The efficacy of chlorine and hot water treatments in killing Salmonella stanley inoculated onto alfalfa seeds was determined. Treatment of seeds containing 10 2 to 10 3 CFU/g in 100-g/ml active chlorine solution for 5 or 10 min caused a significant (P < 0.05) reduction in population, and treatment in 290-g/ml chlorine solution resulted in a significant reduction compared with treatment in 100 g of chlorine per ml. However, concentrations of chlorine of up to 1,010 g/ml failed to result in further significant reductions. Treatment of seeds containing 10 1 to 10 2 CFU of S. stanley per g for 5 min in a solution containing 2,040 g of chlorine per ml reduced the population to undetectable levels (<1 CFU/g). Treatment of seeds in water for 5 or 10 min at 54؇C caused a significant reduction in the S. stanley population, and treatment at >57؇C reduced populations to <1 CFU/g. However, treatment at >54؇C for 10 min caused a substantial reduction in viability of the seeds. Treatment at 57 or 60؇C for 5 min appears to be effective in killing S. stanley without substantially decreasing germinability of seeds. Storage of seeds for 8 to 9 weeks at 8 and 21؇C resulted in reductions in populations of S. stanley of about 1 log 10 and 2 log 10 CFU/g, respectively. The behavior of S. stanley on seeds during soaking, germination, sprouting, and refrigerated storage of sprouts was determined. An initial population of 3.29 log 10 CFU/g increased slightly during 6 h of soaking, by about 10 3 CFU/g during a 24-h germination period, and by an additional 10 CFU/g during a 72-h sprouting stage. A population of 10 7 CFU/g of mature alfalfa sprouts was detected throughout a subsequent 10-day storage period at 5؇C. These studies indicate that while populations of S. stanley can be greatly reduced, elimination of this organism from alfalfa seeds may not be reliably achieved with traditional disinfection procedures. If S. stanley is present on seeds at the initiation of the sprout production process, populations exceeding 10 7 CFU/g can develop and survive on mature sprouts exposed to handling practices used in commercial production and marketing.

Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern. A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B. cereus. The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains. Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml. At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml. Tolerance of vegetative cells of B. cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57. Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively. At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml. The effectiveness of nisin in controlling the growth of psychrotrophic strains of B. cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53. Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B. cereus in nonpasteurized foods held at refrigeration temperatures are warranted.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 g of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 g/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 g/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8°C than at 15°C. Nisin, at a concentration of 5 or 50 g/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15°C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15°C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 g of nisin/ml and stored at 8°C for 14 days; 5 g of nisin/ml was required for inhibition at 15°C. Enterotoxin was not detected in gravy in which less than 5.85 log 10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 g of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8°C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.

The potential for growth of enterotoxigenic Bacillus cereus in reconstituted dry foods is a concern, especially when they are consumed by infants or the immunosuppressed. The ability of a four-strain mixture of spores or vegetative cells of psychrotrophic B. cereus to survive in a commercial, dry infant rice cereal as affected by water activity (a(w); 0.27 to 0.28, 0.52 to 0.55, and 0.75 to 0.78), pH (5.6 and 6.7), and temperature (5, 25, 35, and 45 degrees C) was investigated. The rate of death of vegetative cells in dry cereal stored for 36 weeks was not affected by a(w) or pH. Death of spores in cereal stored at 45 degrees C for up to 48 weeks was enhanced at a(w) 0.78 but was unaffected by pH; loss of viability at 5, 25, and 35 degrees C was largely unaffected by differences in a(w). The effect of temperature (8, 15, 21, and 30 degrees C) on outgrowth of spores of B. cereus inoculated at three levels (0.14, 14, and 133 CFU/g, dry weight basis) into cereal reconstituted with apple juice and commercial pasteurized milk (2% fat) was also studied. Outgrowth of spores did not occur in cereal reconstituted with apple juice. Cereal reconstituted with milk and inoculated with 0.14, 14, and 133 spores per g contained >3 log CFU/g within 24, 9, and 6 h, respectively, at 21 degrees C. Populations in cereal reconstituted with milk and inoculated with 133 CFU of B. cereus spores per g reached 7.11, 7.72, and 7.40 log CFU/g within 12, 48, and 72 h when stored at 30, 21, and 15 degrees C, respectively. The organism grew in cereal reconstituted with milk and held at 8 degrees C for 72 h; however, enterotoxin was not detected. In reconstituted cereal inoculated with 133 spores per g, enterotoxin was detected (detection limit 16 ng/g) after 24, 48, and 72 h at 30, 21, and 15 degrees C, respectively, when the population of B. cereus reached >7 log CFU/g. It is recommended that reconstituted infant foods be either consumed immediately or held at < or = 8 degrees C and consumed within 48 h after preparation.