Aims:  To clarify the diversity of Bacillus subtilis strains in Thua nao that produce high concentrations of products useful in food manufacturing and in health‐promoting compounds. Method and Results:  Production of amylase, protease, subtilisin NAT (nattokinase), and gamma‐polyglutamic acid (PGA) by the Bacillus subtilis strains in Thua nao was measured. Productivity of protease NAT by these strains tended to be higher than by Japanese commercial natto‐producing strains. Molecular diversity of isolated strains was analysed via randomly amplified polymorphic DNA‐PCR fingerprinting. The strains were divided into 19 types, including a type with the same pattern as a Japanese natto‐producing strain. Conclusion:  B. subtilis strains that could be a resource for effective production of protease, amylase, subtilisin NAT, or PGA were evident in Thua nao produced in various regions in northern Thailand. Significance and Impact of the Study:  This study clearly demonstrated the value of Thua nao as a potential resource of food‐processing enzymes and health‐promoting compounds.

Certain Bacillus subtilis strains, such as B. subtilis (natto) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-␥-glutamate (␥PGA). In B. subtilis (natto), ␥PGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS4Bsu1, in the comP gene of a spontaneous ␥PGA-negative mutant of B. subtilis (natto) NAF4. IS4Bsu1 (1,406 bp), the first IS discovered in B. subtilis, encodes a putative transposase (Tpase) with a predicted M r of 34,895 (374 residues) which displays similarity to the Tpases of IS4 family members. Southern blot analyses have identified 6 to 11 copies of IS4Bsu1, among which 6 copies were at the same loci, in the chromosomes of B. subtilis (natto) strains, including NAF4, three commercial starters, and another three ␥PGA-producing B. subtilis (natto) strains. All of the eight spontaneous ␥PGA ؊ mutants, which were derived from five independent NAF4 cultures, had a new additional IS4Bsu1 copy in comP at six different positions within 600 bp of the 5-terminal region. The target sites of IS4Bsu1 were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS4Bsu1 in mutant comP genes. These results indicate that IS4Bsu1 transposes by the replicative mechanism, in contrast to other IS4 members that use the conservative mechanism, and that most, if not all, of spontaneous ␥PGA ؊ mutants appear to have resulted from the insertion of IS4Bsu1 exclusively into comP. The presence of insertion hot spots in comP, which is essential for ␥PGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous ␥PGA ؊ mutation by IS4Bsu1 in B. subtilis (natto).

The inability of chlorine to completely inactivate human bacterial pathogens on whole and fresh-cut produce suggests a need for other antimicrobial washing treatments. Nisin (50 microg/ml) and pediocin (100 AU/ml) individually or in combination with sodium lactate (2%), potassium sorbate (0.02%), phytic acid (0.02%), and citric acid (10 mM) were tested as possible sanitizer treatments for reducing the population of Listeria monocytogenes on cabbage, broccoli, and mung bean sprouts. Cabbage, broccoli, and mung bean sprouts were inoculated with a five-strain cocktail of L. monocytogenes at 4.61, 4.34, and 4.67 log CFU/g, respectively. Inoculated produce was left at room temperature (25 degrees C) for up to 4 h before antimicrobial treatment. Washing treatments were applied to inoculated produce for 1 min, and surviving bacterial populations were determined. When tested alone, all compounds resulted in 2.20- to 4.35-log reductions of L. monocytogenes on mung bean, cabbage, and broccoli, respectively. The combination treatments nisin-phytic acid and nisin-pediocin-phytic acid caused significant (P < 0.05) reductions of L. monocytogenes on cabbage and broccoli but not on mung bean sprouts. Pediocin treatment alone or in combination with any of the organic acid tested was more effective in reducing L. monocytogenes populations than the nisin treatment alone. Although none of the combination treatments completely eliminated the pathogen on the produce, the results suggest that some of the treatments evaluated in this study can be used to improve the microbial safety of fresh-cut cabbage, broccoli, and mung bean sprouts.

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Efficacy of acidified sodium chlorite for reducing the population of Escherichia coli O157:H7 pathogens on Chinese cabbage leaves was evaluated. Washing leaves with distilled water could reduce the population of E. coli O157:H7 by approximately 1.0 log CFU/g, whereas treating with acidified chlorite solution could reduce the population by 3.0 log CFU/g without changing the leaf color. A similar level of reduction was achieved by washing with sodium chlorite solution containing various organic acids. However, acidified sodium chlorite in combination with a mild heat treatment reduced the population by approximately 4.0 log CFU/g without affecting the color, but it softened the leaves. Moreover, the efficacy of the washing treatment was similar at low (4 degrees C) and room (25 degrees C) temperatures, indicating that acidified sodium chloride solution could be useful as a sanitizer for surface washing of fresh produce.

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We investigated the heat resistance of a four-strain mixture of Escherichia coli O157:H7 in raw ground beef in both the absence and presence of white and green tea powders and an apple skin extract. Inoculated meat was cooked using the sous-vide technique, i.e., the meat was packaged in sterile bags and completely immersed in a circulating water bath at low temperature for a period of time. The bags were cooked for 1 h to an internal temperature of 55, 58, 60, or 62.5 degrees C, and then held from 240 min at 55 degrees C to 10 min at 62.5 degrees C. The surviving bacteria were enumerated by spiral plating onto tryptic soy agar overlaid with sorbitol-MacConkey agar. Inactivation kinetics of the pathogens deviated from first-order kinetics. D-values (time, in minutes, required for the bacteria to decrease by 90%) in the control beef ranged from 67.79 min at 55 degrees C to 2.01 min at 62.5 degrees C. D-values determined by a logistic model ranged from 36.22 (D1, the D-value of a major population of surviving cells) and 112.79 (D2, the D-value of a minor subpopulation) at 55 degrees C to 1.39 (D1) and 3.00 (D2) at 62.5 degrees C. A significant increase (P < 0.05) in the sensitivity of the bacteria to heat was observed with the addition of 3% added antimicrobials. D-value reductions of 62 to 74% were observed with apple powder and 18 to 58% with tea powders. Thermal death times from this study will assist the retail food industry to design cooking regimes that ensure the safety of beef contaminated with E. coli O157:H7.

The antibacterial activity of guava (Psidium guajava) and neem (Azadirachta indica) extracts against 21 strains of foodborne pathogens were determined--Listeria monocytogenes (five strains), Staphylococcus aureus (four strains), Escherichia coli O157:H7 (six strains), Salmonella Enteritidis (four strains), Vibrio parahaemolyticus, and Bacillus cereus, and five food spoilage bacteria: Pseudomonas aeroginosa, P. putida, Alcaligenes faecalis, and Aeromonas hydrophila (two strains). Guava and neem extracts showed higher antimicrobial activity against Gram-positive bacteria compared to Gram-negative bacteria except for V. parahaemolyticus, P. aeroginosa, and A. hydrophila. None of the extracts showed antimicrobial activity against E. coli O157:H7 and Salmonella Enteritidis. The minimum inhibitory concentration (MIC) of ethanol extracts of guava showed the highest inhibition for L. monocytogenes JCM 7676 (0.1 mg/mL), S. aureus JCM 2151 (0.1 mg/mL), S. aureus JCM 2179 (0.1 mg/mL), and V. parahaemolyticus IFO 12711 (0.1 mg/mL) and the lowest inhibition for Alcaligenes faecalis IFO 12669, Aeromonas hydrophila NFRI 8282 (4.0 mg/mL), and A. hydrophila NFRI 8283 (4.0 mg/mL). The MIC of chloroform extracts of neem showed similar inhibition for L. monocytogenes ATCC 43256 (4.0 mg/mL) and L. monocytogenes ATCC 49594 (5.0 mg/mL). However, ethanol extracts of neem showed higher inhibition for S. aureus JCM 2151 (4.5 mg/mL) and S. aureus IFO 13276 (4.5 mg/mL) and the lower inhibition for other microorganisms (6.5 mg/mL). No significant effects of temperature and pH were found on guava and neem extracts against cocktails of L. monocytogenes and S. aureus. The results of the present study suggest that guava and neem extracts possess compounds containing antibacterial properties that can potentially be useful to control foodborne pathogens and spoilage organisms.