Determination of free fatty acids in milk and dairy products by reversed-phase HPLC with fluorescence detection

A highly sensitive HPLC with fluorescence detection was developed for the determination of free fatty acids (FFAs) in milk and dairy products. For this purpose, the FFAs were extracted from small amounts of milk (1.0 mL), cheese (0.5 g) and butter (0.5 g) using Sep-Pak cartridge columns, and then derivatized to 9-anthrylmethyl esters with 9-anthryldiazomethane (ADAM). Good separations of the ADAM derivatives of 16 FFAs (C4-C18) that exist in milk, cheese and butter, which permitted quantitative estimation of their individual FFAs, were achieved by HPLC on a C18 column (Cadenza CD-C18, 150 x 3 mm i.d.) using gradient elution with methanol and water. The acid values calculated from the contents of individual FFAs were in good agreement with those obtained by the conventional titration method. These results demonstrate that the present HPLC method is simple, sensitive and precise, and could be utilized widely for determination of the FFAs in milk and dairy products.     

Evaluation of selective plating media for the detection of heat- or freeze-injured Staphylococcus aureus

The efficacy of Baird-Parker (BP) agar, mannitol-salt-egg yolk (MSEY) agar and mannitol salt (MS) agar in detecting Staphylococcus aureus FRI-100 heated at 52 degrees C for 20 min in 100 mmol/L potassium phosphate buffer was determined. Brain heart infusion agar with 1% pyruvate (BHIP agar) supported the highest recovery of injured cells and was used as the control medium. Of the three selective media, significantly higher recovery of heat-injured cells was observed on BP agar than MSEY agar, and the poorest recovery was observed on MS agar (p < 0.05). Low recovery of unheated cells was obtained for MS compared with other media (p < 0.05). A reduction in populations occurred gradually in reagent-grade water stored for 14 days at -20 degrees C. There was no significant difference between BHIP agar and MS agar in the number of freeze-injured cells recovered from 1 to 14 days.     

Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.     

Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.     

Elasto-plastic analysis of orthogonally stiffened plates with initial imperfections under uniaxial compression

In general, the stiffened plates consisting of steel plate elements are unavoidably accompanied by initial imperfections such as residual stresses and initial deflections, which have considerable effects on their ultimate strength. Therefore, it is needed for designing them to develop more rational method taking the ultimate strength influenced by initial imperfections into account rather than the conventional design method being on the basis of the linear elastic buckling theory.From this point of view, this study aims to evaluate rigorously the ultimate strength of orthogonally stiffened plate with initial imperfections under uniaxial in-plane compression. The elasto-plastic finite element method is applied to attain this purpose. By a happy combination of modal analytical technique and conventional finite element method, much reduction of the degree of freedom can be expected to be realized herewith. Some numerical calculations are performed by means of this rigorous method to examine the exactness of the analysis. Moreover, the numerical results are compared with the experimental ones.     

Solution copolymerization of ethylene with α-olefins by a MgCl2 supported TiCl4 catalyst

Summary Copolymerization of ethylene with -olefins, i.e. propylene, butene-1, 4-methyl-pentene-1(4-MP-1), was carried out by a MgCl₂ supported TiCl₄ catalyst in combination with Et₃Al at a temperature as high as 170 °C at which the polymerization system was homogeneous. This catalytic system showed very a high activity and produced copolymers having a density of 0.91–0.94 g/ml. Of these three kinds of comonomers, propylene showed the highest reactivity and caused most frequently the termination of a polymer growing by chain transfer reaction and produced copolymers having the broadest MWD.     

Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein

The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co2+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.     

Effects of processing and cooking on the levels of pesticide residues in soybean samples

The effects of processing and cooking on the levels of pesticide residues in soybean samples were investigated for 14 pesticides in pre-harvest samples. On soaking, the transfer ratios (%, total pesticide residue amount in product/that in soybean) of soaked soybean were greater than 60% for most of the pesticides investigated. The transfer ratio of soymilk ranged from 37% to 92%, and that of tofu ranged from 7% to 63%. The processing factor (Pf, the concentration (mg/kg) of pesticide in product/that in soybean) of tofu ranged from 0.026 to 0.28. These values varied among pesticides. There was a high correlation between the log P(ow) and the transfer ratio of tofu. The test described here should be useful to obtain the transfer ratios of pesticide residues in processing and/or cooking steps.