Preservation of freshness of yellowtail fillet packaged using carbon dioxide-nitrogen gas mixture

Gas-exchange packaging was studied as a method of maintaining the freshness of fish-farm-cultured yellowtail, and its effectiveness was examined. The fish-farm-cultured yellowtail was processed into three layers (two fillets plus bones). Half of these fillets were wrapped using co-extruded ethylene vinyl acetate/polyvinylidene chloride/polyolefin film. The gas used, which was sealed into the packaging, was a mixture of nitrogen and carbon dioxide (N₂/CO₂:60/40). Packages were kept at 5°C. The other half of the fillets were packaged in stretch-wrap made of PVC, for comparison. The effectiveness of both types of packaging on maintaining freshness was examined. Results show that stretch-wrap-packaged yellowtail suffered discoloration of the dark meat sections and a degeneration of quality during the first day of preservation. Discoloration was extremely marked by the 3rd day and the fish was completely valueless as merchandise. In contrast, gas packaging of yellowtail prevented rapid discoloration, maintained taste and texture, and suppressed the growth of bacteria, to give a 3-4-day shelf-life for the fish which was to be used subsequently as sashimi.     

Mechanical Response Properties of He Bilayer Films on an Oscillating Hectorite

We measured the mechanical response of ³ He and ⁴ He bilayer films on a 2D mesoporous Hectorite from 0.1 K to 4 K using 10MHz ultrasound. Below a certain coverage, we observed the slippage of nonsuperfluid films at low temperatures. This behavior was well described by a film-substrate frictional force with a thermally activated process. Furthermore, we found that this slippage is inhibited by the appearance of the fluid state on the inert layer.     

Gene expression property of high-density three-dimensional tissue of HepG2 cells formed in radial-flow bioreactor

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.     

Purification and characterisation of two extracellular acid proteinases from Monascus pilosus

Genus Monascus is one of the most important microorganisms in the fermentation industry in Asia. However, only a little attention has been paid to the proteinases produced by this fungus and their role in the fermentation process. The main objective of this study was to purify and characterise acid proteinases produced by Monascus pilosus. Two acid proteinases (MpiAP1 and MpiAP2) were purified to homogeneity. Both purified enzymes, MpiAP1 and MpiAP2, were monomeric structures with molecular masses of around 43 and 58 kDa, respectively. The former was an acidic non-glycoprotein, whereas the latter was an acidic glycoprotein with 27% carbohydrate content. Although amino-terminal amino acid sequence analysis of both enzymes (MpiAP1 and MpiAP2) of 20 amino acid length showed over 90% similarity, their amino-terminal amino acids were different from each other. Both enzymes were optimally active at 55 °C and at pH 2.5–3.0 against casein or human haemoglobin. The T_1/2 values of MpiAP1 and MpiAP2 were 65 and 70 °C, respectively. Both of the enzymes were completely inhibited by pepstatin A, and markedly by SDS. MoO₃ also showed a partial inhibition of both enzymes. Milk casein and haemoglobin were good substrates for these enzymes. Eleven cleavages were detected using the oxidised insulin B-chain as a peptide for the proteolytic specificity test of MpiAP1, while seven cleavages were detected for MpiAP2.     

Ensiling characteristics and ruminal degradation of Italian ryegrass and lucerne silages treated with cell wall-degrading enzymes

Two experiments were carried out to investigate the effect of added cell wall-degrading enzymes at ensiling on the fermentation and in situ degradation of grass and legume silages. Primary growths of Italian ryegrass (Lolium multiflorum Lam) and lucerne (Medicago sativa L) were wilted and ensiled in laboratory-scale silos with or without enzymes. Silages were opened at 2, 5, 15 and 45 days after storage; the fermentation quality and the contents of cell wall components (NDF, ADF, ADL) were determined. The 45 day silages were subjected to in situ incubation experiments, and the kinetics of DM and NDF degradation was estimated. The enzyme treatment enhanced the lactic acid production (P < 0.01) and reduced the pH value (P < 0.01) of both Italian ryegrass and lucerne silages. The contents of cell wall components, however, were not affected by enzymes, except for NDF of Italian ryegrass silage. The in situ incubation experiments revealed that added enzymes increased the rapidly degradable DM (P < 0.01) and appeared to decrease the rate of degradation of DM and NDF of Italian ryegrass silage. The rapidly degradable DM was not altered when lucerne was treated, but the rate of degradation of NDF was significantly reduced (P < 0.05). These results suggest that although added cell wall-degrading enzymes could improve the preservation of grass and legume silages, the effects on ruminal degradation may be different according to the herbage treated. Enzymatic hydrolysis during ensilage may be restricted to easily digestible cell walls, leaving relatively less digestible components that would be retained in treated silages. © 1999 Society of Chemical Industry     

Fate of coliphage in a wastewater treatment process

The fate of coliphage in a wastewater treatment plant in the central part of Japan was investigated from March to December 2001. A relative abundance of coliphage, 1000-10,000 PFU/ml determined with three different Escherichia coli strains, was detected in the influent. But, no remarkable seasonal change in the phage concentration in the influent was observed during the ten-month test period. Almost ten times higher coliphage concentration was detected by the F+ E. coli strain than by the other two F- strains. The RNA phage was more stable than the DNA phage against aerobic treatment using activated sludge. Most of the phages in the influent and primary settling tank were detected as suspended forms. Anaerobic-aerobic treatment enhanced adsorption of the phage by the solid particles. Almost no phage was detected in the effluent. Aerobic treatment using activated sludge and/or the addition of flocculants such as PAC was effective for the removal of coliphage, an index of enteric viral pollution.     

Introduction of the archaebacterial geranylgeranyl pyrophosphate synthase gene into Chlamydomonas reinhardtii chloroplast

Geranylgeranyl pyrophosphate (GGPP) synthase gene (gds) derived from a thermophilic Archae Sulfolobus acidocaldarius, was introduced into a unicellular green alga Chlamydomonas reinhardtii chloroplast. Heat treatment abolished the prenyltransferase activity of the wild strain, but the activity of the transforment remained. The transformant accumulated gds gene mRNA and translation product.     

Method for determination of aflatoxin M₁ in cheese and butter by HPLC using an immunoaffinity column

A rapid, sensitive convenient method for determination of aflatoxin M? (AFM?) in cheese and butter by HPLC was developed and validated. The method employs a safe extraction solution (mixture of acetonitrile, methanol and water) and an immunoaffinity column (IAC) for clean-up. Compared with the widely used method employing chloroform and a Florisil column, the IAC method has a short analytical time and there are no interference peaks. The limits of quantification (LOQ) of the IAC method were 0.12 and 0.14 μg/kg, while those of the Florisil column method were 0.47 and 0.23 μg/kg in cheese and buffer, respectively. The recovery and relative standard deviation (RSD) for cheese (spiked at 0.5 μg/kg) in the IAC method were 92% and 7%, respectively, while for the Florisil column method the corresponding values were 76% and 10%. The recovery and RSD for butter (spiked at 0.5 μg/kg) in the IAC method were 97% and 9%, and those in the Florisil method were 74% and 9%, respectively. In the IAC method, the values of in-house precision (n=2, day=5) of cheese and butter (spiked at 0.5 μg/kg) were 9% and 13%, respectively. The IAC method is superior to the Florisil column method in terms of safety, ease of handling, sensitivity and reliability. A survey of AFM? contamination in imported cheese and butter in Japan was conducted by the IAC method. AFM? was not detected in 60 samples of cheese and 30 samples of butter.     

2-Formylcinnamaldehyde Formation Yield from the OH Radical-Initiated Reaction of Naphthalene: Effect of NO(2) Concentration

Naphthalene, typically the most abundant polycyclic aromatic hydrocarbon in the atmosphere, reacts with OH radicals by addition to form OH-naphthalene adducts. These OH-naphthalene adducts react with O(2) and NO(2), with the two reactions being of equal importance in air at an NO(2) mixing ratio of ～60 ppbv. 2-Formylcinnamaldehyde [o-HC(O)C(6)H(4)CH═CHCHO] is a major product of the OH radical-initiated reaction of naphthalene, with a yield from the reaction of OH-naphthalene adducts with NO(2) of ～56%. We have measured, on a relative basis, the formation yield of 2-formylcinnamaldehyde from the OH radical-initiated reaction of naphthalene in air at average NO(2) concentrations of 1.2 × 10(11), 1.44 × 10(12), and 1.44 × 10(13) molecules cm(-3) (mixing ratios of 0.005, 0.06, and 0.6 ppmv, respectively). These NO(2) concentrations cover the range of conditions corresponding to the OH-naphthalene adducts reacting ～90% of the time with O(2) to ～90% of the time with NO(2). The 2-formylcinnamaldehyde formation yield decreased with decreasing NO(2) concentration, and a yield from the OH-naphthalene adducts + O(2) reaction of 14% is obtained based on a 56% yield from the OH-naphthalene adducts + NO(2) reaction. Based on previous measurements of glyoxal and phthaldialdehyde from the naphthalene + OH reaction and literature data for the OH radical-initiated reactions of monocyclic aromatic hydrocarbons, the reactions of OH-naphthalene adducts with O(2) appear to differ significantly from the OH-monocyclic adduct + O(2) reactions.