Improvement Of Laccase Production From Ganoderma Lucidum HSD06B And The Application Of Improving The Quality Of Chinese Steamed Bread

In this thesis, a white rot fungus, strain HSD06B, was isolated from a tree full of rot in the pinehurst of Taihang Mountain in Henan Province. The morphological characters of the fungus were examinedand described as follows: Pileus:1.0cm thick; reniform to subcircular; upper surface yellowish brown tolaccate with radial furrows and concentrically sulcate zones. Pore:3-5per mm; suborbicuar; white. Stipe:cylindrical or flattened; laccate; attachment of the stipe to the pileus varied from lateral to nearly central.Hypha: non-septahypha; colorless; has branches;1.9-3.7μm in diameter. Basidiospore: ovoid;yellowish–brown; smooth;5.1-6.3μm×7.9-9.4μm. Based on the analysis of a614bp sequence from therDNA ITS region gene, strain HSD06B was related to Ganoderma lucidum (EU520247.1), and thesimilarity was up to100%. The fungus was identified as a strain of G.lucidum according to itsmorphological characteristics and phylogenetic data.The laccase production by G.lucidum HSD06B was studied under liquid state fermentation withdifferent media and fermentation conditions. Single factor and central composite experimental design wereemployed to optimize the culture medium composition. The optimized liquid state fermentation mediumcontained: glucose38.5g/L, peptone15.0g/L, barn21.1g/L, veratryl alcohol0.8mM/L, polyoxins0.02g/L, KH2PO41.0g/L, MgSO4·7H2O0.5g/L. The optimized culture conditions obtained by single factorexperiment were:10%of inoculation concentration, intial pH5.4,180r/min,30℃of the fermentationtemperature. The highest laccase production of G.lucidum HSD06B cultured in the optimized medium andculture condition is up to50153U/L after7days’cultivation, which was9.7times of previousconditions(5082U/L).Native-PAGE analysis suggested the G.lucidum laccase contains three laccase isoforms. BothLac I and lac II are the main laccase isoforms, and they are much higher than lac III in protein content. Thelaccases from G.lucidum HSD06B were purified by a two-step process: ammonium sulfate precipitation,gel filtration chromatography. After the two-step purification, we obtained mixture of three pure laccases,its purity and yield were4.6folds and38%of the crude laccase respectively. The molecular mass of thelaccases was about Lac I:62.6kDa; Lac II:64.5kDa; Lac III:65.9kDa.Effects of G.lucidum HSD06B laccase on performance of Chinese steamed bread(CSB) were investigated by sensory evaluation, microscopy of CSB and texture profile analysis. Laccase cansignificantly increase the specific volume of CSB. Sensory quality score of CSB treated by laccase ishigher than that of CSB without addition of laccase. The pore size of CSB crumbs supplemented0.5U/glaccase is smaller and regular than that of the control CSB crumbs. Texture profile analysis indicateslaccase significantly improves hardness and chewiness of CSB. However, the springiness and resilience arenot changed. Microstructure analysis showed laccase-treated CSB forms more intensive gluten networkthan that of the CSB without laccase, simultaneously, formation of the large starch granule and starchaggregation were observed. Furthermore, SDS-PAGE analysis indicated that laccase can catalyze wheatprotein cross-linking. The flour with laccase activity of0.5U/g was suitable for CSB making. The laccasehad a significant positive effect on improvement of CSB quality. The above results prove laccase fromG.lucidum HSD06B had a promising applieation in food industry.