Fermentative Quality of Guineagrass Silage by Using Fermented Juice of the Epiphytic Lactic Acid Bacteria (FJLB) as a Silage Additive

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1 87 Fermenttive Qulity of Guinegrss Silge y Using Fermente Juie of the Epiphyti Lti Ai Bteri (FJLB) s Silge Aitive S. Bureenok, T. Nmihir, M. Tmki, S. Mizumhi, Y. Kwmoto* n T. Nk Fulty of Agriulture, University of the Ryukyus, Senru 1, Nishihir-ho, Okinw, , Jpn ABSTRACT : This experiment exmine the hrteristis of fermente juie of epiphyti lti i teri (FJLB) prepre y the ition of gluose, surose n molsses s fermenttion sustrte. The effet of FJLB on the fermenttive qulity n hnges in hemil omposition uring fermenttion of guinegrss silge were lso investigte. The ph vlue of the silges trete with FJLB rpily erese, n rehe to the lowest vlue within 7 ys of strt of fermenttion, s ompre to the ontrol. The numer of lti i teri (LAB) in the trete silges inrese for the first 3 ys, therefter the numer of LAB eline grully up to the en of the experiment. Silges trete with FJLB h lrger popultions of LAB thn the ontrol. Ammoni-nitrogen proution inrese throughout the ensiling perio, whih in the ontrol n no-sugr e FJLB silges were higher thn the other trete silges. Lti i levels vrie with the time of ensiling n mong the silge tretments. For ny sugr FJLB trete silges, the lti i inrese initilly, n then slightly reue to less thn 5 g/kg of ry mtter until 49 ys fter ensiling, exept the silge trete with gluose e FJLB. Nevertheless, lti i ontent of the ontrol erese onstntly from the eginning of ensiling n ws not foun fter 35 ys. Moreover, eti i ontent inrese throughout the ensiling perio. All the FJLB trete silges h signifintly (p<.5) lower ph n mmoni-nitrogen ontent, while signifintly (p<.5) higher lti i ontent n V-sore vlue ompre with the ontrol. This stuy onfirme tht the pplying of FJLB with ny sugr sustrte improve fermenttive qulity of silge. (Asin-Aust. J. Anim. Si. 25. Vol 18, No. : ) Key Wors : Epiphyti Lti Ai Bteri, Guinegrss, Silge, Silge Aitives INTRODUCTION Ensiling hs een known s metho to preserve the moist rops y ontrolling neroi fermenttion. The suess of the ensiling n e hieve when the numer of lti i teri (LAB) is ominnt in the fermenttion, n the tivity of lostrii is restrite. Although it hs een well reognize tht the epiphyti LAB plys n importnt role on silge fermenttion, the numer of epiphyti LAB on the stning rop is tiny n vrile (Muk, 199; Lin et l., 1992), n foun to e lower thn tht of eroi teri, yest n mols (Hellings et l., 1985; Ci et l., 1998). Therefore, n inoultion of LAB in the ensiling proess hs een reommene in orer to mke goo qulity silge. However, the properties of teril strin vry even within the sme speies, n some strins re not effetive in improving fermenttive qulity of silge s well (Woolfor n Swzy, 1984). Reently, mny reserhers hve reporte tht mnipulting numers of the epiphyti LAB n e improve y using fermente juie of epiphyti LAB (FJLB) s silge itive otine y merting rop silge with wter n neroi inuting for 2 ys. Oshim et l. (199) lso reporte tht the pplition of fermente green juie ws more effetive on silge fermenttion thn ommeril lti i teri inoulting in the se of * Corresponing Author: Y. Kwmoto. Tel: , Fx: , E-mil: ysuk@gr.u-ryukyu..jp Reeive Septemer 2, 24; Aepte Jnury 1, 25 iret ut lflf. However, very few reserhes reporte out ensiling of tropil psture speies trete with FJLB. This stuy is purpose to lrify if the pplying FJLB with ition of gluose, surose, n molsses s sustrte woul enhne the fermenttion of the guinegrss silge. MATERIALS AND METHODS FJLB preprtion The FJLB ws prepre from guinegrss (Pnium mximum Jq v. Gtton) efore hrvesting, n 25 g of fresh grss ws merte with 5 ml of istille wter using lener. This ws filtere through sterilize oule heese loths n the filtrte ws e without ny sugr (non-sugr FJLB), with 1% (w/v) gluose n surose (FJLB+1% G n FJLB+1% S), n with 5% (w/v) molsses (FJLB+5% M), respetively. The FJLB ws kept in n inutor t 3 C for 3 ys. Silge mking Guinegrss ws hrveste t flowering stge on Otoer 22, 21 in the experiment fiel, University of the Ryukyus, Okinw Jpn, n hoppe to 1 m piees efore ensiling. The FJLB were e t 1% (v/w) of fresh mteril s silge itives. The ontrol silge ws e with n equivlent mount of istille wter. Approximtely 1 g fresh mtter of trete rop then ws pke into plsti pouh in triplite, n sele with vuum seler (Shrp, Co. Lt, Vuum seler, SQ22). The silos were

2 88 BUREENOK ET AL. Tle 1. Chemil omposition of guinegrss mterils prior to ensiling Item ph.12 Moisture (% FM) 74.3 Dry mtter (%) Buffering pity M.eq.DM 33. WSC (% DM) 3.24 Crue protein (% DM) 7.48 LAB (fu/g FM) Aeroi teri (fu/g FM) FM = fresh mtter, DM = ry mtter. WSC = wter solule rohyrte, LAB = lti i teri. kept t 25 C n smples were tken 1, 3, 7, 14, 21, 35 n 49 ys fter ensiling for hemil nlysis. Chemil nlysis The representtive 2 g fresh mtter of ensile smple ws merte with 7 ml of istille wter n store t 4 C for 12 h. Then the extrt ws filtere using filter pper, the filtrte ws use to etermine the ph, mmoninitrogen (NH 3 -N) n voltile ftty i ontent (VFA) of silge. The ph of silge ws etermine y using ph meter, the NH 3 -N ontent ws mesure y using stem istilltion tehnique, n the orgni i ontent ws mesure y pplying HPLC. Dry mtter ontent of silge ws etermine y oven rying t 7 C to onstnt weight. The ontent of wter solule rohyrte (WSC) ws estimte olorimetrilly using Anthrone metho (Murphy, 1958). Buffering pity of mteril grss ws etermine oring to the metho s esrie y Plyne n MDonl (19). V-sore ws evlute y using the vlues of orgni i ontent n NH 3 -N (Jpn Grssln Frming Forge See Assoition, 1994). Counts of lti i teri One grm of silge ws shken well with 1 ml of sterilize.85% NCl solution, n ws seril ilute. The numer of lti i teri (LAB) in FJLB n the extrt of rop mteril n silges were etermine y ounting the olony forming unit (CFU) fter inution on GYP-CCO 3 gr plte t 35 C for 3 ys (Kozki et l., 1992). Aeroi teri were ounte on gr plte of nutrient roth fter inution t 37 C for 3 ys (Nissui Seiyku Lt., Jpn). Yest n mols were ounte on plte of potto extrose gr (Nissui Seiyku Lt., Jpn) LAB (log 1 fu/g of Fw) LA (g/kg DM) BA(g/kg DM) () 5 Ensiling perio (y) () e Figure 1,,,, e, f. Chnges in the ounts of LAB, ph vlue, LA, AA, BA n NH3-N ontents with ( ) ontrol; ( ) non-sugr FJLB; ( ) FJLB+1% gluose; ( ) FJLB+1% surose; ( ) FJLB+5% molsses uring the ensiling perio. Vlues re mens of three silge smples. Different letters show signifint ifferenes etween tretments t p<.5. fter inution t 3 C for 3 ys. Ensiling perio (y) (e) 2 Ensiling perio (y) ph vlue Sttistil nlysis Dt otine on hemil omposition of silge ensile for 49 ys were nlyze using the GLM proeure of SAS n the signifine of the ifferene mong tretment mens ws etermine y the Dunn s multiple-rnge test (SAS, 1998). RESULTS 4 () Ensiling perio (y) The hemil omposition of mteril prior to ensiling ws shown in Tle 1. The epiphyti lti i teri (LAB) ounts of the extrt of fresh mteril were 1 4 fu/ ml n inrese to the rnge of fu/ml fter inution (Tle 2). The hnges in the numer of LAB ount, ph vlue, AA (g/kg DM) NH 3 -N (g/kg DM) () Ensiling perio (y) 3.5 (f) Ensiling perio (y) Tle 2. The ph vlue, miroorgnism, orgni i n WSC ontine in eh FJLB efore use s silge itives ph LAB Aeroi teri Yest Lti i Aeti i Resiul WSC fu/ml mg/ml Non-sugr FJLB ND..11. FJLB+1% gluose ND FJLB+1% surose ND FJLB+5% molsses ND

3 EFFECT OF FJLB ON FERMENTATIVE QUALITY OF GUINEAGRASS SILAGE 89 lti i n eti i ontents uring silge fermenttion re shown in Figure 1. The numers of LAB ounts inrese rpily n rehe mximum popultion numer within one y fter ensiling for ll sugr FJLB trete silges n then onstntly erese from 1 9 to fu/g until 49 of ensiling perio (Figure 1). The LAB ounts in the ontrol n non-sugr FJLB silges were lower (p<.5) thn the other trete silge in ny ensiling time. The ph vlue of the ontrol silge ws higher (p<.5) thn the other trete silge (Figure 1). The ph of nonsugr FJLB trete silge slightly reue n rehe to 5.5, n no further eline until the en of experiment. In ontrst, the ph of sugr-fjlb trete silges eline rpily on the first y of fermenttion n mintine lmost the sme vlue until 49 of ensiling perio, n the vlues were signifintly (p<.5) lower thn tht of the ontrol n the non-sugr FJLB. In the ontrol n sugr free FJLB trete silges, the lti i ontent slowly inrese up to 3, n onstntly erese n ws not oserve t 35 eventully. Conversely, the other FJLB trete silges h signifintly higher vlue thn the former two silges (p<.5), though the lti i ontent fter 7 slightly erese to less thn 5 g/kg of ry mtter (Figure 1), exept the gluose-fjlb trete silge. The onentrtion of lti i in the molsses-fjlb trete silge ws rmtilly higher (p<.5) thn the other silges t 7. The gluose-fjlb trete silge ontine the lrgest mount of lti i t 49 of ensiling perio (Tle 3). Generlly, eti i ontent ws low t the eginning (Figure 1) n grully inrese until the en of ensiling perio. Nevertheless, fter thir y of ensiling the eti i ontent in the ontrol n non-sugr FJLB were higher (p<.5) thn ll the sugr-fjlb trete silges t ny ensiling time. In se of utyri i, it ws not foun until 14 in the ontrol n 21 in the trete silges of the ensiling perio (Figure 1e). Gluose-FJLB trete silge ws not etete this i in ny ensiling time. NH 3 -N proution steily inrese throughout the ensiling time for ll silges (Figure 1f), though the vlues were less thn 3 g/ kg of DM until 49 fter ensiling. There were no ifferenes mong the ontrol n the non-sugr FJLB trete silges until 14 ys fter ensiling, while ll sugr FJLB trete silges showe the lower (p<.5) onentrtion of NH 3 -N on 49 fter ensiling thn the ontrol n non-sugr FJLB trete silge (Tle 3). Moreover, NH 3 -N ontent in ll sugr trete silges ws not signifintly ifferent. In summry, the ontrol n non-sugr FJLB trete silges showe poor fermenttion with high ph n NH 3 - N ontent, low V-sore n lk of lti i (Tle 3). Preisely, ll the silges trete with FJLB e with ny sugr sustrte revele etter qulity, ompre to respetive opposite experiment result. In ition, gluose ontining FJLB trete silge presente the highest qulity, onsiering the ontent of lti i (p<.5). DISCUSSION Mny reserhers hve reporte tht the guinegrss silges without ny itive revel poor lti i fermenttion with high level of eti i n sre lti i ontent t the en of ensiling time (Niimi n Kwmur, 1998; Imur et l., 21; Sho et l., 24). In this stuy, the pplying of ny sugr trete FJLB s silge itive showe improve the fermenttive qulity of silge with high onentrtion of lti i n low onentrtion of NH 3 -N. In the ontrol n non-sugr FJLB trete silges, the ph vlue ws higher from eginning to en of ensiling perio ompre to trete smple. Moreover, the LAB numer of these silges ws lower thn the trete silges in ny ensiling time. The stuy lerly revele tht ll sugr FJLB silges t the erlier ensiling perio were le to erese ph more rpily n inrese the LAB numer rmtilly in the sme time. Consiering the i ontent, it ws lerly shown tht the sugr trete FJLB use higher lti i formtion. However, it ws foun tht the lti i ontent of ll silges erese with the extension of storge perio. Menwhile, the inresing eti i ontent ws foun uring ensiling perio. There re two evients for these results. Firstly, the popultion of lti i teri h Tle 3. Chemil omposition of 49 ys silge Control Non-sugr FJLB FJLB+1% gluose FJLB+1% surose FJLB+5% molsses SEM 3 ph 5.88, LA 2 (g/kg DM) AA (g/kg DM) BA (g/kg DM) NH 3 -N (g/kg TN) V-sore Men vlues within rows with ifferent supersript letters were signifintly ifferent (p<.5). 2 LA = lti i, AA = eti i, BA = utyri i, NH 3 -N = mmoni-nitrogen. 3 SEM = Stnr error of men.

4 81 BUREENOK ET AL. hnge from homolti to heterolti uring the fermenttion (Shokey et l., 1988). Seonly, when the fresh mteril h low level of WSC ontent or even no more ville rohyrte, LAB were le to utilize lti i n proue more eti i (Lingren et l., 199). Butyri i ontent ws slightly high in non-sugr FJLB silges. This result ws proly ue to the lk of sustrte for the FJLB preominntly grown in the inution perio. Generlly, plnt juie ontins mny kins of miroorgnism suh s LAB, oliform teri n fungi. LAB numer is usully lower thn the other onsuming sugr ompetitor in plnt juie (Hellings et l., 1985; Ci et l., 1998; Yhy et l., 24). In this experiment, when juie ws inute without sugr, these teri oul only use sugr tht present in the plnt juie for their growing. Therefore, LAB oul not grow preominntly with the other teri. Finlly, non-sugr FJLB my ontin the sme numer of LAB or less, ompre to the other teri (Ohmomo et l., 22). In ition, fter pplition of FJLB to mteril rop, these unesirle teri oul ompete to utilize sugr with the LAB, with eresing in the mount of WSC to LAB. This might e expline y high ph n low lti i ontent uring the ensiling perio. Moreover, the growth of utyri i teri oul e stimulte y these onitions s inite high ontent of utyri i t the en of ensiling ws present. This experiment lso showe signifintly ifferent results with the vriety of sugr e in FJLB. It ws foun tht the gluose-e FJLB ws le to erese the ph vlue more rpily thn the others, n fortuntely, lti i ontent onstntly remine uring the ensiling perio. Although, these FJLB were me from the sme rop n the LAB ounts were similr efore the use of silge itives, the ifferene of the fermenttive qulity ws my e ue to the hnge of LAB speies n their perentges in eh sustrte uring inution n fter ensiling. This stuy suggests tht the FJLB trete with gluose proly onsiste of effetive LAB speies with the highest perentge thn tht in other sugr sustrte. CONCLUSION The results oviously onfirme tht using of ny sugr trete FJLB itive somehow woul e one of the wys to improve the fermenttive qulity of silge in the tropil re. Though, the FJLB lso ontin mny kins of miroorgnism n vriety of strins s mentione, further reserhes must e neee to ientify the tul useful speies of teri, n to lrify the mehnism of the teri for improving qulity of silge. ACKNOWLEDGEMENTS The uthors re grteful to Ministry of Eution, Siene, Sport n Culture of Jpn (Monusho) for finnil support. REFERENCES Ci, Y., Y. Benno, M. Ogw, S. Ohmomo, S. Kumi n T. Nkse Influene of Ltoillus spp. from n inoulnt n of Weissell n Leuonosto spp. from forge rops on silge fermenttion. Appl. Envi. Miro. 4: Hellings Ph., G. Bertin n M. Vmelle Effet of lti i teri on silge fermenttion. Proeeings of 15 th Interntionl Grssln Congress, August 24-31, Kyoto, Jpn. pp Imur, Y., T. Nmihir n Y. Kwmoto. 21. Fermenttion qulity of phsey en n guinegrss silges. In Proeeings of 19 th Int.Grss. Congr.,So Pero, So Pulo, Brzil, pp Jpn Grssln Frming Forge See Assoition Guie Book for Qulity Evlution of forge. Tokyo, Jpn (Jpn). pp Kozki, M., T. Uhimur n S. Ok Experimentl Mnul of Lti i teri. Askurshoten, Tokyo, Jpn (Jpn). pp. -1. Lin, C., K. K. Bolsen, B. E Brent, R. A. Hrt, A. M. Feyerherm n W. R. Aimutis Epiphyti miroflor on lflf n whole-plnt orn. J. Diry Si. 75: Lingren, S. E., L.T. Axelsson n R. F. Mfeeters Aneroi L-ltte egrtion y Ltoillus Plntrum. FEMS Miroiol. Lett. : Muk, R. E Preition of lti i teril numers on Luerne. Grss n Forge Si. 45: Murphy, R. P A metho for the extrtion of plnt smple n the etermintion of totl solule rohyrtes. J. Si. Foo Agri. 9: Niimi, M. n O. Kwmur Degrtion of ell wll onstituents of Guinegrss (Pnium mximum Jq.) uring ensiling. J. Jpn. Grssl. Si. (Jpn) 43: Ohmomo, S., O. Tnk, H. K. Kitmoto n Y. Ci. 22. Silge n miroil performne, Ol story ut new prolems. Jpn. Agri. Res. Qurt. 3: Ohshim, M., E. Kimur n H. Yokot A metho of mking goo qulity silge from iret ut lflf y sprying previously fermente juie. Anim. Fee Si. Tehnol. : Plyne, M. J. n P. MDonl. 19. The uffering onstituents of herge n of silge. J. Si. Foo Agri. 17: SAS The SAS systems for winows v.12. SAS Inst. In., Cry, N.C., USA. Sho, T., N. Oh, M. Shimojo n Y. Msu. 24. Effets of ing gluose, sori i n pre-fermente juie on the fermenttion qulity of guinegrss (Pnium mximum Jq.) silge. Asin-Aust. J. Anim. Si. 17: Shokey, W. L., B. A. Dehority n H. R. Conr Effets of

5 EFFECT OF FJLB ON FERMENTATIVE QUALITY OF GUINEAGRASS SILAGE 811 miroil inoulnt on fermenttion of poor qulity lflf. J. Diry Si. 71: Woolfor, M. K. n M. K. Swzy An investigtion into the effet of ultures of lti i teri on fermenttion in silge 1. Strin seletion. Grss n Forge Si. 39: Yhy, M. S., M. Goto, W. Yimiti, B. Smerji n Y. Kwmoto. 24. Evlution of fermenttion qulity of tropil n temperture forge rops ensile with itives of fermente juie of epiphyti lti i teri (FJLB). Asin-Aust. J. Anim. Si. 17: