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.     

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.     

Condensation Reaction Occurring during Plastein Formation by α-Chymotrypsin

An investigation was conducted on myosin and actin-activated heavy meromyosin (HMM) ATPase activities in normal porcine muscle stored for varying periods of time after death. Studies were also made on temperature dependent myosin ATPase, initial burst of ATPase and actin-activated HMM ATPase in normal and in pale, soft and exudative (PSE) porcine muscle. The maximum velocity of acto-HMM ATPase of normal muscle decreased considerably with postmortem time, while the apparent dissociation constant decreased slightly. The maximum velocity of acto-HMM ATPase of postmortem normal muscle was approximately two-times larger than that of the corresponding PSE muscle. However, almost no difference was found in the apparent dissociation constant. The size of the initial burst of phosphate-liberation of myosin prepared from normal muscle was approximately 1.2 mol/mol of myosin and from PSE muscle 0. It is assumed that the lack of contractility of PSE muscle was brought about by two basic myosin malfunctions: one, the irreversible binding of myosin to actin filament and the other, the functional damage of myosin ATPase, responsible for the formation of phosphorylated complex, even when dissociable.     

Studies on Flavor Components in Soybean

Ethanol (1:1) extract of defatted soybean flour was fractionated systematically and the resulting phonolic acid fraction was investigated. This fraction had strong phenol-like flavor and contained at least seven phenolic acids including syringic, vanillic, ferulic, gentisic, salicylic, p-coumaric, and p-hydroxybenzoic acids. The main component among these was syringic acid, which was isolated as 3,5-dinitrobenzoate.

In addition, two isomers of chlorogenic acids, presumably isochlorogenic and chlorogenic acids approximately in a ratio of 1 : 10, were found in this extract. These substances have sour, bitter and astringent flavors.     

Acid-stable α-Amylase of Black Aspergilli

The Acid-stable α-amylase and the acid-unstable α-amylase from Aspergillus niger contained one mole of sulfhydryl group per one mole of enzyme, which probably existed correlating with calcium atom that was essential for the amylase activity.

Iodine reacted at acidic pH specifically with the sulfhydryl group of both enzymes and oxidized it to considerably high degree, since about 4 eq of iodine per mole of sulfhydryl group of both enzymes were consumed. The modification of the sulfhydryl group of the acid-stable α-amylase did not affect the amylase acitvity, while, that of the acid-unstable α-amylase reduced it to 70 per cents intact enzyme. It was difficult to carry out carboxy-methylation of the sulfhydryl group of the acid-stable α-amylase under mild conditions maintaining its activity, but that of the acid-unstable α-amylase was easily achieved.

These facts suggested that some differences existed in the neighborhood of the sulfhydryl group of both enzymes, and that the sulfhydryl group of them was not the active site.     

Acid-stable α-Amylase of Black Aspergilli

Some general properties of the acid-stable dextrinizing amylase of black Aspergillus were investigated comparing with those of Taka-amylase A. The mode of action on starch of this amylase was quite similar to that of Taka-amylase A. Saccharifying degree at red point in starch-iodine color reaction was 5.1% and the limit of starch saccharification was a little over 40 per cent calculated as glucose with both amylases. Maltase activity was absent. Degradation products in the course of starch hydrolysis were also quite similar and they mutarotated downward. So this amylase was decided to be α-type. Thermal stability of the acid-stable α-amylase was higher than that of Taka-amylase A. Its acid stability was much higher than that of Taka-amylase A. Taka-amylase A was inactivated completely at pH 2.2, 37°C, for 30 min, but the acid-stable α-amylase retained 87% of its original activity.

From the amylase preparation of black Aspergillus acid-stable α-amylase and acidunstable α-amylase were separated by gel filtration on sephadex G-100 column. From the acid-unstable α-amylase fraction this enzyme was purified by fractionations with rivanol and acetone, and finally obtained as a homogeneous protein after gel filtration with sephadex G-50. Comparison of some general properties between the two α-amylases was carried out. Catalytic action was quite similar with both enzymes, but dextrinizing unit per mg enzyme protein of the acid-unstable α-amylase was about 5.6 times as large as that of the acid-stable α-amylase. The acid-unstable α-amylase was less heat-stable than the acid-stable α-amylase. Acid stability and pH-activity curve were compared with both α-amylases. High stability of the acid-stable α-amylase in acidic condition was observed, but, in alkaline range, it was more sensitive than the acid-unstable α-amylase.     

Isolation and Characterization of Antitumor Lipopolysaccharide from Proteus mirabilis

Systematic isolation of the cell constituents of Proteus mirabilis RMS–203 was performed to find out localization of antitumor principle only in the lipopolysaccharide (LPS) layer of the cell wall fraction.

LPS with strong antitumor activity was extracted from P. mirabilis RMS–203 by phenol-water method followed by purification on DEAE-Sephadex A–50 column chromatography.

The main components of purified LPS were galactose, hexosamine, 2-keto-deoxy-octonic acid (KDO), myristic acid, β-hydroxymyristic acid and α,ε-diaminopimelic acid.

The minimal effective dose of LPS against Ehrlich solid carcinoma in mice was 0.1~1.0 μg/mouse. LD₅₀ in mice and pyrogenicity in rabbits were 28 mg/kg and 10^?3–10^?5 μg/rabbit, respectively.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.