David R. Farr

High pressure technology in the food industry

Abstract The effects of high hydrostatic pressure (up to 10 000 kg/cm 2 ) on the viability of microorganisms and on protein denaturation have been known for over 90 years. Renewed interest has recently been shown, particularly in Japan where high hydrostatic pressure machines have been developed for food industry research. Efforts are being made to apply high hydrostatic pressure techniques to food technology. At present, the available machines work only for batch processing and have very small working volumes. The problem of achieving high throughput, while minimizing cost and optimizing operational lifetime, remains to be solved.

Ultrastructural localization of β-galactan in the nuclei of the myxomycete Physarum polycephalum

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated β-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei ofP. polycephalum by electron microscopy using a specific “sandwich” technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactin was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasma and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.

An extracellular rennin-like enzyme produced by Physarum polycephalum

Abstract Three new proteases have been detected in the culture fluid of the myxomycete Physarum polycephalum . Production of these enzymes reached a maximum after 120 h. The main protease has been isolated and purified by (NH 4 ) 2 SO 4 fractionation and DEAE cellulose chromatography. The enzyme has a pH optimum of 4.5–5.0 and a temperature optimum of 35 °C. The enzyme has been shown to have a specificity similar to that of rennin (chymosin, EC 3.4.4.3) when acting upon cow κ-casein.

Structure of an α-D-galactosaminoglycan from Physarum polycephalum Spherule walls

Abstract The spherule walls Physarum polucephalum have been reexamined and found to contain 88% of galactosamine (as anhydrogalactosamine), 6.80% of protein, 4.7% of phosphate groups, and a small proportion of acetyl groups (0.5%). Methylation studies indicated that the spherule-wall polysaccharide is a long-chain galactosamino- glycan linked exclusively (1→4) and without phosphate linkages. The specific optical rotation of this unique glycan. [x] D , + 118° (6 M HCI), indicated that it is α- D -linked.

Ultrastructural localization of cell wall teichoic acids in Streptococcus faecalis by means of concanavalin A

Some teichoic acids are known to be partially substituted by α-D-glucopyranosyl residues such as the teichoic acids of Streptococcus faecalis NCIB 8191. They will, therefore, bind specifically the phytohemagglutinin concanavalin A. Concanavalin A labelled with mercury or colloidal gold coated with concanavalin A has been used to mark isolated cell walls in order to localize the teichoic acids at the ultrastructural level. Besides these two direct marking techniques, the indirect concanavalin A-peroxidase technique (localization of peroxidase by the diaminobenzidine method followed by postosmication) has been applied to thin sections of premarked cells. All three methods gave almost identical results, namely, a dense and homogeneous distribution of the cell wall teichoic acids. In control experiments total inhibition was achieved in the presence of methyl-α-D-mannopyranoside. After trichloroacetic acid or alkali extraction of the teichoic acids from isolated walls no marking could be detected.

Factors influencing permeability of the cell membrane of the osmotolerant yeast Saccharomyces rouxii grown in the presence and absence of 18% NaCl. I. Na+/K+-activated Mg2+-dependent ATPase

Abstract It was found that cells of Saccharomyces rouxii contain an ouabain-inhibited ATPase, assumed to be an Na+/K+-activated Mg2+-dependent ATPase, which could serve as a sodium pump protecting the cells in a high salt environment. Twenty-two cell homogenates or supernatants (centrifuged at 3000 × g) grown without added salt in the medium contained sufficient total ATPase activity to liberate (on average) 0.225 μM Pi min−1 mg−1 protein. The percentage of total ATPase inhibited by the addition of ouabain (1 × 10−4 M) varied from 7 to 100%. Cell homogenates or supernatants from cells grown in the presence of 18% NaCl in the media contained sufficient ATPase activity to liberate (on average) 0.114 μM Pi min−1 mg−1 protein, about 50% of the total ATPase activity found in the non-salt grown cells. The percentage of total ATPase activity inhibited by ouabain ranged from 16 to 100%. Although the non-salt-grown cells contained approximately double the total ATPase activity of the salt-grown cells. there was evidence that the percentage of total ATPase that is ouabain sensitive (Na+/K+-activated ATPase) is higher in the salt-grown cells. Also, cells of S. rouxii grown in media without added NaCl, recovered by centrifugation and transferred to media containing 18% NaCl for 16 h and again recovered by centrifugation, homogenized and centrifuged at 10 000 × g contained 61.2% ouabain-sensitive ATPase compared with 21.3% ouabain-sensitive ATPase in the cells before adaptation to the high salt environment.

Isolation and purification of the extracellular ribonuclease from Physarum polycephalum

SummaryThe myxomycete Physarum polycephalum has been reported to produce an extracellular ribonuclease. A quick procedure is described for the isolation and purification of the ribonuclease from the culture supernatant. It involves an initial concentration of the culture supernatant by slow freezing, followed by ammonium sulfate precipitation. The enzyme was adsorbed onto DEAE-cellulose and eluted with a sodium chloride gradient.

Characterization of the semi-purified membrane bound ATPases of Zygosaccharomyces rouxii adapted to 18% NaCl

The yeast (Zygosaccharomyces rouxii) contains a plasma membrane ATPase which exhibits some properties similar to those of the Na+/K+-activated Mg2+-dependent ATPases hitherto reported as existing almost exclusively in animal systems. Cells of Z. rouxii grown under low salt conditions and adapted to media containing 18% NaCl have Mg2+-dependent ATPase activity which is optimum at PH 6.0 and is inhibited by ouabain (10−4 M) in the presence of NaCl and KCl, inhibited by sodium orthovanadate and virtually unaffected by oligomycin. When run through sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), the enzyme appears to contain a subunit ranging from 70000 to 100000 daltons. The mean specific activity (x) from the P6 semipurified membrane fraction of cells grown without added salt was 0.48 μM Pi·mg−1 protein·min− with a batch mean standard error of 0.12. The mean specific activity of similar fractions from 18% NaCl adapted cells was 0.78 μM Pi·mg−1 protein·min−1 with a batch mean standard error of 0.07. It was demonstrated that the enzyme had significant specificity for ATP and had significant dependence upon Mg2+. As much as 75.3% of enzyme activity was inhibited by sodium orthovanadate, a known inhibitor of plasma membrane ATPase. Oligomycin, an inhibitor of mitochondrial ATPase activity, inhibited a maximum of 10.3% of the activity suggesting some mitochondrial contamination. Ouabain (1.6×10−4 M) in the presence of NaCl and KCl (23 mM) showed a significant inhibition of the membrane ATPase activity in one half the cells adapted to 18% NaCl while producing apparent enhancement of ATPase activity in cells grown without salt. The ATPase activity of cells adapted to 18% NaCl was significantly higher than that of cells unexposed to high salt.