Selenium Detection Of Selenium-rich Pleurotus Ostreatus Protein

Selenium(Selenium,Se)is an essential trace element for humans and animals,which cannot be formed in bodies and can only be intaken through diets.There are many forms of selenium in nature,and the absorption and utilization of selenium by organisms are subject to its form.The bioavailability and biological activity of the organic selenium are significantly higher than that of the inorganic selenium.At present,the application research on selenium-enriched pleurotus ostreatus and its active substances has made great progress.Selenium-enriched pleurotus ostreatus and its extracts can be used in food,medicine,additives,etc.The development of products using selenium-enriched pleurotus ostreatus as a raw material has potential commercial value.And qualitative and quantitative methods of the selenium in foods urgently required to be developed and optimized.In this project,the selenium-enriched pleurotus ostreatus powder was used as the raw material to establish and optimize the extraction process and acid hydrolysis conditions of selenium-enriched pleurotus ostreatus protein.At the same time,selenomethionine(SeMet),selenocystine(SeCys)and methylselenocysteine(MeSeCys)were analyzed as target compounds,in selenium-enriched pleurotus ostreatus protein,using high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).Effects of extraction temperature?extraction time and etc.on the morphology and stability of the organic selenium in the seleniumenriched pleurotus ostreatus protein during the extraction process were studied.Conclusions are as follows:Selection and optimization of the selenium-enriched pleurotus ostreatus protein extraction method.In this experiment,selenium-enriched pleurotus ostreatus powder was used as the raw material to extract selenium-enriched pleurotus ostreatus protein by ultrasonic-assisted salt extraction.The extraction temperature,extraction time,protein precipitation p H,liquid-solid ratio and salt concentration were subjected to a single factor experiment.The optimal process conditions for the protein extraction of selenium-enriched pleurotus ostreatus by auxiliary salt extraction method were:extraction time 120 min,salt concentration 0.5mol/L,liquid-solid ratio 30 m L/g,and extraction temperature 50?.Under these conditions,the protein yield was 39.6%.As compared with the cellulase treatment,hemicellulase treatment,ultrasonic-assisted water extraction,alkaline extraction,ultrasonic-assisted alkaline extraction and salt extraction,the ultrasonic-assisted salt extraction method showed a higher efficiency on the protein extraction.The effect of protein extraction method on the extraction rate of selenium protein in selenium-enriched pleurotus ostreatus.The experiment compared the selenium content of proteins obtained by different extraction methods and extraction conditions,and it found that different protein extraction methods have different extraction efficiency on selenium protein extraction from selenium-enriched pleurotus ostreatus.The results showed that the protein obtained by ultrasonic-assisted salt extraction had the highest selenium content,up to 70.56?g/g.Establishment and optimization of HPLC-MS/MS method for the organic selenium detection.In this experiment,the single factor experiment was used to study the effect of acid concentration,acid hydrolysis temperature and acid hydrolysis time on the protein hydrolysis rate and the stability of selenoamino acids.The optimal conditions for acid hydrolysis were determined as follows : hydrochloric acid concentration 4mol/L,acid hydrolysis temperature 80?,acid hydrolysis time48 h.Under the optimal conditions,the hydrolysis rate of the protein was 75%.LC separation of peptides was performed with a Thermo AQUASIL C18 column(150×2.1mm,3?m),and gradient elution was carried out with A solvent(1%acetonitrile aqueous solution 0.1% formic acid)and B solvent(99% acetonitrile aqueous solution 0.1% formic acid).Multi-reaction monitoring(MRM)was selected.The transitions monitored were as follows;methylselenocysteine:m/z183.99?166.96;selenocysteine : m/z 336.92?247.87;selenomethionine : m/z198.00?180.98.The results showed that under the optimal detection conditions,within the concentration range of 20 to 500ng/m L,the linear regression equations for methylselenocysteine,selenocystine and Selenomethionine were A = 18636 c +41678,A=22979c-114002 and A=65986c+282145,respectively.And all linear correlation coefficients R were greater than 0.99.The optimized HPLC-MS/MS method of organic selenium was used to analyze the existence and content of organic selenium in pleurotus ostreatus.The results showed that organic selenium in selenium-enriched pleurotus ostreatus existed in forms of methylselenocysteine,selenocystine and selenomethionine.Selenomethionin e was the most abundant selemoamino acid in selenium-enriched pleurotus ostreatus,with a concentration up to 22.91±0.82?g/g.