Bio fortification of Leek with Selenium Laboratory of Analytical Chemistry & Applied Ecochemistry GHENT UNIVERSITY [In cooperation with ILVO] Promoter: Prof. Filip Tack Co-promoter: Prof. Gijs du Laing By R.V.SRIKANTH.LAVU
Contents � Introduction � Pot experiments (UGent) Aim Experiment Results Discussion � Field experiments ( Field trial ) Aim Experiment Results Discussion � Genetic leek trial (Field trial at ILVO) Aim Results Discussion � Incubation experiments Aim Preliminary results
Introduction � Food crops are the major dietary sources of Selenium (Se) � The content of Se in food depends on the Se content of the soil where plants are grown � Recommended daily dietary uptake of Se for women and men 55 � g and 75 µg per person, respectively � Different Se enriched Allium species have been proposed as dietary supplements [ Clark et al ] � Lots of studies describe ability of Se uptake by Allium species, in particular onion, garlic and chives � For instance, garlic can assimilate Se concentrations above 100 mg/kg plant when they are cultivated in seleniferous soils
Aim Pot experiments � Aim: To study the potential Se uptake and speciation changes in leek with various Se forms and application practices � Three different Se forms Sodium selenate, Sodium selenite and Barium selenate � Three different application approaches Hole, Surface and Mixing application � Four different Se doses 0.2, 1.3, 2.6 and 3.8 µg Se g - 1 soil
Experiment Pot experiments � commercially available Leek ( Allium ampeloprasum var. porrum ) plantlets of Harston variety were purchased � The boxes were filled with 25 kilograms of soil in each box and the soil was completely mixed in mixing rotator with the desired concentration of Se (mixing procedure) � Desired Se concentration was applied into the hole application and on surface of the soil in surface application � After 3 months, plants were removed from boxes and washed first with tap water to remove the surface contaminants, followed by rinsing with deionized water � The entire plant chopped into pieces manually and transferred into polyethylene boxes and shock frozen them immediately with liquid nitrogen and stored at -80°C and then freeze dried by liophilizer and powdered with milling apparatus
Results Pot experiments Table1: Total selenium content of selenium enriched leek. Data represent the mean value of the samples measured in 4 replicates Mixing-Ba- Mixing-Na- selenate Se conc. used for Hole-Selenite Surface-selenite Mixing-selenite selenate samples± enrichment ( � g samples±SD ( � g samples±SD ( � g SD ( � g samples±SD samples±SD ( � g g −1 ) ( � g g −1 ) g −1 ) g −1 ) g −1 ) g −1 ) 0.2 µg/g - 1 soil 21.7±23.9 16.2±8.8 24.1±5.2 102.6±24.4 61.4±40.2 1.3µg/g - 1 soil 37.3±46.5 15.6±11.6 49.7±63.0 313±29.2 63.2±42.1 2.6 µg/g - 1 soil 57.0±27.0 58.0±27.8 71.2±12.7 717±207 255.6±61.3 3.8 µg/g - 1 soil 98.7±42.6 54.9±31.3 103.8±33.2 820±320 288±207
Results Pot experiments Fig. 2 HPLC–ICP MS analysis of a mixture of 7 selenium species using (A) Anion exchange 1. Se-cystine, 2. Se-methyl-Se-cystine, 3. selenite, 4. Se- methionine, 5. γ -glutamyl methyl selenocysteine, 6. selenate, 7. γ -glutamyl selenomethionine (B) ion pairing reversed phase separation 1: selenate, 2: selenite, 3: selenocystine, 4: Semethylselenocysteine, 5: selenomethionine, 6: γ -glutamyl methyl selenocysteine, 8: γ -glutamyl selenomethionine
Results Pot experiments Fig. 3: Overlay of chromatograms of standard containing 7 Se-species anion exchange column (A)Reference standard mixture 1.Se-cystine, 2. Se-methyl-Se-cystine, 3. selenite, 4. Se- methionine, 5. γ -glutamyl methyl selenocysteine, 6. selenate,7. γ -glutamyl selenomethionine (analyses conducted by HPLC-ICP-MS) (B) enzymatic (protease) extract of Se-enriched leek fertilized by Na 2 SeO 3
Discussion Pot experiments � When leek was enriched with Selenate at a each concentration chosen, the total Se content increased significantly � Among the three different Se forms Na 2 SeO 3 recorded lowest uptake when desired Se concentration was applied through mixing application � significant difference was found between the Se content in different Se forms applied and Se application treatments � The biomass seems no effect on Se concentration applied in all the treatments
Discussion Pot experiments � Approximately 42.8% of inorganic selenium was detected while the organic species did not exceed the 20.6% value when treated with Na 2 SeO 4 � The higher inorganic Se concentration when treated with Na 2 SeO 4 indicates a lower inorganic to organic conversion � The total uptake of Se when compared with two different treatments Se(IV) and Se(VI) more organic species conversion took place in Se(IV) found in this study is in agreement with other papers reported in this topic with selenized allium plants Se form Secys MeSecyst Semet Se(IV) Se(VI) No. of unknown applied % % % % % species Na 2 SeO 4 0.6 11.3 8.0 0.7 42.8 2 Na 2 SeO 3 1.3 17.8 28.1 <0.1 26.8 3 BaSeO 4 0.8 7.4 15.4 <0.1 39.2 5
Aim Field experiment � Aim: To study the Se uptake by Se enriched leek and factors effects the Se uptake
Experiment Field experiment � A total of 17 plots was chosen for this study � Two different varieties of leek harston and poulton were used � A plot of 9 m 2 with the field was selected and half of plants in the selected plot was fertilized with Se concentration of 0.5 mg/plant (75 g/ha -1 ) Se to each plant and another half was used as control � white and green part was separated and then white part (most consumable part) was chopped into pieces and transferred into polythene bags, dried at 55° C and dry weights were recorded. The dried samples were then grinded with grinding apparatus � Three plants was analyzed from treated and non treated (white part)
Results Field experiment � Average soil Se concentrations: 0.29±0.08 mg kg -1 at 0-30 cm , 0.22±0.07 mg kg -1 at 30-60 cm Table 3 Se concentration in white part (mg.kg -1 ) Control (n=3) Treatment (n=3) Lowest 0.06±0.01 0.09±0.02 Highest 0.15±0.02 0.54±0.29
Field experiment Results Fig. 4 Se concentrations (mg.kg 1 ) in non-treated (blue) versus Se-treated plants (red, 500 µg Se/plant); H: harston variety and P: poulton variety
Discussion Field experiment � We used approx. 75 g ha −1 and this increased the concentration from 0.09±0.02 to 0.54±0.29 mg kg −1 � Comparison with literature data: � foliar treatment of wheat with Na 2 SeO 3 at 10 g and 20 g ha −1 increased foliar wheat concentrations to 0.094 ± 0.015 and 0.192 ± 0.088 mg kg −1 � Application of 10 g ha −1 Na 2 SeO 3 increased the Se concentrations of pasture crops from 0.04 to 0.06-0.1 mg kg −1 � Bio mass was recorded lower in Se treated plants were observed in 13 plot out of 17 plots
Aim Genetic leek trial � Aim: To evaluate the Se uptake and speciation changes in transgenic leek under Se enriched conditions-field study � 27 genetic leek varieties were studied with two different Se concentrations (sodium selenite) � Sodium selenate & selenite was applied to each plant into the hole after the plant lets were placed into the hole � The fully grown plants were sampled and white part of three plants from each variety chosen for analysis
Aim Incubation experiments � Aim: Study Se bioavailability and species transformations during gastrointestinal digestion of reference compounds, Se-enriched food (crops and supplements), and feed � Current experiment: Study pre-systemic (microbial) metabolism of Se species during gastrointestinal transit through the colon by incubating Se reference compounds and food supplements in a simulator of the human gastrointestinal tract
Incubation experiments stomach colon Small Stomach Ascendending intestine Transverse Descending Fig 6: Simulator of the Human Intestinal Microbial Ecosystem (SHIME)
Preliminary results Incubation experiments Table 5: Identified Se species and number of unidentified Se-containing chromatographic peaks in different gastrointestinal digestions steps when incubating pure Se compounds and Se-containing food supplements Name of the Stomach Small Colon Stomach Small Colon compound intesti intesti spiked ne ne Selenate - - - - - 2 Selenite - - Secys , - - 4 SeMet Semet - - SeMeCys - 1 2 Se(IV), γ - Se(IV), γ - Secys - - - 1 glut- glut- cyst , cyst , SeMe SeMe Cys Cys Basic vitamin - - - - - 2 (Selenate) Altisa (SeMet) - - SeMeCys - 1 2
Preliminary results Incubation experiments A Fig 6: Typical chromatograms obtained after incubating selenite under gastric (A) and colon (B) conditions
Preliminary results Incubation experiments � During gastric and small intestine incubation, no interspecies conversions were observed for the two inorganic species selenate and selenite � However, selenate and selenite were converted into Se-Met, Se-Cyst and unidentified compounds after 48 hours of colon incubation � The organic compound Se-Cyst was subject to interspecies conversions already starting within the small intestine � Species occurring in the food supplements “Altisa” (Se-Met) and “Basic vitamin” (selenate) are subject to the same conversions as the coinciding pure species in the reference solutions; the food supplement matrix does not seem to affect Se transformations
Recommend
More recommend
