Determination Of Biogenic Amines And Isolation Of Histamine-producing Bacteria In Aquatic Foods

Biogenic amines(BAs),a category of important physiological active substances,are mainly formed through the microbial decarboxylation of specific free amino acids in aquatic products.It is affirmed that the ingestion of aquatic foods containing high levels of BAs may result in severe toxicological symptoms for human body.In this dissertation,a simple pre-column derivatization method was established for the simultaneous determination of BAs in aquatic products by high-performance liquid chromatograph(HPLC).The formation of BAs and the growth of microbial floras in aquatic products during the storage were also investigated.Moreover,the species of histamine-producing bacteria in blue scad were isolated and identified.This dissertation is mainly consisted of three sections described as follows:Section one,a method of determining the concentrations of several BAs in aquatic products by HPLC coupled with pre-column dansyl chlorid derivation was established.Under the optimized experimental conditions,seven BAs(including tryptamine,putrescine,cadaverine,histamine,tyramine,spermidine and spermine)could well be separated within 20 min,and the average recoveries tested by adding standards ranged from 90.3% to 102%.The detection limits of the method ranged from 0.02 to 0.26 mg/kg for the seven BAs.Results also showed that putrescine,cadaverine,histamine and tyramine were the dominant BAs in nine species of aquatic products,but the concentrations of BAs in most of the samples were low,except for the histamine(577 mg/kg)in some of blue scad.Section two,the formation of BAs and the growth of microbial floras in aquatic products during the storage were investigated.Changes of BAs in blue scad(Decapterus maruadsi),golden thread(Nemipterus virgatus),belt fish(Trichiurus haumela)and octopus(Octopus vulgaris)separately stored at ? 20 ° C,0 ° C,4 ° C and 25 ° C for different time were measured.Furthermore,the growth of microbial floras during the storage at 4°C and 25°C were analyzed.Results showed that the levels of most BAs significantly increased with the increase of storage time and temperature(p<0.05),while no significant change was observed during the storage at ?20°C(p>0.05).In addition,during the storage at 4 and 25°C,the amounts of microbial floras in blue scad and octopus significantly increased with the increase of storage time and temperature(p<0.05).It can be found simultaneously that the growth of microbial floras strongly and positively correlated with the formation of putrescine,cadaverine,histamine and tyramine during the storage,except for histamine in octopus.Section three,the species of histamine-producing bacteria in blue scad were isolated and identified.The histamine-producing bacteria were isolated from blue scad using a histamine-producing bacterium isolation medium,and their abilities of producing histamine and other BAs were determined by the method of HPLC.In addition,the identification of the histamine-producing bacteria was performed by the sequence of 16 S rDNA and the constructed neighbor-joining phylogenetic tree.Results showed that nine histamine-producing bacteria(H1~H9)were isolated from blue scad,and H8 strain was found to be the strongest histamine producer with the ability of producing 115 mg/L histamine in trypticase soy broth supplemented with 1.0% L-histidine,while other strains were the weak histamine producers(13.0~20.4 mg/L).In addition,except for histamine,the nine strains were able to produce other BAs,such as putrescine,cadaverine,and tyramine,etc.Results also showed that most of the histamine-producing bacteria were Gram negative strains,which were identified as Arthrobacter bergeri(H1),Pseudomonas sp.(H2,H5 and H6),Psychrobacter sp.(H3),Shewanella baltica(H4 and H7),Aeromonas salmonicida(H8 and H9),respectively.In conclusion,the concentrations of BAs in most iced aquatic products were low;frozen temperature can effectively prevent the formation of BAs;putrescine,cadaverine,hisamine and tyramine can be used as the freshness indexes for aquatic products;most of the histamine-producing bacteria isolated from blue scad were Gram negative strains,in which the strain of Aeromonas salmonicida was the strongest histamine-producing bacterium.