Development of Enzymatic Synthesis of γ-Glutamylcarnosine and Its Effects on Taste.

γ-Glutamyl peptides have amide bonds between the γ-carboxy group of glutamic acid and the amino group of amino acids or peptides. Some of these γ-glutamyl peptides are known as kokumi substances. Kokumi substances enhance the taste, mouthfulness, thickness, and continuity of the dish. γ-Glutamylcarnosine (γ-l-glutamyl-ß-alanyl-l-histidine) is a γ-glutamyl peptide, and this peptide has been suggested as a kokumi substance; however, its effects on taste have not been evaluated directly. As γ-glutamylcarnosine is not available commercially, the conditions for its enzymatic synthesis using a γ-glutamyltranspeptidation reaction of γ-glutamyltranspeptidase of Escherichia coli was optimized. The synthesized peptide was purified with a Dowex 1 × 8 column, and its structure was identified by mass spectrometry and NMR spectroscopy. This is the first report of the enzymatic synthesis of γ-glutamylcarnosine. Using this purified preparation, its effects on the sense of taste were investigated. However, the effects of γ-glutamylcarnosine on the sense of taste were not detected except for increased bitterness.

Umami compounds enhance the intensity of retronasal sensation of aromas from model chicken soups.

We examined the influence of taste compounds on retronasal aroma sensation using a model chicken soup. The aroma intensity of a reconstituted flavour solution from which glutamic acid (Glu), inosine 5'-monophosphate (IMP), or phosphate was omitted was significantly lower (p<0.05) than that of the model soup. The aroma intensity of 0.4% NaCl solution containing the aroma chicken model (ACM) with added Glu and IMP was significantly higher (p<0.05) than that of 0.4% NaCl solution containing only ACM. The quantitative analyses showed that adding monosodium glutamate (MSG) to aqueous aroma solution containing only ACM enhanced the intensity of retronasal aroma sensation by 2.5-folds with increasing MSG concentration from 0% to 0.3%. Sensation intensity using an umami solution with added MSG and IMP was significantly higher than that with only MSG when the MSG concentration was 0.05%, 0.075%, or 0.1%. However, it plateaued when MSG concentration was beyond 0.3%.

Characterization of the key aroma compounds in pork soup stock by using an aroma extract dilution analysis.

The aroma extract dilution analysis of an extract prepared from pork stock and subsequent experiments led to the identification of 15 aroma-active compounds in the flavor dilution factor range of 64-2048. Omission experiments to select the most aroma-active compounds from the 15 odor compounds suggested acetol, octanoic acid, δ-decalactone, and decanoic acid as the main active compounds contributing to the aroma of pork stock. Aroma recombination, addition, and omission experiments of these four aroma compounds in taste-reconstituted pork stock showed that each compound had an individual aroma profile. A comparison of the overall aroma between this recombined mixture and pork stock showed strong similarity, suggesting that the key aroma compounds had been successfully identified.

Characterization of the key aroma compounds in beef extract using aroma extract dilution analysis.

Aroma extract dilution analysis (AEDA) of an ether extract prepared from beef extract (BE) and subsequent identification experiments led to the determination of seven aroma-active compounds in the flavor dilution (FD) factor range of 32-128. Omission experiments to select the most aroma-active compounds from the seven aroma compounds suggested that 2,3,5-trimethyl pyrazine, 1-octen-3-ol, 3-methylbutanoic acid, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone were the main active compounds contributing to the aroma of BE. Aroma recombination, addition, and omission experiments of the four aroma compounds in taste-reconstituted BE showed that each compound had an individual aroma profile. A comparison of the overall aroma between this recombination mixture and BE showed a high similarity, suggesting that the key aroma compounds had been identified successfully.

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA.

Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.

A novel gene delivery system in plants with calcium alginate micro-beads.

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.