Rikimaru Hayashi

Effects of high hydrostatic pressure on characteristics of pork slurries and inactivation of microorganisms associated with meat and meat products.

Pork slurries inoculated with various test microorganisms were prepared and subjected to high hydrostatic pressure at 1000 to 6000 atm for 10 min at 25 degrees C to examine for the pressure effects on characteristics of the slurries and the inactivation of the microorganisms associated with meat and meat products. Pressure treatment at higher than 3000 atm caused coagulation and discoloration of the pork slurries. Harder and more white coagulants were obtained by increasing the pressure. Pressure treatment at 3000 to 6000 atm killed all the microorganisms tested by more than 6-log colony-forming units (cfu)/g except Bacillus cereus spores. Gram-negative microorganisms were more labile to pressure than Gram-positive ones. Campylobacter jejuni, Pseudomonas aeruginosa, Salmonella typhimurium and Yersinia enterocolitica were inactivated at pressures higher than 3000 atm; Escherichia coli, Saccharomyces cerevisiae and Candida utilis at pressures higher than 4000 atm; Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis at 6000 atm. Only less than one-log cfu/g of B. cereus spores were inactivated at 6000 atm. Ultraviolet absorption spectra and acridine orange staining suggested that E. coli became permeable and leaked cytoplasmic RNA at lower pressure than S. aureus. From the present findings, the authors propose high hydrostatic pressure treatment as a promising means of preparing wholesome meat and meat products.

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment.

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild‐type RNase A and three mutants—F120A, F120G, and F120W—were analyzed up to a 1.4 Å resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild‐type RNase A, except for the C‐terminal region of F120G with a high B‐factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild‐type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild‐type RNase A, and those of F120W and F120G appeared to be “inside out” compared with that of wild‐type RNase A. Only ∼1 Å change in the distance between Nε2 of His12 and Nδ1 of His119 causes a drastic decrease in kcat, indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.

High pressure in bioscience and biotechnology: pure science encompassed in pursuit of value

A fundamental factors, pressure (P), is indispensable to develop and support applications in the field of bioscience and biotechnology. This short sentence describes an example how high pressure bioscience and biotechnology, which started from applied science, stimulates challenges of basic science and pure science in the biology-related fields including not only food science, medicine, and pharmacology but also biochemistry, molecular biology, cell biology, physical chemistry, and engineering.

Functional and structural roles of constituent amino acid residues of bovine pancreatic ribonuclease A

In protein engineering, wherein the goal is desirable function and high conformational stability, the characteristics of each constituent amino acid residue are important, in terms of the overall characteristics of the target protein. Bovine pancreatic riobonuclease A (RNase A) is, historically, one of the most intensively analyzed proteins, and a considerable amount of information is available on amino acid-level information. Such data would serve to aid the understanding of relationships between the distribution of various amino acid residues in the protein molecule and the unique structure and/or functions of RNase A. This review summarizes the thus-far clarified roles of 38 of the total 124 amino acid residues which comprise RNase A, with respect to protein function, stability, and folding.

Determination of methionine sulfoxide in protein and food by hydrolysis with p-toluenesulfonic acid.

Methionine sulfoxide in peptides and proteins was determined by use of 3 N p-toluenesulfonic acid as a hydrolyzing agent. Samples were hydrolyzed at 110 degrees C for 22 h in an evacuated sealed tube and analyzed for amino acid content. Amino acid analysis showed that the recovery of methionine sulfoxide from a synthetic peptide and its mixture with proteins was consistently better than 90%. The recovery of all other amino acids except tryptophan was complete, and was similar to that observed after hydrolysis with 6 N HCl. The presence of carbohydrates had no effect on the yield. Thus, the present procedure can be used for general and simultaneous determination of methionine sulfoxide as well as other amino acids in proteins.

Action of yeast proteinase C on synthetic peptides and poly-α,l-amino acids

Abstract Yeast proteinase C liberates carboxy-terminal amino acids from a wide variety of N-acyl dipeptides which have aromatic, neutral, acidic or basic amino acid in the carboxy-terminal end. Noticeably, carboxy-terminal or internal proline in peptides can also be liberated. The enzyme has esterase activity for N- acetyl- l -tyrosine ethyl ester and amidase activity for carbobenzoxy dipeptide amides. The enzyme rapidly hydrolyses poly-α, l -glutamic acid at pH 4.2, liberating only glutamic acid, while poly- l -lysine and poly- l -proline are never hydrolysed. All these activities are powerfully inhibited by p-chloromercuribenzoate (PCMB) or DFP. Poly- l -lysine inhibits both peptidase activity for carbobenzoxy-Glu-Tyr and esterase activity for acetyl- l -tyrosine ethyl ester.

An overview of the use of high pressure in bioscience and biotechnology

Abstract The successful application of pressure as a parameter in bioscience and biotechnology, the background, industrial establishment, and developmental activities of high pressure food in food processing in Japan, where it is widely recognized that the most notable progress has been made, is described. It is also emphasized that such progress is naturally extended to bioscience in general.

Advances in high pressure food processing technology in Japan

Publisher Summary This chapter summarizes recent research and developmental activities in Japan on high pressure technology for processing foods. High pressure is useful for the purpose of cooking, processing, sterilizing, and preserving food, as is high temperature. The advantage of high pressure lies in the fact that it avoids the destruction of the covalent bonds and retains natural flavor, tastes, color, and nutrients. High pressure is useful to inactivate enzymes, to gelatinize starches, to sterilize microorganisms, and to kill insects and parasites without accompanying destruction of nutrients and without changing flavor and taste. The prominent use of high pressure sterilization is in partially prepared foods or oven-ready foods. Pressure treatment preserves flavor, taste and natural nutrients, but bacterial spores are not killed. Hence, those foods require chilled transportation. An advantage of using both pressure and temperature together lies in its ability to lead efficient and economical industrial applications. The new preservable sake with white color and fresh flavor, instead of brown color and heated smell, has been brought about in the Japanese market at a reasonable cost by the minimum use of an expensive high pressure machine for the treatment of a small amount of the precipitate.

Purification of Yeast Proteinases:Part III. Isolation and Physicochemical Properties of Yeast Proteinase A and C

yeast was demonstrated by the chromatography on DEAE-Sephadex A-50, and proteinase A and C were isolated and purified by a relatively simple procedure, which was mainly conducted by repeating the chromatography and alcohol fractionation. The final preparation of proteinase C was found to be homogeneous by various physical criteria and crystallized from alcohol solution. On the other hand, although the preparation of proteinase A also showed homogeneous in chromatographic and ultracentrifugal analyses, the result of elec trophoresis disclosed the heterogeneity of the preparation. As the results of the chemical and physicochemical analyses, both enzymes showed large contents of carbohydrate, higher molecular weights and acidic isoelectric points, which seemed to be characteristic to the