Ethanol-mediated variations in cellular fatty acid composition and protein profiles of two genotypically different strains of Escherichia coli O157:H7.

Two strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30 degrees C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of cis-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The trans-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast, cis-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of E. coli O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.

Isoflavone transformation during soybean koji preparation and subsequent miso fermentation supplemented with ethanol and NaCl.

Soybeans were soaked with water for 4 h, steam-cooked, inoculated with the conidia of Aspergillus oryzae, and incubated for 3 days for koji preparation. The koji was then mixed with water-soaked and steam-cooked soybeans (1:2, w/w), ground into paste, and supplemented with 15% ethanol and 12.5% NaCl or 3% ethanol and 6% NaCl for miso fermentation at 30 degrees C. Daidzin, genistin, daidzein, and genistein contents were extracted from the lyophilized and pulverized soybean powder or from the miso homogenate by a developed one-tube procedure and analyzed with an HPLC. After water soaking, daidzein and genistein contents increased markedly, whereas daidzin and genistin contents decreased. Further increases of daidzein and genistein contents and decreases of daidzin and genistin contents were observed after koji mold growth. During fermentation, fungal and lactic acid bacterial (LAB) growth in the miso products was inhibited, whereas soluble protein contents increased much more rapidly in the low-salt miso products supplemented with 3% ethanol and 6% NaCl than the other products. When the 4- and 8-week-fermented miso products were cooked with tofu for sensory evaluation, flavor ratings of the low-salt products were higher than that of a popular commercial product. In both products, the most daidzins and genistins were hydrolyzed after 4 weeks of fermentation. The hydrolytic enzymes contributing to isoflavone transformation originated from soybeans after water soaking and from koji with mold growth. It was of merit that the low-salt fermented products were fairly acceptable in flavor rating and rich in daidzein and genistein contents after 4 weeks of fermentation.

Survival of staphylococcus aureus and Escherichia coli as affected by ethanol and NaCl.

Growth of three strains of Staphylococcus aureus and two strains of Escherichia coli on nutrient agar (NA) supplemented with ethanol and NaCl was investigated. S. aureus did not grow on NA containing > or =10% ethanol (wt/wt) combined with > or =0% NaCl (wt/wt), or 7.5% ethanol combined with 7.5% NaCl. Neither E. coli nor E. coli O157:H7 grew on NA containing > or =7.5% ethanol combined with > or =0% NaCl, 5% ethanol combined with > or =2.5% NaCl, or > or =5% NaCl combined with > or =0% ethanol. It is apparent that NaCl enhanced the inhibitory effect of ethanol on growth of S. aureus and E. coli When cells were suspended in nutrient broth containing 12.5, 20, or 40% ethanol combined with NaCl, viable cells decreased with an increase of ethanol concentration. Ethanol sensitivity among strains and between genera varied in a limited range. When the cells were exposed to 20% ethanol in combination with 5% NaCl, S. aureus and E. coli lost viability after 30 and 10 min, respectively. When treated with 40% ethanol combined with > or =0% NaCl, all test strains lost viability within 5 min.

Apparatus used for small-scale volatile extraction from ethanol-supplemented low-salt miso and GC-MS characterization of the extracted flavors.

An extraction apparatus was equipped with a nitrogen-flushing vessel to purge volatiles from a 10-g miso prepared solution at 40 degrees C, a reflux condenser to recover water, a coiled cold-trap to separate ethanol in advance, and a glass-lined stainless (GLS) trap filled with Tenax TA for flavor adsorption. Volatiles in the GLS tube were released with a thermal desorption device and condensed with a Micro-cryo trap prior to connection with GC and GC-MS for characterization. After analysis, a broad volatile profile comprising 9 categories of functional group and 97 identified compounds was achieved. As affected by ethanol supplementation for miso fermentation, most volatiles except alcohols and acetals in the low-salt products fermented with 5% NaCl and 7.5% ethanol were higher than those in the control products fermented with 9% NaCl and 0% ethanol and the high-ethanol supplemented products fermented with 5% NaCl and 15% ethanol. It reveals that supplementation of ethanol in miso at an appropriate level not only enabled a low-salt miso fermentation but also enhanced flavor formation.

A modified chemical procedure for rapid determination of glucosamine and its application for estimation of mold growth in peanut kernels and koji.

One-step hydrolysis of chitin to release glucosamine for quantitation was achieved by combining a chitin-containing sample (10-200 mg of sample size) in a test tube with 1 mL of 10 M HCl followed by vacuum treatment for 10 min, incubation at 28 degrees C for 30 min, replenishment with 3 mL of deionized water, nitrogen flushing, screw capping, and heat treatment at 140 degrees C for 60 min. A phosphate buffer solution (pH 12.5, 0.2 M) was effective in pH stabilization and enhancing colorimetric determination of glucosamine content. When the modified procedure was applied to analyze glucosamine content in the mycelia of various molds, glucosamine content varied mainly depending on mold species. In estimations of mold growth of the uninoculated peanut kernels incubated under a humidified condition for 5 weeks, cooked rice and soybean inoculated with conidia of Aspergillus oryzae for koji preparation, logarithms of the internal mold populations and glucosamine contents both increased with increases of incubation time. The modified procedure provided a rapid and reliable estimation of mold growth in various substrates.

Fermentation of low-salt miso as affected by supplementation with ethanol.

Steam-cooked soybeans and rice koji were combined (1:1, w/w), mixed with 5% (w/w) NaCl and ground into a fine paste. Samples (30 g) were deposited in nylon/polyethylene plastic bags and supplemented with 10 ml of aqueous ethanol solutions to give concentrations of 0, 2.5, 5, 7.5, 10, 15, 20, and 25% ethanol. Mixtures were homogenized, sealed, and incubated at 28 degrees C for eight weeks. Mold populations were less than 3 log10 CFU/g in all miso products after four weeks of fermentation. Yeast populations increased to 6.1 log10 CFU/g in the control (0% added ethanol) during the first week of fermentation and remained stable throughout the eight-week fermentation period. Yeasts were not detected in products containing 5-25% ethanol. Populations of lactic acid bacteria (LAB) increased to 6 log10 CFU/g after one week of fermentation in products containing 0 and 2.5% ethanol. However, after eight weeks of fermentation, LAB populations in all products were less than 4 log10 CFU/g. Rapid decreases in pH occurred only in products supplemented with 0 or 2.5% ethanol. Percentages of soluble protein in miso products containing various ethanol concentrations during the eight-week fermentation period revealed that protease activity was still active or not greatly inhibited in products supplemented with less than 10% ethanol. In comparison, koji enzymes were comparatively less affected by ethanol than were populations of molds, yeasts, and LAB. Total soluble carbohydrate and glucose contents were higher in products supplemented with 5, 7.5 and 10% ethanol than in other products. Discoloration (browning) during fermentation occurred most rapidly in products supplemented with 5 or 7.5% ethanol. Sensory evaluation of the low-salt (5%) product supplemented with 7.5% ethanol and fermented for eight weeks revealed normal or enhanced flavor ratings compared to ratings for a commercial product.

Estimation of fungal infection of peanut kernels by determination of free glutamic Acid content.

Peanut kernels (Tainan 9, a Spanish cultivar) inoculated with Aspergillus parasiticus, A. flavus, A. niger, or A. ochraceus as well as noninoculated kernels were incubated in a humidified environment (relative humidity, 100%) at 25(deg)C for 7 weeks. Internal fungal populations and changes in moisture and sucrose content and free amino acid composition of the kernels were determined periodically. Fungal populations determined by using A. flavus and A. parasiticus agar and rose bengal chlortetracycline agar as enumerating media were closely correlated. Moisture content in the kernels increased from 5.8 to 20.4% (dry basis), and changes in individual free amino acid contents varied, depending upon the incubation time and type of fungus used as an inoculum. In the early infection period (up to 5 weeks), sucrose contents and logarithms of threonine and tyrosine contents increased while logarithms of free glutamic acid content decreased linearly with incubation time. A negative linear relationship was further obtained between logarithms of fungal populations and the logarithm of free glutamic acid content (R(sup2) > 0.80) of the infected peanut kernels.

Variation of iron, copper, free fatty acid content and lipoxygenase activity in peanut kernels subjected to various pretreatments and roasting.

Peanut kernels subjected to pretreatment including rehydration, blanching and dehydration, and untreated kernels were roasted at 160 degrees C for times ranging from 0 to 90 min. For both peanuts, the iron content in oil and specific lipoxygenase activity in defatted peanut flour decreased, free fatty acid content increased and copper content changed insignificantly with roasting time. Changes of iron content, lipoxygenase activity and free fatty acid content were more significant in untreated peanuts than in pretreated peanuts. At each roasting time, iron, copper and free fatty acid contents in the oils and lipoxygenase activities in the defatted flours prepared from untreated peanuts were higher than in the oils and flours prepared from pretreated peanuts.

Characteristics and application of immobilized papain in a continuous-flow reactor.

Papain was immobilized on an anion-exchange resin by physical adsorption followed by cross-linking with glutaraldehyde. The activity and rate of heat inactivation of the enzyme was determined at 37 to 95 degrees C. Activity increased with an increase in temperature up to 85 degrees C, then decreased slightly at 95 degrees C. Activation of the enzyme by cysteine in 2 mM EDTA increased proportionally with an increase in concentration from 0.004 to 0.12 M. Levels of dissolved oxygen in solutions of EDTA containing cysteine in a closed system and with nitrogen purging in the absence of cysteine in an open system were monitored at 51 degrees C. Both the presence of cysteine and purging with nitrogen were effective in depleting dissolved oxygen. The final oxygen level was essentially independent of cysteine concentrations of 0.01, 0.02, and 0.10 M, and was reached in 95, 85, and 55 min, respectively. However, final levels of oxygen were dependent upon the flow rate of nitrogen from 1 to 10 SLPM and were reached within 15 min. Immobilized papain in a continuous-flow stirred-tank (300 rpm) reactor with a 4-liter reaction volume accompanied by nitrogen purging of 4 SPLM was used to hydrolyze a 1.5% casein solution in the presence of 0.01 M cysteine in 2 mM EDTA at 51 degrees C. The reaction rate was 1 liter/h and activity was measured based on the content of trichloroacetic acid-soluble protein in the outflow. Papain activity was not observed to decrease during a 30-h reaction period.

Hygroscopic Characteristics of Peanut Components and Their Influence on Growth and Aflatoxin Production by Aspergillus parasiticus.

Sound inshell runner-type peanuts, manually damaged inshell peanuts, shells, sound kernels, deskinned kernels and skins were stored in separate flasks under an atmospheric relative humidity of 100% at 28°C. After 5 d, water was adsorbed at levels of 1.2, 1.7, 3.9, 0.9, 1.0 and 9.5 g/100 g dry material, respectively. Surface disinfected components were inoculated with conidiospores of Aspergillus parasiticus NRRL 2999 and incubated under the same conditions. The time required for visible growth of the fungus was 8, 6, 4, 12, 10 and 3 d, respectively. The time for appearance of the conidiospores was 14, 10, 6, 16, 13 and 6 d. After a 3-wk incubation period, aflatoxin levels in peanut components were 111.4, 159.1, 4.4, 58.7, 99.0 and 1.5 µg/g, respectively.