Antimicrobial Activity of Kumazasa (Sasa albo-marginata)

The organic solvent extract of Kumazasa leaves (Sasa albo-marginata) showed antimicrobial activity against bacteria, fungi and yeast. Kumazasa at a concentration of 0.2-1.0% showed stronger antimicrobial activity than potassium sorbate or sodium benzoate at the same concentration. Both acidic and phenolic fractions of the extract showed strong antimicrobial activity. Thirty acidic and phenolic compounds were identified by GC and GC-MS analysis. Acetic, propionic, benzoic, phenylacetic, salicylic, 3-hydroxybenzoic and o-anisic acids, and guaiacol, phenol, 4-ethylphenol, xylenol and 4-vinylphenol were the main components. It was estimated that these components play an important role in the formation of the antimicrobial activity of Kumazasa extract.     

Studies on Proteolysis in Stored Muscle

Changes in the nonprotein nitrogenous compounds produced from rabbit skeletal muscle (L. dorsi) by proteolysis were investigated.

The value of trichloroacetic acid soluble nitrogen, ninhydrin positive materials and phenol reagent positive materials increased during storage at low and high temperature. Changes in bound and free amino acid contents produced by proteolysis during storage were assayed by amino acid analyzer. Most of free amino acids except taurine increased remarkably. Amounts of asparatic acid, glutamic acid, glycine, β-alanine and histidine were increased after hydrolysis as compared with those before hydrolysis.

By using five kinds of Dowex 50 columns, changes in the distributive patterns of the nonprotein nitrogenous compounds were also studied.     

Effect of Cathepsin D Treatment on ATPase Activity of Rabbit Myofibril

The effect of cathepsin D and pepsin treatment on rabbit myofibril was studied by measuring the amount of proteolytic products and Mg-enhanced ATPase activity.

When myofibril was treated with cathepsin D at 3°C and pH 5.0 or 5.5, a little but detectable amount of nonprotein nitrogenous compounds was released. However, there was no change in ATPase activity of myofibril, though treated with cathepsin D of higher units than assumed to be in muscle.

When myofibril was treated with pepsin under the same condition as used above, there was an increase in KCl-concentration dependence of ATPase activity followed by a decrease in the maximal value of ATPase activity.

From the present results, it was concluded that cathepsin D might not take a main role on the post-mortem degradation of myofibril.     

Serratia Protease

The substrate specificity of Serratia protease was determined using various synthetic substrates. The enzyme did not participate in the hydrolysis of di- and tri-peptides except benzoylglycylleucinamide which was split at a limited rate into hippuric acid and leucinamide. The enzyme action on larger peptides was also studied. The enzyme cleaved the gly-leu bond in eledoisin related peptide and the gly-phe bond in bradykinin. The enzyme split oxidized insulin B-chain at twelve different peptide bonds.     

Serratia Protease

Protease from a strain of Serratia contained one gram atom of zinc ion per mole and the zinc ion was essential for the activity. Also zinc-free apoenzyme was isolated as a crystalline form from the native-enzyme. Several metalloenzymes were prepared by the addition of corresponding metal ions to the apoenzyme. Studies on activities toward the hydrolysis of casein showed that relative activities of native- (zinc), cobalt- and manganese-enzyme were 1.0, 1.2 and 0.8, respectively. Toward the hydrolysis of hippurylleucinamide, however, specific activity of cobalt-enzyme was about 10 times that of the native- (zinc-) enzyme. Spectroscopic studies did not reveal any significant differences in conformations among native-enzyme, apoenzyme and the other metalloenzymes.     

Studies on Flavor Components in Soybean

Aliphatic carbonyl compounds in soybean were studied. Volatile carbonyl compounds in defatted soybean flour were identified as methanal, ethanal, n-hexanal, 2-propanone, 2- pentanone, 2-heptanone, 2-heptenal, and 2,4-decadienal, while those in raw soybean as ethanal, n-hexanal, and 2-propanone. Four kinds of non-volatile carbonyl compounds were found in defatted soybean, two of which seemed to be carbonyl ester and carbonylic acid. It is probable that the compounds in defatted soybean are mostly the secondary products derived from autoxidation of the residual fatty acids and esters in the defatting process and/or during the storage thereafter. n-Hexanal in raw soybean amounts to approximately 10 p.p.m., which is, owing to its extremely low flavoring threshold, likely to be one of the main components of the green bean flavor.     

Applying Proteolytic Enzymes on Soybean

An indole derivative having blue fluorescence was produced in cooked soybean digested at 37°C for 24 hr with an acid proteinase Molsin (optimum pH: 2.8) from Aspergillus saitoi or a usual acid proteinase pepsin (optimum pH: 1.6) from beef stomach. This indole derivative was identical with a condensation product from l-tryptophan and n-hexanal. Based on MS, NMR, IR and UV spectrometry, the condensation product was identified as l-pentyl-2, 3, 4, 9-tetrahydro-lH-pyrido [3, 4-b]-indole-3-carboxylic acid [trivial name: 1-pentyl-l, 2, 3, 4-tetrahydro-2-carboline carboxylic acid-(3)].

Data were presented of the formation of the above indole derivative and of the resulting consumption of l-tryptophan and n-hexanal.

The possible ocurrence of the formation of Harmala alkaloids, i.e. 2-carboline derivatives, through in vitro digestion of soybean with acid proteinases was discussed.

A carbonyl-trapping ability of l-tryptophan was suggested.     

Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Applying Proteolytic Enzymes on Soybean

Soy proteins were incubated with a microbial acid protease (Molsin) under the following condition: substrate concentration, 1%; enzyme-substrate ratio (by weight), 1/100; pH, 2.8; and temperature, 40°C—flavor components and related impurities are removable from crude soy-protein concentrates by their incubation for 2 hr under the above condition. The acid-precipitated fraction of soy protein incubated for 2 hr with Molsin (i.e. 2 hr-proteolyzate) showed the following composition: 10% trichloroacetic acid (TCA) insoluble fraction, 47.52%; 10% TCA soluble peptide fraction, 52.02%; and free amino acid fraction, 0.46%. Gel filtration of the 2 hr-proteolyzate gave an elution pattern showing its molecular weight distribution.

In the process of the incubation of the acid-precipitated protein, the 10% TCA insoluble fraction showed increase in amino nitrogen content, its solubility in a phosphate buffer increased to change at 6 hr, and a hydrophobic amino acid share in this fraction increased gradually.

In vitro digestibility of the acid-precipitated fraction were improved and the lipoxygenase activity in this fraction decreased through the Molsin treatment.

Ultracentrifugal analysis showed a decreasing tendency of the cold-insoluble fraction of soy protein during its incubation with Molsin. Optical rotatory dispersion and circular dichroism study elucidated conformational changes in this fraction during its incubation either with or without Molsin.