672 Journal of Food Protection, Vol. 55, No. 9, Pages 672-677 (September 1992) Copyright©, International Association of Milk, Food and Environmental Sanitarians 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).