Ecuatorian brown rice. Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Juan de la Cierva 3, Madrid, Spain.

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1 1 2 Assessment on proximte composition, ietry fier, phytic ci n protein hyrolysis of germinte Ecutorin rown rice 3 4 Ptricio J. Cáceres, Cristin Mrtínez-Villlueng, Loures Amigo c n Jun Fris * Escuel Superior Politécnic el Litorl (ESPOL), Cmpus Gustvo Glino Velsco, km 30,5 Ví Perimetrl, Guyquil, Ecuor Institute of Foo Science, Technology n Nutrition (ICTAN-CSIC), Jun e l Cierv 3, Mri, Spin. c Institute of Foo Science Reserch, CIAL (CSIC-UAM), Nicolás Crer 9, Cmpus e Cntolnco, Mri, Spin *J. Fris is the person whom corresponence shoul e resse Institute of Foo Science, Technology n Nutrition (ICTAN-CSIC), Jun e l Cierv 3, Mri, Spin. E-mil: fris@ictn.csic.es Telephone numer: Fx numer:

2 Astrct ( ) Germinte rown rice (GBR) is consiere helthier thn rown rice (BR) ut its nutritive vlue hs een hrly stuie. Since nutritive qulity of GBR epens on genetic iversity n germintion conitions, six Ecuorin BR vrieties were germinte t 28 n 34 ºC for 48 n 96 h in rkness n proximte composition, ietry fier frctions, phytic ci content s well s egree of protein hyrolysis n peptie content were stuie. Protein, lipis, sh n ville crohyrte rnge %, %, % n 71.6 to 84.0%, respectively, in GBR seelings. Totl ietry fier increse uring germintion ( %), with lrge proportion of insolule frction, while phytic ci ws reuce noticely. In generl, protein hyrolysis occurre uring germintion ws more ccuse t 28 ºC for 48 h. These results suggest tht GBR cn e consume irectly s nutritive stple foo for lrge popultion worlwie contriuting to their nutritionl requirements Keywors: Brown rice, germintion, proximte composition, ietry fier, phytic ci, protein hyrolysis Introuction Rice (Oryz stiv L.) is the most importnt stple foo in mny Asin n South Americn countries, ut its consumption is wiespre ll over the worl proviing the energy n proteins over hlf the worl s popultion. Rice prouction hs grown steily in recent yers, expecting to chieve worl prouction of 512 million tons y 2016 [1]. In Ecuor, rice is the min energy provier since it is the primry component of the min y course. Different vrieties of long grin rice supplie y the Ecuorin government n privte enterprises re provie n frmln hs recently met locl emn to overprouction [2]. BR contriutes not only with the sic nutrients of polishe rice ut lso with ioctive compouns n ntioxints tht re concentrte in the rn, eing prticulrly rich in linoleic ci, -minoutyric ci, tocopherols, tocotrienols, -oryznol n phenolic compouns, s well s functionl proteins, unsponifiles lipis n ietry fier tht mke BR ttrctive eyon its nutritionl qulity [3]. The interest of soking n germintion of BR is enormously incresing nowys, since it is eing emonstrte tht the helth enefits of BR re enhnce with germintion [4]. It hs een recently shown tht 2

3 germinte rown rice (GBR) exerts chemopreventive n immunomoultory ctivity [5], suppresses inflmmtory responses [6], inhiits ipogenesis [7] n ttenutes hyrogen peroxie-inuce oxitive stress [8]. Germintion strts with short soking or steeping process in wter where grin rouse from ormncy. During wter uptke, the ry sees restore their metolic ctivities leing to iochemicl, nutritionl n sensoril chnges. See germintion is promote y the regultion of ifferent proteins involve in storge reserve egrtion, iosynthesis of germintion-promoting hormones, etoxifiction n efense n reinforcement of cell wlls [9]. Germintion results in increse reucing sugrs, reuce mylose n strch grnules ecme smller n less homogeneous [10]. Grin storge proteins re prtilly hyrolyze to pepties n mino cis improving protein igestiility n technofunctionl properties [11]. Soking n germintion process le to reuce phytic ci content [12] tht results in higher minerl ioccesiility [13,14]. Likewise, ietry fier (DF) content increses [15] n the sustntil higher content of insolule fier cn provie potentil enefits in the prevention of ietic vsculr complictions [16]. Recent stuies hve shown the ioctive compoun enhncement uring BR germintion [14,17] n its effect on humn helth [4]. However, to our knowlege, there re no systemtic stuies ressing the effect of ifferent germintion conitions on proximte composition n relte nutritive ttriutes in ifferent BR vrieties. Therefore, the ojective of this work ws to evlute the effect of ifferent germintion conitions on proximte composition, ietry fier n phytic ci content, s well s the protein hyrolysis n peptie content in ifferent Ecuorin BR vrieties. The results will ientify germintion conitions with higher nutritive potentil n will contriute to expn foo compositionl tse showing GBR s n excellent nutritive foo for improving the helth sttus of the popultion worlwie. Mteril n Methos Plnt mterils Experimentl BR cultivr GO39839 (coe GO) n three commercil vrieties INIAP 14, INIAP 15 n INIAP 17 (coe 14, 15 n 17) were provie y the Ntionl Autonomous Institute of Agriculturl Reserch from Ecuor (INIAP, Ecuor), whilst commercil BR vrieties SLF09 n F50 (coe 09 n 50) were supplie 3

4 y foo processor (Procesor Ncionl e Alimentos C.A., INDIA-PRONACA). All vrieties were grown in the cost re of Ecuor (Guyquil) n showe prticulr fetures such s long grin n trnslucent center. Smple Preprtion Germintion processes were crrie out s escrie previously [17]. Briefly, 50 g of BR grins were firstly wshe in 0.1% (v/v) soium hypochlorite n then imie in eionize wter (1:5, w/v) t 28 C for 24 h. Susequently, grins were rine n plce in germintion cin moel EC (Snijers Scientific, Netherlns) in reltive humiity >90 % for 48 h n 96 h t 28 C n 34 C n rkness. Smples were freeze-rie, groune, n store in sele plstic gs uner vcuum conitions t 4 ºC until further nlysis. Every germintion tch ws performe in triplicte. Determintion of Proteins, Ft, Ash, Dietry Fier n Aville Crohyrtes Nitrogen ws etermine ccoring to AOAC [18] n protein content ws clculte using 5.95 s conversion fctor. Ft (AOAC ), sh (AOAC ) n solule, insolule n totl DF (AOAC , AOAC n AOAC , respectively) were lso etermine [19]. Aville crohyrtes were estimte y ifference: 100 (% proteins + % ft + % wter + % ietry fire + % sh) [20]. Determintion of Phytic Aci The ccurte photometricl Hug n Lntzsch s etermintion of phytic ci phosphorous ws use [21]. Asornce ws re t 540 nm in microplte reer (BioTek Instruments, Winooski, VT, USA) (BioTek Instruments). Determintion of Degree of Proteolysis n Peptie Content Degree of proteolysis ws etermine y the nlysis of totl n relese free mino groups (FAG) in germinte smples. FAG were mesure in BR n GBR y ition of 2,4,6-trinitroenzenesulphonic ci (TNBS) s previously escrie [22]. Totl FAG were lso etermine in BR n GBR smples previously hyrolyze using 6N HCl for 24 h t 130 ºC. Degree of proteolysis ws clculte s follows: DH (%) = [(FAG in germinte grin-fag in rw grin)/ Totl FAG] x100 Sttisticl nlysis 4

5 Vlues re expresse s men ± SD from inepenent germintion experiments nlyze in uplicte. ANOVA n Duncn s multiple rnge comprisons were use to ssess ifferences t confience level of 95% (P 0.05). Level of significnce for temperture n time effects were clculte y sttisticl-t for ech component (P 0.05) n positive or negtive correltions were ientifie y the regression coefficient sign. Sttisticl nlyses were performe y Sttgrphics Centurion XVI softwre, version (Sttisticl Grphics Corportion, Rockville, MD). Results n iscussion Effect of germintion conitions on the proximte composition of BR vrieties Figure 1 shows the proximte composition of crue, soke (S) n germinte (GBR) rown rice. In crue grins, protein content rnge from 7.9 to 9.9 % ry mtter (.m.) (Figure 1A). These vlues were slightly higher thn those previously reporte in commercil BR [14,23]. Differences in BR protein content coul e expline y intr-vrietl genetic iversity, ephoclimtic conitions n hrvesting/storge mngement. BR soking (24h t 28ºC) i not ffect the protein content with exception of cv. GO (8% reuction, Figure 1A). Protein losses uring rice soking hve een previously reporte [13] n higher temperture le to lower protein content. Germintion process ffecte ifferently the protein content epening on BR vriety. In generl, germintion i not cuse relevnt chnges in protein content; however, vr. 50 germinte t 28 ºC for 4 ys showe significnt increse (11%). In generl, the lrgest protein content ws oserve in GBR vr. 14, 15 n 09. BR cv. GO n vr. 14 showe negtive correltion for germintion time n positive correltion for temperture (P 0.05) n vr. 17 showe only negtive correltion for germintion time (P 0.05). Decrese protein content is relte to increse mino ci content s consequence of proteses ctivtion [24]. This effect cuses protein soluiliztion n its further leching uring ricle protrusion [13]. In ition, other stuies showe n increse protein content in germinte sees ttriute to protein iosynthesis uring germintion, therefore, the protein content epens on the lnce etween protein egrtion n protein iosynthesis uring germintion [23]. The content of ville crohyrtes in crue BR rnge from %.m. (Figure 1B), results tht gree with the literture [14,23]. BR soking cuse 10% increse in the crohyrtes content (P 0.05) for cv. 5

6 GO, while it remine unchnge in vr. 14, 17 n 50 n it unerwent slight ecrese in vr. 15 n 09 (P 0.05). Germintion rought out ecrese in the ville crohyrte content in ll vrieties stuie. Accoring to sttisticl nlysis, crohyrtes content ws negtively ffecte (P 0.05) y oth germintion time n temperture n only vr. 09 showe negtive correltion (P 0.05) with germintion time. In generl, GBR cv. GO n INIAP vrieties provie lrger ville crohyrtes thn INDIA-PRONACA ones. Chnges in the ville crohyrtes cn e ttriute to the increse ctivity of enogenous α-mylse uring germintion [14,24] cusing the hyrolysis of ntive strch n of the relese of reucing sugrs tht re use s source of energy for the growing seeling [10]. The long-term consumption of GBR in type 2 ietes ptients s stple foo ws useful in improving loo glucose n lipi levels [25]. Commercil INDIA-PRONACA vr. 09 n 50 presente higher lipi content (2.9 n 3.6 %.m.) thn INIAP vr. 14, 15 n 17 ( %.m.), n cv. GO contine n intermeite lipi vlue (2.65 %.m) (Figure 1C), vlues tht re consistent with previous stuy [26] tht foun plmitic, oleic n linoleic cis s the mjor ftty cis (80%). BR soking le to lipi increse in vr. 15 n 09 (P 0.05) n noticele lipi reuction (P 0.05) in cv. GO n vr. 17 n 50. Consequently, uring germintion lipi content unerwent ifferent ehvior epening on BR vriety n germintion conitions. Temperture ws positively correlte with lipi content (P 0.05) in ll BR vrieties, except for vr. 15, while germintion time ws lso positively correlte with lipi content for cv. GO n vr. 15, 17 n 50. Decrese lipi content oserve fter germintion hs een scrie to increse lipse ctivity to generte energy uring seeling growth [27]. On the other hn, uring germintion the iosynthesis of lipis occurs, s it hs een escrie for unsturte n polyunsturte ftty cis [23]. In ition, γ-oryznol is the principl component of unsponificle lipi frction [28] tht, long with tocopherols n tocotrienols, contriutes to the nutritive qulity n helth promoting properties ssocite to GBR [4]. In generl, INDIA-PRONACA GBR vr. 09 provie lrger lipi content thn those from INIAP vrieties n cv. GO. Ash content of stuie BR vrieties rnge from 1.3 to 1.5%.m. (Figure 1D), results tht flls within those reporte in the literture [14,23]. Ash content ecrese (P 0.05) in soke BR, except of vr. 09 where no significnt ifferences were foun. During germintion, time n temperture ffecte (P 0.05) sh content n 6

7 negtive correltions were foun for cv. GO n INIAP vrieties, whilst only germintion time ffecte negtively (P 0.05) to INDIA-PRONACA ones. Decrese sh content coul e expline y lixivition losses uring soking n wtering, [23], nevertheless GBR still hve consierle mount of sh which reflects its minerl content, eing INDIA-PRONACA vrieties the ones with the lrgest sh content. Effect of Germintion on the Contents of Solule n Insolule Dietry Fier Tle 1 shows the content of totl ietry fier (TDF) n their solule (SDF) n insolule (IDF) frctions in Ecuorin crue, soke (S) n germinte (GBR) rown rice. TDF i not excee 5 %.m. in commercil INIAP vr. 14, 15, 17 n cv. GO, whilst INDIA-PRONACA vrieties provie roun 8 %.m, n SDF contriute with 72-75% n 66-68%, respectively. TDF content epens on BR vriety n hrvesting conitions [29]. Although the soking process le to slight (P 0.05) increse of SDF n IDF in some stuie BR vrieties, the highest increse ws oserve fter germintion (Figure 2). SDF n IDF content of INIAP vrieties were positively correlte (P 0.05) with germintion time n temperture n the highest TDF vlues were otine for GBR vr. 14 n 17 t 34 C for 96h (10 g/100g.m.). INDIA-PRONACA vrieties were positively ffecte y germintion conitions n mximum TDF contents were reche in GBR vr. 09 t 28 C for 48h n GBR vr. 50 t 28 ºC for 96h (13.6 n 13.6 %.m., respectively). It is noteworthy tht GBR vr. 15 suffere the lrgest increse in SDF n IDF uring germintion t 34 ºC for 96 h (from 2.9 to 5.1 n 0.9 to 3.7%.m., respectively), lthough the mximum vlues were foun in vr. 09 n 50 (Figure 2) in which SDF n IDF contriute similrly to TDF content (45-55%). It hs een shown tht TDF increses uring BR germintion [23]. In ition, germintion impct on fier frctions n SDF/IDF rtio epens on the vriety n processing conitions [30,31]. It hs een shown tht the content of IDF cn suppress post-pnril glucose level fter intke of soke BR [16]. Effect of Germintion on the Content of Phytic Aci The content of phytic ci in crue, soke (S) n germinte (GBR) vrieties is collecte in Tle 2. Levels of phytic ci in crue BR were rther similr (from 1.15 %.m in cv. GO to 1.5 %.m. in vr. 15). These results mtch with previous t foun in BR vr. Kenjin [32] n re lower thn those in BR cv. Ilpumyeo [26]. Phytic ci content ecrese (P 0.05) fter soking in most Ecuorin vrieties (from 15 % in 7

8 vr. 50 to 48 % in vr. 15), whilst no significnt ifferences were oserve in BR vr. 09. Germintion temperture n time negtively correlte with phytic ci content in ll vrieties, except for vr. 09 in which phytic ci ws only ffecte y germintion time (P 0.05). Interestingly, the lowest phytic ci content ws otine t 34 C for 96 h in ll Ecuorin BR vrieties where losses etween 32% for vr. 09 n 80% for cv. GO were foun. This effect hs een ttriute to increse enogenous phytse ctivity tht releses phosphorous n lower myoinositol phosphtes n, itionlly to phytic ci lixivition n further leching into wter [11]. From the nutritionl point of view, phytic ci is ssocite with the minerl-relte eficiency in humns, s well s protein n lipi vilility. However, phytic ci coul contriute to fight ginst vriety of cncers, ietes, therosclerosis n coronry hert iseses [33]. Effect of Germintion on the Degree of Protein Hyrolysis Figure 2A shows the egree of protein hyrolysis (DH) in soke (S) n germinte (GBR) rown rice vrieties. In generl, proteolysis ws negligile uring soking. Lrger germintion time n temperture le to higher proteolysis. The highest DH vlue (58.5%) ws oserve in vr. 50 followe y vr. 17 (48.9%) n cv. GO (48. 0%). Most of BR vrieties showe positive correltion etween DH n germintion time n temperture (P 0.05). On the contrry, DH in cv. GO correlte negtively with germintion temperture. During germintion proteolysis occurre s consequence of increse protese ctivity tht results in the relese of pepties n free mino cis [24]. These effects re reporte to improve protein igestiility of BR [14]. In orer to confirm this fct, peptie content in crue BR n their respective processe grins ws otine (Figure 2B). Soking le to slight (P 0.05) ecrese in peptie content, except for vr. 17 tht showe significnt (P 0.05) increse n vr. 50, where no significnt ifferences were foun. Germintion temperture n time ffecte positively (P 0.05) peptie content, except for vr. 09 in which germintion temperture ws negtively correlte with peptie content (P 0.05). INDIA-PRONACA vr. 09 showe the lrgest peptie content tht cn e scrie to its lrge protein content (Figure 1A) n noticele DH (Figure 2A). In this vriety the lrgest peptie content ws otine fter 96 h of germintion (3.24 n 4.7 g/100g.m t 28 n 34 ºC, respectively). Bioctive pepties my e relese s consequence of proteolysis tht tkes plce uring see germintion [34]. So fr, enzymtic hyrolysis n fermenttion re the iotechnologicl processes explore to prouce ntioxint pepties from rice 8

9 proteins [35], however, no informtion hs een foun on rice germintion n relte ioctive mino ci sequences. Thus, our group is currently performing further reserch to ientify pepties from GBR with iologicl ctivities. Conclusions This stuy shows tht germintion of rown rice is nturl process of improving its nutritionl qulity; however, the extent of these positive effects epens on BR vriety, germintion time n temperture. Since germinte rown rice is promising foo choice to form prt of helthy n sustinle iets of the popultion worlwie, cost-effective germintion conitions such s 28 ºC n 48 h re recommene to enhnce rown rice nutritionl qulity ttriutes. Acknowlegments P.J.C. is inete to the Ecuorin government through the SENESCYT (Secretrí Ncionl e Eucción Superior, Cienci, Tecnologí e Innovción, Ecuor) support on his octorl stuies. We lso cknowlege to INIAP (Instituto Ncionl e Investigciones Agropecuris of Ecuor) n to INDIA- PRONACA (Prouctor Ncionl e Alimentos C.A.) for proviing the BR sees. We re grteful to M.F. Romero n A. Glrz for their technicl ssistnce. This work ws prtly fune y AGL Project from the Ministry of Economy n Competitiveness (MINECO, Spin). References 1. OECD-FAO Agriculturl Outlook chsooiqdeoaugfos puso--prue cli-semills-e-rroz-y-miz&cti=18:noticis&itemi= Shrif MK, Butt MS, Anjum FM, Khn SH (2014) Rice Brn: A Novel Functionl Ingreient. Crit Rev. Foo Sci. Nutr. 54: Wu F, Chen H, Yng N, Dun X, Jin Z, Xu X (2013) Germinte rown rice enhnces ntioxint ctivities n immune functions in ge mice. Cerel Chem. 90 (6): Wng SY, Chen CW, Lin YT, Chng CY (2012) Inhiitory effect on humn leukemic U937 cell growth n immunomoultory ctivity of germinte rown rice. J. Foo Drug Nl. 20:

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13 303 g/100g.m. c c c A e f c e e c c c B C c c e c D Figure 1. Content of protein (A), ville crohyrtes (B), ft (C) n sh (D) of crue, soke (S) n germinte (GBR) rown rice grins. Brs inicte 13 men vlues (g/100 g ry mtter) n error lines inicte stnr evitions. The sme letter inicte no significnt ifference mong men vlues within rice vriety (P 0.05 ccoring to Duncn s test).

14 Tle 1. Phytic ci content (g/100g.m) in crue, soke (S) n germinte (GBR) Ecuorin rown rice. Tretment Temperture Time cv. GO vr. 14 vr. 15 vr. 17 vr. 09 vr. 50 (º C) (h) Crue ±0.02 f 1.35±0.06 c 1.52±0.04 e 1.26±0.0 f 1.19±0.04 e 1.25±0.05 c S ±0.02 e 0.83± ± ±0.03 e 1.16±0.03 e 1.06±0.03 GBR ± ± ±0.03 c 0.68± ±0.03 c 1.00± ± ± ± ± ± ± ±0.02 c 0.50± ±0.05 c 0.53±0.06 c 1.04± ± ± ± ± ± ± ±0.08 Dt re the men vlues ± stnr evition of three inepenent experiments (n=3). The sme superscript inictes no significnt ifference mong men vlues within column (P 0.05 ccoring to Duncn s test). 14

15 Tle 2. Dietry fier content (g/100g.m) in crue, soke (S) n germinte (GBR) Ecuorin rown rice. Tretment Temperture Time cv. GO vr. 14 vr. 15 vr. 17 vr. 09 vr. 50 (ºC) (h) Solule ietry fier Crue ± ± ± ± ± ±0.16 S ± ± ± ± ± ±0.13 GBR ± ±0.08 c 3.08± ± ±0.08 c 5.81±0.05 Insolule ietry fier ±0.07 c 4.30± ± ± ± ± ±0.11 c 4.30± ±0.19 c 3.89±0.07 c 6.23±0.03 c 6.20±0.06 c ± ±0.12 e 5.11± ± ±0.07 c 6.30±0.08 c Crue ± ± ± ± ± ±0.10 S ± ± ± ± ± ±0.04 GBR ±0.15 c 2.75± ±0.19 c 2.78±0.13 c 7.47±0.17 e 6.78±0.12 c ± ±0.10 c 3.76± ± ± ± ±0.03 e 3.66± ± ±0.08 e 5.85±0.05 c 5.85± ±0.08 e 4.94±0.08 e 3.70± ±0.08 f 7.02± ±0.09 Dt re the men vlues ± stnr evition of three inepenent experiments (n=3). The sme superscript inictes no significnt ifference mong men vlues within column (P 0.05 ccoring to Duncn s test). 15

16 %DH e e e e A cv. GO vr. 14 vr. 15 vr. 17 vr. 09 vr. 50 B 16 Figure 2. Pnel A: Degree of hyrolysis (%DH) of soke (S) n germinte Ecuorin rown rice for 48 h (48) n 96 h (96). Pnel B: Peptie content (g/100g.m.) in crue, soke (S) n germinte (GBR) Ecuorin rown rice. The sme letter inictes no significnt ifference within vriety (P 0.05, ccoring to Duncn s test).

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