杏鲍菇菌糠总黄酮的提取分离及部分生物活性研究

发布时间：2018-03-22 16:03

本文选题：杏鲍菇　切入点：菌糠　出处：《延安大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着杏鲍菇产业的飞速发展,杏鲍菇菌糠也日益增多,通常对其处理的方法是丢弃、堆积、焚烧、作为再生产的原料等,这些处理都不能有效合理的解决菌糠问题,因此,菌糠的合理利用成为了制约工厂化发展和生态文明建设面临的一项挑战。研究表明,菌糠中含有来自培养料、菌丝体和菌丝代谢产物中的大量的营养物质及生物活性成分。本研究以杏鲍菇菌糠为原料,以总黄酮含量为指标,采用超声辅助法对杏鲍菇菌糠中总黄酮进行提取、分离纯化、抗氧化活性、抑菌活性及其稳定性研究,旨在为杏鲍菇菌糠的综合利用提供理论依据。研究内容和结果如下:(1)通过显色反应以及HPLC色谱法定性试验确定菌糠提取物中含有黄酮类化合物之后,分别进行单素试验,在单因素试验的基础上,设计4因素3水平的正交试验对杏鲍菇菌糠总黄酮的提取条件进行了优化,得到提取最佳工艺条件为:乙醇浓度为70%,料液比1:35 g/mL,超声破碎时间20 min,水浴温度为70℃。在该条件下杏鲍菇菌糠总黄酮得率为2.852±0.040 mg/g,且RSD=1.39%,表明该工艺比较稳定。(2)通过对6种树脂的吸附和解吸效果考察,选择AB-8型大孔树脂对杏鲍菇菌糠总黄酮进行分离纯化。在单因素试验基础上,分别对吸附和解吸过程进行正交优化试验,得到杏鲍菇菌糠总黄酮的纯化工艺为:上样液流速为2.0 mL/min、上样液pH为6、上样液浓度0.25 mg/mL、脱液流速为3.0 mL/min、洗脱液浓度90%、洗脱液体积为4.5 BV。发现,总黄酮含量由纯化前的8.049%提高到30.37%,纯度提高了3.77倍;且通过对AB-8树脂重复使用性能的考察发现,重复利用7次后吸附率保持在50%以上,表明AB-8树脂适合于杏鲍菇菌糠总黄酮的分离。(3)以VC为对照,对杏鲍菇菌糠总黄酮的总还原能力以及对DPPH、羟基自由基、超氧阴离子三种自由基的清除能力考察发现:当杏鲍菇菌糠总黄酮的浓度大于1.2 mg/mL时,总黄酮的总还原能力比VC强;另外,在试验选择浓度范围内,杏鲍菇菌糠总黄酮对DPPH的清除率为68.51%;对羟基自由基的清除率为50.38%;对超氧阴离子自由基的清除率为56.06%,其中杏鲍菇菌糠总黄酮对DPPH和羟基自由基的清除能力均比VC强,对超氧阴离子的清除能力弱于VC,说明杏鲍菇菌糠总黄酮具有良好的抗氧化活性。(4)采用打孔法对常见的4种细菌和3种真菌进行抑菌试验,得到杏鲍菇菌糠总黄酮对7种病原菌的最小抑菌浓度分别为:大肠杆菌MIC为40 mg/mL,白色葡萄球菌MIC为20 mg/mL,伤寒杆菌MIC为20 mg/mL,福氏痢疾杆菌MIC为40mg/mL,西瓜腐烂病菌MIC为40 mg/mL,小麦赤霉病MIC为40 mg/mL,棉花枯萎菌MIC为20 mg/mL,说明杏鲍菇菌糠总黄酮具有良好的抑菌活性。(5)通过对光照、温度、pH以及金属阳离子对杏鲍菇菌糠总黄酮稳定性影响的考察,结果表明:杏鲍菇菌糠总黄酮溶液在4℃和20℃存放10 h、pH为6存放24h、避光环境中存放7 d吸光度变化均不大;K+和Na+对吸光度的影响变化也不大,而Cu2+和Fe3+对溶液吸光度有不同程度的影响。
[Abstract]:With the rapid development of mushroom industry, eryngiibacterial chaff is also increasing, usually on the handling method is discarded, accumulation, incineration, as raw materials for reproduction, these treatments are not effective to solve the problem of reasonable WMLE, therefore, reasonable use of SMS has become a challenge for restricting industrialization development and the construction of ecological civilization. The study shows that the fungus bran contains from the culture medium, a large number of physical and biological activity of nutrients and metabolites in mycelium hyphae. In this study, eryngiibacterial chaff as raw materials, with total flavonoids content as index, the ultrasonic assisted method of total flavonoids in p.eryngii extraction, purification, antioxidant activity, antibacterial activity and stability study, aiming to provide a theoretical basis for the comprehensive utilization of p.eryngii. Research contents and results are as follows: (1) by chromogenic reaction and HPLC chromatography After the determination of flavonoids in extract of WMLE test were single hormone test, on the basis of single factor test, orthogonal experiment design of 4 factors and 3 levels of the extraction conditions of total flavonoids from p.eryngii were optimized to get the optimal conditions for the extraction of ethanol concentration 70%, ratio of material to liquid 1:35 g/mL, ultrasonic time 20 min, water bath temperature of 70 degrees. In p.eryngii under the condition of total flavonoids was 2.852 + 0.040 mg/g, and RSD=1.39%, showed that the process was stable. (2) through the adsorption and desorption effect of 6 kinds of resin of choice AB-8 macroporous resin total flavonoids of p.eryngii were isolated. Based on the single factor experiment, respectively on the adsorption and desorption process were studied by orthogonal test, obtained total flavonoids from p.eryngii purification: the sample liquid flow rate is 2 mL/min, the pH of sample was 6, sample solution The concentration of 0.25 mg/mL, and the liquid flow rate is 3 mL/min, eluent concentration was 90%, the elution liquid found a volume of 4.5 BV., total flavonoids content increased from 8.049% to 30.37% before purification, the purity increased 3.77 times; and through the investigation of repeated use performance of AB-8 resin was reused for 7 times, the adsorption rate remained at 50% above, that separation of AB-8 resin suitable for total flavonoids from p.eryngii. (3) compared with VC, the total reducing capacity for total flavonoids of eryngiibacterial chaff and on DPPH, hydroxyl radical, superoxide anion free radical scavenging ability of three kinds of study found: when the concentration of total flavonoids of p.eryngii more than 1.2 mg/mL, the total reducing capacity of total flavonoids was stronger than that of VC; in addition, in the test concentration range, eryngiibacterial chaff total flavonoids the scavenging rate of DPPH was 68.51%; the hydroxyl radical scavenging rate was 50.38%; the superoxide anion free radical scavenging rate was 56. 06%, the total flavonoids of p.eryngii scavenging of DPPH and hydroxyl radicals were stronger than VC, the superoxide anion scavenging ability than VC, indicating that the total flavonoids of p.eryngii had good antioxidant activity. (4) in the bacteriostatic test against 4 bacteria and 3 fungi using common punch method, get the minimum inhibitory concentration of total flavonoids of eryngiibacterial chaff on 7 pathogens were Escherichia coli MIC 40 mg/mL, MIC 20 mg/mL for Staphylococcus aureus, Salmonella typhi MIC 20 mg/mL Shigella flexneri MIC 40mg/mL, MIC 40 mg/ watermelon rot pathogen mL, wheat scab MIC 40 mg/mL, MIC 20 mg/mL of cotton wilt Fusarium, that total flavonoids of eryngiibacterial chaff have better antibacterial activity. (5) based on illumination, temperature, study, effects of pH and metal cations on p.eryngii stability of flavonoids showed that p.eryngii general Huang Tongrong The liquid stored at 10 h for 4 and 20 degrees, pH for 6, 24h for storage, and no change in absorbance for 7 d in the light avoiding environment. K+ and Na+ had little influence on absorbance, while Cu2+ and Fe3+ had different degrees of influence on solution absorbance.

【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ28;X712

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