Optimization Of Lentinus Edodes Liquid Medium Under Se-enriched Condition And Extraction Process Of Se-polysaccharide

Selenium is considered one of the essential trace elements, and is prevention and treatment of tumors, cancer and other diseases. Selenium content in the diet by the local environmental factors is very obvious. Most of the crowd in china is in a state of selenium.Lentinus edodes play an important role in people’s diet. L.edodes can transform inorganic selenium into organic selenium, so as to promote their uptake and utilization of the human body. Se-enriched liquid spawn of L.edodes can be obtained from se-enriched liquid fermentation of L.edodes, and can promote Se-enriched L.edodes cultivation industry standardization, scale and mechanization. The fermentation broth, mycelium and fructification of Se-enriched L.edodes can be used to produce Se-polysaccharide, Se-proteins and related drugs, health supplies and so on. Currently, there is few report at Se-enriched liquid fermentation process of L.edodes and no document at Se-polysaccharide extraction process from Se-enriched liquid spawn of L.edodes. Therefore, the exploration of Se-enriched liquid fermentation of L.edodes and extraction process of Se-polysaccharide has high research value.The experimental material this study used was the large-scale cultivation of L.edodes in Qin-Ba mountain areas, medium temperature type L.edodes strains（‘808#’）, low temperature type L.edodes strains（‘9608#’） and high temperature type L.edodes strains（‘Q12’）. L.edodes strains was Se-domesticated by plate culture method. The experiments of initial experimental design of Plackett-Burman, path of steepest ascent, Box-Behnken design and response surface analysis were adopted to bring Lentinus edodes liquid medium under Se-enriched condition. The experiments of single factor, Box-Behnken design and response surface analysis were adopted to bring the process parameters of Se-polysaccharide extraction. The main research methods and results are as follows:1.Domestication of Se-enriched L.edodes strains: The best Na2 Se O3 concentration of L.edodes strains（‘808#’, ‘9608#’ and ‘Q12’） were selected to provide theoretical basis for theliquid seed production of Se-enriched L.edodes strains. Using tissue culture method, L.edodes strains（‘808#’, ‘9608#’ and ‘Q12’） were Se-domesticated by plate culture method in Na2 Se O3concentration of 0.00¹10.00 mg/L. Based on mycelial growth, low concentrations of Na2 Se O3 promoted the growth of mushroom mycelium and high concentrations of Na2 Se O3had strong inhibition to the growth of mushroom mycelium. The best Na2 Se O3 concentration of L.edodes strains（‘808#’, ‘9608#’ and ‘Q12’） were 30.00 mg/L, 20.00 mg/L and 30.00 mg/L.2.L.edodes（‘808#’） liquid medium under Se-enriched condition. Adopting mycelial biomass of L.edodes as major index, the important factors influencing mycelial biomass which identified by initial experimental design of Plackett-Burman were soybean flour,KH₂PO₄ and Mg SO₄·7H₂O. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and obtain optimal values that bring the optimal liquid medium: soybean flour 55.00 g, corn meal20.00 g, glucose 6.00 g, peptone 3.00 g, yeast extract 2.00 g, KH₂PO₄ 2.39 g, Mg SO₄·7H₂O1.00 g, Na2 Se O3 30.00 mg/L. And under the condition of initial p H natural, temperature 28 ℃,agitation speed of rotary shaker 160 r/min, culture time 20 days, the mycelial biomass of Se-enriched L.edodes（‘808#’） was 69.26 g/L.3.L.edodes（‘9608#’） liquid medium under Se-enriched condition. Adopting mycelial biomass of L.edodes as major index, the important factors influencing mycelial biomass which identified by initial experimental design of Plackett-Burman were yeast extract,potatoes, wheat bran and KH₂PO₄. The path of steepest ascent was undertaken to approach the optimal region of the four significant factors. Box-Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and obtain optimal values that bring the optimal liquid medium: potatoes 288.78 g, wheat bran64.56 g, maltose 15.00 g, yeast extract 10.00 g, KH₂PO₄ 0.80 g, Mg SO₄·7H₂O 2.00 g,Na2 Se O3 20.00 mg/L. And under the condition of initial p H natural, temperature 28 ℃,agitation speed of rotary shaker 160 r/min, culture time 20 days, the mycelial biomass of Se-enriched L.edodes（‘9608#’） was 66.34 g/L.4.L.edodes（‘Q12’） liquid medium under Se-enriched condition. Adopting mycelial biomass of L.edodes as major index, the important factors influencing mycelial biomasswhich identified by initial experimental design of Plackett-Burman were brown sugar, corn meal and KH₂PO₄. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were adopted to further investigate the optimal liquid medium: corn meal 39.74 g, brown sugar15.70 g, wheat bran 15.00 g, powdered beef 5.00 g, KH₂PO₄ 0.80 g, Mg SO₄·7H₂O 1.00 g,Na2 Se O3 30.00 mg/L. And under the condition of initial p H natural, temperature 28 ℃,agitation speed of rotary shaker 160 r/min, culture time 20 days, the mycelial biomass of Se-enriched L.edodes（‘Q12’） was 57.56 g/L.5. The extraction process of Se-polysaccharide in Se-enriched L.edodes fermentation broth. Adopting mycelial biomass and other factors of Se-enriched L.edodes as major index,L.edodes（‘9608#’） was determined as experimental strains to research the extraction process of Se-polysaccharide. Based on single factor experiments, Box-Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and obtain optimal values that bring the extraction process parameters of Se-polysaccharide. The optimal extraction conditions were achieved and listed as follows:ethanol precipitation time 15.45 h, ethanol concentration 82.30%, p H 7.95. Under the optimal conditions, Se-polysaccharide yield was 3.23 g/L, selenium content was 87.59 μg/g Se-polysaccharide.