Isolation of Saccharomyces Spp from Manure of Beef Bali Cattle as a Probiotics Properties and has CMC-ase Activity to Improve Nurient Quality of Rice Bran.

I solation of Saccharomyces Spp

from Manure of Beef Bali Cattle as a

Probiotics Properties and has

CMC-ase Activity to I mprove Nurient

Quality of Rice Bran

By

Desak Putu M as Ari Candraw ati,

D.A. W armadew i, and Igng Bidura

I SSN 0970- 4973 ( Print)

I SSN 2319- 3077 ( Online/ Electronic)

J. Biol. Chem. Research

Volume 31 ( 1) 2 014 Pages No. 39 - 52

Journal of

Biological and

Chemical Research

( An I nternational Journal of Life Sciences and Chemist ry)

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I SSN 09 70- 4973 ( Print)

I SSN 23 19- 3077 ( Online/ Electronic)

Desak Putu Mas Ari Candraw ati I gng Bidura

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RESEARCH PAPER Received: 09/ 11/ 2013 Revised: 20/ 12/ 2013 Accepted: 03/ 01/ 2014

I solat ion of Saccharomyces Spp from Manure of

Beef Bali Catt le as a Probiot ics Propert ies and

has CMC- ase Act ivity to I mprove Nurient Qualit y

of Rice Bran

Desak Putu M as Ari Candraw ati, D.A. W armadew i, and Igng Bidura

Faculty of Animal husbandry, Udayana University, Denpasar-Bali, Jl. PB. Soedirman, Denpasar, Indonesia

ABSTRACT

This experiment w as carried out to st udy both on the CM C-ase activity and probiotics agent of Saccharomyces spp isolation from manure of beef Bali cattle samples can be used in order to alleviate the negative effect of rice bran as feed. Eighteen of Lohma n Brow n laying hens w as assigned to three treatments in a completely randomized design. Each treatment has six replications with one bird per replication (individual cage). The treatments w ere (i) unferment ed rice bran as control; (ii) fermented rice bran by Saccharomyces spp.S-6 isolate; (iii) fermented rice bran by Saccharom yces spp.S-7 isolatse, respectively. The report on the first experiment show ed that five isolates of Saccharomyces spp (Sc.S-5; S-6; S-7; S-8; and S-9) w ere isolated from manure of beef Bali cattle samples in the first experiment. The w hole isolates of Saccharomyces spp show ed resistant grew on both in different temperature (100-450C), acid conditions (2,5-4,5), and bile salt. But only tw o isolates (Sc.S-6 and S-7) w ere pot ensial as probiotics sources and has CM C-ase activity. The study show ed that fermentation of rice bran using both of Saccharomyces spp.S-6 and S-7 culture could improve significant differences (P<0.05) on digestibility of its dry matter (DM ), organic matter (OM ), crude protein (CP), crude fibre (CF), and increased its metabolizable energy of rice bran. The content of dry matter, organic mat ter, and gross energy of rice bran w ere no effected significantly different (P>0.05) by ferment ation. On the other hand, fermentation caused increasing crude protein (CP) and crude fibre (CF) of the rice bran. It w as concluded that tw o isolates (Sc.S-6 and S-7) w ere isolated from manure of beef Bali cattle samples, both w ere the potensial as a probiotics sources and has CM C-ase activity and its utilization in the rice bran fermentation could increa se nutrient composition and digestibilit y of rice bran.

Key w ords: Saccharomyces Spp., Probiotics, Crude Fiber, Digestibility, and Rice Bran.

INTRODUCTION

During recent years, numerous st udies have been undert aken t o obt ain scient ific evidences for beneficial effect of Saccharomyces spp as prom ising probiotic. Such beneficial effect s are considered t o include t he prot ect ion from pat hogen, enhancem ent of the im mune syst em, antimut agenic and ant icarinogenic effect s and the reduct ion of serum cholest erol. Chen et al. (2005) report ed t hat diet ary supplem ent at ion of com plex probiotic slight ly improved digest ibilit y of nut rient s. The use of Saccharomyces cerivisiae (Sc.) cult ure as a probiot ics sourches in poult ry product ion as becom e an area of great interest , because cont inued use of probiot ics in anim al feeds m ay result in t he presence of antibiotics residues in anim al product s (Han et al., 2001). Gut microfloral enzymes are beneficial to t he nut rition of t he host because t hey increase t he digest ion of nut rient s, esp ecially in t he low er int est ine. Previous experim ent s show ed that t he inclusion of microorganisms in the diet s improved feed conversion efficiency and digest ibilit y (Chen et al., 2005). Piao et al. (l999) show ed that 0, 10% yeast added to a diet could reduce anim al w ast es. Yeast cult ure improved posphorus ut ilizat ion in growing chickens. Inconsist ent report s about the effect of probiotics m ay be due t o several aspect s such as st rains of bact eria, dose l evel, diet com posit ion, feeding st rat egy, feed form, and int eract ion w ith other diet ary feed addit ives (Chesson, 1994). Rice bran is a by-product of rice m illing indust ry, w hich is abundantly available (approxim at ely 10% of paddy w eight ) during t he harvest ing season. Unfort unat ely this by-product cont ains t oxic factors such as t rypsin inhibitor, phyt ic acid as phyt at e, and high cont ent of crude fiber. These ant i-nut ritive fact ors have been report ed by Kahlique et al. (2003) causing reduct ion of feed int ake and depress performance of poult ry. These t oxic fact ors are phyt ic acid as phyt at e and crude fiber (CF). These ant i-nut ritive fact ors have been report ed t o reduce feed int ake and depress performance of poult ry. Bach Knudsen (2001) report ed t hat CF has been defined as t he com plex macrom oleculer subst ances in food plant s that are not degraded by mam malian digest ive enzym es. Wit h the except ion of lignin, all of the m at erials called CF are carbohydrat es in nature. CF is thought t o mediat e prot ect ive effect s on t he colonic epit helium t hrough t heir ferm ent at ion product s and fecal bulking capacit y (Wang et al., 2004). Feeding high fiber result ed in a low ered rat e of lipogenesis and t endency of an increased capacit y to ut ilize acet yl-CoA in pigs (Zhu et al., 2003). Non st arch polysaccharides (NSP) are t he carbohydrat e component s of CF and are t he predominant subst rat es for anaerobic ferm ent at ion. The potency of rice bran as energy source for poult ry depends on it s cell w all cont ent, degree of microbial ferm ent at ion in poult ry large intest ine, absorption and production of t he volat ile fat t y acid (Wang et al., 2004). The pot ensial of rice bran as energy sources for poultry depends considerably on such fact ors as cell wall cont ent, degree of m icrobial ferm ent at ion in t he large int est ine, and ext ent of absorption and utilizat ion of the volat ile acids produced (Kahlique et al., 2003). The use of rice bran in poult ry diet s has been highly limit ed by t he presence of phyt ic acid and ot her minor ant i-nut rit ional factors like t annin and non-st arch polysaccharides (NSP). The high levels of NSP in rice bran are lim iting it s unrest rict ed use in poult ry feeding.

These NSPs are know n t o increase t he gut viscosit y, reduce nut rient absorpt ion in the intest ine and affect ed indirect ly t he growt h and perform ance of birds (Rhames et al. 2006; Cao et al., 2003). M any st udies have clearly dem onst rat ed t hat , supplem ented of probiotics t o diet s rich in NSP result s in a significant reduction in t he int est inal viscosit y, enhances energy, and prot ein ut ilization (Bidura et al., 2012; Bidura et al., 2009).

Probiot ics are defined as the viable microorganisms t hat exhibit a beneficial effect on healt h of the host by improving it s int est inal microbial balance. Yeast culture is one of t he most ext ensively st udied probiotic. Tradit ionally, S.cerevesiae have been used for food product s such as baking indust ries, “ t ape” , and are considered as organisms t hat can be used also for a pharmaceut ical (Ahmad, 2005). The use of antibiot ics as routine feed additives has been baned in sam e count ries because of public concern over possible antibiot ics residual effect s and the developm ent of drug-resistent bact eria. Probiotics have been int roduced as an alt ernative t o ant ibiot ics; how ever, t heir effect s on poult ry product ion are not consist ent , result ing in uncert aint ies and skept icism for developm ent of the product s. There are many t ypes of probiot ic preparat ions in the market . M any st udies have been conducted to t est t he afficacy of such preparat ions on anim al growt h and performance. Several st udies w ith broiler have indicat ed that probiotics preparat ions im prove live w eight gain and feed conversion rat e (Bidura et al., 2012; 2009; 2008; Sut arpa et al., 2011). Hong et al. (2004) report ed t hat ferment at ion of feed using Aspergilus oryzae increased digest ibilit y of it s DM and CP.

Therefore, it is susgest ed t hat ferm ent ed of rice bran feeding by Saccharomyces spp isolat es as a CM C-ase akct ivit y (isolat ion from manure of beef Bali cat tle) can be used in order t o alleviat e t he negative effect of rice bran as feed.

M ETERIAL AND M ETHODS

Animals and experimental design

Eight een of Lohman Brown laying hens w as assigned t o three t reat m ent s in a complet ely randomized design. Each t reat ment has six replications w ith one bird per replicat ion (individual cage). All of t he birds were fed experim ent al diet s for t w o days. The t reat m ent s w ere (i) unferm ent ed rice bran as control; (ii) ferment ed rice bran by Saccharomyces spp.S-6 isolat e; (iii) ferm ent ed rice bran by Saccharomyces spp.S-7 isolat e; respect ively. The object ives of t his st udy are t o det ermine t he nut rient digest ibilit y and t he M E value of rice bran using Lohm an Brownn laying hens at 42 w eeks of age.

Probiotics properties

Acid tolerance: to det erm ine t he t rancsit t olerance t o low pH, the m et hod of Corzo and

Gilliland (1999) was used with slight m odificat ions. St rain was grow n in M RS brot h at 370C

for 24 h. A 0.5 ml aliquot of t he bact erial culture w as inoculat ed in 10 m l of phosphat e buffered saline adjust ed t o pH 2 with 4 N HCl. Phosphat e buffer w as prepared by

dissolving NaCl (9 g/ l), Na2HPO42H2O (9 g/ l), and KH2PO4 (1.5 g/ l) in dest iled w at er. The

initial bact erial concent ration w as 106-8 cfu/ m l. Cult ure w as incubat ed at 370C. Aft er 0;

0.5; 1; 2; and 4 h incubation, cell w ere serially dilut ed t enfold in 0.1 M sodium phosphat e

buffer (pH 7.2), and t he viable cells w ere enumerat ed on M RS agar plat e at 370C for 2

days.

Bile resistance: The resist ance t o bile w as exam ined according to m et hod of Hyronim us et al. (2000). Each st rain w as inoculat ed in M RS brot h w ith 0.5 or 1% (w/ v) bile salt

(Difco). Cultures w ere incubat ed at 370C for 24 h, and t he numbers of viable cell w ere

det erm ined and compared t o a cont rol (w it hout bile salt ) on M RS agar plat es.

CM C-a se activity: W eighed as m uch as 11 g of OM EA and 3 g of CM C-ase, t hen dissolved in distilled w at er. Heat in a w at erbat h and aft er it did st relilisasi on autoklav. Refrigerat e

at a t em perat ure of 45-50 0C, t hen poured on Pet ri dishes and left t o solidify. Isolat es t hat

have been cultured in a nut rient brot h for 24 hours. Take a paper disk wit h t w eezers and t hen dipped in a solution of nut rient broth t hen put on a pet ri dish cont aining Om ea media and CM C-ase. Leave for 24 hours. Aft er 24 hours of clear zone w idth measurem ent s are gen erat ed by using a vernier caliper. The size of t he clear zone and the apparent absence of clear zone, an indicator of the abilit y of the microbes t o break down cellulose, as w ell as fast and slow arise t he clear zone (VanDevoorde and Verst raet e, 1987; Kanti, 2007).

Fermented of Rice Bran

The isolat e of Saccharomyces spp.S-6 and S-7 w hich has been approved from bile salt and poult ry digest ive t ract in vit ro t est could assim ilat e cholest erol for probiotics agency and t w o isolat es have CM C-ase act ivit y. The st udy w as carried out at t he Bioscience Laboratory of Udayana Universit y, Bali, Indonesia. Ferm ent at ion of com mercial rice bran

w as prepared as follow s. Comercial rice bran w as used. Approxim at ely 0.20% (2 x 106

spores) Saccharomyces spp. S-6 and S-7 isolat e cult ure w as added to 100 g of st eam ed rice bran. Then, w ater w as added t o bring t he product t o 50% cont ent and left up t o 2

days for ferm ent at ion. Aft er t hat , ferment ed rice bran was dried at 450C for six hours and

t hen it was ground for analysis. Unferm ent ed rice bran w as also ground for it s chem ical analysis.

Retention and excretion of nutrients

In order t o det ermine t he nut rient digest ibilit y and met abolizable energy (M E) value of t he rice bran. The amount of rice bran used w as 50 g. This amount as based on preliminary assays w it h Lohman brow n laying hens using rice bran. All the birds w ere deprived of feed for 24 h t o ensure t hat their aliment ary canals w ere empt y from feed residues. They w er e t hen force-fed w it h the specific amount of rice bran (ferment ed and unferment ed). St ainless st eel

funnel w it h 40 cm st em was used in force f eeding t echnique (M ust afa et al., 2004). Wat er w as

available ad libit um during t he experiment al period. The t ot al excr et a w ere collect ed in

plast ic t rays. The excret a samples w ere frozen, allow ed coming to equilibrium w it h the at mospheric moisture, w eighed, and groun through a 1 mm sieve. Samples of excret a and rice bran w ere subject ed t o appropriat e analysis t o det ermine DM , OM , CP, CF, and energy, respect ively.

Laboratory analyses

Dry mat t er (DM ), organic matt er (OM ), CP and ash det erm inat ions w ere done according t o the Assocciat ion of Official Analyt ical Chem ist s (l994). The CP cont ent of t he diet s w as det erm ined using t he Kjeldahl procedure (AOAC, 1994). Crude fibre in t he feeds w as det erm ined using t he procedure of Van Soest et al. (l991) on oven-dried samples. Gross energy (GE) w as m easured wit h an adiabat ic oxygen bomb calorim et er (Parr, USA),

Calculations

The data w ere used t o calculat e AM E value according to t he follow ing form ulat e (M ust afa et al., 2004): AM E (apparent m et abolizable energy) = IE – FE. Where IE = ingest ed energy; FE = fecal energy voided by the fed birds.

St atistical Analysis

All dat a w ere subject ed t o a one-w ay analysis of variance t est (St eel and Torrie, 1989). St at ist ical significances among t reat m ent means w ere det erm ined by m et hod of New M ult iple Range Test of Duncan when t he F value w as significant at 5% level

RESULTS

Resilience isolat e Saccharomyces spp. t he low pH is one of t he charact erist ics required t o be met by a candidat e or a probiot ic that could be developed into a pot ential probiotic. On t his t est , t he m edium pH w as adjust ed t o 1.5, 3.0, 4.5, and 6.0 by using HCl (hydrochloric acid), because HCl has charact erist ics similar t o stom ach acid. Resist ant to highly acidic nature needs t o be ow ned by t he candidat e probiotic, because t he applicat ion lat er, t his probiot ic candidat e must pass very acidic st omach conditions, before reaching t he colon.

Observat ions as present ed in Table 1. In this st udy, as m any as 10 isolat es Saccharomyces spp. isolat ed from catt le feces w as t est ed resist ance t o various pH, at pH 1.5, 3.0, 4.5, and 6. Two isolat es w ere unable t o grow at pH 1.5, w hich isolates t he S-1 and S-2. Saccharomyces spp.S-1 isolat es even sim ply could not grow at all pH t reat m ents given. Only 8 isolat es w ere able t o grow w ell, nam ely Saccharomyces spp.S-4 t o S-10 isolat es. There is a t endency of t he higher pH, especially at pH 6, most of t he isolat es has decreased t he number of colony life. Saccharomyces spp colonies grow w ell at pH 3, even isolat e S-3 and S-5 Saccharomyces spp colony number reached 5.531 and 5.093 log colony per gram .

Table 1. The number of colony isolates Sa ccharomyces spp (Log colony/ g) at various pH.

Code Isolat e pH

1.5 3 4.5 6

S1 - - - -

S2 - 3.98 3.88 3.32

S3 3.45 5.53 4.41 4.13

S4 4.33 3.65 3.58 4.56

S5 4.12 5.09 4.99 4.12

S6 4.19 4.35 4.36 4.40

S7 4.29 4.42 3.99 3.58

S8 4.06 3.617 4.318 3.86

S9 4.31 4.41 3.95 3.92

S10 4.16 4.33 3.97 3.87

Descript ions: S-1 s / d S-10 is a Saccharomyces spp isolat es w ere isolat ed from manure of beef Bali cat tle

In the t est of resist ance t o bile salt s as seen in Table 2 show s, that the t en isolat es w ere able to survive and grow on a medium cont aining bile salt s at concent rat ions of 0.2 m M and 0.4 m M , whereas at a concent rat ion of 0.6 m M no isolat es can survive. M icrobial isolat es resist ance t o bile salt s is used t o assess t he abilit y t o survive t he digest ive t ract isolat es cont ained bile salt s on the surface of t he int est ine.

Probiot ics w ill be dealing w ith the environment in t he sm all int est ine, in which t here are bile or bile salt s are rem oved by t he liver t hrough t he gall bladder, aft er successfully passing the acidic condit ions in the st om ach. Therefore, in t he process of developing new probiotic, or a new probiotic candidat e m ust be able t o pass t he t est of resist ance t o bile or bile salt s w ere perform ed in vit ro. Based on t he nat ure of t he resist ance show n by som e isolat es, st rai-indicat es t hat t he st rain has t he pot ential to be developed int o a pot ent ial probiot ic.

Table 2. Ability of yeast Sa ccharomyces spp isolates w ere resistant living in bile salts.

Isolat e Bile salt concent rat ion (absorbance)

0.2 mM 0.4 mM 0.6 mM

S1 + (0.244) + (0.169) - (0.067)

S2 + (0.248) + (0.168) - (0.059)

S3 + (0.253) + (0.163) - (0.081)

S4 + (0.252) + (0.158) - (0.064)

S5 + (0.243) + (0.166) - (0.073)

S6 + (0.224) + (0.144) - (0.036)

S7 + (0.192) + (0.133) - (0.031)

S8 + (0.145) + (0.151) - (0.051)

S9 + (0.126) + (0.132) - (0.044)

S10 + (0.138) + (0.149) - (0.035)

Descript ion:

- : A <0.1 (not bear bile salt )

+ : A 0.1 - 0.5 (a bile salt -resist ant )

+ + : A 0.5 - 1.0 (bile salt -resist ant )

+ + + : A> 1.0 (highly resist ant of bile salt s)

S-1 s / d S-10 is a Saccharomyces spp isolat es were isolat ed from m anure of beef Bali cat tle

CM C-ase t est is t o t est t he abilit y of t he yeast Saccharomyces spp isolat es in degrading crude fiber. It can be m easured from t he result ing clear zone diam et er (Table 3). From t he result s of t his st udy w ere only yeast Saccharomyces spp isolat es S-5, S-6, S-7, S-8, and the yeast Saccharomyces spp.S-9 who has the abilit y t o digest fiber. Looks yeast Saccharomyces spp isolat es S-6 has the most wide clear zone, while the S-8 has a clear zone at least .

This m eans t hat isolat es 6 has kem am puam in digest ing crude fiber t han the highest S-8. M ore det ailed, clear zone diamet er and difference generat ed by t he t w o isolat es are presented in Figure 1.

Table 4 show s the nut rient the cont ent of crude prot ein and gross energy w ere slight ly increased by ferm ent at ion. On the ot her hand ferm ent at ion caused decreasing dry mat t er (DM ), organic M at t er (OM ), and crude fibre (CF) of the rice bran. These result s indicat ed t hat carbohydrat e w as used for m icrobial growt h and the reduct ion of dry mat t er. The result indicat ed that all of nut rient digest ibilit y of ferm ent ed rice bran by Saccharomyces spp culture w ere increased significantly (P<0,05) different rat her t han unferm ent ed rice bran.

Figure 1. Saccha romyces spp.S-8 isolates zone is smaller than the isolate Saccharomyces

spp.S-6 (right).

Table 3. CM C-ase activity test yeast Saccha romyces spp isolates based on the diameter of the clear zone caused.

Isolat Clear zone diam et er (cm )

S-1 -

S-2 -

S-3 -

S-4 -

S-5 3.60

S-6 4.51

S-7 4.00

S-8 1.85

S-9 3.80

S-10 -

Descript ion: S-1 s / d S-10 is a Saccharomyces spp isolat es w ere isolat ed from m anure of beef Bali cat tle.

J. Biol. Chem. Research. 45 Vol. 3 1, No. 1: 39-5 2 ( 2014 ) Isolate S8

Isolate S6

Table 4 show s t he nut rient digest ibility and m etabolizable energy of unferm ent ed rice bran (UFR) and ferm ent ed rice bran (FR) ingredient . The digest ibilit y of crude prot ein, organic m at t er, and crude fiber, were slight ly increased significant ly different (P<0,05) by t he ferm ent at ion, bot h by Saccharomyces spp.S-6 and S-7, respectively. The met abolizable energy of FR w as increased significantly different (P<005) than met abolizable energy of UFR. Ferm ent ed of feeding ingredient w ere caused increased of crude prot ein, organic m at t er, and crude fibre digest ibilities, resvect ively t han UFR ingredient s.

Chem ical com posit ion and nut rient digest ibilit y of rice bran (ferm ent ed compared t o t he unferm ent ed) w ere show n in Table 4 as below:

Table 4. Chemical composition and nutrient digestibility of unfermented and fermented rice bran by Saccha romyces Spp.S-6 and S-7 isolates culture (in % Dry M atter).

Rice Bran

Paramet ers

Unfer-ment ed (UFR)

Ferm ent ed by S-6 Isolat e (FR)

Ferm ent e d by S-7 Isolat e

SEM

Chemical composit ion:

Dry M at t er (%) 89.75a 89.64a 89.62a 0.453

Organic M att er (%) 90.93a 90.87a 90.22a 0.318

Crude Prot ein (%) 11.88b 14.69a 14.78a 0.149

Crude Fibre (%) 15.96b 17.65a 18.19a 0.285

Gross Energy (Kcal/ kg) 3258.31a 3272.22a 3274.97a 44.528

Nut rient digest ibilit y (%):

Dry mat t er digest ibilit y (%) 25.48c 28.24b 29.81a 0.185

Organic M at t er digest ibilit y (%) 26.16b 30.07a 30.25a 0.314

Crude Prot ein digest ibilit y (%) 37.38b 51.77a 53.23a 1.106

Crude fibre digest ibilit y (%) 15.92c 24.10b 26.29a 0.252

M et abolizable energy (Kcal/ kg) 1696.38b 1898.44a 1940.69a 19.512

Not e:



UFR = Unferm ent ed rice bran; FR = Ferm ent ed rice brang by 0.20% yeast cult ure



The different superscript at the sam e row is significantly different (P<0.05)

DISCUSSION

The result s show ed t hat only 5 isolat es of Saccharomyces spp pot ent ial as probiotic agent s and have t he CM C-ase act ivit y. To five isolat es show ed resist ance t o acid and bile salt s t hat is charact erist ic of probiot ic bacteria. Saccharomyces is a unicellular

microorganisms lacking chlorophyll, grew well at 30oC and pH 4.8. pH value in t he

gast roint est inal t ract of poult ry in each sect ion, are: crop (4.5), provent riculus (4.4),

gizzard (2.6), duodenum (5.7-6.0), jejunum (5.8), ileum (6.3), colon (6.3), and ceca (5.7) (Hidayat , 2010). Som e advant ages of Saccharomyces ferm ent at ion process, t hese microorganisms are rapidly proliferat ing, resist ant to high alcohol cont ent , resist ant t o high t em perat ures, has held st eady and rapid nature of adapt at ion. According t o Ahm ad

(2005), t emperat ure opt imum environm ent for yeast grow t h is 25-300C and a m axim um

t em perat ure of 35-470C. Som e advant ages Saccharomyces microorganisms in the

ferm ent at ion process t hat is rapidly proliferat ing, resist ant t o high alcohol cont ent , resist ant to high t emperat ures, has held st eady and rapid nature of the adaptat ion. CM C-ase enzym e act ivit y (endo-1,4-bet a-glukonase) is a clear zone around the colony indicat ed that the m icrobes have a st rong ext racellular cellulase enzyme activit y. The size of t he clear zone and the apparent absence of clear zone, an indicator of the abilit y of the microbes t o break down cellulose, as w ell as fast and slow arise t he clear zone (Van Devoorde and Verstraet e, 1987). Ut ama et al. (2006) report ed t hat t he yeast Saccharomyces cerevisiae is yeast capable of producing t he enzym es am ylase and selulolase, so as t o increase t he digest ibilit y of protein and crude fiber such as cellulose and hemicellulose, as has been overhauled in t he form of a sim ple monosaccharide. Cellulolyt ic yeast s capable of producing t he enzyme 1,4 bet endo-glukonase, 1,4 bet a-exo glukonase, and bet a-glucosidase t hat can degrade com ponent s of crude fiber into soluble carbohydrat es (How ard et al., 2003) According Ut am a et al. (2006), ferm ent at ion feed purpose is t o improve t he usabilit y of feed and eliminat ion of ant i- nut rit ional subst ances, as well as ut ilize biom ass form ed. Rice bran ferm ent ed w ith yeast w ill be able t o increase t he m icrobial biomass, so t hat the crude prot ein cont ent of rice bran increased. Also report ed that the ferm ent at ion process and the product is affect ed by t he t ype and number of microbes, subst rat t ypes, pH, and t em perat ure during t he ferm ent at ion process. Biom ass is a form of mass from t he biological processes of microorganisms. M icroorganisms capable of convert ing mat erial int o prot eins. The ferm ent at ion process has the object ive t o produce a product (m at erial feed) t hat have a nut rient cont ent, t ext ure, bet t er biological availabilit y, and reduce subst ance antinut risi. Suparjo et al. (2003) st at ed that t he ferm ent at ion of rice bran with 0.2% Aspergillus niger cultured for 72 hours can m arkedly increase prot ein and phosphorus cont ent of rice bran, on the cont rary low er crude fiber cont ent and acid phyt at rice bran.

According Sumarsih et al. (2007), the longer t he st orage t im e of feed ingredient s w hen ferm ent ed by Trichoderma viride, t he m ore t he loss of dry mat t er and organic m att er mat erial, inst ead increasing t he crude prot ein cont ent and m aterials decreased crude fiber cont ent m at erial. Also report ed that the longer t he ferment at ion process or mat erials st orage t ime, t he increased dry m at t er digest ibilit y and organic m att er mat erials.

According t o Jaelani et al. (2008), the increase in M E cont ent of palm kernel cake (palm kernel cake/ m eal) as a result of ferm ent at ion by the fungus T. reesei of 1824.13 kcal/ kg t o 1930.44 kcal/ kg suspect ed because of t he degradat ion of m annan polysaccharides exist in palm kernel by fungus T. reesei into simpler form s (m onosaccharides) t hat produces enough energy value bet t er t han in the form of polysaccharides mannan.

The sam e t hing w as report ed also by Sabini et al. (2000) which st at es t hat t he fungus T. reesei is able t o degrade a polysaccharide mannan mannot riosa, m annobiosa, and monnosa. According to Jaelani et al. (2008), ferment ed palm kernel cake can m arkedly increase t he crude prot ein cont ent compared to palm kernel cake w it hout ferm ent at ion. Table 3 show s t he chem ical composit ion of unferm ent ed rice bran (UFR) and ferm ent ed rice bran (FR) ingredient . The digest ibilit y both of crude prot ein, organic m at t er, crude fibre, and met abolizable energy w ere slight ly increased by S.cerevisiae ferm ent at ion. S. cerevisiae can not effect on crude fiber digest ibility of rice bran. Becouse, among t he cell w all polysaccharides of rice bran known as nonst arch polysaccharides (NSP) are celluloses, pect ins, and oligosaccharides. NSPs can not be degraded enzymit ically in t he digest ive syst em s of t he birds due t o the lacking of enzymes degrading the NSPs in t heir digest ive syst ems (Choct , 2002). The digest ibilit y of crude prot ein, crude fiber, and M E w ere slight ly increased by t he ferm ent at ion. These result s indicat ed that carbohydrat es other t han fibres w ere used for m icrobial grow t h (Saccharomyces cerevisiae) and the reduct ion of nit rogen free ext ract result ed in increased concent ration of t he other com ponent s. Yi et al. (l996) report ed t hat supplement at ion of m icrobial in diet s im proved N ret ent ion in broiler chickens and in vit ro digest ibilit y of veget able prot ein. Also, Chen et al. (2005) report ed that addit ion of 0.20% complex probiot ic (L.acidophilus and S.cerivisae) in basal diet s w ere inceased digest iblit ies of dry matt er.

Ferm ent ed of rice bran ingredient by ragi (Saccharomyces cereviseae) had bet t er digest ibilit ies, because Saccharomyces cereviseae in the gast roint est inal t ract can part of an probiot ics sourches. Also, Piao et al. (l999), suggest ed t hat probiot ics in the gast ro intest inal t ract can improve prot ein and energy ret ent ion on t he body of birds. These fungal are effect ive in degrading of t he complex com pounds such as b et a-glucans and arabinoxylans (Bedford and Classen, 1992).

Bioferm ent at ion of rice bran by Saccharomuces spp.S-6 and S-7 isolat es cult ure had bet t er digest ibility, because Saccharomuces spp culture in the gast ro int est inal t ract can t o be part of probiotic sources. Saccharomyces cerevisiae as part of probiot ic could im prove prot ein and energy ret ent ion in t he gast ro intest inal t ract of the birds (Piao et al., l999). Cho et al. (2007) report ed that supplement at ion of m icrobe in diet could improve t he bioavailabilit y of diet ary. Wang et al. (2004) report ed t hat t he inclusion of fiber sources such as w heat bran or pot at o st arch reduced t he m aint enance of energy requirem ent . These fungi are effect ive in degradating the complex compounds such as bet a-glucans and arabinoxylans (Dubey, 2006). Chen et al. (2005) report ed that diet ary supplement at ion of complex probiot ic slight ly improved digest ibilit y of nut rient s. Inconsist ent report s about the effect of probiotics m ay be due t o several aspect s such as st rains of bact eria, dose level, diet composition, feeding st rat egy, feed form , and interact ion with other diet ary feed addit ives (Chesson, 1994).

The high level of non-st arch polysaccharides (NSP) in rice bran is limit ing it s unrest rict ed use in poult ry feeding. The NSP is know n increasing t he gut viscosit y, reduce nut rient absorption in t he int est ine and affect indirect ly t he grow t h and perform ance of bird (Rham es et al., 2006; Cho et al., 2007).

M any st udies have clearly demonst rat ed t hat t he addition of probiot ics culture or enzym es t o diet s rich in NSP result ed in a significant reduction of the int est inal viscosit y, enhances energy and prot ein utilization. Wang et al. (2004) report ed that degree of microbial ferm ent at ion in the large int est ine improves t he bioavailabilit y of diet ary. Yi et al. (l996) report ed that supplem ent at ion of m icrobial in diet s improved N ret ent ion in broiler chickens and in vit ro digest ibilit y of veget able prot ein. Also, Chen et al. (2005) report ed t hat addit ion of 0.20% complex probiotic (L. acidophilus and S. cerivisae) in basal diet s increased digest iblit y of DM and CP. Hong et al. (2004) report ed t hat ferment at ion of feed using Aspergilus oryzae increased digest ibilit y of it s DM and CP. The inclusion of soluble diet ary fiber (w heat bran) increased daily NSP excret ed from feces (Wang et al., 2004; Suprapt i et al., 2008), increased bot h of m et abolizable energy and crude prot ein cont ent s of palm kornel meal (Jaelani et al., 2008). Chen et al. (2005) report ed that diet ary supplem ent at ion of complex probiot ic slightly improved digest ibilit y of nut rient s.

CONCLUSION

It w as concluded t hat t here are t wo isolate (Sc.S-6 and S-7) w ere isolat ed from manure of beef Bali cat tle sam ples, bot h w ere t he pot ensial as a probiot ics sources and has CM C-ase akct ivit y and it s ut ilizat ion in the rice bran ferm ent at ion could increase nut rient com posit ion and digest ibilit y of rice bran.

ACKNOW LEDGEM ENTS

Authors are ent irely grat eful for the chem ical analyses of sample by st aff of Nut rit ion Laboratory, Fakult y of Anim al Husbandry, Udayana Universit y, Denpasar-Bali. The authors also w ant t o thank the Ditbinlit abmas, Dirjen Dikt i, Depdiknas in Jakart a for funding t his research. A part of the present st udy w as financially support ed by Ditbinlit abm as, Dirjen Dikt i, Depdiknas in Jakart a.

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Corresponding author: Dr. Igng Bidura, Faculty of Animal Husbandry, Udayana University, Denpasar-Bali, Jl. PB. Soedirman, Denpasar, Indonesia

Em ail:dsk_candraw at i@yahoo.co.id bidura_unud@yahoo.com

Phone / Fax. 0361-702771

gizzard (2.6), duodenum (5.7-6.0), jejunum (5.8), ileum (6.3), colon (6.3), and ceca (5.7) (Hidayat , 2010). Som e advant ages of Saccharomyces ferm ent at ion process, t hese microorganisms are rapidly proliferat ing, resist ant to high alcohol cont ent , resist ant t o high t em perat ures, has held st eady and rapid nature of adapt at ion. According t o Ahm ad (2005), t emperat ure opt imum environm ent for yeast grow t h is 25-300C and a m axim um t em perat ure of 35-470C. Som e advant ages Saccharomyces microorganisms in the ferm ent at ion process t hat is rapidly proliferat ing, resist ant t o high alcohol cont ent , resist ant to high t emperat ures, has held st eady and rapid nature of the adaptat ion. CM C-ase enzym e act ivit y (endo-1,4-bet a-glukonase) is a clear zone around the colony indicat ed that the m icrobes have a st rong ext racellular cellulase enzyme activit y. The size of t he clear zone and the apparent absence of clear zone, an indicator of the abilit y of the microbes t o break down cellulose, as w ell as fast and slow arise t he clear zone (Van Devoorde and Verstraet e, 1987). Ut ama et al. (2006) report ed t hat t he yeast Saccharomyces cerevisiae is yeast capable of producing t he enzym es am ylase and selulolase, so as t o increase t he digest ibilit y of protein and crude fiber such as cellulose and hemicellulose, as has been overhauled in t he form of a sim ple monosaccharide. Cellulolyt ic yeast s capable of producing t he enzyme 1,4 bet endo-glukonase, 1,4 bet a-exo glukonase, and bet a-glucosidase t hat can degrade com ponent s of crude fiber into soluble carbohydrat es (How ard et al., 2003) According Ut am a et al. (2006), ferm ent at ion feed purpose is t o improve t he usabilit y of feed and eliminat ion of ant i- nut rit ional subst ances, as well as ut ilize biom ass form ed. Rice bran ferm ent ed w ith yeast w ill be able t o increase t he m icrobial biomass, so t hat the crude prot ein cont ent of rice bran increased. Also report ed that the ferm ent at ion process and the product is affect ed by t he t ype and number of microbes, subst rat t ypes, pH, and t em perat ure during t he ferm ent at ion process. Biom ass is a form of mass from t he biological processes of microorganisms. M icroorganisms capable of convert ing mat erial int o prot eins. The ferm ent at ion process has the object ive t o produce a product (m at erial feed) t hat have a nut rient cont ent, t ext ure, bet t er biological availabilit y, and reduce subst ance antinut risi. Suparjo et al. (2003) st at ed that t he ferm ent at ion of rice bran with 0.2% Aspergillus niger cultured for 72 hours can m arkedly increase prot ein and phosphorus cont ent of rice bran, on the cont rary low er crude fiber cont ent and acid phyt at rice bran.

According Sumarsih et al. (2007), the longer t he st orage t im e of feed ingredient s w hen ferm ent ed by Trichoderma viride, t he m ore t he loss of dry mat t er and organic m att er mat erial, inst ead increasing t he crude prot ein cont ent and m aterials decreased crude fiber cont ent m at erial. Also report ed that the longer t he ferment at ion process or mat erials st orage t ime, t he increased dry m at t er digest ibilit y and organic m att er mat erials.

According t o Jaelani et al. (2008), the increase in M E cont ent of palm kernel cake (palm kernel cake/ m eal) as a result of ferm ent at ion by the fungus T. reesei of 1824.13 kcal/ kg t o 1930.44 kcal/ kg suspect ed because of t he degradat ion of m annan polysaccharides exist in palm kernel by fungus T. reesei into simpler form s (m onosaccharides) t hat produces enough energy value bet t er t han in the form of polysaccharides mannan.

The sam e t hing w as report ed also by Sabini et al. (2000) which st at es t hat t he fungus T. reesei is able t o degrade a polysaccharide mannan mannot riosa, m annobiosa, and monnosa. According to Jaelani et al. (2008), ferment ed palm kernel cake can m arkedly increase t he crude prot ein cont ent compared to palm kernel cake w it hout ferm ent at ion. Table 3 show s t he chem ical composit ion of unferm ent ed rice bran (UFR) and ferm ent ed rice bran (FR) ingredient . The digest ibilit y both of crude prot ein, organic m at t er, crude fibre, and met abolizable energy w ere slight ly increased by S.cerevisiae ferm ent at ion. S. cerevisiae can not effect on crude fiber digest ibility of rice bran. Becouse, among t he cell w all polysaccharides of rice bran known as nonst arch polysaccharides (NSP) are celluloses, pect ins, and oligosaccharides. NSPs can not be degraded enzymit ically in t he digest ive syst em s of t he birds due t o the lacking of enzymes degrading the NSPs in t heir digest ive syst ems (Choct , 2002). The digest ibilit y of crude prot ein, crude fiber, and M E w ere slight ly increased by t he ferm ent at ion. These result s indicat ed that carbohydrat es other t han fibres w ere used for m icrobial grow t h (Saccharomyces cerevisiae) and the reduct ion of nit rogen free ext ract result ed in increased concent ration of t he other com ponent s. Yi et al. (l996) report ed t hat supplement at ion of m icrobial in diet s im proved N ret ent ion in broiler chickens and in vit ro digest ibilit y of veget able prot ein. Also, Chen et al. (2005) report ed that addit ion of 0.20% complex probiot ic (L.acidophilus and S.cerivisae) in basal diet s w ere inceased digest iblit ies of dry matt er.

Ferm ent ed of rice bran ingredient by ragi (Saccharomyces cereviseae) had bet t er digest ibilit ies, because Saccharomyces cereviseae in the gast roint est inal t ract can part of an probiot ics sourches. Also, Piao et al. (l999), suggest ed t hat probiot ics in the gast ro intest inal t ract can improve prot ein and energy ret ent ion on t he body of birds. These fungal are effect ive in degrading of t he complex com pounds such as b et a-glucans and arabinoxylans (Bedford and Classen, 1992).

Bioferm ent at ion of rice bran by Saccharomuces spp.S-6 and S-7 isolat es cult ure had bet t er digest ibility, because Saccharomuces spp culture in the gast ro int est inal t ract can t o be part of probiotic sources. Saccharomyces cerevisiae as part of probiot ic could im prove prot ein and energy ret ent ion in t he gast ro intest inal t ract of the birds (Piao et al., l999). Cho et al. (2007) report ed that supplement at ion of m icrobe in diet could improve t he bioavailabilit y of diet ary. Wang et al. (2004) report ed t hat t he inclusion of fiber sources such as w heat bran or pot at o st arch reduced t he m aint enance of energy requirem ent . These fungi are effect ive in degradating the complex compounds such as bet a-glucans and arabinoxylans (Dubey, 2006). Chen et al. (2005) report ed that diet ary supplement at ion of complex probiot ic slight ly improved digest ibilit y of nut rient s. Inconsist ent report s about the effect of probiotics m ay be due t o several aspect s such as st rains of bact eria, dose level, diet composition, feeding st rat egy, feed form , and interact ion with other diet ary feed addit ives (Chesson, 1994).

The high level of non-st arch polysaccharides (NSP) in rice bran is limit ing it s unrest rict ed use in poult ry feeding. The NSP is know n increasing t he gut viscosit y, reduce nut rient absorption in t he int est ine and affect indirect ly t he grow t h and perform ance of bird (Rham es et al., 2006; Cho et al., 2007).

M any st udies have clearly demonst rat ed t hat t he addition of probiot ics culture or enzym es t o diet s rich in NSP result ed in a significant reduction of the int est inal viscosit y, enhances energy and prot ein utilization. Wang et al. (2004) report ed that degree of microbial ferm ent at ion in the large int est ine improves t he bioavailabilit y of diet ary. Yi et al. (l996) report ed that supplem ent at ion of m icrobial in diet s improved N ret ent ion in broiler chickens and in vit ro digest ibilit y of veget able prot ein. Also, Chen et al. (2005) report ed t hat addit ion of 0.20% complex probiotic (L. acidophilus and S. cerivisae) in basal diet s increased digest iblit y of DM and CP. Hong et al. (2004) report ed t hat ferment at ion of feed using Aspergilus oryzae increased digest ibilit y of it s DM and CP. The inclusion of soluble diet ary fiber (w heat bran) increased daily NSP excret ed from feces (Wang et al., 2004; Suprapt i et al., 2008), increased bot h of m et abolizable energy and crude prot ein cont ent s of palm kornel meal (Jaelani et al., 2008). Chen et al. (2005) report ed that diet ary supplem ent at ion of complex probiot ic slightly improved digest ibilit y of nut rient s.

CONCLUSION

It w as concluded t hat t here are t wo isolate (Sc.S-6 and S-7) w ere isolat ed from manure of beef Bali cat tle sam ples, bot h w ere t he pot ensial as a probiot ics sources and has CM C-ase akct ivit y and it s ut ilizat ion in the rice bran ferm ent at ion could increase nut rient com posit ion and digest ibilit y of rice bran.

ACKNOW LEDGEM ENTS

Authors are ent irely grat eful for the chem ical analyses of sample by st aff of Nut rit ion Laboratory, Fakult y of Anim al Husbandry, Udayana Universit y, Denpasar-Bali. The authors also w ant t o thank the Ditbinlit abmas, Dirjen Dikt i, Depdiknas in Jakart a for funding t his research. A part of the present st udy w as financially support ed by Ditbinlit abm as, Dirjen Dikt i, Depdiknas in Jakart a.

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Corresponding author: Dr. Igng Bidura, Faculty of Animal Husbandry, Udayana University, Denpasar-Bali, Jl. PB. Soedirman, Denpasar, Indonesia

Em ail:dsk_candraw at i@yahoo.co.id bidura_unud@yahoo.com

Phone / Fax. 0361-702771