EFFECTS OF DIFFERENT LIGHT ILLUMINATION ON SPROUT YIELD AND BIOSYNTHESIS OF ASCORBIC ACID IN FABA BEAN (VICIA FABA L.)

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1 Legume Res., 37 (2) : , 2014 DOI: /j AGRICULTURAL RESEARCH COMMUNICATION CENTRE EFFECTS OF DIFFERENT LIGHT ILLUMINATION ON SPROUT YIELD AND BIOSYNTHESIS OF ASCORBIC ACID IN FABA BEAN (VICIA FABA L.) Yuwei Luo*, Xueming Zi, Weihu Xie 1, Zhenping Ho, Xioxio Jin nd Yun Wng College of Horticulture, Jinling Institute of Technology, , Nnjing, P. R. Chin Received: Accepted: ABSTRACT The effects of different light illumintion on the scorbic cid content nd growth of fb ben sprouts were investigted. Among the six light qulities studied, ultrviolet light hd the highest promoting effect on the scorbic cid content in fb ben sprouts, incresing it by 78.3 per cent compred with the drkness control. Experiments with different durtions of ultrviolet nd red light illumintion in dy showed tht 12 h ultrviolet (500 Lx) nd 12 h red (1000 Lx) light diurnl cycles hd the highest promoting effects on both the scorbic cid level nd fresh weight of fb ben sprouts. The results indicted tht germintion of fb bens under 12 h ultrviolet nd 12 h red light diurnl cycles ws n effective process for incresing the yield nd enhncing the nutritionl qulity of fb ben sprouts. Key words: Ascorbic cid, Fb ben sprout, Germintion, Light illumintion. INTRODUCTION The fb ben (Vici fb L.) is one of the oldest known crops nd rnks sixth in production mong the vrious legumes grown in the world (Concepcion et l., 1998). Fb ben is n importnt source of protein, complex crbohydrtes, minerls, vitmins, dietry fibre nd ntioxidnts (Fernndez et l., 1996). This fvourble nutritionl profile, together with the excellent qulity of fb ben protein nd the good vilbility of its crbohydrtes, mkes the use of this pulse suitble for lrge prt of the world s popultion minly in developing countries (Rehmn et l., 2001). However, fb ben seeds contin mny ntinutritionl fctors such s phytic cid nd enzyme inhibitors. Therefore vrious processes hve been investigted to improve the nutritionl qulity of fb bens. Sprouting is the prctice of soking, drining nd leving seeds until they germinte nd begin to sprout. It hs been identified s n inexpensive nd effective technology for improving the nutritionl qulity of cerels nd grin legumes. As wter is introduced, enzyme inhibitors re disbled nd the seed explodes to life (Fris et l., 1995). Germintion unfolds, nd enzymes trigger elborte biochemicl chnges (Zielinski et l., 2005). In previous study, we found tht germintion could improve the iron nd zinc biovilbility (Luo et l., 2009). In cse of white kidney bens nd chickpes, sprouting improved the protein/mino cid digestibility by decresing nti-nutritionl fctors nd incresing the true/pprent protein/ mino cid digestibility (Rubio et l., 2002; Schhulze et l., 1997). According to Lorenz (1980) the prctice of sprouting of cerel grins nd legume hs become populr in the western world. These could be used in mny different foods including brekfst items, slds, soups, csseroles, pst, nd bked products. It hs been recently reported tht germintion under different type of illumintion hs significnt effect on biosynthesis of scorbic cid nd sprout yield of chickpe (Khttk et l., 2007). But there is little of literture bout the impct of such tretment on scorbic cid content in fb ben. Ascorbic cid hs mny importnt biologicl functions in both plnts nd nimls (Ghosh et l., 1997; Jimenez et l., 1997). Humns re unble to synthesise scorbic cid nd re thus entirely * Corresponding uthor s e-mil: lyw@jit.edu.cn 1 Nnjing Institute of Environmentl Science of Ministry of Environmentl Protection, , Nnjing, P. R. Chin

2 156 LEGUME RESEARCH- An Interntionl Journl dependent on dietry sources to meet their needs (Nishikimi nd Ygi, 1996). Plnt foods re the min dietry sources of scorbic cid in Chin nd most other developing countries. Although scorbic cid is universl constituent of fruits nd vegetbles, the sesonl vilbility of fruits nd vegetbles nd the significnt losses of the vitmin which occur during storge, preprtion nd cooking men tht scorbic cid remins one of the few nutrients in which the diet cn be deficient (Dvey et l., 2000). Therefore enhncing scorbic cid contents in vegetbles nd fruits is beneficil for humn helth. The present reserch work ws conducted to investigte the impct of light illumintion, including vrious light qulities nd lternte illumintion with different lights, on the scorbic cid content nd yield of fb ben sprouts. MATERIALS AND METHODS Fb ben (Qidou 2, cultivted in Jingsu province nd hrvested in 2012) ws purchsed from locl mrket of the sme btch in Nnjing City(Nnjing Jingsu Province). Germintion: Fb bens (500 g) of identicl size nd weight were selected, rinsed in distilled wter nd drined. They were then plced in distilled wter (25± 3 o C) nd soked over night in the drk for 8 h t 25± 3 o C. After pouring off the soking wter, the seeds were rinsed in wter (20± 3 o C) for bout 5min, spred evenly on try lined with bsorbent pper nd then plced in controlled environment chmber t 28 o C (90 95per cent reltive humidity). The seeds were rinsed with wter (20± 3 o C) once dy in the morning. The seeds were showered with distilled wter (25± 3 o C) for bout 5min nd left to drin completely. Smples were tken out from the chmber t intervls of 24, 48, 72, 96, 120, 144, 168 nd 192 h. The smples from ech tretment were weighed for fresh weight nd then divided into two portions. One ws used for scorbic cid nlyses, while the other ws stored t -20 o C for enzyme nlyses fter snp-freezing. Light exposure: The selected fb bens were divided evenly into six groups, ech group consisting of two trys of fb bens, one of which ws illuminted by light (white, blue, ultrviolet, ornge, red nd fr-red light, respectively) distributed so s to give uniform flux density t try level. The bsorbent pper lining the trys ws wtered twice dily to prevent ny drying-out of the sprouts during their period of illumintion. The other trys of fb bens were inserted into blck polyethylene lightproof bgs nd trnsferred into the sme illuminted controlled environment chmber to serve s nonilluminted drkness controls, except for the tretment with ultrviolet light, where the drk control ws trnsferred into nother chmber without ultrviolet light. Fluorescent tubes (40W, Philip Shnghi, Chin) were used s white light source. Respective colored bulb (40W, Philip, Shnghi, Chin) were used s per illumintion tretments. The trys were distributed under the light so s to give uniform flux density to ech try. The sme flux density were obtined by turning on the fixed number of light sources nd by djusting fixed distnces between the lmps nd the test mterils. Different flux densities were obtined by turning on different numbers of lmps nd by djusting the distnce between the lmps nd the test mterils. Flux densities were mesured using quntum sensor (ZDZ, Xuelin, Shnghi, Chin). Ascorbic cid determintion: Fresh smples from ech tretment were tken for scorbic cid estimtion. The direct colorimetric method ws used for the mesurement of this vitmin which is bsed on the mesurement of the extent to which 2,- dichlorophenol indophenol solution is decolorized by scorbic cid in smple extrcts nd in stndrd scorbic cid solutions. Since interfering substnces reduce the dye slowly, rpid determintion would be mesuring minly the scorbic cid. Extrction nd ssy for GLDH: L-Glctono- lctone dehydrogense (GLDH, EC ) ws extrcted nd ssyed s described by Ob(Ob, Ishikw & Nogmi, 1994). Fb bens sprouts (10 g) were ground in 50 ml of extrction buffer (30 mm - (Nmorpholino) propnesulfonic cid (MOPS), ph 7.5, 2 g L -1 bovine serum lbumin (BSA), 4mM Cys, 0.35M mnnitol, 10 g L -1 PVP-40, 25 mm N 4 P 2 O 7 nd 2 mm ethylene dimine tetrcetic cid (EDTA)), filtered through four lyers of cheesecloth nd centrifuged t 2200 g for 5 min. (NH 4 ) 2 SO 4 (200 g L -1, 5ml) ws dded to the superntnt, incubted t 4 o C for 30 min under continuous stirring nd centrifuged t g for 20min. The pellet contini ng membrne-bound protei ns ws

3 resuspended in 1 ml of wshing medium without BSA. GL DH ctivity ws mesured in the resuspended pellet fter semipurifiction step through Sephdex G-25. GLDH ctivity ws ssyed by the reduction of Cyt c t 550nm ( = 21 mm -1 cm -1 ) in medium consisting of 50 mm Tris- HCl, ph 8.0, 60 M Cyt c, 1.5 g L -1 Triton X-100 nd bout g of smple protein. The ctivity of GLDH ws determined s mol Cyt min -1 mg -1 fb bens sprouts (fresh weight). Sttisticl nlysis: Dt were nlysed with SPSS 13.0 for windows XP. The men nd stndrd devition of mens were clculted. The dt were nlysed by one-wy nlysis of vrince (ANOVA). Duncn s multiple rnge test ws used to seprte mens. Significnce ws ccepted t probbility P < RESULTS AND DISCUSSION Biosynthesis of scorbic cid in fb bens sprouts during germintion: Ascorbic cid hs been directly implicted in the modultion of plnt growth, including the erly stge of germintion of embryos (Citterio et l., 1994; Tommsi et l., 2001). The effects of germintion tretments on the scorbic cid level in fb bens re summrised in Tble 1. The results show tht scorbic cid, not detected in unsprouted fb bens, incresed significntly during germintion. This is in greement with observtions on Pinus pine L., which showed Germintion time (dys) Ascorbic cid (mg kg -1 fresh weight) Vol. 37, No.2, tht scorbic cid ws not found in dry seeds but incresed during germintion owing to the rectivtion of its biosynthesis (Tommsi et l., 2001). In order to confirm the biosynthesis of scorbic cid in fb bens during germintion, the ctivity of the key enzyme in scorbic cid biosynthesis ws investigted (Tble 1). GLDH, which ctlyses the oxidtion of L-glctono-1,4- lctone to scorbic cid, the lst step of the scorbic cid biosynthetic pthwy (Wheeler et l., 1998), hs been proved to be one of the key enzymes in scorbic cid biosynthesis (Ob et l., 1995). Tble 1 indictes tht the ctivity of GLDH in fb bens sprouts incresed significntly during germintion, ccompnied by increses in scorbic cid level. This confirms tht the ccumultion of scorbic cid in fb bens during germintion ws due to the rectivtion of its biosynthesis. Ascorbic cid hs mny importnt biologicl functions in both nimls nd plnts. It ws reported tht germintion process, in prticulr under ultrviolet illumintion hs significnt promoting effect on the scorbic cid level in soyben nd inhibiting effect on sprout growth (Ghosh et l., 1997). They observed red light to be effective in enhncing sprout yield while in our drk study, fluorescence nd -rys were more effective in this regrd. The results of this study were well in TABLE 1: Chnges in scorbic cid content, GLDH ctivity nd totl fresh weight of fb ben sprouts during germintion Sig(df= 8) ND 24.8±1.2 g 36.7±6.7 f 112.4±10.1 e 157.8±21.2 c 236.8± ±14.7 b 142.1±10.8 d 110.5±5.8 e GLDH(μmol min -1 mg -1 ) ND 1.68±0.23 f 4.92±0.72 e 8.68±0.84 d 12.65±0.56 b 14.88± ±1.18 b 14.12± ±1.56 c Totl fresh weight (g) 500±12 968±37 g 1123±63 f 1516±63 e 1860±50 d 2200±68 c 2480±102 b 2880± ±100 : Ech vlue is n verge of three determintions. The vlues were obtined from fb bens germinted in drkness t 28 ºC. ND, not detected. Mens followed by different letters within the sme column re significntly different from ech other (P < 0.05).

4 158 LEGUME RESEARCH- An Interntionl Journl greement with those of Ferndez nd Berry (1988) who reported significnt increse in scorbic cid during chickpe germintion. It ws lso reported by Riddoch et. l., (1998) tht mny species of pulses produced significnt quntities of vitmin C up to five dys germintion in both light nd drk, while cooking cused mrked decrese in this vitmin. Yng et l. (2001) reported tht the concentrtions of scorbic cid, vitmin E nd bet-crotene stedily incresed in whet with incresing germintion time, reching their peks fter 7 dys. Significnt increse in the content of scorbic cid of different cerels nd legumes seeds hs lso been reported by Hrmuth-Hoene et l., (1987). Effects of light qulity on scorbic cid level nd growth of fb bens sprouts: The biosynthesis of scorbic cid in plnts is highly responsive to wide vriety of environmentl fctors. Severl groups hve reported tht plnts increse their scorbic cid levels in response to different light intensity (Foyer et l., 1991; Logn et l., 1996), but they did not provide informtion bout the effects of different light types on biosynthesis of scorbic cid. The effects of red, fr-red, white, blue, ornge nd ultrviolet light on the scorbic cid level nd growth of fb bens sprouts were depicted in Tble 2. The results showed tht ultrviolet significntly (P < 0.01) incresed the promotion of biosynthesis of scorbic cid in fb ben sprouts but limited their growth. While red light hd the highest promoting effect on the yield of fb bens sprouts, incresing the totl fresh weight by 30 per cent compred with the drkness control. This ws in greement with observtions on cllus of Sussure medus Mxim which showed tht ultrviolet light hd promoting effect on the contents of secondry products nd red light hd promoting effect on cell growth (Zho et l., 1999). The chnges in the ctivity of GLDH in fb bens sprouts germinted under different lights exhibited the sme trend s the chnges in scorbic cid level (Tble 2). This indicted tht the ctivity of the key enzyme in scorbic cid biosynthesis ws regulted by different light type. Effects of intensity of ultrviolet nd red light on scorbic cid level nd fresh weight of fb bens sprouts: The effect of intensity of illumintions is depicted in Tble 3 nd Tble 4. This study showed tht scorbic cid level of fb bens sprouts incresed significntly when the intensities of ultrviolet ws 500 Lx. While 1000 Lx of red light hs the biggest promotion on fb ben totl fresh weight. So 500 Lx ultrviolet nd 1000 Lx red light were selected for the lternte illumintion tretments. Effects of lternte illumintion with ultrviolet nd red light on scorbic cid level nd fresh weight of fb bens sprouts: Ultrviolet light hd the highest promoting effect on the scorbic cid level in fb ben sprouts but significntly inhibited their growth (Tble 2). In contrst, red light promoted the growth of fb ben sprouts but hd significnt TABLE 2: Effects of light qulity on scorbic cid content, GLDH ctivity nd totl fresh weight of fb ben sprouts Light qulity Ascorbic cid (mg kg -1 fresh weight) GLDH(μmol min -1 mg -1 ) Totl fresh weight (g) Drkness 236.8±16.4 b 14.88±2.10 b 2200±68 c Red light 116.8±21.6 c 7.68±0.56 c 2860±80 Fr-red light 88.6±17.2 d 6.21±0.47 d 2660±70 b Ornge light 110.5±12.6 c 7.21±0.84 c 2300±90 c Blue light 240.8±15.2 b 15.24±1.21 b 1950±80 d White light 235.4±14.6 b 15.92±1.04 b 2250±80 c Ultrviolet 421.6± ± ±40 d Sig(df= 6) Sig= : Ech vlue is n verge of three determintions. The vlues were obtined from fb bens germinted for 5 dys in continuous drkness or with illumintion t 28 ºC. The illumintion intensity of different light qulities ws 1000 Lx. Mens followed by different letters within the sme column re significntly different from ech other (P < 0.05).

5 inhibiting effect on their scorbic cid level (Tble 2). In order to obtin the highest scorbic cid level nd highest fresh weight of fb ben sprouts, the effects of lternte illumintion with ultrviolet nd red light were investigted (Tble 5). The results showed tht 12 h ultrviolet nd 12 h red light diurnl cycles hd the highest promoting effects on both the scorbic cid level nd fresh weight of fb ben sprouts. The scorbic cid content nd totl fresh weight of fb ben sprouts germinted under such conditions were mg kg 1 fresh weight nd 2420 g, which were nd 10.0 per cent higher thn the drkness control vlues respectively. CONCLUSION This study demonstrted tht the ermintion process, in prticulr under ultrviolet illumintion, TABLE 3: Effects of intensity of ultrviolet on scorbic cid content nd totl fresh weight of fb ben sprouts. Vol. 37, No.2, hd significnt promoting effect on the scorbic cid content in fb bens. In the germintion process the increses in scorbic cid level were considered to be consequence of the rectivtion of scorbic cid biosynthesis undergone in the seeds during germintion. Germintion of fb bens under ultrviolet nd red light diurnl cycles ppers to be n effective process for incresing the yield nd enhncing the nutritionl qulity of fb bens sprouts. ACKNOWLEDGEMENTS ` This work ws supported by Ntionl Sci ence Foundtion of Chin ( , ) nd Qing Ln Project. TABLE 4: Effects of intensity of red light on scorbic cid content nd totl fresh weight of fb ben sprouts. Light intensity Ascorbic cid (mg Totl fresh weight L igh t Ascorb ic cid (mg Totl fresh (Lx) kg -1 fresh weight) (g) i nten sity (Lx) kg -1 fresh weight) weight (g) Sig(df= 4) 370±16.8 c 380±15.2 c 440± ±12.6 b 400±16.1 b Sig= ± ± ±65 c 1950±90 b 1650±85 d Sig= :Ech vlue is n verge of three determintions. The vlues were obtined from fb bens germinted for 5 dys t 28 ºC. Mens followed by different letters within the sme column re significntly different from ech other (P < 0.05). TABLE 5: Effects of lternte illumintion with ultrviolet (UV) nd red (RL) light on fb ben growth, GLDH ctivity nd scorbic cid content Sig(df= 4) 118±10.2 c 12 5±11.5 b 15 0± ±13.8 b 116±14.1 c ±12 0 d 2880±110 c 3150±80 b 3350± ±10 0 d : Ech vlue is n verge of three determintions. The vlues were obtined from fb bens germinted for 5 dys t 28 ºC. Mens followed by different letters within the sme column re significntly different from ech other (P < 0.05). UV/RL diurnl Ascorbic cid (mgkg -1 fresh GLDH(μmol min -1 mg -1 ) Totl fresh weight (g) cycle (h/h) weight) 0/ e 12.6 e 2150 c 4/ d 24.2 d 2250 b 8/ c 27.8 c / / b b 2120 c 20/ b 24/ c c 2050 d Sig(df= 6) : Ech vlue is n verge of three determintions. The vlues were obtined from fb bens germinted for 5 dys t 28 ºC. The illumintion intensities of ultrviolet nd red light were 500 nd 1000 Lx respectively. Mens followed by different letters within the sme column re significntly different from ech other (P < 0.05).

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