Inhibition of Clostridium sporogenes PA 3679 and Natural Bacterial Flora of Cooked Vacuum-Packaged Bratwurst by Sodium Acid Pyrophosphate and Sodium Tripolyphosphate With or Without Added Sodium Nitrite

Survival and growth of inoculated Clostridium sporogenes PA3679 and natural aerobic and anaerobic bacterial flora were studied in refrigerated (5°C) and subsequently temperature abused (24–25°C), cooked, vacuum-packaged bratwurst containing 0.5% sodium acid pyrophosphate (SAPP) or sodium tripolyphosphate (STPP) with or without sodium nitrite. Phosphates alone or combined with nitrite did not affect aerobic bacterial counts but resulted in reduced clostridial and anaerobic counts at 5°C. Upon temperature abuse, inhibition of all bacteria by SAPP was significant (P<0.05) for up to 48 hr and greatly enhanced by 100 ppm but not by 50 ppm sodium nitrite, whereas STPP lost its antimicrobial properties after 24 hr. Soluble orthophosphate levels had a positive correlation with bacterial inhibition in SAPP-treated bratwurst.     

Pyruvate Metabolism in Saccharomyces cerevisiae

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.     

N-nitrosamine and mutagenicity formation in Chinese salted fish after digestion.

Salted and dried fish (Nemipterus virgatus), acquired from Hong Kong, was treated with 0.43-110 mM nitrite during in vitro digestion using gastric enzymes and the volatile N-nitrosamine content and mutagenicity on Salmonella typhimurium TA100 assayed without concentration. N-Nitrosodimethylamine (NDMA; the only nitrosamine detected) formation was second order in nitrite concentration. When 10 g of fish was treated with 6.96 mM nitrite, 394 nM NDMA was formed. Thiocyanate was catalytic for NDMA formation at nitrite concentration greater than 0.87 mM and when the ratio of thiocyanate to nitrite was greater than 1. Approximately a 50% inhibition in NDMA formation by ascorbic acid was seen when the ratio of ascorbate to nitrite was approximately 2 or greater and the nitrite concentration was 1.74 mM. Mutagenicity increased with increasing nitrite concentration but the addition of thiocyanate did not increase mutagenicity over nitrite alone. Ascorbate increased mutagenicity even though NDMA formation was inhibited. Even at nitrite concentrations greater than 100-fold higher than expected in vivo, there was insufficient NDMA formed to account for the observed mutagenicity. These data do not exclude the possibility that the observed mutagenicity was due to non-volatile N-nitroso compounds, however, this possibility seems unlikely given the effects of ascorbate and thiocyanate which would be expected to inhibit and enhance non-volatile N-nitroso compound formation.     

Mitochondrial enzyme pathways and their possible role during curing.

The velocity of oxidation of exogenous ferrocytochrome c by nitrite under anaerobic conditions in the presence of skeletal muscle mitochondria is dependent upon pH over at least the range 5.6-6.7, increasing markedly as the pH is lowered. A product of the reaction is the complex formed between nitric oxide and ferricytochrome c. At levels up to 20 mM, nitrite inhibits aerobic cytochrome oxidase action; at higher concentrations, however, a partial resuscitation of the oxidation of ferrocytochrome c occurs, the enhancement of reaction velocity being considerably greater at pH 6.0 than at 6.5. Mitochondrial respiration is also inhibited by nitrite but no similar resurgence was, however, observed and thus the oxidation of ferrocytochrome c by high levels of nitrite is considered to be a direct non-enzymic action. Under anaerobic conditions, the rate of increase of the velocity constant of the oxidation of ferrocytochrome c with nitrite concentration in the presence of muscle mitochondria similarly decreased with rise of pH over the same range. The permeability of the muscle mitochondrion to nitrire has been demonstrated by swelling studies and by the rapid conversion of endogenous ferrocytochrome a3 into its nitrosyl-derivative. Over longer periods of anaerobic incubations of mitochondria with nitrite, oxidation of endogenous cytochromes occurs with the formation of nitrosylferricytochrome c. Above a nitrite concentration of 0.3 mM, the mitochondrial enzyme system probably involved is increasingly inhibited but by a concentration of 30 mM a direct non-enzymic oxidation has intervened. Commercial vacuum packed bacons were examined by electron microscopy. Mitochondria were clearly recognisable although they contained fewer cristae than those observed in fresh meat.     

Effect of sulfite or nitrite on the ATP content and the carbohydrate metabolism in yeast

Low concentrations of sulfite or nitrite (about 0.5 mmol) when applied at pH 3.6, caused a rapid and drastic decrease of the concentration of ATP in yeast cells. Under these conditions, alcoholic fermentation was inhibited by sulfite and to a lesser extent by nitrite. Ethanol consumption under aerobic conditions was shown to be more sensitive to nitrite than to sulfite. This indicates a higher sensitivity of respiratory processes to nitrite than to sulfite. Among 15 enzyme activities assayed in extracts from yeast cells after incubation with sulfite or nitrite, glyceraldehyde-3-phosphate dehydrogenase was shown to be the most sensitive. Analysis of the steady-state concentrations of intermediates of alcoholic fermentation in intact yeast cells also implies inhibition by sulfite or nitrite of the glyceraldehyde-3-phosphate dehydrogenase step of fermentation. In contrast to nitrite, sulfite had an additional effect by accumulating the intracellular steady state concentration of glyceraldehyde-3-phosphate 10 to 100-fold over the concentration in the absence of sulfite. In vitro studies on the equilibrium catalyzed by triosephosphate isomerase or aldolase confirmed the postulated shift of equilibrium concentrations by a formation of complex of glyceraldehyde-3-phosphate with sulfite.     

EFFECT OF INCORPORATING ADDITIONAL OLEIC ACID INTO THE PLASMA MEMBRANE OF BAKER'S YEAST ON THE PERMEATION OF PYRUVIC ACID

Addition of Tween 80 to the growth medium of baker's yeast (Saccharomyces cerevisiae) under anaerobic conditions resulted in an increased content of 9-cis-octadecenoic acid (oleic acid) in a membrane preparation consisting mainly of plasma membrane. There was no significant effect on the pyruvate decarboxylation capacity of disintegrated cells, but that of intact cells was significantly enhanced, presumably because of an oleate-induced increase in the permeability of the plasma membrane to pyruvate.     

Effects of different inorganic salts and organic nutrient components on the growth of Lentinula edodes (Shiitake) mycelium on solid medium

The influence of 11 inorganic salts and various organic nutrients on the mycelial growth of Lentinula edodes (Berk) Pegler (Shiitake) on solid medium was studied. All of these inorganic salts exerted concentration‐dependent inhibitory effects. Nitrite caused the strongest inhibition: 8 mM nitrite inhibited the growth completely. The inhibitory effect of nitrite could be reduced by the addition of ascorbic acid or a black tea extract. The effects of the addition of the different organic nutrients were investigated in order to optimize the composition of the culture medium. The medium containing malt extract (15 g l^?1), starch (3 g l^?1) and oak wood chips (20 g l^?1) proved best for mycelial growth. Growth was not promoted by components with a high organic nitrogen content. Copyright © 2003 Society of Chemical Industry     

A pyruvate dehydrogenase-based amperometric biosensor for assessing pungency in onions (Allium cepa L.)

A disposable prototype electrochemical biosensor was constructed using pyruvate dehydrogenase immobilised on mediated Meldolas Blue electrodes to determine pungency in onions (Allium cepa L.). The optimum operating potential was +50 mV vs. Ag/AgCl reference/counter electrode. The biosensor was able to differentiate between mild and pungent bulbs with pyruvate concentrations ranging between ≈4 and 8 mM in freshly extracted juices. Resolution was 0.5 mM and thus was comparable to the standard Schwimmer and Weston-based colorimetric assay currently employed by industry for determining pungency in macerated onion tissue.     

Asymmetric reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate coupled with nitrate reduction by Paracoccus denitrificans

We found that the asymmetric reduction of ethyl acetoacetate (EAA) to ethyl (R)-3-hydroxybutyrate (EHB) by Paracoccus denitrificans could be induced by nitrate addition under anaerobic conditions. However, the addition of electron donors such as glucose, ethanol, and methanol together with nitrate did not stimulate EHB production from EAA. When the reaction was carried out under optimum conditions (cell concentration, 10 g-d.w.l(-1); EAA, 150 mM; NO3-, 100 mM), after 8 h of reaction 49 mM of EHB with an optical purity of 98.9% e.e. was produced. Furthermore, a fed-batch reaction was carried out with an intermittent addition of nitrate and EAA to reduce substrate inhibition. The linear reduction of EAA to EHB proceeded for 40 h after initiation of the reaction, and finally 124 mM of EHB with an optical purity of 88.7% e.e. was produced after 104 h.     

Effect of growing bacteria isolated from food on staphylococcal growth and thermonuclease activity

The effect of growing Bacillus subtilis, Streptococcus faecalis var. liquefaciens, Escherichia coli and Lactobacillus plantarum on staphylococcal growth and thermonuclease (TNase) activity was investigated in liquid media and in foods. Growth of S. aureus at 37°C for 24 h under aerobic conditions was not inhibited by the four test strains. However, staphylococcal TNase activity decreased by 70 and 80% in the presence of B. subtilis and S. faecalis var. liquefaciens respectively. Staphylococcal growth and TNase activity were strongly inhibited by L. plantarum under anaerobic conditions at pH 5.5 but not at pH 7.0. Furthermore, optimal TNase production by S. aureus occurred in cooked meat medium containing 0.5 to 5.0% NaCl. TNase production significantly decreased at higher concentrations of NaCl. In the presence of B. subtilis and S. faecalis var. liquefaciens. TNase activity decreased at NaCl levels of 0.5 to 5.0% but not at NaCl concentrations>5.0%. TNase activity was also inhibited by growing B. subtilis and S. faecalis var. Liquefaciens at pH 5.0 to 7.0. The rate of inhibition increased with increasing pH. TNase activity was not inhibited after 48 h incubation at 20° in the presence of B. subtilis and S. faecalis var. liquefaciens but significant inactivation could be demonstrated at 25° to 37°C. The results obtained with artificially contaminated, sterile food samples were similar to those obtained with brain-heart infusion broth, but the degree of decrease in TNase activity in food was much lower than that in brain-heart infusion broth.     

Role for Fe(III) minerals in nitrate-dependent microbial U(IV) oxidation

Microbiological reduction of soluble U(VI) to insoluble U(IV) is a means of preventing the migration of that element in groundwater, but the presence of nitrate in U(IV)-containing sediments leads to U(IV) oxidation and remobilizaton. Nitrite or iron(III) oxyhydroxides may oxidize U(IV) under nitrate-reducing conditions, and we determined the rate and extent of U(IV) oxidation by these compounds. Fe(III) oxidized U(IV) at a greater rate than nitrite (130 and 10 microM U(IV)/day, respectively). In aquifer sediments, Fe(III) may be produced during microbial nitrate reduction by oxidation of Fe(II) with nitrite, or by enzymatic Fe(II) oxidation coupled to nitrate reduction. To determine which of these mechanisms was dominant, we isolated a nitrate-dependent acetate- and Fe(ll)-oxidizing bacterium from a U(VI)- and nitrate-contaminated aquifer. This organism oxidized U(IV) at a greater rate and to a greater extent under acetate-oxidizing (where nitrite accumulated to 50 mM)than under Fe(II)-oxidizing conditions. We showthatthe observed differences in rate and extent of U(IV) oxidation are due to mineralogical differences between Fe(III) produced by reaction of Fe(II) with nitrite (amorphous) and Fe(III) produced enzymatically (goethite or lepidocrocite). Our results suggest the mineralogy and surface area of Fe(III) minerals produced under nitrate-reducing conditions affect the rate and extent of U(IV) oxidation. These results may be useful for predicting the stability of U(IV) in aquifers.     

ANTIOXIDANT ACTIVITY OF FERMENTED BERRY JUICES AND THEIR EFFECTS ON NITRIC OXIDE AND TUMOR NECROSIS FACTOR-ALPHA PRODUCTION IN MACROPHAGES 264.7 GAMMA NO(–) CELL LINE

Serratia vaccinii, a novel bacterium isolated from blueberry microflora, increased the phenolic content of berry juices, and thus increased antioxidant activities. The fermentation capacity of Serratia was investigated with Saskatoon berries, cranberries, strawberries and grapes in both aerobic and anaerobic conditions. It was shown to be compatible with wine yeast in anaerobic fermentations, producing wine with high antioxidant activity. The effects of fermented berry juices were tested on lipopolysaccharide/inferon‐gamma‐activated macrophages 264.7 NO(–). Data indicated that fermented berry juices strongly inhibited activated‐macrophage NO production but induced tumor necrosis factor‐alpha production.     

Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX

The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation–reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.     

Effect of processing of corn for production of masa,tortillas and tortilla chips on the scavenging capacity of reactive nitrogen species

The effect of processing conventional and pigmented corn into masa, tortilla and tortilla chips on the ability to scavenge nitric oxide (NO) and peroxynitrite (ONOO^?) was investigated. The level of retention of nitric oxide and peroxynitrite (mediated for inhibition of nitrite formation scavenging capacity for masa, tortilla and tortilla chips) ranged 56.2–78.2%, 67.4–45%, 40.3–62.0% (for nitric oxide) and 38.6–81.7%, 23.3–47.7%, 19.3–67.2% (for inhibition of nitrite formation), respectively. The antinitrosative activities were affected significantly (P > 0.05) by nixtamalisation, but not by processing masa into tortilla and tortilla chips (P > 0.05). The yellow variety and its corresponding products showed the greatest capacity to scavenge nitric oxide and inhibition of nitrite formation among conventional varieties and the purple variety ranked highest among the pigmented varieties.     

Soil column evaluation of factors controlling biodegradation of DNT in the vadose zone

High concentrations of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) are present in vadose zone soils at many facilities where explosives manufacturing has taken place. Both DNT isomers can be biodegraded under aerobic conditions, but rates of intrinsic biodegradation observed in vadose zone soils are not appreciable. Studies presented herein demonstrate that nutrient limitations control the onset of rapid 2,4-DNT biodegradation in such soils. In column studies conducted at field capacity, high levels of 2,4-DNT biodegradation were rapidly stimulated by the addition of a complete mineral medium but not by bicarbonate-buffered distilled deionized water or by phosphate-amended tap water. Biodegradation of 2,6-DNT was not observed under any conditions. Microcosm studies using a DNT-degrading culture from column effluent suggest that, after the onset of 2,4-DNT degradation, nitrite evolution will eventually control the extent of degradation achieved by two mechanisms. First, high levels of nitrite (40 mM) were found to strongly inhibit 2,4-DNT degradation. Second, nitrite production reduces the solution pH, and at pH levels below 6.0, 2,4-DNT degradation slows rapidly. Under conditions evaluated in laboratory-scale studies, 2,4-DNT biodegradation enhanced the rate of contaminant loss from the vadose zone by a factor of 10 when compared to the washout due to leaching.     

Cellular and transcriptional response of Pseudomonas stutzeri to quantum dots under aerobic and denitrifying conditions

Pseudomonas stutzeri was exposed to quantum dots (QDs) with three different surface coatings (anionic polymaleic anhydride-alt-1-octadecene (PMAO), cationic polyethylenimine (PEI), and carboxyl QDs) under both aerobic and anaerobic (denitrifying) conditions. Under aerobic conditions, toxicity (assessed per growth inhibition) increased from PMAO to carboxyl to PEI QDs. The positive charge of PEI facilitated direct contact with negatively charged bacteria, which was verified by TEM analysis. Both PMAO and PEI QDs hindered energy transduction (indicated by a decrease in cell membrane potential), and this effect was most pronounced with PEI QDs under denitrifying conditions. Up-regulation of denitrification genes (i.e., nitrate reductase narG, periplasmic nitrate reductase napB, nitrite reductase nirH, and NO reductase norB) occurred upon exposure to subinhibitory PEI QD concentrations (1 nM). Accordingly, denitrification activity (assessed per respiratory nitrate consumption in the presence of ammonia) increased during sublethal PEI QD exposure. However, cell viability (including denitrification) was hindered at 10 nM or higher PEI QD concentrations. Efflux pump genes czcB and czcC were induced by PEI QDs under denitrifying conditions, even though Cd and Se dissolution from QDs did not reach toxic levels (exposure was at pH 7 to minimize hydrolysis of QD coatings and the associated release of metal constituents). Up-regulation of the superoxide dismutase (stress) gene sodB occurred only under aerobic conditions, likely due to intracellular production of reactive oxygen species (ROS). The absence of ROS under denitrifying conditions suggests that the antibacterial activity of QDs was not due to ROS production alone. Overall, this work forewarns about unintended potential impacts to denitrification as a result of disposal and incidental releases of QDs, especially those with positively charged coatings (e.g., PEI QDs).     

Behaviour of Listeria monocytogenes in the presence of Listeria innocua during storage of minced beef under vacuum or in air at 0°C and 10°C

Minced beef was inoculated with low levels (1·2–1·7 log₁₀cfu g⁻¹) of Listeria monocytogenes or Listeria innocua, or a combination of the two strains. Inoculated samples were stored at 0 or 10°C under two packaging atmospheres (aerobic and vacuum) for up to 28 days and surviving organisms recovered on Palcam Agar. The only significant increases in numbers of Listeria spp. occurred in samples held at 10°C under aerobic conditions. In vacuum packs, growth of both strains was inhibited. Under aerobic conditions meat pH increased from an initial value of pH 5·85 to c. 8·85 within 28 days. The pH of vacuum packaged meat declined to c. 4·95 during the same period. These differences in pH may be related to differences in the nature and effects of different background microflora that were observed to develop under each of these packaging conditions.Pseudomonas spp. predominated in aerobically stored beef, whereas in vacuum packed beef lactic acid bacteria predominated. No significant differences were observed between the growth rates of Listeria spp. inoculated into beef mince in pure and mixed culture. This suggests that the more frequent prevalence of Listeria innocua than Listeria monocytogenes in meat and meat products is not due to overgrowth or inhibition of the pathogen (Listeria monocytogenes) by the non-pathogen(Listeria innocua) during low-temperature storage.     

Effect of Sodium Erythorbate on Residual Nitrite, pH and Bacterial Growth in Liver Sausage Formulated with Different Levels of Mechanically Separated Pork

Some microbiological, chemical, and physical characteristics of liver sausage formulated with 0, 25, 50, and 75% mechanically separated pork (MSP), 156 ppm sodium nitrite and 0 or 550 ppm sodium erythorbate were studied during refrigerated storage (5°C, 6 days) and subsequent elevated-temperature holding (20–24°C, 48 hr). Growth of inoculated Clostridium sporogenes was inhibited by 156 ppm nitrite in all sausages even after 48 hr at 20–24°C, regardless of MSP level. Growth of aerobic mesophiles and facultative anaerobes at 20–24°C was also inhibited in sausage formulated with 156 ppm nitrite and 550 ppm erythorbate. Erythorbate addition and increased MSP levels resulted in higher Hunter a color values. Residual nitrite, pH values, and total iron content of liver sausage increased with increasing MSP content.     

Antimicrobial Activity of Sodium Bicarbonate

Sodium bicarbonate (SB) inhibited the growth of bacteria and yeasts in agar media model systems under certain conditions. Escherichia coli, Lactobacillus plantarum, Staphylococcus aureus, and Pseudomonas aeruginosa aerobic plate counts were reduced 10,000-fold by 0.12M (1% w/v) SB. Saccharomyces cerevisiae and Hansenula wingei were more sensitive; counts were reduced 100,000-fold by 0.06M SB. Potassium bicarbonate was equally inhibitory, but equimolar sodium chloride had no effect, ruling out osmotic- and sodium-mediated mechanisms of inhibition. The bicarbonate ion was identified as the probable cause of SB-mediated inhibition although, in some cases, pH elevation played a significant role.