Submerged fermentation of Pleurotus citrinopileatus mycelia with high ergothioneine content and its physiological activities

• Main Authors: Shin-Yi Lin, 林欣儀
• Other Authors: Jeng-Leun Mau
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/49565249095224069222

博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 104 === Ergothioneine is a sulfur containing amino acid that functions as an oxidative damage and anti-inflammation in organisms. Ergothioneine is produced only by fungi and few mycobacteria. It cannot be biosynthesized in human body, and is only ingested from dietary sources. Mushrooms are a primary source of ergothioneine, especially the Pleurotus spp. Pleurotus citrinopileatus (golden oyster) is an edible mushroom, also known as Yu-huang-mo in Chinese. Many reports indicate that this mushroom have shown biological and pharmacological activities, such as antitumor, immunoenhancing, antioxidant and antimutagenic activities. The optimization of the submerged culture of the culinary-medicinal P. citrinopileatus was studied using a one-factor-at-a-time, two-stage stimulation and central composite rotatable design (CCRD) to produce mycelia with high ergothioneine content. Pleurotus citrinopileatus mycelium was prepared with high ergothioneine content (HEPM) and its proximate composition, non-volatile taste components, antioxidant properties and antiphotoaging were studied. In order to identify the biological origin of the strain, the molecular identification method had been used to confirm the P. citrinopileatus based on the nucleotide sequences of ITS rDNA. The total length of ITS rDNA sequences was 650 bp. In BLAST search, the first to15th sequences in the Gene Bank had the higher identification of the published P. citrinopileatus sequence. Based on the results and morphology, we could confirm the strain in this study was P. citrinopileatus. The optimal culture conditions for mycelia harvested at day 22 were found to be 25oC, inoculation ratio of 5%, 2% glucose, 0.5% yeast extract and the adjustment of the initial pH value to 10; and the biomass and ergothioneine content was 8.28 g/L and 88.49 mg/L, respectively. We could obtain higher ergothioneine P. citrinopileatus mycelia after three amino acid precursor added. The addition of amino acid precursor had increased the ergothioneine content of mycelia with the addition of cysteine being the most effective. In addition, the results obtained from CCRD showed that the recommended combination for cysteine, histidine, and methionine were 8, 4, and 0.5 mM, respectively. The experimental maximal ergothioneine content was 99.64 mg/L. With the addition of complex precursors and under the optimal culture conditions, mycelia harvested at days 16-20 showed higher ergothioneine content. Accordingly, the information obtained had been used to produce mycelia with high ergothioneine content. The ergothioneine contents of fruiting body, HEPM and RPM were 3.89, 14.57 and 0.37 mg/g dw, respectively. HEPM contained higher dietary fiber, soluble polysaccharide and ash contents but lower carbohydrate, reducing sugar, fiber and fat contents than RPM. However, HEPM contained less amount of total sugars and polyols (47.43 mg/g dw). Besides, HEPM showed more intensive umami taste. In addition, the antioxidant properties of 70% ethanolic extracts from P. citrinopileatus fruiting body, HEPM and LEPM were investigated. On the basis of EC50 values obtained, the extract from HEPM showed the most effective antioxidant activity, reducing power and scavenging ability whereas fruiting bodies showed the most effective antioxidant activity, chelating ability and Trolox equivalent antioxidant capacity. Contents of ergothioneine and total phenols of HEPM were 61.22 and 60.65 mg/g, respectively. Overall, the extract of HEPM was good in antioxidant properties and high amount in components. We investigated whether or not the protective effects of HEPM 70% ethanolic extract against ultraviolet B (UVB) irradiation in human skin fibroblast cell culture (Hs68). The cell viability, cell cycle, protected oxidative and expressed of matrix metalloproteinases (MMPs) and Type-Ι procollagen were studied. The cell protective effect of HEPM on UVB-induced cytotoxicity had been investigated. HEPM 70% ethanolic extract at 5 μg/mL was shown 81.95% cell viability at UVB 300.0 mJ/cm2 irradiation. HEPM extract treatment at 5 to 1000 μg/mL dose-dependent improved the cell viability of UVB-irradiated. The result indicated that HEPM extract did not exhibit a cytotoxic effect. HEPM extract at 100 μg/mL was a potent scavenger of UVB induced reactive oxygen species (ROS) and inhibited ROS level by 96.43%. Treatment with HEPM extract rescued Hs68 cells from UVB-induced apoptosis. That also decreased in the expression of MMP-1, whereas increased in the production of Type-Ι procollagen. In this study, we focused on the assessment of metabolic differences of mushroom fruiting bodies and mycelia. We used NMR and PCA model to compare metabolites from fruiting bodies and mycelia. After NMR analysis, 55 water-soluble metabolites from fruiting bodies and mycelia had identified and quantified. Among them, 39 metabolites detected were significantly different between HEPM and RPM samples. In HEPM, concentrations of cystine, histidine, leucine, proline and trehalose were higher than those detected in RPM, whereas in RPM, concentrations of 4-aminobutyrate, arginine, glutamine, glucose and trigonelline were higher than those in HEPM. The highest concentration in fruiting bodies of P. citrinopileatus was UDP-N- acetylglucosamine that is involved in the synthesis of chitin. A metabolic pathway map of P. citrinopileatus mycelium clearly showed the effect in metabolites after amino acid precursor had added. Overall, our results indicated that the addition of precursor increased the ergothioneine content of P. citrinopileatus mycelia with the addition of mix amino acid solution being the most effective. HEPM had high bioavailable compounds, essential amino acids and biological compounds. The extract from HEPM was good in antioxidant properties and anti-photoaging ability than that from fruiting bodies and RPM. HEPM could be used for the development of anti-photoaging products.