Fermentation Preparation And Biological Activity Investigation Of Octopamine

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 typeâ…¡diabetes.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 merely0.01⁰.03%, and in fish sauce,however, relative abundant （0.01⁰.1%）. 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^-1after15-20days of brewage. It reachedeven more than7mg·mL^-1in 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 fermentationCytochrome 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 sauceIn 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^-1citric acid-0.02mol·L^-1sodium 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 miceIn 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 activityIn the previous literature the biological activity of octopamine focused on thepreventive function on obesity and typeâ…¡diabetes. 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.