巴西革耳Lentinus striguellus发酵特性及菌丝体多糖的研究
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摘要
本文确立了巴西革耳(Lentinus striguellus)发酵最适培养基组成及适宜的摇瓶发酵条件，并对发酵获得的菌丝体营养成分进行分析。应用色谱法等技术从巴西革耳菌丝体热水提取粗多糖中分离纯化了两个主要的组分均一的杂多糖LS-Ⅰ、LS-Ⅱ-b。用TLC、GC、UV、IR、高碘酸氧化与Smith降解、~1H-NMR、~(13)C-NMR等技术基本阐明了其理化性质及主要结构特征。并采用Wolfrom方法对LS-Ⅰ进行分子修饰，获得硫酸化多糖S-LS-Ⅰ，同时应用UV、IR、Sephadex G-200凝胶柱层析、~(13)C-NMR等技术研究LS-Ⅰ硫酸化后结构上的变化。对LS-Ⅰ进行急性毒性试验，研究结果表明，实验小鼠在大剂量状况下未出现死亡，也未有明显毒性反应出现的一些指标变化。初步探讨了LS-Ⅰ、LS-Ⅱ-b、S-LS-Ⅰ三种多糖对经免疫抑制剂CY处置小鼠的免疫功能影响，结果提示，三种多糖均具有增强小鼠免疫功能的活性。
The optimal conditions for submerged fermentation culture of Lentinus striguellus and the nutrition components in the mycelia were explored. The extraction, purification and physicochemical characterization of polysaccharides in mycelia ofLentinus striguellus were also studied. The molecular modification and immunity functions of the polysaccharides also been examined.
The research established the suitable fermentative conditions which affect the mycelia growth of Lentinus striguellus. High level of crude proteins, total carbohydrates, reduced sugar, vitamins and minerals in the mycelia has been tested. The total amino acid is as high as 12.21% involving 4.81% essential amino acid.
The crude polysaccharides were extracted from mycelia with hot water. Two main fractions LS- I - LS- II -b were obtained after eluting from DEAE-cellulose and Sephadex G-100 columns. Sephadex G-200 chromatography and cellulose acetate film electrophoresis showed that the two fractions were homogeneous. Each of them contained about 92.96%^ 78.15% of total sugar. UV and infrared spectroscopy determination revealed typical absorption of polysaccharide and a-pyranglycoside linkage. The results indicated that both of them are heteropolysaccharide. LS-1 is protein free while LS-II-b contains 16.5% protein.
The purified preparations were characterized by means of Sephadex G-200, optical rotation, TLC, GC, periodate oxidation, Smith degradation, 'H-NMR, 13C-NMR etc. The results are showed as follows:
LS-I : [α]20=+63.330(H2O); Mw=1.15xl04Da; sugar constituents (in a molar ratio): Ara:Fuc:Xyl:Fru:Man:Glc=0.04:0.02:0.06:2.78:0.67:1.79; main chain: α-(l→6) linkage; side chains: α-(l→4) or α-(l→2) linkage.
LS-II-b: [α]20=+73.670(H2O); Mw=3.88 x 103 Da; sugar constituents(in a molar
ratio): Fru:Man:Glc(2.99:0.83:5.51); main chain: α-(l→3)-linked Glc and α-(l→3)-linked Fru residues; side chains: α-(l→6), (l→4) or/and (l→3)-linked Glc, Fru and Man residues.
LS- I was chemically modified by chlorosulfonic acid-pyridine method. A modified compound (S-LS- I ) with 2.01 substituted degree was obtained. The molecular weight of S-LS-1 was estimated to be 7.03xl03Da by Sephadex G-200 column chromatography. The UV and IR spectrum indicated that S-LS-1 has typical absorptions of polysaccharide, -S-O- and -SO3. High reactivity of hydroxyls on C-6, C-2 and C-4 position of 13C-NMR indicated that the sulfates were located on the carbon-6, carbon-2 and carbon-4.
The acute toxicity of LS-I was studied. Mice of Kun Ming strain were given LS- I through celiac injection at 125mg/kg dosage which is 30-125 times of the clinical dosage of the same kind of medicines. The result indicated that there were no any significant toxic effects to the tested animals.
The immunity functions of LS-1, LS-II -b and S-LS-1 were investigated on the immunosuppressive mice by injecting cyclophosphamide(CY). The results showed that three polysaccharides could significantly increased the index of spleen weight and leucocyte counts of peripheral blood, improved the phagocytic function of mononuclear-macrophage, and enhanced serum lysozyme activity of the immunosuppressive mice, which suggested their immunity functions.
The optimal conditions for submerged fermentation culture of Lentinus striguellus and the nutrition components in the mycelia were explored. The extraction, purification and physicochemical characterization of polysaccharides in mycelia ofLentinus striguellus were also studied. The molecular modification and immunity functions of the polysaccharides also been examined.
The research established the suitable fermentative conditions which affect the mycelia growth of Lentinus striguellus. High level of crude proteins, total carbohydrates, reduced sugar, vitamins and minerals in the mycelia has been tested. The total amino acid is as high as 12.21% involving 4.81% essential amino acid.
The crude polysaccharides were extracted from mycelia with hot water. Two main fractions LS- I - LS- II -b were obtained after eluting from DEAE-cellulose and Sephadex G-100 columns. Sephadex G-200 chromatography and cellulose acetate film electrophoresis showed that the two fractions were homogeneous. Each of them contained about 92.96%^ 78.15% of total sugar. UV and infrared spectroscopy determination revealed typical absorption of polysaccharide and a-pyranglycoside linkage. The results indicated that both of them are heteropolysaccharide. LS-1 is protein free while LS-II-b contains 16.5% protein.
The purified preparations were characterized by means of Sephadex G-200, optical rotation, TLC, GC, periodate oxidation, Smith degradation, 'H-NMR, 13C-NMR etc. The results are showed as follows:
LS-I : [α]20=+63.330(H2O); Mw=1.15xl04Da; sugar constituents (in a molar ratio): Ara:Fuc:Xyl:Fru:Man:Glc=0.04:0.02:0.06:2.78:0.67:1.79; main chain: α-(l→6) linkage; side chains: α-(l→4) or α-(l→2) linkage.
LS-II-b: [α]20=+73.670(H2O); Mw=3.88 x 103 Da; sugar constituents(in a molar
ratio): Fru:Man:Glc(2.99:0.83:5.51); main chain: α-(l→3)-linked Glc and α-(l→3)-linked Fru residues; side chains: α-(l→6), (l→4) or/and (l→3)-linked Glc, Fru and Man residues.
LS- I was chemically modified by chlorosulfonic acid-pyridine method. A modified compound (S-LS- I ) with 2.01 substituted degree was obtained. The molecular weight of S-LS-1 was estimated to be 7.03xl03Da by Sephadex G-200 column chromatography. The UV and IR spectrum indicated that S-LS-1 has typical absorptions of polysaccharide, -S-O- and -SO3. High reactivity of hydroxyls on C-6, C-2 and C-4 position of 13C-NMR indicated that the sulfates were located on the carbon-6, carbon-2 and carbon-4.
The acute toxicity of LS-I was studied. Mice of Kun Ming strain were given LS- I through celiac injection at 125mg/kg dosage which is 30-125 times of the clinical dosage of the same kind of medicines. The result indicated that there were no any significant toxic effects to the tested animals.
The immunity functions of LS-1, LS-II -b and S-LS-1 were investigated on the immunosuppressive mice by injecting cyclophosphamide(CY). The results showed that three polysaccharides could significantly increased the index of spleen weight and leucocyte counts of peripheral blood, improved the phagocytic function of mononuclear-macrophage, and enhanced serum lysozyme activity of the immunosuppressive mice, which suggested their immunity functions.
引文
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