Study On The Screening, Taxonomic Analysis And Application Of One Novel, Salt-Tolerant And High Efficient Biogenic Amines Degrading Bacterium

Biogenic amines, as a kind of hazardous substances in food especially infermented food, have attracted worldwide attentions. Exploring effective technologyfor the reduction of biogenic amines in foods has important implications for foodsafety and human health. Degrading biogenic amines by microorganisms is apromising method for the control of biogenic amines in foods. The screening,identification, biogenic amines degrading properties, safety evaluation and applicationof one novel biogenic amines degrading bacterium are systematically studied in thisdissertation.(1) The sample preparation method, and the high performance liquidchromatography method using fluorescence detector and pre-column derivatizationwith dansyl chloride are established for determining multiple biogenic amines in fishsauce. The effectiveness of the proposed methods is verified by some indexes. Atpresent, in the active industrial standard and exposure draft of national standardregarding commercially Chinese fish sauce, there is not any limit standard aboutbiogenic amines. Biogenic amines in commercially Chinese fish sauce are analyzedby the established methods above. The results show that among the thirty five samplesdetected, twenty samples contain more than50mg/kg histamine, twenty one samplescontain more than100mg/kg tyramine and ten samples contain more than1000mg/kg total biogenic amines. It indicates that biogenic amines are ubiquitous incommercially Chinese fish sauce.(2) The strain SWA25with the properties of salt-tolerance, eight biogenicamines degrading ability and not accumulation of any biogenic amines by itself isscreened from fermented fish sauce sample. The strain SWA25shows closestrelationship with H.xianhensis CGMCC1.6848T, but the16S rRNA gene sequences similarity and DND hybridization between these two strains are only96.5%(less than97%) and30.7%(less than50%), respectively. It indicates that the strain SWA25maybe a novel species of the genus Halomonas. The results of polyphasic taxonomicanalysis confirm that the strain SWA25is exactly a novel species of the genusHalomonas. According to the location where the strain SWA25is isolated, the nameHalomonas shantousis sp.nov. is proposed.(3) The biogenic amines degrading properties of H.shantousis SWA25is studied.The growth of H.shantousis SWA25is strongly suppressed by tryptamine when theconcentration is more than200mg/kg. The results show that the reduction of biogenicamines is mainly because of the degradation by amine oxidase distributing on the cellmembrane, not the adsorption or bioaccumulation mechanisms. Under certainconditions, H.shantousis SWA25can degrade all the tryptamine, phenylethylamineand tyramine in nine hours, and degrade66.7%of histamine、42.9%of putrescine、52.4%of cadaverine、48.0%of spermidine and42.0%of spermine in20hours. In theenvironment of pH ranging from6.0to8.0, temperature ranging from30°C to40°C,NaCl concentration ranging from0.0%to3.0%or ethanol concentration ranging from0.0%to2.0%, H.shantousis SWA25shows the highest biogenic amines degradingactivity. In the environment of pH ranging from4.0to6.0, temperature ranging from20°C to30°C or NaCl concentration ranging from3.0%to15.0%, H.shantousisSWA25can keep most of its biogenic amines degrading activity. The system containsmore than2%ethanol can strongly inhibit the biogenic amines degrading activity ofH.shantousis SWA25.(4) The safety of H.shantousis SWA25is evaluated. The results of acute toxicitytest show that the maximum tolerable dose of mouse is more than15g/kg·bw,meaning that H.shantousis SWA25is nontoxic. The results of the inherited toxic tests(including Ames test, mouse bone marrow micronucleus test and mouse spermatidmalformation test) indicate that there is no genetic toxicity in H.shantousis SWA25.There is no subchronic toxicity observed in H.shantousis SWA25during thirty daysfeeding test. Therefore, H.shantousis SWA25can be safely applied to the foodindustry as a functional starter culture. (5) H.shantousis SWA25is applied to the fermentation of Setipinna taty fishsauce as a functional starter culture for biogenic amines degradation. This studied isalso carried out to evaluate the biogenic amines degrading capacity of H.shantousisSWA25in food matrix of high salt content. Comparing with the control group,H.shantousis SWA25can reduce64.5%of histamine,59.2%of tyramine,71.0%ofcadaverine,63.4%of tryptamine,68.2%of phenylethylamine,22.0%of putrescineand55.3%of total biogenic amines, but does not influence the content of spermidineand spermine which are trace biogenic amines in fish sauce. Meanwhile, H.shantousisSWA25does not significantly interfere with the pH values, NaCl concentrations, totalsoluble nitrogen, amino acid nitrogen and total volatile basic nitrogen duringfermentation, and does not change the free amino acids composition and sensoryflavor when the fermentation is ended. H.shantousis SWA25can significantlyincrease the amount of total aerobic microorganisms, inhibit the growth of tyramineand histamine producing bacteria, but does not influence the growth of putrescine andcadaverine producing bacteria. Therefore, H.shantousis SWA25can be used as afunctional starter culture for biogenic amines degradation in Setipinna taty fish saucefermentation.(6) The silver carp sausage was produced by compound fermentation withH.shantousis SWA25(used as a functional starter culture for biogenic aminesdegradation) and L.plantarum CICC20718(used for pH decrease in sausage). Thisstudied is also carried out to evaluate the biogenic amines degrading capacity ofH.shantousis SWA25in food matrix of low pH value. Comparing with the controlgroup without any starter culture, compound fermentation can reduce the pH valuequickly, increase the titratable acid content, water content, total free amino acidscontent and water activity, inhibit the formation of total volatile basic nitrogen, andpromote the whiteness, hardness, chewiness, springiness and the acceptability ofsilver carp sausage products. The compound fermentation can also increase theamount of total aerobic microorganisms and lactic acid bacteria, and reduce theamount of yeast, enterobacteria and pseudomonas. In addition, the compoundfermentation can reduce87.2%of histamine,77.0%of tyramine,70.0%of putrescine, 66.0%of cadaverine,67.5%of tryptamine,73.9%of phenylethylamine and74.4%oftotal biogenic amines. When the compound fermentation group compares with thegroup using L.plantarum CICC20718as the only starter culture, we can infer thatL.plantarum CICC20718plays a leading role in the changes of physicochemicalproperties, and both L.plantarum CICC20718and H.shantousis SWA25play animportant role in the reduction of biogenic amines in silver carp sausage. Besides,H.shantousis SWA25is crucial to the reduction of tyramine. Therefore, the compoundfermentation with L.plantarum CICC20718and H.shantousis SWA25can be appliedto produce silver carp sausage with low biogenic amines content.At present, most of the biogenic amines control technologies are proposed by themechanism of inhibiting or inactivating microorganisms. These technologies are notstable, cannot be used to fermented foods or degrade already exist biogenic amines.The safe strain obtained in this dissertation can efficiently degrade multiple biogenicamines and adapt to the complex food matrix of high salt content or low pH value.This study provides an effective way to the biogenic amines reduction in foods, andlays a foundation for the development of functional starter cultures for biogenicamines degradation.