Bio fortification of Leek with Selenium

Laboratory of Analytical Chemistry & Applied Ecochemistry

GHENT UNIVERSITY

[In cooperation with ILVO]

By

R.V.SRIKANTH.LAVU

Promoter: Prof. Filip Tack

Co-promoter: Prof. Gijs du Laing

� Introduction� Pot experiments (UGent)

AimExperimentResultsDiscussion

� Field experiments ( Field trial )AimExperimentResultsDiscussion

� Genetic leek trial (Field trial at ILVO)AimResultsDiscussion

� Incubation experiments AimPreliminary results

Contents

� 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 µg 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

Introduction

� 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- 1soil

Pot experimentsAim

� 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 liquidnitrogen and stored at -80°C and then freeze dried by liophilizer and powdered with milling apparatus

Pot experimentsExperiment

Pot experimentsResults

Se conc. used forenrichment (µg

g−1)

Hole-Selenite samples±SD

(µg g−1)

Surface-selenitesamples±SD (µg

g−1)

Mixing-selenitesamples±SD (µg

g−1)

Mixing-Na-selenate

samples±SD (µg g−1)

Mixing-Ba-selenate samples±SD (µg

g−1)

0.2 µg/g- 1soil 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

Table1: Total selenium content of selenium enriched leek. Data represent the mean value of the samples measured in 4 replicates

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 byNa2SeO3

� When leek was enriched with Selenate at a each concentration chosen, the total Se content increased significantly

� Among the three different Se forms Na2SeO3 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

Pot experimentsDiscussion

� Approximately 42.8% of inorganic selenium was detected while theorganic species did not exceed the 20.6% value when treated with Na2SeO4

� The higher inorganic Se concentration when treated with Na2SeO4

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

Pot experimentsDiscussion

Se form applied

Secys %

MeSecyst %

Semet%

Se(IV) %

Se(VI) %

No. of unknownspecies

Na2SeO4 0.6 11.3 8.0 0.7 42.8 2

Na2SeO3 1.3 17.8 28.1 <0.1 26.8 3

BaSeO4 0.8 7.4 15.4 <0.1 39.2 5

� Aim: To study the Se uptake by Se enriched leek and factors effects the Se uptake

Field experimentAim

� A total of 17 plots was chosen for this study

� Two different varieties of leek harston and poulton were used

� A plot of 9 m2 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)

Experiment 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

Results

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.kg1) in non-treated (blue) versus Se-treated plants (red, 500 µg Se/plant); H: harston variety and P: poulton variety

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 Na2SeO3 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 Na2SeO3 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

Field experimentDiscussion

� 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 Genetic leek trial

� Aim: Study Se bioavailability and species transformations duringgastrointestinal 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 experimentsAim

stomachSmall intestine

colon AscendendingTransverse Descending

Stomach

Fig 6: Simulator of the Human Intestinal Microbial Ecosystem (SHIME)

Incubation experiments

Preliminary results

Name of the compound spiked

Stomach Small intestine

Colon Stomach Small intestine

Colon

Selenate - - - - - 2

Selenite - - Secys ,SeMet

- - 4

Semet - - SeMeCys - 1 2

Secys - Se(IV), γ-glut-cyst, SeMeCys

Se(IV), γ-glut-cyst, SeMeCys

- - 1

Basic vitamin (Selenate)

- - - - - 2

Altisa (SeMet) - - SeMeCys - 1 2

Table 5: Identified Se species and number of unidentified Se-containing chromatographicpeaks in different gastrointestinal digestions steps when incubating pure Se compounds and Se-containing food supplements

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

Preliminary results Incubation experiments

� Confirmative experiment of Colon microbial community role in speciation:

When Se reference compounds were spiked to autoclaved colon

suspension, no species conversion was observed for selenate, selenite

and Se-met after 48 hours of incubation; for Se-Cyst, four peaks were

identified

� Future prospective:

Further experiments with Se fortified food crops will be studied with

these type of incubation experiments

Preliminary results Incubation experiments