章鱼胺的发酵制备及生物活性研究
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摘要
章鱼胺(Octopamine)作为重要的生物胺，在生物体内起了神经介质的作用，近年的研究发现它是一种天然的β3-肾上腺素能受体激动剂，有调节代谢与能量平衡的功效，在肥胖症的治疗和II型糖尿病的防治上有潜在的价值。章鱼胺可以单独或与其它原料联合用于减肥与糖尿病的治疗，可以作为食品、健康食品原料乃至临床药物的辅助制剂。枳实和鱼露是天然章鱼胺的主要来源，但枳实中章鱼胺的含量仅为0.01～0.03％，而鱼露中相对丰富的章鱼胺含量（0.01～0.1%），使从鱼露中提取章鱼胺开发健康产品成为可能。本课题旨在利用水产品加工的废弃物制备速酿鱼露，从中提取章鱼胺，并研究章鱼胺的生物活性。本论文利用智利外海茎柔鱼（Dosidicus gigass）的内脏为原料，采用保温、加曲保温、加酶保温等不同速酿方法制备鱼露，测定了鱼露酿造过程中章鱼胺含量的变化；并参照鱼露中章鱼胺的生成路径，尝试以酪胺为底物生物转化生成章鱼胺。本论文研究从鱼露中提取章鱼胺的有效方法，以探讨从鱼露中提取章鱼胺开发健康产品的可能性。在章鱼胺的应用实验中，研究了章鱼胺对营养肥胖小鼠的减肥作用。最后以章鱼胺为原料合成了十种章鱼胺衍生物，测定了它们的抗氧化活性，并与章鱼胺的前体化合物酪胺对比，以期对章鱼胺的生物活性做进一步研究。
1.鱼露酿制过程中章鱼胺的产生
以智利外海茎柔鱼内脏为原料，采用保温、加曲保温、加酶保温等不同速酿方法制备鱼露，测定鱼露在不同酿制阶段的pH值、氨基酸态氮和章鱼胺含量，并对最终样品作感官评价。结果表明：酿制20d内，pH值变化显著，上升速度较快，从弱酸性升至中性或弱碱性；在此期间，氨基酸态氮的变化也较为显著，其浓度增加了15mg·mL~(-1)以上，20d后趋于稳定；鱼露酿制前期，章鱼胺含量逐渐增加，15-20d时，达到最大值，高于5mg·mL~(-1)，加酶保温速酿的样品中章鱼胺含量甚至超过7mg·mL~(-1)。20d后，鱼露中章鱼胺的含量呈下降趋势。为与速酿鱼露作对比，本研究测定了常温自然酿制鱼露在酿制过程中pH值、氨基酸态氮和章鱼胺含量的变化。结果显示，在常温自然酿制鱼露中的各种变化速度较速酿鱼露缓慢得多。30d后，pH值仍保持在酸性范围内，氨基酸态氮的浓度仅增加了5mg·mL~(-1)左右，章鱼胺含量为3.5mg·mL~(-1)，仅为加酶保温速酿样品中章鱼胺含量峰值的一半。
本研究利用茎柔鱼的不可食部分酿制鱼露，可以发现在酿制过程中章鱼胺含量明显提高，表明酿造过程可有效提高章鱼胺含量，这可为水产品加工的下脚料和废弃物的综合利用提供一条途径。本研究对于酿制半年以上的保温速酿鱼露样品中的章鱼胺含量也进行了测定，发现仍然高达4.5mg·mL~(-1)以上。说明鱼露可长期保持较高的章鱼胺水平。若将速酿鱼露做为提取章鱼胺的原料，可任选保温、加曲保温、加酶保温三种速酿方法之一，20d的酿制时间即可。
感官评定的结果表明保温和加曲保温速酿的样品在气味上有较浓的鱼香气，而加酶保温速酿的样品腥气较重，气味不佳。对保温速酿鱼露中组胺和重金属含量测定的结果表明其中组胺及砷、镉的含量均已超标，因此，从安全性考虑，用智利外海茎柔鱼内脏酿制的鱼露不适于直接作为调味品食用。
2.酪胺发酵生物合成章鱼胺
鱼露在漫长的发酵过程中，细胞色素P450酶系（cytochrome P450s，CYP450）对章鱼胺的生物合成起了关键的作用。文献资料显示在研究细胞色素P450酶的活性时往往要使用价格昂贵的超速冷冻离心机提取动物肝脏中的微粒体。本文参照鱼露中章鱼胺的生成路径，用高速冷冻离心机提取了含有细胞色素P450酶的草鱼肝脏S-9部位，并以之为酶源，以维生素C为氢载体，异烟肼为单胺氧化酶抑制剂，以酪胺为原料生物转化生成章鱼胺。提取得到的S-9部位冷藏在4℃条件下，其中的细胞色素P450酶活性只能保持4小时左右，而-25℃冷藏条件下其活性可保持8-10天。生物合成实验的结果显示氢载体对生物合成章鱼胺是必需的，反应1h产生的章鱼胺为879.66μg/4.5mL反应液，章鱼胺的生物合成率为21.4%，酪胺代谢率为90.0%。这表明以草鱼肝脏S-9部位为酶源生物转化生成章鱼胺是可行的，但由于P450酶系的非专一性，章鱼胺的生物合成率较低，有待进一步改进。
3．鱼露中章鱼胺的分离和提取
鱼露中章鱼胺的含量较为丰富，高于其他许多水产品。从鱼露中有效提取章鱼胺并进行精制，可以为进一步开发利用天然章鱼胺打下基础。从智利外海茎柔鱼内脏速酿鱼露中提取章鱼胺，可使废弃物得以综合利用，减少资源浪费和环境污染。本文中采用静态吸附和动态吸附相结合的方法，综合考虑吸附量、解吸率等因素，研究硅胶分离提取鱼露中章鱼胺的吸附性能和洗脱参数。结果表明：60～100目硅胶对鱼露中章鱼胺有较好的吸附分离效果，静态饱和吸附量达3.837mg·g~(-1)(干硅胶），30%的乙醇水溶液为最佳洗脱剂，洗脱率达90.48％；60～100目硅胶对鱼露中章鱼胺动态吸附的最佳上样量为4BV，用1.1BV洗脱液可基本洗脱完全。得到的洗出液采用HPLC分离系统，在流动相0.02mol·L~(-1)柠檬酸-0.02mol·L~(-1)磷酸二氢钠（7∶3），PH=3、流速10mL min~(-1)、检测波长274nm的条件下被色谱柱SepaxHP-C18柱(21.2mm250mm，10μm)精制，真空冷冻干燥后得到白色粉末状固体。将此白色固体与标准品章鱼胺在同样的条件下进行质谱分析，可证明得到的白色粉末状固体为纯度较高的天然章鱼胺。
4.章鱼胺和市售减肥药对营养性肥胖小鼠减肥作用的比较
在章鱼胺的应用实验中，本实验比较了章鱼胺和市售减肥药对营养性肥胖小鼠的减肥作用。采用高脂饲料喂养4周构建营养性肥胖小鼠模型。再将小鼠分组喂养4周后发现，饲料中加入一定剂量章鱼胺和减肥药的小组中小鼠体重、Lee s指数、脂肪湿重、脂肪系数、血清总胆固醇(TC)、甘油三脂(TG)与模型组存在显著性差异(P <0.05)。以上结果表明章鱼胺对营养性肥胖小鼠有一定的减肥降脂作用。
5．章鱼胺衍生物的合成及抗氧化活性研究
关于章鱼胺的生物活性，文献报道大多为肥胖症的治疗和II型糖尿病防治方面的研究。既往研究指出，章鱼胺的前体化合物酪胺具有较高的抗氧化活性，且与其浓度呈正相关。本文以章鱼胺为原料合成十种衍生物，并对它们进行核磁共振氢谱和质谱定性分析，确证其结构。为比较章鱼胺、酪胺及章鱼胺衍生物的抗氧化活性，本文测定了这些物质对DPPH自由基、超氧阴离子自由基、羟基自由基的清除率。研究结果表明，章鱼胺及其两种衍生物OA07和OA08，即N-正十一烷基甲酰基-2-羟基-2-(4-羟基苯基)-乙胺和N-(2-硝基-3-甲基苯甲酰基)-2-羟基-2-(4-羟基苯基)-乙胺，清除DPPH自由基、超氧阴离子自由基和羟基自由基的能力均高于酪胺；另一章鱼胺衍生物OA05，即N-(4-硝基苯甲酰基)-2-羟基-2-(4-羟基苯基)-乙胺具有较高的超氧阴离子自由基清除能力，值得进一步关注和研究。
由于国内目前有关章鱼胺的研究报告或产品较少，本课题的研究有可能进一步推动对章鱼胺的研究，为更好地生产和应用章鱼胺提供一定的理论依据和研究基础。本课题组之前的研究发现鳀鱼（Engraulis）和七星鱼（Channa asiatica）鱼露原液中章鱼胺含量较高，达到0.05-0.1%左右，考虑到我国当前鱿鱼加工业中存在资源利用率低和环境污染严重两个问题，本研究以智利外海茎柔鱼内脏为原料制备鱼露并探讨从中提取章鱼胺的有效方法，为天然章鱼胺的制取提供实验依据。同时，化学合成了章鱼胺的衍生物，将它们及章鱼胺的抗氧化活性与章鱼胺的前体化合物酪胺进行对比，旨在对章鱼胺的生物活性做进一步研究，拓展章鱼胺的开发和利用领域。
As an important biogenic amine, octopamine plays a role of neurotransmitter inorganism. Recent researches revealed that it is a natural β3-adrenergic receptor agonist(β3-ARA), which has effects on metabolism regulatinon and energy balance.Octopamine also has an important preventive function on obesity and typeⅡdiabetes.As raw materials of food and health food or auxiliary preparation for clinical medicine,octopamine can be used independently or jointly with other materials for weight lossand diabetes treatment. Natural octopamine mainly exists in Fructus aurantiiImmaturus (FaI), but the content in FaI is merely0.01~0.03%, and in fish sauce,however, relative abundant (0.01~0.1%). It is possible to extract octopamine fromfish sauce and develop health products. This paper aims at the rapid brewage of fishsauce by the waste of aquatic products, extraction of octopamine, and study on thebiological activity of octopamine and its derivatives. In this paper, visceral ofDosidicus gigass was used as raw material to produce fish sauce by3quick brewingmethods of insulation, adding either koji or enzyme under insulation. The change inoctopamine content during brewage was investigated. As the substrate, tyramine wasbioconverted to octopamine according to the formation path of octopamine in fishsauce. An effective method was studied to extract octopamine in fish sauce in order tofind the possibility of developing health food. In application experiments ofoctopamine, its effect on the anti-obesity in nutritive obesity mice was studiedcompared with weight loss pills. Finally, ten kinds of octopamine derivatives weresynthesized. For further study of the biological activity of octopamine, the antioxidantactivity of octopamine and its dereivatives were determined, and compared with that oftyramine, the precursor compound of octopamine.
1. Octopamine production during fish sauce brewing process
Fish sauce was brewed from the viscera of Dosidicus gigass in this study. Toshorten brewage period, methods such as insulation, adding enzyme or koji underinsulation was adopted. The changes in pH value, amino acid nitrogen and content ofoctopamine during brewage were investigated. The sensory characteristics were alsoevaluated. The results showed that in the first20days, the pH value increasedobviously and changed from acidic to neutral or weak alkaline, while the amino acidnitrogen rised significantly and had an increased by15mg·mL~(-1). They all becamestable after20days of brewage. Initially, the content of octopamine ascended graduallyand climbed to the top of more than5mg·mL~(-1)after15-20days of brewage. It reachedeven more than7mg·mL~(-1)in fish sauce added enzyme. After20days, the octopaminecontent showed a trend of decrease. With the speedy brewing fish sauce forcomparison, the changes in pH value, amino acid nitrogen and content of octopaminein natural brewing fish sauce were also measured. The results showed that all theabove changes in natural brewing fish sauce were much slower than those of in quickbrewing ones. After30days of natural brewage, the pH value was still acidic, theamino acid nitrogen increased only by5mg·mL~(-1). Meanwhile, the content ofoctopamine reached3.5mg·mL~(-1), which was only half of the content peak in fishsauce added enzyme.
In this study inedible parts of Dosidicus gigass was used to make fish sauce. Itcan be found that octopamine content improved effectively in the brewing process,which indicates that octopamine will be produced during brewage. Maybe this is a newway for comprehensive utilization of waste during aquatic products processing. Theoctopamine content in fish sauce after half year s insulation brewage was rather stableand more than4.5mg·mL~(-1), which demonstrated that the content of octopamine in fishsauce can keep at high level for a long time. If only for octopamine extraction,20daysof brewage is enough, and any one of the3quick brewing methods can be chosen.
The results of sensory evaluation showed that the smell of fish sauce addedenzyme was the most unpleasant among the three kinds of samples. The content ofhistamine and heavy metal in insulation brewing fish sauce were determined. Theresult indicated that the content of histamine, As and Cd were higher than national standard. Therefore, from the security considerations, the fish sauce is not suitable forhuman consumption as a condiment directly.
2. Biotransformation from tyramine to octopamine by fermentation
Cytochrome P450enzyme system (cytochrome P450s, CYP450) plays a key rolein the biosynthesis of octopamine during the long fermentation process of fish sauce.Most literature displayed that expensive speeding refrigerated centrifuge was oftenused to extract microsome from animal liver in the study of cytochrome450activities.In this study, hepatic S-9place containing cytochrome450in the liver of Grass Carpwas extracted by a high speed refreigerated contrifuge. When the hepatic S-9place wasstored at4℃, the cytochrom P450activity can only keep about4h, while it can bemaintained for8-10days at-25℃. According to the formation path of octopamine infish sauce, tyramine was bioconverted to octopamine with Grass Carp hepatic S-9place as the resource of cytochrome P450, ascorbic acid as hydrogen carrier andisoniazid as monoamine oxidase inhibitor. The results showed that hydrogen carrier isnecessary for the biosynthesis of octopamine, and the octopamine obtained after1hcatalysis was879.66μg in4.5mL reaction liquid. The biosynthesic rate of octopamineand metabolic rate of tyramine were21.44%and90.03%, respectively. This indicatedthat it is feasible to produce octopamine when hepatic S-9place of Grass Carp beingtaken as resource of enzyme. But because of the non-specifity of cytochrome P450thebiosynthesic rate of octopamine was relative low, which need to be further improved.
3. Octopamine separation and extraction in fish sauce
In fish sauce octopamine content is rather rich, higher than many other aquaticproducts. Extraction and refining of octopamine in fish sauce can lay the foundationfor further development and utilization of natural octopamine. It is a way ofcomprehensive utilization of aquatic litter to process fish sauce by squid viscera. It alsocan reduce the waste of resources and environmental pollution. The adsorption andelution parameters was studied during separation and extraction of octopamine in fishsauce, while the static and dynamic adsorption was combined and adsorption anddesorption rate were also be considered. The results demonstrated that60～100meshsilica gel has high efficiency in separate adsorption with the static saturated adsorption of3.837mg g-1(dry silica gel).30%ethanol was as eluent with its elutive power90.48%. The optimal value of the dynamic adsorption of octopamine in fish sauce on60～100mesh silica gel was4BV, and when the volume of eluent was1.1BV, theeluting process was basically completed. The eluate was refined by a HPLC separationsystem. A SepaxHP-C18column (21.2mm x250mm,10μm) was used. The mobilephase was0.02mol·L~(-1)citric acid-0.02mol·L~(-1)sodium dihydrogen phosphate (7:3,v/v) and detection wavelength was274nm. White solid powder was obtained aftervacuum freeze drying. This white powder and octopamine standard was analized byMass spectrometer in the same condition. It can be proved that the white powder washigh purity natural octopamine by almost the same spectra.
4. Comparison of Anti-obesity effect of octopamine and commercial weight loss pillson nutritive obesity mice
In application experiments, the effect of octopamine on the anti-obesity innutritive obesity mice was studied compared with commercial weight loss pills. Thealimentary obesity model was established by high fat diet for4weeks. Then the micewere grouped feeding for another4weeks. The body weight, Lee s index, fat wetweight, FC and the content of TC and TG in groups with a certain dose of octopamineor diet pills added in the feed were significantly different from those in alimentaryobesity model group (P <0.05). The above results demonstrated that there is a certainweight and fat reducing effect of octopamine on nutritional obesity mice.
5. Synthesis of octopamine derivatives and study on antioxidant activity
In the previous literature the biological activity of octopamine focused on thepreventive function on obesity and typeⅡdiabetes. Past researches indicated thattyramine, the precursor compound of octopamine, has comparative high antioxidantactivity which is positively related to its concentration. In this paper ten derivatives ofoctopamine were synthesized and their chemical structure were confirmed by Nuclearmagnetic resonance and mass spectroscopy. The antioxidant activity of octopamine,tyramine as well as octopamine derivatives were then tested by investigating theirscavenging effect against DPPH free radicals, superoxide anion radicals and hydroxylfree radicals. It it showed that the scavenging rate of octopamine and its two derivatives, OA07and OA08against the above three kinds of radicals were higherthan that of tyramine. OA05, another octopamine derivative had prominent scavengingeffect against superoxide anion radicals, which is worthy of further attention and study.
At present there are less research report or product about octopamine. This papermay promote further research on octopamine and provide theoretical basis andresearch foundation for production and application of octopamine. Earlier studiesfound that octopamine content of fish sauce made from Engraulis and Channa asiaticais rather high, at around0.05-0.1%. Considering the actual conditions of low utilizationrate of resources and environmental pollution in squid processing, visceral ofDosidicus gigass was used to for preparation of fish sauce. Moreover, an effectivemethod was studied to extract octopamine in fish sauce. This also provides theexperimental basis for the industrial production of natural octopamine. Meanwhile,octopamine derivatives were synthesized. The antioxidant activity of octopamine andits derivatives were detected and compared with that of tyramine, the precursorcompound of octopamine. Further study on biological activity of octopamine is desiredto expand its field of development and utilization.
1.鱼露酿制过程中章鱼胺的产生
以智利外海茎柔鱼内脏为原料，采用保温、加曲保温、加酶保温等不同速酿方法制备鱼露，测定鱼露在不同酿制阶段的pH值、氨基酸态氮和章鱼胺含量，并对最终样品作感官评价。结果表明：酿制20d内，pH值变化显著，上升速度较快，从弱酸性升至中性或弱碱性；在此期间，氨基酸态氮的变化也较为显著，其浓度增加了15mg·mL~(-1)以上，20d后趋于稳定；鱼露酿制前期，章鱼胺含量逐渐增加，15-20d时，达到最大值，高于5mg·mL~(-1)，加酶保温速酿的样品中章鱼胺含量甚至超过7mg·mL~(-1)。20d后，鱼露中章鱼胺的含量呈下降趋势。为与速酿鱼露作对比，本研究测定了常温自然酿制鱼露在酿制过程中pH值、氨基酸态氮和章鱼胺含量的变化。结果显示，在常温自然酿制鱼露中的各种变化速度较速酿鱼露缓慢得多。30d后，pH值仍保持在酸性范围内，氨基酸态氮的浓度仅增加了5mg·mL~(-1)左右，章鱼胺含量为3.5mg·mL~(-1)，仅为加酶保温速酿样品中章鱼胺含量峰值的一半。
本研究利用茎柔鱼的不可食部分酿制鱼露，可以发现在酿制过程中章鱼胺含量明显提高，表明酿造过程可有效提高章鱼胺含量，这可为水产品加工的下脚料和废弃物的综合利用提供一条途径。本研究对于酿制半年以上的保温速酿鱼露样品中的章鱼胺含量也进行了测定，发现仍然高达4.5mg·mL~(-1)以上。说明鱼露可长期保持较高的章鱼胺水平。若将速酿鱼露做为提取章鱼胺的原料，可任选保温、加曲保温、加酶保温三种速酿方法之一，20d的酿制时间即可。
感官评定的结果表明保温和加曲保温速酿的样品在气味上有较浓的鱼香气，而加酶保温速酿的样品腥气较重，气味不佳。对保温速酿鱼露中组胺和重金属含量测定的结果表明其中组胺及砷、镉的含量均已超标，因此，从安全性考虑，用智利外海茎柔鱼内脏酿制的鱼露不适于直接作为调味品食用。
2.酪胺发酵生物合成章鱼胺
鱼露在漫长的发酵过程中，细胞色素P450酶系（cytochrome P450s，CYP450）对章鱼胺的生物合成起了关键的作用。文献资料显示在研究细胞色素P450酶的活性时往往要使用价格昂贵的超速冷冻离心机提取动物肝脏中的微粒体。本文参照鱼露中章鱼胺的生成路径，用高速冷冻离心机提取了含有细胞色素P450酶的草鱼肝脏S-9部位，并以之为酶源，以维生素C为氢载体，异烟肼为单胺氧化酶抑制剂，以酪胺为原料生物转化生成章鱼胺。提取得到的S-9部位冷藏在4℃条件下，其中的细胞色素P450酶活性只能保持4小时左右，而-25℃冷藏条件下其活性可保持8-10天。生物合成实验的结果显示氢载体对生物合成章鱼胺是必需的，反应1h产生的章鱼胺为879.66μg/4.5mL反应液，章鱼胺的生物合成率为21.4%，酪胺代谢率为90.0%。这表明以草鱼肝脏S-9部位为酶源生物转化生成章鱼胺是可行的，但由于P450酶系的非专一性，章鱼胺的生物合成率较低，有待进一步改进。
3．鱼露中章鱼胺的分离和提取
鱼露中章鱼胺的含量较为丰富，高于其他许多水产品。从鱼露中有效提取章鱼胺并进行精制，可以为进一步开发利用天然章鱼胺打下基础。从智利外海茎柔鱼内脏速酿鱼露中提取章鱼胺，可使废弃物得以综合利用，减少资源浪费和环境污染。本文中采用静态吸附和动态吸附相结合的方法，综合考虑吸附量、解吸率等因素，研究硅胶分离提取鱼露中章鱼胺的吸附性能和洗脱参数。结果表明：60～100目硅胶对鱼露中章鱼胺有较好的吸附分离效果，静态饱和吸附量达3.837mg·g~(-1)(干硅胶），30%的乙醇水溶液为最佳洗脱剂，洗脱率达90.48％；60～100目硅胶对鱼露中章鱼胺动态吸附的最佳上样量为4BV，用1.1BV洗脱液可基本洗脱完全。得到的洗出液采用HPLC分离系统，在流动相0.02mol·L~(-1)柠檬酸-0.02mol·L~(-1)磷酸二氢钠（7∶3），PH=3、流速10mL min~(-1)、检测波长274nm的条件下被色谱柱SepaxHP-C18柱(21.2mm250mm，10μm)精制，真空冷冻干燥后得到白色粉末状固体。将此白色固体与标准品章鱼胺在同样的条件下进行质谱分析，可证明得到的白色粉末状固体为纯度较高的天然章鱼胺。
4.章鱼胺和市售减肥药对营养性肥胖小鼠减肥作用的比较
在章鱼胺的应用实验中，本实验比较了章鱼胺和市售减肥药对营养性肥胖小鼠的减肥作用。采用高脂饲料喂养4周构建营养性肥胖小鼠模型。再将小鼠分组喂养4周后发现，饲料中加入一定剂量章鱼胺和减肥药的小组中小鼠体重、Lee s指数、脂肪湿重、脂肪系数、血清总胆固醇(TC)、甘油三脂(TG)与模型组存在显著性差异(P <0.05)。以上结果表明章鱼胺对营养性肥胖小鼠有一定的减肥降脂作用。
5．章鱼胺衍生物的合成及抗氧化活性研究
关于章鱼胺的生物活性，文献报道大多为肥胖症的治疗和II型糖尿病防治方面的研究。既往研究指出，章鱼胺的前体化合物酪胺具有较高的抗氧化活性，且与其浓度呈正相关。本文以章鱼胺为原料合成十种衍生物，并对它们进行核磁共振氢谱和质谱定性分析，确证其结构。为比较章鱼胺、酪胺及章鱼胺衍生物的抗氧化活性，本文测定了这些物质对DPPH自由基、超氧阴离子自由基、羟基自由基的清除率。研究结果表明，章鱼胺及其两种衍生物OA07和OA08，即N-正十一烷基甲酰基-2-羟基-2-(4-羟基苯基)-乙胺和N-(2-硝基-3-甲基苯甲酰基)-2-羟基-2-(4-羟基苯基)-乙胺，清除DPPH自由基、超氧阴离子自由基和羟基自由基的能力均高于酪胺；另一章鱼胺衍生物OA05，即N-(4-硝基苯甲酰基)-2-羟基-2-(4-羟基苯基)-乙胺具有较高的超氧阴离子自由基清除能力，值得进一步关注和研究。
由于国内目前有关章鱼胺的研究报告或产品较少，本课题的研究有可能进一步推动对章鱼胺的研究，为更好地生产和应用章鱼胺提供一定的理论依据和研究基础。本课题组之前的研究发现鳀鱼（Engraulis）和七星鱼（Channa asiatica）鱼露原液中章鱼胺含量较高，达到0.05-0.1%左右，考虑到我国当前鱿鱼加工业中存在资源利用率低和环境污染严重两个问题，本研究以智利外海茎柔鱼内脏为原料制备鱼露并探讨从中提取章鱼胺的有效方法，为天然章鱼胺的制取提供实验依据。同时，化学合成了章鱼胺的衍生物，将它们及章鱼胺的抗氧化活性与章鱼胺的前体化合物酪胺进行对比，旨在对章鱼胺的生物活性做进一步研究，拓展章鱼胺的开发和利用领域。
As an important biogenic amine, octopamine plays a role of neurotransmitter inorganism. Recent researches revealed that it is a natural β3-adrenergic receptor agonist(β3-ARA), which has effects on metabolism regulatinon and energy balance.Octopamine also has an important preventive function on obesity and typeⅡdiabetes.As raw materials of food and health food or auxiliary preparation for clinical medicine,octopamine can be used independently or jointly with other materials for weight lossand diabetes treatment. Natural octopamine mainly exists in Fructus aurantiiImmaturus (FaI), but the content in FaI is merely0.01~0.03%, and in fish sauce,however, relative abundant (0.01~0.1%). It is possible to extract octopamine fromfish sauce and develop health products. This paper aims at the rapid brewage of fishsauce by the waste of aquatic products, extraction of octopamine, and study on thebiological activity of octopamine and its derivatives. In this paper, visceral ofDosidicus gigass was used as raw material to produce fish sauce by3quick brewingmethods of insulation, adding either koji or enzyme under insulation. The change inoctopamine content during brewage was investigated. As the substrate, tyramine wasbioconverted to octopamine according to the formation path of octopamine in fishsauce. An effective method was studied to extract octopamine in fish sauce in order tofind the possibility of developing health food. In application experiments ofoctopamine, its effect on the anti-obesity in nutritive obesity mice was studiedcompared with weight loss pills. Finally, ten kinds of octopamine derivatives weresynthesized. For further study of the biological activity of octopamine, the antioxidantactivity of octopamine and its dereivatives were determined, and compared with that oftyramine, the precursor compound of octopamine.
1. Octopamine production during fish sauce brewing process
Fish sauce was brewed from the viscera of Dosidicus gigass in this study. Toshorten brewage period, methods such as insulation, adding enzyme or koji underinsulation was adopted. The changes in pH value, amino acid nitrogen and content ofoctopamine during brewage were investigated. The sensory characteristics were alsoevaluated. The results showed that in the first20days, the pH value increasedobviously and changed from acidic to neutral or weak alkaline, while the amino acidnitrogen rised significantly and had an increased by15mg·mL~(-1). They all becamestable after20days of brewage. Initially, the content of octopamine ascended graduallyand climbed to the top of more than5mg·mL~(-1)after15-20days of brewage. It reachedeven more than7mg·mL~(-1)in fish sauce added enzyme. After20days, the octopaminecontent showed a trend of decrease. With the speedy brewing fish sauce forcomparison, the changes in pH value, amino acid nitrogen and content of octopaminein natural brewing fish sauce were also measured. The results showed that all theabove changes in natural brewing fish sauce were much slower than those of in quickbrewing ones. After30days of natural brewage, the pH value was still acidic, theamino acid nitrogen increased only by5mg·mL~(-1). Meanwhile, the content ofoctopamine reached3.5mg·mL~(-1), which was only half of the content peak in fishsauce added enzyme.
In this study inedible parts of Dosidicus gigass was used to make fish sauce. Itcan be found that octopamine content improved effectively in the brewing process,which indicates that octopamine will be produced during brewage. Maybe this is a newway for comprehensive utilization of waste during aquatic products processing. Theoctopamine content in fish sauce after half year s insulation brewage was rather stableand more than4.5mg·mL~(-1), which demonstrated that the content of octopamine in fishsauce can keep at high level for a long time. If only for octopamine extraction,20daysof brewage is enough, and any one of the3quick brewing methods can be chosen.
The results of sensory evaluation showed that the smell of fish sauce addedenzyme was the most unpleasant among the three kinds of samples. The content ofhistamine and heavy metal in insulation brewing fish sauce were determined. Theresult indicated that the content of histamine, As and Cd were higher than national standard. Therefore, from the security considerations, the fish sauce is not suitable forhuman consumption as a condiment directly.
2. Biotransformation from tyramine to octopamine by fermentation
Cytochrome P450enzyme system (cytochrome P450s, CYP450) plays a key rolein the biosynthesis of octopamine during the long fermentation process of fish sauce.Most literature displayed that expensive speeding refrigerated centrifuge was oftenused to extract microsome from animal liver in the study of cytochrome450activities.In this study, hepatic S-9place containing cytochrome450in the liver of Grass Carpwas extracted by a high speed refreigerated contrifuge. When the hepatic S-9place wasstored at4℃, the cytochrom P450activity can only keep about4h, while it can bemaintained for8-10days at-25℃. According to the formation path of octopamine infish sauce, tyramine was bioconverted to octopamine with Grass Carp hepatic S-9place as the resource of cytochrome P450, ascorbic acid as hydrogen carrier andisoniazid as monoamine oxidase inhibitor. The results showed that hydrogen carrier isnecessary for the biosynthesis of octopamine, and the octopamine obtained after1hcatalysis was879.66μg in4.5mL reaction liquid. The biosynthesic rate of octopamineand metabolic rate of tyramine were21.44%and90.03%, respectively. This indicatedthat it is feasible to produce octopamine when hepatic S-9place of Grass Carp beingtaken as resource of enzyme. But because of the non-specifity of cytochrome P450thebiosynthesic rate of octopamine was relative low, which need to be further improved.
3. Octopamine separation and extraction in fish sauce
In fish sauce octopamine content is rather rich, higher than many other aquaticproducts. Extraction and refining of octopamine in fish sauce can lay the foundationfor further development and utilization of natural octopamine. It is a way ofcomprehensive utilization of aquatic litter to process fish sauce by squid viscera. It alsocan reduce the waste of resources and environmental pollution. The adsorption andelution parameters was studied during separation and extraction of octopamine in fishsauce, while the static and dynamic adsorption was combined and adsorption anddesorption rate were also be considered. The results demonstrated that60～100meshsilica gel has high efficiency in separate adsorption with the static saturated adsorption of3.837mg g-1(dry silica gel).30%ethanol was as eluent with its elutive power90.48%. The optimal value of the dynamic adsorption of octopamine in fish sauce on60～100mesh silica gel was4BV, and when the volume of eluent was1.1BV, theeluting process was basically completed. The eluate was refined by a HPLC separationsystem. A SepaxHP-C18column (21.2mm x250mm,10μm) was used. The mobilephase was0.02mol·L~(-1)citric acid-0.02mol·L~(-1)sodium dihydrogen phosphate (7:3,v/v) and detection wavelength was274nm. White solid powder was obtained aftervacuum freeze drying. This white powder and octopamine standard was analized byMass spectrometer in the same condition. It can be proved that the white powder washigh purity natural octopamine by almost the same spectra.
4. Comparison of Anti-obesity effect of octopamine and commercial weight loss pillson nutritive obesity mice
In application experiments, the effect of octopamine on the anti-obesity innutritive obesity mice was studied compared with commercial weight loss pills. Thealimentary obesity model was established by high fat diet for4weeks. Then the micewere grouped feeding for another4weeks. The body weight, Lee s index, fat wetweight, FC and the content of TC and TG in groups with a certain dose of octopamineor diet pills added in the feed were significantly different from those in alimentaryobesity model group (P <0.05). The above results demonstrated that there is a certainweight and fat reducing effect of octopamine on nutritional obesity mice.
5. Synthesis of octopamine derivatives and study on antioxidant activity
In the previous literature the biological activity of octopamine focused on thepreventive function on obesity and typeⅡdiabetes. Past researches indicated thattyramine, the precursor compound of octopamine, has comparative high antioxidantactivity which is positively related to its concentration. In this paper ten derivatives ofoctopamine were synthesized and their chemical structure were confirmed by Nuclearmagnetic resonance and mass spectroscopy. The antioxidant activity of octopamine,tyramine as well as octopamine derivatives were then tested by investigating theirscavenging effect against DPPH free radicals, superoxide anion radicals and hydroxylfree radicals. It it showed that the scavenging rate of octopamine and its two derivatives, OA07and OA08against the above three kinds of radicals were higherthan that of tyramine. OA05, another octopamine derivative had prominent scavengingeffect against superoxide anion radicals, which is worthy of further attention and study.
At present there are less research report or product about octopamine. This papermay promote further research on octopamine and provide theoretical basis andresearch foundation for production and application of octopamine. Earlier studiesfound that octopamine content of fish sauce made from Engraulis and Channa asiaticais rather high, at around0.05-0.1%. Considering the actual conditions of low utilizationrate of resources and environmental pollution in squid processing, visceral ofDosidicus gigass was used to for preparation of fish sauce. Moreover, an effectivemethod was studied to extract octopamine in fish sauce. This also provides theexperimental basis for the industrial production of natural octopamine. Meanwhile,octopamine derivatives were synthesized. The antioxidant activity of octopamine andits derivatives were detected and compared with that of tyramine, the precursorcompound of octopamine. Further study on biological activity of octopamine is desiredto expand its field of development and utilization.
引文
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