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The process of extracting seleno-protein from the selenium enriched Lyophyllum decastes mycelia cultured in a 20 L fermentor was optimized, and the effects of selenium enrichment on both types and contents of amino acids in Lyophyllum decastes mycelia were analyzed. Single factor tests and Box-Benhnken central combined response surface test were used to optimize process of extracting seleno-protein from Lyophyllum decastes mycelia. The content of protein was determined by 3,3'-diaminobenzidine spectrophotometry. The types and contents of amino acids in mycelia protein before and after selenium enrichment were compared by means of amino acid analyzer. The results showed that the optimal conditions of extracting seleno-protein from mycelia of Lyophyllum decastes were extraction temperature of 64 °C, extraction time of 60 min, liquid-solid ratio of 200:1 g/mL, and extraction times of 2. The protein extraction rate was 75.13%, and the content of selenium in mycelia was 63.87 μg/g. The amino acid composition were analyzed by means amino acid score (AAS) and chemical score (CS), and the nutritional value of the protein in the selenium-enriched Lyophyllum decastes mycelia was evaluated. The varieties of amino acids in the selenium-enriched Lyophyllum decastes mycelia were abundant and the content of essential amino acids for human body was 17.20 g/100 g, 19.75% higher than that in the non-selenium-ecriched Lyophyllum decastes mycelia. The ratio of EAA/NEAA was 0.51, close to the recommended value proposed by WHO, and the values of both AAS and CS were close to the those in the model protein. In summary, the protein extraction rate could be improved by optimizing the extraction process, and there was selenium in the protein from selenium-enriched mycelia, which promoted the increase of amino acid content. The nutritional value of protein in selenium-enriched mycelia was higher than that in non-selenium-riched mycelia, and selenium-enrichec mycelia had potential edible and application value. [ABSTRACT FROM AUTHOR]