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

Abstract

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.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 (K 2 HPO 4 ⅐ 3H 2 O, 0.5 g; MgSO 4 ⅐ 7H 2 O, 0.2 g; NaCl, 0.2 g; MnSO 4 ⅐ H 2 O, 0.2 g; FeCl 3 , 0.002 g; D-mannitol, 10 g; distilled H 2 O, 1 liter) plus 1% NaNO 3 and 1% proline in a 2-liter flask. Cultures were grown for 18 to 22 h at 37°...

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