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响应面法优化六妹羊肚菌边角料中麦角硫因的提取工艺
Optimization of extraction process of ergothioneine from Morchella sextelata scraps by response surface methodology
, ({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}为提高六妹羊肚菌边角料中麦角硫因的得率，以麦角硫因得率为评价标准，分析了提取温度、乙醇浓度、液料比、提取时间等单因素对麦角硫因提取量的影响，在单因素实验的基础上通过响应面法对六妹羊肚菌边角料中麦角硫因的提取工艺条件进行了优化。单因素和响应面法得到的麦角硫因最佳提取工艺条件：液料比为40:1 (体积质量比)、乙醇浓度为50%、提取温度为60 ℃、提取时间为20 min。以该条件进行验证实验所得麦角硫因提取量为(0.236±0.000 887) mg/g，与预测值0.235 mg/g无显著差异(误差值0.426%)，表明优化得到的工艺具有有效性和可靠性。本研究确定了六妹羊肚菌边角料中麦角硫因的最佳提取工艺，为六妹羊肚菌边角料的开发利用提供基础数据。
The effect of extraction temperature, ethylalcohol concentration, liquid to material ratio and extraction time on the extraction yield of ergothioneine in M. sextelata scraps were studied by using the content of ergothioneine obtained as indicator, and the response surface methodology based on single factor experiment was applied to optimize the technological process of extraction. The results obtained by single factor experiment and response surface methodology indicated that liquid to material ratio of 40:1 (mL/g), ethylalcohol concentration of 50%, extraction temperature of 60 ℃, and extraction time of 20 min were the optimum extraction condition. Under this optimized condition, the extraction yield of ergothioneine was (0.236±0.000 887) mg/g, no significantly difference was found as compared with estimated value 0.235 mg/g (error value 0.426%), indicating that the optimized process was effective and reliable. This study provides basic data for utilization of M. sextelata scraps.
六妹羊肚菌 / 边角料 / 麦角硫因 / 响应面法 / 含量测定 {{custom_keyword}} /
Morchella sextelata / scrap / ergothioneine / response surface methodology / content determination {{custom_keyword}} /
表1 qRT-PCR引物联序列Table 1 Primers’ sequences for qRT-PCR |
| Gene | Forward primer (5ʹ→3ʹ) | Reverse primer (3ʹ→5ʹ) |
|---|---|---|
| GAPDH | CTCCTCCTGTTCGACAGTCA | CGACCAAATCCGTTGACTCC |
| Claudin-1 | CACCGTCTGTGTTTGAGCA | CAAACCACCGCTTACAGATG |
| Occludin | GACTATGTGGAAAGAGTTGAC | ACCGCTGCTGTAACGAG |
| ZO-1 | TTCACGCAGTTACGAGCAAG | TTGGTGTTTGAAGGCAGAGC |
图1 棕榈酸和银耳多糖对Caco-2细胞活力和完整性的影响 A：不同浓度TPs对细胞活力影响；B：TPs和PAd对细胞活力影响；C：TPs和PAd对细胞完整性的影响. PAd组与CON组相比，#P<0.05，##P<0.01；Pad+TPs处理组与PAd组相比，*P<0.05，**P<0.01. 下同Fig. 1 Effects of palmitic acid (PAd) and Tremella fuciformis polysaccharides (TPs) on cell viability and TEER value of Caco-2. A: Effects of TPs concentration on cell viability; B: Effects of TPs and PAd on cell viability; C: Effects of TPs and PAd on cell integrity. PAd vs. CON, #P<0.05 and ##P<0.01; PAd+TPs treatment group vs. PAd, *P<0.05 and **P<0.01. The same below. |
图4 银耳多糖对棕榈酸诱导的紧密连接蛋白mRNA表达量的影响 A：Claudin-1水平；B：Occludin水平；C：ZO-1水平Fig. 4 Effect of TPs on PAd-induced tight junction protein mRNA expression. A: Claudin-1 level; B: Occludin level; C: ZO-1 level. |
图5 银耳多糖对棕榈酸诱导的紧密连接蛋白表达量的影响 A：Western blot检测Claudin-1、Occludin、ZO-1的蛋白条带；B：Claudin-1、Occludin、ZO-1的相对表达量Fig. 5 Effects of TPs on PAd-induced tight junction protein expression. A: Protein bands of Claudin-1, Occludin and ZO-1 were detected by Western blot. B: Relative expression of Claudin-1, Occludin, and ZO-1. |
图6 银耳多糖对TRL4/MyD88/NF-κB信号通路的影响 A：Western blot检测TLR4、MyD88、p-NF-κB的蛋白条带；B：TLR4、MyD88、p-NF-κB的相对表达量Fig. 6 Effects of TPs on TRL4/MyD88/NF-κB signaling pathway. A: Protein bands of TLR4, MyD88, and p-NF-κB were detected by Western blot. B: Relative expression of TLR4, MyD88, and p-NF-κB. |
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PDF(876 KB)
表1 qRT-PCR引物联序列
图1 棕榈酸和银耳多糖对Caco-2细胞活力和完整性的影响 A：不同浓度TPs对细胞活力影响；B：TPs和PAd对细胞活力影响；C：TPs和PAd对细胞完整性的影响. PAd组与CON组相比，#P<0.05，##P<0.01；Pad+TPs处理组与PAd组相比，*P<0.05，**P<0.01. 下同图2 银耳多糖对棕榈酸诱导的Caco-2细胞氧化性的影响 A：CAT水平；B：SOD水平；C：MDA水平图3 银耳多糖对棕榈酸诱导的Caco-2细胞炎症因子释放量的影响 A：IL-1β水平；B：IL-6水平；C：TNF-α水平图4 银耳多糖对棕榈酸诱导的紧密连接蛋白mRNA表达量的影响 A：Claudin-1水平；B：Occludin水平；C：ZO-1水平图5 银耳多糖对棕榈酸诱导的紧密连接蛋白表达量的影响 A：Western blot检测Claudin-1、Occludin、ZO-1的蛋白条带；B：Claudin-1、Occludin、ZO-1的相对表达量图6 银耳多糖对TRL4/MyD88/NF-κB信号通路的影响 A：Western blot检测TLR4、MyD88、p-NF-κB的蛋白条带；B：TLR4、MyD88、p-NF-κB的相对表达量
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