Selection of lactic acid bacteria as a probiotic and evaluated its performance on gnotobiotic catfish Clarias sp

This study aimed to select lactic acid bacteria (LAB) as a potential probiotic that producing anti-microbial compounds in order to treat motile aeromonads septicemia disease caused by Aeromonas hydrophila on catfish Clarias sp. and evaluated its performance on gnotobiotic catfish. The in vitro assay was done to select several LAB isolates based on antagonistic activity against pathogenic bacteria. The selected isolate was tested in vivo to observe their ability to improve growth performances of catfish. The study was conducted with five treatments consists of K(normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test). The results showed that the addition of Pediococcus pentosaceus E2211 as selected probiotic could increase survival rate, specific growth rate, and immune response towards infection of A. hydrophila. The best survival rate after challenge test was obtained in Np and Gp treatments (88.46%), followed by G treatment (65.38%), while the K+ was only 53.84%. The conclusion of this study was P. pentosaceus E2211 potentially used as a probiotic candidate for normal and gnotobiotic catfish. The presence of normal microflora with P. pentosaceus E2211 in Np treatment showed the best probiotic performance with daily growth rate 3.28%, feed conversion ratio 1.79, and total intestinal bacteria reached 108 CFU/mL significantly different from other treatments (P<0.05).


INTRODUCTION
Catfish Clarias sp. is one of the leading commodities in freshwater fish with high demand and is already reared by the society.That high demand will be demanding in increasing catfish production to fulfill the demand (KKP, 2014).The increasing production of catfish intensively often experiences a variety of problems, one of them is disease attack.The disease that often attacks the catfish is motile aeromonads septicemia (MAS) disease, also known as redness blotches disease caused by A. hydrophila bacterial infection (Kusdarwati et al., 2017).Lukistyowati and Kurniasih (2012) stated that A. hydrophila affected freshwater fish rearing and often cause outbreaks of disease with a high mortality rate (80-100%) in a short time (1 to 2 week).According to Anyanwu et al. (2015), the mortality range of catfish infected by A. hidrophila could reach 90% of mortality.Wahjuningrum et al. (2013) also reported that the infection of A. hydrophila on the density of 10 4 CFU/mL can cause a mortality of juvenile catfish up to 50% of the population.
The use of antibiotics for MAS disease control continuously appear a variety of negative impacts such as the pathogen resistance and the accumulation of toxic residues (Hao et al., 2014).Therefore, it takes an alternative prevention for MAS disease that is effective, secure, inexpensive, environmental friendly and has no negative effects on consumers.
An effort that can be done to prevent the MAS disease is using lactic acid bacteria (LAB) as probiotics candidate (control agent) on the catfish (Suskovic et al., 2010).The species of lactic acid bacteria that has been widely utilized and has been successfully commercialized as a probiotic were from the genera of Lactobacillus and Bifidobacterium.This two species of bacteria has known as probiotics candidate that are safe and able to produce bacteriocin metabolite to inhibit the growth of pathogens and increase the host immunity (Hegarty et al., 2016;Gomez et al., 2016) therefore it can be utilized as an alternative to replace the antibiotics.
Lactic acid bacteria are the gram-positive bacteria, non-spore forming bacteria, anaerobic or facultative, rod-shaped or rounded bacteria, and produce lactic acid as an end product during the fermentation of carbohydrates process (Quinto et al., 2014).Tambekar and Bhutada (2010) added that the agent of anti-bacteria like lactic acid can inhibit the growth of pathogenic bacteria because it can lower the pH, therefore, the pathogenic bacteria is difficult to survive.In this study, the performance of LAB as probiotics was observed in normal catfish and gnotobiotic catfish (Situmorang et al., 2014).According to Martin et al. (2016), the gnotobiotic animal is a group of germ-free animal and has no microorganisms living both inside and outside their body to control the microbial specifically so that the interactions between the host and the microbial can be seen clearly.In gnotobiotic catfish, the existence of good bacteria both outside their body and in their digest tracts was being abolished or reduced by using various types of antibiotics, therefore, the role of probiotic bacteria can be evaluated.This study aimed to select a few isolates of the LAB and to evaluate its performance through the catfish feed in normal catfish and in gnotobiotic catfish as an effort to control the A. hydrophila infection.

Isolate of bacteria
The bacteria that used in this study were LAB isolates of Lactobacillus plantarum E1211, Pediococcus pentosaceus E1222, P. pentosaceus E2211, P. pentosaceus E5, P. pentosaceus E7, and P. pentosaceus E8, were a collection from Laboratory of Animal Biotechnology, Research Center for Biological Resources and Biotechnology (PPSHB), Bogor Agricultural University and A. hydropliha isolates that obtained from a collection of Fish Health Laboratory, Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural University.

The pathogenicity test
The pathogenicity test carried out through in vitro test in blood agar plate.LAB isolates streaked on blood agar plate and incubated at 27-28 °C for 24 hours.The pathogenicity marked from the formation of a clear zone around the colonies.Isolates that did not form a clear zone around the colonies were then selected to be tested further.

Aeromonas hydrophila
The inhibited activity of LAB isolates towards A. hydrophila known on its size of the inhibited zone that was formed by using agar spot assay (Klose et al., 2010).Each of LAB isolate inoculated in 50 mL of liquid medium of man ragosa sharpe (MRS) and incubated at 37 °C for 10 to 14 hours (the optical density reached 0.8), then it centrifuged at 7000 rpm for 20 minutes to obtain free cell supernatant, it neutralized the pH (pH reached 6.5) by using 1 N NaOH.Meanwhile, the culture of A. hydrophila (with a density of 10 6 CFU/mL) poured in trypticase soy agar (TSA) plate.After it became solid, agar spot assay on the media is made by using sterilized straw (with autoclave for 15 minutes at 121 °C and pressure of 15 lbs).Then free cell supernatant of volume 25, 50, 75, and 100 µL inserted into each spot and incubated at 30 °C for 24 hours, observed, and measured the inhibited zones by using the ruler.

The growth curve
The bacterial growth curve aimed to find out the bacterial growth phases and to determine the harvesting time of the cells.A single colony bacteria inoculated into 20 mL of sterile media MRSB and incubated at 37 °C for 10 minutes.Then 10 mL of the cultured bacteria inoculated into 90 mL of sterile MRSB medium and incubated at 37 °C.The growth of bacteria was observed every one hour by measuring the optical density based on absorbance value using a spectrophotometer at a wavelength of 620 nm.

The mixture of selected lactic acid bacteria in feed and the viability test
The feed sterilized using an autoclave at 121 °C and 15 lbs of pressure for 15 minutes.The density of probiotics that added to feed was 10 8 and 10 10 CFU/mL.The determination of probiotics density was based on the results of the calculation of total plate count (TPC) after several hours of incubation, then was diluted using phosphate buffer saline (PBS).The selected lactic acid bacteria inoculated on MRSB medium and incubated at 37 °C.The cultured bacteria was then centrifuged at 7000 rpm for 20 minutes, the cells were taken and re-suspended with PBS in which obtained the concentrations of 10 8 and 10 10 CFU/mL.As many as 1% of probiotics and 2% of egg whites as the adhesive sprayed onto the feed.After LAB was being mixed in feed, the viability of the bacteria tested through it took 1 g of feed to be diluted into 9 mL of 0.85% NaCl and 0.1 mL of bacterial suspension streaked on MRS solid medium and incubated for 24 hours at 37 °C, and then the colonies of the bacteria were calculated.

Aeromonas hydrophila towards the catfish
The pathogenicity of A. hydrophila in catfish was tested by determining the lethal dose 50 (LD50) that indicated by the bacterial density caused 50% of fish mortality.The catfish injected with A. hydrophila (10 5 , 10 6 , 10 7 , and 10 8 CFU/ mL) through intramuscular injection and reared for 10 days in the aquarium (sizing 60×30×40 cm, containing 10 L of water with a stocking density of each aquarium was 10 fishes).The observation is done every day to obtain the mortality of fish.The concentration of A. hydrophila that was obtained from the LD50 test as much as 10 6 CFU/mL used as the infection dose of A. hydrophila upon the challenge test of catfish.

The experimental preparation of normal catfish and gnotobiotic catfish
The experimental fish that used in this study had two treatments i.e. normal catfish and gnotobiotic catfish.The preparation for gnotobiotic catfish was done by adding different types of antibiotics into the water, they were 250 mg/L of ampicillin, 125 mg/L of rifampicin, and 250 mg/L of chloramphenicol for the reduction of bacteria in the outside part of the fish body.After that, the fish was being fast for 24 hours, until then they were given feed that containing 100 µg/mL of rifampicin for the reduction of the bacteria in the digestive tract for 4-5 days (Afrilasari, 2017).The utilization of rifampicin is based on the sensitivity result of catfish's intestinal bacteria that showed the intestinal bacteria of catfish tend to be sensitive to rifampicin more than other antibiotics.

The preparation of experimental feed
The feed used for the study were commercial pellet 781 (from the production of PT.Central Pangan Pertiwi) with 31-33% of protein content.The probiotic dose that used was as much as 1% in 100 g of total feed (Wang, 2007).The feed preparation included spraying the probiotics as much as 1% with a density of 10 8 CFU/mL in the feed and the addition of 2% of egg white as an adhesive.

In vivo test in catfish
Catfish that used in the study was from catfish farmers in Dramaga, Bogor.Fish acclimated to experimental condition for a week before being given the treatment.The average weight was 8.76 g and reared in aquariums sizing 60×30×40 cm 3 with a density of 15 fishes/aquarium.The fish reared for 30 days and fed three times a day (in the 08.30a.m, 14.00 a.m and 19.30 pm, night) with feeding rate of 4-5% of fish biomass.The water quality in aquarium kept through siphoning and changing the water as much as 30% from total volume of the aquarium every two days.
After 30 days of rearing, the challenge test was done through intramuscular injection of A. hydrophila into the fish body.The fish rearing during the challenge test lasted for 10 days.This study consisted of five treatments, they were K-(normal catfish fed with feed without probiotic and not tested challenge), K+ (normal catfish fed with feed without probiotic and tested challenge), Np (normal catfish fed with feed added with probiotics and tested challenge), G (gnotobiotic catfish fed with feed without probiotic and tested challenge), and Gp (gnotobiotic catfish fed with feed added probiotics and tested challenge).

Growth parameters Survival rate (SR)
The survival rate of the experimental fish was observed at the end of the study that is calculated by using the following formula: SR = Survival rate (%) Nt = The total fish at the end of the study No = The total fish at the initiation of the study

Specific growth rate (SGR)
The specific growth rate of the fish calculated based on the average weight of every 10 days during the fish rearing.The specific growth rate of the fish is calculated by using the following formula: SGR = Specific growth rate (%/day) Wt = The average weight of fish at the end of study (g) Wo = The average weight of fish at the initiation of the study (g) t = Days of rearing

Feed conversion ratio (FCR)
The feed conversion ratio was observed during the rearing by calculating the amount of eaten feed.The feed conversion ratio is calculated using the following formula (Huisman, 1987): FCR = Feed conversion ratio F = The amount of eaten feed (g) Wt = The average weight of fish at the end of study (g) Wo = The average weight of fish at the initiation of the study (g) D = The average weight of dead fish (g)

The total amount of intestinal bacteria
The total amount of bacteria in the digestive tract of fish is calculated by using TPC (total plate count) method.The calculation of the total amount of intestinal bacteria was done after 30 days of rearing and after 10 days of challenge test.The digestive tract of catfish as much as 1 g crushed and dissolved in 10 mL of sterile PBS, then it homogenized by using vortex and 0.85% NaCl for serial dilution.The sample that has been diluted was scattered on TSA plate for obtaining total intestinal bacteria, in the other hand, MRSA used for total intestinal of LAB and MRSA with 50 µg/mL addition of rifampicin used for total probiotics bacteria, is incubated for 24 hours at room temperature, and the total of colonies was counted.

Hematological parameters Total erythrocyte
Blood samples were sucked by using a pipette with a scale of 0.5 and was added with Hayem's solution up to 101.The blood and Hayem's solution in the pipette were shacked for 3-5 min in eight form until it got the homogeneity.The first drop was disposed and the following was dropped slowly to haemocytometer, covered it with a cover glass, and observed it by using a microscope.The calculation was performed in five different points of view of haemacytometer.The total erythrocyte is calculated using the following formula:

Total leukocyte
Blood samples were sucked by using a pipette with a scale of 0.5 and was added with Turk's solution up to the scale of 11.The blood and Turk's solution in the pipette were shacked for 3-5 min in eight form until it got the homogeneity.The first drop was disposed and the following was dropped slowly to haemocytometer, covered it with a cover glass, and observed it by using a microscope.The calculation was performed in five different points of view of haemacytometer.The total leukocyte is calculated using the following formula:

Haemoglobin level
Haemoglobin level (Hb) measurement was carried out through Sahli method that converts blood into haematic acid (Gillet et al., 2009).Blood samples were sucked by using Sahli pipette to scale of 0.2 mL, transferred it into Hb-meter tube that already filled with HCl 0.1 N to scale of 10 (red colored), stirred it, and rested for 3-5 min.Afterward, an aquades was added until the blood and HCl had the similar color to the indicator of Hb-meter.The scale of matched color signified the haemoglobin level.

Phagocytic activity of fish
The phagocytic activity could be obtained from the capability of leukocyte cell to phagocyte the pathogen cell.Firstly, Staphylococcus aureus bacteria were cultured in tryptic soy broth (TSB) medium for 18 hours.After that, 10 6 CFU/mL of bacteria and fish blood was taken as much as 50 µL of it, mixed it, and was incubated at room temperature for 20 min.Then the blood dropped slowly into slide glass, withdrawn it by using another slide glass with forming 45°, and finally drained it well.That sliding glass soaked with methanol for 10 min and drained it again before being wash with Giemsa solution for 10 min.After all, it rinsed gently by using aquades and drained it again.Furthermore, it was observed by using a microscope.The phagocytic activity is calculated using the following formula:

Data analysis
Survival rate data, specific growth rate, feed conversion ratio, the total number of bacteria, and the hematological parameters analyzed by analysis of variance (ANOVA).If it was a significant difference, it continuously performed with Duncan tested on α = 0.05.The data were analyzed by using SPSS 15.0 software.

Pathogenicity test
In vitro tests in blood agar medium aimed to find out the haemolysis capability of bacteria isolates (Mangunwardoyo et al., 2010).The result of pathogenicity test from the sixth LAB isolates showed that all of that isolates in this study did not form a clear zone which on blood agar medium (Figure 1).It showed that all of six bacteria isolates were nonhaemolytic bacteria (able break down the erythrocytes).Suardana et al. (2014) reported that in some isolates, the formation of clear zone indicates the virulence of bacteria to break down the erythrocytes by extracellular bacteria protein substance known as haemolysin as an expression of its phenotype.
The formation of widely clear zones on the blood agar medium showed the β-haemolysis activity in bacteria isolates.The result of this study was in accordance with Damodharan et al. (2015) that stated LAB does not generally have a haemolytic activity.

Aeromonas hydrophila
The antagonistic test aims to find out at the ability of LAB isolates in producing antimicrobial compounds to inhibit the growth of A. hydrophila.The LAB isolates can produce various protein (polypeptide) substance that has antimicrobial activity i.e bacteriocin during the growth period (Zacharof & Lovitt, 2012).The antagonistic activity was characterized by the formation of inhibitory zones around the spot on media after it giving LAB free cell supernatant at low pH and neutral pH (pH 6.5).The LAB free cell supernatant treatment that was not neutralized aimed to maintain acidic conditions of organic acid compounds, while the neutralized Figure 1.The result of pathogenicity test from six LAB isolates on blood agar medium treatment of free cell supernatant aimed to eliminate the antibacterial influence of organic acid so that the active antibacterial substance was only the organic compounds (Puspita, 2011).All of LAB isolates showed the inhibitory activity against A. hydrophila towards low pH, yet it has not shown the inhibitory activity at neutralized pH (Table 1).This was likely due to bacteriocin that produced was still not able to inhibit A. hydrophila in the density of 10 6 CFU/ mL or the experimental bacterial isolates tend to produce organic acids.According to Khunajakr et al. (2008), LAB strain has the ability to produce organic acids that can potentially apply as probiotics.Lactic acid is able to weaken the permeability of bacteria especially in outer membranes of gram-negative bacteria through breaking down its part.
The repeat test conducted on selected three LAB isolates rely on its inhibitor zone.It was done by increasing the viscosity of supernatant concentration that produced on neutralized pH conditions.The observation result from the index of supernatant inhibitor zone against A. hydrophila showed the highest was LAB P. pentosaceus E2211, moreover, it was selected as probiotics candidate for catfish on the next experimental stages (Table 2).Rosyidah et al. (2013) reported that P. pentosaceus E2211 is one of LAB that was successfully isolated from the fermentation process (immersion) of corn spontaneously for 48 hours.P. pentosaceus E2211 is a gram-positive bacteria with coccusshaped and has negative catalase.

The growth curve of selected bacterial isolates
The curve of growing bacteria was observed through the increasing of optical density towards the time (Figure 2).The growth curves of P. pentosaceus E2211 has a lag phase (adaptation) about two hours of incubation period.The adaptation phase is an adjustment phase of the bacteria that already moved into a new medium with any different substrate and conditions of its environment (White, 2007).The time of this phase was varied, depending on the ability of bacteria to adapt to the surrounding environment.The second hour until the sixth hour of the cells entered the log phase (exponential) was characterized by increasing the OD significantly, the seventh hour to 18 th -hour, the cells entered stationary phase and at 19 thhour and so on, the cells experienced the death phases, which was characterized by decreasing value of OD.Exponential phase is the phase of bacteria active growth, moreover, the cells mass increase exponentially towards the time, while the stationary phase is a stop phase of cells growth (White, 2007) with bacterial growth that tends to go slow and remain.The death phase is the phase of decreasing the number of bacterial cells because the bacteria dies after running out of some nutrients and it is on the incompatible environmental conditions i.e an existence of toxic matter from bacterial metabolism during incubation (Klose et al., 2010).According to the growth curve that obtained from this study, the cells harvesting time for the next cells production on the next stages are performed on the exponential phase, or at the sixth hour.

The viability of probiotic bacteria in fish feed
The ability of probiotic bacteria to survive is known through testing the viability of probiotic in fish feed.The viability of fresh probiotics cultures during preparation and storage is the main thing in probiotic preparation (Wang et al., 2008) moreover the probiotics can be applied and provide benefits to its host.The density of probiotics P. pentosaceus E2211 that added into the feed for viability test is 10 8 and 10 10 CFU/ml (Table 3).Afrilasari et al. (2017) reported that the results of viability test of probiotic Bacillus megaterium PTB 1.4 (the density of 10 8 CFU/ mL) that added into feed is able to survive with a cell density of 10 6 CFU/mL and significantly affect the total intestinal bacteria of catfish, while Zare et al. (2015) reported that the addition of probiotics Pediococcus acidilactici with density of 10 10 CFU/ml also give the significant effects towards the total number of LAB in intestine.
The viability of LAB isolates P. pentosaceus E2211 was still well even though it was either experienced a decreasing in the total number of bacteria after mixing feed.The decrease in the total number of bacteria due to the number of probiotics that added to feed as much as 1% in 100 g gained 0.01% of probiotics.It showed that P. pentosaceus can be applied as a probiotic in fish through the fish feed.P. pentosaceus has been widely applied as a feed additive in the United States, around Europe, China, Thailand, Australia, and New Zealand (Lim & Tan, 2009).
According to the results of the test, the viability of P. pentosaceus E2211 in feed, the density of P. pentosaceus E2211 that used in the in vivo test of catfish was 10 8 CFU/mL.

Aeromonas hydrophila towards the catfish
The virulence of A. hydrophila can be known through the LD50 value is produced, it was the observation to a dose that capable to kill 50% of the fish population.According to Rey et al. (2009), the infection of A. hydrophila can cause clinical symptoms after several hours of infection and the mortality began after 7 hours.The infection further will cause mortality after 12-24 hours.As for the densities of A. hydrophila used in the observation of LD50 test towards the catfish were 10 5 , 10 6 , 10 7 , and 10 8 CFU/mL (Table 4).
Figure 2. The growth curve of selected isolate P. pentosaceus in MRSB medium for 19 hours at 37 °C.The arrow showed the limit of bacterial growth zenith Table 3.The viability of Pediococcus pentosaceus E2211 in fish feed

Treatments
The initial addition of bacteria (CFU/mL) The total probiotic bacteria after 30 min of mixing (CFU/mL) The 1 st viability test 10 8 5.4 x 10 6 The 2 nd viability test 10 10 1.3 x 10 9 According to the fish mortality pattern, after the fish injected with A. hydropila, it can be seen that the higher the density of A. hydrophila is injected then so does the mortality rate of fish.The density of bacteria that cause 50% of mortality in fish population within 5 days of incubation was 10 6 CFU/mL.According to LD50 test that has been performed, it obtained that the concentration of A. hydrophila was 10 6 CFU/ mL used for challenge test.Triyaningsih et al. (2014) stated that the LD50 value can indicate the level of virulence from the bacteria.Bacteria that has a LD50 value between 10 4.5 -10 5.5 CFU/ mL belongs to a high virulent group of bacteria; an LD50 value between 10 5.5 -10 7 CFU/mL belongs to a virulent group of bacteria; and LD50 more than 10 7 CFU/mL is an avirulent group of bacteria.

The growth performance of catfish
The growth performance of catfish after in vivo test was showed through its survival rate after 30 days of rearing, the specific growth rate, and the feed conversion ratio.The survival rate of catfish at all the treatments during the 30 days of rearing was 100%.This showed that the treatment of gnotobiotic and probiotic during this study was safe for catfish.Probiotic treatment produced the highest SGR compared to other treatments, but the FCR was not significantly different (P>0.05) with other treatments (Figure 3; Table 4).The specific growth rate of all the treatments tended to experience an increasing at 10, 20, and 30 days of rearing.During 10 days of rearing, the SGR value of gnotobiotic catfish (G and Gp) was lower than the controls (K-and K+).This indicated that the gnotobiotic catfish was still in its recovery time after various antibiotics treatment so that the result of the SGR value was still low.At 20 th day of rearing, there was a significant difference between the Np treatment (2.70 ± 0.24) with K-treatment (2.12 ± 0.14).The highest SGR value after 30 days of rearing was Np treatment that significantly different (P < 0.05) with four other treatments.
Np treatment was a treatment feed with the addition of probiotic P. pentosaceus E2211.This is indicated that the presence of probiotics P. pentosaceus E2211 was capable to associate with intestinal microflora in increasing the nutrients absorption thus increasing the specific growth rate of fish.The improvement of fish growth after probiotic treatment occurred due to the increase of appetite and digestibility of fish.The increasing of fish digestibility that fed with probiotics occurred because the probiotic synthesized the extracellular enzymes such as protease, amylase, and lipase, and a gr owth factor such as vitamins, fatty acids, and amino acids (Sahu et al., 2008).Xing et al. (2013) reported that fed supplementation of P. pentosaceus (10 9 CFU/g) in cobia fish (Rachycentron canadum) for two weeks showed a better growth than the control.The higher growth in probiotic treatment associated with intestinal bacteria of experimental fish.The highest total number of intestinal bacteria, total LAB, and total probiotic of P. pentosaceus E2211 obtained at Np treatment that significantly different (P<0.05) with the other treatments (Table 6).This was because in addition to the capability of bacteria to colonize optimally, the probiotic P. pentosaceus E2211 was also capable to associate with LAB microflora in the catfish's intestine so it experienced an increasing the amount of LAB in the intestine and suppressed the pathogenic bacteria.According to Hegarty (1999) the ability of probiotics in improving the performance of the growth of its host can be seen from the microbial community in its intestine.The balance of the intestinal microflora can cause the probiotic reported that the addition of the LAB probiotic Lactobacillus sp. is able to increase the number of intestinal bacteria at the end of rearing.
The existence of LAB probiotic can balance the digestion microbes through growth induction of good bacteria and inhibit pathogenic bacteria through the of lactic acid production that is bactericidal (Askarian et al., 2011).The LAB group can also produce acetic acid, hydrogen peroxide, and bacteriocin that can suppress the growth of competitors bacteria (Castex et al., 2008;Maeda et al., 2013).Moslehi et al. (2016) reported that P. pentosaceus is capable to colonize well in the digestive tract of Siberian sturgeon (Acipenser baerii Brandt, 1869) with the highest total of P. pentosaceus in intestine obtained at the treatment with doses of 2×10 9 CFU/g.The ability of bacteria to colonize well in the digestive tract of fish caused by the P. pentosaceus has the ability to survive to pass the fish gut with low pH and bile salt with an alkaline pH (Maji et al., 2016).

Aeromonas hydrophila
The survival rate of catfish after challenge test at the probiotics treatment (Np and Gp) was significantly different (P<0.05)compared with the treatment without probiotics (Figure 4).The Np and Gp treatment can reach the survival rate of 88.46% higher compared to K+ (53.84%) and G (65.38%).This was because the probiotics P. pentosaceus E2211 produces an antibacterial substance against A. hydrophila that was shown in inhibit zone at antagonistic test against A. hydrophila.Antimicrobial activity of LAB against pathogenic bacteria is due to the production of organic acids, hydrogen peroxide, and bacteriocin (Vesterlund, 2009).Damodharan et al. (2015) reported that the antimicrobial activity of P. pentosaceus KID7 against gram-positive and gram-negative bacteria comes from organic acid production.P. pentosaceus has an activity of anti-A.hydrophila through the production of the extracellular product (ECP) that produces the maximum antagonistic activity in inhibit zone as much as 9 to 10 mm, and it can still be maintained at pH 7-9.According to Soltani et al. (2015), with the anti-A.hydrophila in P. pentosaceus, it helps the probiotics to pass through the digestive tract of fish and colonize dominate in the intestine of fish thus suppressing the growth of A. hydrophila and other various pathogens in the intestine while enhancing the immunity of fish so that it can increase the survival rate of fish higher than Note: The numbers that followed with the same superscripts in the same column showed significantly different according to Duncan test at a confidence level of α 0.05 Figure 3.The specific growth rate (SGR) of catfish at 10, 20, 30 days of rearing.K-(normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test).The same superscripts showed the significantly different result according to Duncan test at a confidence level of α 0.05 the fish without probiotics.
The survival rate after challenge test was higher on catfish fed with probiotic treatment (Np and Gp) than the K+ treatment (Figure 5a, b, c, and d), it was also supported by the hematological parameters (total erythrocytes, total leukocytes, haemoglobin, and phagocytic activity after challenge test).The existence of probiotics P. pentosaceus E2211 can assist and accelerate the recovery process of fish by increasing the non-specific immune response and the resistance against MAS.The fish haematological parameters in this study were been in normal range of the total erythrocyte (1.3×10 6 cells/mm 3 ), the total leukocytes (2.15×10 4 cells/mm 3 ), and the haemoglobin (6.10 g/dL) (Svobodova & Vyukusova, 1991;Takashima & Hibiya 1995).Soltani et al. (2015) reported that the addition of Lactococcus lactis probiotics (JF831150) proved to be able to increase total leukocyte, erythrocytes, and haemoglobin of Persian sturgeon (Acipenser persicus).The total erythrocytes and haemoglobin that was higher at the addition of probiotics treatment than K+ indicated that P. pentosaceus E2211 was able to maintain the fish health status, by keeping the total erythrocytes and haemoglobin in the normal range after A. hydrophila infection.The high amount of total leukocytes and phagocytic activity in Gp and Np treatment showed that the existence of probiotic was able to induce the production of leukocytes in a massive amount against A. hydrophila infection.The existence of normal microflora in association with P. pentosaceus E2211 in Np treatment was able to increase the immunity of fish better than P. pentosaceus E2211 in Gp treatment.Sukenda et al. (2016), stated that the increase of total leukocytes caused by the response from the fish body against infectious diseases.Leukocyte cell is a phagocytes cell to prevent unwanted bacteria and spread the virulence factors in the fish body.The part of leukocyte 46.5 ± 15.97 b 0.00 ± 0.00 a 7.396 ± 1.24 a Note: K-(normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test).The numbers that followed with the same superscripts in the same column showed significantly different according to Duncan test at a confidence level of α 0.05.
Figure 4.The survival rate of catfish after 10 days of challenge test.K-(normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test)..The same superscripts showed the significantly different result according to Duncan test at a confidence level of α 0.05 cell that according to with the function are monocytes and neutrophils that indicated by the value of phagocytic activity (Utami et al., 2015).Monocytes and neutrophils are the components of blood cells that generate superoxide anion (O2 -), hydrogen peroxide (H2O2), nitric oxide (NO), peroxide nitrite (ONOO -), hypochlorite acid (HOCl), and hydroxyl radical (OH -) that have the high ability to kill microbes (Ellis, 2001).

CONCLUSION
The selected LAB isolates of P. pentosaceus E2211 is potential for probiotics candidate in normal catfish and gnotobiotic catfish (Clarias sp.), that indicated by the survival rate of Np and Gp treatment after challenge test, it was 88.46%, while K+ and G were only reached 53.84% and 65.38%.The performance of P. pentosaceus E2211 as probiotics was showed through its ability to maintaining the survival rate, the specific growth rate, the total intestinal bacteria, and the immune response of catfish to A. hydrophila infection.The existence of normal microflora in association with P. pentosaceus E2211 in Np treatment showed the best performance of probiotic with 3.28%/ day of the specific growth rate, 1.78 of the feed conversion ratio, and the total intestinal bacteria reached 10 8 CFU/mL.

Figure 5 .
Figure 5.The hematological parameters of catfish after 10 days of challenge test: (a) The total erythrocytes; (b) The total leukocytes; (c) Haemoglobin; (d) Phagocytic activity.K-(normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test).The same superscripts showed the significantly different result according to Duncan test at a confidence level of α 0.05.

Table 1 .
The index of supernatant inhibitor zone from the six isolates of lactic acid bacteria against Aeromonas hydrophila

Table 2 .
The index of supernatant inhibitor zone towards increasing the viscosity 25 times from the three isolates of lactic acid bacteria against Aeromonas hydrophila

Table 4 .
The determination of lethal dose 50 (LD50) of Aeromonas hydrophila towards the catfish

Table 5 .
The survival rate and feed conversion ratio of catfish feed after 30 days of rearing

Table 6 .
The total amount of bacteria in the digestive tract of catfish after 30 days of rearing