Diversity Of Toxin Production And Influencing Mechanism Of Prorocentrum Lima

Prorocentrum lima（P.lima）was not only a common harmful algae,but also one of the main dinoflagellates that produced diarrheal shellfish.In experiments,the production of toxin had been proved to be inversely proportional to nutritional restriction,when the proportion of nutritional limit was higher than a certain proportion.Toxin production increased,while temperature,nutrients,light and other factors also affected the toxin production of P.lima.In order to analyze the differences and influencing factors of toxin production of P.lima in different environments,the studies on the growth and toxin production of P.lima in recent years were reviewed in this paper.It was found that the total amount of toxin produced by toxin-producing strains varied greatly under different culture conditions.At the same time,it was of great significance to explore the effects of environmental factors on toxin production of P.lima.In this study,taking P.lima as the research object,the physiological relationship between region and P.lima was analyzed,and a DSTs fingerprint database based on P.lima from different regional sources was established.According to the results,two typical P.lima strains（3XS and SHG strains）were selected to explore the effects of light intensity and light cycle on their growth and toxin production.2-¹³C-glycine was labeled by isotope labeling,which provided a new method and idea for the study of the synthesis and metabolic pathway of DSTs and its esterified toxins.Finally,through the optimization of esterification hydrolysis conditions and simulated digestion,it was found that there was a difference between the traditional monitoring methods and the actual digestion hydrolysis rate.The main contents were as follows:（1）Ten typical P.lima strains were selected from four major coastal waters of China（Yellow Sea,Bohai Sea,South China Sea and East China Sea）and subcultured in the laboratory.The growth physiological indexes were analyzed and the growth curves and toxin production curves were drawn.At the same time,the HRMS screening and identification methods of OA,DTX1 and their derivatives in P.lima were established.24 kinds of DSTs components were successfully identified using a variety of parent ions and rich daughter ion fragments.By using this method,the toxin metabolism spectra of the main P.lima in the four coastal areas of China were drawn,the toxins were classified according to their characteristics,and the effects of different regions on their growth and toxin production were analyzed.At the same time,the diversity of P.lima toxin production was explored and studied by the rich toxin metabolism spectra of different P.lima algae strains.（2）Two typical algae strains SHG and 3XS with great differences in toxin production were selected as the research objects to explore the changes of esterified toxin content,growth status,and pigment content of P.lima in different illumination and light cycles.Combined with the fluorescence imaging map of the microscope,the effects of light on the toxin production and physiology of P.lima were revealed.Multi-group science was used to analyze the law of substance accumulation and related metabolite transformation during the growth of algae cells and to clarify the change law and key regulation mechanism of light on the production of P.lima esterified toxins.Light was an important environmental factor,and both light intensity and light time affected the growth of algae.It was found that the photosynthetic systems of the two algae were damaged under the influence of strong light,and the shortening of light duration could restore them to a certain extent,but could not return to the normal level.The change of pigment content proved that strong light caused stress to its photosynthesis,and the change of the light cycle could slow down its effect.Both light intensity and light cycle could affect the production of toxins.Strong light and prolonged light time could promote the synthesis of toxins and their esterified states,but the effects on different algae strains were slightly different.（3）Using 2-¹³C-glycine as the material,¹³C stable isotope tracer technique combined with high resolution mass spectrometry was used to study the synthesis and metabolic pathway of diarrheal shellfish toxin and its diol esterified toxin.SHG strain and 3XS strain were used as research objects.High resolution mass spectrometry was used to identify toxins and ¹³C isotopes.Glycine as a source of added nitrogen had little effect on the growth of algae cells and significantly increased the total amount of DSTs produced by the two strains of algae.The results of isotope labeling showed that all DSTs were labeled with ¹³C-glycine,and the toxin production had no significant change before and after labeling.At the same time,high-resolution mass spectrometry found that the abundance of isotope isomers of DSTs was labeled,in which the esterification state was more affected by labeling,and the second-order mass spectrometry clearly showed the labeling of fragment ions and dehydration peaks.This method intuitively illustrated that glycine could be used as a toxin-producing donor,and the stable isotope labeling method was used to realize the synchronous labeling of esterified state for the first time,which was helpful for the further study of the formation mechanism of diarrheal shellfish poisoning.（4）In this chapter,the algae strain SHG,with a high content of esterified DSTs,was selected to further explore and optimize the traditional alkaline hydrolysis conditions of OA and its esterified state by orthogonal experiment.At the same time,a variety of esterases were used to explore the effects on OA and esterified states.Finally,the static in vitro human digestion scheme was used to investigate the effect of human digestion on OA under the influence of the shellfish matrix.It was found that the increase of temperature,time,and alkali concentration increased the degree of hydrolysis,and the reaction had a strong temperature dependence,with temperature having the most severe effect.When the hydrolysis temperature was 76°C,the hydrolysis time was 50 min,and the concentration of Na OH was 2.5 mol/L,the degree of esterification hydrolysis of OA was the highest.The hydrolysis ability of the three kinds of esterifying enzyme commonly used in scientific research was weaker than alkaline hydrolysis.Among them,the hydrolysis ability of Bacillus subtilis esterase and porcine liver esterase was slightly stronger,while that of acetylcholinesterase was very limited.The hydrolysis ability of simulated digestion was stronger than that of single enzyme,but there was still a gap between its ability and alkaline hydrolysis.It was proved that there were some errors in the existing monitoring methods to estimate the toxicity of DSTs by the alkali-thermal reaction,which may lead to the accumulation of highly toxic toxins in the human body,and the re-estimation of the toxicity of diarrhoeal shellfish toxins was worthy of further discussion.