Degradation of myofibrillar proteins is one of the causes of post-mortem muscle softening of fish. In order to clarify the endogenous proteases responsible for fish muscle softening, we injected specific protease inhibitors into the duct of Cuvier of live red sea bream to suppress the endogenous protease activities under similar physiological conditions. After sacrificing the fish, we confirmed the effects of protease inhibitors on degradation of myofibrillar components by western blot analysis during storage at 25℃. Degradation of myosin heavy chain (MHC) and β-connectin were significantly suppressed by leupeptin, diisopropyl fluorophosphate (DFP) (serine protease inhibitors), and o-phenanthroline (a metalloproteinase inhibitor). Hydrolysis of α-actinin was inhibited by E-64 (a cysteine protease inhibitor). Degradation of troponin I was suppressed by leupeptin, DFP, o-phenanthroline, and E-64. The limited degradation of tropomyosin was inhibited by DFP and o-phenanthroline. Our results suggested that endogenous serine proteases and metalloproteinases were involved in degradation of most of the myofibrillar components (MHC, β-connectin, troponin I, and tropomyosin) while α-actinin was hydrolyzed only by cysteine proteases in red sea bream muscle.
A rapid and simple method for the simultaneous determination of the dipeptide type sweetners, those are aspartame (APM), neotame (NE) and advantame (AD), in various foods by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) was developed. The sweetners were extracted from foods with 0.01 mol/L hydrochloric acid in ultrasonic bath, and the extract was loaded on a strata-X-C, cation-exchange and reverse-phase cartridge. The HPLC separation was performed on a Inertsil ODS-4 column (5 μm, 4 mm i.d. × 150 mm) with ultraviolet detection, using 0.01 mol/L phosphate buffer (pH3.5)-acetonitrile as a mobile phase. The LC/MS separation was performed on a InertSustain C18 column (3 μm, 2.1 mm i.d. × 50 mm) with a mobile phase of 0.01 mol/L ammonium formate-formic acid buffer (pH3.5)-methanol, and MS detection with positive ion electrospray ionization. In HPLC, the quantification limit of three sweetners was 0.001 g/kg. In LC/MS, the limit of APM was 0.005 g/kg, and that of AD and NE was 0.001 g/kg. The mean recoveries from foods fortified at the levels of 0.02 and 0.20 g/kg were 87.8-106.3% in HPLC, and 64.0-128.9% in LC/MS.
The Pacific oyster was cultured off the coast of Konagai along Isahaya Bay, Nagasaki Prefecture using the conventional suspension method (CSM) and the single seed method (SSM). By using the SSM, oysters were said to have good appearance and taste better than those by CSM, although no comparison report was found on free amino acid constituents affecting the taste between the two different culture methods of CSM and SSM. We investigated the taste-associated free amino acids of the oysters obtained from the same district in December 2012 and March 2013 and cultured using either of the two methods. Besides, we made another comparison of the free amino acid constituents between the two collection periods of winter and spring. Consequently, we observed that the mean total levels of amino acids in oysters from both collection periods was higher in oysters cultured using the SSM than in those using the CSM. Especially in December 2012, we found that the mean total level of free amino acids was significantly higher in the SSM-cultured oysters by ca. 36% than in the CSM-cultured oysters. The level of glutamic acid, related to umami, was significantly higher in oysters cultured using the SSM than the CSM by ca. 70% and 81% in December 2012 and March 2013, respectively. At both collection periods, serine, glycine, alanine, and β-alanine, which were related to sweetness, showed higher levels in the oysters cultured using the SSM than the CSM. Especially in December 2012, the levels of threonine, serine, alanine, and β-alanine were significantly higher in the SSM-cultured oysters than in the CSM-cultured oysters by ca. 139%, 167%, 78% and 81%, respectively. In March 2013, the levels of glycine and β-alanine were significantly higher by ca. 26% and 32%, respectively. These findings exhibited the SSM-cultured oyster had stronger tastes in umami and sweetness than the CSM-cultured oyster. On the other hand, oysters obtained in March showed higher level of total free amino acid and contained more free amino acids related to umami and sweet tastes than those in December during the same harvest season in Konagai. Accordingly, the results correspond to the feedback of consumers that the oyster cultured using SSM tastes better than that using CSM, and further, that the spring oyster tastes better and richer than the winter oyster, which might be due to the abundance of amino acids that impart the sweet and umami tastes.
We compared the taste components of Pacific oysters (Crassostrea gigas) cultured using the single seed method (single seed oysters) off the coast of Konagai (Nagasaki Prefecture, Japan) that were cultured for different periods. Single seed oysters that had been farmed up to the first harvest year (first-year oysters), the procedure typical in this area, and those that had been kept for an additional summer and into the second harvest year (second-year oysters) were compared. We analyzed taste components including free amino acids, ATP and related compounds, and glycogen levels in the oysters that were obtained in the different harvest seasons. The experiments were repeated at 4 different times between February 2013 and February 2014 because of chemical constituent fluctuations under various environmental conditions. First, we used 2-way ANOVA to analyze all groups of the data collected in the experimental replicates and then we independently evaluated differences between the 2 groups, first- and second-year oysters, using t-tests. The second-year single seed oysters showed significantly higher values of whole body weights and soft-tissue weights than those of first-year oysters. However, the soft tissue ratios, meaning soft tissue per gram of the whole body weight including the shell, were significantly lower in second-year oysters than first-year oysters, which suggested that second-year oysters had less edible contents. Significantly higher glycine, alanine, and serine levels were observed in first-year oysters than in second-year oysters, indicating that first-year oysters might be more sweet-tasting. In contrast, no significant differences in AMP, IMP, and glycogen levels were observed between the 2 groups of single seed oysters. Moreover, in a taste evaluation test of the 2 oyster groups performed in January 2014, different culture periods had little or no effect on taste, except for their better shape and larger size.
Ginger (Zingiber officinale) is well-known spice and cultured on a temperate region. We attempted to standardize the ginger using 1H-NMR-metabolomics because the standardization of the ginger has been provided by the quantity of one or a small number of compounds detected in ginger. The score plot of principal component analysis (PCA) using 1H-NMR of the ginger aqueous extract showed some outliers. The results of conducting PCA to the ginger extract except the outliers showed that there are differences between Kintoki species and Amami native species, and between China L5 species and Sanshu Kochi species, China L4 species. Furthermore, we tried to OPLS-DA to evaluate the varietal variation in chemical components. The results showed that sucrose, glucose, alanine, arginine, asparagine, malic acid and gingerol are important factors for the classification of the ginger.