Classification of a Bacterial Isolate, from Pozol, Exhibiting Antimicrobial

1123

Journal of Food Protection, Vol. 63, No. 8, 2000, Pages 1123–1132 Copyright ᮊ, International Association for Food Protection

Classiﬁcation of a Bacterial Isolate, from Pozol, Exhibiting Antimicrobial Activity against Several Gram-Positive and Gram-Negative Bacteria, Yeasts, and Molds†

P. RAY, C. SANCHEZ, D. J. O’SULLIVAN, AND L. L. McKAY*

Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108, USA

MS 00-12: Received 11 January 2000/Accepted 4 April 2000 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021

ABSTRACT

A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product. This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators. By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identiﬁed as members of the genus Bacillus, possibly a variant of Bacillus subtilis. While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identiﬁed for production in broth. The compound(s) was heat stable (121ЊC for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E. The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae. The inhibitory compound(s) could possibly serve as a food biopreservative.

Current consumer trends for minimally processed of their food preservation, but also due to certain mysticism foods with a long shelf life, coupled with concerns over the associated with their consumption. possible ill effects from chemical preservatives, have stim- Pozol is one such example. It is an indigenous fer- ulated the search for new, natural antimicrobial substances mented maize dough that dates back to the Mayan civili- effective against foodborne spoilage and pathogenic micro- zation (25). The Mayans used pozol to combat intestinal organisms (4). Potential natural biopreservatives being ex- disorders and as a poultice for wounds and skin infections amined for exploitation by the food industry include lytic (26). Herrera and Ulloa (7), in studying the microbiology enzymes, plant antimicrobials, and bacteriocins (21). Much of pozol during the 1970s, isolated a bacterium that was of the research on bacteriocins has concentrated on those thought to be responsible for pozol’s putative antagonistic produced by lactic acid bacteria. The presumed safety of properties, as the organism exhibited a broad spectrum of these bacteriocins is due to their production by microor- inhibition against many gram-positive and gram-negative ganisms generally regarded as safe and their sensitivity to bacteria, yeasts, and molds. This isolate was initially char- proteolytic enzymes present in the gastrointestinal tract of acterized as Agrobacterium azotophilium. However, DNA– humans. Therefore, they have potential for being used as rRNA hybridization studies conducted by DeSmedt and biopreservatives in place of some currently used chemical Ley in 1977 (5) questioned its identity when the isolate additives (6, 9, 13). However, a drawback of using these exhibited a higher degree of homology to gram-positive bacteriocins is their narrow spectrum of activity and their bacteria than to Agrobacterium sp. To our knowledge, no ineffectiveness against gram-negative bacteria, yeasts, and one has attempted to explore further the antagonistic prop- molds. Therefore, as stated by Klaenhammer (12), there is erties of the pozol isolate since its initial isolation in the a demand for natural biopreservatives that are effective in 1970s. controlling a broad range of food spoilage and pathogenic Therefore, the objectives of this investigation were to bacteria, yeasts, and molds. As a result of this current in- conﬁrm the identity of the pozol isolate, determine its an- terest, many culture collections have been extensively timicrobial spectrum, examine the conditions for production screened for microorganisms capable of producing natural of the antagonistic compound(s), and investigate its chem- biopreservatives (20). Another source of new isolates ca- ical nature. This information could then possibly be used pable of producing biopreservatives could be indigenous in assessing the antimicrobial compound as a biopreserva- fermented foods that are primarily known only within their tive for use in enhancing the safety and extending the shelf country of origin. Some of these indigenous fermented life of our food supply. foods have endured the passage of time, not only because MATERIALS AND METHODS * Author for correspondence. Tel: 612-624-3090; Fax: 612-625-5272. † Published as paper number 001180007 of the contribution series of the Bacteria, yeasts, and molds and their maintenance. The Minnesota Agricultural Experiment Station based on research conducted original pozol isolate, designated in our laboratory as Aa91, was under project 18-062. obtained from Universidad Nacional Autonoma de Mexico, Mex- 1124 RAY ET AL. J. Food Prot., Vol. 63, No. 8

ico City, Mexico. We isolated CS93 from fresh pozol obtained in TABLE 1. Response of organisms to the antimicrobial com- Villahermosa, Tabasco, Mexico. Ten grams of pozol was diluted pound(s) produced by a Bacillus sp. isolated from pozol in 10 ml 0.85% NaCl, vigorously mixed, and plated on N-free Reaction Lipman and Basic 77 Fred and Waksman agar plates (25) to obtain to isolated colonies. Colonies were purified by streaking on nutrient inhibi- Zone sizea agar (Difco Laboratories, Detroit, Mich.) and then grown in nu- Indicator organism tor(s) (mm) trient broth at 37ЊC and tested for production of antimicrobial compound(s) against indicator bacteria. Bacterial strains used in Molds this study to determine the activity spectrum of CS93 are de- Alternia sp. ϩb 22 scribed in Table 1. All strains were maintained as frozen stock Eurotium sp. ϩ 19 cultures in 11% reconstituted nonfat dry milk at Ϫ60ЊC. Working Absidia sp. ϩ 34 stock cultures of pozol isolates were kept at 4ЊCinnutrient agar. Thaminidium sp. ϩ 20 Mold and yeast cultures used (Table 1) were maintained on malt Fusarium graminearum ϩ 19 Њ Њ extract agar (Difco) slants at 4 C and were grown at 25 C for 5 Fusarium sporoditicus ϩ 19 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021 days before use in inhibition assays. Micrococcus luteus and Esch- Fusarium popae ϩ 29 erichia coli V517 were selected as routine indicator organisms. Fusarium culorum ϩ 29 Strains were grown aerobically in nutrient broth at 37ЊC, stored Fusarium solanii ϩ 20 at 4ЊC, and subcultured every 2 weeks. Fusarium moniliforme ϩ 21 Fusarium oxysporum ϩ 22 Bacteriocin activity assays. Screening for the antimicrobial Penicillium expansum ϩ 18 compound(s) was performed using three methods: the flip streak Pencillium roquefortii ϩ 31/54c method (2), the membrane filter method (11), and the well dif- Penicillium digitatum ϩ 19 fusion assay (24). Agar concentration used varied with the meth- Penicillium patulin ϩ 25 od: 1.5% for the flip streak and well diffusion assays, and 0.8% Penicillium cyclopium ϩ 21 for the membrane filter method. Soft agar overlays used in the Penicillium citrinum ϩ 19 well diffusion and membrane filter assays were prepared with the Aspergillus niger ϩ 18 appropriate growth medium and 0.75% agar. In all three methods Aspergillus parasiticus Ϫ 0/23c the plates were incubated for the specific temperature and time Aspergillus clavatus ϩ 22 appropriate for the indicator organism used. Plates were examined Aspergillus terrus ϩ 22 for the presence or absence of a clear zone of inhibition against Aspergillus ochraceous ϩ 21 the indicator organism. Antimicrobial activity was quantified into Aspergillus candidus ϩ 21 arbitrary units (AU/ml) using the well diffusion assay. One AU Aspergillus versicolor ϩ 18 was defined as the reciprocal of the highest serial twofold dilution Aspergillus flavus ϩ 21 showing a zone of inhibition of the indicator organism (24). Yeasts Polymerase chain reaction of 16S rRNA. To obtain geno- Kluveromyces sp. ϩ mic DNA of CS93 and Aa91, the strains were grown in 10 ml Saccharomyces cerevisiae ϩ Њ nutrient broth for 18 h in a 37 C rotary shaker at 200 rpm. A 1.5- Schiziosaccharomyces sp. ϩ ml aliquant was centrifuged and the pellet was washed in 1 ml of Yogurt contaminant #2 ϩ ␮ sterile H2O. Cells were resuspended in 200 lofsterile H2O, and 0.5 volume of acid-washed glass beads (106 ␮morfiner) (Sigma Gram-positive bacteria Chemical Co., St. Louis, Mo.) was added to the tube. Tubes were Lactococcus lactis IFPL 359 ϩ placed in a MiniBeadbeater-8 (Biospec Products, Bartlesville, Lactobacillus plantarum IFPL 935 ϩ Okla.) for 10 s at homogenization speed. The tubes were micro- Listeria monocytogenes ϩ centrifuged, the supernatant was diluted 10-fold, and 1 ␮l was Staphylococcus aureus FF265 ϩ used in a polymerase chain reaction (PCR). S. aureus 196E Ϫ The primers used for amplification of bacterial 16S rRNA S. aureus FRI-100 Ϫ were selected from the literature (14). The sequence for the primer S. aureus 35 Ϫ correlating to positions 519 to 536 was CAGCWGCCGCGGTA Bacillus subtilis 6633 ϩ ATWC; the sequence ACGGGCGGTGTGTRC corresponded to Bacillus cereus 35 ϩ positions 1,392 to 1,406. In these sequences W ϭ AorTandR B. cereus TJL-8 ϩ ϭ AorG. B. cereus N9 ϩ ␮ The PCR mixture included 200 mM of each dNTP, 2.0 lof Gram-negative bacteria 10ϫ PCR buffer with MgCl , 0.25 ␮l Taq polymerase (all from 2 Escherichia coli V517 ϩ Perkin Elmer, Foster City, Calif.), 12 mM primer, 1 ␮l template, E. coli O157:H7 #1 ϩ and sterile H Otoatotal volume of 20 ␮l. The reaction was run 2 E. coli O157:H7 #2 ϩ under the following conditions in a Stratagene Robocycler PCR Helicobacter pylori ϩ machine: 35 cycles of 92ЊC for 1 min, 35ЊC for 1 min, 68ЊC for Salmonella typhimurium ϩ 1.5 min, and 1 cycle of 68ЊC for 5 min. Samples were analyzed Pseudomonas fragi ϩ on a 1% NuSieve GTG agarose (FMC Bioproducts, Rockland, Maine) gel in 1ϫ Tris-Acetate-EDTA buffer for 2 h at 7 V/cm. a Size of clear zone of inhibition against the indicator organism. Gels were stained in ethidium bromide (0.5 ␮g/ml) and examined b ϩ indicates a clear zone of inhibition against the indicator or- under ultraviolet light to visualize the banding patterns. ganism. The nucleotide sequences of the PCR products were deter- c Size of zone that includes area with no growth plus area where mined using a 373A automated DNA sequencer with a Prism growth was observed but sporulation did not occur. J. Food Prot., Vol. 63, No. 8 ANTIMICROBIAL(S) OF A POZOL ISOLATE 1125

Ready Reaction Dye Deoxy Terminator Cycle Sequencing kit quickly cooled and was evaluated for retention of the antimicro- (Applied Biosystems, Norwalk, Conn.). The sequence was ana- bial activity (17). lyzed using the DNAStar software package (DNAStar Inc., Mad- ison, Wis.). The sequence of the PCR fragment from CS93 and Effect of the antimicrobial compound(s) on indicator or- the 16S rRNA sequence of all Bacillus strains found in GenBank ganisms. To examine the effect of the active compound(s) against were used to construct a phylogenetic tree using the DNAStar indicator organisms in a broth system, the following assay was program. The phylogenetic tree was generated using the neighbor- conducted. A 1% inoculum of exponentially growing cells ϭ joining method. (OD600nm 0.6) from E. coli V517, M. luteus, or Saccharomyces cerevisiae was added to 9.9 ml of active culture supernatant. E. Triplicate arbitrary-primed PCR analysis. Bacteria were coli and M. luteus were grown at 37ЊC (220 rpm) in Luria-Bertani fingerprinted using a rapid and sensitive procedure termed tripli- or M17 broth, respectively. S. cerevisiae was grown in potato cate arbitrary-primed (TAP) PCR (3). A crude cell lysate was dextrose broth at 25ЊC (220 rpm). Control treatments included the prepared as previously described. PCR mixtures were prepared in indicator strain inoculated into nutrient broth, active supernatant a volume of 60 ␮l, containing 200 mM of each dNTP, 6.0 ␮lof treated with 1 mg/ml pronase E for 1 h, and active supernatant ϫ ␮ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021 10 buffer without MgCl2,5mMMgCl2,14mMprimer, 2 l treated with 1 mg/ml pronase E that was previously heat treated ␮ template, and 3.0 l Taq polymerase (Perkin Elmer). The reaction for 10 min in boiling water. The OD600nm was determined at ap- mixture was divided into six aliquots of 10 ␮l and amplified under propriate time intervals. the following conditions in a Stratagene Robocycler: 1 cycle at To determine the viability of the indicator cells after treat- 92ЊC for 1 min; 40 cycles at 92ЊC for 30 s; 35ЊC, 37ЊC, 39ЊC, ment with the active compound(s), a second set of experiments 40ЊC, 42ЊC, and 44ЊC for 1 min; 68ЊC for 1.5 min; and 1 cycle was conducted. Indicator strains were grown as described above, of 68ЊC for 5 min. The samples were run on a 1% gel in 1ϫ Tris- and 1% of the exponentially growing cells was added to 9.9 ml Acetate-EDTA buffer. active supernatant. Samples were taken every 15 min for 1 h and then at hourly intervals. Samples were serially diluted, and CFU/ Biochemical and physiological characteristics. CS93, ml was determined on the appropriate growth medium. Aa91, and Bacillus subtilis 6633 were tested for sugar metabolism using the API 50 CHB test kit (bioMerieux, Lyon, France) ac- Lytic effect of active compound(s) against E. coli V517. cording to manufacturer’s directions. A series of phenotypic tests Cell lysis was measured by collecting E. coli V517 cells grown Њ used to identify Bacillus sp. was conducted on CS93, Aa91, and to an OD600nm of 0.45 in 10 ml nutrient broth at 37 C (220 rpm). B. subtilis 6633 according to standard methods (23). After centrifugation, the pellets from individual tubes were resuspended in 10 ml of either active supernatant or nutrient broth. Influence of carbohydrate on growth and production of Samples were taken at intervals and measured at OD . antimicrobial compound(s). Various carbohydrates were tested 600nm for their ability to support the growth of CS93 and for production RESULTS of the antimicrobial compound(s) (1). Nutrient agar was individ- ually supplemented with 1% of the following carbohydrates: mel- Isolation and spectrum of inhibition of CS93. The izitose, lactose, glucose, fructose, sucrose, raffinose, xylose, sac- broad spectrum of the antagonistic compound(s) originally charose, potato starch, sorbose, arabinose, dextrin, mannitol, cel- reported by Ulloa and Herrera (26) to be produced by Aa91 libiose, cornmeal, corn starch, and maseca (lime-treated corn sparked interest in both the compound(s) and the bacterium. meal). We obtained Aa91 from Ulloa and Herrera to investigate Production studies in broth. Culture supernatants of CS93 its antagonistic behavior. Results revealed that Aa91 had an grown at 37ЊCin100 ml nutrient broth supplemented with man- attenuated spectrum of inhibition compared to the original nitol in a 500-ml flask (200 rpm) were collected hourly. Growth findings (22). This could have been due to the maintenance of CS93 was followed by monitoring the pH and OD at 600 nm. of the organism on agar slants at room temperature for over The samples were centrifuged, filter sterilized (0.45 ␮m pore size 20 years. filter), and used for antimicrobial activity assays. In an attempt to find an organism exhibiting the original spectrum of inhibition, we examined fresh pozol from Effects of enzymes, pH, and heat treatment. Sensitivity of Mexico and isolated a strain, presumably related to Aa91 the active substance to proteolytic and other enzymes was tested on active cell-free supernatant (15). Samples of 100 ␮l were tested that was designated CS93. Of several colonies capable of after 0-, 1-, and 3-h treatments with 1 mg/ml final concentration growing on nitrogen-free agar medium (a characteristic re- of the following enzymes in water: RNase, DNase I, catalase, ported for Aa91 (25)), only CS93 exhibited antagonism lipase, ␣-amylase, trypsin, ␣-chymotrypsin, pronase E, and pro- against the indicator E. coli V517. This isolate also had tease K (Sigma). Inhibitory activity of the treated samples was similar morphological characteristics as Aa91 (results not compared to respective controls (without enzymes) for the zone shown). of growth inhibition against the indicator bacteria. Control treat- The flip streak method was initially used to determine ments also consisted of enzyme solutions without antimicrobial a spectrum of inhibition of CS93 (Table 1). Growth of all compound(s). indicator strains tested was inhibited except for three strains Sensitivity of the active substance(s) to different pH values of Staphylococcus aureus and the mold Aspergillus par- was determined by adjusting the pH of the supernatant from pH asiticus. Aa91 differed from CS93 in its inability to inhibit 2to12with NaOH or HCl and incubating for 1 h. The pH of the samples was adjusted back to 7 and tested against the indicator the Absidia sp., Fusarium graminerium, and the Kluvero- strains (10). myces sp. Both Aa91 and CS93 exhibited two zones of Thermal stability of the antimicrobial compound(s) was de- inhibition against Aspergillus, Thamnidium, and Penicilli- termined by autoclaving cell-free supernatant containing the active um. A clear zone of complete mold growth inhibition was substance(s) at 121ЊC for 15 min. The autoclaved solution was observed followed by a zone of white mycelium where 1126 RAY ET AL. J. 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FIGURE 1. Inhibition of mold growth by Aa91 in malt extract agar containing 4% maseca. Three zones are distinguished: a central zone of complete inhibition, a zone of white mycelium (inhibition of sporulation), and a zone of characteristic growth.

sporulation was inhibited, followed by a zone of character- TAP PCR of CS93, Aa91, and B. subtilis 6633. TAP istic mycelial growth (Fig. 1). CS93 was able to inhibit DNA fingerprinting permits the differentiation of closely growth of B. subtilis 6633 and was also inhibited by B. related strains, and it was used to determine the similarity subtilis 6633, a subtilin producer. Subtilin is a lantibiotic of CS93 to Aa91 and of both strains to B. subtilis 6633 active against gram-positive bacteria. The mutual inhibition (Fig. 3). The pozol isolates shared almost identical finger- of these two bacteria suggests that CS93 may not produce prints and had many bands in common. However, the fin- subtilin and is producing a different compound effective gerprint from B. subtilis 6633 was significantly different against gram-positive bacteria. Although the number of mi- from these two strains and had few bands in common with croorganisms examined was limited, it is clear that CS93 them. exhibits a broad spectrum of activity against gram-positive Physiological and biochemical characteristics of and gram-negative bacteria as well as yeasts and molds. Aa91 and CS93. Although Ulloa et al. (26) described Aa91 Preliminary results have also demonstrated that CS93’s as a gram-negative coccal bacilli, our results indicated that compound(s) inhibits the outgrowth of B. subtilis 6633 both pozol isolates were highly Gram variable. Further- spores (data not shown). more, both strains were found to produce endospores, a Phylogenetic analysis of CS93. Ulloa et al. (26) de- characteristic of Bacillus sp. scribed Aa91 as a gram-negative, nitrogen-fixing, coccal Results of the API CHB 50 test kit indicated that CS93, bacilli capable of producing a broad-spectrum antimicrobial Aa91, and B. subtilis 6633 had identical carbohydrate fer- compound(s). They identified Aa91 as an Agrobacterium mentation patterns that correlated to the manufacturer’s re- azotophilium. DeSmedt and Ley (5) however, found Aa91 ported pattern for B. subtilis. Phenotypic tests (catalase pos- to have higher DNA homology to several gram-positive itive, Voges-Proskauer test positive, no growth in anaerobic bacteria like Bacillus sp. than to A. azotophilium strains. To agar, and hydrolysis of starch) commonly used to identify clarify the identity of Aa91, as well as the new isolate Bacillus sp. also indicated that CS93 and Aa91 were related CS93, an internal fragment of the 16S rRNA from both to B. subtilis. strains was amplified using PCR and then directly se- Production of the antimicrobial compound(s) on a quenced. The sequences were entered into a Blast search solid surface. Initial studies on production of the antimi- for identification purposes and construction of a phyloge- crobial compound(s) by CS93 were conducted utilizing netic tree using 16S rRNA sequences of all the Bacillus agar medium, as production was not observed using broth strains in GenBank. The 16S rRNA sequence results agreed systems. Using the membrane filter technique, the effect of with the findings of DeSmedt and Ley (5) that Aa91 was agar concentration on production was examined by growing a gram-positive bacterium sharing DNA homology to Ba- CS93 on nutrient agar plates supplemented with 0.3% man- cillus sp. Sequence analysis also indicated that CS93 was nitol and 0.5 to 1.5% agar. Zones of inhibition were evident identical to Aa91 and closely related to B. subtilis (Fig. 2). at all agar concentrations, but the growth and zone size J. Food Prot., Vol. 63, No. 8 ANTIMICROBIAL(S) OF A POZOL ISOLATE 1127 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021

FIGURE 2. Phylogenetic tree comparing the 16S rRNA sequence from CS93 with the 16S rRNA sequences from all Bacillus sp. reported in GenBank. Sequences were aligned, and the phylogenetic tree was constructed using the neighbor-joining method in DNAStar.

increased with decreasing levels of agar. At agar concen- Growth occurred on all carbohydrates tested except for trations below 0.75%, CS93 overgrew the membranes after sorbose (Table 2). Growth on D-mannitol exhibited the larg- 12 h, and it was not possible to test for the inhibitory com- est zone at 4.8 cm. Dextrin, lactose, sucrose, melizitose, pound(s). corn starch, mannose, glucose, and saccharose gave zones 1128 RAY ET AL. J. Food Prot., Vol. 63, No. 8 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021

FIGURE 3. DNA ﬁngerprint analysis of Aa91, CS93, and B. subtilis 6633 at 35, 37, 39, 40, 42, and 44ЊC. Lanes 1 to 6, Aa91; lanes 7to12, CS93; lanes 13 to 18, B. subtilis 6633; lane 19, molecular mass marker.

ranging from 2.5 to 3.1 cm. Raffinose, potato starch, xylose, and maseca yielded the smallest zones of inhibition, all 1.5 cm. TABLE 2. Growth of CS93 and production of its antimicrobial Production studies in broth. After identifying param- Њ compound(s) against E. coli V517 at 37 Conmedium supple- eters that resulted in large zones of inhibition in a solid mented with carbohydrates system such as reduced agar concentration, temperature, Growth of CS93 after: medium composition, and carbohydrate source, attempts to Zone size produce the antimicrobial compound(s) in a liquid system Carbohydrates 12 h 48 h (cm) were initiated. Initial studies concentrated on the growth Melizitose ϩa ϩ 2.5 medium, as components in commercial media have been Lactose ϩ ϩ 2.5 reported to influence the production of a variety of anti- Glucose Ϫ ϩ 3.1 microbial compounds (27). For example, staphylococcin is Fructose Ϫ ϩ NDb not released in a diffusible form unless the producing or- Sucrose ϩ ϩ 2.5 ganism is grown in tryptose soy broth or in yeast extract Raffinose ϩ ϩ 1.5 broth (16). Once CS93 was identified as a Bacillus sp., the ϩ ϩ Xylose 1.5 degree of aeration during growth was examined. After ϩ ϩ Saccharose 3.1 growing CS93 in flasks at different speeds and using vari- Potato starch ϩ ϩ 1.5 ous broth volumes, aeration parameters allowing production Sorbose Ϫ Ϫ ND Arabinose Ϫ ϩ ND in a liquid system were identified. CS93 was then grown Њ Dextrin Ϫ ϩ ND at 37 Cin100 ml nutrient broth supplemented with 1% Mannitol ϩ ϩ 4.8 mannitol in a 500-ml flask (200 rpm). Figure 4 illustrates Cellibiose ϩ ϩ ND the change in pH, optical density, and production of the Corn meal Ϫ ϩ ND inhibitory compound(s). Production was detected at 8 h, Corn starch ϩ ϩ ND after the pH began to increase. Activity units against M. Maseca ϩ ϩ 1.5 luteus ranged from 20 AU at 8 h to 640 AU at 25 h, and a ϩ, visible growth and Ϫ,novisible growth. against E. coli, activity units ranged from 20 AU at9hto b ND, not determined. 320 AU at 18 h. At 6, 12, 24, and 35 h the inhibitory J. Food Prot., Vol. 63, No. 8 ANTIMICROBIAL(S) OF A POZOL ISOLATE 1129 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021

FIGURE 4. Changes in pH (top), and OD, and production of the antimicrobial compound(s) during the growth of CS93 in nutrient broth at 37ЊC (220 rpm). Inhibition of M. luteus (shaded bars) and E. coli V517 (dotted bars) was tested. Activity units were determined by multiplying the reciprocal of the last dilution with activity by the amount of serially diluted membrane-ﬁltered broth used in the well diffusion assay. spectrum was retested against a variety of indicator strains broth, spent medium treated with pronase E, and spent me- by using the well diffusion assay. Results indicated that the dium treated with heat-denatured enzyme. E. coli grew in compound(s) produced in broth at the four time periods was nutrient broth and spent medium treated with pronase E. also active against gram-positive and gram-negative bac- However, no change in OD was observed when this strain teria, yeasts, and molds. This suggested that the same com- was grown in spent medium or spent medium ϩ heat-de- pound(s) was being produced in both agar and broth sys- natured enzyme (Fig. 5C). Results suggested a bactericidal tems. activity against gram-negative bacteria as the number of E. coli cells was reduced by 6 log cycles in the spent medium Effect of enzymes, pH, and heat treatment on the but increased in the control medium (Fig. 5F). S. cerevisiae inhibitory compound(s). The sensitivity of the antimicro- was also unable to grow in the spent medium or spent me- bial compound(s) to various enzymes, heat treatments, and dium ϩ heat-denatured pronase E but did grow in nutrient pH values was assessed by examining for retention or loss broth and in spent medium containing pronase E (Fig. 5B). of inhibitory activity after treatment (Table 3). Activity The inability to grow was a fungistatic effect, as the number against gram-positive and gram-negative bacteria, yeasts, of viable yeast cells was constant over 8 h in the spent and molds was retained after exposure to all enzymes ex- medium but cell number increased if the inhibitory com- cept pronase E. The antimicrobial substance(s) was heat pound(s) was absent (Fig. 5E). The inhibition of M. luteus stable even after treatment at 121ЊC for 15 min and exhib- was bacteriostatic (Fig. 5A and 5D). Unlike E. coli and S. ited activity over the pH range of 3 to 11. cerevisiae, growth of M. luteus in spent medium ϩ pronase Effect of antimicrobial compound(s) on indicator E was not comparable to that in nutrient broth. This may organisms. E. coli, M. luteus, and S. cerevisiae were used have been due to only partial inactivation of the antimicro- to examine whether the inhibitory compound(s) produced bial(s) resulting in residual levels of the active com- bactericidal or bacteristatic effects. Growth of the indicator pound(s). In addition, of the three indicators used, M. luteus strains was monitored in nutrient broth, spent nutrient was the most sensitive to the inhibitory compound(s). 1130 RAY ET AL. J. Food Prot., Vol. 63, No. 8

TABLE 3. Factors affecting activity of the antimicrobial com- subtilis 6633 in genetic and phenotypic characteristics, their pound(s) produced by CS93 using the well-diffusion assay against growth on nitrogen-free agar and the ability to produce a E. coli V517 broad-spectrum antimicrobial compound(s) coupled with Treatment Antimicrobial activity remaining the differences in the TAP PCR ﬁngerprints suggest that CS93 and Aa91 may be new variants of B. subtilis. Enzymes In the past decade several Bacillus sp. have been re- ␣-Chymotrypsin ϩ ported to produce proteinaceous antimicrobial compounds ϩ Proteinase K such as tochicin (19), coagulin (8), and cerein (18). These Pronase E Ϫ bacteriocins are effective against a variety of gram-positive Trypsin ϩ ␣-Amylase ϩ bacteria; however, they are ineffective against gram-nega- Lipase ϩ tive bacteria, yeasts, and molds. An exception is the anti- DNase I ϩ microbial compound produced by Bacillus pumilus that was

RNase ϩ reported to be effective against a wide range of molds (17). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021 Catalase ϩ The isolates CS93 and Aa91 are examples of a Bacillus sp. pH with a broad spectrum of inhibition against gram-positive 2 Ϫ and gram-negative bacteria, yeasts, and molds. There are 3 ϩ numerous potential applications for such a compound in 4 ϩ agriculture, in the food industry to increase the safety and 5 ϩ shelf life of foods, as well as in the pharmaceutical industry. 6 ϩ The antimicrobial compound(s) produced by CS93 was 7 ϩ heat stable and active over a wide pH range. This coincides ϩ 8 with reports of other antifungal metabolites that are resis- 9 ϩ tant to acidic and basic conditions (17). Proteolytic enzyme 10 ϩ 11 ϩ pronase E was able to inactivate the antimicrobial com- 12 Ϫ pound(s), suggesting the compound(s) is proteinaceous. Treatment with lipase A did not cause any loss of activity. Heat This result suggests that a lipid moiety is not involved in 50ЊC ϩ the active portion of the antimicrobial compound(s). The 70ЊC ϩ 100ЊC ϩ compound(s) also appeared to lack sugar residues, based on ␣ 121ЊC ϩ the resistance to -amylase (17). Catalase also had no effect on the inhibitory properties, thus eliminating hydrogen per- oxide as the active inhibitory agent. A bactericidal action was found against E. coli V517 Lytic effect of the inhibitory compound(s) against while a bacteriostatic and a fungistatic action was found E. coli V517. A bacteriolytic action was observed when E. against M. luteus and S. cerevisiae, respectively. Most bac- coli V517 cells were resuspended in spent medium con- teriocins such as pediocin and nisin have a bactericidal ac- taining the inhibitory compound(s) (Fig. 6). The OD de- creased from 0.45 to 0.03 in 3 h, whereas turbidity in- tion on sensitive bacteria. Their proposed mode of action creased in either nutrient or enzyme-treated spent broth. is by binding to specific receptors on sensitive bacterial cell Therefore, the bactericidal action resulted in concomitant walls and then forming pores through the membrane re- cell lysis of E. coli V517. sulting in an efflux of ions and destablization of the proton motive force within the cell (20). The mechanism causing DISCUSSION the bactericidal action against E. coli by the active com- A potentially novel antibacterial compound(s) pro- pound(s) of CS93 is not known. duced by CS93, isolated from fresh pozol, was partially Further investigation into the effect of the antimicrobial characterized. Pozol isolates CS93 and Aa91 were identi- compound(s) revealed that lysis occurred when E. coli fied as Bacillus sp. and appear to be related closely to B. V517 was treated with spent medium. This phenomenon subtilis. CS93 and Aa91 had high homology to B. subtilis has also been attributed to coagulin against Bacillus coa- 6633 based on 16S rRNA analysis; however, B. subtilis gulans CIP (8). Lysis was proposed to result from the in- 6633 exhibited significantly different banding patterns fluence of the cationic properties of the antimicrobial pep- when a fingerprint analysis was compared to those of the tides on the activity of autolytic enzymes. two pozol isolates. CS93 and Aa91 appeared to be closely In summary, CS93 produces a broad-spectrum anti- related, based on the presence of many common bands in microbial compound(s) effective against gram-positive and the fingerprints. This is especially interesting as Ulloa and gram-negative bacteria, yeasts, and molds. The com- Herrera isolated Aa91 in 1972, and CS93 was isolated from pound(s) is proteinaceous, heat stable, and active over a fresh pozol in our laboratory in 1994 (22). Both strains wide pH range. Identification of the compound(s) is cur- possessed the unusual ability to grow on nitrogen-free me- rently under investigation, including determination of the dium and produced a broad-spectrum antimicrobial com- mode of action against other organisms, optimization of pound(s). While the pozol isolates appeared similar to B. growth medium components, and evaluation of effective- J. Food Prot., Vol. 63, No. 8 ANTIMICROBIAL(S) OF A POZOL ISOLATE 1131 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/8/1123/1673934/0362-028x-63_8_1123.pdf by guest on 26 September 2021

FIGURE 5. Effect of CS93’s active compound(s) on the growth of M. luteus (A), S. cerevisiae (B), and E. coli (C). Indicator strains were grown in nutrient broth (Ⅵ), spent medium (Ⅺ), spent medium ϩ pronase E (ⅷ), spent medium ϩ heat-treated pronase E (⅜). OD (600 nm) was monitored over time. Mode of action of CS93’s active compound(s) against M. luteus (D), S. cerevisiae (E), and E. coli (F). The CFU/ml of the indicator strains was determined after inoculation into nutrient broth (Ⅵ) and spent medium (Ⅺ).

ness against spoilage and pathogenic microorganisms in food systems. ACKNOWLEDGMENTS

This project was funded in part by the Kraft General Foods Chair in Food Science, Minnesota–South Dakota Foods Research Center, Min- nesota Agricultural Experiment Station and Quest International. P. Ray was partially funded by the Land O’ Lakes John Brandt Memorial Fel- lowship. The authors are grateful for the technical expertise of Dr. Denis Twomey and Dr. Ebenezer Vedamuthu. REFERENCES

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