| Straw mushroom(Volvariella volvacea Bull.Sing)is an edible fungus originated from China and grown at high temperature and high humidity areas.It has a tender texture with delicious taste,rich nutrition,including various functional ingredients such as polysaccharides,proteins and triterpenoids.To develop the functional products and improve the added values of V.volvacea,it is essential to systematically investigate the high-efficiency extraction technology,fine structure characterization,activity in vitro and in vivo and possible bioactive mechanism of polysaccharides extracted from V.volvacea.Hence,based on the current researches,the presnt study would aim to: 1)develop and optimize the ultrasound-assisted process parameters for maximum yield of polysaccharides from the fruiting bodies of V.volvacea at egg stage,2)compare the primary structural properties of polysaccharides extracted by conventional and ultrasound-assistant methods,3)investigate the purification and fine structural characterization of a novel α-glucan from V.volvacea fruiting bodies,and 4)finally elucidate the immunomodulatory activity and potential mechanism of the α-glucan on macrophage RAW264.7 cells.The main obtained results are as follows:(1)The ultrasound-assisted process parameters were developed and optimized for maximum yield of polysaccharides from the fruiting bodies of V.volvacea at egg stage.One-factor-at-a-time experiment results showed that ultrasound power,extraction temperature,ultrasound time and liquid to material ratio showed the significant effect on the crude polysaccharide yield.And ultrasound power 90-210 W,extraction temperature 40-60 °C,ultrasound time 20-40 min,liquid to material ratio 10:1-30:1 were selected for further optimization by using response surface methodology.A maximum polysaccharide yield of 8.28±0.23% was obtained under the optimized conditions of ultrasound power of 175 W,extraction temperature of 57 °C,ultrasound time of 33 min,and the ratio of liquid to raw material of 25:1,respectively.Compared with the yield of hot water extraction(7.56±0.28%),the ultrasound-assistance favored the polysaccharides extraction from V.volvacea for its higher polysaccharide yield and efficiency.(2)The primary structural properties of polysaccharides from ultrasound-assisted extraction and hot-water extraction were compared.Results indicated that the content of crude polysaccharide obtained by ultrasound-assisted extraction(UAE-VVP)was 65.82%,which was higher than that of hot-water extraction(HWE-VVP)about 60.78%.Two polysaccharides contained glucose,mannose and galactose,but the glucose content of UAE-VVP was 75.63%,higher than that of HWE-VVP about 69.12%.UAE-VVP contained three components of 255.42 kDa,2.69 kDa and 0.1167 kDa,accounting for 24.55%,46.87% and 28.58%,respectively,while HWE-VVP contained two components of 357.56 kDa and 2.47 kDa,accounting for 92.08% and 7.92%,respectively.No absorption peaks appeared at 260 nm and 280 nm in UV spectra,proving extremely sacnty content of protein and nucleic acid.In addition,FT-IR spectra of UAE-VVP and HWE-VVP were similar with the typical characteristic polysaccharide peaks.(3)Purification and fine structural characterization of a novel α-glucan UAE-VVPI-a from V.volvacea fruiting bodies were investigated.The UAE-VVP polysaccharides were purified by DEAE-Sepharose and Sephadex G-100 column chromatography,wherein the UAE-VVPI component accounted for 85.74%.The contents of polysaccharide and protein in four components UAE-VVPI-a,UAE-VVPI-b,UAE-VVPII and UAE-VVPIII were 98.2%,82.5%,8.7% and 9.2%,0.8%,5.1%,73.2%,and 78.9%,respectively.UAE-VVPI-a significantly stimulated the release of NO(18.29 μM).The molecular weight of UAE-VVPI-a was about 143.56 kDa;UV spectra showed no absorption peak at 260 nm and 280 nm,indicating no protein and nucleic acid existed.By GC analysis,monosaccharide composition of UAE-VVPI-a was only glucose;FT-IR spectra showed that UAE-VVPI-a was a kind of polysaccharide with α-pyranoside bond;Periodate oxidation,Smith degradation analysis indicated the existance of 1→,1→6,1→4 or 1→4.6;Methylation and NMR analysis confirmed the backbone chain structure of →4)-α-Glcp-(1→,branched of →4,6)-α-Glcp-(1→ and branched at O-6,with the ratio of 1:8:1.The 3D-structure of UAE-VVPI-a was observed by Congo-red experiment,AFM and SEM scan analysis.Results showed that UAE-VVPI-a contained a triple-helix structure,and the multiple molecular chains were intertwined and aggregated,showing a small spiral rod structure.(4)The immunomodulatory activity and potential mechanism of UAE-VVPI-a on macrophage RAW264.7 cells were evaluated and elucidated.Results showed that UAE-VVPI-a concentration ranged from 31.25 to 500 μg/mL showed no significant effect on the proliferation.UAE-VVPI-a could significantly promote the ability of neutral red phagocytosis.Moreover,the secretion of NO(7.76-32.52 μM),IL-6(48.43-150.27 pg/mL),IL-1β(20.26-46.19 pg/mL)and TNF-α(348.32-820.32 pg/mL)were induced with a dose-dependent manner.Confirmed by qRT-PCR,mRNA levels of iNOS,IL-6,IL-1β and TNF-α were all increased,respectively.In addition,Western blot analysis indicated that UAE-VVPI-a might stimulate the expression of p38,JNK and ERK through MAPK signaling pathways. |
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