葡萄酒抗氧化活性测定方法研究
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摘要
目前,测定葡萄酒抗氧化活性的方法较多,但没有统一标准的测定方法以及方法的测定参数。本文对葡萄酒抗氧化活性的测定方法进行了研究,确定出了两种适合葡萄酒抗氧化活性测定方法,ABTS法和CUPRAC法,并对其测定条件和葡萄酒样品稀释倍数进行了研究。本研究得到以下结果:
1福林-肖卡比色法(FC)测定的红葡萄酒样的总酚含量在(1402±71 mg/L)～(3130±228 mg/L),桃红葡萄酒的总酚含量在(495±25 mg/L)～(1086±134 mg/L),白葡萄酒样的总酚含量在(189±12 mg/L)～(387±31 mg/L),红葡萄酒的总酚含量高于桃红葡萄酒高于白葡萄酒,不同葡萄品种酿造的酒之间也有差异。
2不同方法间相关性差异比较大,最低值为MC/TBARS(-0.080),最高值为TPI/CUPRAC (0.963)。TPI、ORAC、DPPH、ABTS和CUPRAC五种检测方法之间,相关性均达到极显著水平,最低为ORAC/ABTS(0.845),最高为TPI/CUPRAC(0.963)。羟自由基和超氧自由基清除能力与以上五种方法之间的相关性也具有显著性,最低HRSA/ABTS(0.628),最高值为TPI/SRSA(0.886)。SRSA与各方法相关性高于HRSA与其它方法的相关。TBARS和MC与其它各方法之间的相关性均很弱,尤其是MC法与各方法间的相关性均为最低值。因此TPI、ORAC、DPPH、ABTS和CUPRAC五种检测方法测定的葡萄酒抗氧化结果具有一致性,而其它方法一致性较差。
3 ABTS法测定葡萄酒的抗氧化活性时,红葡萄酒的稀释倍数为0.2:10～0.4:10,桃红葡萄酒的最稀释倍数为1:10～4:10,白葡萄酒的稀释倍数为3:10～7:10,反应时间为2～5 min。优化的ABTS法,以分光光度计为仪器,测定葡萄酒的抗氧化活性时,回收率在94.5～105.0%之间,回收率较高,同天检测的相对标准偏差(RSD)在4.7～6.8%,隔天检测的RSD在4.7～8.8%,可以用于葡萄酒抗氧化活性的常规测定。
4 CUPRAC法测定葡萄酒的抗氧化活性时,反应温度为室温,反应时间为30 min,缓冲液的pH为7.0,Cu2+和Nc的加入量均为0.5 mL。分光光度计测定时,红葡萄酒的最佳稀释倍数为0.4:10 ~0.6:10,桃红葡萄酒的为2:10 ~ 6:10,白葡萄酒的为4:10 ~ 6:10 ;酶标仪测定时,红葡萄酒的最佳稀释倍数为0.08:10 ~0.12:10,桃红葡萄酒的为0.4:10 ~1.2:10,白葡萄酒的为0.8:10 ~ 1.2:10。采用优化的CUPRAC法,以分光光度计为仪器,测定葡萄酒的抗氧化活性时,回收率在95.0～104.0%之间,同天检测的相对标准偏差(RSD)在3.8～6.7%,隔天检测的RSD在4.3～8.4%。
There is no uniform method and for mensurating the antioxidant activity of wines, although many methods are workable. The determination method for antioxidant activity of wines was studied in this paper, two antioxidant activity mensurating methods-ABTS method and CUPRAC method were confirmed, and the condition of mensuration and dilution of wines were studied too. Main results were as follows:
1 Total phenol index (TPI) of wine was mensurated by Folin-Ciocalteu colorimetric method, the TPI of red wines range from 1402±71 mg/L to 3130±228 mg/L, rose wines 495±25 mg/L to 1086±134 mg/L and white wines 189±12 mg/L to 387±31 mg/L, red wines have highest TPI,the differentia was also existed between different grape cultivars.
2 The correlation between different methods was marked, the lowst value was MC/TBARS (-0.080), and the highest value was TPI/CUPRAC (0.963). The correlation between TPI、ORAC、DPPH、ABTS and CUPRAC were most remarkable, and the lowest was ORAC/ABTS (0.845),and the highest was TPI/CUPRAC (0.963). HRSA and SRSA also had remarkable correlation with TPI、ORAC、DPPH、ABTS and CUPRAC, the lowest was HRSA/ABTS (0.628),and the highest was TPI/SRSA (0.886).The correlation of SRSA with other methods was higher than that of HRSA. The correlation of TBARS and MC with other methods were tiny, especially the correlation of MC with other methods was lowest. Therefore, the consistency were existed between the results of antioxidant activity determined by TPI、ORAC、DPPH、ABTS and CUPRAC methods, and the consistency of other methods were worse.
3 When ABTS method was used to mensurate antioxidant activity, red wines dilution were range from 0.2:10 to 0.4:10, rose wines 1:10 to 4:10 and white wines 0.2:10 to 0.4:10, the reaction time was 2 to 5 min. When the antioxidant activity of wine was mensurated by the optimal ABTS method with the spectrophotometer as the measuring instrument, the percent recovery was range from 94.5% to 105.0%, relative standard deviation (RSD) of the same day was 4.7% to 6.8%, and RSD of every other day was 4.7% to 8.8%. So, this method can be used as a conventional method to mensurate the antioxidant activity of wines.
4 When CUPRAC method was used to mensurate antioxidant activity, the reaction temperature was room temperature, the reaction time was 30 min, the pH of buffer solution was 7.0 and the addition of Cu(Ⅱ) and Nc both was 0.5 mL. Red wines dilution were 0.4:10 to 0.6:10,rose wines were 2:10 to 6:10 and white wines were 4:10 to 6:10 for spectrophotometer mensuration. Red wines dilution were 0.08:10 to 0.12:10,rose wines were 0.4:10 to 1.2:10 and white wines were 0.8:10 to 1.2:10 for microplate reader mensuration. When the antioxidant activity of wine was mensurated by the optimal CUPRAC method with the spectrophotometer as the measuring instrument, the percent recovery was range from 95.0% 104.0%, RSD of the same day was 3.8% to 6.7%, and RSD of every other day was 4.3% to 8.4 %.
1福林-肖卡比色法(FC)测定的红葡萄酒样的总酚含量在(1402±71 mg/L)～(3130±228 mg/L),桃红葡萄酒的总酚含量在(495±25 mg/L)～(1086±134 mg/L),白葡萄酒样的总酚含量在(189±12 mg/L)～(387±31 mg/L),红葡萄酒的总酚含量高于桃红葡萄酒高于白葡萄酒,不同葡萄品种酿造的酒之间也有差异。
2不同方法间相关性差异比较大,最低值为MC/TBARS(-0.080),最高值为TPI/CUPRAC (0.963)。TPI、ORAC、DPPH、ABTS和CUPRAC五种检测方法之间,相关性均达到极显著水平,最低为ORAC/ABTS(0.845),最高为TPI/CUPRAC(0.963)。羟自由基和超氧自由基清除能力与以上五种方法之间的相关性也具有显著性,最低HRSA/ABTS(0.628),最高值为TPI/SRSA(0.886)。SRSA与各方法相关性高于HRSA与其它方法的相关。TBARS和MC与其它各方法之间的相关性均很弱,尤其是MC法与各方法间的相关性均为最低值。因此TPI、ORAC、DPPH、ABTS和CUPRAC五种检测方法测定的葡萄酒抗氧化结果具有一致性,而其它方法一致性较差。
3 ABTS法测定葡萄酒的抗氧化活性时,红葡萄酒的稀释倍数为0.2:10～0.4:10,桃红葡萄酒的最稀释倍数为1:10～4:10,白葡萄酒的稀释倍数为3:10～7:10,反应时间为2～5 min。优化的ABTS法,以分光光度计为仪器,测定葡萄酒的抗氧化活性时,回收率在94.5～105.0%之间,回收率较高,同天检测的相对标准偏差(RSD)在4.7～6.8%,隔天检测的RSD在4.7～8.8%,可以用于葡萄酒抗氧化活性的常规测定。
4 CUPRAC法测定葡萄酒的抗氧化活性时,反应温度为室温,反应时间为30 min,缓冲液的pH为7.0,Cu2+和Nc的加入量均为0.5 mL。分光光度计测定时,红葡萄酒的最佳稀释倍数为0.4:10 ~0.6:10,桃红葡萄酒的为2:10 ~ 6:10,白葡萄酒的为4:10 ~ 6:10 ;酶标仪测定时,红葡萄酒的最佳稀释倍数为0.08:10 ~0.12:10,桃红葡萄酒的为0.4:10 ~1.2:10,白葡萄酒的为0.8:10 ~ 1.2:10。采用优化的CUPRAC法,以分光光度计为仪器,测定葡萄酒的抗氧化活性时,回收率在95.0～104.0%之间,同天检测的相对标准偏差(RSD)在3.8～6.7%,隔天检测的RSD在4.3～8.4%。
There is no uniform method and for mensurating the antioxidant activity of wines, although many methods are workable. The determination method for antioxidant activity of wines was studied in this paper, two antioxidant activity mensurating methods-ABTS method and CUPRAC method were confirmed, and the condition of mensuration and dilution of wines were studied too. Main results were as follows:
1 Total phenol index (TPI) of wine was mensurated by Folin-Ciocalteu colorimetric method, the TPI of red wines range from 1402±71 mg/L to 3130±228 mg/L, rose wines 495±25 mg/L to 1086±134 mg/L and white wines 189±12 mg/L to 387±31 mg/L, red wines have highest TPI,the differentia was also existed between different grape cultivars.
2 The correlation between different methods was marked, the lowst value was MC/TBARS (-0.080), and the highest value was TPI/CUPRAC (0.963). The correlation between TPI、ORAC、DPPH、ABTS and CUPRAC were most remarkable, and the lowest was ORAC/ABTS (0.845),and the highest was TPI/CUPRAC (0.963). HRSA and SRSA also had remarkable correlation with TPI、ORAC、DPPH、ABTS and CUPRAC, the lowest was HRSA/ABTS (0.628),and the highest was TPI/SRSA (0.886).The correlation of SRSA with other methods was higher than that of HRSA. The correlation of TBARS and MC with other methods were tiny, especially the correlation of MC with other methods was lowest. Therefore, the consistency were existed between the results of antioxidant activity determined by TPI、ORAC、DPPH、ABTS and CUPRAC methods, and the consistency of other methods were worse.
3 When ABTS method was used to mensurate antioxidant activity, red wines dilution were range from 0.2:10 to 0.4:10, rose wines 1:10 to 4:10 and white wines 0.2:10 to 0.4:10, the reaction time was 2 to 5 min. When the antioxidant activity of wine was mensurated by the optimal ABTS method with the spectrophotometer as the measuring instrument, the percent recovery was range from 94.5% to 105.0%, relative standard deviation (RSD) of the same day was 4.7% to 6.8%, and RSD of every other day was 4.7% to 8.8%. So, this method can be used as a conventional method to mensurate the antioxidant activity of wines.
4 When CUPRAC method was used to mensurate antioxidant activity, the reaction temperature was room temperature, the reaction time was 30 min, the pH of buffer solution was 7.0 and the addition of Cu(Ⅱ) and Nc both was 0.5 mL. Red wines dilution were 0.4:10 to 0.6:10,rose wines were 2:10 to 6:10 and white wines were 4:10 to 6:10 for spectrophotometer mensuration. Red wines dilution were 0.08:10 to 0.12:10,rose wines were 0.4:10 to 1.2:10 and white wines were 0.8:10 to 1.2:10 for microplate reader mensuration. When the antioxidant activity of wine was mensurated by the optimal CUPRAC method with the spectrophotometer as the measuring instrument, the percent recovery was range from 95.0% 104.0%, RSD of the same day was 3.8% to 6.7%, and RSD of every other day was 4.3% to 8.4 %.
引文
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