Analysis of residual solvents in natural flavorings by headspace GC using the standard addition method

Headspace GC using the standard addition method has been developed for the simultaneous determination of organic solvents in natural flavorings. The procedure can be outlined as follows: an aliquot of the sample is transferred to a 10 mL vial. To each vial, a DMSO solution containing solvents at different concentrations is added as the standard solution. The vials are kept at 50 degrees C (for automatic injection) or 40 degrees C (for hand-operated injection) for 40 minutes. One mL of the vapor phase in each vial is injected into a gas chromatograph equipped with an Aquatic-2 column (0.25 mm i.d. x 60 m). To evaluate this method, we conducted a performance study in collaboration with 10 laboratories, using ginger oleoresin. We analyzed 6 solvents (methanol, 2-propanol, acetone, dichloromethane, hexane, and 1,1,2-trichloroethene) for which the maximum residue limits are established in Japan's Specifications and Standards for Food Additives. Methanol and acetone existed in the ginger oleoresin, so only the other that four kinds of solvents were added to it. Eight of the laboratories used automatic injection, while the remaining two used hand-operated injection. Statistical analyses were conducted on the data obtained from the 8 laboratories. Repeatability standard deviations (RSDr) ranged from 4.3 to 11.4%, and reproducibility standard deviations (RSDR) ranged from 8.4 to 19.0%.     

Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2

A novel N-acyl-d-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic d-amino acids with the highest preference for N-acetyl-d-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The k(cat) and K(m) values for NADF were 41?s(-1) and 2.5?mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1?mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20?mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/β-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.     

Characteristic diagrams of coal-derived liquids: the correlation between aromaticity and atomic HC ratios

Characteristic diagrams, which are very useful in studies of the reaction pathways in coal liquefaction processes and to characterize coal-derived materials, are presented. Using model compounds, aromatic carbon fraction, f_a, and aromatic proton fraction, f_aH, are plotted against atomic $\text{H}\text{C}$ ratio. The experimental results of an anthracene oil and its hydrogenated oils are plotted on these diagrams, and the usage and the validity of the charts are verified.     

Physicochemical Properties of Sweetpotato Starches with Different Gelatinization Temperatures

Physicochemical properties of five sweetpotato starches differing in gelatinization temperature were examined. The gelatinization temperature of Koganesengan starch, an ordinary cultivar of sweetpotato in Japan, was 73.6°C, whereas those of the other starches were measured to be 71.6°C for Kyukei 96162–1, 65.8°C for Kyushu No.127, 63.9°C for Kyukei 240, and 54.9°C for Quick Sweet. Some relationships of the primary structural properties with the gelatinization temperature have been found. As the gelatinization temperature decreased: i) the content of phosphate groups attached to the glucosyl residues decreased, ii) the amylose content, which was determined as difference in long chains of debranched original starch and of its amylopectin, decreased, iii) the proportion of unit chains with DP > 100 in the amylopectin fraction increased, iv) the proportion of unit chains with DP 6 to 10 in the amylopectin fraction increased, whereas that of unit chains with DP 12 to 24 decreased, v) the B‐type crystallinity of the starch granules was enhanced, and vi) the proportion of longer chains constituting each Nägeli amylodextrin increased. Moreover, it was found that thin pastes of the low temperature‐gelatinizing starches retrograded slower during cold storage than the ordinary starch. Among the starches, Quick Sweet starch granules, having the lowest gelatinization temperature, were digested rapidly by pancreatin.     

Reaction of lithium naphthalene with carboxylic acids containing the dichlorocyclopropane ring system

Carboxylic acids containing the dichlorocyclopropane ring system react with lithium naphthalene to give dechlorinated products. For example, from the reaction of 9,10-dichloromethylene octadecanoic acid and lithium naphthalene, a mixture of 9,10-nonadecadienoic acid as a main product and unidentified minor products were obtained.     

Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.     

Protective effect of lutein after ischemia-reperfusion in the small intestine

Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesise lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. There is little information about the effects of intake of lutein in tissues other than the eyes. The aim of this study was to clarify the protective effect of lutein against oxidative injury using ischemia-reperfusion (I/R) model rats and to determine the relationship between pharmacokinetics and antioxidant activity of lutein. Intestinal I/R was induced by 30-min occlusion of the superior mesenteric artery followed by 60-min reperfusion. After 60 min of reperfusion, intestinal tissue was used for analysis of Evans blue dye extravasation, lipid peroxidation and myeloperoxidase activity. Lutein administered before I/R had a significant protective effect against oxidative injury.     

Development and evaluation of qualitative detection methods for unapproved genetically modified rice (LLRice)

We developed a specific method to extract DNA from rice grain samples and modified the qualitative real-time PCR method provided by Bayer Co., Ltd. for reliable detection of the genetically modified (GM) rice variety, LLRice601, which has not undergone safety assessment for regulatory approval in Japan. Moreover, we conducted a data analysis to confirm the results obtained with real-time PCR. The yields of DNA extracted from powdered samples of rice grains were almost equal among 5 different varieties of rice, and there was no significant difference in the yield over three days. Reliable results were obtained using 50 ng of the extracted DNA as the template for real-time PCR. To examine the adequacy of the methods, we organized an interlaboratory study with the participation of 2 laboratories, in which 80 test samples were analyzed in a blinded manner. The statistical analysis revealed no significant difference in the Ct value for the endogenous gene of the DNA samples and for the targeted DNA sequence of 0.1% samples. The limit of detection of the method was approximately 0.1%. Analysis of the fluorescence intensity of the PCR-amplified product of the construct-specific DNA sequence suggested that it may be reasonable to judge a sample as positive when a Ct value of less than 40 is obtained.     

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.