碩士 / 國立高雄海洋科技大學 / 水產食品科學研究所 / 106 / Marine algae possess unique amino acids not seen in terrestrial plants. Agonists of glutamate receptors, kainic acid (KA) and domoic acid (DA) are a representative example of such amino acids. These excitatory amino acids were originally separated as an anthelminthic principal from the red algae “makuri” Digenea simplex (Murakami et al. 1953) and “hanayanagi” Chondria armata (Daigo, 1959), respectively. A strong overdose of either KA or DA causes dysmnesia by destroying the CA3 region of the hippocampus, which coordinates memory in the cerebrum (Strain and Taker 1991). DA is the causative substance of amnesic shellfish poisoning (ASP) that occurred in Canada in 1987, where people were poisoned due to eating mussels, which had become toxic by ingesting and accumulating DA originally produced by the diatom Pseudo-nitzschia pungens forma multiseries (Bates et al., 1989; Wright et al., 1989). Because DA causes human food poisoning as well as abnormal behavior and death of animals, a lot of studies have been conducted mainly with diatoms to examine the distribution, transfer/accumulation via the food chain, biosynthetic pathway, and human intoxication mechanism of DA (Bates et al. 1989; Perl et al. 1990; Ramsey et al. 1998; Kotaki et al. 1999; Savage et al. 2012). However, little information is available on the influence of environmental factors on growth and KA/DA production of the above red algae, or on the production mechanism and physiologic functions of KA/DA in the red algae. To contribute to the elucidation of these points, some fundamental studies on the KA/DA-producing red algae were conducted.
In Chapter I, various marine algae (a total of 69 samples) inhabiting Japan and Taiwan were collected and screened for KA/DA by a liquid chromatography-mass spectrometry (LC-MS). Contents of related excitatory amino acids, glutamic acid (Glu) and aspartic acid (Asp), were also investigated by an automated amino acid analyzer. As a result, 16–2158 µg/g dry wt of KA was detected in 5 of 12 algae from Ishigaki Island, 3 of 8 algae from Yakushima Island, and 3 of 32 algae from Xiaoliuqiu. Nine (9) of the 11 algae in which KA was detected was different species from D. simplex. DA was detected only in 4 algae from Yakushima Island. Two (2) of the 4 algae were different species from C. armata, and the DA content (6 and 29 µg/g dry wt) was much lower than that of C. armata (2815 and 4431 µg/g dry wt). Glu was contained in all algae except for one sample with mean and maximum content of 494 and 2680 µg/g dry wt, respectively. Asp was detected in 48 of 69 algae, whose mean Asp content (112 µg/g dry wt) was much lower than that of Glu. Two D. simplex had Glu contents of 264 and 547 µg/g dry wt, whereas two C. armata contained higher Glu contents with 1428 and 2680 µg/g dry wt. A strong positive correlation (r = 0.98, p < 0.05) was observed between DA contents and Glu contents in the 4 algae in which DA was detected.
In Chapter II, D. simplex was cultured with various culture media to clarify the nutritional conditions to produce KA. Unlike C. armata (Jiang et al., 2014), D. simplex was insensitive to excessive manganese, and grew best (mean growth rate ~800% for 25 days) in modified PES medium (mPES; seawater + nitrate, phosphate, iron, trace metals, vitamins, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]- ethanesulfonic acid) prepared with autoclaved seawater. LC-MS analysis of the algal extracts revealed that the KA content of the explants cultured with mPES or N•P•Fe medium (seawater + nitrate, phosphate, and iron) was somewhat higher than that of wild specimens (1748–2378 µg/g vs 1562 µg/g). The 1H-nuclear magnetic resonance (1H-NMR) spectrum of the KA extracted and purified from pooled explants was indistinguishable from the previously reported KA spectrum (Todeschi et al., 1997).
In Chapter III, growth and KA production of D. simplex irradiated with monochromatic light were investigated to explore how environmental light condition affects the KA production. D. simplex explants originally collected from Ishigaki Island, and successively subcultured thereafter in our laboratory were used as materials. Small branches cut from the explants were cultured with mPES at 21°C for 42 days under photosynthetically active radiation of 80 µmol/m2/s provided by cool-white fluorescent lamps (White), or by Blue (470 nm), Green (530 nm), or Red (660 nm) light-emitting diodes (LEDs) with a photoperiod of 12:12 light:dark. The results indicated that The growth rate of explants was different depending on the light wavelength; the rate of Green (298% at 28 d and 511% at 42 d in average) was similar to that of White (329% at 28 d and 525% at 42 d), but the rate of Red (497% at 28 d and 526% at 42 d) was higher than that of Green or White at 28 d. The growth rate of Blue was generally lower than that of Green or White, being only 374% at 42 d. In contrast, no significant difference due to light wavelength was observed in KA production, i.e., the total KA of explant significantly increased during the culture period, and reached about 9-fold that at the beginning of culture (68 μg/individual) at 42 d irrespective of the light wavelength.
In Chapter IV, a culture experiment similar to that of the previous chapter was conducted using C. armata explants, which was originally collected from Hanasezaki, Kagoshima Prefecture, and successively subcultured in our laboratory. Small thalli or rhizoids cut from the explants were cultured for 42 days with the same conditions as those of the previous chapter. As a result, the growth of C. armata was much worse than that of D. simplex; the growth rate (in average) at 42 d was 121% in White, and 139% and 173% in Blue and Green. Moreover, no growth was observed in Red (growth rate, 91%), in which D. simplex showed best growth in the previous chapter. The total DA (initial 119 µg/individual) also fluctuated during the culture period, and finally increased to 139 and 159 µg/individual in Blue and Green, but decreased to 87 µg/individual in White at 42 d. In Red, the total DA content gradually decreased, and became about half (45 µg) that at the beginning of culture.
As a conclusion, fundamental data to create distribution profiles of excitatory amino acids in marine algae could be obtained in the present study. Moreover, we succeeded in establishing laboratory culture of D. simplex, and could clarify a part of the influence of light quality given on the growth and KA production of D. simplex.
| Identifer | oai:union.ndltd.org:TW/106NKIM0084002 |
| Date | January 2018 |
| Creators | Chisato Urata, 浦田千里 |
| Contributors | Yung-Hsiang Tsai, Osamu Arakawa, 蔡永祥, 荒川修 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 81 |
Page generated in 0.1237 seconds