Ar ticulos cientificos. Caracterizacién de productos fibroliticos comerciales utilizados en la alimentacion de rumiantes Characterization of commercial fibrolytic products for ruminant diets Laura Ramirez Cancino" Emilio Aranda lhafee"* Germén David Mendoza Martinez" Ls LandoisPalencia'™* Luis Alberto Miranda Romero: Marla Magdalena Crosby Galvan* Abstract Enzymatic activity (IU, international units) was determined for three enzymatic complexes (A, B, and C), at pH 5.5 and 6.5, using pH 102 crystalline cellulose, carboxymethyl cellulose (CMC), and oat xylan as substrate. Activity was higher (P < 0.05) at pHi 6.5. Cellulalytic activity was higher (P < 0.05) with crystalline cellulase and CMC with enayme A (53.68 and 56.879, IU). “The activity was higher (P < 0.05) with enzymes A and ® (222.169 y 229.319, UI) with xylan ‘The degradation half time (h) was similar (P > 0.05) among enzymes (A 4.02 h, B 4.18 h and C 4.37 h). Jn vitro digestion of crystallin cellulose and CMC was higher (P < 0.05), with ‘enzyme C (75.69%), and for CMC was higher with enzymes C and B (99.99% and 87.049 9) land different between enzymes C and A (99.99% and 79.54b%). When exogenous enzymes were incubated with ruminal microbes, a high enzymatic activity with purified substrates was observed, which does not necessarily corresponds to a highest digestibility, presumably ‘due to the differences on the degradation rate among them (A 17.229%, 8 16.5720% and C 415.850%), ‘ey words: ENZYMES, ACTIVITY, CELLULOSE, HEMICELLULOSE, XYLAN. Resumen Se determind la actividad enzimética (unidades internacionales, U1) de tres complejos enaimstcos (A, 8, ¥ C), 2 pH 5.5 65, utllzando como sustrato caulosa cristalina DH 102, Carboxiret ezluloca (CHC) y xylan de avena, Se observe mayor actividad (P < 0.05) a pH 6:5 {a actividad celuoitica fue mayor (P< 105) para celulesa crstaina y CMC con la enaima A (63.688 y 56.878, UD). La actividad fue mayor (P< 0.05) para las enzimas Ay B (222.168 228.31, Ul) con xylan. El tempo medio de degradacion fue similar (P< 0.08) entre enaimas (A, 4.02; 8, 4.18 nC, 4.37). La dlgestibiidad Invitro dela celulosa cristina fue mayor ('< 0.05) para la enzinia€ (75.696), para CMC fue mayor con la enzimas C y B (09.9 ¥ 87.028009) ysdferente entre Ia enzima Cy A (99.98 y 70.540). Al incuba las enzimas ‘xégenas con microorganismos ruminales se observé que una mayor actividad enzimatica en Sustrotos puros no necesariamente corresponde a una mayor digestibildad, posblemente Por [a diterancia en la tasa de degradacion entre ells (A, 17229; B, 16.579; y C, 15.8509), Palabras clave: ENZIMAS, ACTIVIDAD, CELULOSA, HEMICELULOSA, XYLAN. eco demande 2004, acetado 7 de sei de 2004, ‘rgruma, de Cannes, Congo te Fongrundss Carretera isico‘encco, km 95.5, Monti, $6290, Estado de cacy nc. Te 0109959650200 ant 12, Fas 962.0079, coma mandomenpor me» imreeDcolpon mS ‘Resinlgos ae ‘Shteede Hin de Rusnotn, Celi de Portgradandor, Campus Tabasco, Castane S08, Fraccionaints Lat Rese ‘Una te 80570, Cadena Taboo, Tel 01 (51) 29098, eal eareeacipos mk “Sie Evin, Clg etn ee oceans cy sista enon 302 9-00 S007 ami laienagtonnact hoping Vea Mee, 36 (1)2008Introduction rnzymes can be used as feeds to" increase i ‘ligestiaiy| Tes alo needed 1 fenayenes to identi re i ‘To determine the enzymatic activity, i vo ruminal Adogradation ofthe enzymes and in stm digestion of purified substrates, the following commercial come plexes were used: enzyme A (Fibrozyme, Alltech), ‘which contain enzymes from Aspe nigerand Tiek- xerma srde, enzyme B with cellulases and glucanases, and the enzyme C, that contains hemicelullases and cellulases (industrial hemicelulase, Enmes). Enzymatic activity ‘Thesubstratesused to valuate enzymatic activity were ‘xysallin cellulose pl 102 carboxymethilellulose (CMC)" and oatsglan” ln? wl ofbulfersolution pl ori 2unlof sodium phosphate bulfer (pH 6.5, 01 M) were dissolved 2% erysaline cellulose, 2% CMC, or 1% oatsylan, The enzymes were disoled in 2m of buffer soltion pH 5 and 63 at 2% and 1%, t0 obtain a relation enzyme-substrate v'¥ or weight. The preparation was incubated at 38°C for one hour: Enzymatic activity was determined by reducing sugars using dinitrosalieylie acd (RADSL);"* one 1 lof RADSL acid wasaclded to 2ml ofthe sample and incubated in water bath at 95°C by 15 m, a blank with ADSL. was alo included. Then tnbes were cooled with tap water and centrifuged (S00 g) for 20m, Anbydrate glucose was used for the standard curve; ilormede actividad enzimatica comeretates, los cuales no Material y métodos Para _determinar la actividad enzimetica, la ddegradacién ruminal i sit de las enzimas y la digest in sil de sustratos puros, se utlizron complejos enrimiticos comerciales: ensima A Gibrazyme, Allech), que contienen enzimas de Asporpilus niger y Tichoderma viride; encima B, que contiene celukasas y glucanasas; y"ensima C, que contiene hemicelulaas y celulasas (hemicelulasa de ‘uso industrial, Enmes) Actividad enzimatica Los susuatos utlzados para evaluar la actividad ewimatica fueron eelulost cristina, pH 102 carboximetl ccfulos” y xylan de avena"” En 2 ml de una solncién amortiguadora pHT 5.5 0 en 2 ml amortiguados en pH 6.5 ce sodo-osfato al 0.1M, se dlivolvi 2% de celulosa eristalina, 2% de CMC 0 1% para xylan, Las easimas se disoieron en 2 al de solucién amortiguadora pl 5.5 y 6.5 al 2% y 1% para obtener una relacién enzima-sustrato v/ 0 peso/s. La preparacién se incubé a 29°C por una hora. Teoquan SA oe iam. Meco Sento embaryo. csmg NsO11/100 allowing 10 vest for $0'm, Bovine feram albumin se used as standard at 780 nm te"cneyinatic activity was defined ae polypropylene which contained the same protein con centration for cach enzyme proict (25.8 ul), of the enzyme A (571% of protein, 152 ul of the commer ial product), B (827% of protein, 312 ul ofthe com- ‘mercial product) and C (6.7% of protein, 385 pl of commercial product.” The N-NHb values were cor- rected with blank (ruminal lid and artifical al cof McDougall). Incubation hours were 0 2, 4,6, 12, and 24 hours. Fermentation was stopped with IICI (50%) and the N-NHs concentration was determined by the McCullough method.” ‘A completely randomized block design was used," ting das of incubation (3 ays) asa criteria of block. with the Tukey tes." A simple regression analses La actividad enim: mediante rallies Droteina y se dejo reposar dur Tefrigerador. La muestra fe centrifgada por 10 min (O00) Hlsedimento se dgolvi en 2 lee NOH IN, yyee-agte con el vorex hasta disolver Ia mutes Voneriormente se tomaroin 0.2 nt de ba esta nina de bovine ‘de 7a) nn, Loe la por micromoles Degradacién in vitro de las enzimas incubar con microorganismos ruminales in vitro. El liquido ruminal se obtuvo de dos vaquillas Holstein {PV £00 kg), con canulas ruminales aimentadas con alfalfa El inéculo se extra con una bomba de vacio ‘equipada con filirs, dos horas antes de si el alimenio, y se deposit6 ent un tern previamente gascado con CO:, que se mantuvo a 39°C, La relacidn liquido ruminal y saliva artificial ‘de McDougall fue de 1:1 (15:15 ml), la incubacion se realia6 en tubos de 60 ml de polipropileno que contenian la misma concentracién de proteina para cat producto enzimatico (25:8 yl), de fa enzima A duc comercial), B Vit Mex, 36 (I) 20053Gri720.GH/rate of degradation), were 0-688 14 ‘mean value of the natal log of 2. assuming’ hat ‘of th legradation 2] and the rate of degradation is the slope lime and natural fog of N-NED. The slopes ‘ere compared with a confidence interval In vitro digestibility of purified substrates ith accli (360 mg). CMC (500 mag) and sola (O00 mg) using the same cnzymatic activity accoring to te substrate Samples were incubated at 30°C by 12 and 24 hours in Bem and 40 p por (PITS), maintaining a 11 ratio (15: were wasived and dried by 24h and weighted. Hy differences in weight, substrate dis- Sppearance wae dever mined Mca Blocking criteria the jon (days). The GUM procedure was uscd for ve analyses! and the Tukey text 10 compare A higher ensymatic activity (P < 0.05) was found at pil 65 dan at pl 5.5 (Lable 1), with the exception of dhe enzyme € when was incubated with crystalline cellulose and CMC. When crystalline cellulose was incubated, the major activity (P< 0.05), was with the enzyme A, in comparison to the enzymes Band C. In incubations with CMG, the enzyme A showed greater activity (P< 0.05), that the enzymes B and G (pH 6. and the enzyme C had the higher activity t pl 5.5 (Table 1) In the pt 6.5 solution, the enzymes A and Chua higher activity (P-© 0.05) om xan ham with enzyme B; however, when xylan was incubated at pHL 5.5, the activity (P< 0.05) was higher with enzyme A, than enzymes Band € (Table 1 In vitro degradation of the enzymes ‘There were no differences (P > 0.05) on ammonia nitrogen concentration among enzymes; and its con- centration (N-NHs) increased as incubation time was (S.27% de proveina, 312 yl del producto comercia) ¥€ (oi Se de proteina, 385 yl del producto Comercial)! Low valores de N-NHs fueron corregidos sedetuno con HCL ai 50% Ia concentracign N-NIs se determing por el metodo He MeCalongh. Se utilizd un disefio de bloques completamente ala lo come criteria te blewueo low dias Ge incubacion (tres dias). EI analisis de ¥ ol proc se compe Tukey" Serealiz6u deters fendimticos en ef medio ruminal, considerando una ‘inctiea de primer orden, estimando cl tiempo medio de degradacion (11/20.608/tara de degradacion), fasa de degradacion es la pendiente de la ecuacion de regret ef ventpo pel Lin del NN Tas" pendiontss se compararon con intervalos de Digestibilided in vitro de sustratos puros con adicién de enzimas Utiliando Ios valores de In actividad enrimstiea (Cuadeo 1 lcelilosa cristal 100 mg) ERIC: (500 mg) y xslan {G00 mg); mando. Ly mma actividad enzimtien de acuerdo con el sistrato, Las mitestras fueron incubadas a 84°C por 12 y 24 horas en bolsas de poliseda (3.5 x 8 em y 40 de diimewro de poro), {que se colocaron ttibos de 50 ml de polipropileno {que contenian liguido ruminal y saliva artificial de McDougall (pH 6.8), manteniendo una relaci6n 1:1 (15:15 m). Las bolsas fueron lavadas ysecadas por 2+ horas en estnfa (60°C) y pesadas. Por diferencia de peso se determiné la desaparicién del strato, Los resultados se analizaron con un diseiio ‘estadistico de bloques completamente al azar, usando ‘como eriterio de blogueo les dias de incubacion (dos dias), Se us6 el provedimiento GLM para el ani ‘de varianza' y la prueba de Tukey para comparar las medias." Resultados Actividad enzimatica Se encontrs mayor actividad encimitica (P < 0.05) en pil 65 que a pil 5.5 (Cuadro 1), excepto conENZYMATIC ACHIVETY jansl OF GLUCOSE EQUIVALENTS FORMED BY min PERE mg ptt mo of ucose utvatntfommed by sia por ss sae aa 13" os 35 wane sar paar 29 ESTIBILITY (4) OF THE ENZIMES AND PRUFIED SUBTSR ATES WITHTHE 2 inoe sare 199 bt azoe fso8 7 2 7837 yas oer 162 958 $708" 999" ast eos 2608 eae ase 24 7990 yaa 120 Rate of enzyme 2" 1os7* iss ose degradation C1) "= 05) Ma WE GTRSTONA TRG wn he Sas We AS RGIS GSTS ‘ihe personage of degradation wa ze. “Hal time of degradation of the enzyme Vet, Méx., 36 (1) 2005(bie 2) In vitro di ity of pure substrates with sudhtion ofensymes nvr digestibility of crystalline cellulose w (P= 003), with the enzyme C at 12 fey was higher (P2005) for # han enzsines A and B (Table 2). Discussion Enzymatic activity atic activity aries among commercial products peat ‘hat contain (= Found in the present research are similar to those described by Nrereko ef af with other commercial ‘cnymes (Depot 10, Ligticell 2300, Muitifectxylansce ‘and Sumizeme). Rosults presented in Table | indicate that enzymes ‘A.and B had lower activity at pH 5.5 with the three substrates and the enzyme C only with xylan, which ‘could be explained in the case of incubations ‘erytalline cellulose and CMC, to the fact of the ibrolytic enzymes had an optimum pll around neutrality; in that respect, Morgavi «ta pointed ou that the maximum activity with cellulases and xsla- fied by Krause al’ measuring the activity of eel lases (81 IU) and xylanases (4.4 TU). Inthe present surly, the enzymatic activity during the incubate ‘of CMC and xylan was affected at pil 5.5; however, the enzyme A had the major activity regarding to the ‘enzymes B and C with the three substrates. In rola- tionship to his, Morgavie found thatthe cel lases and xylanases were not affected by acid pH: it ‘onsima A, respecto de tae cenzimas By C (Cadre 1). Degradacién in vitro de las enzimas tayor (P00) para ‘para By © (Cuadro 2) con adicién de enzimas atom similares, pero la enzima A presents menor digestibilidad con respectoa la envima C (Cad 2), uando se incubd por 12 h el xylan, ladigestbildad fue mayor (P < 0.03) para la enzima C, seguida por fa enzima A y finalmente la envima B, a las 24h de subacién la digestiblidad fue mayor para la enzima Con respectoa las enzimas Ay B (P< 0.05) (Cuadro 2). Discusion Actividad enzimatica La actividad ensimitica entre productos comerciales varia de acuerdo con el tipo de enrima (xilanasas, Cchulasas, hemicelulasas te.) que contengan,~el pl del medio y el tipo de sustrato iilizado, Los valores cencontrados en el presente trabajo son similares a los que describen Nsereko et al.” con otras enzimasnas been reported that enzymes from fungal « lave opttnal conditions i lene pH and elevated ter peratures (Rode A af)” which ay explain why he ‘nated septum p flv bacteria 'Nerescko eat© observed that the enzymes produced by the organisms, it has been reported that during purification of xylanase from Iipex laceus (Polyponss tulpiferae), duree wypes of xyla- tases were obtained, from which two did not hydro Iyze xylan. Celluloftic enzymes from enzyme A had a greater activity chan enzymes Band C when were incubated at acid of neutral pI In vitro degradation of the enzymes ‘The N-NHs concentration was constant up to 12 h incubation, increasing a 24 of incubation. Simkar results reported Pinon oa! in a in ni tly, whet incubated Fibroryme by 12 h however, at 4b incu bation, the N-NHE concentration remained constant ‘This difference can be related to dierent factors lke ‘Pigwea 1: Dipamice de desradacion ruminal tro ESSE Spins snsmatsos ami of re ruminal degradation of thee comerciales (Depot 40, Liguicell 2500, Mateifect ‘Sslanse y Sumieye). fas enemas Ay B. " fend por Keatie ef nee strinaa ta seve ste selene CLOW wits GEA Ul). Biel presente trabajo, ta acivdad Snsimatica. al inewbar de CMC y sana pit nary, Ia envio A ine aor io. anterior, Monga t anterior puiera explicar por qué la enzima C wo fue afectada en pH 5.5 cirando se incubé celuloss cristalina y CMC, ya que el pH 6ptimo para bacterias ccelulotitieas es de 6.5." Neeteko eta” observaron que la actividad en ‘ica varia mucho entre productos enzimaticos, aunque se originen de sin mismo microorganism, lo cial puede deberse a las enzimas que contenga el producto Y¥ su habilidad para ajustarse al sitio activo de cada sustrato; los resultados cuando se incubé celulosa cristalina y CMC en pl¥ 6.5, fueron que Ia enrima A ttuvo mayor actividad con respecto a las enzimas By c Por otra parte, los resultados de bx incubscion del xylan indican que las enrimas Ay C contienen nayor cantidad de xilosidasa y xikanasa que la en B Se han deserito valores menores en la actividad Vek Mis, 36(1) 20057inal uid, level of enzyme ‘and variation anong inoculum donors? The rate of Slegradation of the ‘oat treater lor the enzyme C wih respect ta the enzyme A; regarding that, has been observed that the s0- ‘of exogenous enzymes in Fuminal environment Siyntalline ctlalowe: In vitro digestibility of pure substrates with addition of enzymes Sitienences im aigesbaiey Inajor cellulelysie netivity by symergiam with vaminal oe. (Table 2) can be due toa by synergism differences in digestibili jor cellulolyie act microbes. I has been reported that im ail incubations of CMC with ruminal fuid and purified cellulose, the digestion of CMC is increased." Xylan digestibility ‘was major for the enzyme C at 12 and 24 h incuba- tion, even when both have the same xylanase activity than che enzyme A, in presence of ruminal microbes their performance results different. ‘On the basis ofthe evaluation of exogenous fibro- Itic enzymes in the present study, stand out that the results of the enzymatic using purified sub- strates do not necessarily corresponds to a major in vino digestibility in presence of ruminal microbes, which can be explained by the differences on degra- dation rate beween the enzymes and by the syner- «gism with ruminal microbes among them. ca de ta xilanana pairs (90.5 4 198 UL). sc puede explicar porguc las enzi ‘varias ailanasas:y ‘Cnganinemon distinte "xiaton diferencias en las enzimas producidas por foe ‘organismos, se ha informacdo que al purifiar xilanasas Cire taceus (Pobporus tubferae) ee obsanieton toe pow de silanasan, de las cuales dar no hideokeabsa ras de In oni we as enzimae Ih y aplt nett ci Degradacién in vitro de las enzimas respec te tase Sin embargo, en ‘el presente estudio. el inset buiwo ster de la alimentacion, pero cl ewe que permanecieron las enrimas con Tos. sustratos Tue suficiente para observar altas digestibilidades a cheepeion de Ir ensima Iveuando se sncubo celulost excepcién de Ta enzima B cuando se incubé celulosa cristina Digestibilidad in vitro de sustratos puros con adicién de enzimas Morgavi etal observaron disminucién en la actividad. de ensimas fibroliticas comerciales, obtenidas de “Trichoderma ongibrachatum, a lash de incubacién con liguido ruminal. Las enzimas A y C incrementaron la digestibitidad de la celulosa crisalina de 12 2 24 h incubacidn, lo cual puede atrbuirse a mayor actividad celuloltica y esabiidad de las ensimas. AL respecto, Pinos et al.® encontraron que enzimas fibrolitcas exigenas glucosiladas tienen efecis posiivos en la degradacion in stro de componentes de la pared celular, pero en el caso de la enzima B no prese actividad celullitica sobre la celulosa cristalina, queReferencias 1 Krause M, Beauchemin KA, Rede IM, Faer Bl, Norgaard P. Bbrobte enzyme (eatment of basley srain and source of forage in highvgrain diets fed rowing cate. J Anion Ses 1908;70:29122020, 2 Morgasi PD, Beauchemin AK, Neereko IV, Rode ML, Meallster AT, lwaasa DA, of af. Resistance of feed enzymes to proteolytic inacivation by rumen smieroorganiune ane yatrointentinal proteases JAP Sei 2001 :70-1691-4680, 3. Wallace JR, Wallace AIS, McKain N, Neereko 1Y, Haarinell FG. Influence of supplementary fibrotic nye on the fermentation of conn sd gras slags by mixed ruminal microorganisms in vit, Anim Sci 2001;79:1905-1916. 4. Konda T, Amano ¥, Nisizawa K. Purification and opertien of two endori-e 7 xylanases fen Ieper actus (Patporus tulip). J Biochess 198508. 1545- S54, sereko LY, Monsasi PD, Rode ML, Beauelemin AK. MeAlliter AT, Effects of fungal enzyine preparations fom hyutrobis and subsequent degradation of alfalfa Ina fiber by mined rumen inctoonganians éeitm, ‘Ania Feed So Tels 2000881584170. {8 Officer DI. Feed Enzymes, In: Farm Animal Metabolism © Dn851903788/3788eh19 pa 2000-148, Summer JB. Diniuosalicsic acid: a reagent for the estimation of sgar in normal and diahetic urine. J Biol Chea 1021:47.7.8, & Miller GL, Bhim R, Glennon WE, Burton AL Meanutements of carhoximethycelllawe atvity Aral Biochem 1960,9:197 ©. Lowey OH, Rowebroug NJ, Farr AL, Randall Rf Protein measurement with the Fin phen! reagent. puede debersea sm aja actividad ceoitica (Cadro 1). La enzima € presents la mayor a digesbiidad st las 12 h com respecte a las enzimas A y B. Sin cembargo, 4 las 21 h de incubacion la digestibilidad de las enimas C y B fue similar y mayor a fa de la fenzima C com respecto ala enim A. La diferencias en las digesilidades (Cuadro 2) pueden deberse a tena mayor actividad celulalitiea por sinergimno com Jos miroorganismos ruminal. Se ha notificado que al incuhar in sii CMC con liquido ruminal y celilosa pura se incrementa Ia digestibilidad de la CMC." La digestibilidad del alan fue mayor para la enema Ca las 12 y 24 th de incubacish, que aunque Genen wisn actividad silanssiea que Is enrima A, en presencia fe microorganismos rminalcs st compartamiento resulta diferente Conbase en evahiacin delasenzimasfibroiticas cexigenas en el presente esnidio, se destaca que los resultadosde a aetvidad enimtica uibizando susta- {os pros no necesatiamente corresponden 4 mayor digestibilidad in efroen presenciade microorganismos ruminales, fo cual puede deberse a la diferencia en I tasa de degradacin entre las enzimas y por el Snergano cot los mieroorganismonruminales para 1H Morgavi PD, Beauchemin AK, Nsereko LY, Rode ML, Twaasa DA, Yang ZW, oat Spnergy between ruminal ibrolytic csneymes and eneymnes frown Tichderna Langiracinnn, J Dairy Si 2000;881310-1321 15.Rode LM, Yang WZ, Beauchemin KA. Fibrolytic ‘enzyme supplements for dairy coms in cary lactation. [Dairy Si 1900;92:2121-2126. 16.Pinos RJM, Mendoza GD, Biscena G, Cobos P Efecto pared celular de heno de alfalfa (Medien sativa) 0 de