The Study Of Mycelium Submerged Fermentation Of Hunong Lingzhi No.1 Variety And Its Active Polysaccharides

Ganoderma lucidum as a valuable medical fungi has a long history in China,it has been found more than 2000 years ago for its medicinal value.As one of the main active ingredients,polysaccharides get much attention for their activities of anti-tumor,antioxidant and immunoregulatory recently.Some reports also showed the ganoderma lucidum polysaccharide by liquid submerged fermentation were similar to the fruit bodies.Because it was not influenced by season,the production cycle was shorter,and the cost of fermentation was cheaper than planting,furthermore,the production efficiency was improved,it has become an important means to obtain the ganoderma lucidum polysaccharide compounds.The purpose of this study was to offer technique support,included screening of ganoderma lucidum strains,optimum of submerged fermentation parameters,and optimizing strategy for fermentation tank cultivating,also immune activity test of crude polysaccharide.There are four parts in this dissertation.Study on the screening of strains,the optimum of submerged fermentation parameters,optimizing strategy for fermentation tank cultivating,and immune activity test of crude polysaccharide were discussed in this dissertation.1.Screening high yield intracellular polysaccharide strains and kinetics study of liquid submerged fermentation by Ganoderma lucidumScreening 72 strains of Ganoderma lucidum preserved in laboratory based on synthetic intracellular polysaccharide of liquid submerged fermentation.Two indicators including mycelium biomass,intracellular polysaccharide were analyzed based on intracellular polysaccharide yield.The results showed that the highest yield strains were G0041,G0069 and G0119.After a detailed study on the fermentation process of G0119,the maximum mycelium biomass was 22.48 g/L,maximum intracellular polysaccharide content was 18.25 g/100g,and intracellular polysaccharide yield was 1.48 g/L.In addition,the dynamic equation of G0119 was built to obtain the equation and parameter of mycelial growth,intracellular polysaccharide generated and the glucose consumption.The establishment of the equation provided a better basis for the optimization of subsequent research for Ganoderma lucidum liquid submerged fermentation.2.Strategy of multi-stage aeration rate for intracellular polysaccharide productionThe effect of aeration rate on the mycelial growth of Ganoderma lucidum G0119,specific mycelial growth rate,polysaccharide production,specific polysaccharide production rate,and p H,dissolved oxygen changes were investigated in seven parallel6 L batch fermenters.The result revealed that the aeration rate had significant impacts on the growth of G.lucidum G0119 and production of intracellular polysaccharide.The mycelium biomass was the highest when aeration rate was 9 L/min,reached 15.42g/L.The maximum yield of the intracellular polysaccharide reached 2.10 g/L under6L/min.According to the results,the following control strategies were studied:the early stage of the fermentation for 0 to 31.2 hours aeration rate for 6 L/min,31.2 to 43hours aeration rate for 9 L/min,43 to 55 hours aeration rate for 6 L/min,after 55 hours aeration rate for 5 L/min.The mycelium dry weight reached 16.35 g/L,6.03%higher than 9 L/min aeration rate.The yield of the intracellular polysaccharide reached 2.24g/L,6.67%higher than the aeration rate of 6 L/min.3.Process optimization for intracellular polysaccharide of Ganoderma lucidum mycelium in liquid submerged fermentationWith high yield intracellular polysaccharide of Ganoderma lucidum mycelium as the goal,first of all,the effect of carbon source,nitrogen source and phosphate on mycelium biomass and intracellular polysaccharide production were explored in single factor experiments,determination of optimal medium were glucose,yeast powder 01and KH₂PO₄.Secondly,the optimum levels of glucose,yeast powder 01,KH₂PO₄,p H and inoculation amount that were determined by Single factor experiments and Box-Benhnken design combined with response surface analysis were 40g/L,12 g/L,3g/L,5.5 and 10 m L/100m L.Under the optimal condition,the intracellular polysaccharide predictive production in optimized medium reached 2.03 g/L,experiment result was 1.98 g/L,the error is 2.46%,and it was with in the permissible range.To do further training in the 6 L fermentation tank,mycelium biomass was25.31 g/L,intracellular polysaccharide yield was 2.59 g/L,which was 27.59%higher than the predicted value.Combined with the optimization results in 6 L batch fermenters,the results in 50 L batch fermenters were consistent to 6 L batch fermenters,the mycelium biomass maximum value was 25.20 g/L,intracellular polysaccharide yield was 2.65 g/L.It showed that the optimization process could effectively improve the yield of Ganoderma lucidum mycelium,and the process was feasible.4.The influence of intracellular and extracellular polysaccharides from liquid submerged fermentation of Ganoderma lucidum on the macrophages-activation activityThe high performance size exclusion chromatography equipped with multiple angle laser light scattering detector,viscostar detector and refractive index detector(HPSEC-MALLS-VS-RI)was adopted to determine the molecular weight and characteristics of intracellular polysaccharide and extracellular polysaccharide from liquid submerged fermentation.Analysis results indicated that intracellular polysaccharide component 1 Mw.High molecular weight was 4.695×106 D,the mass fraction was 58%,the extracellular polysaccharide component 2 of Mw.High molecular weight was 5.554×104 D,the mass fraction was 94.9%.Activity test results showed that two samples exhibited activities on stimulating RAW 264.7 macrophages to release NO.And present a certain concentration dependence.