| Auricularia auricula is a homologous fungus of medicine and food,which has unique nutritional value and health care,and is one of the four edible fungi in the world.As the second largest variety of fungi in China,the ann UVl output of Auricularia auricula has increased year by year.Polysaccharide is an important active component in Auricularia auricula,which has many physiological activities such as anti-tumor,anti-oxidation,antibacterial,anti-inflammatory and immunomodulatory,and has great market development value and application prospect.The yield of Auricularia auricula polysaccharide plays an important role in the development of its related products,and the breeding of high-yielding polysaccharide strains is an important way to increase the yield of Auricularia auricula polysaccharide.In this study,Atmospheric and Room Temperature Plasma(ARTP)mutagenesis technology was used to mutate the protoplasts of the original strains(Heiwei 15 and Heiwei danpian),and a new strain of Auricularia auricula with high polysaccharide yield was selected.The physical and chemical properties,in vitro antioxidant and in vitro immune activity of crude polysaccharides in mycelia were compared between the high-yielding strains and the original strains.The main results are as follows:(1)Using Auricularia auricula protoplast yield as eval UVtion index,optimization experiments were carried out by single factor and response surface method.The results showed that the optimum conditions for protoplast preparation were enzymatic time of3.7 h,enzymatic temperature of 32 ℃,enzyme concentration of 1.6 % and age of 7 d,and the yield of protoplasts could reach 22.13×107 CFU/m L.(2)Using Heiwei 15 and Heiwei danpian as starting strains,single or iterative mutagenesis of ARTP was used to screen the superior strains with high yield of polysaccharides.672 and 243 mutagenesis strains were obtained through screening indexes such as plate growth rate,antagonism test and polysaccharide yield,and further screening was conducted to obtain the target strains with high yield of polysaccharides.APTP strains A13,A276,A421,D-56 and D-60 yielded 66.89%,61.59%,40.39%,70.69% and 81.97% more polysaccharides,respectively.(3)The infrared spectrum,monosaccharide composition and molecular weight of crude polysaccharides in mycelia of the original strain Heiwei danpian,Heiwei 15 and mutagenic strains D-56,D-60 and A13,A276 and A421 were determined,respectively.It was found that the monosaccharide composition and molecular weight of crude polysaccharides in mycelia of the mutagenic strains were different from those of the original strain.(4)To compare the antioxidant activity of crude polysaccharides from Auricularia auricula mycelium before and after mutagenesis.Heiwei 15,A13,A276 and A421 mycelium crude polysaccharides: The IC50 values of DPPH· scavenging were 0.747,0.621,0.535 and 1.726 mg/m L,and the IC50 values of O2·-scavenging were 5.816,5.867,5.669 and 5.080 mg/m L,respectively.The IC50 values of OH· scavenging were0.090,0.118,0.113 and 0.101 mg/m L,respectively.Crude polysaccharides from Heiwei danpian,D-56 and D-60 mycelium: The IC50 values of DPPH· scavenging were 0.621,0.586 and 0.519 mg/m L,respectively;the IC50 values of O2·-scavenging were 5.607,5.421 and 5.807 mg/m L,respectively;the IC50 values of OH·scavenging were 0.131,0.129 and 0.129 mg/m L,respectively.The scavenging ability of crude polysaccharide from mycelium of mutagens A13,A276,A421,D-56 and D-60 was close to that of positive control Vc,and the crude polysaccharide from mycelium had better OH·scavenging activity.(5)To compare the difference of immune activity of crude polysaccharide in Auricularia auricula mycelium before and after mutagenesis.The results showed that compared with the negative control group,crude polysaccharide from mycelium of the original strain and mutagenic strain could stimulate the secretion of TNF-α,IL-6 and IL-1β of macrophage RAW264.7 at the concentration of 50,200 and 500 μg/m L(P<0.05),and the effect was close to or even exceeded the positive control LPS.Heiwei15 series strain: Compared with the original strain,the crude polysaccharide of A13 and A276 mycetes better promoted IL-6 secretion of RAW264.7 when the crude polysaccharide concentration was 500 μg/m L(P<0.05).A276 could promote IL-1βsecretion of RAW264.7 at the concentrations of 200 and 500 μg/m L(P<0.05).The crude polysaccharide of D-56 and D-60 mycelium had a good effect on the secretion of TNF-α and IL-6 of RAW264.7 when the concentration was 500 μg/m L compared with the original strain(P<0.05).At the concentration of 200 and 500 μg/m L,D-56 and D-60 had better IL-1β secretion(P<0.05).The results showed that the crude polysaccharides of A276,D-56 and D-60 mycelium had good immune activity in vitro. |
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