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

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I solation of Saccharomyces Spp
from Manure of Beef Bali Cattle as a
Probiotics Properties and has CMCase Activity to I mprove Nutrient
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
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J. Biol. Chem. Research. Vol. 31, No. 1: 39- 52 ( 2014)
( An I nternational Journal of Life Sciences and Chemistry)
Ms 30/ 2/ 160/ 2013, All rights reser ved

I SSN 09 70-4973 ( Print)
I SSN 23 19-3077 ( Online/ Electronic)

Desak Putu Mas Ari Candraw ati

I gng Bidura

http:// www.jbcr.in
jbiolchemres@gmail.com

info@jbcr.in
Received: 09/ 11/ 2013

Revised: 20/ 12/ 2013

RESEARCH PAPER
Accepted: 03/ 01/ 2014

I solation of Saccharomyces Spp from Manure of
Beef Bali Cattle as a Probiotics Properties and
has CMC-ase Activity to I mprove Nutrient Quality
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
0
0
spp show ed resistant grew on both in different temperature (10 -45 C), 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 (P0.05) 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.

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Candraw ati, et al., 20 14

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 level, 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.
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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
0
Gilliland (1999) was used with slight m odificat ions. St rain was grow n in M RS brot h at 37 C
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
6-8
0
initial bact erial concent ration w as 10 cfu/ m l. Cult ure w as incubat ed at 37 C. 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

0
buffer (pH 7.2), and t he viable cells w ere enumerat ed on M RS agar plat e at 37 C for 2
days.
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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
0
(Difco). Cultures w ere incubat ed at 37 C 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

0
at a t em perat ure of 45-50 C, 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
6
w as prepared as follow s. Comercial rice bran w as used. Approxim at ely 0.20% (2 x 10
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
0
days for ferm ent at ion. Aft er t hat , ferment ed rice bran was dried at 45 C 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 was 50 g. This amount as based on preliminary
assays wit h Lohman brown laying hens using rice bran. All the birds were 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 wit h the specific amount of rice bran (ferment ed and unferment ed). St ainless st eel
funnel wit h 40 cm st em was used in force f eeding t echnique (M ust afa et al ., 2004). Wat er was
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, allowed coming to equilibrium wit h the
at mospheric moisture, weighed, and groun through a 1 mm sieve. Samples of excret a and
rice bran were 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),
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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.
Statistical 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 the 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
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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)

S1
S2
S3
S4
S5
S6
S7
S8
S9
S10

0.2 mM
+ (0.244)
+ (0.248)
+ (0.253)
+ (0.252)
+ (0.243)
+ (0.224)
+ (0.192)
+ (0.145)
+ (0.126)
+ (0.138)

0.4 mM
+ (0.169)
+ (0.168)
+ (0.163)
+ (0.158)
+ (0.166)
+ (0.144)
+ (0.133)
+ (0.151)
+ (0.132)
+ (0.149)

0.6 mM
- (0.067)
- (0.059)
- (0.081)
- (0.064)
- (0.073)
- (0.036)
- (0.031)
- (0.051)
- (0.044)
- (0.035)

Descript ion:
:
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 .
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This m eans t hat isolat es S-6 has kem am puam in digest ing crude fiber t han the highest S8. 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