红曲抑菌物质橙色素与桔霉素的研究
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摘要
本文从红曲的抑菌作用出发，重点研究了红曲中的橙色素和桔霉素。主要研究内容和结果如下：
(1) 利用滤纸片法和管碟法确定了红曲的抑菌谱：红曲70％乙醇和水溶液浸提物对蜡样芽孢杆菌、枯草芽孢杆菌等革兰氏阳性菌具有较强的抑制作用，对革兰氏阴性菌、霉菌和酵母菌没有抑制效果。
(2) 利用吸附柱分离三种色素，并用TLC法纯化，刮取相应的色素点，70％乙醇溶解，浓缩，进行抑菌实验，证实橙色素是主要的抑菌物质，对细菌具有较强的抑制效果，其抑菌性与其吸光度呈正比变化，橙色素对酵母菌和霉菌无抑制效果。
(3) 对橙色素进行了初步研究，橙色素可以氧化为黄色素，且氧化剂的浓度越大，黄色素生成率越高；氮源是影响橙色素产量的一个主要因素，以NH_4Cl为氮源，确定其最适添加量为0.01％。
(4) 确定了薄层层析双相展开的方法检测红曲中的桔霉素，70％乙醇提取液的展开剂分别为氯仿：丙酮(9：1)，甲苯：乙酸乙酯：甲酸(6：3：1)；水溶液提取液的展开剂为二氯甲烷：丙酮(9；1)，氯仿：甲醇：水(16：6：0.5)。该方法的最小检出量可达0.1微克，回收率和重现性均较好，且不需要复杂的提取过程，方法简单、易行，适于生产厂家使用。
(5) 利用光照、微波和氧化剂等处理方式，研究了减少红曲产品桔霉素的方法。
The dissertation focuses on the antibiotic substances produced by Monascus, orange pigment and Citrinin. The main research contents and results are as follows:
(1) Disc papers and Oxford cups were used to ascertain bacteriostatic effect of Hongqu. Using 70% ethanol and water as solvent, the extraction inhibited Bacillus subtilis, Bacillus cereus and Staphylococcus aureus and had no effect on E.coli, Salmonella. Typhi, yeasts and algae.
(2) Hongqu contains three pigments, such as red, orange, and yellow pigments. The three pigments were separated by absorption column chromatograph. Then, TLC was used to purify them, and antibiotic experiments were made to define which was the main antibiotic substance. Results showed, orange pigment was the major antibiotic substance, and it could inhibit bacteria ,but had no effect on yeasts and algae. The diameter of the inhibition zone was directly proportional to the value of absorption of orange pigment.
(3) Orange pigment could be oxidized with hydrogen peroxide to yellow pigment . Concentration of H2O2 was proportional to creation of yellow pigment. The quantity of orange pigment was influenced by the nitrogen source of the culture medium .
(4) TLC method was set up to detect Citrinin in Hongqu. Extraction by 70% ethanol , developments were performed, in one direction, with chloroform -acetone (9:l,v/v),and then toluene-ethyl acetate-formic acid (6:3:1, v/v/v). Samples was extracted by water, the extraction was developed in dichloromethane-acetone(9:1,v/v), then chloroform -methanol -water (16:6:0.5) .Minimize quantity of Citrinin detected is 0.1 u g.
(5) Study on decrease of Citrinin in Hongqu was conducted.
(1) 利用滤纸片法和管碟法确定了红曲的抑菌谱：红曲70％乙醇和水溶液浸提物对蜡样芽孢杆菌、枯草芽孢杆菌等革兰氏阳性菌具有较强的抑制作用，对革兰氏阴性菌、霉菌和酵母菌没有抑制效果。
(2) 利用吸附柱分离三种色素，并用TLC法纯化，刮取相应的色素点，70％乙醇溶解，浓缩，进行抑菌实验，证实橙色素是主要的抑菌物质，对细菌具有较强的抑制效果，其抑菌性与其吸光度呈正比变化，橙色素对酵母菌和霉菌无抑制效果。
(3) 对橙色素进行了初步研究，橙色素可以氧化为黄色素，且氧化剂的浓度越大，黄色素生成率越高；氮源是影响橙色素产量的一个主要因素，以NH_4Cl为氮源，确定其最适添加量为0.01％。
(4) 确定了薄层层析双相展开的方法检测红曲中的桔霉素，70％乙醇提取液的展开剂分别为氯仿：丙酮(9：1)，甲苯：乙酸乙酯：甲酸(6：3：1)；水溶液提取液的展开剂为二氯甲烷：丙酮(9；1)，氯仿：甲醇：水(16：6：0.5)。该方法的最小检出量可达0.1微克，回收率和重现性均较好，且不需要复杂的提取过程，方法简单、易行，适于生产厂家使用。
(5) 利用光照、微波和氧化剂等处理方式，研究了减少红曲产品桔霉素的方法。
The dissertation focuses on the antibiotic substances produced by Monascus, orange pigment and Citrinin. The main research contents and results are as follows:
(1) Disc papers and Oxford cups were used to ascertain bacteriostatic effect of Hongqu. Using 70% ethanol and water as solvent, the extraction inhibited Bacillus subtilis, Bacillus cereus and Staphylococcus aureus and had no effect on E.coli, Salmonella. Typhi, yeasts and algae.
(2) Hongqu contains three pigments, such as red, orange, and yellow pigments. The three pigments were separated by absorption column chromatograph. Then, TLC was used to purify them, and antibiotic experiments were made to define which was the main antibiotic substance. Results showed, orange pigment was the major antibiotic substance, and it could inhibit bacteria ,but had no effect on yeasts and algae. The diameter of the inhibition zone was directly proportional to the value of absorption of orange pigment.
(3) Orange pigment could be oxidized with hydrogen peroxide to yellow pigment . Concentration of H2O2 was proportional to creation of yellow pigment. The quantity of orange pigment was influenced by the nitrogen source of the culture medium .
(4) TLC method was set up to detect Citrinin in Hongqu. Extraction by 70% ethanol , developments were performed, in one direction, with chloroform -acetone (9:l,v/v),and then toluene-ethyl acetate-formic acid (6:3:1, v/v/v). Samples was extracted by water, the extraction was developed in dichloromethane-acetone(9:1,v/v), then chloroform -methanol -water (16:6:0.5) .Minimize quantity of Citrinin detected is 0.1 u g.
(5) Study on decrease of Citrinin in Hongqu was conducted.
引文
[1] 傅金泉编著，中国红曲及其实用技术[M]，中国轻工业出版社，1998
[2] 陈彦霖，李昭蓉，陈建州等．红曲菌种的研究开发与应用[J]．食品工业月刊(台)．1998，30(7)：1-10
[3] 秦人伟等．红曲防腐剂的研制[J]．精细与专用化学品．2000，23：16-17
[4] Wong H,Koehler PE., Producfion of red water-soluble Monascus pigments[J]. J Food Sci. 1983,48:1200-1203
[5] Hajjaj H,Klaebe A,Production and identification of N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus rubber and the occurrence of electron donor-acceptor complexes in these red pigments[J]. Appl. Environ Microbiol. 1997,63:2671-2678
[6] Lin TF, Yakushijin K, Buchi GH, Demain AL. Formation of water-soluble Monascus red pigment biological and semi-synthetic processes[J]. J Ind Microbiol. 1992,9:173-179
[7] 毛宁，陈松生．红曲霉有效生理活性物质及其应用[J]．中国调味品．1995，4：8-11
[8] P Juzlova. Secondary metabolites of the fungus Monascus: a review[J]. J.Industrial Microbio. 1996,16:163-170
[9] Yongsmith B,S Krairak and R Bavavoda. Production of yellow pigments in submerged culture of a mutant of Monascus spp[J]. J Ferment Bioeng. 1994,78:223-228
[10] Lin TF, Demain, AL. Leucine interference in the production of water-soluble red Monascus pigment[J]. Arch Microbiol. 1994,162:114-119
[11] Lin TF, Yakushijin K, Buchi GH, Demain AL. Formation of water-soluble Monascus red pigment biological and semi-synthetic processes[J]. J Ind Microbiol. 1992,9:173-179
[12] Carels M and D Shepherd.. The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture[J]. Can J Microbiol. 1997,23:1360-1372
[13] Yongsmith B,W Tabloka, W Yongmanitchai and R Bavavoda.. Culture conditions for yellow pigment formation by Monascus spKB 10 grown on cassava medium[J]. World J Microbiol Biotechno. 1993,19:85-90
[14] L.Martinkova, Biological activity of polyketide pigments produced by the fungus Monascus[J]. J.Applied Bacteriology. 1995,79:609-616.
[15] Shen Z,Yu P,Su M, et al. a prospective study on Zhitai capsul in the treatment of primary hyperlipidemia[J]. Nat. Med. J.China. 1996,26:156-157
[16] 路秀玲。红曲霉固态发酵生产生理活性物质Monacolin K的研究[M]．天津轻工业学院硕士学位论文，2000
[17] Wong HC and YS Bau. Pigmentation and antibacterial activity of fast neutron- and X-ray-induced strains of Monascus purpureus Went[J]. Plant Physiol. 1977,60:578-581
[18] P.J.Blanc et al; Characterization of Monascidin A from Monascus as Citrinin[J]. International Journal of Food Microbiology. 1995,27:201-213.
[19] Sako, F., kobayashi, N.Watabe, Induction of γ-glutamyltranspeptidase and glutathione S-transferase in cultured fetal rat hepatocytes by laccaic acid and Monascus pigments[J]. Chem. -Biol. Interactions. 1983,44:17-26.
[20] Bilgrami, K.S., Sinha, S.P., Nephrotoxic and hepatotoxic effects of citrinin in mice[J]. Proc. Indian Natl. Sci. Acad. 1988,54:35-37
[21] 田军等．红曲多糖的化学结构分析[J]．南京中医药大学学报．1998，7：35-38
[22] 迁启介．红曲调节血压作用[J]．日本酿造协会杂志．1994，89(3)：207-211
[23] 远藤章．特开昭56-138116．1981．113-115；56-115717．1981．101-104；56-150013．1981．91-94
[24] 董盛明等．红曲霉抑菌作用的探讨[J]．中国调味品．1991，1：11-13
[25] Leistner L, J Fink-Gremmels and J Dresel.. Monascus-exatract as a possible alternataiveto nitrite in meats[M]. 37th Int Congr Meat Sci Technol Proc. 1991,3:1252-1256.
[26] 徐尔尼，许杨，刘文群．红曲霉发酵液抑菌作用的探讨[J]．中国畜产与食品．1998，1：11-12
[27] 王柏琴，杨浩彬，刘克，红曲在肉类中的应用[J]．食品工业科技．1997，21(6)：29-32
[28] 远藤章，特开昭56-150013，1981，91-94
[29] 远藤章，发酵与工业，1985，43(6)：552-554
[30] 童群义，高孔荣，周正宏．红曲发酵液的抑菌[J]．食品工业科技．1997，5：25-37
[31] 角田广，上野芳夫等著，孟昭赫等译，《真菌毒素图解》，人民卫生出版社，1977
[32] A.Ciegler, R.F.Vesonder, et al. Applied and Environmental Microbiology[J]. 1977,33(4):1004-1006
[33] 李云，阎学秋，李牧秋．红曲中桔霉素的研究[J]．食品与发酵工业．1999，26(3)：82-86
[34] Blanc P J, M.O.Loret, A.Santerre and G.Goma., Production of citrinin by
various species of Monascus, Bioteehnol Lett, 1995,17:291-294.
[35] Hawksworth, D.L. and Pitt, J.I. A new taxonomy for Monascus species based on the cultural microsopical characters[J]. Aust. J.Bot. 1983,31:51-61
[36] 李燕萍，许杨，徐尔尼．红曲霉的抑菌活性物质与桔青霉素[J]．卫生研究．1999，27(8)：75-78
[37] Hassen. Hajjaj, Alain Klaebe; Biosynthetic Pathway of Citrinin in the Filamentous Fungus Monascus ruber as Revealed by 13C Nuclear Magnetic Resonanace[J]; Appl. Environ. Icrobiology. 1999,65(1):311-314
[38] Chernozemsky, I.N.Balkan endemic nephropathyand the associated tumours of the urinary system: a summary of epidemiological features in Bulgaria[J]. In Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. 1991,3:3-4
[39] Terry Vrabcheva, Ewald Usleber: Co-occurrence of Ochratoxin A and Citrinin in Cereals from Bulgarian villages with a History of Balkan Endemic Nephropathy[J]; J.Agric. Food Chem; 2000,48:2483-2488
[40] 许赣荣，傅伞泉．红曲产品在欧洲的合法地位、菌种分类及有害物质桔霉素——当今欧洲红曲生产及应用透视[J]．酿酒科技．1998，90(6)：16-18
[41] R.Dietrich, E.Usleber, E.Martlbauer, eet al. Evidence of the nephrotoxical mycotoxin citrinin in foodstuff and by Monascus ssp[J]. Produced foodstuff colors. 1994,5:47-50
[42] 河端俊治(日)编：《实用食品卫生》[M]，北京大学出版社，1992
[43] 许赣荣，傅金泉，红曲产品的合法化、菌种分类及有害物质桔霉素——当今欧洲红曲生产及应用的焦点[J]，中国酿酒，1998，5：6-8
[44] 袁森泉，广东饲料[J]，1999，3(12)：24-26
[45] 罗惠云，粮食真菌毒素的去毒方法[J]，粮食知识，1998，5：46-47
[46] 张治锬编：《抗生素药品检验》[M]，人民卫生出版社，1987
[47] 中华人民共和国国家标准《GB4926-85食品添加剂红曲米》
[48] 夏书华等，有关红曲色素的提取及提高其色价的研究[J]。北京农学院学报，1999，1(14)：44-46
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[50] K.霍斯泰特曼(瑞士)编：《制备色谱技术—在天然产物分离中的应用》[M]，科学出版社，2000
[51] 史坚编：《现代柱色谱分析》[M]，上海科学技术文献出版社，1986
[52] 傅若农编：《色谱分析概论》[M]，化学工业出版社，2000
[53] 陆冰真等编，《薄层层析法在食品分析中的应用》[M]，北京大学出版社，1991
[54] 何丽一编：《平面色谱方法及应用》[M]，化学工业出版社，2000
[55] 吕心泉等，红曲色素提取工艺条件探索[J]，食品添加剂，1991，4：53-57
[56] 常碧影，梁冬生等．氨基酸分析技术的研究与现状[J]．氨基酸杂志，1992，2：29-40
[2] 陈彦霖，李昭蓉，陈建州等．红曲菌种的研究开发与应用[J]．食品工业月刊(台)．1998，30(7)：1-10
[3] 秦人伟等．红曲防腐剂的研制[J]．精细与专用化学品．2000，23：16-17
[4] Wong H,Koehler PE., Producfion of red water-soluble Monascus pigments[J]. J Food Sci. 1983,48:1200-1203
[5] Hajjaj H,Klaebe A,Production and identification of N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus rubber and the occurrence of electron donor-acceptor complexes in these red pigments[J]. Appl. Environ Microbiol. 1997,63:2671-2678
[6] Lin TF, Yakushijin K, Buchi GH, Demain AL. Formation of water-soluble Monascus red pigment biological and semi-synthetic processes[J]. J Ind Microbiol. 1992,9:173-179
[7] 毛宁，陈松生．红曲霉有效生理活性物质及其应用[J]．中国调味品．1995，4：8-11
[8] P Juzlova. Secondary metabolites of the fungus Monascus: a review[J]. J.Industrial Microbio. 1996,16:163-170
[9] Yongsmith B,S Krairak and R Bavavoda. Production of yellow pigments in submerged culture of a mutant of Monascus spp[J]. J Ferment Bioeng. 1994,78:223-228
[10] Lin TF, Demain, AL. Leucine interference in the production of water-soluble red Monascus pigment[J]. Arch Microbiol. 1994,162:114-119
[11] Lin TF, Yakushijin K, Buchi GH, Demain AL. Formation of water-soluble Monascus red pigment biological and semi-synthetic processes[J]. J Ind Microbiol. 1992,9:173-179
[12] Carels M and D Shepherd.. The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture[J]. Can J Microbiol. 1997,23:1360-1372
[13] Yongsmith B,W Tabloka, W Yongmanitchai and R Bavavoda.. Culture conditions for yellow pigment formation by Monascus spKB 10 grown on cassava medium[J]. World J Microbiol Biotechno. 1993,19:85-90
[14] L.Martinkova, Biological activity of polyketide pigments produced by the fungus Monascus[J]. J.Applied Bacteriology. 1995,79:609-616.
[15] Shen Z,Yu P,Su M, et al. a prospective study on Zhitai capsul in the treatment of primary hyperlipidemia[J]. Nat. Med. J.China. 1996,26:156-157
[16] 路秀玲。红曲霉固态发酵生产生理活性物质Monacolin K的研究[M]．天津轻工业学院硕士学位论文，2000
[17] Wong HC and YS Bau. Pigmentation and antibacterial activity of fast neutron- and X-ray-induced strains of Monascus purpureus Went[J]. Plant Physiol. 1977,60:578-581
[18] P.J.Blanc et al; Characterization of Monascidin A from Monascus as Citrinin[J]. International Journal of Food Microbiology. 1995,27:201-213.
[19] Sako, F., kobayashi, N.Watabe, Induction of γ-glutamyltranspeptidase and glutathione S-transferase in cultured fetal rat hepatocytes by laccaic acid and Monascus pigments[J]. Chem. -Biol. Interactions. 1983,44:17-26.
[20] Bilgrami, K.S., Sinha, S.P., Nephrotoxic and hepatotoxic effects of citrinin in mice[J]. Proc. Indian Natl. Sci. Acad. 1988,54:35-37
[21] 田军等．红曲多糖的化学结构分析[J]．南京中医药大学学报．1998，7：35-38
[22] 迁启介．红曲调节血压作用[J]．日本酿造协会杂志．1994，89(3)：207-211
[23] 远藤章．特开昭56-138116．1981．113-115；56-115717．1981．101-104；56-150013．1981．91-94
[24] 董盛明等．红曲霉抑菌作用的探讨[J]．中国调味品．1991，1：11-13
[25] Leistner L, J Fink-Gremmels and J Dresel.. Monascus-exatract as a possible alternataiveto nitrite in meats[M]. 37th Int Congr Meat Sci Technol Proc. 1991,3:1252-1256.
[26] 徐尔尼，许杨，刘文群．红曲霉发酵液抑菌作用的探讨[J]．中国畜产与食品．1998，1：11-12
[27] 王柏琴，杨浩彬，刘克，红曲在肉类中的应用[J]．食品工业科技．1997，21(6)：29-32
[28] 远藤章，特开昭56-150013，1981，91-94
[29] 远藤章，发酵与工业，1985，43(6)：552-554
[30] 童群义，高孔荣，周正宏．红曲发酵液的抑菌[J]．食品工业科技．1997，5：25-37
[31] 角田广，上野芳夫等著，孟昭赫等译，《真菌毒素图解》，人民卫生出版社，1977
[32] A.Ciegler, R.F.Vesonder, et al. Applied and Environmental Microbiology[J]. 1977,33(4):1004-1006
[33] 李云，阎学秋，李牧秋．红曲中桔霉素的研究[J]．食品与发酵工业．1999，26(3)：82-86
[34] Blanc P J, M.O.Loret, A.Santerre and G.Goma., Production of citrinin by
various species of Monascus, Bioteehnol Lett, 1995,17:291-294.
[35] Hawksworth, D.L. and Pitt, J.I. A new taxonomy for Monascus species based on the cultural microsopical characters[J]. Aust. J.Bot. 1983,31:51-61
[36] 李燕萍，许杨，徐尔尼．红曲霉的抑菌活性物质与桔青霉素[J]．卫生研究．1999，27(8)：75-78
[37] Hassen. Hajjaj, Alain Klaebe; Biosynthetic Pathway of Citrinin in the Filamentous Fungus Monascus ruber as Revealed by 13C Nuclear Magnetic Resonanace[J]; Appl. Environ. Icrobiology. 1999,65(1):311-314
[38] Chernozemsky, I.N.Balkan endemic nephropathyand the associated tumours of the urinary system: a summary of epidemiological features in Bulgaria[J]. In Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. 1991,3:3-4
[39] Terry Vrabcheva, Ewald Usleber: Co-occurrence of Ochratoxin A and Citrinin in Cereals from Bulgarian villages with a History of Balkan Endemic Nephropathy[J]; J.Agric. Food Chem; 2000,48:2483-2488
[40] 许赣荣，傅伞泉．红曲产品在欧洲的合法地位、菌种分类及有害物质桔霉素——当今欧洲红曲生产及应用透视[J]．酿酒科技．1998，90(6)：16-18
[41] R.Dietrich, E.Usleber, E.Martlbauer, eet al. Evidence of the nephrotoxical mycotoxin citrinin in foodstuff and by Monascus ssp[J]. Produced foodstuff colors. 1994,5:47-50
[42] 河端俊治(日)编：《实用食品卫生》[M]，北京大学出版社，1992
[43] 许赣荣，傅金泉，红曲产品的合法化、菌种分类及有害物质桔霉素——当今欧洲红曲生产及应用的焦点[J]，中国酿酒，1998，5：6-8
[44] 袁森泉，广东饲料[J]，1999，3(12)：24-26
[45] 罗惠云，粮食真菌毒素的去毒方法[J]，粮食知识，1998，5：46-47
[46] 张治锬编：《抗生素药品检验》[M]，人民卫生出版社，1987
[47] 中华人民共和国国家标准《GB4926-85食品添加剂红曲米》
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