients and, as did the early Mayans, in ceremonies promoting the growth and harvest of maize (2, 12, 13). The early Mayans consumed pozol since at least 1560 AD (3, 13). They also used pozol as a medicine to control diarrhea, to reduce fever, and to cure intestinal infections. Pozol was used on wounds as a poultice to prevent infection (16). Pozol was later found to inhibit a number of different bacteria, yeasts, and molds (4).

The maize fermentation that produces pozol is uncontrolled and involves yeasts, molds, and bacteria (12, 14). The primary fermentation organisms are lactic acid bacteria (6). Ampe et al. (1) found that Lactococcus and Leuconostoc spp. were dominant at the start of the fermentation, but Lactobacillus and Streptococcus spp. dominated by the end of the fermentation. Aerobic mesophilic bacteria and Enterobacteriaceae were also present during the fermentation (17).

A number of other bacteria have consistently been found to be part of pozol fermentation, including an organism initially identified as Agrobacterium azotophilium (13). This organism was found to inhibit the growth of a number of gram-positive bacteria, gram-negative bacteria, yeasts, and molds (15).

The inhibitory activity of this organism could account for the early Mayan culture's use of pozol as a medicine and for the inhibitory effect of pozol, as seen in early experiments by Herrera and Ulloa (4). A. azotophilium was, however, misidentified in these early experiments and has since been reisolated, reclassified as Bacillus sp. strain CS93 by 16S rRNA sequencing, and shown to produce broad-spectrum antimicrobial activity (10). The strain was deposited with the Northern Regional Research Laboratory, Peoria, Ill., as NRRL B-21974.

The inhibitory activity of strain CS93 was exhibited over a broad range of pH (3 to 11) and was heat stable (10). Activity against all test organisms was inactivated by pronase E, suggesting that the compound(s) is proteinaceous (10). The objective of this study was to identify the compound(s) responsible for the antimicrobial activity of Bacillus sp. strain CS93 in pozol that could account for the inhibitory effects of pozol and the medicinal significance of this food to the ancient Mayans.

Inhibitory activity was produced in 250 ml of Fred Waksman Basic 77 (K₂HPO₄ · 3H₂O, 0.5 g; MgSO₄ · 7H₂O, 0.2 g; NaCl, 0.2 g; MnSO₄ · H₂O, 0.2 g; FeCl₃, 0.002 g; D-mannitol, 10 g; distilled H₂O, 1 liter) plus 1% NaNO₃ and 1% proline in a 2-liter flask. Cultures were grown for 18 to 22 h at 37°C with shaking at 225 rpm. The cells were removed by centrifugation at 9,000 x g for 10 min, and the supernatant was filtered through a 0.45-µm-pore-size filter and lyophilized for 18 h with a Hetovac VR-1 vacuum concentrator (Heto Lab Equipment A/S Birkerod, Denmark). When 100 mg of lyophilized, active, cell-free CS93 culture supernatant was injected onto a preparative Econosil C₁₈ reverse-phase high-performance liquid chromatography (HPLC) column (250 by 22 mm; Alltech Associates Inc., Deerfield, Ill.), eight peaks were observed (Fig. 1). The seventh peak (Fig. 1, second peak from the right) exhibited activity in a disk assay against bacteria, yeasts, and molds, while the eighth peak (Fig. 1, rightmost peak) exhibited activity against yeasts and molds (Fig. 2). After this purification step, 0.2 mg of the lyophilized peak produced a zone of inhibition that was three times as large as that produced by 1 mg of cell-free supernatant against E. coli and six times as large as that produced by 1 mg of cell-free supernatant versus mold. This suggested that more than one antimicrobial compound is produced by CS93. The eighth peak was collected and separated on an analytical C₁₈ HPLC column. The sample contained peaks that corresponded to the peaks exhibited by a commercial sample of iturin A subjected to the same chromatographic conditions (data not shown). The presence of iturin A was confirmed by using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MS) and comparing the strain CS93 iturin sample spectrum to that of the commercial iturin A sample generated on the same mass spectrometer. Both samples exhibited peaks having mass numbers of m/z 1,043.2, 1,065.2, and 1,079.3; however, the eighth peak was not as concentrated and the peak at m/z 1,057.2 was not present. The latter peak was present following concentration of the sample (Fig. 3), although the peaks from the strain CS93 iturin sample had shifted by 0.5 mass unit owing to recalibration of the instrument. It is the 14 mass unit difference between the peaks that suggests the presence of an iturin, as this difference is characteristic of the two fatty acids of 14 and 15 carbon atoms that are called iturinic acids and are associated with iturin (7).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Preparative reverse-phase (C18) chromatography of ultrafiltered permeate of CS93 cell-free supernatant. Lyophilized, cell-free supernatant was resuspended at 100 mg/ml in H2O. A 1-ml sample was applied to the column. A gradient elution of 2 to 20% methanol over 8 column volumes was used for separation. The flow rate was 10 ml/min, with detection at 280 nm. Each peak was collected and screened for activity against E. coli and Absidia sp. in a disk assay. Activity was found in the seventh and eighth peaks (second peak from the right and rightmost peak, respectively).

View larger version (98K): [in this window] [in a new window]   FIG. 2. Disk assay of preparative C18 HPLC fractions from Fig. 1. In panel A, the fractions were dried and resuspended in H2O at 100 mg/ml. A 0.2-mg sample was applied to each disk, dried for 20 min, and placed on a nutrient agar plate swabbed with E. coli. Plates were incubated overnight at 37°C. Each disk number corresponds to a peak in Fig. 1; disk C is the H2O control. In panel B, the fractions were treated as in panel A and disks were placed on a potato dextrose agar plate swabbed with Absidia sp. The plates were incubated for 2 days at 25°C. Each disk number corresponds to a peak in Fig. 1; disk C is the H2O control.

View larger version (23K): [in this window] [in a new window]   FIG. 3. (A) Matrix-assisted laser desorption-ionization mass spectra of iturin A (Sigma). (B) The antifungal compound found in preparative reverse-phase HPLC peak 8 of lyophilized supernatant of strain CS93 cells grown in Basic 77.

The seventh peak was screened for activity against E. coli and Absidia sp. (Fig. 2). Peak 7 was then collected and analyzed by HPLC-MS. The electrospray mass spectra were recorded on a Finnigan (San Jose, Calif.) LCQ ion trap mass spectrometer in positive-ion mode. The sample (20 µl in 50 mM formic acid) was loaded onto a Finnigan HPLC apparatus, and separation was conducted with a polyhydroxyethyl aspartamide 200-A pore column (200 by 9.4 mm; PolyLC, Inc., Columbia, Md.) with 50 mM formic acid. This separation introduced the sample into the sprayer. The capillary temperature was 200°C, and the voltage was 650 V. A peak at m/z 271.1 was observed (Fig. 4). This peak was identical to the published electrospray mass spectrum reported for bacilysin (19).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Electrospray mass spectrum of preparative reverse-phase HPLC (C18) peak 7 (Fig. 1) of the lyophilized, cell-free supernatant of CS93 grown in Basic 77. The sample was loaded onto a Finnigan HPLC apparatus for size exclusion chromatography on a polyhydroxyethyl aspartamide column with 50 mM formic acid as the buffer. The sample eluted from the column at 17.69 to 18.18 min. A Finnigan LCQ MS apparatus in positive-ion mode was used to record the mass spectrum. The mass at m/z 271.1 suggested the presence of bacilysin plus a proton.

The only component of bacilysin missing in the MS-MS analysis was alanine. In the initial MS-MS analysis involving bacilysin, the lowest m/z allowed by the mass spectrometer was not low enough to detect alanine. However, the peak at m/z 182.1 suggested that alanine was present, as this peak, if subtracted from m/z 271.1 (the bacilysin peak), would represent a loss of one alanine (89 Da). Further, amino acid analysis of fraction 7 showed alanine to be a primary amino acid and small amounts of anticapsin (tyrosine) to also be present. Therefore, by combining the amino acid analysis and the mass spectral analysis, it was concluded that both anticapsin and alanine were present in fraction 7, and thus the peak at m/z 271.1 represented bacilysin.

The presence of another antimicrobial compound in peak 7 was also evident in the electrospray mass spectrum (Fig. 4). The peak at m/z 289.1 and its isotopic peak at m/z 291.1 represent the mass spectrometric signature for chlorotetaine (8). This compound is structurally similar to bacilysin and is produced by Bacillus subtilis BGSC 1E2 along with bacilysin. As the two inhibitors are structurally similar, Rapp et al. (8) demonstrated that chlorotetaine was not an isolation procedure artifact by purifying bacilysin from other B. subtilis strains. BGSC 1E2 was the only strain to produce both bacilysin and chlorotetaine. Thus, CS93 is only the second strain reported to produce chlorotetaine and the first strain found to produce all three antibiotics (8, 18).

That the antibacterial activity of CS93 was due to the presence of bacilysin and chlorotetaine is also supported by three further observations. First, the inhibitory activity against gram-positive and gram-negative bacteria was inactivated only by pronase E, which is the only protease that inactivates bacilysin (10, 11). Second, the inhibitory activity against E. coli was reversed by the addition of N-acetylglucosamine, a known competitive inhibitor of the activity of bacilysin (5). Third, the activity of peak 7 was much stronger against Absidia sp., the mold indicator, than against E. coli. This higher level of activity against molds is also characteristic of bacilysin (5).

The presence of bacilysin, chlorotetaine, and iturin A may explain the effectiveness of the traditional medicinal uses of pozol by the Mayans (2, 13, 16). These traditional Mayan uses for pozol suggest that both bacilysin and chlorotetaine have potential as biopreservatives in food, as these compounds have been consumed in pozol by indigenous populations for centuries. Both compounds are water soluble and active over a wide pH range, which could allow their application in a wide variety of food products. It is possible that the broad spectrum of activity may not be exhibited in certain foods, as some bacteria were only sensitive to the compounds on minimal medium (5). However, Ray (9) found effective inhibition of E. coli (a 5-log reduction in apple juice) in tests of the CS93 supernatant in both apple juice and meats. Further work on these antimicrobials produced by the pozol isolate might eventually lead to their use in food preservation systems. It would also be of interest to evaluate the presence of these antimicrobials in pozol itself.

	ACKNOWLEDGMENTS

 
We thank Tom Krick and Leann Higgins of the University of Minnesota Mass Spectrometry Consortium for the Life Sciences for help with MS.

This work was funded in part by the Kraft General Food Chair and by Quest International.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108. Phone: (612) 624-3090. Fax: (612) 625-5272. E-mail: lmckay{at}umn.edu.

Present address: Department of Viticulture and Enology, University of California, Davis, Davis, CA 95616-8749.

	REFERENCES

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