生物酶法制备真菌菌丝体多糖的工艺优化及生物活性研究
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摘要
本文将9种酶提取真姬菇、齿耳菌和乳牛肝菌菌丝体多糖的效果进行了比较;并首次利用均匀设计优化了真姬菇菌丝体多糖的提取条件;对获得的真姬菇、齿耳菌菌丝体多糖进行了抗肿瘤和抗氧化活性的检测;同时比较了真姬菇胞壁多糖和胞内多糖的生物活性。
溶壁酶是提取3种真菌菌丝体多糖产量最高的酶类,其多糖产量分别是14.78±0.22 mg/g干菌丝、16.66±1.60 mg/g干菌丝、27.49±1.89 mg/g干菌丝,比对照提高了164.36%、156.68%、141.19%。在真姬菇和乳牛肝菌菌丝体多糖的提取中,中性蛋白酶A是最经济适合的酶类,其多糖产量分别为8.52±0.27 mg/g干菌丝、20.67±1.86 mg/g干菌丝,比对照提高了56.48%、44.85%;而在齿耳菌菌丝体多糖的提取中,中性蛋白酶A的多糖产量是7.39±0.16 mg/g干菌丝,比对照提高了13.86%,但与对照产量无差异显著性(p>0.05)。
通过单因素实验获得了中性蛋白酶A酶解制备真姬菇菌丝体多糖的适宜酶用量、酶解时间、酶解温度、提取时间、提取温度和提取次数;采用均匀设计方法获得了中性蛋白酶A制备菌丝体多糖的最佳条件为:在2%酶用量下,43.05℃酶解4h,100℃提取1h,提取1次。该优化条件下多糖产量的预测值为16.65 mg/g干菌丝,验证实测值为15.73±0.15 mg/g干菌丝,优化条件下获得的多糖产量比优化前的产量提高75.0%,比热水提取法和微波辅助提取法分别提高122.5%和104.8%,表明酶解法是简便易行的高产制备真姬菇菌丝体多糖的方法。
利用MTT法检测了真姬菇菌丝体多糖的抗肿瘤活性。果胶酶、酸性蛋白酶、纤维素酶A提取的菌丝体多糖对癌细胞(SGC7901)的抑制率显著高于其它酶及对照提取的多糖(p<0.05),其IC50分别是0.343 mg/mL、0.397 mg/mL、0.430 mg/mL。中性蛋白酶A提取的多糖的抗肿瘤活性的IC50是0.984 mg/mL。真姬菇胞壁多糖和胞内多糖的抗肿瘤活性试验表明,发挥抗肿瘤作用的多糖主要位于菌丝体胞内。
不同酶类提取的齿耳菌菌丝体多糖的抗肿瘤活性试验表明,纤维素酶B提取的多糖的活性显著高于其他酶类提取的多糖(p<0.05),其抗肿瘤活性的IC50为0.863 mg/mL。
抗氧化活性试验中,木瓜蛋白酶、果胶酶、中性蛋白酶A和B提取的真姬菇菌丝体多糖的抗氧化活性较高,他们的羟基自由基清除能力的EC50分别是51.98μg/mL、55.76μg/mL、63.56μg/mL、62.49μg/mL;超氧阴离子自由基清除能力的EC50分别是153.77μg/mL、195.61μg/mL、252.68μg/mL、219.85μg/mL。酶类对真姬菇胞壁多糖的抗氧化活性无影响。多糖的抗氧化活性成分主要存在于菌丝体胞内。
木瓜蛋白酶提取的齿耳菌菌丝体多糖的总抗氧化能力,清除羟基自由基能力,超氧阴离子自由基清除能力均比其他酶提取的多糖高(p<0.05),其羟基自由基清除能力、超氧阴离子自由基清除能力的EC50分别是59.42μg/mL、74.78μg/mL。
9 evaluated enzymes were selected to extract polysaccharide from fungal mycelia, and then uniform design was utilized for optimaization of technology for high yield polysaccharides from Hypsizigus marmoreus mycelia. the anti-tumor and antioxidant activity of polysaccharide from fungal mycelia was discussed, and the bio-activity of the polysaccharide intro-mycelia and in the wall of mycelia was also discussed.
It can get the hignest yield polysaccharides from Hypsizigus marmoreus, Suillus sp and Mycoleptodonoides.sp mycelia by lywallzyme, 14.78±0.22 mg/g, 16.66±1.60 mg/g, 27.49±1.89 mg/g, 164.36%, 156.68%, 141.19% higher than control. It was concluded that Neutral protease A was the most cost efficient among 9 evaluated enzymes when extraction of polysaccharide from mycelia of Hypsizigus marmoreus and Suillus sp, 8.52±0.27 mg/g, 20.67±1.86 mg/g, 56.48%, 44.85% higher than control.
The single factor and uniform design experiment was conducted to optimize the conditions, and a regression model were constructed to predict polysaccharide yield from Hypsizigus marmoreus mycelia. The highest polysaccharide yield was obtained when 2% (w/w) of neutral protease was added into the hydrolytic reaction and incubated for 4 h at 43oC, and then extracted one time at 100 oC for 1 h. Using the regression model, the predicted polysaccharide yield was 16.65 (mg/g DMW), which was very close to the measured value of 15.73±0.15 (mg/g DMW). Thus the prediction equation was validated to accurately estimate polysaccharide yield. When following the optimized procedures, polysaccharide yield was raised 75.0% from that prior to optimization. Compared to hot-water extraction and microwave assisted extraction methods, enzymatic extraction method increased polysaccharide yield by 122.5% and 104.8%, respectively. It was concluded that enzymatic extraction is a simple, high yield method to prepare polysaccharide from Hypsizigus marmoreus mycelia.
The anti-tumor activity of polysaccharides from Hypsizigus marmoreus mycelia by different enzymatic hydrolysis.has been observed. The test indicated that the anti-tumor activity of polysaccharides extracted by different enzymatic is different. The anti-tumor activity of polysaccharides from Hypsizigus marmoreu mycelia by pectinase, cellulase and acid protease is higher than by other enzymes. The polysaccharides from mycelia by pectinase, cellulose, acid protease and neutral protease 1 can obviously kill the tumor cell of SGC7901, 0.343 mg/mL, 0.397 mg/mL, 0.430 mg/mL, and 0.984 mg/mL respectively in 50% death rate. The anti-tumor activity of polysaccharides in mycelia is higher than polysaccharides from the wall of mycelia.
The anti-tumor activity of polysaccharides from Mycoleptodonoides sp mycelia by cellulose B is higher than by other enzymes, it can obviously kill the tumor cell of SGC 7901 in 50% death rate with the dosage of 0.863 mg/mL. The antioxidant activity of polysaccharides from mycelia by different enzymatic hydrolysis.is also different. The antioxidant activity of polysaccharides from mycelia by pectinase, papain, neutral protease A and neutral protease B is higher than by other enzymes. Polysaccharides extracted by papain pectinase, neutral protease A and neutral protease B can obviously scavenge hydroxyl free radical, 51.98μg/mL,55.76μg/mL , 62.49μg/mL , 63.56μg/mL respectively in 50% scavenging rate. Polysaccharides extracted by them can obviously scavenge superoxide anion free radical, 153.77μg/mL,195.61μg/mL,219.85μg/mL,252.68μg/mL respectively in 50% scavenging rate.
The antioxidant activity of polysaccharides in mycelia is higher than polysaccharides from the wall of mycelia.
The antioxidant activity of polysaccharides from Mycoleptodonoides sp mycelia by papain is higher than by other enzymes, it can obviously scavenge hydroxyl free radical and superoxide anion free radical, with the dosage of 59.42μg/mL, 74.78μg/mL respectively in 50% scavenging rate.
溶壁酶是提取3种真菌菌丝体多糖产量最高的酶类,其多糖产量分别是14.78±0.22 mg/g干菌丝、16.66±1.60 mg/g干菌丝、27.49±1.89 mg/g干菌丝,比对照提高了164.36%、156.68%、141.19%。在真姬菇和乳牛肝菌菌丝体多糖的提取中,中性蛋白酶A是最经济适合的酶类,其多糖产量分别为8.52±0.27 mg/g干菌丝、20.67±1.86 mg/g干菌丝,比对照提高了56.48%、44.85%;而在齿耳菌菌丝体多糖的提取中,中性蛋白酶A的多糖产量是7.39±0.16 mg/g干菌丝,比对照提高了13.86%,但与对照产量无差异显著性(p>0.05)。
通过单因素实验获得了中性蛋白酶A酶解制备真姬菇菌丝体多糖的适宜酶用量、酶解时间、酶解温度、提取时间、提取温度和提取次数;采用均匀设计方法获得了中性蛋白酶A制备菌丝体多糖的最佳条件为:在2%酶用量下,43.05℃酶解4h,100℃提取1h,提取1次。该优化条件下多糖产量的预测值为16.65 mg/g干菌丝,验证实测值为15.73±0.15 mg/g干菌丝,优化条件下获得的多糖产量比优化前的产量提高75.0%,比热水提取法和微波辅助提取法分别提高122.5%和104.8%,表明酶解法是简便易行的高产制备真姬菇菌丝体多糖的方法。
利用MTT法检测了真姬菇菌丝体多糖的抗肿瘤活性。果胶酶、酸性蛋白酶、纤维素酶A提取的菌丝体多糖对癌细胞(SGC7901)的抑制率显著高于其它酶及对照提取的多糖(p<0.05),其IC50分别是0.343 mg/mL、0.397 mg/mL、0.430 mg/mL。中性蛋白酶A提取的多糖的抗肿瘤活性的IC50是0.984 mg/mL。真姬菇胞壁多糖和胞内多糖的抗肿瘤活性试验表明,发挥抗肿瘤作用的多糖主要位于菌丝体胞内。
不同酶类提取的齿耳菌菌丝体多糖的抗肿瘤活性试验表明,纤维素酶B提取的多糖的活性显著高于其他酶类提取的多糖(p<0.05),其抗肿瘤活性的IC50为0.863 mg/mL。
抗氧化活性试验中,木瓜蛋白酶、果胶酶、中性蛋白酶A和B提取的真姬菇菌丝体多糖的抗氧化活性较高,他们的羟基自由基清除能力的EC50分别是51.98μg/mL、55.76μg/mL、63.56μg/mL、62.49μg/mL;超氧阴离子自由基清除能力的EC50分别是153.77μg/mL、195.61μg/mL、252.68μg/mL、219.85μg/mL。酶类对真姬菇胞壁多糖的抗氧化活性无影响。多糖的抗氧化活性成分主要存在于菌丝体胞内。
木瓜蛋白酶提取的齿耳菌菌丝体多糖的总抗氧化能力,清除羟基自由基能力,超氧阴离子自由基清除能力均比其他酶提取的多糖高(p<0.05),其羟基自由基清除能力、超氧阴离子自由基清除能力的EC50分别是59.42μg/mL、74.78μg/mL。
9 evaluated enzymes were selected to extract polysaccharide from fungal mycelia, and then uniform design was utilized for optimaization of technology for high yield polysaccharides from Hypsizigus marmoreus mycelia. the anti-tumor and antioxidant activity of polysaccharide from fungal mycelia was discussed, and the bio-activity of the polysaccharide intro-mycelia and in the wall of mycelia was also discussed.
It can get the hignest yield polysaccharides from Hypsizigus marmoreus, Suillus sp and Mycoleptodonoides.sp mycelia by lywallzyme, 14.78±0.22 mg/g, 16.66±1.60 mg/g, 27.49±1.89 mg/g, 164.36%, 156.68%, 141.19% higher than control. It was concluded that Neutral protease A was the most cost efficient among 9 evaluated enzymes when extraction of polysaccharide from mycelia of Hypsizigus marmoreus and Suillus sp, 8.52±0.27 mg/g, 20.67±1.86 mg/g, 56.48%, 44.85% higher than control.
The single factor and uniform design experiment was conducted to optimize the conditions, and a regression model were constructed to predict polysaccharide yield from Hypsizigus marmoreus mycelia. The highest polysaccharide yield was obtained when 2% (w/w) of neutral protease was added into the hydrolytic reaction and incubated for 4 h at 43oC, and then extracted one time at 100 oC for 1 h. Using the regression model, the predicted polysaccharide yield was 16.65 (mg/g DMW), which was very close to the measured value of 15.73±0.15 (mg/g DMW). Thus the prediction equation was validated to accurately estimate polysaccharide yield. When following the optimized procedures, polysaccharide yield was raised 75.0% from that prior to optimization. Compared to hot-water extraction and microwave assisted extraction methods, enzymatic extraction method increased polysaccharide yield by 122.5% and 104.8%, respectively. It was concluded that enzymatic extraction is a simple, high yield method to prepare polysaccharide from Hypsizigus marmoreus mycelia.
The anti-tumor activity of polysaccharides from Hypsizigus marmoreus mycelia by different enzymatic hydrolysis.has been observed. The test indicated that the anti-tumor activity of polysaccharides extracted by different enzymatic is different. The anti-tumor activity of polysaccharides from Hypsizigus marmoreu mycelia by pectinase, cellulase and acid protease is higher than by other enzymes. The polysaccharides from mycelia by pectinase, cellulose, acid protease and neutral protease 1 can obviously kill the tumor cell of SGC7901, 0.343 mg/mL, 0.397 mg/mL, 0.430 mg/mL, and 0.984 mg/mL respectively in 50% death rate. The anti-tumor activity of polysaccharides in mycelia is higher than polysaccharides from the wall of mycelia.
The anti-tumor activity of polysaccharides from Mycoleptodonoides sp mycelia by cellulose B is higher than by other enzymes, it can obviously kill the tumor cell of SGC 7901 in 50% death rate with the dosage of 0.863 mg/mL. The antioxidant activity of polysaccharides from mycelia by different enzymatic hydrolysis.is also different. The antioxidant activity of polysaccharides from mycelia by pectinase, papain, neutral protease A and neutral protease B is higher than by other enzymes. Polysaccharides extracted by papain pectinase, neutral protease A and neutral protease B can obviously scavenge hydroxyl free radical, 51.98μg/mL,55.76μg/mL , 62.49μg/mL , 63.56μg/mL respectively in 50% scavenging rate. Polysaccharides extracted by them can obviously scavenge superoxide anion free radical, 153.77μg/mL,195.61μg/mL,219.85μg/mL,252.68μg/mL respectively in 50% scavenging rate.
The antioxidant activity of polysaccharides in mycelia is higher than polysaccharides from the wall of mycelia.
The antioxidant activity of polysaccharides from Mycoleptodonoides sp mycelia by papain is higher than by other enzymes, it can obviously scavenge hydroxyl free radical and superoxide anion free radical, with the dosage of 59.42μg/mL, 74.78μg/mL respectively in 50% scavenging rate.
引文
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