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Bacillus tomato juice Agar: a special selective medium for aciduric microorganisms.

ABSTRACT--Of four selective media, Bacillus Tomato Juice Agar (BTJA) was the best medium to detect and enumerate aciduric, pH-elevating Bacillus, which cause non-effervescent spoilage of under processed tomatoes. One hundred five samples were collected randomly from garden soil or natural vegetative materials. Samples were swabbed with sterilized cotton or mixed with peptone water and then inoculated into selective medium BTJA, containing phytone, tomato juice, sodium chloride, and bromcresol purple. The medium was adjusted to pH 4.3 then sterilized at 121[degrees]C for 10 min. Sodium sorbate 0.015% was used to inhibit growth of fungi. Comparative growth of the isolates studied in different selective media (BTJA, PBBA, and PBBT) indicates that the most effective was BTJA. Therefore, BTJA was used to isolate, identify frequency in natural environment, and compare the Bacillus strains that elevate the pH of acid food products. The new medium may have future applications in the isolation and enumeration of other acidurics including yeast-like microorganisms.

Key words: bacillus, aciduric, effervescence, spore-formers, bacteriology-media, tomatoes.

INTRODUCTION

Certain strains of Bacillus coagulans elevate the pH of tomato juice and thus pose a health hazard during spoilage of home-canned tomato products (Anderson 1984, Shamsudin 1984, Al-Dujaili and Anderson 1991). Fields et al. (1977) obtained an isolate from spoiled, under processed home-canned green beans that subsequently increased the pH of tomato serum broth. The isolate was presumptively identified as B. coagulans 065-T-08, but no inference was drawn to its potential as a botulinal hazard in tomato products. Anderson (1984) found the Fields' strain (FS) not only grew well anaerobically in heat processed tomato juice, but elevated juice pH from 4.40 to 5.05 during 6 days of incubation at 35[degrees]C. Shamsudin (1984) indicated that the purple broth base (PBB) could be used for rapid detection of contaminants (acid and nonacid producing microorganisms) found in spoiled acid food products. The results also showed that the PBB with or without glucose has potential as an indicator medium and provided good growth. The purpose of the current investigation was to develop a selective medium that could be utilized to survey for these spore-bearer bacteria in nature. Because the bacteria in question raise pH of acid media, use of a pH indicator was evaluated to determine if an acid to alkaline color change could serve as presumptive presence of these isolates in contaminated plant and food samples.

MATERIALS AND METHODS

Sampling

For isolations from plant leaf surfaces, moistened sterile swabs were individually rubbed over 10x10 mm leaf surface areas and then streaked onto Petri plates (15x150 mm) of prepared isolation medium. All vegetation was selected from common accessible garden areas unless otherwise mentioned (Table 1). Soil was sampled at random from on-campus floral beds, home gardens and the University farms. At each site, approximately 3 kg of soil were collected and well mixed. Twenty grams of each sample were carefully mixed in the laboratory on the day of collection. A 1 g subsample was transferred to 10 ml sterile 0.1% peptone water diluent. From this mixture, 0.1 ml was transferred to each of 3 plates of selective isolation medium and streaked over the plate. This plating procedure was repeated in triplicate for each soil sample.

Nearly spherical potatoes and tomatoes of about baseball size were selected at random from freshly harvested produce from the university farms. Two potatoes and 2 paired tomatoes served as individual subsets of each. These were individually swabbed with sterile cotton swabs (moistened previously with 0.1% peptone water). The 2 swabs from the potatoes of each subset or those from each of the tomatoes were separately streaked on prepared isolation medium.

Isolation Medium

Because the desired isolates would be aciduric or acidophilic, a selective medium, Bacillus Tomato Juice Agar (BTJA), was developed for isolation (Table 2). As an adjunctive, we chose commercial tomato juice which, in our experience, gave faster growth of reference cultures than basal medium acidified with either citric or hydrochloric acids. Sodium sorbate was evaluated at various dilutions and effectively employed as an inhibitor for yeast and molds. Bromcresol purple was incorporated to indicate pH elevation during growth. Potassium sorbate, at levels of 0.100, 0.050, 0.025, 0.020, 0.015, 0.0145, 0.0140, 0.010, and 0.005% (g/liter) was aseptically added to the autoclaved media. Fields' strain of B. coagulans and unidentified fungi were inoculated and incubated for 48 h at 44.5[degrees]C. Growth of bacteria and fungi were observed within 48 h. The newly developed BTJA was used extensively throughout the isolation phase.

Comparative Growth of Isolates in Acidified PBB and BTJA

Acidulant materials included tomato juice, citric acid monohydrates, and hydrochloric acid. These acidic products were added to bring PBB (Table 3) and BTJA to pH 4.3 before autoclaving. The pH level of each medium was measured prior to autoclaving with an Orion Research 611 digital meter. The meter (Fisher model SO-B-1071 pH meter, Fisher Scientific, Pittsburgh, PA) was standardized before every pH reading by two certified buffer solutions of pH 7.0 and 4.0, respectively. All media were then autoclaved at 121[degrees]C for 10 min and were separately inoculated with 5 selected isolates. All plates were incubated at 44.5[degrees]C for 24 and 48 h.

Criteria for Isolation

Desired colonies were those that grew on BTJA and produced zones of purple in the yellow, acidified medium. Single colonies with a purple margin and background were carefully picked for re-streaking on BTJA if they had a dry, flake-like appearance similar to the reference Fields' strain (FS). All isolates were examined by Gram and spore stains, as were control cultures.

Control Cultures

B. coagulans 065-7-08, B. licheniformis B110, and B. coagulans NRS 54 were used as reference cultures. All cultures were grown aerobically on trypticase soy agar slants (TSA) (BBL Microbiology Systems, Cockeysville, MD) or BTJA at 44[degrees]C. Stock cultures were maintained at 7[degrees]C in 16x125 mm screw cap tubes on TSA slants.

Morphology

One to two day old subcultures grown on TSA were examined for colonial morphology. Cell morphology of isolates was determined with light and also by phase contrast microscopy (Nikon Optiophon, Tokyo, Japan). For the latter, cells were added to a pre-coated agar slide. After placement of a cover glass, cells were observed under oil immersion-phase contrast condition.

RESULTS AND DISCUSSION

Twenty-four isolates similar to the Field's strain were obtained from natural sources: 7 strains from 35 subsets of raw potatoes, 5 from 11 subsets of raw tomatoes, and 2 from home-canned tomatoes. The latter were water-bathed, cooked in quart canning jars. All together, isolates were obtained from each of the 9 sites tested (Table 1). The 24 isolates showed remarkable resemblance. All produced flake-like pellicles in TSB cultures after 2 days incubation at 44[degrees]C. Morphologically and physiologically, they were quite similar to FS, yet considerably different from any of the flat-sour strains of B. coagulans or the cultures of B. licheniformis in our possession (Al-Dujaili and Anderson 1991). All isolates produced powder-dry, flake-like colonies with irregular margins within 24 h; whereas, 24 h colonies of B. licheniformis B 110 were white and gummy or mucoid in texture. Flat-sour colonies of B. coagulants NRB 54 appeared moist, flat and had round regular margins (Table 3).

Purple broth base (PBB) medium used in this study (Table 4) was supplemented individually with one of three acidulants (tomato juice, citric acid, and hydrochloric acid) to compare growth among the isolates and for visual detection of any change in pH during growth (Table 5). No growth of 6 isolates was observed after 24 h. In PBB containing citric or hydrochloric acids, isolates become more strongly aerobic, usually producing slight, visible colonies. There was restricted growth of Bacillus strains associated with using either citric or hydrochloric acids as an acidulant. The inhibitory action of the acids was due to the undissociated acids, which can pass through the bacterial membrane and affect the permeability of the bacterial cells (Lynch 1988, Shamsudin 1984, Rice and Pederson 1953).

When compared to the PBB without tomato juice, tomato juice as an acidulant in PBB produced good growth of all isolates (Table 5). Data also indicate that tomato juice could be used as an acidulant and a supplemental enrichment source of carbohydrate, vitamins, and minerals for the Bacillus strains. Isolates grow rapidly, usually within 24 h, resulting in a color change of the medium from yellow to purple. An observed change in the color of the medium may be due to the fermentation of a carbohydrate from tomato juice and a decrease in the pH below 4.8 or when the pH increases to 6.8. Since there is no variation between the sample data and Field's strain, no statistical analysis is necessary.

When phytone was substituted for gelatone (pancreatic digest of gelatin), increased growth of isolates was obtained. Phytone peptone (BBL) is an enzymatic hydrolysate of soybean meal and acts as a nitrogen source for the cultivation of a large variety of organisms including fungi and bacteria (DIFCO Manual 1984). Results of the present study (Table 6) show that the substitution of phytone for gelatone, increased the growth of isolates, with or without tomato juice present in the medium. Moreover, the ability of isolates to proliferate in BTJA suggests that our isolates may be able to grow at the same pH as in ripe or overripe tomatoes that have relatively high digested carbohydrate content.

Findings from present cultural studies substantiate those of other investigations (Fields et al. 1977, Shamsudin 1984, Anderson 1984) described below. Shamsudin (1984) reported that PBB with or without glucose supported good growth of B. coagulans strain 064-T-08 and enabled visual detection of pH changes occurring during fermentation of glucose by B. coagulans strains. Anderson (1984) found that tomato juice samples initially at pH 4.5 supported good growth and that pH was elevated to 5.0 after 6 days incubation at 35[degrees]C. Fields et al. (1977) used tomato serum to isolate B. coagulans strains that elevate pH of serum from 4.20 to 4.75. The elevation occurred after 5 days of incubation at 35[degrees]C.

The organic acids in tomato juice have been identified by several investigators and include mainly citric and malic acids with very small amounts of acetic, lactic, and oxalic acid (Rice and Pederson 1953, Stern et al. 1942). The inhibitory effects of these acids have been determined for different species of bacteria, and in several types of media, with considerable differences in the order as well as the extent of inhibitory activity (Palop et al. 1997, Roberts and Hoover 1996, Sandoval et al. 1992). Rice and Pederson (1953) concluded that the inhibitory activity of supplemental citric acid incorporated in tomato juice was low when pH was held at a pH of 4.4. Cultures of B. coagulans were able to grow in 0.8% citrate at pH 4.4, but there was an inhibitory effect at pH below 4.4. Therefore, the Rice and Pederson study supports the results of the present study in which tomato juice can be used as an acidulant in BTJA, with no inhibitors for aciduric spore-former Bacillus strains at pH 4.4 (Table 7).

The BTJA medium was challenged with the unidentified fungal isolate from soil samples. This contaminated medium was used to determine if potassium sorbate would adequately serve as a fungal inhibitor. Sorbate was necessary to control yeast and fungi, and the effective level was determined by trial and error. Nine various concentrations of potassium sorbate, 0.100 to 0.005% were evaluated using 3 mold cultures before deciding on 0.015 g/liter. Fungal growth was completely inhibited without any effect on the growth of strain 064-6-08 at the 0.015% potassium sorbate (Table 7). Potassium sorbate is an antimicrobial agent in a variety of foods (Sofas and Busta 1981). The acid serves to extend the storage life of such products as butter, cheese, meats, cereals, and bakery items, some fruits, berry products, vegetable products, and other foods (Anonymous 1977, 1978, 1979). Therefore, we used potassium sorbate (0.015%) as an antifungal agent in BTJA (Table 2). Actodione was tried as a fungistat and found ineffective. The effectiveness of sorbates depends on many factors such as pH, concentration of sorbate, and type of microorganisms. There was a wide range of fungi isolated from strawberries and tomatoes that was inhibited by 0.05% sorbate in tomato juice medium (Furia 1972). The yeast and filamentous fungi were inhibited in media containing 0.1% sorbate at pH 4.5. The lactic acid bacteria were inhibited at this concentration of sorbate at pH 3.5 (Furia 1972). Flores et al. (1988) also found that potassium sorbate at 0.05% delayed initiation of growth and sporulation by Aspergillus ochraceus 0L24 in yeast extract-sucrose broth at 35[degrees]C.

CONCLUSIONS

A new, reliable, selective medium was developed to isolate Bacillus that elevates tomato juice pH. In BTJA, the tomato juice provided acidity for selectivity and additional nutrient for growth of aciduric spore-bearers. Other adjunct acids, e.g. citric and hydrochloric, did not support initial good growth of the FS or our reference cultures of Bacillus. Potassium sorbate was necessary to control yeast and fungi, and the effective level was determined by trial and error. Potassium sorbate at a level of 0.015% provides the optimum inhibition of the growth of fungi with no inhibition of the growth of bacteria. A new differential selective medium, BTJA, may have applications for isolation and enumeration of other aciduric microflora, especially acid oxidizers and perhaps lactic. This medium might also be used for study of film yeast in brewing because yeast (i.e., Saccharomyces uvarum) are used in brewing to produce alcohol. Moreover, this medium could be used for rapid detection of lactic contaminants found in acid food products such as tomato products, pickles, and sauerkraut. In a quality-control laboratory, BTJA could be used to identify survivors of inadequately canned foods for aerobic or anaerobic plate count as a MPN procedure due to its acid-base indicator and selectivity spore-formers of interest in food quality monitoring and research.
TABLE 1. Origin and isolation frequency of Bacillus strains from
various natural sources.

 Designated
 Source Frequency number

Soil 4/20 2-86
 5-87
 25-87
 29-87
Whole-canned tomatoes (a) 2/2 1-86
 (Home canning) 4-86
Potatoes (b) 7/35 P[O.sub.1]-87
 P[O.sub.2]-87
 P[O.sub.3]-88
 P[O.sub.4]-88
 P[O.sub.5]-88
 P[O.sub.6]-88
 P[O.sub.7]-88
Tomatoes (b) 5/11 [T.sub.1]-88
 [T.sub.2]-88
 [T.sub.3]-88
 [T.sub.4]-88
 [T.sub.5]-88
Unwashed lettuce (c) 1/4 L-88
Dried pepper powder (c) 1/4 3-86
Green bean leaves (d) 1/3 10-86
Squash leaves (d) 1/4 11-86
Weed leaves (c) 2/23 24-86
 30-86

(a) Morgantown vicinity, from a homemaker's kitchen.

(b) West Virginia University farms.

(c) Retail market, Morgantown.

(d) Home garden, Robert Anderson.

(e) Flower bed, Pensacola.

TABLE 2. Composition of purple broth base (PBB).

 Ingredient Quantity

Pancreatic digest of gelatin 10.0 g/L
NaCl 5.0 g/L
Bromcresol purple 0.02 g/L
Distilled water 1,000 ml

TABLE 3. Morphology of Bacillus isolates.

 B. coagulans
 Characteristics NRS 54 Isolates

Gram's stain + +
Rods
 Width, [micro]m 0.6-1.0 0.60-0.96
 Length, [micro]m 2.5-5.0 2.5-5.0
Colonies
 Color Grayish-white Dirty-cream
 Wetness Moist Powder dry
 Appearance Flat and translucent Flake-like
 Margins Round Irregular, wavy
Sorangium swollen + +
Spore shape Ellipsoid Ellipsoid
 (Round-oval) (Round-oval)
Spore position Central terminal Central terminal

 B. licheniforms
 Characteristics B 110

Gram's stain +
Rods
 Width, [micro]m 0.6-0.8
 Length, [micro]m 1.5-3.0
Colonies
 Color Dull, white, opaque
 Wetness
 Appearance Attached strongly
 Margins Irregular mucoid
Sorangium swollen -
Spore shape Ellipsoid
 (Round-oval)
Spore position Central terminal

TABLE 4. Composition of Bacillus Tomato Juice Agar (BTJA), adjusted to
pH 4.35 before autoclaving.

 Ingredient Quantity

Phytone TM peptone 10.0 g/L
Bromcresol purple 0.02 g/L
NaCl 5.0 g/L
Agar 30.0 g/L
Potassium sorbate 0.15 g/L
Tomato juice 400 ml
Distilled water 600 ml

TABLE 5. Comparative 24 and 48 h growth of Bacillus strains on Purple
Broth Base (PBB) with tomato juice, citric acid, or hydrochloric acid
as acidulans.

 Growth (a) on PBB medium

 Strain +Tomato juice +Citric acid +HCl

 Incubation time (h)

 24 48 24 48 24 48

064-T-08 ++ +++ - + - +
1-86 ++ +++ - + - +
2-86 ++ +++ - + - +
3-86 ++ +++ - + - +
P[O.sub.1]-87 ++ +++ - + - +
P[O.sub.2]-87 ++ +++ - + - +

(a) (-) No growth; (+) Pinpoint growth, colonies sizes less than 1 mm;
(++) Moderate surface growth, colonies sizes 1-1.5 mm; (+++) Heavy
surface growth, colonies sizes 1.5-2 mm.

TABLE 6. Comparative 24 and 48 h growth of Bacillus strains on
acidified Purple Broth Base (PBB) and Bacillus Tomato Juice Agar
(BTJA) media.

 Strain Growth (a) on medium

 BTJA PBB + tomato juice

 Incubation time (h)

 24 48 24 48

064-T-08 ++ ++++ ++ +++
1-86 ++ ++++ ++ +++
2-86 ++ ++++ ++ +++
3-86 ++ ++++ ++ +++
P[O.sub.1]-87 ++ ++++ ++ +++
P[O.sub.2]-87 ++ ++++ ++ +++

(a) (++) Moderate growth, colonies size range between 1-1.5 mm;
(+++) Heavy surface growth, colonies sizes range between 1.5-2 mm;
(++++) Very heavy surface growth, colonies sizes > 2 mm.

TABLE 7. Effect of different levels of potassium sorbate as an
inhibitor of growth of bacteria and fungal growth in BTJA.

 Growth (a) after 24 h

Potassium Unidentified
 sorbate B. coagulans fungal
 (g/L) 064-T-08 contamination

 0.100 - -
 0.050 - -
 0.025 - -
 0.020 + -
 0.015 +++ -
 0.0145 +++ +
 0.014 +++ +
 0.010 +++ ++
 0.005 +++ +++

(a) (-) No growth; (+) Pinpoint growth, colonies sizes less than
1 mm; (++) Moderate surface growth, colonies sizes 1-1.5 mm;
(+++) Heavy surface growth, colonies sizes 1.5-2 mm.


LITERATURE CITED

AL-DUJAILI, J. AND R.E. ANDERSON. 1991. Aciduric, pH-elevating Bacillus which cause noneffervescent spoilage of underprocessed tomatoes. J. Food Sci. 56(6):1613.

ANDERSON, R.E. 1984. Growth and corresponding elevation of tomato juice pH by Bacillus coagulans. J. Food Sci. 49:647.

ANONYMOUS. 1977. Outlook expands for use of sorbates to preserve "natural freshness" of foods. Food Proc. 38:46.

ANONYMOUS. 1978. Sorbic acid and potassium sorbate for preserving food freshness and market quality. Monsanto Industrial Chemicals Co., St. Louis, Missouri.

ANONYMOUS. 1979. Sorbic acid as a food preservative. Food Proc. Ind. 48:36.

DIFCO Manual of Dehydrated Culture Media and Reagents. 1984. 10th Ed. DIFCO Laboratory Inc., Detroit, MI.

FIELDS, M.C., A.F. ZAMORA, AND M. BRADSHER. 1977. Microbiological analysis of home-canned tomatoes and green beans. J. Food Sci. 42:931.

FLORES, L.M., L.S. PALOMAR, P.A. Roll, AND L.B. BULLERMAN. 1988. Effect of potassium sorbate and other treatments on the restaurant-type Mexican hot sauce. J. Food Prot. 51:4.

FURIA, T.E. (Ed.) 1972. Handbook of food additives, 2nd ed. CRC Press. Cleveland, OH.

LYNCH, D.J. AND N.N. POTTER. 1988. Effects of organic acids on thermal inactivation of Bacillus stearothermophilus and Bacillus coagulans spores in Frankfurter emulsion slurry. J. Food Prot. 51.475.

PALOP, A., F.J. SALA, AND S. CONDOS. 1997. Occurrence of a highly heat-sensitive spore subpopulation of Bacillus coagulans STCC 4522 and its conversion to a more heat-stable form. Applied Env. Microbiology 63:2246-51.

RICE, A.C. AND C.S. PEDERSON. 1953. Factors influencing growth of Bacillus coagulans in tomato juice I. Size of inoculum and oxygen concentration. Food Res. 18:115.

ROBERTS, C.M. AND D.G. HOOVER. 1996. Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin. J. Applied Bacteriology 81:363-368.

SANDOVAL, J.A., J.A. BARREIRO, AND S. MENDOZA. 1992. Thermal resistance of Bacillus coagulans in double concentrated tomato paste. J. Food Sci. 57:1369-1370.

SHAMSUDIN, M.N. 1984. Characteristics of Bacillus coagulans growth in tomato juice and similar products. M.S. Thesis. West Virginia Univ., Morgantown.

SOFAS, J.N AND F.F. BUSTA. 1981. Antimicrobial activity of sorbate: A review. J. Food Prot. 44:614.

STERN, R.M., G.P. HEGARTY, AND O.B. WILLIAMS. 1942. Defection of Bacillus thermoacidurans (Berry) in tomato juice and successful cultivation of the organism in the laboratory. Food Research. 7:186.
Jameel S. Al-Dujaili
Division of Sciences
Louisiana State University at Eunice
Eunice, LA 70535

Robert Anderson
Division of Plant and Soil Sciences
508 Brooks Hall
West Virginia University
Morgantown, WV 26506
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Date:Jan 1, 2000
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