Asako Narai-Kanayama

Effects of non-fermented and fermented soybean milk intake on faecal microbiota and faecal metabolites in humans

The effects of non-fermented soybean milk (NFSM) and fermented soybean milk (FSM) intake on the faecal microbiota and metabolic activities in 10 healthy volunteers were investigated. Soybean oligosaccharides, raffinose and stachyose were utilized by bifidobacteria except for Bifidobacterium bifidum, but most strains of Escherichia coli and Clostridium perfringens could not use them. During the dietary administration of FSM, the number of bifidobacteria and lactobacilli in the faeces increased (p < 0.05), and clostridia decreased (p < 0.05). Moreover, the concentrations of faecal sulphide were decreased (p < 0.01) in the intake of FSM. During the dietary administration of NFSM, the number of bifidobacteria tended to increase. These results indicate that the consumption of soybean milk, especially FSM, is related to improvement of the intestinal environment.

High level production of bioactive di- and tri-tyrosine peptides by protease-catalyzed reactions

Papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media was efficient for synthesis of oligo-tyrosine peptides having angiotensin I-converting enzyme inhibitory activity. Di-, tri-, and tetra-tyrosine accumulated in the soluble fraction of reaction mixture. On the other hand, the peptide products with degree of polymerization from 5 to 10 were insoluble, yields of which were influenced by initial concentrations of the ester substrate. The precipitated products could be used as substrates for α-chymotrypsin in DMSO/buffer systems producing soluble oligo-tyrosine peptides. In the reaction media containing DMSO at 40-50% (v/v), most of the precipitates were converted to soluble peptides. The two-step enzymatic reaction, that is papain-catalyzed synthesis of Tyr-polymers from L-tyrosine ethyl ester followed by their hydrolytic cleavage by α-chymotrypsin, is expected to be a potent procedure for synthesis of biologically active di- and tri-tyrosine peptides in good yield.

α-Chymotrypsin-catalyzed synthesis of poly-l-cysteine in a frozen aqueous solution

Poly-l-cysteine (PLCys) is drawing attention as a potential sorbent of thiol (SH)-reactive toxic heavy metal ions in the wastewater and polluted soils. However, preparation of PLCys relies on chemically synthesized polymers, in which SH groups must be protected and deprotected prior to use. On the other hand, α-chymotrypsin polymerized l-cysteine ethyl ester in a frozen aqueous solution, provides PLCys with degree of polymerization from 6 to 11 without blocking of SH groups. Kinetic analyses suggested that the acylation of α-chymotrypsin with the initial substrate was a rate-limiting step in the enzymatic polymerization. The peptide yields reached 85% and 65% of SH groups in PLCys were assumed to be free forms. Although detail information on correlation between the state of SH groups and heavy metal adsorption properties of PLCys should be explored in further studies, the present study for the first time proposed an easy method for synthesis of PLCys requiring neither SH-protection nor -deprotection.

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

Theaflavin-3-gallate specifically interacts with phosphatidylcholine, forming a precipitate resistant against the detergent action of bile salt

Abstract Black tea is a highly popular beverage, and its pigments, polymerized catechins such as theaflavins (TFs), are attracting attention due to their beneficial health effects. In this study, to test the inhibitory activities of TFs on the intestinal absorption of cholesterol, we investigated their effects on phosphatidylcholine (PC) vesicles in the absence or presence of a bile salt. (−)-Epicatechin gallate, (−)-epigallocatechin gallate, and TFs formed insoluble complexes with PC vesicles. Galloylated TFs such as TF2A, TF2B, and TF3 precipitated far more than other polyphenols. The subsequent addition of taurocholate redispersed the polyphenol-PC complexes, except that a large amount of TF2A remained insoluble. After incubation with taurocholate-PC micelles, TF2A elevated the turbidity of the micelle solution, providing red sediments. The TF2A-specific effect was dependent on the PC concentration. These results suggest that TF2A interacts with PC and aggregates in a specific manner different from catechins and other TFs. Specific interaction of TF2A with PC vesicles and the effects of subsequent addition of taurocholate.

The effect of soymilk intake on the fecal microbiota, particularly Bifidobacterium species, and intestinal environment of healthy adults: a pilot study

The influence of soymilk on the fecal microbiota, particularly Bifidobacterium species, and metabolic activities were investigated in eight healthy adult humans. During the soymilk intake period, the number of bifidobacteria in feces was significantly higher (p<0.05) on day 14 of the soymilk intake period than before the intake period, whereas that of Enterobacteriaceae was significantly lower (p<0.05) on days 7 and 14 of the soymilk intake period than before the intake period. In an investigation of Bifidobacterium at the species or group level, the numbers of all species and groups studied slightly increased during the soymilk intake period. These results show that the intake of soymilk may contribute to improving the intestinal environment.

Mechanistic investigation of capability of enzymatically synthesized polycysteine to cross-link proteins

Background Previously, we had reported that α-chymotrypsin–catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6–11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials. Methods This study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70 °C to avoid PLCys-independent, β-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC. Conclusions Addition of urea and thermal treatment at 60 °C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker. General significance Enzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.