Determination of Antioxidant Activity of Agrocybe chaxingu Fermentation Broth by a Modified Fenton Reaction Method
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摘要: 以番红为氧化还原指示剂，用Fenton反应法，通过研究番红用量、样液用量、pH值和维生素C对反应体系吸光值的影响，改进Fenton法以建立一种茶薪菇发酵产物抗氧化性的快速检测方法。改进的条件为：pH7.0~7.5，520 μg·mL-1的番红溶液由0.5 mL变成2.0 mL，样液由7.0 mL变成0.5 mL，30 min内显色稳定；在最佳条件下，以维生素C作为羟自由基清除剂阳性对照，为茶薪菇发酵产物的抗氧化性评价提供标准。结果表明：茶薪菇胞内、胞外的羟自由基清除率分别达48.62%~65.43%（维生素C当量72.62~90.22 mmol·L-1）、57.59%~72.82%（维生素C当量82.02~97.96 mmol·L-1），证实了改进Fenton法的实用性，且所用试剂均无毒副作用，可快速测定茶薪菇发酵产物的抗氧化性。Abstract: Saffron was used as the redox reaction indicatorin the rapid determination of the antioxidant activity of the fermentation broth of Agrocybe chaxinguHuang. Fenton reaction was modified for the methodology development by comparing the effect of safranin usage, sample volume, pH, and vitamin C on the light absorbance measured on a spectrophotometer. The optimized conditions included pH 7.0-7.5 with an increased volume on the 520 μg·mL-1safranin solution from 0.5 mL to 2.0 mL anda much-reduced usage on the sample solution from 7.0 mL to 0.5 mL. The absorbance reading remained constant within 30 min. Under the conditions, vitamin C was applied as a standard hydroxyl radical scavenger to evaluate the antioxidant activity of A. chaxingu broth. The results showed that the hydroxyl radical-scavenging ability of the A. chaxingu intracellular metabolites was 48.62%-65.43% (equivalent to vitamin C 72.62-90.22 mmol·L-1), and that of the extracellular metabolites, 57.59%-72.82% (equivalent to vitamin C 82.02-97.96 mmol·L-1). The newly established rapid determination method was, thus, confirmed for its validity. In addition, there were no toxic reagentsapplied in the testing that would cause any safety concerns.
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Key words:
- spectrophotometry /
- fenton reaction /
- hydroxyl radicals (·OH) /
- saffron /
- antioxidant
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图 1 番红体积对体系吸光值的影响
Figure 1. Effect of applied volume of saffron on spectro-photometric absorbance measurement
图 2 茶薪菇发酵液体积对体系吸光度影响
Figure 2. Effect of applied volume of extracellular metabolites of A. chaxingu on absorbance measurement
图 3 维生素C的羟自由基清除率标准曲线
Figure 3. Standard curve of hydroxyl radical scavenging rate with vitamin C
表 1 不同样液、番红体积的羟自由基清除率
Table 1. Hydroxyl radical scavenging rates with application of varied sample and saffron volumes
项目 番红体积2.0 mL 番红体积3.0 mL 清除率比值R1 发酵液体积0.5 mL 36.79 39.49 1.07 发酵液体积1.0 mL 66.03 60.47 0.92 清除率比值R2 1.79 1.53 注：清除率比值R1为番红体积3.0 mL时清除率与番红体积2.0 mL时清除率的比值，R1的理论值为1；清除率比值R2为发酵液体积1.0 mL时清除率与发酵液体积0.5 mL时清除率的比值，R2的理论值为2。 
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表 2 酸碱度对体系吸光值的影响
Table 2. Effect of pH on absorbance measurement
ΔpH 5.5 6.0 6.5 7.0 7.4 7.5 8.0 8.5 pH0 5.50 6.00 6.50 7.00 7.39 7.49 8.02 8.51 pH1 4.43 5.05 6.08 6.78 7.05 7.23 7.69 7.86 pH 1.07 0.95 0.42 0.22 0.34 0.26 0.33 0.65 A520 0.163 0.212 0.253 0.322 0.341 0.345 0.365 0.384 ΔA 0.138 0.089 0.048 0.021 0.040 0.044 0.064 0.083 注：A=0.301；ΔA=|A520-A|；ΔpH=pH1-pH0。
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表 3 显色稳定性
Table 3. Stability of reaction color for spectrophotometry measurement
吸光值 放置时间/min 0 5 10 15 20 25 30 A520 0.342 0.337 0.334 0.333 0.332 0.331 0.331 A520 0.459 0.449 0.447 0.445 0.444 0.442 0.442
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表 4 茶薪菇菌丝体提取液的羟自由基清除率
Table 4. Hydroxyl radical scavenging rate of hot water extracts of A. chaxingu mycelia
项目 配方Ⅰ 配方Ⅱ 配方Ⅲ 配方Ⅳ 清除率/% 65.43±2.96a 55.84±2.75b 51.06±0.97c 48.62±1.57c 维生素C当量/(mmol·L-1) 90.22 80.18 75.18 72.62 注：(1)配方Ⅱ~Ⅳ为在液体培养基配方Ⅰ基础上，添加2%、4%、8%油茶枝粉浸提液；(2)同行中不同小写字母表示差异显著(P＜0.05)；(3)维生素C当量：与样品羟自由基清除率相等时维生素C的浓度，下表同。
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表 5 茶薪菇发酵液的羟自由基清除率
Table 5. Hydroxyl radical scavenging rate of extracellular metabolites of A.chaxingu
项目 配方Ⅴ 配方Ⅵ 配方Ⅶ 配方Ⅷ 清除率/% 69.18±5.28a 57.59±2.76b 72.82±3.87a 60.22±5.13a 维生素C当量/(mmol·L-1) 94.16 82.02 97.96 84.77 注：配方Ⅴ、Ⅵ为在液体培养基配方Ⅰ的基础上分别去掉麦麸、玉米粉，Ⅶ、Ⅷ在Ⅴ的基础上添加2%、4%油茶枝粉浸提液。
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