ADENOSINE TRIPHOSPHATE ENHANCES THE FLUORESCENCE OF 4′,6-DIAMIDINO-2-PHENYLINDOLE (DAPI)-LABELED ESCHERICHIA COLI O157:H71

Live cells of E. coli O157:H7 were labeled by 4′,6-diamidino-2-phenylindole (DAPI) in buffers of different pH. The extent of labeling was relatively insensitive to pH in the range of 6.5 to 9.5. The fluorescence intensity of ± 10⁴ DAPI-labeled bacteria per mL in optical cuvettes could be detected by a luminescence spectrometer. With a fluorescence microplate reader attachment, less than 10³ of labeled bacteria could be measured. DAPI-labeling inhibited the growth and respiratory activities of the bacteria. The addition of 0.5 to 6 mM concentrations of ATP induced a substantial increase in the fluorescence of labeled bacteria. Maximal enhancement by ATP was observed from bacteria still maintaining low levels of physiological activities. The enhancement favored more alkaline media with pH greater than 9. A replacement of ATP with ADP or AMP diminished the extent of enhancement. Other triphosphate nucleotides did not enhance fluorescence of DAPI-labeled bacteria. Comparable ATP enhancements were also observed with Pseudomonas alcaligenes and Shewanella putrefaciens. Solubilization/destruction of cell membranes of labeled bacteria by detergents essentially eliminated the ATP enhancement. Absorption and fluorescence spectroscopic measurements indicated that ATP could interact with free and bound DAPI. These results suggest that observed ATP enhancement in fluorescence intensity of DAPI labels in intact cells may be applied to increase the sensitivity of microorganism detection.     

BAKING PERFORMANCE AND CONSUMER ACCEPTABILITY OF RAW AND EXTRUDED COWPEA FLOUR BREADS

Cowpea flour was used to partially replace wheat flour in yeast bread, using automatic household‐type bread machines for mixing, proofing and baking. Loaves containing 15 or 30% extruded cowpea flour weighed more (683.4 g) than loaves from other treatments (641.1–652.6 g). The 100% wheat had the highest loaf volume (2.58 L) and the 30% extruded cowpea the lowest (1.64 L). Cowpea flour breads contained more protein (13.9–15.4%) than the 100% wheat (4.1% fat, 12.5% protein). Bread made with 15% extruded cowpea flour was not different (P < 0.05) from the all‐wheat control in sensory quality and acceptability. Hedonic ratings for the control and 15% extruded cowpea flour ranged from 6.6 (like slightly) to 7.4 (like moderately) for all sensory attributes. The least liked samples contained either 30% raw or 30% extruded cowpea flour, receiving ratings for all attributes ranging from 4.8 (disliked slightly) to 6.2 (liked slightly). Overall, 15% extruded cowpea flour demonstrated successful bread making performance without compromising sensory quality.     

Investigation into the use of a carbocyanine dye for the rapid detection of lipopolysaccharide associated with the low temperature spoilage of meat

The possibility for the use of a carbocyanine dye assay for the detection of lipopoly-saccharide (LPS) associated with low temperature bacterial spoilage of meat was evaluated. The assay was only capable of detecting Pseudomonus spp. when the viable count exceeded l0⁸ colony of forming units (CFU) per ml or per g. Pre-treatment of cell suspensions with EDTA, dimethylsulphoxide or by sonication with the aim of releasing LPS failed to increase the sensitivity of the assay. It was hence concluded that the carbocyanine dye assay is of little use for the detection of incipient meat spoilage.     

Potential value of the Limulus lysate assay for the measurement of meat spoilage

Using a microtitre plate method the Limulus lysate endotoxin assay was shown to be capable of detecting Pseudomonas spp. at viable cell concentrations of 10²-l0³/ml. The changes in endotoxin concentration, microbial flora, pH and ERV of laboratory-produced minced beef and minced pork stored at 4°C were monitored. The endotoxin concentration was shown to increase with the onset of spoilage. Factors were derived enabling an estimate of AMC to be made from the endotoxin concentration determined by the Limulus lysate assay. The use of the microtitre plate system significantly reduces the cost of the Limulus lysate assay.