| Stromata of Shiraia bambusicola were used as folk drug in China, and the main active chemicals of them were hypocrellins. In this study, a hypocrellin-producing strain was isolated and identified as Shiraia sp.. The main components of the pigments were isolated and identified. Pigment-producing conditions of solid-state fermentation and physiological characteristics of the strain were also investigated. The results are as follows:1) A hypocrellin-producing strain named SUPER-H168 was isolated using PDA plates, and identified with molecular biological methods (18S rDNA and ITS-5.8S rDNA). According to the results of cluster analysis (91% and 88% supported with S. bambusicola, respectively), the strain was proved to belong to the genus Shiraia. Small pycnidia (3-5μm×10-20μm) and conidia (<1μm) were also found, which were not reported before. Results of compositions analysis showed that it was very different between the mycelia and the stromata. However, contents of hypocrellins were very close to each other.2) Pigments produced by strain SUPER-H168 in solid-state fermentation were isolated by preparation RP-HPLC and six main components were found. Four of them were identified with NMR and LC-MS. The data showed that these four components were hypocrellin A, hypocrellin B, hypocrellin C and elsinochrome C. Elsinochrome C was firstly isolated from the pigments produced by Shiraia genus.3)While the growth of Shiraia sp. SUPER-H168 needed adequate nutrition, it was hard on those of poor nutrition substrates (e.g. rice bran, bagasse, sawdust, bamboo chips, straw, orange peel and corncob). Some grains (e.g. rice, millet, wheat, black rice, corn, sorghum and buckwheat) and byproduct (e.g. wheat bran) could be used for pigments production. When the soybean and soybean meal were used as substrates, the strain grew well, but no hypocrellins was detected. It was revealed that the pigment production was related to the Starch/Protein ratio (S/P) of the substrates, the pigments was produced by the strain when the ratio was more than 1.4. Further more, the percentage contents of the pigments were also influenced by the substrates. Hypocrellin A was the main composition (usually more than 40%) of the pigments, and there was no HB detected while millet and sorghum were used as substrates. The result showed thatα-amylase and protease activities of fermented substrates were both relatively low. Although the variation tendency of theα-amylase activity was similar to that of reducing sugar content, and thus could reflect the biomass change in a certain period, there was no significant relationship between these two enzymes and pigment production.4)Corn grits were selected as the fermentation substrate for optimization in the Erlenmeyer flasks (250 mL). The pigment- producing conditions were preliminarily optimized by one-factor-at-a-time method, followed by further optimization via response surface methodology. The optimal parameters were obtained as: corn grits (1.0-1.2 mm) 25 g, wheat bran 5 g, initial moisture content of 50%, initial pH 7.0, NaNO3 0.261 g, ZnSO4·7H2O 0.054 g and glucose 1.5 g, incubation temperature 30℃, relative humidity 80%-90% and incubation period of 18 d. Under these conditions, the yield of pigments could reach 14.08 mg/g dry solid, which was almost 2 folds of the initial yield. It was also found that nitrogen sources and their addition levels had significant effect on the pigment production. In a certain extent, NaNO3, beef extract and yeast extract could promote pigment production, while urea, some ammonium salts ((NH4)2SO4, NH4Cl and NH4NO3) and peptone had strong inhibition on the pigment production. Metal ions also had certain effects on the colorant yield. Zn2+, Na+ and K+ could promote pigment production while Mn2+ inhibited the pigment secretion. The kinetic models of solid-state fermentation for mycelial growth, pigment production and substrate (residual sugar) consumption were developed based on the experimental results.5) The physiological characteristics during the pigment production process were studied using liquid-state fermentation. It was found that the oxidation level was relatively high (the malondialdehyde content increased), indicating that the strain was under oxidative stress induced by the pigments during the pigment production period. T-AOC (total antioxidant capacity) level varied slightly, thus suggested that the antioxidant mechanisms might be activated to maintain the balance of both intracellular and extracellular oxidative/reductive state. Furthermore, pigment production could be promoted by 40μmol/L (final concentration) of hydrogen peroxide and white light illumination at a PFD (photon flux density) of 40μE/m2/s both on the PDA plates and in the Erlenmeyer flasks. The results showed that a certain oxidative stress could promote pigment production. It was supposed that pigment secretion might be regulated by a kind of positive feedback mechanism, which was triggered by the oxidative stress induced by the pigment under illumination.
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