Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Determination of theaflavins including methylated theaflavins in black tea leaves by solid-phase extraction and HPLC analysis

A quantitative method for four theaflavins and two methylated theaflavin derivatives in black tea leaves was developed by solid-phase extraction and a high-performance liquid chromatographic method with photodiode array detection. The theaflavins in black tea leaves were extracted three times with 40 vol 50% aqueous ethanol (mg dry tea powder/mL) containing 2% ascorbic acid. The ethanol extracts were diluted 4-fold with distilled water. All diluted extracts were directly applied to the solid-phase C18 cartridge column without concentration. The fraction of theaflavins was obtained by 40% ethanol extraction after rinsing with water followed with 15% ethanol extraction. An aliquot of theaflavins after concentration was injected onto an ODS C18 reversed-phase column, and four theaflavins and two methylated theaflavins were sufficiently separated by a linear gradient system using distilled water and acetonitrile with 0.5% acetic acid. This analytical method is sensitive for the determination of a small amount of methylated theaflavins, since various interfering substances produced during the fermentation process were eliminated in advance by solid-phase extraction. Using this analytical method, we also demonstrated that methylated theaflavins were easily produced during the manufacture of black tea.     

Maturity and reproduction of goneplacid crab <Emphasis Type="Italic">Carcinoplax vestita</Emphasis> (Decapoda,Brachyura) in Tokyo Bay

ABSTRACT:     Sexual maturity, morphological sexual dimorphism, and reproduction of the goneplacid crab Carcinoplax vestita were investigated in Tokyo Bay, Japan, from November 2002 to October 2003. The puberty molt in males was evidenced by changes in the relative size of the chelipeds and merus of the walking legs, and was estimated to occur at a size range of 13.20–18.85 mm carapace length. Post-pubertal females were identified by the relative size of the abdomen and puberty was estimated to occur at a size range of 12.81–15.46 mm carapace length. Sexual dimorphism in C. vestita was observed in all features that showed secondary sexual characteristics. Monthly changes of gonad index in males, and of seminal receptacle index and occurrence of sperm plugs in females were synchronized, and indicated that mating was intense in spring. Ovaries began developing in March. Ovigerous females were found in all months except December, but were clearly more abundant between August and October. Fecundity ranged from 7800 to 57 000 mature oocytes per female per batch and was highly correlated with body size. The results suggest that some females may spawn more than one batch per year.     

Bioavailability of amino acids chelated and glass embedded zinc to rainbow trout, Oncorhynchus mykiss, fingerlings

A 2 × 4 factorial experiment was conducted to determine the bioavailability of zinc (Zn) from amino acids chelated (Zn–Am) and glass embedded Zn (Zn–Gl) as sources for rainbow trout, Oncorhynchus mykiss , fed practical type diets. Two levels of Zn (20 and 40 mg kg⁻¹) were supplemented to the diets using either zinc sulphate (Zn–Sf), zinc methionine (Zn–Mt), Zn–Am or Zn–Gl. Rainbow trout with an average weight of 2 g were fed the experimental diets for 15 weeks. Growth and feed gain ratio (FGR) were not significantly influenced by the dietary Zn content and forms. Alkaline phosphatase (ALP) activity for both levels of Zn–Am was significantly higher than that of Zn–Sf and Zn–Gl at 20 mg supplementation. In another experiment, fish of about 95 g were fed the same experimental diets to determine the absorption of Zn and it was found to be significantly higher from Zn–Am compared with the rest. Retention from Zn–Am at 20 mg was significantly higher than the rest, excluding Zn–Sf. The results suggest that the availability of Zn from Zn–Am might be superior among the sources compared.     

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.     

Genetic identification of native populations of fluvial white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> in the upper Tone River drainage

ABSTRACT:   Stocking of exogenous, hatchery-reared white-spotted charr Salvelinus leucomaenis has been conducted throughout much of their range in Honshu Island, Japan, to increase angling opportunities. Although the native charr populations are thought to have declined because of hybridization with introduced fish, their distribution and genetic status have been uncertain. Fine population structures of charr in the upper Tone River drainage were examined using mitochondrial DNA and microsatellite analyses so as to clarify the presence of native populations. One common mtDNA haplotype was detected in all populations in the Ohashi River and Watarase River, and four and one tributary populations were monomorphic for such haplotypes, respectively. However, several haplotypes, considered to have originated from stocked hatchery fish, were observed in the stocked and the remaining populations. Judging from the genetic integrity over a fine geographic scale, the former were considered as indicative of native populations and the latter as admixtures with hatchery fish. Comparisons of genetic diversity, deviations from the Hardy–Weinberg equilibrium, principal component analysis, and relatedness estimations based on microsatellite DNA can also provide evidence for distinguishing native populations from those influenced by hatchery fish.