Effect of Light Emitting Diode to Sunflower Sprouts.

Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.Effect of Light Emitting Diode to Sunflower Sprouts.

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURAL AND FORESTRY

VU THI HOA Topic title :

THE EFFECT OF LIGHT EMITTING DIODE TO SUNFLOWER

SPROUTS

BACHELOR THESIS

Study Mode : Full-time

Major : Food technology

Faculty : Biotechnology and food technology

Batch : 2013-2017

Thai Nguyen, 24/ 06 /2016

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

VU THI HOA Topic title :

THE EFFECT OF LIGHT EMITTING DIODE TO SUNFLOWER

SPROUTS BACHELOR THESIS Study Mode : Full-time

Major : Food Technology

Faculty : Biotechnology and Food Technology

Batch : 2013-2017

Supervisors : Assoc Prof Dr Songsin Photchanachai

Msc Pham Thi Tuyet Mai

Thai Nguyen, 24/ 06/2016

DOCUMENTATION PAGE WITH ABTRACT

Thai Nguyen University of Agriculture and Forestry

Thesis Title Effect of Light Emitting Diode to Sunflower

Sprouts Supervisor(s) Assoc Prof Dr Songsin Photchanachai

Msc Pham Thi Tuyet Mai Abstract:

This study evaluated the antioxidant properties of sunflower sprouts (Helianthus annuus L.)

as affected by Light Emitting Diode (LED) illumination Five to six days old sprouts were supplied with the lights, including: white LED, blue LED (460 nm), red LED (630 nm), sunlight, fluorescent light and dark light for 12 h, 36 h, in a controlled environment with the temperature, relative humidity and light intensity of 25 0 C, 65-70% and 50-60 µmol/m 2 s respectively Blue LED light resulted in the enhanced DPPH, vitamin C, blue light also increased phenolic content at 36 h light Chlorophyll content did not change significantly Red LED light decreased phenolic and vitamin C contents, DPPH and chlorophyll contents also lower than that treated with sunlight (control) White light enhanced vitamin C but decreased phenolic content Dark light reduced total antioxidant activity, chlorophyll, vitamin

C and phenolic contents.

Key words: Sunflower, LED lights, antioxidant activity,

phenolic, anthocyanin, vitamin C,

me through my internship, providing useful advice for the improvement of this work

I would like to acknowledge my teachers at TUAF, Msc Trinh Thi Chung and the whole teachers at Faculty of Biotechnology and Food Technology were created a good practice environment for us

I wish to express my sincere thanks to my introductor researcher Chalinee Songkajorn who interesting and instructed during my time here Big thanks also go to Nipada Ranmeechai and Naruchon Tantharapornrerk for guidance and immense knowledge to me and special to all members of Seed Lab for their kind assistance, they have been supportive and keen friends

Finally, I would like to express my thanks to my family for their support in my education Also thanks to everyone in Postharvest Technology Laboratories for helping me in conducting the experiment

CONTENTS

PART 1.INTRODUCTON 1

1.1.Background 1

1.2Objectives 2

1.3 Scope of research 2

1.4 Hypothesis 2

PART 2 MATERIAL AND METHODS 3

2.1 Plant materials and growth conditions 3

2.2 Experiment design 3

2.2.1 Experiment 1: 3

2.2.2 Experiment 2 3

2.2.3 Statistical analysis 4

2.3 Measurement of parameters 4

2.3.1 Measurement of total weight 4

2.3.2 Determination of chlorophyll 4

2.3.3 Determination of total ascorbic acid content (Vitamin C) 4

2.3.4 Determination of total antioxidant activity (DPPH) 5

2.3.5 Determination of total phenolic content 5

PART3 RESULTS 6

3.1 Effect of LED blue and different light on quality eating and antioxidant activity compound 6

3.1.1 Chlorophyll pigments 6

3.1.2 Total phenolic content 7

3.1.3 Antioxidant activity 7

3.1.4 Ascorbic acid content 8

3.2 Effect different LEDs on quality eating and antioxidant activity compound 9 3.2.1 Chlorophyll pigments 9

3.2.2 Total phenolic content 10

3.2.3 Antioxidant activity 11

3.2.8 Ascorbic acid content 12

PART 5 CONCLUSION 13

REFERENCES 14

LIST OF FIGURES

Figure 1: Effect of sun light, dark light, blue LED and fluorescent on chlorophyll a and

b, in cotyledon of sunflower sprouts after grown for 6 days The different letters on the columns indicate that values are significantly different (P<0.01)

Figure 2: Effect of sun light, dark light, blue LED and fluorescent on total phenolic content of sunflower sprouts after grown for 6 days The different letters on the

columns indicates that values are significantly different (P<0.01)

Figure 3: Effect of sun light, dark light, blue LED and fluorescent on antioxidant activity (DPPH) of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly different (P<0.01)

Figure 4: Effect of sun light, dark light, blue LED and fluorescent on ascorbic acid

content of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly different (P<0.01)

Figure 5: Effect of sun light, white LED, red LED and blue LED on chlorophyll a and

b and total chlorophyll in cotyledons of sunflower sprouts after grown for 6 days The different letters on the columns indicate that values are non-significantly different Figure 6: Effect of sun light, white LED, red LED and blue LED on total phenolic content of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly different (P<0.05)

Figure 7: Effect of sun light, white LED, red LED and blue LED on antioxidant

activity (DPPH) of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly different (P<0.01)

Figure 8: Effect of sun light, white LED, red LED and blue LED on ascorbic acid

content of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly different (P<0.01)

Figure9: Standard curve of ascorbic acid at 540 nm

Figure10: Standard curve of Gallic acid at 765 nm for determination of phenolic content

Figure11: Illustrative images of sunflower sprouts for 3 days grown in dark and more

36h under sun light, dark light, blue LED and fluorescent

Figure12: Illustrative images of sunflower sprouts for 5 days grown in dark and more

12h under sun light, dark light, blue LED and fluorescent

LIST OF TABLES

Table1: Effect of sun light, dark light, blue LED and fluorescent on the accumulation

of pigments chlorophyll a and b, total chlorophyll, DPPH in cotyledon of sunflower sprout

Table2: Effect of sun light, dark light, blue LED and fluorescent on the accumulation

of vitamin C and phenolic in cotyledon of sunflower sprout

Table3: Effect of sun light, white light, red light and blue light on the accumulation of pigments chlorophyll a and b, DPPH in cotyledon of sunflower sprout

Table4: Effect of sunlight, white light, red light and blue light on the accumulation of Vitamin C and phenolics in cotyledon of sunflower sprout

Table5: Total fresh weight of sunflower sprout for 3 days grown in the dark and more 36h under sun light, dark light, blue LED and fluorescent

Table6: Total fresh weight of sunflower sprout for 5 days grown in the dark and more 12h under sun light, blue light, red light and white light

Table7: List of chemical reagents and equipment/tools need to be used

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PART 1 INTRODUCTON 1.1 Background

Sunflower (Helianthus annuus L.) is one of the few crop species that originated

in North America (most originated in the Fertile Crescent, Asia or South or Central America Sunflowers is a common name refering to its peculiarity of being heliotropic, which means it is able to orient itself to the solar star Sunflower roots can explore deeper soil layers so it can grow on dry and sunny places Sunflowers sprouts is an excellent source of vitamin E, B, C and minerals It also contains a high amount of protein, bioactive compounds and antioxidants It improves digestion, brain power, immune system, and the functioning of the cardiovascular systems Aside from that, it prevents heart disease and cancer Sunflowers are usually consumed by two ways: sunflower seeds and sunflower oil But nowadays people use more vegetables under sprouts in their diet, due to easy growing at home without spending a lot of area and time With its excellent nutrients, sunflower microgreens help keep our blood healthy, reduce inflammation, calms the nervous system, aid in the heart health, and support cellular recovery (Julie Daniluk, 2011) Moreover, germinated edible seeds are an excellent source of dietary phenolic antioxidant (Bolívar A and Luis Cisneros,2010) therefore; natural antioxidants from the microgreens have attracted increasing interests due to their safety for the consumer (Samuoliene G et al., 2011)

Light is one of the most important environmental factors, which acts on plants

as the sole source of energy (Samuoliene G et al., 2011) Light as an important signal influences the transition from etiolated to de-etiolated state, a stimulus for plant development biosynthesis of cell component and gene expression throughout the life cycle of plant (Wu M.C et al.,2007) Temperature and light are important sources for plant growth they have ability to promote germination and increase nutrients But, natural temperature and light is not enough for the development Light-emitting diodes (LEDs) have many advantages compared to other light sources It contributes

in the accumulation of bioactive and antioxidant compounds in plants during the photosynthesis (Dsouza et al, 2015) Using LEDs in food production is a best solution

to provide the freshness, safety and nutritious food to maintain a healthy and active

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life for people Therefore, this study evaluated the effect of LED providing different lighting spectra on antioxidant properties of sunflower microgreens

1.2Objectives

To evaluate the effect of LEDs on antioxidant components, total antioxidant

activity, chemical compositions and eating quality of sunflower microgreens

1.3 Scope of research

This research compared between the light conditions for sunflower micreogreen throughout chemical properties The different light, wavelength used including: White light, Red (630nm), Blue (460nm), sunlight, dark light and fluorescent light Experiment 1 were sunlight, dark, fluorescent lights and blue light on sunflower microgreens while experiment 2 tested for sunlight red, blue and white lights The chemical properties of sunflower microgreens focused on total ascorbic acid content (TAAC), total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, total chlorophyll a and b contents

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PART 2 MATERIAL AND METHODS 2.1 Plant materials and growth conditions

Sunflower (Helianthus annuus L.) seeds were obtained from a market in

Bangkok Seeds were kept at 40C during experiment in the Seed Laboratory, Division

of Postharvest Technology, King Mongkut’s University of Technology Thonburi Seeds were soaked and incubated prior to sowing in the plastic basing containing moist coconut coir (fiber) Sunflower seeds (250g) were grown in the plastic basin, watered three times by spraying throughout the growing period LEDs, fluorescent, sunlight and dark lights were used

2.2 Experiment design

2.2.1 Experiment 1:

The sunflower seeds were prepared and grown as described in 2.1 in a plastic basin, 1basin for each treatment and conducted four treatments shown below The basins were covered with plastic canvas plastic for 3 days, after 3days and spayed with water The seedlings were transferred to the four treatment following for experiment 1: Treatment1: planting under darkness for 36 hours (control treatment)

Treatment2: planting under sunlight for 36 hours

Treatment3: planting under fluorescent light for 36 hours

Treatment4: planting under blue LED light for 36 hours

After 6 days, sunflower microgreens were cut then packed in zip bags and stored at 40C to keep the quality of the microgreens and prepared for analysis

2.2.2 Experiment 2

The sunflower seeds were prepared and grown as described in 2.1 in plastic basin, one basin for each treatment and performed four treatments They were covered with plastic canvas plastic for five days After that, the plastic canvas was opened open and spray water The following were the treatments for experiment 2:

Treatment1: planting under sunlight for 12 hours (control treatment)

Treatment2: planting under white light for 12 hours

Treatment3: planting under red light for 12 hours

Treatment3: planting under blue light for 12 hours

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2.2.3 Statistical analysis

The results were analyzed using the SAS program version 9.0 (SAS Institute Inc., Cary, NC, USA) at 95% confidence level and means were separated by Ducan’s Multiple Range Test (DMRT) at 5% level of significance

2.3 Measurement of parameters

2.3.1 Measurement of total weight

The total weight of sunflower microgreens was measured in grams (g) by electronic balance with three decimals

2.3.2 Determination of chlorophyll

Chlorophyll content was determined according to the methods of Moran (1982) using spectrophotometer (UV-1800, Shimadzu, Japan) Extracts were prepared using the cotyledons only with an approximate weight of five g (fresh weight)/ per replicate and added with 20mL of N,N Dimethylformamide Each treatment had eight replicates Then, the solution was homogenized at 8.4 rpm and incubated at 40C for 24h in dark condition Then, the homogenate was filtered to remove solid The sample was diluted at a proportion of 1:40( 1 sample: 40 N-N ) The optical density was measured using a UV-1800 spectrophotometer chlorophyll a is 664nm (OD664) and chlorophyll b is 647nm (OD647) Determined chlorophyll from the following equations (Lichtenthaler and Wellburn, 1983; Zhang et al., 2009, as cited in H Li et al, 2012): Chl.a (mg/g)=(12.72*OD664-2.59*OD647 )V/1000W

Chl.b (mg/g)=(22.88*OD647-4.67*OD664 )V/1000W

Chl.(a+b) (mg/g)= Chl.a+Chl.b

2.3.3 Determination of total ascorbic acid content (Vitamin C)

Vitamin C was determined by DNPH method (Kapur et al., 2012)

Chemical preparation: 5% metaphosphoric acid (metaphosphoric 5g + distilled water), 2% Thiourea solution( thiourea 2g in 5% metaphosphoric acid), 0,02% indophenol (20mg 2,6 indophenol + 100mL distilled water), 85% sulfuric acid , 2% 2,4- Dinotrophenyl hydrazine(DNP) (2g DNP +10N H2SO4 100ml) and standard ascorbic acid

Sample preparation: Five g fresh sample was weighed, added with 20ml of 5% metaphosphoric, homogenized in plastic tube (IKA T25, Ultra-Turrax, Japan) and

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placed in an ice bath Then, the sample was filtered The sample was extracted, take 0.4mL filtrate and mix with 0.2mL of 0,02% indophenol and held for 2-3 minutes Next, 0.4mL of 2% thiourea solution and 0.2mL of 2% DNP(except blank sample) were added to the solution and shaken After that, it was incubated at 500C for 1h in hot water bath When finished, the sample were taken out and added with 1mL of 85%

H2SO4 then incubated at room temperature for 30minutes The absorbance was measured at 540nm using spectrophotometer (UV-1800, Shimadzu, Japan) The standard used were ascorbic acid and metaphosphoric with concentration at 0, 20, 40,

60, 80, 100 mg/L

2.3.4 Determination of total antioxidant activity (DPPH)

Total antioxidant activity will be determined by DPPH method (Hussian, Suradkar, Javaid, Akram and Parvez, 2015)

Chemical preparation: working solution was prepared by DPPH and 95% ethanol

Extracts preparation: Five g sample (wet weight) was added with 20mL of 85% ethanol and homogenized using homogenizer at 8.4rpm in ice bath Then, it was centrifuged at 12,000rpm for 10minutes to obtain the supernatant After that, 0.15mL supernatant was taken, mixed with 2.85mL working solution and incubated in dark condition for 30min Absorbance was measured at 515nm in a UV-Vis spectrophotometer(Shimadzu, UV-1800, Japan)

DPPH radical scavenging (%)=[(Aworking solution- Asample)]/Aworking solution

2.3.5 Determination of total phenolic content

Phenolic content was measured following the Folin-Ciocalteu method of Singleton et al(1999) The steps to create supernatant solution was the same analysis with DPPH analysis 0.02 mL supernatant was take out and 1.6mL distilled water was added Then, 0.1mL of 100% Folin-ciocalteu phenol solution mixed with 0.2mL of 20% sodium carbonate was added Next, it was incubated at 400C for 30 min in water bath When finished, the sample was taken out and placed at room temperature for 15 min and was measured using double beam UV-Vis spectrophotometer (Shimadzu, UV, Japan) at fixed wavelength of 765nm The standard used was garlic acid with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1 mg/L

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PART3 RESULTS 3.1 Effect of LED blue and different light on quality eating and antioxidant activity compound

3.1.1 Chlorophyll pigments

The effect of sunlight, dark light, blue LED and fluorescent on accumulation of chlorophyll a, b and total chlorophyll in cotyledon of sunflower sprout was showed in Figure 1 Interestingly, fluorescent light has the most ability to produce chlorophyll pigments Chlorophyll a, b and total chlorophyll contents were 13.78, 5.19 and 18.97 mg/100g FW, respectively There was significant difference between different lights Chlorophyll a, b and total chlorophyll under dark light whichshowed the lowest due to light absence, seedling require light for photosynthesis and initiate photo morphogenesis (Tiaz and Zeiger, 2010)

0 2 4 6

Figure 1 Effect of sunlight, dark light, blue LED and fluorescent on chlorophyll a and

b and total chlorophyll in cotyledon of sunflower microgreens after grown for 6 days The different letters on the columns indicate that values are significantly different (P≤0.01)

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3.1.2 Total phenolic content

The results of the total phenolic content ranged between 21.51 to 28.94 mmol GAE/100g FW The highest total phenolic content was under blue LED (28.94 mmol GAE/100g FW) This may relate to the abiotic stress, (Rivero et al., 2001) reported that thermal stress accumulates phenolic in tomato and water melon plants

Whereas results were not significant among sunlight, dark and fluorescent lights

Figure 2 Effect of sunlight, dark light, blue LED and fluorescent on total phenolic content of sunflower sprouts after grown for 6 days The different letters on the columns indicates that values are significantly difference (P≤0.01)

23.06b

21.51b

28.94a

22.69b