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.
(An International Journal of Life Sciences and
Chemistry)
Ms 30/2/160/2013, All rights reserved
ISSN 0970-4973 (Print)
ISSN 2319-3077 (Online/Electronic)
http:// www.jbcr.in
jbiolchemres@gmail.com
info@jbcr.in
RESEARCH PAPER
Received: 09/11/2013
Revised: 1/12/2013
Accepted :9/12/2013
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
Desak Putu Mas Ari Candrawati, D.A. Warmadewi, and
IGNG Bidura
Faculty of Animal husbandry, Udayana University, Denpasar-Bali, Jl.
PB. Soedirman, Denpasar, Indonesia
ABSTRACT
This experiment was carried out to study both on the CMC-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
Lohman Brown laying hens was assigned to three treatments in
a completely randomized design. Each treatment has six
replications with one bird per replication (individual cage). The
treatments were (i) unfermented rice bran as control; (ii)
fermented rice bran by Saccharomyces spp.S-6 isolate; (iii)
fermented rice bran by Saccharomyces spp.S-7 isolatse,
respectively. The report on the first experiment showed that
five isolates of Saccharomyces spp (Sc.S-5; S-6; S-7; S-8; and S9) were isolated from manure of beef Bali cattle samples in the
first experiment. The whole isolates of Saccharomyces spp
showed resistant grew on both in different temperature (1000
45 C), acid conditions (2,5-4,5), and bile salt. But only two
isolates (Sc.S-6 and S-7) were potensial as probiotics sources
and has CMC-ase activity. The study showed that fermentation
of rice bran using both of Saccharomyces spp.S-6 and S-7
culture could improve significant differences (P0.05) by fermentation. On the other hand,
fermentation caused increasing crude protein (CP) and crude
fibre (CF) of the rice bran. It was concluded that two isolates
(Sc.S-6 and S-7) were isolated from manure of beef Bali cattle
samples, both were the potensial as a probiotics sources and
has CMC-ase activity and its utilization in the rice bran
fermentation could increase nutrient composition and
digestibility of rice bran.
Key words: Saccharomyces Spp., Probiotics, Crude Fiber,
Digestibility, and Rice Bran.
Published by Society for Advancement of Science®
INTRODUCTION
During recent years, numerous studies have been undertaken to obtain
scientific evidences for beneficial effect of Saccharomyces spp as
promising probiotic. Such beneficial effects are considered to include
the protection from pathogen, enhancement of the immune system,
antimutagenic and anticarinogenic effects and the reduction of serum
cholesterol. Chen et al. (2005) reported that dietary supplementation of
complex probiotic slightly improved digestibility of nutrients.
The use of Saccharomyces cerivisiae (Sc.) culture as a probiotics
sourches in poultry production as become an area of great interest,
because continued use of probiotics in animal feeds may result in the
presence of antibiotics residues in animal products (Han et al., 2001).
Gut microfloral enzymes are beneficial to the nutrition of the host
because they increase the digestion of nutrients, especially in the lower
intestine. Previous experiments showed that the inclusion of
microorganisms in the diets improved feed conversion efficiency and
digestibility (Chen et al.,2005). Piao et al. (l999) showed that 0,10%
yeast added to a diet could reduce animal wastes. Yeast culture
improved posphorus utilization in growing chickens. Inconsistent
reports about the effect of probiotics may be due to several aspects
such as strains of bacteria, dose level, diet composition, feeding
strategy, feed form, and interaction with other dietary feed additives
(Chesson, 1994).
Rice bran is a by-product of rice milling industry, which is abundantly
available (approximately 10% of paddy weight) during the harvesting
season. Unfortunately this by-product contains toxic factors such as
trypsin inhibitor, phytic acid as phytate, and high content of crude fiber.
These anti-nutritive factors have been reported by Kahlique et al.
(2003) causing reduction of feed intake and depress performance of
poultry. These toxic factors are phytic acid as phytate and crude fiber
(CF). These anti-nutritive factors have been reported to reduce feed
intake and depress performance of poultry. Bach Knudsen (2001)
reported that CF has been defined as the complex macromoleculer
substances in food plants that are not degraded by mammalian
digestive enzymes. With the exception of lignin, all of the materials
called CF are carbohydrates in nature. CF is thought to mediate
protective effects on the colonic epithelium through their fermentation
products and fecal bulking capacity (Wang et al., 2004). Feeding high
fiber resulted in a lowered rate of lipogenesis and tendency of an
increased capacity to utilize acetyl-CoA in pigs (Zhu et al., 2003). Non
starch polysaccharide (NSP) are the carbohydrate components of CF
and are the predominant substrates for anaerobic fermentation.
The potency of rice bran as energy source for poultry depends on its
cell wall content, degree of microbial fermentation in poultry large
intestine, absorption and production of the volatile fatty acid (Wang et
al., 2004). The potensial of rice bran as energy sources for poultry
depends considerably on such factors as cell wall content, degree of
microbial fermentation in the large intestine, and extent of absorption
and utilization of the volatile acids produced (Kahlique et al., 2003). The
use of rice bran in poultry diets has been highly limited by the presence
of phytic acid and other minor anti-nutritional factors like tannin and
non-starch polysaccharides (NSP). The high levels of NSP in rice bran is
limiting its unrestricted use in poultry feeding. These NSPs are known
to increase the gut viscosity, reduce nutrient absorption in the intestine
and affected indirectly the growth and performance of birds (Rhames et
al. 2006; Cao et al., 2003). Many studies have clearly demonstrated
that, supplemented of probiotics to diets rich in NSP results in a
significant reduction in the intestinal viscosity, enhances energy, and
protein utilization (Bidura et al., 2012; Bidura et al., 2009).
Probiotics are defined as the viable microorganisms that exhibit a
beneficial effect on health of the host by improving its intestinal
microbial balance. Yeast culture is one of the most extensively studied
probiotic. Traditionally, S.cerevesiae have been used for food products
such as baking industries, tape , a d are o sidered as orga is s that
can be used also for a pharmaceutical (Ahmad, 2005). The use of
antibiotics as routine feed additives has been baned in same countries
because of public concern over possible antibiotics residual effects and
the development of drug-resistent bacteria. Probiotics have been
introduced as an alternative to antibiotics; however, their effect on
poultry production are not consistent, resulting in uncertainties and
skepticism for development of the products. There are many types of
probiotic preparations in the market. Many studies have been
conducted to test the afficacy of such preparations on animal growth
and performance. Several studies with broiler have indicated that
probiotics preparations improve live weight gain and feed conversion
rate (Bidura et al., 2012; 2009; 2008; Sutarpa et al., 2011). Hong et al.
(2004) reported that fermentation of feed using Aspergilus oryzae
increased digestibility of its DM and CP.
Therefore, it is susgested that fermented of rice bran feeding by
Saccharomyces spp isolates as a CMC-ase akctivity (isolation from
manure of beef Bali cattle) can be used in order to alleviate the
negative effect of rice bran as feed.
METERIAL AND METHODS
Animals and experimental design
Eighteen of Lohman Brown laying hens were assigned to three
treatments in a completely randomized design. Each treatment has six
replications with one birds per replication (individual cage). All of the
birds were fed experimental diets for two days. The treatments were (i)
unfermented rice bran as control; (ii) fermented rice bran by
Saccharomyces spp.S-6 isolate; (iii) fermented rice bran by
Saccharomyces spp.S-7 isolate; respectively. The objectives of this study
is to determine the nutrient digestibility and the ME value of rice bran
using Lohman Brownn laying hens at 42 weeks of age.
Probiotics properties
Acid tolerance: to determine the trancsit tolerance to low pH, the
method of Corzo and Gilliland (1999) was used with slight
modifications. Strain were grown in MRS broth at 370C for 24 h. A 0.5
ml aliquot of the bacterial culture was inoculated in 10 ml of phosphate
buffered saline adjusted to pH 2 with 4 N HCl. Phosphate buffer was
prepared by dissolving NaCl (9 g/l), Na2HPO42H2O (9 g/l), and KH2PO4
6-8
(1.5 g/l) in destiled water. The initial bacterial concentration was 10
0
cfu/ml. Culture were incubated at 37 C. After 0; 0.5; 1; 2; and 4 h
incubation, cell were serially diluted tenfold in 0.1 M sodium phosphate
buffer (pH 7.2), and the viable cells were enumerated on MRS agar
0
plate at 37 C for 2 days.
Bile resistance: The resistance to bile was examined according to
method of Hyronimus et al. (2000). Each strain was inoculated in MRS
broth with 0.5 or 1% (w/v) bile salt (Difco). Culture were incubated at
0
37 C for 24 h, and the numbers of viable cell were determined and
compared to a control (without bile salt) on MRS agar plates.
CMC-ase activity: Weighed as much as 11 g of OMEA and 3 g of CMCase, then dissolved in distilled water. Heat in a waterbath and after it
did strelilisasi on autoklav. Refrigerate at a temperature of 45-50 0C,
then poured on Petri dishes and left to solidify. Isolates that have been
cultured in a nutrient broth for 24 hours. Take a paper disk with
tweezers and then dipped in a solution of nutrient broth then put on a
petri dish containing Omea media and CMC-ase. Leave for 24 hours.
After 24 hours of clear zone width measurements are generated by
using a vernier caliper. The size of the clear zone and the apparent
absence of clear zone, an indicator of the ability of the microbes to
break down cellulose, as well as fast and slow arise the clear zone
(VanDevoorde and Verstraete, 1987; Kanti, 2007).
Fermented of Rice Bran
The isolate of Saccharomyces spp.S-6 and S-7 which has been approved
from bile salt and poultry digestive tract in vitro test could assimilate
cholesterol for probiotics agency and two isolates have CMC-ase
activity. The study was carried out at the Bioscience Laboratory of
Udayana University, Bali, Indonesia. Fermentation of commercial rice
bran was prepared as follows. Comercial rice bran was used.
6
Approximately 0.20% (2 x 10 spores) Saccharomyces spp. S-6 and S-7
isolate culture was added to 100 g of steamed rice bran. Then, water
was added to bring the product to 50% content and left up to 2 days for
fermentation. After that, fermented rice bran was dried at 45 0C for six
hours and then it was ground for analysis. Unfermented rice bran was
also ground for its chemical analysis.
Retention and excretion of nutrients
In order to determine the nutrient digestibility and metabolizable
energy (ME) value of the rice bran. The amount of rice bran used was
50 g. This amount as based on preliminary assays with Lohman brown
laying hens using rice bran. All the birds were deprived of feed for 24 h
to ensure that their alimentary canals were empty from feed residues.
They were then force-fed with the specific amount of rice bran
(fermented and unfermented). Stainless steel funnel with 40 cm stem
was used in force feeding technique (Mustafa et al., 2004). Water was
available ad libitum during the experimental period
The total excreta were collected in plastic trays. The excreta samples
were frozen, allowed to come to equilibrium with the atmospheric
moisture, weighed, and groun through a 1 mm sieve. Samples of
excreta and rice bran were subjected to appropriate analysis to
determine DM, OM, CP, CF, and energy, respectively.
Laboratory analyses
Dry matter (DM), organic matter (OM), CP and ash determinations were
done according to the Assocciation of Official Analytical Chemists
(l994). The CP content of the diets was determined using the Kjeldahl
procedure (AOAC, 1994). Crude fibre in the feeds were determined
using the procedure of Van Soest et al. (l991) on oven-dried samples.
Gross energy (GE) was measured with an adiabatic oxygen bomb
calorimeter (Parr, USA),
Calculations
The data were used to calculate AME value according to the following
formulate (Mustafa et al., 2004): AME (apparent metabolizable energy)
= IE – FE. Where IE = ingested energy; FE = fecal energy voided by the
fed birds.
Statistical Analysis
All data were subjected to a one-way analysis of variance test (Steel and
Torrie, 1989). Statistical significances among treatment means were
determined by method of New Multiple Range Test of Duncan when
the F value was significant at 5% level.
RESULTS
Resilience isolate Saccharomyces spp. the low pH is one of the
characteristics required to be met by a candidate or a probiotic that
could be developed into a potential probiotic. On this test, the medium
pH was adjusted to 1.5, 3.0, 4.5, and 6.0 by using HCl (hydrochloric
acid), because HCl has characteristics similar to stomach acid. Resistant
to highly acidic nature needs to be owned by the candidate probiotic,
because the application later, this probiotic candidate must pass a very
acidic stomach conditions, before reaching the colon.
Observations as presented in Table 1. In this study, as many as 10
isolates Saccharomyces spp. isolated from cattle feces was tested
resistance to various pH, at pH 1.5, 3.0, 4.5, and 6. Two isolates were
unable to grow at pH 1.5, which isolates the S-1 and S-2. Saccharomyces
spp.S-1 isolates even simply could not grow at all pH treatments given.
Only 8 isolates were able to grow well, namely Saccharomyces spp.S-4
to S-10 isolates. There is a tendency of the higher pH, especially at pH 6,
most of the isolates has decreased the number of colony life.
Saccharomyces spp colonies grow well at pH 3, even isolate 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 Saccharomyces spp (Log
colony/g) at various pH.
Code Isolate 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
Descriptions: S-1 s / d S-10 is a Saccharomyces spp isolates were
isolated from manure of beef Bali cattle
In the test of resistance to bile salts as seen in Table 2 shows, that the
ten isolates were able to survive and grow on a medium containing bile
salts at concentrations of 0.2 mM and 0.4 mM, whereas at a
concentration of 0.6 mM no isolates can survive. Microbial isolates
resistance to bile salts is used to assess the ability to survive the
digestive tract isolates contained bile salts on the surface of the
intestine.
Probiotics will be dealing with the environment in the small intestine, in
which there are bile or bile salts are removed by the liver through the
gall bladder, after successfully passing the acidic conditions in the
stomach. Therefore, in the process of developing new probiotic, or a
new probiotic candidate must be able to pass the test of resistance to
bile or bile salts were performed in vitro. Based on the nature of the
resistance shown by some isolates, strai-indicates that the strain has
the potential to be developed into a potential probiotic.
Table 2. Ability of yeast Saccharomyces spp isolates were resistant
living in bile salts.
Isolate
Bile salt concentration (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)
Description:
- : A 1.0 (highly resistant of bile salts)
S-1 s / d S-10 is a Saccharomyces spp isolates were isolated from
manure of beef Bali cattle
CMC-ase test is to test the ability of the yeast Saccharomyces spp
isolates in degrading crude fiber. It can be measured from the resulting
clear zone diameter (Table 3). From the results of this study were only
yeast Saccharomyces spp isolates S-5, S-6, S-7, S-8, and the yeast
Saccharomyces spp.S-9 who has the ability to digest fiber. Looks yeast
Saccharomyces spp isolates S-6 has the most wide clear zone, while the
S-8 has a clear zone at least. This means that isolates S-6 has
kemampuam in digesting crude fiber than the highest S-8. More
detailed, clear zone diameter and difference generated by the two
isolates are presented in Figure 1.
Isolate
S8
Isolate
S6
Clear zone
Figure 1. Saccharomyces spp.S-8 isolates zone is smaller than the
isolate Saccharomyces spp.S-6 (right).
Table 3. CMC-ase activity test yeast Saccharomyces spp isolates based
on the diameter of the clear zone caused.
Isolat
Clear zone diameter (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
Description: S-1 s / d S-10 is a Saccharomyces spp isolates were isolated
from manure of beef Bali cattle
Table 4 shows the nutrient the content of crude protein and gross
energy were slightly increased by fermentation. On the other hand
fermentation caused decreasing dry matter (DM), organic Matter (OM),
and crude fibre (CF) of the rice bran. These results indicated that
carbohydrate was used for microbial growth and the reduction of dry
matter. The result indicated that all of nutrient digestibility of
fermented rice bran by Saccharomyces spp culture were increased
significantly (P
Chemistry)
Ms 30/2/160/2013, All rights reserved
ISSN 0970-4973 (Print)
ISSN 2319-3077 (Online/Electronic)
http:// www.jbcr.in
jbiolchemres@gmail.com
info@jbcr.in
RESEARCH PAPER
Received: 09/11/2013
Revised: 1/12/2013
Accepted :9/12/2013
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
Desak Putu Mas Ari Candrawati, D.A. Warmadewi, and
IGNG Bidura
Faculty of Animal husbandry, Udayana University, Denpasar-Bali, Jl.
PB. Soedirman, Denpasar, Indonesia
ABSTRACT
This experiment was carried out to study both on the CMC-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
Lohman Brown laying hens was assigned to three treatments in
a completely randomized design. Each treatment has six
replications with one bird per replication (individual cage). The
treatments were (i) unfermented rice bran as control; (ii)
fermented rice bran by Saccharomyces spp.S-6 isolate; (iii)
fermented rice bran by Saccharomyces spp.S-7 isolatse,
respectively. The report on the first experiment showed that
five isolates of Saccharomyces spp (Sc.S-5; S-6; S-7; S-8; and S9) were isolated from manure of beef Bali cattle samples in the
first experiment. The whole isolates of Saccharomyces spp
showed resistant grew on both in different temperature (1000
45 C), acid conditions (2,5-4,5), and bile salt. But only two
isolates (Sc.S-6 and S-7) were potensial as probiotics sources
and has CMC-ase activity. The study showed that fermentation
of rice bran using both of Saccharomyces spp.S-6 and S-7
culture could improve significant differences (P0.05) by fermentation. On the other hand,
fermentation caused increasing crude protein (CP) and crude
fibre (CF) of the rice bran. It was concluded that two isolates
(Sc.S-6 and S-7) were isolated from manure of beef Bali cattle
samples, both were the potensial as a probiotics sources and
has CMC-ase activity and its utilization in the rice bran
fermentation could increase nutrient composition and
digestibility of rice bran.
Key words: Saccharomyces Spp., Probiotics, Crude Fiber,
Digestibility, and Rice Bran.
Published by Society for Advancement of Science®
INTRODUCTION
During recent years, numerous studies have been undertaken to obtain
scientific evidences for beneficial effect of Saccharomyces spp as
promising probiotic. Such beneficial effects are considered to include
the protection from pathogen, enhancement of the immune system,
antimutagenic and anticarinogenic effects and the reduction of serum
cholesterol. Chen et al. (2005) reported that dietary supplementation of
complex probiotic slightly improved digestibility of nutrients.
The use of Saccharomyces cerivisiae (Sc.) culture as a probiotics
sourches in poultry production as become an area of great interest,
because continued use of probiotics in animal feeds may result in the
presence of antibiotics residues in animal products (Han et al., 2001).
Gut microfloral enzymes are beneficial to the nutrition of the host
because they increase the digestion of nutrients, especially in the lower
intestine. Previous experiments showed that the inclusion of
microorganisms in the diets improved feed conversion efficiency and
digestibility (Chen et al.,2005). Piao et al. (l999) showed that 0,10%
yeast added to a diet could reduce animal wastes. Yeast culture
improved posphorus utilization in growing chickens. Inconsistent
reports about the effect of probiotics may be due to several aspects
such as strains of bacteria, dose level, diet composition, feeding
strategy, feed form, and interaction with other dietary feed additives
(Chesson, 1994).
Rice bran is a by-product of rice milling industry, which is abundantly
available (approximately 10% of paddy weight) during the harvesting
season. Unfortunately this by-product contains toxic factors such as
trypsin inhibitor, phytic acid as phytate, and high content of crude fiber.
These anti-nutritive factors have been reported by Kahlique et al.
(2003) causing reduction of feed intake and depress performance of
poultry. These toxic factors are phytic acid as phytate and crude fiber
(CF). These anti-nutritive factors have been reported to reduce feed
intake and depress performance of poultry. Bach Knudsen (2001)
reported that CF has been defined as the complex macromoleculer
substances in food plants that are not degraded by mammalian
digestive enzymes. With the exception of lignin, all of the materials
called CF are carbohydrates in nature. CF is thought to mediate
protective effects on the colonic epithelium through their fermentation
products and fecal bulking capacity (Wang et al., 2004). Feeding high
fiber resulted in a lowered rate of lipogenesis and tendency of an
increased capacity to utilize acetyl-CoA in pigs (Zhu et al., 2003). Non
starch polysaccharide (NSP) are the carbohydrate components of CF
and are the predominant substrates for anaerobic fermentation.
The potency of rice bran as energy source for poultry depends on its
cell wall content, degree of microbial fermentation in poultry large
intestine, absorption and production of the volatile fatty acid (Wang et
al., 2004). The potensial of rice bran as energy sources for poultry
depends considerably on such factors as cell wall content, degree of
microbial fermentation in the large intestine, and extent of absorption
and utilization of the volatile acids produced (Kahlique et al., 2003). The
use of rice bran in poultry diets has been highly limited by the presence
of phytic acid and other minor anti-nutritional factors like tannin and
non-starch polysaccharides (NSP). The high levels of NSP in rice bran is
limiting its unrestricted use in poultry feeding. These NSPs are known
to increase the gut viscosity, reduce nutrient absorption in the intestine
and affected indirectly the growth and performance of birds (Rhames et
al. 2006; Cao et al., 2003). Many studies have clearly demonstrated
that, supplemented of probiotics to diets rich in NSP results in a
significant reduction in the intestinal viscosity, enhances energy, and
protein utilization (Bidura et al., 2012; Bidura et al., 2009).
Probiotics are defined as the viable microorganisms that exhibit a
beneficial effect on health of the host by improving its intestinal
microbial balance. Yeast culture is one of the most extensively studied
probiotic. Traditionally, S.cerevesiae have been used for food products
such as baking industries, tape , a d are o sidered as orga is s that
can be used also for a pharmaceutical (Ahmad, 2005). The use of
antibiotics as routine feed additives has been baned in same countries
because of public concern over possible antibiotics residual effects and
the development of drug-resistent bacteria. Probiotics have been
introduced as an alternative to antibiotics; however, their effect on
poultry production are not consistent, resulting in uncertainties and
skepticism for development of the products. There are many types of
probiotic preparations in the market. Many studies have been
conducted to test the afficacy of such preparations on animal growth
and performance. Several studies with broiler have indicated that
probiotics preparations improve live weight gain and feed conversion
rate (Bidura et al., 2012; 2009; 2008; Sutarpa et al., 2011). Hong et al.
(2004) reported that fermentation of feed using Aspergilus oryzae
increased digestibility of its DM and CP.
Therefore, it is susgested that fermented of rice bran feeding by
Saccharomyces spp isolates as a CMC-ase akctivity (isolation from
manure of beef Bali cattle) can be used in order to alleviate the
negative effect of rice bran as feed.
METERIAL AND METHODS
Animals and experimental design
Eighteen of Lohman Brown laying hens were assigned to three
treatments in a completely randomized design. Each treatment has six
replications with one birds per replication (individual cage). All of the
birds were fed experimental diets for two days. The treatments were (i)
unfermented rice bran as control; (ii) fermented rice bran by
Saccharomyces spp.S-6 isolate; (iii) fermented rice bran by
Saccharomyces spp.S-7 isolate; respectively. The objectives of this study
is to determine the nutrient digestibility and the ME value of rice bran
using Lohman Brownn laying hens at 42 weeks of age.
Probiotics properties
Acid tolerance: to determine the trancsit tolerance to low pH, the
method of Corzo and Gilliland (1999) was used with slight
modifications. Strain were grown in MRS broth at 370C for 24 h. A 0.5
ml aliquot of the bacterial culture was inoculated in 10 ml of phosphate
buffered saline adjusted to pH 2 with 4 N HCl. Phosphate buffer was
prepared by dissolving NaCl (9 g/l), Na2HPO42H2O (9 g/l), and KH2PO4
6-8
(1.5 g/l) in destiled water. The initial bacterial concentration was 10
0
cfu/ml. Culture were incubated at 37 C. After 0; 0.5; 1; 2; and 4 h
incubation, cell were serially diluted tenfold in 0.1 M sodium phosphate
buffer (pH 7.2), and the viable cells were enumerated on MRS agar
0
plate at 37 C for 2 days.
Bile resistance: The resistance to bile was examined according to
method of Hyronimus et al. (2000). Each strain was inoculated in MRS
broth with 0.5 or 1% (w/v) bile salt (Difco). Culture were incubated at
0
37 C for 24 h, and the numbers of viable cell were determined and
compared to a control (without bile salt) on MRS agar plates.
CMC-ase activity: Weighed as much as 11 g of OMEA and 3 g of CMCase, then dissolved in distilled water. Heat in a waterbath and after it
did strelilisasi on autoklav. Refrigerate at a temperature of 45-50 0C,
then poured on Petri dishes and left to solidify. Isolates that have been
cultured in a nutrient broth for 24 hours. Take a paper disk with
tweezers and then dipped in a solution of nutrient broth then put on a
petri dish containing Omea media and CMC-ase. Leave for 24 hours.
After 24 hours of clear zone width measurements are generated by
using a vernier caliper. The size of the clear zone and the apparent
absence of clear zone, an indicator of the ability of the microbes to
break down cellulose, as well as fast and slow arise the clear zone
(VanDevoorde and Verstraete, 1987; Kanti, 2007).
Fermented of Rice Bran
The isolate of Saccharomyces spp.S-6 and S-7 which has been approved
from bile salt and poultry digestive tract in vitro test could assimilate
cholesterol for probiotics agency and two isolates have CMC-ase
activity. The study was carried out at the Bioscience Laboratory of
Udayana University, Bali, Indonesia. Fermentation of commercial rice
bran was prepared as follows. Comercial rice bran was used.
6
Approximately 0.20% (2 x 10 spores) Saccharomyces spp. S-6 and S-7
isolate culture was added to 100 g of steamed rice bran. Then, water
was added to bring the product to 50% content and left up to 2 days for
fermentation. After that, fermented rice bran was dried at 45 0C for six
hours and then it was ground for analysis. Unfermented rice bran was
also ground for its chemical analysis.
Retention and excretion of nutrients
In order to determine the nutrient digestibility and metabolizable
energy (ME) value of the rice bran. The amount of rice bran used was
50 g. This amount as based on preliminary assays with Lohman brown
laying hens using rice bran. All the birds were deprived of feed for 24 h
to ensure that their alimentary canals were empty from feed residues.
They were then force-fed with the specific amount of rice bran
(fermented and unfermented). Stainless steel funnel with 40 cm stem
was used in force feeding technique (Mustafa et al., 2004). Water was
available ad libitum during the experimental period
The total excreta were collected in plastic trays. The excreta samples
were frozen, allowed to come to equilibrium with the atmospheric
moisture, weighed, and groun through a 1 mm sieve. Samples of
excreta and rice bran were subjected to appropriate analysis to
determine DM, OM, CP, CF, and energy, respectively.
Laboratory analyses
Dry matter (DM), organic matter (OM), CP and ash determinations were
done according to the Assocciation of Official Analytical Chemists
(l994). The CP content of the diets was determined using the Kjeldahl
procedure (AOAC, 1994). Crude fibre in the feeds were determined
using the procedure of Van Soest et al. (l991) on oven-dried samples.
Gross energy (GE) was measured with an adiabatic oxygen bomb
calorimeter (Parr, USA),
Calculations
The data were used to calculate AME value according to the following
formulate (Mustafa et al., 2004): AME (apparent metabolizable energy)
= IE – FE. Where IE = ingested energy; FE = fecal energy voided by the
fed birds.
Statistical Analysis
All data were subjected to a one-way analysis of variance test (Steel and
Torrie, 1989). Statistical significances among treatment means were
determined by method of New Multiple Range Test of Duncan when
the F value was significant at 5% level.
RESULTS
Resilience isolate Saccharomyces spp. the low pH is one of the
characteristics required to be met by a candidate or a probiotic that
could be developed into a potential probiotic. On this test, the medium
pH was adjusted to 1.5, 3.0, 4.5, and 6.0 by using HCl (hydrochloric
acid), because HCl has characteristics similar to stomach acid. Resistant
to highly acidic nature needs to be owned by the candidate probiotic,
because the application later, this probiotic candidate must pass a very
acidic stomach conditions, before reaching the colon.
Observations as presented in Table 1. In this study, as many as 10
isolates Saccharomyces spp. isolated from cattle feces was tested
resistance to various pH, at pH 1.5, 3.0, 4.5, and 6. Two isolates were
unable to grow at pH 1.5, which isolates the S-1 and S-2. Saccharomyces
spp.S-1 isolates even simply could not grow at all pH treatments given.
Only 8 isolates were able to grow well, namely Saccharomyces spp.S-4
to S-10 isolates. There is a tendency of the higher pH, especially at pH 6,
most of the isolates has decreased the number of colony life.
Saccharomyces spp colonies grow well at pH 3, even isolate 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 Saccharomyces spp (Log
colony/g) at various pH.
Code Isolate 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
Descriptions: S-1 s / d S-10 is a Saccharomyces spp isolates were
isolated from manure of beef Bali cattle
In the test of resistance to bile salts as seen in Table 2 shows, that the
ten isolates were able to survive and grow on a medium containing bile
salts at concentrations of 0.2 mM and 0.4 mM, whereas at a
concentration of 0.6 mM no isolates can survive. Microbial isolates
resistance to bile salts is used to assess the ability to survive the
digestive tract isolates contained bile salts on the surface of the
intestine.
Probiotics will be dealing with the environment in the small intestine, in
which there are bile or bile salts are removed by the liver through the
gall bladder, after successfully passing the acidic conditions in the
stomach. Therefore, in the process of developing new probiotic, or a
new probiotic candidate must be able to pass the test of resistance to
bile or bile salts were performed in vitro. Based on the nature of the
resistance shown by some isolates, strai-indicates that the strain has
the potential to be developed into a potential probiotic.
Table 2. Ability of yeast Saccharomyces spp isolates were resistant
living in bile salts.
Isolate
Bile salt concentration (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)
Description:
- : A 1.0 (highly resistant of bile salts)
S-1 s / d S-10 is a Saccharomyces spp isolates were isolated from
manure of beef Bali cattle
CMC-ase test is to test the ability of the yeast Saccharomyces spp
isolates in degrading crude fiber. It can be measured from the resulting
clear zone diameter (Table 3). From the results of this study were only
yeast Saccharomyces spp isolates S-5, S-6, S-7, S-8, and the yeast
Saccharomyces spp.S-9 who has the ability to digest fiber. Looks yeast
Saccharomyces spp isolates S-6 has the most wide clear zone, while the
S-8 has a clear zone at least. This means that isolates S-6 has
kemampuam in digesting crude fiber than the highest S-8. More
detailed, clear zone diameter and difference generated by the two
isolates are presented in Figure 1.
Isolate
S8
Isolate
S6
Clear zone
Figure 1. Saccharomyces spp.S-8 isolates zone is smaller than the
isolate Saccharomyces spp.S-6 (right).
Table 3. CMC-ase activity test yeast Saccharomyces spp isolates based
on the diameter of the clear zone caused.
Isolat
Clear zone diameter (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
Description: S-1 s / d S-10 is a Saccharomyces spp isolates were isolated
from manure of beef Bali cattle
Table 4 shows the nutrient the content of crude protein and gross
energy were slightly increased by fermentation. On the other hand
fermentation caused decreasing dry matter (DM), organic Matter (OM),
and crude fibre (CF) of the rice bran. These results indicated that
carbohydrate was used for microbial growth and the reduction of dry
matter. The result indicated that all of nutrient digestibility of
fermented rice bran by Saccharomyces spp culture were increased
significantly (P