Mild hypothermia promotes the viability of in vitro-produced bovine blastocysts and their transcriptional expression of the cold-inducible transcription factor Rbm3 during in vitro culture.

In this study, we evaluated the effects of holding in vitro-produced bovine blastocysts under mild hypothermia (33°C or 35°C), by examining viability and hatching rates of day 7 blastocysts (day 0: in vitro fertilization) cultured for 6 days and transcriptional expression of cold-inducible transcription factors Cirp and Rbm3, implicated in mild hypothermia-induced cellular protection against various types of stress. In the normothermic control (38.5°C), viability of the embryos decreased rapidly after day 10, and most samples were degenerated on day 13. However, mild hypothermia, particularly at 33°C, resulted in maintenance of high embryonic survival rates until day 13 (77.1% on day 13) and significant increases in transcriptional expression of Rbm3 in day 11 embryos compared with those at 38.5°C. Thus, our results suggested that upregulation of Rbm3 may occur in response to mild hypothermia in many bovine embryos, providing insights into the effects of mild hypothermia on embryo quality.

Change of thermal inactivation of Clostridium botulinum spores during rice cooking.

Cooked and packed rice (CaPR), a popular rice product in Japan, is distributed with oxygen-absorbing agents and without refrigeration. When the final product was inoculated with spores of several strains of proteolytic Clostridium botulinum at a dose of 10(3) spores per g (2 x 10(5) spores per package) and incubated at 30 degrees C, the bacteria grew and produced neurotoxins in 40 days. To simulate more realistic cases of contamination, the same dose of spores was inoculated before the cooking process. When cooked at 100 degrees C for 30 min, a small number of spores survived and the toxins were detected in some of the samples after incubation for 180 days. However, when cooked at 100 degrees C for 15 min immediately followed by 105 degrees C for 15 min, neither survivors nor the toxins were detected during incubation for 270 days after cooking. Even when inoculated with 10(5) spores per g of one of the most heat-resistant strains, 213B, viable spores were not detected after cooking. The inactivation by these heating conditions in different media indicated that the spores were inactivated >1,000-fold more in rice suspension than in cooked meat medium or phosphate buffer. It was therefore suggested that rice contains component(s) that facilitates thermal inactivation of C. botulinum.

Chlorine sensitivity of feline calicivirus, a norovirus surrogate.

The sensitivity to free chlorine of feline calicivirus (FCV), a norovirus surrogate, was examined relative to chlorine demand. When a crude suspension of FCV was treated with a sodium hypochlorite solution containing 10 microg/ml free chlorine, the extent of the decrease of viral infectivity clearly depended on the volume of the reaction mixture. The apparent sensitivity of FCV to free chlorine increased with the reduction of host cell debris, indicating that chlorine demand must be minimized to know the true sensitivity of the virus. We therefore partially purified the viruses from the host cell components and found that the infectivity of FCV was reduced by more than log 4.6 by 5 min of treatment with 300 ng/ml free chlorine.

Plant Trials of Bacon Made with Lactic Acid Bacteria, Sucrose and Lowered Sodium Nitrite.

Bacon prepared with 40 and 80 mg/kg (ppm) sodium nitrite, 0.7% sucrose and a culture of Pediococcus acidilactici (Wisconsin Process), and control bacon prepared with 120 ppm sodium nitrite and no added sucrose or bacterial culture were produced at three commercial bacon production plants. Sodium chloride, phosphate and sodium ascorbate (or sodium erythorbate) levels, as well as other processing conditions such as pumping rate, smokehouse temperature and time, forming and slicing conditions, were those normally used by each plant. Randomly selected samples of each lot were used for a challenge experiment with Clostridium botulinum (types A and B), with ca. 1,000 heat-shocked spores/g of bacon inoculated on each slice, vacuum packaged and incubated at 27°C. Samples were taken periodically up to 56 d of incubation and examined for the presence of botulinal toxin. The challenge experiment revealed that test bacon was substantially greater in antibotulinal properties than the control bacon. Residual nitrite levels of test bacon were lower than those of the control bacon, as were nitrosamines formed upon frying. Average N-nitrospyrrolidine level was 8.6 µg/kg (ppb) in the control, <2.7 ppb in the 80-ppm nitrite product, and <1.6 ppb in the 40-ppm nitrite product. This study indicates that bacon commercially prepared by the Wisconsin Process with 40 or 80 ppm sodium nitrite has a lesser risk of nitrosamine and botulinal toxin formation than bacon prepared with 120 ppm sodium nitrite and no added sucrose and lactic acid bacteria.

Sensory Characteristics of Reduced Nitrite Bacon Manufactured by the Wisconsin Process.

Bacon with a culture of lactic acid-forming bacteria, Pediococcus acidilactici , plus 0.7% sucrose and 40 or 80 mg sodium nitrite/kg (Wisconsin Process), and control bacon with 120 mg sodium nitrite/kg but no lactic acid bacteria and sucrose, were produced at three commercial bacon plants under production conditions. The bacon was stored under refrigeration for 5 to 8 wk, then subjected to sensory analyses by an experienced sensory panel. Quantitative descriptive visual analysis was performed on uncooked as well as cooked samples, and the cooked samples were served for quantitative descriptive sensory analysis. Results indicated that the test bacon with reduced amounts of sodium nitrite was as acceptable as the control bacon with no sugar and lactics, with the 80 mg/kg nitrite-bacon being the most preferred of all. These results and the results of botulinal challenge and nitrosamine tests indicate that the test process can be a satisfactory alternative to processing bacon by the conventional procedure with 120 mg sodium nitrite/kg.

Evaluation of the Bacteriological Safety of Low-Salt Miso.

Studies were done to evaluate the safety of three different low-salt (2.36 to 5.79% NaCl) misos inoculated with different bacterial pathogens. Clostridium botulinum types A and B (inoculum level of ca. 120 spores/g) did not produce toxin in any of the misos within 18 wk at 25°C. Staphylococcus aureus , Salmonella typhimurium and Yersinia enterocolitica (inoculum level of ca. 103 to 104 CFU/g) progessively died in all of the misos held at either 10 or 25°C. The miso samples, which were obtained from Japan (3.75 and 5.79% NaCl) and California (2.36% NaCl), had water activities of 0.843, 0.835 and 0.875, respectively, and pH values of 5.26, 5.30 and 4.73, respectively. Results indicate that low-salt misos with these properties are not likely to be bacteriological health risks.

Evaluation of the Microbiological Safety of Tempeh Made from Unacidified Soybeans.

Studies were done to evaluate the safety of tempeh made from unacidifed soybeans and inoculated with different bacterial pathogens. Pathogens were added to either the soybeans before fermentation by Rhizopus oligosporus or the tempeh after fermentation and steaming. In the latter method, the inoculated products were incubated at several different temperatures (5, 10, 15 and 25°C). Clostridium botulinum (types A and/or B) toxin was produced in 2 d during the fermentation and within 5 d at 25°C or 4 wk at 15°C in tempeh inoculated and incubated in vacuum packages after fermentation and steaming. Staphylococcus aureus grew very well (>6-log10 CFU/g increase) in 2 d during the fermentation, and grew from ca. 103 CFU/g to 108 CFU/g in 7 d at 25°C and 21 d at 15°C in tempeh inoculated after fermentation and steaming. Staphylococcal enterotoxins were detected in some of these samples. Salmonella typhimurium also grew well during the fermentation (>6-log10 CFU/g increase in 1 d), but grew relatively slowly at 25 and 15°C in tempeh inoculated after fermentation and steaming. Yersinia enterocolitica grew very well (>6-log10 CFU/g increase) in 1 d during the fermentation, and also grew well in tempeh inoculated after fermentation and steaming, with a >6 log10 CFU/g increase in 2 d at 25 or 15°C and 5 d at 10°C. Results of these studies indicate the need for maintaining: (a) a high level of sanitary practices during production and (b) good refrigeration (≤5°C) of the product following fermentation until it is used.

Evaluation of the Microbiological Safety of Tofu.

Studies were done to evaluate the safety of tofu inoculated with different bacterial pathogens and held at different temperatures. Clostridium botulinum (type A and/or B) toxin was produced in tofu after 1 and 3 wk of storage at 25 and 15°C, respectively, but not within 6 wk at 5 or 10°C. Compared with water-packed tofu, vacuum-packaged tofu did not increase the risk of botulinal toxin production at 15°C. Staphylococcus aureus and Salmonella typhimurium grew at similar rates at 10, 15 and 25°C, but decreased in number at 5°C. Staphylococcal enterotoxin was not produced within 4 wk at 10°C, 8 d at 15°C, or 2 d at 25°C, even though an S. aureus population of >107 CFU/g was present in most samples analyzed. Staphylococcal enterotoxin was detected in a 5-d sample held at 25°C. Toxin production may have been delayed because of the low amount of oxygen in water-packed tofu. Yersinia enterocolitica grew at all temperatures evaluated (5, 10, 15 and 25°C), including a 4- to 6-log10 increase within 14 d at 5°C. Results of these studies substantiate the need for: (a) implementing a high level of sanitary practices during tofu production, (b) pasteurizing tofu after packaging, and (c) properly refrigerating tofu during storage and display.

Toxin Production by Clostridium Botulinum in Media at pH Lower Than 4.6.

Clostridium botulinum types A and B were able to produce toxin in media containing high concentrations of proteins at pH well below 4.6. A medium containing 15% pork, 0.5% yeast extract, 0.5% glucose and 0.05% cysteine-HC1 at an initial pH between 4.30 and 4.36 produced a toxic sample in as early as one week of incubation at 30 C. Toxin production occurred only when large amounts of precipitated protein were present. The presence of other microorganisms was not required for botulinal toxin production. Media prepared with hydrolyzed protein did not have precipitates and did not support production of botulinal toxin at pH lower than 4.6. Since large amounts of precipitated protein seemed necessary for botulinal toxin production, the possibility of the presence of small, localized pockets of higher pH values within the matrices of precipitated protein could not be ruled out.

Challenge of Pasteurized Process Cheese Spreads with Clostridium botulinum Using In-Process and Post-Process Inoculation.

A study was done to evaluate the antibotulinal safety of pasteurized process cheese spreads and to compare two different published methods of inoculation of cheese spreads with Clostridium botulinum spores. Pasteurized process cheese spreads of various compositions were challenged with approximately 1,000 spores per g of C. botulinum types A and B. Two different methods of challenge were tested: (a) an "in-process" or "hot" inoculation in which a spore suspension was added to hot cheese spread in a cooker during agitation, and (b) a "post-process" or "cold" inoculation in which 0.1 ml of heat-shocked (80°C, 10 min) spore suspension was added to cheese spread already packed in glass jars and stirred. Certain products that were thought to have an adequate margin of safety by hot challenge studies became toxic when challenged by the cold method. Experiments to check localization of the spores in cold-inoculated cheese spread produced results suggesting that the concentration of the inoculum plus the localized diluting effect of added water in the cold-inoculated cheese spread probably account for the discrepancy between the two procedures.

A Challenge of Pasteurized Process Cheese Spread with Clostridium botulinum spores.

Pasteurized process cheese spread with pimientos, packaged in glass jars, inoculated during processing with 1000 spores of Clostridium botulinum per gram, developed neither gas nor toxin at 52 or 54% moisture when sodium phosphate was used as the emulsifier. When sodium citrate was the emulsifier, the product developed gas at 52% moisture and became toxic at 54%. At 58% moisture the product became gassy and toxic with either emulsifier. Since the product is commercially manufactured at 52% moisture with phosphate emulsifier, a substantial margin of safety exists.