基于钴配合物干预下的马氏珠母贝优质珍珠培育过程研究
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摘要
珍珠,历来被视作奇珍至宝,它象征纯真、完美、尊贵和权威,与璧玉并重。高品质、珠宝级珍珠一直在国内外高端市场供不应求,因此提高珍珠品质已是国内外珍珠研究领域的热点。尽管目前改进的培育方法及加工技术从一定程度上提高了珍珠的质量,但皆有不同程度的缺憾或局限性。为了探讨高品质珍珠的培育方法,本文以马氏珠母贝为研究对象,利用自己合成的新人工干预物质2-丙基-4,5-咪唑二甲酸合钴(Ⅱ)/[Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO,通过藻、贝、珠代谢传递途径,探究优质珍珠培育的新方法。从而为我国优质珍珠的研究与开发提供新的思路。
本文的主要研究工作及结果如下:
1.马氏珠母贝化学成分分析
(1)采用高效液相色谱仪(HPLC)检测出马氏珠母贝贝肉中17种氨基酸,其中必需氨基酸(Lys、Thr、Phe、Val、Leu、Ile、Met)含量占总氨基酸的32.0%,呈味氨基酸(Arg、Ala、Gly、Asp、Glu)含量占总氨基酸的56.2%。贝壳中检测出10种氨基酸,必需氨基酸含量占总氨基酸含量的52.0%,呈味氨基酸含量占总氨基酸含量的41.8%。
(2)采用气相色谱-质谱联用仪(GC-MS)对马氏珠母贝贝肉氨基酸水解液梅拉德反应产物进行检测,共分离出18种化合物,其中含有乙酸乙酯、特丁基对苯二酚、二乙二醇丁醚醋酸酯、柏木醇4种香味物质,占总含量的46.57%。
(3)马氏珠母贝贝肉和贝壳的脂溶性组分采用气相色谱-质谱联用仪(GC-MS)分析检测,鉴定出的主要成分是烷烃化合物。贝壳与贝肉脂溶性成分有6种组分是相同的,分别是二乙基癸氧基硼烷、二十烷、3,8-二甲基十一烷、3,7-二甲基壬烷、二十一烷、邻苯二甲酯异丁酯。此外,马氏珠母贝贝肉的脂溶性组分中,棕榈酸的含量最高,为58.78%。
(4)采用电感耦合等离子体原子发射光谱法(ICP-AES)对马氏珠母贝贝肉和贝壳矿物质进行分析,贝壳与贝肉中Zn、Mg、Fe三种矿物质的含量均比较高,其中常量元素Mg在贝肉和贝壳中含量均最高,在贝肉中Mg含量高达95.80mg/Kg,贝壳中Mg的含量高达514.00mg/Kg。
2.钴配合物诱导合成及表征
以2-丙基-4,5-咪唑二甲酸为配体化合物,与Co(NO_3)_2合成了一种新型钴配合物[Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO,并对其结构进行了表征。该晶体的对称单元是由一个位于对称中心的二价钴离子、两个去质子化的2-丙基-4,5-咪唑二甲酸、两个配位水和两个游离的N,N-二甲基甲酰胺组成,中心离子二价钴离子与两个N,O双齿的配体和两分子结合水进行配位,形成配位数为六的扭曲的八面体结构。游离的N,N-二甲基甲酰胺通过N-H...O和O-H...O氢健相互作用与配合物连接并形成的一个二维的空间平面。
3.钴配合物对扁藻毒理性研究
运用评价化学品毒性藻类测试的标准实验方法,得到钴配合物和钴离子对扁藻生长的最小无显著差异浓度(LNOEC)分别为5.0mg/L,1.25mg/L,抑制扁藻生长的96h半效应浓度(96h-EC50)分别为31.92mg/L和7.16mg/L。实验结果表明无论从LNOEC还是从96h-EC50考虑,都证明抑制扁藻生长的毒性由大到小的顺序是钴离子﹥钴配合物。
4.钴配合物在藻、贝、珠传递过程示踪研究
基于藻—贝—珠的传递途径,研究钴配合物和钴离子的传递效应。采用稳定同位素质谱仪(IRMS)和电感耦合等离子体原子发射光谱法(ICP-AES)对15N和钴元素分析,探讨分析扁藻对钴配合物的富集情况,以及钴配合物和钴离子在珍珠层、外套膜、珍珠囊的传递效果。结果表明,扁藻对钴配合物的富集效应,由直线提高到逐渐平稳,在第3d天达到最大吸附量。富集钴配合物和钴离子的扁藻,喂养马氏珠母贝,结果发现钴配合物中的15N和钴元素可以随摄食时间的增加,在外套膜、珍珠囊富集,最终可以代谢分泌到马氏珠母贝的珍珠层中。
5.钴配合物对贝体碱性磷酸酶活性的影响
采用对硝基苯磷酸二钠法测定钴配合物和钴离子对碱性磷酸酶(ALP)的活性,结果发现两者对马氏珠母贝外套膜中的ALP均有一定的激活作用,且钴离子的激活作用大于钴配合物,两者对珍珠质的分泌均有一定的促进作用。研究ALP的酶学性质,对于深入探讨珍珠形成的机理,为寻找适宜的酶激活剂提高珍珠的产量和质量具有重要的指导意义。
6.钴配合物对珍珠层质结构的影响
钴配合物及钴离子通过藻—贝—珠途径传递后培育的珍珠,和钴配合物体外处理,及未经处理的珍珠的光泽度及电镜扫描结构图对比分析,结果发现钴配合物对珍珠的光泽度具有良好的优化和修饰效果。由此可知,珍珠光泽度与其表面结构的好坏有直接关系,即珍珠表面的层状结构越明显,文石晶体形状越规则,结构越致密,表面缺陷越少,则珍珠光泽就越好。
Pearl has always been regarded as treasure of the treasure, it is a symbol of purity, perfection, dignity and authority, both with jade. High quality, grade pearl jewelry has been in short supply in the domestic and international high-end market. Therefore, it is paid attention to improve pearl quality pearl at home-and-abroad research field. Although cultivation methods and processing technology has improved to some extent, and improved the quality of pearls, but have different degrees of defects or limitations, failing to achieve high-quality pearls in the stabilization and efficient training. In this paper, the methods of cultivating high-quality pearls were studyed, as research object of Pinctada martensii. With the use of the new synthetic human intervention material, 2 - propyl 4,5 - imidazo acid cobalt(Ⅱ)/[Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO, new methods of cultivating high-quality pearls were explored through algae, oyster pearl and pearl pathways. It will provide new ideas for the research and development of high-quality pearl in China.
Main studies and conclusion summarized as follows:
1.Analysis of the chemical composition in Pinctada martensii
(1)Seventeen amino acids in meat of Pinctada martensii were detected by high performance liquid chromatography(HPLC), essential amino acids (Lys, Thr, Phe, Val, Leu, Ile, Met) accounting for 32.0% of the total amino acids and flavor amino acids (Arg, Ala, Gly, Asp, Glu) accounting for 56.2% of the total aminl acids. Nine amino acids were detected in shell, essential amino acids accounting for 52.0% of the total amino acids and flavor amino acids accounted for 41.8% of the total amino acids.
(2)Eghiteen Maillard reaction products of hydrolyzate amino acid in the meat of Pinctada martensii were analyzed by gas chromatography-mass spectrometer(GC-MS). Four flavors, including ethyl acetate, t-butyl hydroquinone, diethylene glycol ether acetate and cedar, accounted for 46.57% of the total content.
(3)Fat-soluble components in the meat and shell of Pinctada martensii were analyzed by gas chromatography - mass spectrometer (GC-MS), identificated alkane compounds in major components. Six fat-soluble ingredients in shell and meat are the same, namely, diethyl-decanoic oxygen borane, 20 alkyl, 3,8 - dimethyl undecane, 3,7 - dimethyl-nonyl alkanes, 21 alkane, methyl isobutyl phthalate.
(4) By inductively coupled plasma atomic emission spectrometry (ICP-AES) minerals were analyzed in the meat and shell of Pinctada martensii. The contents of Zn, Mg, Fe minerals were higher in the shell and meat. Mg in the flesh and shells were the highest, amounting to 95.80mg/Kg in the meat and up to 514.00mg/Kg in the shell.
2. Synthesis and characterization of Cobalt complex. A novel cobalt complex [Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO was synthesized with 2 - propyl 4, 5 - dicarboxylate imidazole and Co (NO3)2, and its structure was characterized. In the symmetric unit, it consists of anti-divalent cobalt ion in the center, two deprotonated 2-propyl 4,5- imidazole -2-carboxylic acids, two coordinated waters and two free of N, N- dimethylformamide compositions. The central ion of divalent cobalt ion with two N, O bidentate ligand and two coordinated water molecules form a coordination number of six of the distorted octahedral structure.
3. Study on toxicity of cobalt complex to Platymonas subcordiformis. A standard method of algal bioassay for evaluatingthe toxicity of toxic chemicalwas applied in the experiments. The lowest no observable effect concentrations (LNOEC) of cobalt complex and cobalt ion on the growth of Platymonas subcordiformis were found to be 5.0mg/L and 1.25mg/L, and 96h-EC50s were obversed to be 31.92mg/L and 7.16mg/L, respectively. According to both LNOEC and 96h-EC50 calculated, the toxicities of these compounds on inhibitingthe growth of Platymonas subcordiformis increased in the order cobalt ion>cobalt complex.
4. Tracing study on the tranfer process of cobalt complex in algae,oyster pearl and pearl pathway. The tansfer effect of cobalt complex and cobalt ion were studyed based on algae - oyster shell - pearl pathway. 15N and cobalt elements were analyzed with stable isotope mass spectrometry and inductively coupled plasma atomic emission spectrometry, to explore the enrichment effect on Platymonas subcordiformis to cobalt complex and transfer effect of cobalt complex and cobalt ion in the pearl layer, mantle and pearl sac. The results show that the enrichment effect on Platymonas subcordiformis to cobalt complex is the line gradually increased to stable, up to maximum adsorption in the third day. Platymonas subcordiformis enriched cobalt complex and cobalt ion were used to feed Pinctada martensii, and the results are 15N and cobalt elements can be increased with feeding time in the mantle and pearl sac, finally which can be passed to pearl layer.
5. Effects of cobalt complex on the activity of akaline phosphatase in oyster pearl. Cobalt complex and cobalt ion on the activity of ALP were determined by nitrophenyl phosphate disodium method. It was found both can active ALP on the mantle of Pinctada martensii, and cobalt ion is more active than cobalt complex. Besides, Cobalt complex and cobalt ion can promote the secretion of nacre. It has important guidance that find a suitable enzyme activator to improve the yield and quality of peals through studying the enzymatic properties of ALP and the depth of the formation mechanisms of pearl.
6. Effects of Cobalt complex on the structure of pearl nacre. Pearl cultivated based on cobalt complexe and cobalt ion in algae - oyster pearl - pearl passway, pearl in vitro cobalt complexe and untreated pearl were respectively scanned by electron microscope, and results showed that cobalt complexe have good optimization and modification effect to pearl gloss. The relationshipin between pearl gloss and surface structure of pearl is that the layer structure of pearl surface is more obvious, aragonite crystal shape more regularly arranged, structure more compact, surface defects less, and the better is pearl gloss.
本文的主要研究工作及结果如下:
1.马氏珠母贝化学成分分析
(1)采用高效液相色谱仪(HPLC)检测出马氏珠母贝贝肉中17种氨基酸,其中必需氨基酸(Lys、Thr、Phe、Val、Leu、Ile、Met)含量占总氨基酸的32.0%,呈味氨基酸(Arg、Ala、Gly、Asp、Glu)含量占总氨基酸的56.2%。贝壳中检测出10种氨基酸,必需氨基酸含量占总氨基酸含量的52.0%,呈味氨基酸含量占总氨基酸含量的41.8%。
(2)采用气相色谱-质谱联用仪(GC-MS)对马氏珠母贝贝肉氨基酸水解液梅拉德反应产物进行检测,共分离出18种化合物,其中含有乙酸乙酯、特丁基对苯二酚、二乙二醇丁醚醋酸酯、柏木醇4种香味物质,占总含量的46.57%。
(3)马氏珠母贝贝肉和贝壳的脂溶性组分采用气相色谱-质谱联用仪(GC-MS)分析检测,鉴定出的主要成分是烷烃化合物。贝壳与贝肉脂溶性成分有6种组分是相同的,分别是二乙基癸氧基硼烷、二十烷、3,8-二甲基十一烷、3,7-二甲基壬烷、二十一烷、邻苯二甲酯异丁酯。此外,马氏珠母贝贝肉的脂溶性组分中,棕榈酸的含量最高,为58.78%。
(4)采用电感耦合等离子体原子发射光谱法(ICP-AES)对马氏珠母贝贝肉和贝壳矿物质进行分析,贝壳与贝肉中Zn、Mg、Fe三种矿物质的含量均比较高,其中常量元素Mg在贝肉和贝壳中含量均最高,在贝肉中Mg含量高达95.80mg/Kg,贝壳中Mg的含量高达514.00mg/Kg。
2.钴配合物诱导合成及表征
以2-丙基-4,5-咪唑二甲酸为配体化合物,与Co(NO_3)_2合成了一种新型钴配合物[Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO,并对其结构进行了表征。该晶体的对称单元是由一个位于对称中心的二价钴离子、两个去质子化的2-丙基-4,5-咪唑二甲酸、两个配位水和两个游离的N,N-二甲基甲酰胺组成,中心离子二价钴离子与两个N,O双齿的配体和两分子结合水进行配位,形成配位数为六的扭曲的八面体结构。游离的N,N-二甲基甲酰胺通过N-H...O和O-H...O氢健相互作用与配合物连接并形成的一个二维的空间平面。
3.钴配合物对扁藻毒理性研究
运用评价化学品毒性藻类测试的标准实验方法,得到钴配合物和钴离子对扁藻生长的最小无显著差异浓度(LNOEC)分别为5.0mg/L,1.25mg/L,抑制扁藻生长的96h半效应浓度(96h-EC50)分别为31.92mg/L和7.16mg/L。实验结果表明无论从LNOEC还是从96h-EC50考虑,都证明抑制扁藻生长的毒性由大到小的顺序是钴离子﹥钴配合物。
4.钴配合物在藻、贝、珠传递过程示踪研究
基于藻—贝—珠的传递途径,研究钴配合物和钴离子的传递效应。采用稳定同位素质谱仪(IRMS)和电感耦合等离子体原子发射光谱法(ICP-AES)对15N和钴元素分析,探讨分析扁藻对钴配合物的富集情况,以及钴配合物和钴离子在珍珠层、外套膜、珍珠囊的传递效果。结果表明,扁藻对钴配合物的富集效应,由直线提高到逐渐平稳,在第3d天达到最大吸附量。富集钴配合物和钴离子的扁藻,喂养马氏珠母贝,结果发现钴配合物中的15N和钴元素可以随摄食时间的增加,在外套膜、珍珠囊富集,最终可以代谢分泌到马氏珠母贝的珍珠层中。
5.钴配合物对贝体碱性磷酸酶活性的影响
采用对硝基苯磷酸二钠法测定钴配合物和钴离子对碱性磷酸酶(ALP)的活性,结果发现两者对马氏珠母贝外套膜中的ALP均有一定的激活作用,且钴离子的激活作用大于钴配合物,两者对珍珠质的分泌均有一定的促进作用。研究ALP的酶学性质,对于深入探讨珍珠形成的机理,为寻找适宜的酶激活剂提高珍珠的产量和质量具有重要的指导意义。
6.钴配合物对珍珠层质结构的影响
钴配合物及钴离子通过藻—贝—珠途径传递后培育的珍珠,和钴配合物体外处理,及未经处理的珍珠的光泽度及电镜扫描结构图对比分析,结果发现钴配合物对珍珠的光泽度具有良好的优化和修饰效果。由此可知,珍珠光泽度与其表面结构的好坏有直接关系,即珍珠表面的层状结构越明显,文石晶体形状越规则,结构越致密,表面缺陷越少,则珍珠光泽就越好。
Pearl has always been regarded as treasure of the treasure, it is a symbol of purity, perfection, dignity and authority, both with jade. High quality, grade pearl jewelry has been in short supply in the domestic and international high-end market. Therefore, it is paid attention to improve pearl quality pearl at home-and-abroad research field. Although cultivation methods and processing technology has improved to some extent, and improved the quality of pearls, but have different degrees of defects or limitations, failing to achieve high-quality pearls in the stabilization and efficient training. In this paper, the methods of cultivating high-quality pearls were studyed, as research object of Pinctada martensii. With the use of the new synthetic human intervention material, 2 - propyl 4,5 - imidazo acid cobalt(Ⅱ)/[Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO, new methods of cultivating high-quality pearls were explored through algae, oyster pearl and pearl pathways. It will provide new ideas for the research and development of high-quality pearl in China.
Main studies and conclusion summarized as follows:
1.Analysis of the chemical composition in Pinctada martensii
(1)Seventeen amino acids in meat of Pinctada martensii were detected by high performance liquid chromatography(HPLC), essential amino acids (Lys, Thr, Phe, Val, Leu, Ile, Met) accounting for 32.0% of the total amino acids and flavor amino acids (Arg, Ala, Gly, Asp, Glu) accounting for 56.2% of the total aminl acids. Nine amino acids were detected in shell, essential amino acids accounting for 52.0% of the total amino acids and flavor amino acids accounted for 41.8% of the total amino acids.
(2)Eghiteen Maillard reaction products of hydrolyzate amino acid in the meat of Pinctada martensii were analyzed by gas chromatography-mass spectrometer(GC-MS). Four flavors, including ethyl acetate, t-butyl hydroquinone, diethylene glycol ether acetate and cedar, accounted for 46.57% of the total content.
(3)Fat-soluble components in the meat and shell of Pinctada martensii were analyzed by gas chromatography - mass spectrometer (GC-MS), identificated alkane compounds in major components. Six fat-soluble ingredients in shell and meat are the same, namely, diethyl-decanoic oxygen borane, 20 alkyl, 3,8 - dimethyl undecane, 3,7 - dimethyl-nonyl alkanes, 21 alkane, methyl isobutyl phthalate.
(4) By inductively coupled plasma atomic emission spectrometry (ICP-AES) minerals were analyzed in the meat and shell of Pinctada martensii. The contents of Zn, Mg, Fe minerals were higher in the shell and meat. Mg in the flesh and shells were the highest, amounting to 95.80mg/Kg in the meat and up to 514.00mg/Kg in the shell.
2. Synthesis and characterization of Cobalt complex. A novel cobalt complex [Co(C_8H_8N_2O_4)_2(H_2O)_2]·2C_3H_7NO was synthesized with 2 - propyl 4, 5 - dicarboxylate imidazole and Co (NO3)2, and its structure was characterized. In the symmetric unit, it consists of anti-divalent cobalt ion in the center, two deprotonated 2-propyl 4,5- imidazole -2-carboxylic acids, two coordinated waters and two free of N, N- dimethylformamide compositions. The central ion of divalent cobalt ion with two N, O bidentate ligand and two coordinated water molecules form a coordination number of six of the distorted octahedral structure.
3. Study on toxicity of cobalt complex to Platymonas subcordiformis. A standard method of algal bioassay for evaluatingthe toxicity of toxic chemicalwas applied in the experiments. The lowest no observable effect concentrations (LNOEC) of cobalt complex and cobalt ion on the growth of Platymonas subcordiformis were found to be 5.0mg/L and 1.25mg/L, and 96h-EC50s were obversed to be 31.92mg/L and 7.16mg/L, respectively. According to both LNOEC and 96h-EC50 calculated, the toxicities of these compounds on inhibitingthe growth of Platymonas subcordiformis increased in the order cobalt ion>cobalt complex.
4. Tracing study on the tranfer process of cobalt complex in algae,oyster pearl and pearl pathway. The tansfer effect of cobalt complex and cobalt ion were studyed based on algae - oyster shell - pearl pathway. 15N and cobalt elements were analyzed with stable isotope mass spectrometry and inductively coupled plasma atomic emission spectrometry, to explore the enrichment effect on Platymonas subcordiformis to cobalt complex and transfer effect of cobalt complex and cobalt ion in the pearl layer, mantle and pearl sac. The results show that the enrichment effect on Platymonas subcordiformis to cobalt complex is the line gradually increased to stable, up to maximum adsorption in the third day. Platymonas subcordiformis enriched cobalt complex and cobalt ion were used to feed Pinctada martensii, and the results are 15N and cobalt elements can be increased with feeding time in the mantle and pearl sac, finally which can be passed to pearl layer.
5. Effects of cobalt complex on the activity of akaline phosphatase in oyster pearl. Cobalt complex and cobalt ion on the activity of ALP were determined by nitrophenyl phosphate disodium method. It was found both can active ALP on the mantle of Pinctada martensii, and cobalt ion is more active than cobalt complex. Besides, Cobalt complex and cobalt ion can promote the secretion of nacre. It has important guidance that find a suitable enzyme activator to improve the yield and quality of peals through studying the enzymatic properties of ALP and the depth of the formation mechanisms of pearl.
6. Effects of Cobalt complex on the structure of pearl nacre. Pearl cultivated based on cobalt complexe and cobalt ion in algae - oyster pearl - pearl passway, pearl in vitro cobalt complexe and untreated pearl were respectively scanned by electron microscope, and results showed that cobalt complexe have good optimization and modification effect to pearl gloss. The relationshipin between pearl gloss and surface structure of pearl is that the layer structure of pearl surface is more obvious, aragonite crystal shape more regularly arranged, structure more compact, surface defects less, and the better is pearl gloss.
引文
[1]邓陈茂,童银洪.南珠养殖与加工技术[M].北京:中国农业出版社,2005:1-4.
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