Failure of nisin to inhibit outgrowth of Clostridium botulinum in a model cured meat system.

Up to 550 ppm (550 micrograms/ml) of nisin in combination with 60 ppm (60 micrograms/ml) of nitrite failed to prevent outgrowth of Clostridium botulinum spores in pork slurries adjusted to pH 5.8. Reducing the pH enhanced nisin activity. Proteolytic and nonproteolytic type B spores were equally resistant to nisin.     

Failure of nisin to inhibit outgrowth of Clostridium botulinum in a model cured meat system

Up to 550 ppm (550 micrograms/ml) of nisin in combination with 60 ppm (60 micrograms/ml) of nitrite failed to prevent outgrowth of Clostridium botulinum spores in pork slurries adjusted to pH 5.8. Reducing the pH enhanced nisin activity. Proteolytic and nonproteolytic type B spores were equally resistant to nisin.     

Growth from Spores of Clostridium perfringens in the Presence of Sodium Nitrite

The method by which sodium nitrite may act to prevent germination or outgrowth, or both, of heat-injured spores in canned cured meats was investigated by using Clostridium perfringens spores. Four possible mechanisms were tested: (i) prevention of germination of the heat-injured spores, (ii) prior combination with a component in a complex medium to prevent germination of heat-injured spores, (iii) inhibition of outgrowth of heat-injured spores, and (iv) induction of germination (which would render the spore susceptible to thermal inactivation). Only the third mechanism was effective with the entire spore population when levels of sodium nitrite commercially acceptable in canned cured meats were used. Concentrations of 0.02 and 0.01% prevented outgrowth of heat-sensitive and heat-resistant spores, respectively. Nitrite-induced germination occurred with higher sodium nitrite concentrations.     

The effect of nisin-sodium chloride interactions on the outgrowth of Bacillus licheniformis spores

The effect of salt (NaCl) on the efficacy of nisin in preventing outgrowth of Bacillus licheniformis spores was determined in Plate Count Agar (PCA). An equivalent liquid medium was used for heat activation. Nisin and salt were added to the heat-activation medium, the PCA, or both. The spores were extremely sensitive to nisin; outgrowth were completely inhibited in salt-free media when 10 iu/ml of nisin was present in both the heat-activation and the growth media or when 100 iu/ml nisin was present in either the heat-activation and the growth medium. In media supplemented with 1% salt, outgrowth occurred from 1% of spores exposed to 100 iu/ml nisin in either the heat-activation or the growth medium. A 3% salt supplement was necessary before detectable outgrowth occurred when both the heat-activation and the growth media contained 100 iu/ml nisin. Salt appears to antagonize the sporicidal action of nisin by interfering with nisin adsorption onto the spore.     

Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present.     

Effect of processing variables on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted meat cured with sorbic acid and sodium nitrite.

The effects of the initial pH and a "short pump" on the outgrowth of Clostridium sporogenes PA 3679 spores in comminuted cured pork were studied. Fresh ground pork was cured with salt, sugar, phosphate, ascorbate, and varying amounts of sodium nitrite and sorbic acid. The product was comminuted and inoculated with 1,000 spores of C. sporogenes per g. The meat was stuffed into 1-ounce (ca. 28.4-g) aluminum tubes, cooked to 58.5 degrees C, cooled, and incubated at 27 degrees C to observe for swells. Product cured with 0.2% sorbic acid in combination with 40 ppm sodium nitrite (40 microgram/g) had better clostridium inhibition than did product cured with 120 ppm nitrite within a pH range of 5.0 to 6.7. The sorbic acid-40 ppm nitrite combination also gave better clostridial protection than did the 120 ppm nitrite alone when reduced amounts of curing ingredients were present.     

Causes of variation in botulinal inhibition in perishable canned cured meat.

Final internal processing temperatures within the range of 63 to 74 degrees C did not alter the degree of botulinal inhibition in inoculated perishable canned comminuted cured pork abused at 27 degrees C. Adding hemoglobin to the formulation reduced residual nitrite after processing and decreased botulinal inhibition. Different meats yielded different rates of botulinal outgrowth when substituted for fresh pork ham. Pork or beef heart meat showed no inhibition of the Clostridium botulinum inoculum even with a 156-microgram/g amount of sodium nitrite added to the product. This effect appears to be one of stimulating outgrowth, since residual nitrite depletion was not measurably altered.     

Causes of variation in botulinal inhibition in perishable canned cured meat.

Final internal processing temperatures within the range of 63 to 74 degrees C did not alter the degree of botulinal inhibition in inoculated perishable canned comminuted cured pork abused at 27 degrees C. Adding hemoglobin to the formulation reduced residual nitrite after processing and decreased botulinal inhibition. Different meats yielded different rates of botulinal outgrowth when substituted for fresh pork ham. Pork or beef heart meat showed no inhibition of the Clostridium botulinum inoculum even with a 156-microgram/g amount of sodium nitrite added to the product. This effect appears to be one of stimulating outgrowth, since residual nitrite depletion was not measurably altered.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

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 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/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 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees 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 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram 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 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Role of Curing Agents in the Preservation of Shelf-stable Canned Meat Products

Experiments were conducted to gain a better understanding of the mechanism by which sodium chloride, sodium nitrate, and sodium nitrite supplement the action of heat in preserving canned cured meat products. Heated spores of putrefactive anaerobe 3679h were less tolerant of all three curing agents in the outgrowth medium than were unheated spores. When the curing agents were added to the heating menstruum, but not to the outgrowth medium, sodium chloride and sodium nitrate tended to protect the spores against heat injury, but sodium nitrite did not. When the spores were both heated and cultured in the presence of the curing agents: (i) nitrate and salt increased the apparent heat resistance at low concentrations (0.5 to 1%) but decreased it at concentrations of 2 to 4%; (ii) nitrite was markedly inhibitory, especially at pH 6.0. At the normal pH of canned luncheon meats (approximately 6.0), nitrite appears to be the chief preservative agent against spoilage by putrefactive anaerobes.     

Effect of calcium carbonate and iron on the availability and uptake of iron,manganese, phosphorus and calcium in pea (Pisum sativum L.)

Summary The effect of CaCO₃ and iron on the availability of iron, manganese phosphorus and calcium was studied in the greenhouse on pea (Pisum sativum L.) crop on a light textured soil, which was marginal in exchangeable calcium. Addition of calcium carbonate caused significant increase in dry matter yield with no added iron at both the stages of crop growth. But yeild intended to decrease with 8% CaCO₃ at 75 days of crop growth. Dry matter yield also increased with the addition of iron upto 10 ppm at 45 days and upto 5 ppm at 75 days. The iron concentration and uptake decreased with the increase in CaCO₃ and increased with the application of iron at both the stages of crop growth. The application of iron and CaCO₃ decreased concentration and uptake of phosphorus significantly at both the stages.Like phosphorus, concentration and uptake of manganese also decreased with the increase in added CaCO₃ upto 8% and iron upto 20 ppm at 45 and 75 days. The concentration of calcium increased with the addition of CaCO₃ to the extent of 50 and 40% with 8% CaCO₃ at 45 and 75 days, while the uptake of calcium increased more than 3 folds at 45 days and more than 2 folds at 75 days. The concentration of calcium decreased with the application of iron upto 20 ppm but the uptake at 45 days increased upto 10 ppm and at 75 days upto 5 ppm and then decreased.The concentration of Fe, P and Ca decreased at 75 days and that of Mn increased while the uptake of all these nutrients increased at 2nd stage due to higher dry matter.     

Influence of various mixtures of inhaled toluene and xylene on the biological monitoring of exposure to these solvents in rats.

The present study was undertaken to describe the influence of simultaneous exposure by inhalation to toluene and xylene on some aspects of their respective metabolic disposition. Adult male rats were exposed acutely (5 h) to 75, 150, and 225 ppm of toluene or xylene and to various mixtures of these solvents: toluene (75 ppm) and xylene (225 ppm), toluene (150 ppm) and xylene (150 ppm), toluene (225 ppm) and xylene (75 ppm). Compared with single exposure, simultaneous exposure resulted in lower amounts of excreted hippuric acid (20-30%) and methylhippuric acids (4-40%) in urine over a period of 24 h, even though significant differences were seen only with the toluene (150 ppm) and xylene (150 ppm) combination. In addition, increased concentrations of solvents in blood (toluene, 230%; xylene, 500%) and in brain (toluene, 230%; xylene, 320%) were found during the immediate post-exposure period. Simultaneous exposure also enhanced the pulmonary elimination of both solvents (toluene, 190-240%; xylene, 340-650%). Influence of repeated simultaneous exposure (9 days) was investigated for the toluene (150 ppm) and xylene (150 ppm) combination and the results compared with those of repeated exposure to each solvent administered singly. Under these conditions, repeated simultaneous exposure decreased the excretion of urinary metabolites, but only after the first exposure. On the other hand, simultaneous exposure resulted in significantly higher concentrations of toluene (210%) and xylene (240%) in blood throughout the entire 9-day exposure period. These results strongly suggest mutual metabolic interactions (inhibition) between toluene and xylene that affect the metabolic disposition of both solvents and ultimately the biological monitoring of data of exposure to a combination of solvents in rats.     

Iron metabolism in iron-deficient male quail

1. Male quails submitted 20 and 120 days to a low iron diet (7 ppm) were compared to female laying quails, exposed for 30 days to the same low iron regime, in order to compare the response of the iron metabolic control under a single (erythropoiesis) or a doubled (erythropoiesis and egg formation) iron demand. 2. Iron deposit in storage organs, the classical hematology and the intestinal iron absorption were analyzed in these animals. 3. In males, after 120 days, the iron deposits were reduced 50 and 75%, but hematological values (hematocrit and hemoglobin concentration) were normal, although in laying quails, after 30 days, an anemic condition was evident in both blood parameters and iron deposits, provoking an iron deficient erythropoiesis. 4. The enhancement of the intestinal iron uptake, confirms the anemic character of these birds.     

Detection of nitrate/nitrite bioavailability in wastewater using a luxCDABE-based Klebsiella oxytoca bioluminescent bioreporter

In the present study, we have constructed a bioluminescent bioreporter for the assessment of nitrate/nitrite bioavailability in wastewater. Specifically, an approximately 500-bp DNA fragment containing a nitrate/nitrite-activated nasR-like promoter (regulating expression of genes encoding nitrite reductase in the genus Klebsiella) was fused upstream of the Vibrio fischeri luxCDABE gene cassette in a modified mini-Tn5 vector. Characterization of this strain, designated W6-1, yielded dose-dependent increased bioluminescence coincident with increased nitrate, nitrite, and ammonium added to the growth medium from 1 to 11 ppm. Bioluminescence in response to nitrogen species addition was light dependent up to 10, 7, and 8 ppm with nitrate, nitrite, and ammonium, respectively. This response was linear in the range from 1 to 8 ppm for nitrate (R2 = 0.98), 1 to 6 ppm for nitrite (R2 = 0.99), and 1 to 7 ppm for ammonium (R2 = 0.99). A significant bioluminescent response was also recorded when strain W6-1 was incubated with slurries from aged, nitrate/nitrite contaminated wastewater. Thus, bioreporter strain W6-1 can be used to elucidate factors that constrain the use of nitrate/nitrite in wastewaters.     

Developmental toxicity studies in Crl:CD (SD) rats following inhalation exposure to trichloroethylene and perchloroethylene

The potential for trichloroethylene (TCE) and perchloroethylene (PERC) to induce developmental toxicity was investigated in Crl:CD (SD) rats whole-body exposed to target concentrations of 0, 50, 150 or 600 ppm TCE or 0, 75, 250 or 600 ppm PERC for six hours/day, seven days/week on gestation day (GD) 6-20 and 6-19, respectively. Actual chamber concentrations were essentially identical to target with the exception of the low PERC exposure level, which was 65 ppm. The highest exposure levels exceeded the limit concentration (2 mg/L) specified in the applicable test guidelines. Maternal necropsies were performed the day following the last exposure. Dams exposed to 600 ppm TCE exhibited maternal toxicity, as evidenced by decreased body weight gain (22% less than control) during GD 6-9. There were no maternal effects at 50 or 150 ppm TCE and no indications of developmental toxicity (including heart defects or other terata) at any exposure level tested. Therefore, the TCE NOEC for maternal toxicity was 150 ppm, whereas the embryo/fetal NOEC was 600 ppm. Maternal responses to PERC were limited to slight, but statistically significant reductions in body weight gain and feed consumption during the first 3 days of exposure to 600 ppm, resulting in a maternal NOEC of 250 ppm. Developmental effects at 600 ppm consisted of reduced gravid uterus, placental and fetal body weights, and decreased ossification of thoracic vertebral centra. Developmental effects at 250 ppm were of minimal toxicological significance, being limited to minor decreases in fetal and placental weight. There were no developmental effects at 65 ppm.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Sources and Sinks for Ammonia and Nitrite on the Early Earth and the Reaction of Nitrite with Ammonia

An analysis of sources and sinks for ammonia and nitrite on the early Earth was conducted. Rates of formation and destruction, and steady state concentrations of both species were determined by steady state kinetics. The importance of the reaction of nitrite with ammonia on the feasibility of ammonia formation from nitrite was evaluated. The analysis considered conditions such as temperature, ferrous iron concentration, and pH. For sinks we considered the reduction of nitrite to ammonia, reaction between nitrite and ammonia, photochemical destruction of both species, and destruction at hydrothermal vents. Under most environmental conditions, the primary sink for nitrite is reduction to ammonia. The reaction between ammonia and nitrite is not an important sink for either nitrite or ammonia. Destruction at hydrothermal vents is important at acidic pH's and at low ferrous iron concentrations. Photochemical destruction, even in a worst case scenario, is unimportant under many conditions except possibly under acidic, low iron concentration, or low temperature conditions. The primary sink for ammonia is photochemical destruction in the atmosphere. Under acidic conditions, more of the ammonia is tied up as ammonium (reducing its vapor pressure and keeping it in solution) and hydrothermal destruction becomes more important.     

Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria.

Nisin, produced by Lactococcus lactis subsp. lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum. This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria. After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested. Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested. These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria. Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.     

Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria.

Nisin, produced by Lactococcus lactis subsp. lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum. This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria. After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested. Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested. These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria. Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.     

EFFECTS OF CULTURE CONDITIONS ON HYDROGENASE ACTIVITY OF SCENEDESMUS D3

The effects of culture conditions and of culture ages upon hydrogenaseactivity in Scenedesmus D₃ was investigated with nitrite andbenzoquinone as acceptors. It was noticed that level of ironin the culture medium played an important part in the developmentof hydrogenase activity. The cells grown on a medium of relatively high level of iron(10^–4 M) showed higher activity both in nitrite and benzoquinonereduction than those grown on a medium of relatively low levelof iron (10^–5 M). In the former case, hydrogenase activityappeared even after a short incubation with hydrogen (30 min). When the cells with low hydrogenase activity were transferredto a medium containing 10^–;4 M iron and the activity wasmeasured every 24 hours, the following facts were observed. (a) Nitrite reduction activity increased markedly during thefirst 24 hours and then decreased gradually during further cultivation.Furthermore, a parallelism was observed between nitrite reductionactivity and cell size. (b) On the other hand, the pattern of change in quinone reductionactivity was somewhat different from that of nitrite reduction.This activity appeared only after 3 days' cultivation, reacheda maximum at 4th or 5th day and then decreased. However, thepresence of glucose in the culture medium suppresses the appearanceof quinone reduction activity, while the nitrite reduction activitywas less markedly affected. ¹Present address: Department of Experimental Radiology, Facultyof Medicine, Kyushu University, Fukuoka.