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Determination of Lactic Acid Bacteria Viability
In the Small Intestine of Catfish (Pangasius
djambal) by using a Radioisotope of 32P
Irawan Sugoro1, Dimar Fairuz2, Ania Citraresmini1, and Adria Prilianti Murni1
1
Center for Application of Isotope and Radiation, National Nuclear Energy Agency
Jl. Lebak Bulus Raya No. 49, Jakarta, Indonesia
2 Departement of Biology – Faculty of Science and Technology UIN Syarif Hidayatullah
Jakarta, Indonesia
ARTICLE INFO
AIJ use only:
Received date
Revised date
Accepted date
Keywords:
Catfish
Viability
Probiotic
Lactic acid bacteria
Radioisotope of 32P
ABSTRACT
The viability is important to be determined, as its probiotic potency in ther small
instestine of fish. The result can be used as a basis to determine the feeding
frequency of probiotic when applied to fish. The aim of this study is to gain
information about Lactic Acid Bacteria (LAB) viability’s in the fish small intestine,
by using 32P isotope technique. Catfish (Pangasius djambal) was used as a tested
fish, and LAB with code of P2.1 PTB as the subject of the experiment. Before the
viability tested, LAB has been labelled with radioisotope 32P, then mixed into
catfish feed. The viability could be determined through the activity of 32P counted.
The result showed that percentage of LAB viabilty in the saml intestine of catfish
was declined until the day of 7. Percentage of LAB viability was decreased at an
amount of 30% at the days of 3. Based on this result, the frequency feeding of LAB
P2.1 PTB is could be done for every 3 days.
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INTRODUCTION
The role of probiotic and its mechanism
of action in the aquaculture is continuously
being researched and developed. This is to
consider with its potency to improve the
quantity and quality of aquaculture production.
Probiotics are living microorganisms which
provide health benefit to the host, when taken
in sufficient quantities [1]. The benefits of
probiotics for aquaculture are improving the
immune system [2], improve feed efficiency
and protein retention [3] and enhance the
growth rate [4].
One of the terms for microorganisms to
be used as probiotics is the ability to colonize
the intestinal epithelial cells, thus preventing or
reducing the colonization of pathogenic
microorganisms [5]. Research on the
application of probiotics to fish in general is
still centered on its ability to increase the
growth rate of fish and the antimicrobial
effects. However, research about probiotic
viability in the digestive tract of its host is still
rarely found.
The ability of microorganisms to
colonize the host’s digestive tract can be
determined by the viability test using a
radioisotope tracer compound. Radioisotope
used in this study is radioisotope phosporus-32
(32P). The 32P had a unique trait, as a label and
radiation source, therefore these compounds
can be used as tracer in the experiments of
molecular biology for animals, plants and
bacteria [6]. Phosphorus-32 is a beta-emitter
(1.71 MeV) with a half-life of 14.3 days, which
1
is able to replace the phosphorus compound
where the cellular components made up, such
as cell membranes, RNA, DNA, and ATP as
energy cell [7] [8]. Thus it makes radioisotopes
of 32P compounds easier to binds with
microorganism cell in the viability test.
This study aims to determine the
viability of Lactic Acid Bacteria (LAB) in the
small intestine of catfish to gain the empirical
data about its potency as probiotics. The result
of viability test can also be used as a basis to
determine the feeding frequency of probiotic
when applied to fish. A lactic acid bacterium
(LAB) with code of P2.1 PTB was used in this
experiment. This bacterium was isolated from
cat fish intestine [9].
EXPERIMENTAL METHODS
Growth Curve Analysis
A lactic acid bacterium (LAB) was
inoculated into NB media in the Erlenmeyer.
The cultures were incubated with a shaker
incubator for 0, 2, 4, 8, 12, 18, 24 to 30 hours
for measure the number of cells by using of
spectrophotometer (λ600 nm) and pH of media.
Measurement of % incorporation of 32P
Measurement of % incorporation was
performed at the same time with measurement
of growth curve. A 100 µL of 32P (KH232PO4;
10 mCi) was mixed into each of Erlenmeyer
flask which also contained NB and LAB. Each
of Erlenmeyer was coded and was incubated
using shaker incubator for 0, 2, 4, 8, 12, 18, 24
and 30 hours. Each isolate in NB media was
taken 5 ml and transferred into a tube. Each of
sample tubes was centrifuged at 10,000 rpm,
and then rinsed with distilled water 2 times.
The supernatant was separated to measure its
activity with Liquid Scintillation Counter
(LSC). Percentage of incorporation was
determined by using the following formula :
% incorporation = (A-B)/A x (100%)
Notes:
A : activity of 32P in media on day of 0 (cpm)
B : activity of 32P in media on day of t (cpm)
Determination of Viability using 32P
Lactic Acid Bacteria culture with the
highest % incorporation in NB medium was
inoculated into 100 ml of NB medium. The 100
µL of 32P was added and incubated
subsequently at room temperature with 120
rpm agitation until the peak time of
incorporation. After that, the supernatant and
pellet of LAB culture was separated
(precipitation) by centrifuging at 10,000 rpm
and temperature of 40C, then rinsed with
distilled water for 2 times. The pellet was
diluted again with distilled water and mixed
with the feed. This mixture was left for 2 hours.
The pellets were inserted into an aquarium that
already contains 20 cat fishes with length of
16-19 cm. The proportion of feed given was
3% of fish’s body weight/fish. Observations
were conducted on days 0, 1, 3, 7 and 14.
Bacterial viability was known by
detecting the presence of LAB-labelled 32P in
small intestine. It was cut from the end of the
ventriculous to the base of the large intestine
and the sample was soaked in 5 ml of HCl 35%.
The mixture was homogenized with a vortex
and centrifuged at a speed of 5000 rpm. The
activity of supernatant was measured with LSC.
Viability of the bacteria was determined by
using the following formula :
% viability = A/B x (100%)
Notes:
A : activity on day of t (cpm)
B : activity on day of 0 (cpm)
RESULTS AND DISCUSSION
Growth Curve
The growth curve of LAB showed
there’s no adaptation phase in its phase (Figure
1). The bacteria was directly come into
exponential phase. It could be due to the same
media in inoculum and growth culture, hence
the adaptation could be in shorter time. After
2
12 hours, the bacteria were entered stationary
phase until the end of incubation (30 hours) but
the decline or death phase was not occured.
continue to divide. When the cells die, the
phosphorous will be released back into the
media and absorbed again by the living cells.
Fig. 1. Comparison of cell growth with a pH value.
Fig. 2. % incorporation of
32
P on bacteria cells.
During the bacteria growth, pH of
media was decreased, for until 4 hours
incubation. After that, the pH was increased
until 18 hours. It’s indicating that the bacteria
utilized nutrition in media. Acid condition
could be occured because of the degradation of
organic compound that producing volatile fatty
acid such as acetic, lactic, and propionic acid.
Degradation of protein such as peptone, meat
extract and yeast extract, which was contained
in NB, producing ammonia and have become
the causing of base condition in media [10].
The pH value is very effecting for the
metabolism of bacteria, especially for enzyme
rate [11].
Fig. 3. Mechanism of bacteria labelled by radioisotope of
% Incorporation of 32P
% incorporation of 32P in bacteria cell
was increasing during incubation (Figure 2).
The highest of % incorporation was occured at
the hour of 12th at the observation. This time
can be used as a reference for further studies to
measure cell viability in the catfish intestine.
The curve pattern showed similarity
with the pattern of growth curve (Figure 1). It
was suspected that during the division,
incorporated radioisotope of 32P has become
the part of cell such as DNA, membrane and
ATP (Figure 3). This isotope only could be
absorbed when the cells are still alive and still
32
P.
Determination of LAB Viability
The percentage of LAB viability in the
small intestine of catfish was declined until the
day of 7th (Figure 4). Percentage of LAB
viability was decreased less than 30% at the
day of 3rd. If the number of LAB cells
decreased, it will effecting the performance
rate of probiotic for the fish growth. We are
expecting the LAB’s probiotic function will
not be exhausted in the gut.
The occurence of this condition could
be due to the bacterial release with the
excretion of feces, therefore only a small
3
amount could be colonized. It can be shown
that until the day of 7th, the viability of LAB
was 5%. Other possibility is the bacteria were
died because it lost the ability to compete with
indigenous bacteria [12]. It showed that these
isolates were able to survived and colonized
the small intestine of catfish. Description on
how the bacteria could be viable can be seen in
the figure 5.
The results of this study showed that
isolates of LAB with code P2.1 PTB had
potentially become a candidate probiotic. In
addition, the decrease of LAB viability on the
day of 3rd could become the reference for the
feeding frequency. It could be done for every 3
days. The application of LAB based on the
viability data in the small intestine is useful for
the reduction in production costs due to
administration that probiotics must be given
every day.
CONCLUSION
The viability of Lactic Acid Bacteria
(LAB) could be determined by using a
radioisotope of 32P. The frequency feeding of
LAB with code P2.1 PTB was done for every 3
days.
Fig. 4. Viability of LAB in the small intestine of catfish by using
a radioisotope of 32P.
Catfish
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