Taxonomy And Phylogeny Of Inonotus Linteus Complex And Liquid Fermentation Of I. Sanghuang

"Sanghuang" is a popular medicinal mushroom complex in traditional Chinese medicine, and was previously recorded as Phellinus linteus (Berk.&M.A. Curtis) Teng. But for a long time, the taxonomy and phylogeny of this complex has been controversial and ambiguous. Currently studies and products on medicinal mushroom called "sanghuang" in full swing, but at the same time the phenomenon of species confusion is increasingly. It has greatly restricted the scientific exploitation, healthy and sustainable development of this precious medicinal mushroom. So the research on a plenty of specimens from East Asia, American and Africa was carried out to determine the morphological and phylogenetic relationships and their geographical distribution and hosts. In addition, the study about liquid fermentation, bioactive components and antioxidant activity in vitro were conducted on Inonotus sanghuang Sheng H. Wu, T. Hatt.&Y.C. Dai.The Inonotus linteus complex comprised several closely related species characterized by pileate basidiomes, a heterogeneous hyphal system with a monomitic context and a dimitic hymenophoral trama. Setae are from hymenia and basidiospores are broadly ellipsoid to subglobose, yellowish and thick-walled. Researches to understand this complex have been particularly notable in East Asia. In the present paper, the complex was studied at a larger scale, including samples from Mesoamerica. Inonotus linteus, an epitype of this species being designated here, is circumscribed as a narrow sense based on morphological and phylogenetic data, and this has lead to delimiting a new species I. cubensis Y.C. Dai, Decock&L.W. Zhou from Mesoamerica, and provides evidence for another two new additional species, I. alpinus Y.C. Dai&X.M. Tian and I. zonatus Y.C. Dai&X.M. Tian from East Asia. Phylogenetic analyses of ITS sequences also confirmed the status of I, baumii T. Wagner&M. Fisch.,I. linteus (Berk.&M.A. Curtis) Teixeira,I. lonicericola (Parmasto) Y.C. Dai,I. lonicerinus (Bondartsev) Sheng H. Wu, Y.C. Dai&T. Hatt.,I. vaninii (Ljub.) T. Wagner&M. Fisch.,I. sanghuang Sheng H. Wu, T. Hatt.&Y.C. Dai,I. weigelae T. Hatt.&Sheng H. Wu and I. weirianus (Bres.) T. Wagner&M. Fisch. as distinct taxa, while I. tenuicontextus L.W. Zhou&W.M. Qin is considered to be a synonym of I weigelae. A key to eleven species in the complex is provided.For I. sanghuang growing exclusively on Morus, response surface methodology (RSM) was used to optimize the liquid fermentation medium. Plackett-Burman design (N=12) was used to examine the main factors affecting the mycelial biomass production from corn flour, glucose, peptone, yeast extract, bran, KH2PO4、MgSO47H2O and pH. The results showed that corn flour, yeast extract, bran and pH were selected as the main factors and the path of steepest ascent model was used to determine the optimum condition of mycelial biomass production. The four-factor, three-level Box-Behnken design (BBD) was employed to optimize the mycelial biomass. Under the optimized composition of culture medium (38.96g/L of corn flour,25.00g/L of glucose,3.75g/L of peptone,4.15g/L of yeast extract,20.55g/L of bran,1.25g/L of KH2PO4,0.625g/L of MgSO47H2O and pH6.39), the predicted and expected maximal mycelial biomass reached17.60g/L and18.33±0.86g/L, respectively. The optimized mycelial biomass was2.5times higher than the original mycelial biomass (7.29g/L).The crude extracts of intercellular polysaccharide(IPS) and exopolysaccharides (EPS) produced by liquid fermentation of I. sanghuang were extracted by the way of water extraction followed by ethyl alcohol deposition. Two components (IPS1and IPS2) of IPS and two components (EPS1and EPS2) of EPS were isolated by a gel-filtration chromatography of Sephadex G-100. The results about the contents and DPPH radical scavenging activities of these six polysaccharides showed that there were no significant correlation between the polysaccharide contents and DPPH radical scavenging activities for all the six samples. The DPPH radical scavenging rates were enhanced with the rise of the polysaccharide contents of IPS, IPS1and IPS2in the concentration range of6.25μg/mL to500μg/mL. EPS, EPS1and EPS2showed no obvious DPPH radical scavenging activity in the test concentration range.In order to probe into the effects of two-stage cultivation (consisted of an initial10days shake cultivation and then30days static culture) on the accumulation of I. sanghuang liquid fermentation metabolites, the contents change of total flavonoids, total phenolic compounds and polysaccharides in mycelia, as well as DPPH and hydroxyl radical scavenging activities of IS-EtOH and IS-H2O, were investigated during the whole period of two-stage cultivation. The results demonstrated that the contents of total flavonoids and total phenolic compounds in mycelia was found to increase dramatically by37.92%and77.27%, respectively, after10days and15days static culture. The results also implied that the richness of total flavonoids compounds increased dramaticly during the two-stage cultivation. The results of HPLC analysis were conjectured that rutin, eriodictyol, naringenin and sakuranetin may be contained in mycelia. The high free radical scavenging activity of IS-EtOH was resulted from the presence of phenolic-type compounds (total flavonoids and total phenolic compounds).