Liquid Fermentation Of Irpex Lacteus Fr. And Antitumor Activity Of Proteoglycan

Medicinal fungi is a kind of medication which has treatment,prevention or inhibition to disease and no toxicity to human and health maintenance.Fungal polysaccharide is one of the main active ingredient in medicinal fungi,and has obvious effects on strengthening the body immunity,anti-tumor,anti-oxidation and anti-virus activity.The reseach of fungal polysaccharides has become one of the development of new drugs.Because of the lack of wildlife resources and the difficulties of artificial cultivation,the submerged fermentation technology to produce medicinal fungi will have a widest potential application.Some of medicinal fungi have been achieven the industrialization of culture in submerged fermentation.Irpex lacteus Fr.belongs to the non-Aphyllophorales polyporaceae Irpex Fr.,which is a wood decay fungi.It is born in the dead wood.Irpex lacteus Fr.is the raw materials of "Shenyan Kang",which is an immunosuppressant to chronic glomerulonephritis caused by immune dysfunction.However,research on Irpex lacteus Fr.,both at home and abroad is rarely carried out.For its in-depth development and application,this paper systematically study the liquid fermentation conditions,polysaccharide extraction process,as well as its anti-tumor activity in vitro.The sort of carbon and nitrogen source and metal ions were optimized by single factor tests,which were lactose,yeast extract powder,potassium nitrate,Na⁺ and Ca²⁺. The effect on the dry weight of medium was investigated with the Plackett-Burman design.Three most important factors,lactose,yeast extract powder,magnesium sulfate,for dry weight of medium were selected from total of 8 factors.Then the concentration of lactose, yeast extractive,magnesium sulfate in the medium was investigated by means of the path of steepest ascent tests and Response Surface Methodology(RSM) based on a three-level three-factor central composite design.The results shown that the most significant factor is lactose,followed yeast extract powder.Optimized values of the factors were determined to be lactose 29.34 g/L,yeast extractive 17.13 g/L,magnesium sulfate 0.64 g/L,phosphate potassium 0.30 g·L,sodium chloride 5.85×10^-4 g/L, calcium chloride 9.10×10^-4 g/L,potassium nitrate 0.50 g/L,vitamin B₁ 0.10 g/L and a maximum dry weight of mycelium of 9.67 g/L was obtained.The shake-flask culture conditions were investigated by means of single factor tests.It is initial pH 5.0,age of inoculum 5 d,inoculum level 7%,liquid volume 60 mL/250 mL,shaking speed 150 r/min,culture temperature 26℃,fermentation time 5 days,the dry weight of mycelium of 14.31 g/L increased about 47%,mycelium polysaccharides production of 1.89 g/L.One-factor tests and Response Surface Methodology(RSM) were applied to optimize the hot water extraction and ultrasound extraction conditions of Irpex lacteus Fr.mycelium polysaccharides.Experiment factors and levels were first selected by one-factor tests.On the basis of this,extracting temperature,extracting time the ratio of water to sample,were chosen as the factors of hot water extraction in RSM and each factor in the design was studied at three different levels.The optimum hot water extraction conditions were confirmed as follows:extracting temperature 94.7℃, extracting time 3.3 h,the ratio of water to sample 62:1(mL:g).Extracting temperature has the most important effect on polysaccharides production followed by the ratio of water to sample.The extraction rate of polysaccharides under the optimum hot water extraction conditions could reach the highest value of 18.66%with two-times extraction.Ultrasound extracting time,ultrasound power and the ratio of water to sample were chosen as the factors of ultrasound extraction in RSM and each factor in the design was studied at three different levels.The optimum ultrasound extraction conditions were confirmed as follows:ultrasound extracting time 184 s,ultrasound power 410 W,the ratio of water to sample 48:1(mL:g),sequence of each factor:the ratio of water to sample＞ultrasound extracting time＞ultrasound power.The extraction rate of polysaccharides under the optimum ultrasound extraction conditions could reach the highest value of 19.16%with two-times extraction.Ultrasound extraction could greatly shorten the extraction time and reduce the ratio of water to sample,but the extraction rate of polysaccharides was slightly higher than hot water extraction.MTT assay was used to study the effect of Irpex lacteus Fr.exo and inner polysaccharides on various human tumor cells proliferation in vitro.The results showed that exo-polysaccharides couldn't inhibit the proliferation of tumor cells in vitro or inhibitory activity didn't have concentration-dependent.Inner polysaccharides significantly inhibited the proliferation of Hela,ZR-75-30 and MCF-7 in vitro. Inhibitory activity of the hot water extracted and ultrasound extracted inner polysaccharides in different concentrations and different time was studied by MTT assay.Hot water extracted polysaccharides anti-Hela,ZR-75-30,as well as the proliferation of MCF-7 in vitro the smallest IC₅₀ were 403.88μg/mL,121.00μg/mL and 439.21μg/mL,and ultrasound extracted polysaccharides the smallest IC₅₀ were 225.08μg/mL,163.95μg/mL and 326.36μg/mL.The results showed that the inhibitory activity of ultrasound extracted polysaccharides on Hela and MCF-7 in vitro was stronger than hot water extracted polysaccharides,while on ZR-75-30, slightly less than hot water extracted polysaccharides.