Enrichment Of Exogenous Inorganic Selenium In Duckweed And Its Protein-binding Forms

Objective:Selenium is one of the essential trace elements that are vital to human health,and both selenium deficiency and selenium overdose in human body can lead to related diseases and seriously endanger human health.The human body mainly through dietary supplementation of selenium,and plant-based diet is one of the main sources of selenium supplementation.Duckweed is a protein-rich aquatic floating plant,which can be a good source of protein supplement in animal feed.Accordingly,in this paper,duckweed was mixed with two exogenous inorganic selenium in culture,focusing on the effect of exogenous inorganic selenium on protein and protein selenium content in duckweed and the accumulation mechanism of selenium binding with protein in duckweed,to provide some scientific basis for the development of selenium-rich duckweed as a new selenium-rich plant protein material.Methods:In this study,the samples of duckweed were cultured and analyzed by tolerance test and nutritional test.Five selenium levels were set for duckweed tolerance experiment:0.0,1.0,2.0,5.0 and 10.0 mg/L.The exogenous inorganic selenium types were Na₂SeO₃and Na SeO₄,and the duckweed species were Landoltia punctata.The culture cycle was 7 days.Four selenium levels were set in the duckweed nutrition experiment:0.0,1.0,2.0 and 5.0 mg/L.The types of exogenous inorganic selenium were Na₂SeO₃and Na SeO₄,and the types of duckweed were Landoltia punctata and Spirodela polyrhiza.The culture cycle was also 7 days.After the culture experiment,duckweed was harvested immediately for the determination of physiological indexes and selenium-related indexes.The fresh weight of duckweed was determined by weighing method.SOD activity was determined by NBT method,POD activity was determined by guaiacol method,and CAT activity was determined by UV absorption method.The chlorophyll and carotenoid in duckweed were extracted by 95%ethanol.The contents of chlorophyll and carotenoid were determined by ultraviolet spectrophotometer under specific wavelength.The improved sequential extraction method was used to extract the protein of duckweed,and the biuret reagent method was used to determine the protein content.The total selenium and protein selenium contents of duckweed were determined by hydride generation-atomic fluorescence method（HG-AFS）.At the same time,stepwise regression method was used to analyze the factors affecting the content of Se in protein.Results:（1）Low concentration（1,2 mg/L）of Na₂SeO₃could promote the growth of duckweed,and when Na₂SeO₃concentration exceeded 5 mg/L,it could inhibit the growth of duckweed（P<0.05）.1 mg/LNa₂SeO₄promoted the growth of duckweed,while Na₂SeO₄concentration higher than 2 mg/L inhibited the growth of duckweed（P<0.05）.The promotion effect of 1 mg/L Na₂SeO₃on duckweed growth was more obvious than that of Na₂SeO₄（P<0.05）,and the inhibitory effect of 5 mg/L and 10mg/L Na₂SeO₃and Na₂SeO₄on duckweed growth was not significantly different（P>0.05）.（2）The SOD,POD and CAT enzyme activities of duckweed first increased and then decreased with the increase of Na₂SeO₃concentration.The activities of three antioxidant enzymes in 5 mg/L treatment group were the highest,and the SOD activity in 5 mg/L treatment group was significantly higher than that in control group and other treatment groups（1,2,10 mg/L）（P<0.05）.Compared with the control group,the activities of POD enzyme and CAT enzyme had no significant difference（P>0.05）.With the increase of Na₂SeO₄concentration,SOD activity of duckweed gradually increased,while POD and CAT activities showed a trend of increasing first and then decreasing.The SOD activity of 10 mg/L treatment group was significantly higher than that of control group and 1 mg/L treatment group,the POD activity of 1mg/L treatment group was significantly higher than that of control group and 10 mg/L treatment group（P<0.05）,but there was no significant difference in CAT activity between treatment groups and control group（P>0.05）.（3）The total selenium content of duckweeds increased with the increase of exogenous inorganic selenium concentration,and the total selenium content of Na₂SeO₃and Na2SeO₄treatment groups was much higher than that of control group（P<0.05）.Compared with the two different inorganic selenium treatments,the total selenium content of duckweed in Na₂SeO3 treatment group was higher than that in Na₂SeO₄treatment group,and there was no significant difference in total selenium content between the two inorganic selenium treatments（P>0.05）.（4）In Na₂SeO₃treatment group of Landoltia punctata,the contents of chlorophyll a,b and total chlorophyll decreased gradually with the increase of their concentrations,while the contents of carotenoid were almost unchanged.The contents of chlorophyll a,b and total chlorophyll in Na₂SeO₄treatment group were close to those in control group,and the contents of carotenoid increased with the increase of Na₂SeO₄concentration.The contents of chlorophyll a,b,carotenoid and total chlorophyll in Na₂SeO₃treatment groups of Spirodela polyrhiza were very close to those in the control group.The contents of chlorophyll a,b and chlorophyll in 5 mg/L Na₂SeO₄treatment group were significantly lower than those in control group（P<0.05）,but there was no difference in the content of carotenoid in Na₂SeO₄treatment groups compared with control group（P>0.05）.（5）The proportion of albumin and globulin in the two treatments was much higher than that of the other two proteins.The contents of albumin,globulin and prolamin in each treatment group of Landoltia punctata and Spirodela polyrhiza were very close.The gluten content of Spirodela polyrhiza was significantly higher than that of Landoltia punctata in all treatment groups（P<0.05）.Inorganic selenium supplementation significantly increased the contents of total selenium and protein selenium in Landoltia punctata and Spirodela polyrhiza（P<0.05）.Albumin selenium and gluten selenium are the main forms of selenium in Landoltia punctata and Spirodela polyrhiza There were no significant differences in the content of four protein selenium and total selenium in Landoltia punctata and Spirodela polyrhiza（P>0.05）.（6）There was a positive correlation between total selenium content and total protein selenium content（P<0.01）,and there was also a positive correlation between total selenium content and four protein selenium content in（P<0.01）in Landoltia punctata and Spirodela polyrhiza.And there was also a positive correlation between selenium content of four proteins in Landoltia punctata and Spirodela polyrhiza（P<0.01）.Protein content was the mediating variable in the process of total selenium affecting protein selenium content,and the mediating effect was partial mediating.Conclusion:The suitable concentration range of exogenous inorganic selenium for duckweed growth is 0-5 mg/L.Low concentration of inorganic selenium can promote the growth of duckweed,but high concentration of inorganic selenium may inhibit the growth of duckweed,and duckweed has a stronger tolerance to Na₂SeO₃.When exogenous inorganic selenium was used to culture duckweed,1-2 mg/L Na₂SeO₃was helpful to promote the growth of duckweed,improve the activity of antioxidant enzymes.The concentration of albumin and globulin were higher than other species in Landoltia punctata and Spirodela polyrhiza.Exogenous inorganic selenium can increase the contents of protein selenium and total selenium of two duckweeds.Albumin selenium and glutelin selenium are the main forms of protein selenium of two duckweeds.With the same concentration of inorganic selenium added exogenously,the growth status of Spirodela polyrhiza is better than that of Landoltia punctata,so Spirodela polyrhiza is more suitable for the development of selenium-enriched protein biomaterials,considering that the growth condition of Spirodela polyrhiza is better than that of Landoltia punctata.The correlation analysis of each index of the duckweed and the mediating effect analysis of the factors influencing the protein selenium content of the duckweed showed that the protein content of the Landoltia punctata was the mediating variable influencing the protein selenium content,and its mediating effect was partially mediated.