Evaluation of Anaerobic Growth of Bacillus Licheniformis and Bacillus Subtilis in Tomato Juice

Evaluation of Anaerobic Growth of Bacillus licheniformis and Bacillus subtilis in Tomato Juice

J. H. RODRIGUEZ, M. A. COUSIN*, and P. E. NELSON

Department of Food Science, Purdue University, West Lafayette, Indiana 47907 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 (Received for publication December 27, 1991)

ABSTRACT Odlaug and Pflug (21) reported similar results for Aspergil lus gracilis and C. botulinum coinoculated into tomato Conditions responsible for growth and pH elevation by se juice. lected strains of Bacillus subtilis and Bacillus licheniformis were studied in order to assess the potential hazard of metabiosis Bacillus species are commonly found on raw fruits occurring between Clostridium botulinum and mesophilic Bacil and vegetables. There has been concern over their ability lus species in aseptically packaged tomato juice. The effects of to cause an increase in pH since Knaysi and Gunsalus (14) the initial tomato juice pH on the growth of these Bacillus strains found that Bacillus subtilis or Bacillus licheniformis caused were evaluated. Cultures of B. licheniformis previously identified the pH of broth to increase to greater than 9.0. B. by Fields et al. (6) were reclassified as B. subtilis because none licheniformis has been isolated from home-canned toma grew under strict anaerobic conditions nor used propionate. B. toes (6,19). Fields et al. (6) reported that 14 of the subtilis did not grow under strict anaerobic conditions but could Bacillus isolates were able to raise the pH of tomato juice use oxygen if present in the environment. None of the B. subtilis serum to 4.8 or higher when grown aerobically. Montville or B. licheniformis strains grew at pH 4.0 or 4.2. Only B. subtilis 075-T-09 and B. licheniformis 64-83-46 strains isolated from and Sapers (18) used the strains isolated and identified by acidic foods were able to grow at pH 4.4 in tomato juice. Fields et al. (6) and found that they raised the pH to Anaerobically, B. licheniformis strains did not grow in tomato greater than 5.2 in aerobic conditions. Anderson (7) re juice or tomato juice with added nitrate and thiamine and even ported that a Bacillus coagulans strain could raise the pH showed a loss of viability. Both B. licheniformis and B. subtilis of tomato juice to over 5.0 after 6 d at 35°C under aerobic require oxygen for growth in tomato juice at pH 4.4. conditions. Bacillus species produce heat-resistant spores and are common contaminants of soil and water, as are Clostridium Foodbome botulism is an intoxication caused by the species. The species of most concern in metabiosis are B. ingestion of food contaminated with a neurotoxin produced subtilis and B. licheniformis. B. subtilis is classified as an by Clostridium botulinum. Botulism is generally considered aerobe and B. licheniformis as a facultative anaerobe (7,25). a hazard in low acid (pH > 4.6) canned foods since C. Previous research has shown that strains of these two botulinum does not grow and produce toxins at pH levels Bacillus species can grow in tomato juice under aerobic below 4.6. Most outbreaks of botulism have resulted from conditions. The purpose of this research was to study the home-canned low acid foods. Odlaug and Pflug (20) re anaerobic growth of selected strains of B. subtilis and B. ported that tomato products were implicated in 17 of 34 licheniformis in tomato juice. This information will aid in outbreaks of botulism involving home-canned foods from assessing the potential for C. botulinum to grow in canned 1899 to 1975. In 1915, commercially canned tomato catsup tomato products if the pH is raised above 4.6. was implicated in a botulism case (20).

Metabiosis is a term used to identify a condition where MATERIALS AND METHODS one microorganism can alter the environment thereby al lowing another previously restricted microorganism to grow. Test microorganisms The concern with acid foods is the elevation of the pH and Cultures of Bacillus were obtained from M. Fields (Univer the decrease of the oxygen levels to conditions where C. sity of Missouri, Columbia), T. J. Montville (U.S. Department of botulinum can grow and produce toxin. In recent years, Agriculture, Philadelphia, PA), the National Food Processors there has been concern over the metabiotic interactions Association (NFPA, Berkeley, CA), and the American Type Culture Collection (ATCC, Rockville, MD) (Table 1). Cultures between aciduric spoilage microorganisms and C. botuli from T. J. Montville and M. Fields were originally isolated by num. Huhtanen et al. (73) reported that Cladosporium and Fields et al. (6) from home-canned tomatoes. B. licheniformis 64- Penicilliurn species could increase the pH to near neutral in 83-46 was originally isolated from acidified onions by NFPA. tomato juice. C. botulinum spores then germinated, out Cultures were maintained on nutrient agar (Difco Laboratories, grew, and produced toxin in this moldy tomato juice. Detroit, MI) slants at 4°C with transfers every 3 months.

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992 ANAEROBIC GROWTH IN TOMATO JUICE 673

TABLE 1. Index of strains of Bacillus used in this study. ppm nitrate. After the dry ingredients were dissolved in distilled water, the medium was heated and continually flushed with Name when Strain Source oxygen-free nitrogen. The oxygen was removed by passing the received code No. nitrogen through a copper-filled oxygen removing furnace (Sargent Welch, Springfield, NJ). Resazurin was added to the medium (0.8 B. licheniformis' 075-T-09 Montville ml of 0.025% solution per 200 ml), and the medium was boiled B. licheniformis' 015-T-03 Montville and flushed with nitrogen until the resazurin turned from blue to B. licheniformis' 110-T-05 Montville pink to colorless. The medium was cooled and continually flushed B. licheniformis' 011-T-ll Montville with nitrogen. The pH was adjusted to 7.0 with 6 N HC1. Cysteine B. licheniformis* 042-T-09 Fields HC1 (.05%, Sigma Chemical Co.) was added to each flask and the B. licheniformis* 093-T-04 Fields pH rechecked. The medium was dispensed into anaerobic culture B. licheniformis* 024-T-10 Fields tubes (Bellco, Vineland, NJ) that were flushed with nitrogen and B. licheniformis' 042-T-04 Fields stoppered with rubber stoppers. The tubes were placed into a B. licheniformis* 097-T-005 Fields clamp press (Bellco) and autoclaved for 15 min at 121°C. After B. licheniformis* 015-T-06 Fields cooling, those tubes containing media that remained colorless B. licheniformis* 090-T-15 Fields were used. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 B. subtilis 015-T-032 Fields Spores of B. subtilis 075-T-09 and a type strain of B. B. subtilis 049-T-09 Fields licheniformis NRRL 1264 were quickly thawed, and diluted in B. licheniformis 64-83-46 NFPA prereduced dilution blanks that were made by adding cysteine B. licheniformis NRRL 1264 Montville HC1 (0.5%) to 0.1% peptone water. The prereduced blanks were B. licheniformis 14580 ATCC dispensed and autoclaved as described for the medium. Serial B. licheniformis 8480 ATCC dilutions were made so that the medium would contain approxi 5 B. subtilis E6051 ATCC mately 10 spores per ml. The spores were added to the tubes of medium in an anaerobic glove box and restoppered. The tubes * Reclassified as B. subtilis and will be referred to as B. subtilis were heated at 80°C for 10 min to activate the spores. After for the remainder of this manuscript. cooling in ice water, the tubes were incubated at 32°C. Growth was assessed by reading the absorbance at 660 nm in a Spectronic 20 (Bausch and Lomb, Rochester, NY) after 1, 2, 3, and 6 d. An aerobic culture served as a control for each nitrate level. Verification of species Biochemical tests (Table 2) were used to verify the identity of all Bacillus strains. All media were prepared and inoculated Aerobic growth of Bacillus strains in tomato juice at different pH according to the reference (Table 2) except for the following values modifications: (a) For growth at 55°C, nutrient agar slants were Tomato juice with no added NaCl and a pH of 4.4 was prewarmed in water at 55°C. Each tube was inoculated with a canned at Purdue University. Approximately 600 ml were each single streak up the center of the slant. All tubes were placed into adjusted to pH 4.0, 4.2, 4.4, 4.6, 4.8, and 5.0 with citric acid or a beaker of tempered water in the incubator, (b) For growth at 1 N NaOH. Each medium was dispensed into tubes in 9-ml 60°C, prewarmed nutrient broth was inoculated with a loopful of portions and autoclaved at 121 °C for 15 min. The pH of the a 24-h nutrient broth culture. All tubes were placed into a beaker tomato juice was rechecked after autoclaving. No readjustment of tempered water in the incubator, (c) For growth in sodium was needed. chloride (NaCl), 10 and 12% NaCl were added to nutrient broth. B. licheniformis ATCC 14580, ATCC 8480, and NFPA 64- (d) Tagatose (0.5% tagatose, Sigma Chemical Co., St. Louis, MO) 83-46 and B. subtilis ATCC E6051, and 075-T-09 from 24-h was added to phenol red agar base (BBL, Cockeysville, MD). (e) nutrient broth cultures were inoculated into 9-ml tomato juice Rhamnose (1% rhamnose, Sigma Chemical Co.) was added to tubes at each pH, and 8 tubes each of B. licheniformis ATCC phenol red agar base (BBL). 14580 and ATCC 8480 and B. subtilis ATCC E6051 and 12 tubes each of B. licheniformis NFPA 64-83-46 and B. subtilis 075-T- Spore production 09 were incubated at 32°C. The inoculum level of each strain was A mineral medium of Donnellan et al. (5) was used for determined by spread plate counts of the inoculum on nutrient agar incubated at 35°C for 24 to 36 h. An uninoculated tube of sporulation. The mineral medium contained: FeCl2 or FeCl3, tomato juice served as the control for each pH. 0.0036 mM; MgCl2, 0.041 mM; MnCl,, 0.1 mM; NH4C1, 10 mM; _ Na,S04, 0.75 mM; KH2P04, 0.5 mM; CaCl2, 10 mM; NH4N03, Growth was assessed visually after 1, 2, 3, and 4 weeks for 1.2'mM; D-Glucose, 10 mM; L-Glutamic Acid, 10 mM. One liter the presence of an obvious pellicle. Media from tubes where no of medium was dispensed into 2-L flasks and autoclaved at 121°C apparent growth was observed after 4 weeks were plated onto for 15 min. The pH was adjusted to 7.0 with 1 N NaOH. Bacterial nutrient agar and incubated at 35°C for 24 to 36 h. Also, the final cultures were inoculated into the medium from nutrient agar pH was taken after 4 weeks. slants. The medium was incubated at 32°C with shaking until Anaerobic growth of Bacillus strains in tomato juice and nutrient greater than 90% spores were present, as determined by phase- broth plus glucose at different pH values contrast microscopy. This usually required 2 to 3 d. Spores were B. licheniformis ATCC 14580, NFPA 64-83-46, and B. harvested by centrifugation at 10,000 x g for 10 min and washed subtilis 075-T-09 were inoculated from nutrient agar slants into 10 times with sterile distilled water (23). The final pellet was 100 ml of nutrient broth in 250-ml screw-neck flasks and placed resuspended in approximately 100 ml sterile distilled water and in a shaking incubator at 35°C for 24 h. Serial dilutions were aliquots were frozen and stored at -20°C until used. made so that the final concentration of cells in the tomato juice 4 Anaerobic growth of Bacillus spores in the presence of nitrate would be approximately 10 cells per ml. The inoculum level of Strict anaerobic culture methods were used throughout this each strain was determined by making serial dilutions of 1 ml of study (12). The basal medium of Smith et al. (24) for nitrate inoculum and doing triplicate spread plates onto nutrient agar. Final inoculum levels are given in Table 3. Tomato juice at pH reduction was used. Potassium nitrate (KN03) was added to 200 ml of medium to final concentrations of 0, 5, 50, 500, and 5000 4.0, 4.2, 4.4, 4.6, 4.8, and 5.0 (adjusted with citric acid or 1 N

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992 674 RODRIGUEZ, COUSIN AND NELSON

TABLE 2. Results from biochemical tests for strains of B. subtilis and B. licheniformis.

Biochemical tests2

i o fi. subtil lis' 075-T-09 J ------+k + +/- 042-T-04 ------+ + + +

110-T-05 + Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 093-T-04 ______+ - + +/- 011-T-ir ------+ + +/- 015-T-03m ------+ + + 024-T-10 ------+ + 042-T-09 ------+ - + - 097-T-005 ------+ + +/- +/- 015-T-06 ------+ + + +/- 090-T-15 ------+ + + + fi. subtilis ATCC E6051 + + + . 049-T-09 ------+ - 015-T-032 ------+/- +/

B. licheniformis NRRL 1264m + + + + + + + + + + ATCC 14580 + + + + + + + + + + ATCC 8480 + + + + + + + + + + 64-83-46" + + + + + + + + + +

Catalase, Voges Proskauer, and starch hydrolysis were positive, and hippurate hydrolysis and rhamnose were negative for all strains. Rhamnose was done according to Logan and Berkeley (15) and all others, according to Gordon et al. (9) and Gordon (8). Gordon et al. (9). Nutrient broth. O'Donnell et al. (22). Hanakova-Bauerova et al. (10) and Mac Faddin (16). Logan and Berkeley (15). Burdon et al. (4). Burdon (J). Fields except those designed with other letters or ATCC numbers. No growth. + = growth. +/- = growth but less than for +. T. Montville. National Food Processors Association.

NaOH) and nutrient broth plus 1% glucose adjusted to pH 4.4 and divided between the two anaerobic jars with two tubes per jar for 4.8 with citric acid and pH 7.0 were inoculated for this experi pH 4.4, 4.8, and 7.0 for each microorganism. ment. A tube of tomato juice, which was inoculated with 1 ml After 12 d, one anaerobic jar was opened, the tubes were sterile distilled water, served as the control for each pH. An visually assessed for growth, and plate counts were made on aerobic culture of each pH and each microorganism served as a nutrient agar. The final pH was recorded. control for the inoculum. After inoculation, the tubes of tomato juice were divided Anaerobic growth of Bacillus strains in tomato juice with added between two anaerobic jars with GasPak systems (BBL, nitrate, or thiamine Cockeysville, MD) and incubated at 35°C with replications per jar Approximately 105 cells per ml of fi. licheniformis strains as follows: for B. licheniformis 64-83-46, three tubes each of pH 64-83-46 and ATCC 14580 and B. subtilis strains 075-T-09 and 4.0, 4.2, 4.4, 4.6, 4.8, and 5.0; for B. licheniformis 14580, two 093-T-04 were added to sterilized tomato juice at pH 4.4 (adjusted tubes each of pH 4.4, and 4.8; and for B. subtilis 075-T-09, two with 1 N NaOH), pH 4.4 plus 0.5% KN03, and pH 4.4 plus tubes sach of pH 4.4, 4.6, and 4.8. Tubes of nutrient broth were 0.001% thiamine HC1 (Sigma Chemical Co.). The inoculum level

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992 ANAEROBIC GROWTH IN TOMATO JUICE 675 TABLE 3. Colony forming units (CFU) per ml of tomato juice at time 0 incubated aerohically (A), anaerobically (AN), and anaerobically with added nitrate and thiamine (NT).

Culture A CFU/ml AN CFU/ml NT CFU/ml

B. licheniformis ATCC 14580 2.5 x 105 3.3 x 104 5.8 x 105 B. licheniformis ATCC 8480 1.8 x 105 NAa NA B. licheniformis 64-83-46 3.0 x 104 3.8 x 104 9.2 x 104 B. subtilis ATCC E6051 6.7 x 103 NA NA B. subtilis 075-T-09 1.3 x 103 8.8 x 103 2.2 x 105 B. subtilis 093-T-09 NA NA 8.7 x 10s

a NA-not applicable.

of each culture was determined by making plate counts of the TABLE 4. Comparison of B. subtilis and B. licheniformis (8,25). inoculum onto nutrient agar. Uninoculated tubes of each medium

served as controls. A culture of each microorganism that was B. subtilis B. licheniformis Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 inoculated into each medium and incubated aerohically served as a control for the inoculum. The tubes were divided between two Rods anaerobic jars with GasPak systems (BBL, Cockeysville, MD) Width (m) 0.7-0.8 0.6-0.8 and incubated at 35°C. Length (m) 2-3 1.5-3 After 3 weeks, one jar was opened, the tubes were observed Gram + +' + for growth, and the pH was recorded. After 8 weeks, the second Motility + + anaerobic jar was opened, the medium in the tubes was visually Catalase + + assessed for growth, and plate counts from each tube were Anaerobic - + determined on nutrient agar incubated at 35°C for 24 to 36 h. The Temperature-growth final pH was recorded for the medium in each tube. max 40-55°C 50-55°C min 5-20°C 15°C RESULTS AND DISCUSSION Voges Proskauer + + Growth in Identification of Bacillus species 7% NaCl + + The results of all biochemical tests to identify B. media, pH 5.7 + + subtilis or B. licheniformis are presented in Table 2. The Hydrolysis of cultures isolated and identified by Fields et al. (6) as B. starch + + hippurate licheniformis were reclassified as B. subtilis based upon - - Use of their inability to grow anaerobically, lack of arginine propionate - + dihydrolase, and lack of utilization of propionate, tagatose, citrate + + rhamnose, and maltose (Table 4). Anaerobic growth was a Acid from characteristic of the type strain of 5. licheniformis and the glucose + + NFPA isolate, B. licheniformis 64-83-46; however, the arabinose + + isolates from Fields et al. (6), Montville (77), and Montville xylose + + and Sapers (18) did not grow anaerobically and would not mannitol + + b be of concern in canned foods. maltose - + D-tagatosec - + rhamnosec - + Spore production Reduction of nitrate + + A medium by Donnellan et al. (5) proved to be a good Arginine dihydrolased - + sporulation medium for both 5. subtilis and B. licheniformis. Use of a mineral medium increases sporulation because it + = 85% to 100% of strains positive for character. would more likely produce spores similar to those found in Burdon (3). nature. B. subtilis produced a characteristic olive-green Logan and Berkeley (15). spore mass in this medium, while spores of B. licheniformis Hanakova-Bauerova et al. (JO). were white.

Anaerobic growth of Bacillus spores in the presence of oxygen. Maintaining strict anaerobic conditions was diffi nitrate cult even in the special tubes used in this research. B. No growth was observed for B. subtilis in a basal licheniformis NRRL 1264 grew in all levels of nitrate. As medium containing nitrate when strict anaerobic conditions the concentration of nitrate increased, so did the turbidity. were maintained; however, when the slightest amount of Based on these results, B. subtilis cannot grow in the total oxygen entered the system, some growth occurred as a absence of oxygen but is proficient at utilizing residual pellicle on the top of the medium. The presence of oxygen oxygen that may be present in an environment and B. was indicated by the tubes turning from colorless to pink. licheniformis can grow anaerobically. Broman et al. (2) and When growth would occur in the pink tubes, they would Gibson and Gordon (7) reported that anaerobic growth of quickly turn back to colorless as the bacteria used up the B. licheniformis was enhanced when nitrate was in the

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992 676 RODRIGUEZ, COUSIN AND NELSON medium. B. subtilis grows only slightly under anaerobic production. Where no visual growth was present, there was conditions when nitrate is present (7,11,14,25). no change in pH. Restricted anaerobic growth for B. subtilis was prob ably due to the efficiency of this microorganism in using any available oxygen (11,14,17). B. subtilis has a very fast growth rate, and anaerobic systems such as stabs in butt slants allow some oxygen to diffuse into the medium. Establishment of anaerobic conditions in an anaerobic jar can take a few hours and even longer for oxygen dissolved in the medium to be eliminated. During this time, growth could occur and lead to misinterpretation of a microorganism's oxygen requirements. A strict anaerobic environment must be maintained to correctly label a micro organism as aerobic or anaerobic because microorganisms

vary in their ability to grow at reduced oxygen partial Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 pressures. [j Control | B. licheniformis 84-83-46 § B. subtilis 075-T-09

I B. subtilis EB0S1 | B. licheniformis 14580 [j B. licheniformis 8480 Aerobic growth of Bacillus strains in tomato juice at different pH values Figure 2. Average final pH of tomato juice, initially adjusted to Inoculum levels of the bacteria differed according to pH 4.4 to 5.0, that had growth of B. licheniformis and B. subtilis their growth in nutrient both. Table 3 shows the initial after 4 weeks at 32°C. concentration of each bacterium in the tomato juice. After 24 h it was apparent that plate counts to determine how At pH values of 4.0, 4.2, and at other pH values where much the cell number had increased would not be possible no pellicle was present, the cultures were unrecoverable because these bacteria formed a heavy pellicle on the after 4 weeks from the tomato juice by plate counts. This surface of the tomato juice indicating a strongly aerobic indicated a loss of viability due to the acidic conditions. type of growth. Montville and Sapers (18) also reported that tomato juice No growth was visible in any tubes of tomato juice at was bactericidal to B. licheniformis strain NRRL 1264. pH 4.0 or 4.2 for any strains after 4 weeks at 32°C. At pH The ability of B. subtilis 075-T-09 and B. licheniformis 4.4, growth was observed only in tubes of tomato juice 64-83-46 to tolerate acid conditions may be carried in only inoculated with B. licheniformis 64-83-46 and B. subtilis a few cells in the population. These cells may grow and 075-T-09 (Fig. 1). Both of these cultures were originally metabolize thus changing the pH and allowing other cells isolated from acid products, and their capacity to grow at to grow. The differences between acid-tolerant and acid- pH 4.4 and raise the pH indicates a character not shared by sensitive strains may be affected by many factors. This the type cultures tested. research did not conclusively show why these differences were noted.

Anaerobic growth of Bacillus strains in tomato juice and nutrient broth plus glucose at different pH values Cultures were chosen because of their aerobic growth in tomato juice at pH values of 4.4 to 4.8. The initial inoculum levels in tomato juice are presented in Table 3. After 12 d, one anaerobic jar was opened. The other anaerobic jar was left for a longer period of time, but the anaerobic environment was lost as indicated by the methyl ene blue indicator and, therefore, was not included in these results. There was no obvious growth in tomato juice for any B. licheniformis 8480 M B. licheniformis 14580 W B. licheniformis 64-83-46 of the cultures after 12 d. The plate counts of tomato juice g B. subtilis 075-T-09 I B. subtilis E6051 revealed a loss of viability for B. licheniformis 64-83-46 at pH 4.0, 4.2, and 4.4 and for ATCC 14580 at pH 4.4 and Figure 1. Percentage of tubes positive for growth of B. 4.8. One out of three tubes of tomato juice inoculated with licheniformis and B. subtilis after 4 weeks at 32°C as indicated B. licheniformis 64-83-46 at pH 4.6 and two out of three by the presence of a pellicle in tomato juice with beginning pH of tubes at pH 5.0 showed no decrease in cell number. There 4.4 to 5.0. were no pH changes for any tubes of tomato juice inocu lated with B. licheniformis 64-83-46 and ATCC 14580 or Where growth was visually apparent, pH increased for B. subtilis 075-T-09. Although there was no pH change or B. subtilis and B. licheniformis, indicating a similar me visual evidence of growth, the cell number of B. subtilis tabolism in tomato juice (Fig. 2). The pH was well above 075-T-09 inoculated into tomato juice at pH 4.4 and 4.8 the lowest value limiting C. botulinum growth and toxin remained constant.

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992 ANAEROBIC GROWTH IN TOMATO JUICE 677 Nutrient broth plus glucose was used to provide a not lose viability in acid media as rapidly as did B. medium that was conducive to anaerobic growth. There licheniformis. was no apparent growth in the nutrient broth plus glucose REFERENCES at pH 4.4 or 4.8 for all three cultures. However, when the pH of the nutrient broth plus glucose medium was lowered 1. Anderson, R. E. 1984. Growth and corresponding elevation of tomato juice pH by Bacillus coagulans. J. Food Sci. 49:647,649. to 4.4, a precipitate formed, which made the visual detec 2. Broman, K., N. Lauwers, V. Stalon, and J. Wiame. 1978. Oxygen tion of turbidity or growth difficult. There was no change and nitrate in utilization by Bacillus licheniformis of the arginase and in the pH of the medium with an initial pH of 4.4 or 4.8. arginine deiminase routes of arginine catabolism and other factors affecting their syntheses. J. Bacterid. 135:920-927. There were no cells recoverable after 12 d by plate counts 3. Burdon, K. L. 1955. Useful criteria for the identification of Bacillus of the nutrient broth plus glucose at pH 4.4 and 4.8 for B. anthracis and related species. J. Bacieriol. 71:25-42. licheniformis 64-83-46 or ATCC 14580. B. subtilis 075-T- 4. Burdon, K. L., J. C. Stokes, and C. E. Kimbrough. 1942. Studies of the common aerobic spore forming bacilli. II. Fermentation reactions 09 did not change in cell number. in agar butt slants. J. Bacteriol. 44:163-168. In the nutrient broth plus glucose at pH 7.0, B. 5. Donnellan, J. E., E. H. Nags, and H. S. Levinson. 1964. Chemically licheniformis 64-83-46 increased in cell number by one-log defined, synthetic media for sporulation and for germination and growth of Bacillus subtilis. J. Bacteriol. 87:332-336. cycle and the pH decreased to 5.45 and 5.5 for the two 6. Fields, M. L., A. R. Zamora, and M. Bradsher. 1977. Microbiologi Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/9/672/1662814/0362-028x-55_9_672.pdf by guest on 26 September 2021 replicates, whereas the cell number for B. licheniformis cal analysis of home-canned tomatoes and green beans. J. Food Sci. ATCC 14580 did not change from the initial inoculum 42:931-934. 7. Gibson, T., and R. E. Gordon. 1974. Endospore-forming rods and level. The pH had decreased slightly to 6.4 in the nutrient cocci. Genus I. Bacillus, pp. 529-550. In R. E. Buchanan and N. E. broth plus glucose at pH 7.0. B. subtilis showed no loss of Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th viability nor did the pH change. The pH of the anaerobic ed. Williams and Wilkins Co., Baltimore. 8. Gordon, R. E. 1977. The genus Bacillus, pp. 319-336. In A. I. Laskin controls did not change. Growth was visible in all aerobic and H. A. Lechevalier (ed.), CRC handbook of microbiology, vol. I, controls, and the pH changed indicating the inoculum was 2nd ed. CRC Press Inc., Cleveland, OH. viable. 9. Gordon, R. E., W. C. Haynes, and C. H. Pang. 1973. The Genus Bacillus. U.S.D.A. Handbook No. 427. Washington, DC. B. subtilis is not as susceptible to loss of viability in 10. Hanakova-Bauerova, E., M. Kocur, and T. Martinec. 1965. Concern acidic conditions as is B. licheniformis because the cells ing the differentiation of Bacillus subtilis and related species. J. may have a mechanism for protection from acid. The pHs Appl. Bacteriol. 28:384-389. 11. Hobbs, G., and T. Cross. 1983. Identification of endospore-forming of 5.45 and 5.5, which were observed for B. licheniformis bacteria, pp. 49-78. In A. Hurst and G. W. Gould (ed.), The bacterial after growth, may be the limiting pH values where this spore, vol. 2. Academic Press, New York. bacterium can grow anaerobically in nutrient broth. 12. Holdeman, L V., E. P. Cato, and W. E. C. Moore. 1977. Anaerobic laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg. Anaerobic growth of Bacillus strains in tomato juice with 13. Huhtanen, C. N., J. Naghski, C. S. Custer, and R. W. Russell. 1976. added nitrate and thiamine Growth and toxin production by Clostridium botulinum in moldy tomato juice. Appl. Environ. Microbiol. 32:711-715. Broman et al. (2) reported that thiamine was necessary 14. Knaysi, G., and I. C. Gunsalus. 1944. A study of the so-called for anaerobic growth of B. licheniformis in the absence of Marburg and the Lawrence and Ford strains of Bacillus subtilis. J. nitrate and improved growth when nitrate was supplied. Bacteriol. 47:381-389. 15. Logan, N. A., and R. C. W. Berkeley. 1984. Identification of Bacillus This experiment tested the ability of thiamine and nitrate to strains using the API system. J. Gen. Microbiol. 130:1871-1882. enhance anaerobic growth of B. licheniformis 64-83-46 and 16. Mac Faddin, J. F. 1980. Biochemical tests for the identification of ATCC 14580 and B. subtilis 093-T-09 and 075-T-09. The medical bacteria, 2nd ed. Williams and Wilkins, Baltimore. 17. Montville, T. J. 1982. Metabiotic effect of Bacillus licheniformis on initial inoculum levels are shown in Table 3. After 3 and 8 Clostridium botulinum: implications of home-canned tomatoes. Appl. weeks, there were no visual signs of growth in any tubes of Environ. Microbiol. 44:334-338. tomato juice. The pH had not changed from 4.38. The 18. Montville, T. J., and G. M. Sapers. 1981. Thermal resistance of spores from pH elevating strains of Bacillus licheniformis. J. Food number of cells still viable after 8 weeks had been reduced Sci. 46:1710-1712, 1715. 2 to 10 cells per ml or less or were not recoverable from 19. Mundt, J. O., J. L. Collins, I. E. McCarty, and R. Bailey. 1978. most tubes by plating methods. Cultures incubated aerobi- Description and microbiology of home-canned tomatoes and tomato juice. J. Food Prot. 41:944-947. cally grew and raised the pH, indicating that the inoculum 20. Odlaug, T. E., and I. J. Pflug. 1978. Clostridium botulinum and acid and medium were satisfactory. foods. J. Food Prot. 41:566-573. Addition of nitrate or thiamine to tomato juice had no 21. Odlaug, T. E., and 1. J. Pflug. 1979. Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis. effect on the anaerobic growth of B. licheniformis or B. Appl. Environ. Microbiol. 37:496-504. subtilis. In summary, B. licheniformis and B. subtilis re 22. O'Donnell, A. G„ J. R. Norris, R. C. W. Berkeley, D. Claus, T. quire oxygen for growth in tomato juice at pH 4.4. For Kaneko, N. A. Logan, and R. Nozaki. 1980. Characterization of Bacillus subtilis. Bacillus pumilus, Bacillus licheniformis, and Bacil growth under anaerobic conditions, some factor other than lus amyloliquefaciens by pyrolysis gas-liquid chromatography deox nitrate or thiamine may be needed in the tomato juice. yribonucleic acid-deoxyribonucleic acid hybridization, biochemical tests, and API systems. Int. J. Syst. Bacteriol. 30:448-459. 23. Roberts, T. A, and A. D. Hitchins. 1964. Resistance of spores, pp. CONCLUSIONS 611-670. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press, New York. B. subtilis strains did not grow in strict anaerobic 24. Smith, N. R., R. E. Gordon, and F. E. Clark. 1952. Aerobic sporeforming bacteria. U.S. Department Agriculture. Monograph conditions; however, these strains could use residual amounts No. 16. U.S. Department of Agriculture, Washington, DC. of oxygen that were in the medium. Neither B. subtilis nor 25. Sneath, P. H. A. 1986. Endospore-forming gram-positive rods and B. licheniformis grew in tomato juice below pH 4.4, and at cocci, pp. 1104-1207. In P. H. A. Snealh, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systemic bacteriology, vol. this pH oxygen was needed for growth. B. subtilis cells did 2. Williams and Wilkins, Baltimore.

JOURNAL OF FOOD PROTECTION, VOL. 55, SEPTEMBER 1992