Stevia rebaudiana Growth and modification of SVgly content by Red LEDs

Transcription

1 Stevia rebaudiana Growth and modification of SVgly content by Red LEDs Prof. Jan M.C. Geuns Lab Functional Biology, KULeuven Kasteelpark Arenberg 31, BUS Heverlee-Leuven BELGIUM

2 Stevia rebaudiana

3 STEVIA: night interruption 5 min red LEDs

4 Stevia vs. steviol glycosides EU: Stevia plant + leaves: Novel Food Not yet authorised in the EU Steviol glycosides, E960 : food additive hence: ADI 0-4 mg/kg SVequivalents

5 Main Sweeteners HO CH 2 OH O HO HOH HO 2 C O HO CH 2 OH O O O HO OH O HO HOH 2 C HO O CH 2 OH O O O HO OH HO OH H H HO HOH 2 C O C=O H O HO HOH 2 C O C=O H O HO OH HO OH Stevioside Rebaudioside A

6 Advantages SVglys/leaves not-toxic no calories heat-stable to 140 C, 1 h; OK for cakes and biscuits non-fermentable not absorbed: too large molecules not degraded by enzymes of gastro-intestinal tract taste enhancing 100 % natural product slight licorice taste, somewhat bitter only small amounts needed (300x sweeter than 0.4 % sugar) leaves can be used as such non-addictive sweetener for children leaves and pure steviol glycosides can be cooked and baked no caries formation

7 Stevioside/Rebaudioside A 1. As sweetener: SMALL doses ( mg/d) Sweetness / purity (> 95 % required) Validated techniques analysis Development of IS 2. Pharmacological effects: LARGE doses ( mg/d Stevioside) - hypertension - type 2 diabetes - anti-carcinogenic - preventive effect on atherosclerosis - radical scavenging 3. Steviol Glucuronide: active principle? Radical scavenging

8 State of the art Stevia rebaudiana is self-incompatible: # genotypes required for viable seed production Viable seeds: for sale at: or seedling production by Rudy Raes, Stevia rebaudiana is a short-day plant (SD) SDs (< 12 h photoperiod) cause rapid flowering, contrary to long-days (LDs) During SDs, LD conditions are simulated with red LED light illumination during the night 8/52

9 Accumulation of steviol glycosides within Stevia rebaudiana General outline: what is known so far? Source: Bondarev et al. (2003)

10 Accumulation of steviol glycosides within Stevia rebaudiana General outline: what is known so far? Leaves >> Flowers > Stems > Seeds >> Roots All organs have the three major steviol glycosides (SVglys), yet proportions can differ in each organ Leaves: SVgly proportions may vary greatly depending on plant age and phase of plant development Different cvs can have totally different composition, e.g., more RebA>>ST

11 How to increase sweetener production? - Longer vegetative growth period - Avoid flowering - Interrupt nights with Red LEDs (660 nm or ± 660) - Choose good cv.

12 EUSTAS Stevia gene bank EUSTAS: European Stevia Association Preservation of different genotypes Selection of good cvs. with high RebA content Selection of frost-resistant cvs. Cvs. adapted to EU weather conditions Cryopreservation possible (?) In vitro stock of all cvs.

13 Some plants in the gene bank

14 Decontamination

15 Decontamination (30 sec EtOH+20 min 2 sodium hypochlorite) min 20 min 30 min surviving dead contaminated

16 Conclusions gene bank MS medium is optimal for multiplication + storage Storage at 5 C is too low, at 25 C too high Storage at 15 C for 1 year is possible (Currently all 11 accessions received are stored in vitro at 15 C in the first in vitro stevia germplasm collection). Longer? Maybe 10 C?

17 Prevention of flowering Stevia: short-day plant flowers after # of SDs South-America: Daylengths h Flowering after wks, hence: up to 5 6 harvest/year Labour-intensive Idea: Night interruption with red LEDs (Light-in-the-box)

18 Stimulation of steviol glycoside accumulation by red light Phytochrome response: principles Light-sensitive pigment found within leaves Sensitive to red (660 nm) and far-red (730 nm) light Regulates the time of flowering, seed germination, elongation of seedlings, etc Pr / Pfr ratio determines if certain processes will take place As daylength is sensed by phytochrome, activation by red light might simulate LDs by prolonging vegetative growth and avoiding flower bud formation

19 19/52

20 20/52

21 SD Stimulation of steviol glycoside accumulation by red light SD + 5 min RED

22

23

24 STEVIA: night interruption 5 min red LEDs Final length: ± 3 m

25 g dry leaf mass / plant mg SVgly / plant Stimulation of steviol glycoside accumulation by red light Results First experiment SD SD+LED Days of incubation Days of incubation SD SD+LED

26 Total leaf production per shoot/plant W9 W25 W35 Leaf dry weight SD 2.3 g - - SD + 5 LED 3.5 g 34.2 g g SVglys SD SD g (2.0 %) 0.15 g g 14.5 g 5 LED (4.3 %) (11.8 %) (11.0 %) 26/52

27 Total production per year W9 W25 W35 1 Year Leaf dry weight SD 2.3 g g SD + 5 LED 3.5 g 34.2 g g g 15x SVglys SD SD + 5 LED 0.05 g (2.0 %) 0.15 g (4.3 %) g 4.0 g 14.5 g (11.8 %) 27/52 (11.0 %) 21.3 g 74x

28 Simulating LDs on a field Belts of red LED lights (660 nm), e.g. every 20 m at a height of 2-3 m Only 5 min illumination is sufficient Advantages Less harvests / year, thus less labour costs Easier for vegetative multiplication (cuttings) Small amounts of electricity needed (solar power!) LEDs have long lifespans (> 20,000 hours): 657 years! Small investment per hectare (< 5,000 euro) Easier extraction due to greater percentage yield Temperate climate: at least two-month prolongation of growth season 28/52

29 Suggestions to increase production Select good cultivar: cfr. Stevia gene bank (EUSTAS); Crossings between cvs. to obtain e.g. large leaves, huge vegetative production, great SVgly content Cold resistant cvs. adapted to our climatic conditions

30 Plant with giant leaves length: 12 cm

31 ? Dream or reality? Plants with giant leaves red LEDs treated, 3 m height g dry leaves per plant 80,000 plants/ha tonnes dry leaves/ha!! 20 % SVgly content SVgly: tonnes/ha (at 1:10 ratio) or tonnes SVglys (at 1:40 ratio)

32 Stevioside/Stevia as sweetener At the moment: ± 40,000 ha fields Production: ± 120,000 t Stevia leaves Or: ± 12,000 t purified sweetener (1:10) Use in Thailand: ± 91 mg/day i.e.: daily ± 350 million people consume Stevia in all its forms Never indications of harmful effects!

33 Culture of Stevia Greenhouse: C Soil: Sandy loam, ph 5-7 Control moisture, sensitive to mold infections Weekly: minerals Note: sometimes Boron-deficiency White fly! Outside: end April till October

34 Mold infections: Alternaria alternata

35 Use of leaves Cut shoots about 10 cm above ground Dry leaves in mico-wave (± 3 min at 800W) Grind leaves to a fine powder Use as such: sweetness: ± 30 x sugar Put leaf powder in tea bag, staple it Boil in water and use extract to sweeten drinks and food

36 Use of leaves Examples of recipes: see booklets Take same amounts of leaves as for Stepa- Sweet which is 30 x sweeter than sugar Make your own soft drink without sugar! Use water extracts of leaves instead

37 Examples of uses

38 Use of leaves Extraction of white purified powder: sophisticated extraction units required EU purity requirement SVglys: 95 % AND: sum of Stevioside + RebaudiosideA 75 % LEAVES + PLANTS : NOVEL FOOD - Not yet authorised in EU; start petition!!! - Blocked in Germany: stability studies required of NF (which compounds????)

39 Billboard in Austria: sugar lobby

40 2. Pharmacological effects (HIGH DOSES) Too good to be true?? 1) Lowering of blood pressure 2) Diabetes type 2 - Lowering blood glucose - Increase of insulin secretion - Effect: [glucose] dependent - Enhancing insulin sensitivity - Decrease of glycagon secretion 3) Anti-carcinogenic effects 4) Anti-atherosclerosis effects 5) ROS-scavenging

41 Beneficial pharmacological effects Leaves have about 10 x better radical scavenging activity than SVglys Consumption of 1 g dry leaves/day might be good in hypertension, type 2 diabetes, atherosclosis, inflammation, some forms of cancer.