胭脂红酸检测方法及色素提取改善
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摘要
胭脂虫(Cochineal)是一类寄生在仙人掌类植物上的资源昆虫,其雌性成虫体内含有一种蒽醌类物质——胭脂红酸,是优质天然染色剂胭脂虫红色素的有效组分,具有较高的经济价值。本论文在近年来国内研究人员对胭脂虫及胭脂虫红色素研究的基础上,开展了HPLC在胭脂红酸检测上的应用,建立了胭脂红酸快速、精确的检验方法。为推进中国胭脂虫红色素产业化,论文从化学组分角度出发,对滇中地区繁养的胭脂虫进行了工业原料质量测评,研发了胭脂虫红色素高效提取方法;初步探索了酶解法在精制胭脂虫红色素上的应用可行性;测试了胭脂红酸清除自由基的能力。
通过比较不同配比的流动相,不同流速和温度对出峰效果的影响,建立胭脂红酸的高效液相色谱检测方法,并与常用的分光光度法作比较,为胭脂红酸的分析检测提供参考依据。通过检测分析、比较胭脂虫(滇中)与胭脂虫(秘鲁)的堆密度、单重虫粒数、吸水率和体积膨胀率等物理指标,以及两地胭脂虫干虫体中四氯化碳提取物、乙醇提取物、石油醚提取物、总蛋白质、胭脂红酸、不溶性糖类和灰分等主要化学组分的含量,评估了我国滇中地区繁养的胭脂虫质量;并对引进国内后在滇中地区放养的不同世代的胭脂虫主要化学组分进行了分析对比,为我国繁养胭脂虫,并进一步开发利用胭脂虫资源提供了理论依据。通过进一步优化了胭脂虫红色素微波辅助提取的工艺参数,以及开发微波-超声波协同萃取胭脂虫红色素的工艺,为胭脂虫红色素的快速提取进一步充实理论依据。应用酶水解法与超滤膜法精制色素,除去胭脂虫红色素中可溶性大分子蛋白质,以达到除去致敏源蛋白质。研究了胭脂红酸作为功能性食品对自由基的清除能力,为利用胭脂虫红色素打下基础。
(1)高效液相色谱检测胭脂红酸试验表明:流动相为甲醇-乙腈-磷酸(0.01%)的混合液(体积比为10:15:75),流速为0.8 mL /min,柱温为20℃,出峰效果最好。纯度为100%的胭脂红酸作为标准样品,绘制标准曲线,280 nm和494 nm的检测波长下分别得回归方程Y=60.7360X,R~2=0.9999;Y=8.6048X,R~2=0.9990,两者线性关系均良好。以280 nm和494 nm为检测波长,使用纯度为100%的胭脂红酸验证仪器的精密度,自制胭脂虫红色素验证稳定性和加标回收率。测得精密度相对标准偏差分别为0.42%和0.54%,稳定性相对标准偏差分别为0.16%和0.32%,加标回收率分别为96.8%和94.3%。以Sigma公司生产的胭脂红酸(纯度为96%)为标样,分别用高效液相色谱测峰面积和用分光光度法测吸光度,分别绘制标准曲线方程得:Y=7.8260X,R~2=0.9988和Y=0.0137X,R~2=0.9997浓度在9.5～95.0μg/mL的范围内线性相关性良好。并分别用高效液相色谱法和分光光度法对10种色素样品进行定量分析,分光光度法所得结果较高效液相色谱结果偏高,但两种方法所得结果经单因素方差分析,显著性指标P值为0.904,即两种方法测定结果差异性不显著。两种方法都可以用于定量分析胭脂红酸样品中的胭脂红酸。
作为工业昆虫原料,我国引进的胭脂虫及不同繁养世代实验表明:胭脂虫(滇中)与秘鲁(胭脂虫的原产地)胭脂虫相比,除堆密度含量差异较小外,其余物理参数和主要化学组分差异性均较大,但滇中地区繁养的胭脂虫虫体中胭脂红酸的含量仍较高,作为生产加工胭脂红酸的原料,仍具有较高的开发利用价值。滇中地区繁养的不同世代的胭脂虫,虽然胭脂虫各组分有所差异,但胭脂红酸含量均较高,不同世代之间差异不显著,因此,三代胭脂虫作为生产加工胭脂虫红色素的原料,均具有较高的开发利用价值。
(2)胭脂虫红色素微波辅助提取实验表明:微波处理前常温预浸泡40min,处理后75℃水浴浸提40min最适宜于色素提取。在单因素试验的基础上,对液料比,微波功率及微波辐射时间进行三因素三水平响应面分析试验,得到最优条件为液料比7.5,微波功率714W,辐射时间73s。最优条件下,合适的提取级数为12次。对胭脂虫红色素进行微波-超声波协同提取,在单因素试验的基础上,对微波功率、处理时间、液料比及超声波功率进行四因素三水平响应面分析试验,得到最优条件为微波功率464W,处理时间为18.48min,液料比为8.23,超声波功率627W。最优条件下,合适的提取级数为4次。微波提取与微波-超声波协同提取法相比,前者能耗较少,但所需时间长,提取级数较多,在生产实践中,可按不同的需求和所具备的条件选择适当的方法。
(3)对提取的胭脂虫红色素进行木瓜蛋白酶水解实验,将水解后的胭脂虫红色素溶液依次透过100KDa、30KDa、10KDa、3KDa及1Kda的超滤膜,分别测定各截留液的体积,溶液中胭脂红酸浓度,含氮物质浓度,计算不同分子截留液中胭脂红酸及含氮物质的含量;并与未经木瓜蛋白酶水解的胭脂虫红色素溶液在相同条件处理后的参数作比较,发现经过木瓜蛋白酶水解,胭脂虫红色素中的大分子蛋白质被一定程度的降解,由于胭脂虫红色素中的致敏性物质多为大分子蛋白质,因此,木瓜蛋白酶水解对清除胭脂虫红色素的致敏性蛋白质,会起到一定的作用。
(4)为了评估胭脂红酸清除自由基能力,以DPPH·、ABTS+·及O_2ˉ·等3种自由基为试验对象,通过比较特定波长下自由基溶液吸光度的变化,评估胭脂红酸清除自由基的能力,并与抗坏血酸清除3种自由基的能力做比较。胭脂红酸清除自由基结果表明:胭脂红酸与抗坏血酸对3种自由基DPPH·、ABTS+及O_2ˉ·均具有清除活性;当胭脂红酸达到一定浓度时,对DPPH·、ABTS+·及O_2ˉ·的清除率分别可达83%、85%及46%左右。表明胭脂红酸是一种对自由基具有一定清除活性的食品添加剂。
Cochineal (Dactylopius coccus Costa) dye can be used in food, medicine and cosmetics in the United States Food and Drug Administration. There are little research on the pigment in our country. Experiment parameters of the extraction of cochineal dye were optimized. For purification method, the method of ultrafiltration membrane and silica gel column chromatograph were used, and determine the main ingredient. The factors affecting pigment stability are discussed.
The main results were as follows:
(1) when mobile phase was consist of acetonitrile, methanol and phosphoric acid (0.01%) (volume ratio 10:15: 75), flow rate was 0.8 mL/min, column temperature at 20℃, peak shape was the best. This mothed used for determined car- minic acid both at 280 nm and 494 nm. Carminic acid(purity:100%) was utilized as standard sample, made standard curve, obtain regression equation Y=8.6048X, R~2=0.9990, and Y=60.7360X,R~2=0.9999, respectively. Both equa-tion’s linear relationship were fine. when determine cochineal red pigment, the result showed that in this method, sam- ple had good stability(determined at 280 nm, RSD=0.16%; at 494 nm, RSD= 0.32%), equipment had high precision(determined at 280 nm, RSD=0.42%; at 494 nm,RSD=0.54%) and sample had high recovery rate(determined at 280 nm, recovery rate was 96.8, at 494 nm was 94.3).compare high-performance liquid chromatography (HPLC) and spectrophotometry to determine the purity of carminic acid samples, these two methods make quantitative analysis of 10 kinds of samples, the result of spectrophotometry measurements is slightly higher than high-performance liquid chromatography, but the results of the two methods no insignificant difference.
Significant differences were exist between cochineal cultivated in middle area of Yunnan province and in Peru, except bulk density. But the carminic acid contents is high. The conclusion is that Cochineal cultivated in middle area of Yunnan province can also used for making carminic acid. Through compare cochineal of three generations, the results show that even there some difference existi in cochineal of three generations, but the difference of carminic acid contents are not significant, so cochineal of three generations can all used for make cochineal dye.
(2) In order to develop a method for microwave-assisted extract cochineal dye, single-factor test was carried out. The results show that cochineals should soak in water for 40min before microwave treatment, and extract with water for 40min after microwave treatment. On the basis of single-factor test, the microwave-assisted extraction process parameters about extraction of cochineal dye were optimized with response surface method. The results suggest that the optimal parameters should as follows: ratios of liquid to material 7.5, microwave power 714W, microwave treatment time 73s. Under this conditon, the best extracting times is 12.
In order to develop a method for microwave - ultrasonic synergistic extract cochineal dye, On the basis of single-factor test (included microwave power, treatment time, ratios of liquid to material and ultrasonic power), optimized extraction process parameters with response surface method, best condition should as follows: microwave power 464W, treatment time is 18.48min, ratios of liquid to material 8.23, ultrasonic power 627W. Under this conditions, the suitable extraction times is 4.
Compare the microwave-assisted extract mathod and microwave-ultrasonic extract mathod, we can find out that use microwave-assisted extract mathod, energy consumption are lower than microwave-ultrasonic extract mathod. But time consumption is longer, and extraction times is more than microwave-ultrasonic extract mathod.
(3) Utilize the papayin hydrolisis cochineal dye, then penetrates the cochineal dye through the 100KDa, 30KDa, 10KDa, 3KDa and the 1Kda ultra filter. Determines the volume of different solution, and determines the carminic acid concentration, the azotic material concentration; And compare with cochineal dye that has not treated with the papayin, the results show that after the papayin hydrolisis, in the cochineal dye, macro-molecule protein degenerated in some extent, because in the cochineal dye, much of sensitization material are the macro-molecule protein, therefore, the papayin hydrolisis can eliminates the sensitization protein of cochineal dye.
(4) To assess the free radical scavenging capacity of carminic acid, the research was carried out to investigate the free radical (including DPPH·, ABTS+·and O_2ˉ·) scavenging activity of it, make comparison of free radical scavenging activity between carminic acid and ascorbic acid. The result showed thatBoth carminic acid and ascorbic acid have free radical scavenging activity for DPPH·, ABTS+·and O_2ˉ·. Carminic acid’s can clean away 83% DPPH·in solution, 85% ABTS+·in solution, 46% of O_2ˉ·in solution. It’s show that carminic acid is a food addictive which has free radical scavenging activity.
通过比较不同配比的流动相,不同流速和温度对出峰效果的影响,建立胭脂红酸的高效液相色谱检测方法,并与常用的分光光度法作比较,为胭脂红酸的分析检测提供参考依据。通过检测分析、比较胭脂虫(滇中)与胭脂虫(秘鲁)的堆密度、单重虫粒数、吸水率和体积膨胀率等物理指标,以及两地胭脂虫干虫体中四氯化碳提取物、乙醇提取物、石油醚提取物、总蛋白质、胭脂红酸、不溶性糖类和灰分等主要化学组分的含量,评估了我国滇中地区繁养的胭脂虫质量;并对引进国内后在滇中地区放养的不同世代的胭脂虫主要化学组分进行了分析对比,为我国繁养胭脂虫,并进一步开发利用胭脂虫资源提供了理论依据。通过进一步优化了胭脂虫红色素微波辅助提取的工艺参数,以及开发微波-超声波协同萃取胭脂虫红色素的工艺,为胭脂虫红色素的快速提取进一步充实理论依据。应用酶水解法与超滤膜法精制色素,除去胭脂虫红色素中可溶性大分子蛋白质,以达到除去致敏源蛋白质。研究了胭脂红酸作为功能性食品对自由基的清除能力,为利用胭脂虫红色素打下基础。
(1)高效液相色谱检测胭脂红酸试验表明:流动相为甲醇-乙腈-磷酸(0.01%)的混合液(体积比为10:15:75),流速为0.8 mL /min,柱温为20℃,出峰效果最好。纯度为100%的胭脂红酸作为标准样品,绘制标准曲线,280 nm和494 nm的检测波长下分别得回归方程Y=60.7360X,R~2=0.9999;Y=8.6048X,R~2=0.9990,两者线性关系均良好。以280 nm和494 nm为检测波长,使用纯度为100%的胭脂红酸验证仪器的精密度,自制胭脂虫红色素验证稳定性和加标回收率。测得精密度相对标准偏差分别为0.42%和0.54%,稳定性相对标准偏差分别为0.16%和0.32%,加标回收率分别为96.8%和94.3%。以Sigma公司生产的胭脂红酸(纯度为96%)为标样,分别用高效液相色谱测峰面积和用分光光度法测吸光度,分别绘制标准曲线方程得:Y=7.8260X,R~2=0.9988和Y=0.0137X,R~2=0.9997浓度在9.5～95.0μg/mL的范围内线性相关性良好。并分别用高效液相色谱法和分光光度法对10种色素样品进行定量分析,分光光度法所得结果较高效液相色谱结果偏高,但两种方法所得结果经单因素方差分析,显著性指标P值为0.904,即两种方法测定结果差异性不显著。两种方法都可以用于定量分析胭脂红酸样品中的胭脂红酸。
作为工业昆虫原料,我国引进的胭脂虫及不同繁养世代实验表明:胭脂虫(滇中)与秘鲁(胭脂虫的原产地)胭脂虫相比,除堆密度含量差异较小外,其余物理参数和主要化学组分差异性均较大,但滇中地区繁养的胭脂虫虫体中胭脂红酸的含量仍较高,作为生产加工胭脂红酸的原料,仍具有较高的开发利用价值。滇中地区繁养的不同世代的胭脂虫,虽然胭脂虫各组分有所差异,但胭脂红酸含量均较高,不同世代之间差异不显著,因此,三代胭脂虫作为生产加工胭脂虫红色素的原料,均具有较高的开发利用价值。
(2)胭脂虫红色素微波辅助提取实验表明:微波处理前常温预浸泡40min,处理后75℃水浴浸提40min最适宜于色素提取。在单因素试验的基础上,对液料比,微波功率及微波辐射时间进行三因素三水平响应面分析试验,得到最优条件为液料比7.5,微波功率714W,辐射时间73s。最优条件下,合适的提取级数为12次。对胭脂虫红色素进行微波-超声波协同提取,在单因素试验的基础上,对微波功率、处理时间、液料比及超声波功率进行四因素三水平响应面分析试验,得到最优条件为微波功率464W,处理时间为18.48min,液料比为8.23,超声波功率627W。最优条件下,合适的提取级数为4次。微波提取与微波-超声波协同提取法相比,前者能耗较少,但所需时间长,提取级数较多,在生产实践中,可按不同的需求和所具备的条件选择适当的方法。
(3)对提取的胭脂虫红色素进行木瓜蛋白酶水解实验,将水解后的胭脂虫红色素溶液依次透过100KDa、30KDa、10KDa、3KDa及1Kda的超滤膜,分别测定各截留液的体积,溶液中胭脂红酸浓度,含氮物质浓度,计算不同分子截留液中胭脂红酸及含氮物质的含量;并与未经木瓜蛋白酶水解的胭脂虫红色素溶液在相同条件处理后的参数作比较,发现经过木瓜蛋白酶水解,胭脂虫红色素中的大分子蛋白质被一定程度的降解,由于胭脂虫红色素中的致敏性物质多为大分子蛋白质,因此,木瓜蛋白酶水解对清除胭脂虫红色素的致敏性蛋白质,会起到一定的作用。
(4)为了评估胭脂红酸清除自由基能力,以DPPH·、ABTS+·及O_2ˉ·等3种自由基为试验对象,通过比较特定波长下自由基溶液吸光度的变化,评估胭脂红酸清除自由基的能力,并与抗坏血酸清除3种自由基的能力做比较。胭脂红酸清除自由基结果表明:胭脂红酸与抗坏血酸对3种自由基DPPH·、ABTS+及O_2ˉ·均具有清除活性;当胭脂红酸达到一定浓度时,对DPPH·、ABTS+·及O_2ˉ·的清除率分别可达83%、85%及46%左右。表明胭脂红酸是一种对自由基具有一定清除活性的食品添加剂。
Cochineal (Dactylopius coccus Costa) dye can be used in food, medicine and cosmetics in the United States Food and Drug Administration. There are little research on the pigment in our country. Experiment parameters of the extraction of cochineal dye were optimized. For purification method, the method of ultrafiltration membrane and silica gel column chromatograph were used, and determine the main ingredient. The factors affecting pigment stability are discussed.
The main results were as follows:
(1) when mobile phase was consist of acetonitrile, methanol and phosphoric acid (0.01%) (volume ratio 10:15: 75), flow rate was 0.8 mL/min, column temperature at 20℃, peak shape was the best. This mothed used for determined car- minic acid both at 280 nm and 494 nm. Carminic acid(purity:100%) was utilized as standard sample, made standard curve, obtain regression equation Y=8.6048X, R~2=0.9990, and Y=60.7360X,R~2=0.9999, respectively. Both equa-tion’s linear relationship were fine. when determine cochineal red pigment, the result showed that in this method, sam- ple had good stability(determined at 280 nm, RSD=0.16%; at 494 nm, RSD= 0.32%), equipment had high precision(determined at 280 nm, RSD=0.42%; at 494 nm,RSD=0.54%) and sample had high recovery rate(determined at 280 nm, recovery rate was 96.8, at 494 nm was 94.3).compare high-performance liquid chromatography (HPLC) and spectrophotometry to determine the purity of carminic acid samples, these two methods make quantitative analysis of 10 kinds of samples, the result of spectrophotometry measurements is slightly higher than high-performance liquid chromatography, but the results of the two methods no insignificant difference.
Significant differences were exist between cochineal cultivated in middle area of Yunnan province and in Peru, except bulk density. But the carminic acid contents is high. The conclusion is that Cochineal cultivated in middle area of Yunnan province can also used for making carminic acid. Through compare cochineal of three generations, the results show that even there some difference existi in cochineal of three generations, but the difference of carminic acid contents are not significant, so cochineal of three generations can all used for make cochineal dye.
(2) In order to develop a method for microwave-assisted extract cochineal dye, single-factor test was carried out. The results show that cochineals should soak in water for 40min before microwave treatment, and extract with water for 40min after microwave treatment. On the basis of single-factor test, the microwave-assisted extraction process parameters about extraction of cochineal dye were optimized with response surface method. The results suggest that the optimal parameters should as follows: ratios of liquid to material 7.5, microwave power 714W, microwave treatment time 73s. Under this conditon, the best extracting times is 12.
In order to develop a method for microwave - ultrasonic synergistic extract cochineal dye, On the basis of single-factor test (included microwave power, treatment time, ratios of liquid to material and ultrasonic power), optimized extraction process parameters with response surface method, best condition should as follows: microwave power 464W, treatment time is 18.48min, ratios of liquid to material 8.23, ultrasonic power 627W. Under this conditions, the suitable extraction times is 4.
Compare the microwave-assisted extract mathod and microwave-ultrasonic extract mathod, we can find out that use microwave-assisted extract mathod, energy consumption are lower than microwave-ultrasonic extract mathod. But time consumption is longer, and extraction times is more than microwave-ultrasonic extract mathod.
(3) Utilize the papayin hydrolisis cochineal dye, then penetrates the cochineal dye through the 100KDa, 30KDa, 10KDa, 3KDa and the 1Kda ultra filter. Determines the volume of different solution, and determines the carminic acid concentration, the azotic material concentration; And compare with cochineal dye that has not treated with the papayin, the results show that after the papayin hydrolisis, in the cochineal dye, macro-molecule protein degenerated in some extent, because in the cochineal dye, much of sensitization material are the macro-molecule protein, therefore, the papayin hydrolisis can eliminates the sensitization protein of cochineal dye.
(4) To assess the free radical scavenging capacity of carminic acid, the research was carried out to investigate the free radical (including DPPH·, ABTS+·and O_2ˉ·) scavenging activity of it, make comparison of free radical scavenging activity between carminic acid and ascorbic acid. The result showed thatBoth carminic acid and ascorbic acid have free radical scavenging activity for DPPH·, ABTS+·and O_2ˉ·. Carminic acid’s can clean away 83% DPPH·in solution, 85% ABTS+·in solution, 46% of O_2ˉ·in solution. It’s show that carminic acid is a food addictive which has free radical scavenging activity.
引文
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