Changes in Protease Activity and Proteins in Naked Oats (Avena nuda L.) during Germination

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1 Asin Journl of Agriulture nd Food Sienes (ISSN: ) Chnges in Protese Ativity nd Proteins in Nked Ots (Aven nud L.) during Germintion Ling-Ling Zhng 1 nd Jin-Guo Xu *1,2 1 College of Life Sienes, Shnxi Norml University, 1 Gongyun Street, Linfen, Chin 2 College of Food Sienes, Shnxi Norml University, 1 Gongyun Street, Linfen, Chin * Corresponding uthor s emil: xjg71 [AT] 163.om ABSTRACT Chnges in protese tivity, the ontent nd omposition of proteins were investigted during germintion of nked ots. Compred with rw grots, n inrese in protese tivity from ot grots ws ontinuously oserved, nd it inresed y 252% nd rehed the mximum t stge G60; the rude protein nd prolmin inresed silly in the ontent, nd they inresed y 10.35%, 18.33% t the end of germintion respetively; while the levels of lumin, gloulin, glutelin, solule nd insolule protein first inresed nd then deresed during germintion. The protese tivity orrelted highly with the ontent of different protein frtions ut gloulin during germintion. Keywords Nked ot, Protein, Protese tivity, Germintion 1. INTRODUCTION Germintion hs een widely used for enturies to soften the kernel struture, to derese ntinutritionl ompounds nd to improve its nutritionl vlue in ens (Alonso et l., 2000) nd some erel seeds (Wu & Wll 1980; Koehler et l., 2007). During germintion, endogenous enzymes re produed or tivted, whih my degrde mromoleulr to smll moleulr sustnes, nd produe some tive sustnes suh s phenolis, γ minoutyri id nd so on (Xu et l., 2009; Xu et l., 2010; Shi et l., 2010). One of the most importnt physil-hemil hnges tht our during germintion is the degrdtion of the protein nd their onversion into solule peptides nd mino ids to provide sustrtes for the plnt s development, whih n result in the hnges in protein ontent nd size distriution. During germintion of ots, the ontent of rude protein inresed nd protein ws degrded to inrese the solule protein ontent nd free mino ids (Tin et l., 2010; Wu 1983; Klose et l., 2009). However, germintion ws lso very tive nd omplex metoli proess tht my derese nutritive vlue of pulses (Nnnn et l., 1990; Rozn et l., 2000; Urno et l., 2005). Wilhelmson et l. (2001) reported tht the hemil omposition of mlted ot seeds hs onern with the onditions nd the level of germintion. Therefore, it is neessry to evlute wht hppens to the hnges in nutrients, phytohemils nd relted properties ffeted y these hnges in ots during germintion, espeilly the hnges of proteins. However, informtion on these spets is very limited in ots, espeilly nked ot ultivrs (Aven nud L.) from Chin. The ojetive of the present study ws to further investigte the effet of highly ontrolled germintion proess on the protese tivity, the hemil omposition nd ontent of proteins, whih tht re required to produe high qulity food sed on nked ots. 2.1 Plnt mterils 2. MATERIALS AND METHODS Biyn II, nked ot ultivrs (Aven nud L.), ws used in the study. The ultivr ws grown in 2012 in ses for growing orgni ot, Shnxi, Chin. The hrvested ot grots were dried to out 10% moisture nd then stored until time of steeping nd germintion. 2.2 Germintion Ot grots were surfe sterilized using 1% solution of sodium hypohlorite for 30 s, nd then they were wshed three times with demonized wter efore steeping. The ot grots were steeped with demonized wter for 12 h t 25 o C, ertion for 1 h every 4 h, nd smpling (S12) ws rried out t the end of steeping. After steeping, the remining ots were drined nd germinted for 72 h t 25 o C nd 95% reltive humidity, nd six smplings were rried out (G12, G24, G36, G48, G60, nd G72), whih took t 12, 24, 36, 48, 60 nd 72 h during the germintion proess. After smpling, smples were immeditely freeze dried nd stored t 40 o C until time of nlysis. Asin Online Journls ( 128

2 Protein ontent (%) Protese tivity (μg/min/g) 2.3 Determintion of protese tivity Asin Journl of Agriulture nd Food Sienes (ISSN: ) The protese tivity ws mesured ording to the method of Hrvey nd Oks (1974) with some modifitions. Two grms of milled ots were mixed, on n ie th, with 10 ml of itri id-disodium hydrogen phosphte uffer (0.02 M, ph 6.0) ontining 5 mm β merptoethnol nd 2.5 mm disodium ethylenedimine tetreti id (EDTA) nd shken with lortory rotry shker t 250 rpm for 20 min t 4 o C. Then the homogente ws entrifuged t g for 20 min t 4 o C, nd the superntnt ws the rude protese extrts. The retion mixture onsisted of 2 ml of rude enzyme liquid nd 2 ml of sustrte (20 g/l sein) ws inuted for 60 min t 40 o C nd then terminted for 5 min t 90 o C, followed y ooling nd entrifugtion (5 000 g, 15min) t 4 C. The sorne ws mesured t 275 nm nd the tivity of protese ws defined s the µg of L-tyrosine produed t 40 o C per minute per grm on dry weight sis (DW). 2.4 Extrtion of protein Two-grm milled ot smples were extrted with 50 ml Tris-HCl uffer (0.05 M, ph 7.8) for 30 min t room temperture y n ultrsoni homogenizer, nd then the homogentes were entrifuged t g for 15 min t 4 o C. After entrifugtion, the superntnts were removed nd extrtion ws repeted two times t the sme onditions. Then djusting the ph vlue of the omined superntnts to out 4.5, the resulting preipittes fter entrifugtion were resuspended in 50 ml of Tris-HCl uffer nd nlyzed for solule protein. The residues for determintion of insolule protein were extrted with 50 ml 0.2% NOH nd then extrts were treted under the sme onditions s the solule protein. Ot protein frtions were extrted ording to the Osorne proedure with some modifitions. Nmely, milled smples were sequentilly extrted with distilled wter, slt solution (2.0% NCl), ethnol (75%, v/v), nd dilute lkli (0.02 M NOH) to yield lumin, gloulin, prolmin, nd glutelin, respetively. Eh frtion ws extrted t 1:10 (w/v) solid-to-solvent rtio. Eh extrtion ws rried out t room temperture for 30 min y n ultrsoni homogenizer nd entrifuged fterwrds t g for 10 min. 2.5 Determintion of protein ontent Totl nitrogen ws determined y the method of Kjeldhl (AOAC 1990). Crude protein ontent ws lulted y N The ontent of protein frtions ws lso determined y the method of Lowry et l. (1951) with ovine serum lumin s the stndrd. 2.6 Sttistil nlysis All results re expressed s men ± SD (n=3). One-wy nlysis of vrine (ANOVA) nd Dunn s test were performed with signifint level eing onsidered t P < Chnges in protese tivity 3. RESULTS AND DISCUSSION The protese tivity of the ot grots t different germintion stges re shown in Figure 1. Compred with rw grots, the levels of protese tivity inresed with the proessing of steeping nd germintion. The protese tivity inresed pproximtely 17% during steeping nd the first 12 hours of germintion (G12). Therefter, rpid inrese (p < 0.05) in the protese tivity ws deteted, nd it inresed y 252% nd rehed the mximum t stge G60 ompred with rw grots, nd then the protese tivity egn to derese. But no differene in the protese tivity ws found from stges G36 to G Rw S12 G12 G24 G36 G48 G60 G72 Germintion time (h) 0 Figure 1: The levels of protese tivity (line) nd rude protein (r) in ots t different stges of germintion Asin Online Journls ( 129

3 Protein ontent (%) 3.2 Chnges in the ontent of rude protein, solule, nd insolule protein Asin Journl of Agriulture nd Food Sienes (ISSN: ) The rude protein inresed grdully during germintion of ots (Figure 1). Nevertheless, the ontent of the rude protein did not hnge signifintly (p > 0.05) from stges S12 to G60, nd it inresed (p < 0.05) y 10.35% t the end of germintion ompred with rw grots. The levels of solule nd insolule protein t different germintion stges re shown in Figure 2. Insolule protein first inresed y 28.41% nd rehed the mximum t stge G48, nd then it deresed y 9.88% t the end of germintion. Similr to insolule protein, the ontent of solule protein first inresed signifintly (p < 0.05) y 59.41% nd rehed the mximum t stge G48, nd then it deresed y 30.09%. In ddition, there ws no distint differene in solule protein during steeping nd the first 24 hours of germintion (G12), though solule protein deresed slightly t S12 nd G24 stges s ompred with rw grots, whih ws different from the hnges of insolule protein solule protein d d d insolule protein 6 4 d d d d d 2 0 Rw S12 G12 G24 G36 G48 G60 G72 Germintion time (h) Figure 2: The ontent of solule nd insolule proteins in ots t different germintion stges 3.3 Chnges in the ontent of protein frtions In generl, erel proteins hve een lssified sed on their soluility (Osorne frtiontion) lumin, gloulin, prolmin nd glutelin frtions. The levels of the different protein frtions in ots during steeping nd germintion re shown in Tle 1. Unlike whet nd some other erels, the highest ontent ws gloulin, followed y glutelin nd prolmin, the lowest lumin in ot seeds, whih hve een otined y some uthors (Shewry 1995; M & Hrwlkr 1984). However, there were some differenes in the ontent of individul protein frtions euse of the differenes in the extrtion solvent nd its onentrtion (Roert et l.,1985). The levels of protein frtions in ots were ll influened y the germintion proess. Compred with rw grots, lumin ontent deresed signifintly y 19.18%, 10.75% t stge S12 nd G12, respetively. Therefter, rpid inrese (p < 0.05) in lumin ontent ws deteted, nd it inresed (p < 0.05) y 53.57% nd rehed the mximum t stge G60 ompred with rw grots (Tle 1), nd then lumin ontent deresed. There ws slight derese in gloulin ontent during steeping, then it inresed y 7.25% nd rehed the mximum t stge G36, nd then it deresed nd ws lower thn tht of rw grots t the lst germintion stge. Prolmin first inresed, nd then it deresed y 4.84% nd rehed the minimum t stge G24. Susequently, prolmin inresed ontinuously nd signifintly y 18.33% nd rehed the mximum t the end of germintion stge. Glutelin first inresed y 14.63% t G24 stge nd then deresed y 24.19% y the end of germintion ompred with rw grots, its ontent from stges G48 to G72 were lower thn tht of rw grots. Tle 1: Content (mg/g DW) of different protein frtions in ots t different germintion stges Alumin Gloulin Prolmin Glutelin Rw grot 15.53±0.54 d 59.35±0.22 d 22.75±0.88 de 35.55±2.52 S ±0.52 e 58.65±0.35 d 24.56±0.64 d 36.06±2.33 G ±0.44 de 62.62± ±0.50 d 38.28±2.04 G ± ± ±0.72 e 40.75±3.45 G ± ± ±0.41 d 37.22±1.88 G ± ± ± ±2.17 Asin Online Journls ( 130

4 Asin Journl of Agriulture nd Food Sienes (ISSN: ) G ± ±0.60 d 26.55± ±1.80 G ± ±0.34 d 26.92± ±1.44 Vlues re represented s men ± stndrd devition of triplites; Different smll letters within the sme olumn indite sttistilly signifint differenes etween the mens of different germintion stges t P < Correltion nlysis To further investigte the their interreltionship, the orreltions mong protese tivity nd the ontent of different protein frtions were estlished in ot grots during germintion, nd orreltion oeffiients (R) re shown in Tle 2. Solule protein onsists hiefly of lumin nd gloulin while insolule protein is minly omposed of prolmin nd glutelin. Correltion nlysis showed tht lumin my e responsile for the inrese of solule protein, nd while the hnge in insolule protein ontent ws determined y the degree of prolmin inresing nd of glutelin deresing. The protese tivity orrelted highly with the ontent of different protein frtions, ut it ws irrelevnt to gloulin, however, there ws signifintly negtive orreltion (p < 0.05) etween glutelin ontent nd the protese tivity, whih indites tht the protese tivity hd gret influene on the hnges in the ontent of different protein frtions. Tle 2: Correltion nlysis etween protese tivity nd different protein frtions SP IP Alumin Gloulin Prolmin Glutelin IP Alumin d Gloulin Prolmin Glutelin d Protese tivity d d d Correltion oeffiient R. Arevitions of solule nd insolule proteins (SP, solule protein; IP, insolule protein). Signifint (p < 0.01). d Signifint (p < 0.05). 4. DISCUSSION In the urrent study, the levels of protese tivity inresed during germintion of ots, whih ws supported y previous reserhes (Klose et l., 2009; Mikol & Jones 2010; Hüner & Arendt 2010) tht the highest inrese in proteolyti tivities ould e deteted during the first three dys of germintion, ut not fter tht. The rude protein inresed grdully during germintion, whih ws in greement with previous report (Tin et l., 2010). However, euse the rude protein ontent in food is ommonly determined y mesuring the totl nitrogen ontent nd multiplying it with n pproprite ftor, its ontent my not e equl to the nturl protein espeilly in the se of germintion, whih ws onfirmed y the following work. Nmely, solule nd insolule protein first inresed nd then deresed during germintion. Urno et l. (2005) reported the protein in pes ws signifintly deresed with the proessing of germintion, while Alonso et l. (2000) reported the inrese in protein from f nd kidney ens during germintion. In the present study, the slight derese in the level of solule protein during germintion ws similr to tht reported y Elmki et l. (1999), whih my result from its loss during steeping of seeds nd lso, utiliztion for the growth nd development of the emryo (Wu et l., 1980), while the inrese of solule protein my result from synthesiztion nd degrdtion of other proteins. Some reported studies demonstrted tht insolule protein deresed during germintion of plnt seeds (Urno et l., 2005; Wnsundr et l., 1999), whih ws in disgreement with the present study. The explntion for this result in present study is proly tht insolule protein is eing degrded while eing produed. In ddition, the insolule protein n e esily otined from ots euse of looser kernel struture during the lter period of germintion, whih my lso e nother use of the inrese of insolule protein. A signifint inrese in lumin frtion ws expeted euse of the ft tht this frtion ontins the mjority of the metolilly enzymes while glutelin frtion deresed signifintly in the lter stge of germintion, whih supported the result otined y Klose et l. (2009), however, there were some differenes from hnges in the ontent of prolmin nd gloulin frtions. In ft, eh of protein frtions extrted ording to the Osorne proedure ws likely omposed of group of different moleulr weight proteins. Consequently, the inrese in level of one protein frtion does not simply men tht the degrdtion of the protein frtion did not tke ple during germintion. 5. CONCLUSION Conlusively, the urrent study indites tht germintion n improve the protese tivity, t sme time whih led to so mny different hnges in proteins ot grins during germintion. However, the hoie of germintion time might e Asin Online Journls ( 131

5 Asin Journl of Agriulture nd Food Sienes (ISSN: ) of gret importne nd germintion for 48 h under highly ontrolled onditions would e suffiient. Besides, euse the physiologil nd iohemil retion resulting from steeping nd germintion of erel seeds is extremely omplex proess ffeted y severl ftors suh s the time, temperture, nd so on. 6. ACKNOWLEDGEMENT This work ws finnilly supported y projet of the Nturl Siene Foundtion of Shnxi Provine, Chin (projet no ). 7. REFERENCES 1. Alonso R, Aguirre A, Mrzo F Effets of extrusion nd trditionl proessing methods on ntinutrients nd in vitro digestiility of protein nd strh in f nd kidney ens. Food Chemistry 68: AOAC Offiil Methods of Anlysis Assoition of Offiil Anlytil Chemists, (15 Ed.). Wshington, DC. 3. Elmki HB, Biker EE, El Tiny AH Chnges in hemil omposition, grin mlting, strh nd tnnin ontents nd protein digestiility during germintion of sorghum ultivrs. Food Chemistry 64: Hrvey BMR, Oks A Chrteristis of n id protese from mize endosperm. Plnt Physiology 53: Hüner F, Arendt EK Studies on the influene of germintion onditions on protein rekdown in ukwhet nd ots. Journl of Institute of Brewing 116: Klose C, Shehl BD, Arendt EK Fundmentl study on protein hnges tking ple during mlting of ots. Journl of Cerel Sienes 49: Lowry OH, Roserough NJ, Frr LA, Rndll RJ Protein mesurement with the Folin phenol regent. Journl of Biologil Chemistry 193: M C-Y, Hrwlkr VR Chemil hrteriztion nd funtionlity ssessment of ot protein frtions. Journl of Agriulturl nd Food Chemistry 32: Mikol M, Jones BL Eletrophoreti nd in solution nlyses of endoproteinses extrted from germinted ots. Journl of Cerel Sienes 31: Nnnn IA, Dixon-Phillips R, MWtters KH, Hung YC Effet of germintion on the physil, hemil, nd sensory hrteristis of owpe produts: flour, pste, nd kr. Journl of Agriulturl nd Food Chemistry 38: Roert LS, Nozzolillo C, Altosr I Chrteriztion of ot (Aven stiv L.) residul proteins. Cerel Chemistry 62: Rozn P, Kuo Y, Lmein F Free mino ids present in ommerilly ville seedlings sold for humn onsumption. A potentil hzrd for onsumers. Journl of Agriulturl nd Food Chemistry 48: Shewry PR Plnt storge proteins. Biologil Reviews 70: Shi H, Nm PK, M Y Comprehensive profiling of isoflvones, phytosterols, toopherols, minerls, rude protein, lipid, nd sugr during soyen (Glyine mx) germintion. Journl of Agriulturl nd Food Chemistry 58: Tin B, Xie B, Shi J, Wu J, Ci Y, Xu T Physiohemil hnges of ot seeds during germintion. Food Chemistry 119: Urno G, Arnd P, Vílhez A, Arnd C, Crer L, Porres JM Effets of germintion on the omposition nd nutritive vlue of proteins in Pisum stivum L. Food Chemistry 93: Wnsundr PKJPD, Shhidi F, Brosnn ME Chnges in flx (Linum usittissmum) seed nitrogenous ompounds during germintion. Food Chemistry 65: Wilhelmson A, Oksmn-Cldentey K-M, Litil A, Suortti T, Kukovirt-Norj A, Poutnen K Development of germintion proess for produing high β-glun, whole grin food ingredients from ot. Cerel Chemistry 78: Wu YV Effet of germintion on ots nd ot protein. Cerel Chemistry 60: Wu YV, Wll JS Lysine ontent of protein inresed y germintion of norml nd high-lysine sorghum. Journl of Agriulturl nd Food Chemistry 28: Xu JG, Tin CR, Hu QP, Luo JY, Wng XD, Tin XD Dynmi hnges in phenoli ompounds nd ntioxidnt tivity in ots (Aven nud L.) during steeping nd germintion. Journl of Agriulturl nd Food Chemistry 57: Xu JG, Hu QP, Dun JL, Tin CR Dynmi hnges in γ-minoutyri id nd glutmte deroxylse tivity in ots (Avenel nud L.) during steeping nd germintion. Journl of Agriulturl nd Food Chemistry 58: Asin Online Journls ( 132