花椒籽黑色素提取纯化及性质研究
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摘要
花椒(Zanthoxylum bungeanum Maxim),是我国特有的食用香辛料之一,在我国栽培历史悠久,分布地域辽阔,品种资源丰富。花椒籽是生产调味料花椒的副产物。花椒籽种皮富含天然黑色素。花椒籽榨油后的饼粕颜色深褐,是提取天然黑色素的优质原料。本研究以花椒籽油粕为原料,研究了花椒籽黑色素提取纯化技术及其理化功能性质。主要研究结果如下:
(1)采用中心组合试验优化了花椒籽黑色素的浸提工艺。结果表明:影响花椒籽黑色素提取效果的因素依次为温度>NaOH浓度>料液比,花椒籽黑色素提取的优化工艺参数为NaOH浓度为1.20mol/L,料液比为1∶24.64,温度为70℃下提取2h,提取两次。所得花椒籽色素粗品为棕黑色无定型粉末,其得率为6.1%,色价为201.62。
(2)采用碱性蛋白酶酶解和Sevag法联用可有效脱除花椒籽黑色素粗品中的蛋白质。碱性蛋白酶脱蛋白条件为温度50℃,pH 9,加酶量为粗色素溶液(浓度为1mg/mL)体积的6%,再用Sevag法处理三次,蛋白质脱除率可达81.23%,花椒籽黑色素色价可提高50%。
(3)经聚酰胺凝胶层析分离纯化所得的花椒籽黑色素色价为364.58,是未纯化前色素粗品色价的1.8倍。纯化后的花椒籽黑色素在紫外光区250～290nm处有弱的吸收肩峰,可见光区无吸收,FT-IR图谱分析其结构含有羧基、羟基和苯环等官能团。
(4)花椒籽黑色素易溶于碱性水溶液中,其水溶液在pH<5.0时产生沉淀,pH>7时稳定性好;在碱性条件下该色素对光、热稳定;耐氧化和耐还原;食盐和蔗糖对其影响微小;K+对色素稳定性没有明显影响,Mn2+对色素具有保护或增色作用,Fe2+,Al3+,Zn2+,Cu2+,Pb2+使色素溶液沉淀;在10～500μg/mL的浓度范围内,花椒籽黑色素体外清除·OH的能力远远强于相同浓度的VC和茶多酚,当浓度为500μg/mL时,黑色素对·OH的清除率已超过90%,远远大于同浓度的VC的37.96%和茶多酚的29.39%。在0.1～1mg/mL的浓度范围内,花椒籽黑色素的总还原力低于相同浓度的VC和茶多酚。花椒籽黑色素稳定性及抗氧化性良好,具有开发潜力。
Zanthoxylum bungeanum Maxim is one kind of special spice in China. It has a long history of cultivation , large geographical distribution and rich variety resource. Zanthoxylum bungeanum seeds are the by-products of pepper sauce. The seeds contain plentiful functional oil, the seed coat contains natural melanin. The greaves which left after exploiting the oil from Zanthoxylum bungeanum seeds are the high-quality raw materials to extract the natural melanin. In order to demonstrate the utilization of this kind of melanin resource, this thesis studied on the extraction, isolation, purification of melanins from Zanthoxylum bungeanum seeds, discussed its stabilities, functional characteristics, and its prime structure.
The main results of this paper:
(1) The second-order regression rotation design was carried out to optimize the extraction processing. The results showed that the sequence of factors that worked on melanin extraction was as below: temperature > NaOH concentration > the ratio of material to reagent; the melanin was suitable to be extracted twice with 1.20mol/ L NaOH at 70℃for 2h, and the ratio of material to reagent was 1∶24.64. 6.1% yield of melanin was obtained, and the color value of melanin extracted from Zanthoxylum bungeanum seeds was 201.62.
(2) The protein from melanin was suitable to be removed by Alcalase-Sevag methods. The optimum conditions that suitable for alcalase enzymolysis to remove the protein from melanin was temperature 50℃, pH 9, enzyme amount 6% (which meant 6% of raw melanin solution volume)and then it should be treated three times by Sevag method.The protein removal rate reached 81.23%, and its color value can be enhanced by 50%.
(3) The melanin extracted from Zanthoxylum bungeanum seeds was refined by polyamide Chromatography. The color value of purified melanin was 364.58, 1.8 times higher, compared with the raw melanin. Based on the experiment, the ingredient with the strongest tinting power was scaned by ultraviolet absorption spectra and infrared absorption spectra. The results showed melanin had a weak absorption platform in the range of 250～290nm wavelength and no absorption in the visible range; FT-IR spectra showed its structure contains hydroxyl group connected to benzene and carboxyl group.
(4) The melanin from Zanthoxylum bungeanum seeds was easy to dissolve in Alkaline water, the aqueous solution got turbid when pH<5.0 and remaind clear and stable when pH>7.0.It was relatively stable to light indoor and heat when the pH >7 for a short time.; the anti-oxidation ability to H2O2 and anti-reduction ability to Na2SO3 were relatively good; the effects of sodium chloride and saccharose on the melanin’s stability were limited; K+ had no significant effect on its stability, Mn2+ had a good protective effect on it, but Fe2+,Al3+,Zn2+,Cu2+,Pb2+ can deposit the melanin; scavenging activity of the melanin on·OH was much higher than that of VC and tea polyphenol but the reducing power was lower than that of later them. In a word, the melanin from Zanthoxylum bungeanum seeds is worth to develop for its high stability and antioxidant activity.
(1)采用中心组合试验优化了花椒籽黑色素的浸提工艺。结果表明:影响花椒籽黑色素提取效果的因素依次为温度>NaOH浓度>料液比,花椒籽黑色素提取的优化工艺参数为NaOH浓度为1.20mol/L,料液比为1∶24.64,温度为70℃下提取2h,提取两次。所得花椒籽色素粗品为棕黑色无定型粉末,其得率为6.1%,色价为201.62。
(2)采用碱性蛋白酶酶解和Sevag法联用可有效脱除花椒籽黑色素粗品中的蛋白质。碱性蛋白酶脱蛋白条件为温度50℃,pH 9,加酶量为粗色素溶液(浓度为1mg/mL)体积的6%,再用Sevag法处理三次,蛋白质脱除率可达81.23%,花椒籽黑色素色价可提高50%。
(3)经聚酰胺凝胶层析分离纯化所得的花椒籽黑色素色价为364.58,是未纯化前色素粗品色价的1.8倍。纯化后的花椒籽黑色素在紫外光区250～290nm处有弱的吸收肩峰,可见光区无吸收,FT-IR图谱分析其结构含有羧基、羟基和苯环等官能团。
(4)花椒籽黑色素易溶于碱性水溶液中,其水溶液在pH<5.0时产生沉淀,pH>7时稳定性好;在碱性条件下该色素对光、热稳定;耐氧化和耐还原;食盐和蔗糖对其影响微小;K+对色素稳定性没有明显影响,Mn2+对色素具有保护或增色作用,Fe2+,Al3+,Zn2+,Cu2+,Pb2+使色素溶液沉淀;在10～500μg/mL的浓度范围内,花椒籽黑色素体外清除·OH的能力远远强于相同浓度的VC和茶多酚,当浓度为500μg/mL时,黑色素对·OH的清除率已超过90%,远远大于同浓度的VC的37.96%和茶多酚的29.39%。在0.1～1mg/mL的浓度范围内,花椒籽黑色素的总还原力低于相同浓度的VC和茶多酚。花椒籽黑色素稳定性及抗氧化性良好,具有开发潜力。
Zanthoxylum bungeanum Maxim is one kind of special spice in China. It has a long history of cultivation , large geographical distribution and rich variety resource. Zanthoxylum bungeanum seeds are the by-products of pepper sauce. The seeds contain plentiful functional oil, the seed coat contains natural melanin. The greaves which left after exploiting the oil from Zanthoxylum bungeanum seeds are the high-quality raw materials to extract the natural melanin. In order to demonstrate the utilization of this kind of melanin resource, this thesis studied on the extraction, isolation, purification of melanins from Zanthoxylum bungeanum seeds, discussed its stabilities, functional characteristics, and its prime structure.
The main results of this paper:
(1) The second-order regression rotation design was carried out to optimize the extraction processing. The results showed that the sequence of factors that worked on melanin extraction was as below: temperature > NaOH concentration > the ratio of material to reagent; the melanin was suitable to be extracted twice with 1.20mol/ L NaOH at 70℃for 2h, and the ratio of material to reagent was 1∶24.64. 6.1% yield of melanin was obtained, and the color value of melanin extracted from Zanthoxylum bungeanum seeds was 201.62.
(2) The protein from melanin was suitable to be removed by Alcalase-Sevag methods. The optimum conditions that suitable for alcalase enzymolysis to remove the protein from melanin was temperature 50℃, pH 9, enzyme amount 6% (which meant 6% of raw melanin solution volume)and then it should be treated three times by Sevag method.The protein removal rate reached 81.23%, and its color value can be enhanced by 50%.
(3) The melanin extracted from Zanthoxylum bungeanum seeds was refined by polyamide Chromatography. The color value of purified melanin was 364.58, 1.8 times higher, compared with the raw melanin. Based on the experiment, the ingredient with the strongest tinting power was scaned by ultraviolet absorption spectra and infrared absorption spectra. The results showed melanin had a weak absorption platform in the range of 250～290nm wavelength and no absorption in the visible range; FT-IR spectra showed its structure contains hydroxyl group connected to benzene and carboxyl group.
(4) The melanin from Zanthoxylum bungeanum seeds was easy to dissolve in Alkaline water, the aqueous solution got turbid when pH<5.0 and remaind clear and stable when pH>7.0.It was relatively stable to light indoor and heat when the pH >7 for a short time.; the anti-oxidation ability to H2O2 and anti-reduction ability to Na2SO3 were relatively good; the effects of sodium chloride and saccharose on the melanin’s stability were limited; K+ had no significant effect on its stability, Mn2+ had a good protective effect on it, but Fe2+,Al3+,Zn2+,Cu2+,Pb2+ can deposit the melanin; scavenging activity of the melanin on·OH was much higher than that of VC and tea polyphenol but the reducing power was lower than that of later them. In a word, the melanin from Zanthoxylum bungeanum seeds is worth to develop for its high stability and antioxidant activity.
引文
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