水溶性红曲黄色素的分离纯化与结构鉴定
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摘要
红曲色素是天然色素的重要组成部分。近年来,随着食品安全意识深入人心,红曲色素以其安全,稳定,低廉的优势而备受关注。然而,我国红曲色素产品单一,生产水平低,品质不稳定等方面,成为了中国红曲产品拓展国际市场的巨大障碍。因此,开发新型天然色素,改善红曲色素品质,提高产品竞争力,成为我国食品添加剂行业的发展重点。
“水溶性红曲黄色素”的开发,拓展了红曲色素产品的应用领域,标志着我国红曲色素产品研发水平进入了一个新阶段。但是,该红曲色素衍生物的分子结构尚不明确,给产品的推广应用及其工业化技术革新带来了一定障碍。本文以分离纯化水溶性红曲黄色素为基础,鉴定并阐明各组分的分子结构为目标;笔者以工业化生产的水溶性红曲黄色素为原料,采用薄层层析法,高效液相色谱法,光电二极管阵列检测器–蒸发光散射检测器联用技术,液相色谱–电喷雾质谱仪联用技术,多级质谱技术,高分辨质谱技术等实验方法,经过了大量的实验,确立了较优的色谱质谱条件,完成了水溶性红曲黄色素6个主体组分的分离纯化与结构鉴定工作。主要研究结果如下:
1.建立了水溶性红曲黄色素的薄层色谱分离条件。以色谱级甲醇为提取剂,三氯甲烷:甲醇=1:1(v/v)为展开剂,展开30分钟,能够有效地分离水溶性红曲黄色素。
2.依据薄层色谱的初次分离得到的不同色带,利用高效液相色谱法进一步分离纯化,并确立了较优的色谱条件。同时,光电二极管阵列检测器–蒸发光散射检测器联用条件的确立,为液质联用仪中的液相色谱部分奠定了基础。采用美国Waters公司Sunfire C18柱(4.6 mm×150 mm,5μm),0.8 ml/min流速,甲醇和水梯度淋洗(见表3.1);蒸发光散射检测器要求气体压力25 psi,喷雾器温度45℃,漂移管温度60℃,增益为1。
3.多种质谱技术的应用,确定了不同组分的分子量和元素组成(见表5.1),从而为各组分分子结构的分析以及裂解途径的推断提供了可靠的信息来源。
该论文的完成,深化了我们对水溶性红曲黄色素结构层次的认识,为加快工业化技术创新,扩大产品的应用推广,稳定产品品质奠定基础。同时,也为相关标准的制定提供依据和参考,为改善人们的生活品质具有促进作用。
Monascus pigment is an important part of the natural pigment. In recent years, with the food safety awareness deeply rooted in us, pigments from Monascus have been much concerned with their security, stability, low-cost advantage. However, some questions such as the product of a single, low quality and instability have made them lost parts of international market. Therefore, to develop new natural pigments has become the focus of development of food additives industry.
The development of "Water-soluble Monascus yellow pigments" may expand the range of these products’usage. At the same time, it indicates that China R & D level of Monascus pigment into a new stage. However, the molecular structure of these pigment derivatives are not clear. And it is a resistance barrier for application of these pigments.The author aims to structure identificating of the Water-soluble Monascus yellow pigments by thin layer chromatography, high performance liquid chromatography, photodiode array detector - evaporative light scattering detector coupled technique, tandem mass spectrometry. Finally, the auther have got 6 main component of Water-soluble Monascus yellow pigment (see Chapter 5, Table 5.1).The main research content were as follows:
1.TLC was established to separate the components of Water-soluble Monascus yellow pigment.With methanol as extracting solvent, chloroform and methanol (v/v) was used as developing solvent of these pigments. After 30 minutes, Water-soluble Monascus pigments can be effectively separated.
2. Based on the crude separation by TLC, the optimum chromatographic conditions of HPLC were established which is with Waters Sunfire C18 column (4.6 mm×150 mm, 5μm), 0.8 ml / min, gradient elution of methanol and water (see Table 3.1) and so on. At the same time, the hyphenated technique of photodiode array detector with evaporative light scattering detector provides a basis for LC-MS.
3. The application of a variety of mass spectrometry techniques provided a reliable source of information for each component of the molecular structure and fragmentation pathway.
The completion of this paper is good at understanding of their structure, expanding product applications. It also will contribute to provide a reference for related standards and to improve the quality of life of people.
“水溶性红曲黄色素”的开发,拓展了红曲色素产品的应用领域,标志着我国红曲色素产品研发水平进入了一个新阶段。但是,该红曲色素衍生物的分子结构尚不明确,给产品的推广应用及其工业化技术革新带来了一定障碍。本文以分离纯化水溶性红曲黄色素为基础,鉴定并阐明各组分的分子结构为目标;笔者以工业化生产的水溶性红曲黄色素为原料,采用薄层层析法,高效液相色谱法,光电二极管阵列检测器–蒸发光散射检测器联用技术,液相色谱–电喷雾质谱仪联用技术,多级质谱技术,高分辨质谱技术等实验方法,经过了大量的实验,确立了较优的色谱质谱条件,完成了水溶性红曲黄色素6个主体组分的分离纯化与结构鉴定工作。主要研究结果如下:
1.建立了水溶性红曲黄色素的薄层色谱分离条件。以色谱级甲醇为提取剂,三氯甲烷:甲醇=1:1(v/v)为展开剂,展开30分钟,能够有效地分离水溶性红曲黄色素。
2.依据薄层色谱的初次分离得到的不同色带,利用高效液相色谱法进一步分离纯化,并确立了较优的色谱条件。同时,光电二极管阵列检测器–蒸发光散射检测器联用条件的确立,为液质联用仪中的液相色谱部分奠定了基础。采用美国Waters公司Sunfire C18柱(4.6 mm×150 mm,5μm),0.8 ml/min流速,甲醇和水梯度淋洗(见表3.1);蒸发光散射检测器要求气体压力25 psi,喷雾器温度45℃,漂移管温度60℃,增益为1。
3.多种质谱技术的应用,确定了不同组分的分子量和元素组成(见表5.1),从而为各组分分子结构的分析以及裂解途径的推断提供了可靠的信息来源。
该论文的完成,深化了我们对水溶性红曲黄色素结构层次的认识,为加快工业化技术创新,扩大产品的应用推广,稳定产品品质奠定基础。同时,也为相关标准的制定提供依据和参考,为改善人们的生活品质具有促进作用。
Monascus pigment is an important part of the natural pigment. In recent years, with the food safety awareness deeply rooted in us, pigments from Monascus have been much concerned with their security, stability, low-cost advantage. However, some questions such as the product of a single, low quality and instability have made them lost parts of international market. Therefore, to develop new natural pigments has become the focus of development of food additives industry.
The development of "Water-soluble Monascus yellow pigments" may expand the range of these products’usage. At the same time, it indicates that China R & D level of Monascus pigment into a new stage. However, the molecular structure of these pigment derivatives are not clear. And it is a resistance barrier for application of these pigments.The author aims to structure identificating of the Water-soluble Monascus yellow pigments by thin layer chromatography, high performance liquid chromatography, photodiode array detector - evaporative light scattering detector coupled technique, tandem mass spectrometry. Finally, the auther have got 6 main component of Water-soluble Monascus yellow pigment (see Chapter 5, Table 5.1).The main research content were as follows:
1.TLC was established to separate the components of Water-soluble Monascus yellow pigment.With methanol as extracting solvent, chloroform and methanol (v/v) was used as developing solvent of these pigments. After 30 minutes, Water-soluble Monascus pigments can be effectively separated.
2. Based on the crude separation by TLC, the optimum chromatographic conditions of HPLC were established which is with Waters Sunfire C18 column (4.6 mm×150 mm, 5μm), 0.8 ml / min, gradient elution of methanol and water (see Table 3.1) and so on. At the same time, the hyphenated technique of photodiode array detector with evaporative light scattering detector provides a basis for LC-MS.
3. The application of a variety of mass spectrometry techniques provided a reliable source of information for each component of the molecular structure and fragmentation pathway.
The completion of this paper is good at understanding of their structure, expanding product applications. It also will contribute to provide a reference for related standards and to improve the quality of life of people.
引文
[1]甘纯玑,彭时尧,苏金为等.红曲色素及其水溶性衍生物的开发[J].中国食品用化学品,1998,(2):35-37.
[2]甘纯玑,彭时尧,苏金为等.水溶性红曲红色素和黄色素的制备.中国发明专利申请号. 96122279.4(1996)
[3]许赣荣.红曲桔霉素的检测及发酵控制技术[D].江南大学,无锡市青山湾2004.
[4] Yii-Lih Lin, Teng-Hsu Wang, Min-Hsiung Lee, et al. Biologically active components and nutraceuticals in the Monascus-fermented rice[J]. Appl Microbiol Biotechnol, 2008( 77):965–973.
[5]广井忠夫.红曲红曲菌的形态和红曲色素的纯化[A].2000东方红曲国际学士研讨会论文集[D].2000:27-35.
[6] Ashok Pandey,Carlos Ricardo 8occol,Christian Larroche. Current Developments in Solid-state Fermentation[M].India: Springer Science+Business Media, 2008.344.
[7] Hassan Hajjaj. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance[J]. Applied Environmental Microbiology,1999, 65(1): 311-313.
[8]甘纯玑,蔡春燕.薄层色谱法分离红曲色素[J].精细化工,1989,6(5):51-53.
[9]张慧娟,陶冠军,陈蕴等.红曲色素的制备及HPLC和LC/MS检测方法[J].食品研究与开发,2006,27(4):112-115.
[10]何丽一,平面色谱方法及应用[M].第2版.北京:化学工业出版社,2005.
[11]云自厚,欧阳津,张晓彤.液相色谱检测方法[M].第2版.北京:化学工业出版社,2005.
[12]汪正范.色谱定性与定量[M].第2版.北京:化学工业出版社,2007.
[13] R. Martin Smith. Understanding mass spectra : a basic approach[M].2nd ed. USA: John Wiley & Sons, Inc.2004.2.
[14]何美玉.现代有机质谱与生物质谱[M].北京:北京大学出版社,2002.
[15] Rolf Ekman, et al. Mass spectrometry : instrumentation, interpretation, andapplications[M].USA: John Wiley & Sons, Inc.2009.28.
[16]汪正范,杨树民,吴侔天等.色谱联用技术[M].第2版.北京:化学工业出版社,2007.
[17]王光辉,熊少祥.有机质谱解析[M].北京:化学工业出版社,2006.
[18]郭芳,郭东川,李钟庆.红曲菌的色素与产生菌[J].微生物学杂志,2004,24(6):11-14.
[19] Birendra N. Pramanik, A. K. Ganguly, Michael L. Gross.Applied Electrospray Mass Spectrometry [M].USA: Marcel Dekker, 2002.
[20]于世林.高效液相色谱方法及应用[M].第2版.北京:化学工业出版社,2005.
[21] Yii-Lih Lin, Teng-Hsu Wang, Min-Hsiung Lee, et al. Biologically active components and nutraceuticals in the Monascus-fermented rice[J]. Appl Microbiol Biotechnol, 2008( 77):965–973.
[22]衣珊珊,沈昌,韩永斌等.红曲色素形成机理及提供色价的途径[J].食品科学,2005,26(7):256-261.
[23]S. S. Teng, W. Feldheim. Analysis of Anka Pigments by Liquid Chromatography with Diode Array Detection and Tandem Mass Spectrometry[J]. Chromatographia. 1998.47(9/10):529-236.
[24]夏明,杜琪珍.高速逆流色谱提取分离红曲色素的研究[J].广州食品工业科技.2003.18(4):5-6.
[25]赵燕,温辉梁,胡晓波.红曲色素及其在食品工业中的应用[J].中国食品添加剂, 2004, (4): 90-93.
[26] Endo A. Monacloin K a new hypochlespe-rolemic agent produced by a Monascus spiecies[J]. Journal of Antibiotics, 1985, 38: 420-422.
[27]沈士秀.红曲的研究、生产及应用[J].食品工业科技,2001, (1): 85-87.
[28] Jun ogihara,Jun kato, et al. Biosythesis of PP-V, a monascorubramine homologue,by Penicillium sp.A Z[J]. Journal of Bioscience and Bioengineering, 2000,90(6): 678-680.
[29] Hassan Hajjaj, Alain Klaebe, etal. Production and identification on N-Glucosylrubropuncta mine and N-Glu- cos ylmonascorubra mine from Monascus ruber and Occurrence of electron Donor Acceptor complexes in these red pigments[J].Applied of Environmental Microbiology, 1997, 63(7): 2671-2678.
[30] Hassan Hajjaj. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance[J]. Applied Environmental Microbiology, 1999, 65(1): 311-313.
[31] Rasheva T, Kujumdzieva A,et al. Lipid production by Monascus purpureus albino strain [J]. Journal of Biotechnology, 1997, 56: 217-224.
[32] Hopwood D A. Genetic contributions to understanding polyketide synthases[J]. Chemistry Review, 1997, (7): 2465-2498.
[33] Hendrickson L,Ray D C, et al. Lovastatin biosynthesis in Aspergillus terreus:characterization of blocked mutants,enzyme activities and a multifunctional polyketide synthase gene[J].Chemistry and Biology, 1999, 6(7): 429-438.
[34]李浩然,杜竹玮,张剑锐.红曲色素稳定性研究[J].食品科学, 2003, 24(11): 59-62.
[35] Fabre C E, Santerre A L, Loret M O, et al. Production and food application of the red pigments of Monascus ruber[J]. J Food Science, 1993, 58(5): 1099-1102.
[36] Lee Y K. Production of Monascus pigments by a solid-liquid state culture method[J]. Journal of Fermentation and Bioengineering, 1995, 79(5): 516-518.
[37] Pastrana L.Estimation of bioprocess variables from Monascus ruber cultures by means of stoichiomet -ric Models[J].Process Biochemistry, 1995, 30(7): 607-613.
[38] Shin C S, Kim H J, Kim M J, et al. Morphological change and enhanced pigment production of Monascus when cocultured with Saccharomyces cerevisiae or Aspergillus oryzae[J].Biotechnology.and Bioengineering, 1998, 59: 576-581.
[39] Suh J H, Shin C S. Physiological analysis on novel coculture of Monascus sp. J101 with Saccharomyces cerevisiae[J]. FEMS Microbiology Letters, 2000, 190: 241-245.
[40] Suh J H, Shin C S. Analysis of the morphologic changes of Monascus sp. J101 cells cocultured with Saccharomyces cerevisiae [J]. FEMS Microbiology Letters, 2000, 193: 143-147.
[41]连喜军,王红娟,王昌禄,等.各类抗氧化剂对红曲红色素中红色素的护色作用[J].中国食品添加剂, 2004, (1):61-62,60.
[42]杨胜利,杨海麟,王龙刚,等.超声波处理对红曲色素产量的影响[J].无锡轻工大学学报, 2003, 22(1): 99-101.
[43] Xijun Lian, Changlu Wang, Kunliang Guo. Identification of new red pigments produced by Monascus rubber[J]. Dyes and Pigments, 2007(73): 121-125.
[44] Sonia Campoy, Angel Rumbero, Juan F. Martín. Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures[J]. Appl Microbiol Biotechnol, 2006(70): 488–496.
[45] P Juzlova, L Martinkova, V Kren. Secondary metabolites of the fungus Monascus: a review[J]. Journal of Industrial Microbiology , 1996(16), 163-170.
[46] Yunquan Zheng, Yawen Xin, Yanghao Guo. Study on the fingerprint profile of Monascus products with HPLC–FD, PAD and MS[J]. Food Chemistry, 2009(113): 705–711.
[2]甘纯玑,彭时尧,苏金为等.水溶性红曲红色素和黄色素的制备.中国发明专利申请号. 96122279.4(1996)
[3]许赣荣.红曲桔霉素的检测及发酵控制技术[D].江南大学,无锡市青山湾2004.
[4] Yii-Lih Lin, Teng-Hsu Wang, Min-Hsiung Lee, et al. Biologically active components and nutraceuticals in the Monascus-fermented rice[J]. Appl Microbiol Biotechnol, 2008( 77):965–973.
[5]广井忠夫.红曲红曲菌的形态和红曲色素的纯化[A].2000东方红曲国际学士研讨会论文集[D].2000:27-35.
[6] Ashok Pandey,Carlos Ricardo 8occol,Christian Larroche. Current Developments in Solid-state Fermentation[M].India: Springer Science+Business Media, 2008.344.
[7] Hassan Hajjaj. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance[J]. Applied Environmental Microbiology,1999, 65(1): 311-313.
[8]甘纯玑,蔡春燕.薄层色谱法分离红曲色素[J].精细化工,1989,6(5):51-53.
[9]张慧娟,陶冠军,陈蕴等.红曲色素的制备及HPLC和LC/MS检测方法[J].食品研究与开发,2006,27(4):112-115.
[10]何丽一,平面色谱方法及应用[M].第2版.北京:化学工业出版社,2005.
[11]云自厚,欧阳津,张晓彤.液相色谱检测方法[M].第2版.北京:化学工业出版社,2005.
[12]汪正范.色谱定性与定量[M].第2版.北京:化学工业出版社,2007.
[13] R. Martin Smith. Understanding mass spectra : a basic approach[M].2nd ed. USA: John Wiley & Sons, Inc.2004.2.
[14]何美玉.现代有机质谱与生物质谱[M].北京:北京大学出版社,2002.
[15] Rolf Ekman, et al. Mass spectrometry : instrumentation, interpretation, andapplications[M].USA: John Wiley & Sons, Inc.2009.28.
[16]汪正范,杨树民,吴侔天等.色谱联用技术[M].第2版.北京:化学工业出版社,2007.
[17]王光辉,熊少祥.有机质谱解析[M].北京:化学工业出版社,2006.
[18]郭芳,郭东川,李钟庆.红曲菌的色素与产生菌[J].微生物学杂志,2004,24(6):11-14.
[19] Birendra N. Pramanik, A. K. Ganguly, Michael L. Gross.Applied Electrospray Mass Spectrometry [M].USA: Marcel Dekker, 2002.
[20]于世林.高效液相色谱方法及应用[M].第2版.北京:化学工业出版社,2005.
[21] Yii-Lih Lin, Teng-Hsu Wang, Min-Hsiung Lee, et al. Biologically active components and nutraceuticals in the Monascus-fermented rice[J]. Appl Microbiol Biotechnol, 2008( 77):965–973.
[22]衣珊珊,沈昌,韩永斌等.红曲色素形成机理及提供色价的途径[J].食品科学,2005,26(7):256-261.
[23]S. S. Teng, W. Feldheim. Analysis of Anka Pigments by Liquid Chromatography with Diode Array Detection and Tandem Mass Spectrometry[J]. Chromatographia. 1998.47(9/10):529-236.
[24]夏明,杜琪珍.高速逆流色谱提取分离红曲色素的研究[J].广州食品工业科技.2003.18(4):5-6.
[25]赵燕,温辉梁,胡晓波.红曲色素及其在食品工业中的应用[J].中国食品添加剂, 2004, (4): 90-93.
[26] Endo A. Monacloin K a new hypochlespe-rolemic agent produced by a Monascus spiecies[J]. Journal of Antibiotics, 1985, 38: 420-422.
[27]沈士秀.红曲的研究、生产及应用[J].食品工业科技,2001, (1): 85-87.
[28] Jun ogihara,Jun kato, et al. Biosythesis of PP-V, a monascorubramine homologue,by Penicillium sp.A Z[J]. Journal of Bioscience and Bioengineering, 2000,90(6): 678-680.
[29] Hassan Hajjaj, Alain Klaebe, etal. Production and identification on N-Glucosylrubropuncta mine and N-Glu- cos ylmonascorubra mine from Monascus ruber and Occurrence of electron Donor Acceptor complexes in these red pigments[J].Applied of Environmental Microbiology, 1997, 63(7): 2671-2678.
[30] Hassan Hajjaj. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance[J]. Applied Environmental Microbiology, 1999, 65(1): 311-313.
[31] Rasheva T, Kujumdzieva A,et al. Lipid production by Monascus purpureus albino strain [J]. Journal of Biotechnology, 1997, 56: 217-224.
[32] Hopwood D A. Genetic contributions to understanding polyketide synthases[J]. Chemistry Review, 1997, (7): 2465-2498.
[33] Hendrickson L,Ray D C, et al. Lovastatin biosynthesis in Aspergillus terreus:characterization of blocked mutants,enzyme activities and a multifunctional polyketide synthase gene[J].Chemistry and Biology, 1999, 6(7): 429-438.
[34]李浩然,杜竹玮,张剑锐.红曲色素稳定性研究[J].食品科学, 2003, 24(11): 59-62.
[35] Fabre C E, Santerre A L, Loret M O, et al. Production and food application of the red pigments of Monascus ruber[J]. J Food Science, 1993, 58(5): 1099-1102.
[36] Lee Y K. Production of Monascus pigments by a solid-liquid state culture method[J]. Journal of Fermentation and Bioengineering, 1995, 79(5): 516-518.
[37] Pastrana L.Estimation of bioprocess variables from Monascus ruber cultures by means of stoichiomet -ric Models[J].Process Biochemistry, 1995, 30(7): 607-613.
[38] Shin C S, Kim H J, Kim M J, et al. Morphological change and enhanced pigment production of Monascus when cocultured with Saccharomyces cerevisiae or Aspergillus oryzae[J].Biotechnology.and Bioengineering, 1998, 59: 576-581.
[39] Suh J H, Shin C S. Physiological analysis on novel coculture of Monascus sp. J101 with Saccharomyces cerevisiae[J]. FEMS Microbiology Letters, 2000, 190: 241-245.
[40] Suh J H, Shin C S. Analysis of the morphologic changes of Monascus sp. J101 cells cocultured with Saccharomyces cerevisiae [J]. FEMS Microbiology Letters, 2000, 193: 143-147.
[41]连喜军,王红娟,王昌禄,等.各类抗氧化剂对红曲红色素中红色素的护色作用[J].中国食品添加剂, 2004, (1):61-62,60.
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