Abstract: Abstract: Tricholoma matsutake (TMWEIF) Singer is one of the most valuable mushrooms, particularly famous for its delicacy in the world. An excellent source of nutrients can be rich in proteins, unsaturated fatty acids, vitamins, dietary fibers, but low in fat. TMWEIF also shares the outstanding properties of anti-tumor, lowering cholesterol, antioxidant, and strengthening the immune system. High-temperature water extraction has been usually applied to extract the immunity active compounds at present. However, these immunity active compounds indeed are prone to be destroyed by high temperatures. In this study, the low-temperature (≤30 °C) water extraction of immunity active factors was performed on the TMWEIF from Changbai Mountain, China, using the response surface method (RSM) under the Box-Behnken center combination design by the Design Expert 10.0 software. The structure and amino acid compositions of the TMWEIF were further characterized by an automatic amino acid analyzer, Fourier transform infrared spectroscopy (FT-IR), circular dichroism spectrum (CD), and ultraviolet spectrum (UV). A single factor experiment were conducted at the liquid-solid ratios (10:1, 20:1, 30:1, 40:1, and 50:1 mL/g), extraction time (30, 60, 90, 120, and 150 min), and extraction temperature (10, 15, 20, 25, and 30 °C). An optimum extract condition was determined for the TMWEIF by RSM, where the NO emission by RAW264.7 macrophages, and the protein yield of extract were taken as the indicators. The results showed that the optimal extraction parameters for the protein yield were the liquid-solid ratio of 45:1 mL/g, extraction time of 140 min, and extraction temperature of 20 °C, where the extract was named TMWEIF-1. Moreover, the optimal extraction parameters for the NO release amount were the liquid-solid ratio of 30:1 mL/g, extraction time of 120 min, and extraction temperature of 20 °C, where the extract was named TMWEIF-2. The extraction parameters for the TMWEIF-2 presented a lower liquid-solid ratio and shorter extraction time than before, indicating an improved production efficiency. The protein yield and NO release amount were 8.07%, 3.68 μg/mL, and 7.06%, 4.11 μg/mL, respectively, for the TMWEIF-1 and TMWEIF-2 under the optimal conditions. There were significant differences (P<0.05) between the TMWEIF-1 and TMWEIF-2 in the protein yield and NO release amount. The measurements were well matched with the predicated values from the models, indicating that the regression equations and models were reliable to predict the influences of factors on the protein yield and NO release amount of the extract. The FT-IR analysis showed that the TMWEIF-1 and TMWEIF-2 exhibited the typical absorption peaks of protein and polysaccharide within the wave number of 4000-500 cm-1. The FT-IR and UV analysis showed that the structures of TMWEIF-1 and TMWEIF-2 were similar, and the main components were protein and polysaccharide. The CD analysis revealed that the secondary structures of protein in the TMWEIF-1 and TMWEIF-2 were mainly α-helix and β-turn, where the higher content of α-helix was observed in the TMWEIF-1, and a larger amount of β-turn in the TMWEIF-2. The polysaccharide contents in the TMWEIF-1 and TMWEIF-2 were 306.95 and 304.98 mg/g, respectively, and the protein contents were 132.14 and 107.78 mg/g, respectively. The polysaccharide was also mainly glucan with α-glucoside, according to the content determination. The amino acids analysis showed that there was no significant difference (P>0.05) in the contents of amino acids in the TMWEIF-1 and TMWEIF-2, where the essential amino acids accounted for 27 %. Both TMWEIF-1 and TMWEIF-2 contained 17 kinds of amino acids rich in glutamate (Glu) and aspartic acid (Asp), but the relative content of Glu of TMWEIF-2 was slightly higher. Besides, there was a higher mean percentage of hydrophobic amino acids and basic amino acids related to the immune activity in the TMWEIF-2 than that in the TMWEIF-1. Consistently, the NO release induced by the TMWEIF-2 was significantly higher than that by the TMWEIF-1. Consequently, the TMWEIF-2 and the extraction parameters are expected to serve as the better industrial application prospect, in terms of extraction efficiency and immune activity. The finding can provide a scientific basis for the further development and utilization of immunity active factors in the wild matsutake functional foods.