@phdthesis{oai:mie-u.repo.nii.ac.jp:00010831,
 author = {鶴田, 小百合},
 month = {Jan},
 note = {application/pdf, In this study, three assays for the rapid detection of ingredients and food additives, including fish roes, poisonous mushrooms, and sweeteners and preservatives in various foods, were developed to secure the safety and quality of food. In chapter 1, a rapid and sensitive TaqMan real-time polymerase chain reaction (PCR) assay was developed for the detection of Pacific cod (Gadus macrocephalus) and capelin (Mallotus villosus) roes in Alaska pollack (Theragra chalcogramma) roe products. Primers and TaqMan minor groove binder (MGB) probes were designed according to the gene encoding cytochrome b and evaluated by the specificity for 3 Alaska pollack roes, 3 Pacific cod roes, 9 capelin roes, and samples from 48 other species. Mitochondrial DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.002 ng/L of DNA. The linearity of the standard curves for the three species ranged from 0.002 ng/L to 20 ng/L of DNA with a square regression correlation (R2) of 1.000. This assay was applied for the detection of Pacific cod and capelin roes in mixture samples: Pacific cod or capelin roes were added to Alaska pollack roes at 0.1%, 1%, and 10%. The threshold cycle (Ct) values were obtained from both the mixture samples at 0.1%. The practical applicability of this assay was examined in 64 samples of Alaska pollack roe products. In all cases, the species detected from the samples corresponded with the species described on the food label. This new assay proved to be specific to the target species, highly sensitive, and applicable to
food samples, making it useful for the rapid detection of Pacific cod and capelin roes in Alaska pollack roe products. In chapter 2, a rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two types of mushrooms: the poisonous mushroom Omphalotus guepiniformis and the edible mushroom Lentinula edodes, both of which have fruiting bodies with a similar morphology. Primers and TaqMan MGB probes were designed according to the internal transcribed spacer 1–5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes, and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.25 pg of DNA. The linearity of both the detection systems ranged from 0.25 pg to 2.5 ng of DNA, with R2 of >0.999. The Ct values for raw and processed (baking, stir-frying, deep-frying, boiling for 30, 60, 2 120, or 180 min or artificial digesting by α-amylase and pepsin) fruiting bodies and for fruiting bodies [1% (w/w)] mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved to be specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for the rapid identification of O. guepiniformis and L. edodes in case of mushroom poisoning. In chapter 3, a rapid and simple assay was developed for the simultaneous determination of 12 sweeteners and 9 preservatives in various foods by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The sweeteners and preservatives were extracted from solid samples with 80% and 50% methanol and from liquid samples with 80% methanol; and this was followed by Oasis WAX cartridge cleanup. The LC separation was performed on an XSelect CSH Phenyl-Hexyl column (5 μm, 2.1 mm × 150 mm) with a mobile phase of 10 mmol/L acetate buffer (pH 4.0)–acetonitrile, and MS detection was performed with negative-ion electrospray ionization. The quantification limits of acesulfame K, alitame, aspartame, cyclamic acid, neotame, saccharin Na, p-hydroxybenzoic acid methyl, p-hydroxybenzoic acid ethyl, p-hydroxybenzoic acid isopropyl, p-hydroxybenzoic acid propyl, p-hydroxybenzoic acid isobutyl, and p-hydroxybenzoic acid butyl were 0.001 g/kg; those of dulcin, glycyrrhizic acid, neohesperidin dihydrochalcone, rebaudioside A, stevioside, sucralose, and benzoic acid were 0.005 g/kg; and those of sorbic acid and dehydroacetic acid were 0.02 g/kg. The mean recoveries from 10 types of foods fortified at the levels of 0.02 and 0.2 g/kg were 70.9%–119.0%, and their relative standard deviations were 0.1%–11.7%. This new assay proved to be highly selective and quantitative for the detection of these 12 sweeteners and 9 preservatives and was applicable to food samples, making it useful for the rapid detection of these sweeteners and preservatives in various foods. In conclusion, the three assays developed in the present study proved that the above mentioned ingredients and food additives are detectable in a rapid and simple manner and are considered to be useful in verifying species described on food labeling of fish roe products, investigating sources of mushroom poisonings, and simplifying the determination of food additives., 本文, 85p},
 school = {三重大学},
 title = {食品中の原材料および食品添加物の迅速な検出法に関する研究},
 year = {2015}
}