Intek Akuakultur.
Volume 6.
Nomor 1.
Tahun 2022.
E-ISSN 2579-6291.
Halaman 103-111 The bacteria Bacillus sp.
Potential Probiotics in the Digestive Tract of Silver Pompano Trachinotus blochii Aminatul Zahra1.
Sri Novalina Amrizal2 Jurusan Budidaya Perairan.
Fakultas Ilmu Kelautan dan Perikanan.
Universitas Maritim Raja Ali Haji Jurusan Teknologi Hasil Perikanan.
Fakultas Ilmu Kelautan dan Perikanan.
Universitas Maritim Raja Ali Haji
INFO NASKAH
Kata Kunci:
Bacillus sp.
Probiotics.
Enzyme.
Silver Pompano
ABSTRAK
Isolation of Bacillus sp.
the potential for probiotics in the digestive tract of Silver Pompano has never been carried out.
Therefore it is necessary to research the isolation and selection of Bacillus sp.
potential probiotics in the digestive tract of Silver Pompano to increase growth and feed Bacteria were isolated from the digestive tract of Silver Pompano to ensure that the isolates obtained were from Bacillus sp.
then gram staining and spore staining were performed on the isolates obtained.
After that, a Proteolytic.
Lipolytic.
Amylolytic Activity test was carried out, then a Resistance Test to Stomach Acid and Bile Salts, as well as a bacterial growth test for 24 hours.
There are six isolates of Bacillus sp.
, which have the potential for probiotics in Silver Pompano's digestive tract, which has the ability of proteolytic, lipolytic, and amylolytic enzyme activity and is resistant to acid and alkaline conditions.
Gedung FIKP Lt.
II Jl.
Politeknik Senggarang, 29115.
Tanjungpinang.
Telp :
Fax.
Email: Aminatulzahra@umrah.
Bakteri Bacillus sp.
Berpotensi Probiotik dalam Saluran Pencernaan Ikan Bawal Bintang Trachinotus blochii Aminatul Zahra1.
Sri Novalina Amrizal2 Aquaculture Department.
Faculty of Marine Science and Fisheries.
Raja Ali Haji Maritime University Fisheries Product Technology Department.
Faculty of Marine Science and Fisheries.
Raja Ali Haji Maritime University ARTICLE INFO
ABSTRACT
Keywords Isolasi Bacillus sp.
yang berpotensi probiotik pada saluran pencernaan ikan bawal bintang belum pernah dilakukan.
Oleh karena itu perlu dilakukan penelitian isolasi dan seleksi Bacillus sp.
probiotik potensial dalam saluran pencernaan ikan bawal bintang untuk meningkatkan pertumbuhan dan efisiensi pakan ikan.
Bakteri diisolasi dari saluran pencernaan ikan bawal bintang untuk memastikan bahwa isolat yang diperoleh berasal dari Bacillus kemudian dilakukan pewarnaan gram dan pewarnaan spora pada isolat yang diperoleh.
Setelah itu dilakukan uji aktivitas proteolitik, lipolitik, amilolitik, kemudian uji resistensi asam lambung dan garam empedu, serta uji pertumbuhan bakteri selama 24 jam.
Terdapat enam isolat Bacillus sp.
berpotensi sebagai probiotik pada saluran pencernaan ikan bawal bintang, yaitu memiliki kemampuan aktivitas enzim proteolitik, lipolitik, dan amilolitik serta tahan terhadap kondisi asam dan basa.
Bacillus sp.
Probiotik.
Enzim.
Bawal bintang Gedung FIKP Lt.
II Jl.
Politeknik Senggarang, 29115.
Tanjungpinang.
Telp :
Fax.
Email: Aminatulzahra@umrah.
INTRODUCTION
Silver pompano is one of the potential aquaculture commodities and has economic value.
In silver pompano aquaculture, there are several problems, one of which is feed.
high feed prices can cost up to 70% of the total production Intek Akuakultur.
Volume 6.
Nomor 1.
Tahun 2022.
E-ISSN 2579-6291.
Halaman 103-111 To overcome this problem, it is necessary to take steps to increase feed efficiency in aquaculture.
Improving feed efficiency can be done by giving Probiotics are food additives in the form of live microorganism cells that have a beneficial effect on the host animal that consumes them through balancing the microorganism flora in the digestive tract, thus helping the digestive system and supporting probiotic bacteria in the digestive tract, especially in the large (Irianto, 2.
Probiotic bacteria can be used as growth promoters to increase feed efficiency by improving enzyme activity and feed digestibility (Akhter et al.
, 2.
Widanarni et al.
, 2015.
Suprayudi et al.
To increase the success of utilization by the host, it is possible to isolate probiotic bacteria from the same species (Widanarni et al.
, 2.
One of the probiotic species that is often used in aquaculture is Bacillus sp.
The bacteria Bacillus sp.
can produce enzymes capable of metabolizing large amounts of fat, protein, and carbohydrates that can effectively increase the activity of digestive enzymes (Han et al.
, 2015.
Liu et al.
, 2.
These bacteria are also known to have the ability to produce spores that are resistant to chemical compounds and physical agents (Slepecky and Hemphill 2.
The bacteria Bacillus sp.
ND2 increased the daily growth rate significantly in catfish and had a better feed conversion ratio than the control treatment (Sumartini et al .
The bacteria Bacillus subtilis BS3.
Bacillus amyloliquefaciens BS4, and Bacillus cereus BS6 isolated from the digestive tract of sunu grouper were able to improve the growth performance of sunu grouper (Astuti 2.
Isolation of Bacillus sp.
potential for probiotics has never been Therefore it is necessary to research the isolation and selection of Bacillus sp.
potential probiotics in the digestive tract of Silver Pompano to increase growth and feed efficiency.
MATERIALS AND METHODS
Isolation and Purification of Probiotic Candidate Bacteria Bacteria are isolated from the digestive tract of Pompano.
The intestine obtained was crushed aseptically, and every 1 gram was dissolved with sterile phosphate-buffered saline solution (PBS.
NaCl 0.
8 g.
KCl 0.
02 g.
Na2HPO4 0.
15 g.
KH2PO 0.
02 g, distilled water 100 mL).
Subsequently, serial dilutions of the samples obtained were carried out from a concentration of 10 -1 to The diluted sample was then incubated at 80AC for 10-15 minutes (Slepecky and Hemphill 2.
The suspension was then inoculated on complete seawater (SWC.
bacto peptone 1.
25 g, yeast extract 0.
25 g, glycerol 3 mL, seawater 750 mL, distilled water 250 mL, bacto agar 15 .
and incubated for 24 hours at temperature 29AC.
The bacterial isolates were then separated based on the colonies that repeatedly grew to obtain pure bacterial cultures.
To ensure that the isolates obtained were from Bacillus sp.
, then gram staining and spore staining were performed on the isolates obtained.
Intek Akuakultur.
Volume 6.
Nomor 1.
Tahun 2022.
E-ISSN 2579-6291.
Halaman 103-111 Gram stain Bacterial preparations were made by dripping one drop of distilled water on the object glass.
The colonies were taken using an ose needle, then smeared into the distilled water on the object-glass.
Bacterial smear preparations were homogenized and spread over the slide.
The object glass were then fixed on a Bunsen until dry.
Fixation is done not directly over the flame but given a distance so that the bacterial cells are not damaged.
The staining procedure for bacterial smear preparations was started by dripping 2-3 drops of Gram A solution .
rystal violet dy.
, left for 1 minute, then washed with running water and air-dried.
In the second stage, 2 drops of Gram B solution .
otassium iodide/KI solutio.
were added, left for 1 minute, then washed with running water and air-dried.
Then 2 drops of Gram C solution .
cetone-alcohol solutio.
were added, left for 30 seconds, then washed with running water and air-dried.
Finally, 2 drops of Gram D solution .
afranin dy.
were added, left for 30 seconds, then passed with running water and air-dried.
The stained bacterial preparations were observed using a microscope with a magnification of 1000 times.
Spore Coloring The bacteria observed were fixed with distilled water, then the bacterial smear was flooded with malachite green and put on a stove for 2-3 minutes.
The dye is inhibited from evaporation and boiling.
Thereafter, the preparations were cooled, rinsed in distilled water and dried.
Safranin was added to the test, waited for 30 seconds, and rinsed off with distilled and dried water.
The preparations were observed under microscopy with a magnification of 1000 times.
Selection of Probiotic Bacteria A series of tests were carried out on the selection of probiotic bacteria in the gastrointestinal tract of Pompano.
The bacterial selection stage to obtain probiotic candidates was carried out following the procedure performed by Widanarni et al.
and includes the following:
Proteolytic.
Lipolytic and Amylolytic Activity Test This test aims to measure the ability of each isolate to produce protease, lipase and amylase enzymes.
Probiotic bacteria candidates were cultured at 29AC for 24 hours on media with the addition of 2% skim milk for proteolytic assay, 2% starch for amylolytic assay, and 2% olive oil for lipolytic assay.
The protease activity was indicated by a clear zone around the isolates grown on agar media enriched with skim milk.
Amylase activity was shown by the colour around the hermit becoming bright yellow on media supplemented with carbohydrates after being flooded with 1% KI solution.
And the results of lipase activity were indicated by the presence of bright green colour in the isolates grown in media enriched with fat sources after being flooded with saturated CuSO4 solution.
Resistance Test to Acid and Alkaline Conditions The resistance of gastric acid and alkaline conditions was used to test bacteria's ability to survive in the stomach, which has a low acidity .
H), and the digestive tract, which has an alkaline pH.
A total of 1 mL of probiotic candidate Intek Akuakultur.
Volume 6.
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E-ISSN 2579-6291.
Halaman 103-111 bacterial suspension was inoculated into a test tube containing 9 mL of sterile SWC broth media with pH 2.
he addition of HCl decreased pH) and pH 7.
H was increased by the addition of NaOH).
At 29oC, the suspension was Using the spread plate method, observations were made at 2, 4, 6, and 8 hours after inoculation by counting the number of colonies that grew.
The difference in colony growth between treatment and control indicated resistance to gastric acid and bile salts .
edia with pH .
The smaller the difference, the more resistant the bacteria obtained are to gastric acid and bile salts.
Bacterial Growth Test This experiment was carried out to determine the growth curve of bacteria in order to obtain the exponential phase.
1 mL of the bacterial isolate was cultured in 9 mL of liquid SWC media and incubated in a water bath shaker for 24 hours at 29AC.
Bacterial growth was monitored every 2 hours by measuring the optical density (OD) with a spectrophotometer at 620 nm.
The density value can be used to calculate the bacteria's growth curve, allowing you to determine the best time to harvest bacteria.
RESULTS
Isolation and Purification of Probiotic Candidate Bacteria Bacteria isolated from the digestive tract of Pompano obtained 15 bacterial Then the results of observations under a microscope there are 13 rodshaped bacteria and three cocci-shaped bacteria.
The results of gram staining were four gram-negative isolates and 11 gram-positive isolates.
In the results of the spore staining, seven isolates could absorb malachite green and eight isolates that could not absorb malachite green.
From these results, further for the enzyme test were bacteria that had the form of bacilli, gram-positive, and had spores, there were six isolates, namely isolates A.
M, and O.
The results of observation of shape, gram test, and spore test presented in Table 1.
Table 1.
Observation of Shape.
Gram Test, and Spore Test Isolate Form Gram Spore Test Basil Positive Spores Basil Positive Spores Basil Positive Spores Basil Positive Non Spore Cocci Negative Non Spore Basil Positive Non Spore Basil Positive Non Spore Basil Positive Non Spore Basil Positive Spores Cocci Positive Non Spore Cocci Negative Non Spore Basil Negative Non Spore Basil Positive Spores Basil Negative Non Spore Basil Positive Spores Intek Akuakultur.
Volume 6.
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Halaman 103-111 Enzyme Test The purpose of the enzyme test was to measure the ability of each isolate to produce protease, lipase and amylase enzymes.
Isolates A.
M, and O could produce protease, lipase, and amylase enzymes.
Enzyme test results are presented in Table 2.
Table 2.
Enzyme Test Results Isolate Protease Lipase Amylase Resistance Test to Acid and Alcaline Conditions The resistance of bacterial isolates to acid changes in the digestive tract is presented in the following figure 1.
OD of Bacteria at pH 7.
Resistance of bacterial isolates to pH 7.
Isolate 2 hours 4 hours 6 hours 8 hours Figure 1.
Resistance of bacterial isolates to pH 7.
5 at various observation The resistance of bacterial isolates to alcaline changes in the digestive tract is presented in the following figure 2.
Intek Akuakultur.
Volume 6.
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E-ISSN 2579-6291.
Halaman 103-111 OD of Bacteria at pH 2.
Resistance of bacterial isolates to pH 2.
Isolate 2 hours 4 hours 6 hours 8 hours Figure 2.
Resistance of bacterial isolates to pH 2.
5 at various observation Bacterial Growth Growth curve of bacteria are shown in Figure 3.
The time obtained is used as a reference for harvesting bacteria.
The highest bacterial growth in isolates A.
M, and O was at 24 hours.
Intek Akuakultur.
Volume 6.
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E-ISSN 2579-6291.
Halaman 103-111 Figure 3.
Growth curve of bacteria isolate DISCUSSION Isolation of bacteria from the gastrointestinal tract of silver pompano produced 15 bacterial isolates that were able to survive after 15 minutes of heat treatment at 80AC.
Heat treatment aims to obtain spore-forming bacteria of the species Bacillus (Slepecky and Hemphill, 2.
From the gram staining results, it is known that 10 of the 15 bacteria obtained from the isolation are gram-positive, rod-shaped bacteria.
The results of spore staining on rod-shaped bacteria showed that 6 out of 10 bacteria obtained could produce endospores, which indicated that the bacteria belonged to the Bacillus sp.
Bacillus sp.
capable of producing spherical, oval or cylindrical endospores.
Endospore formation is very common in this type of bacteria as an environmental survivorship strategy (Toldar, 2.
Bacillus produces spores that are more resistant to disinfectants, drying, and heat than their vegetative cells so that they stay alive for a long time (Kuebutornyea et , 2.
Further examination of probiotic candidates for these eight bacteria showed that the eight bacterial isolates were capable of producing protease, lipase and amylase enzymes.
Extracellular enzymes produced by candidate probiotic bacteria will help enzymes in the digestive tract of the host to hydrolyze nutrients in the feed, and as a result, the process of digestion and absorption in the digestive tract of fish will take place more easily (Afrilasari et al.
, 2.
Resistance to acidic and alkaline conditions is an essential criterion for probiotics to survive in the host gastrointestinal tract (Amraii et al.
, 2.
The probiotic candidate bacteria obtained were able to survive in both acidic and alkaline conditions because the isolates were isolated from the digestive tract of fish, so they had adapted to the very high pH fluctuations.
Bacteria that are resistant to acidic and alkaline pH conditions can live in the digestive tract where these bacteria can help the process of food absorption and can increase fish The digestive tract of fish has a harsh environment because it consists of digestive enzymes, pH variations, and bile salts.
To select candidate isolates of probiotic bacteria, it is important to pay attention to pH tolerance.
In addition, changes in pH ranging from 1 to 7.
8 in the digestive tract of fish occurred during pepsin activity, and pH values higher than 7.
8 occurred during lipid activity.
The potential of bacterial isolates to tolerate low pH is important in selecting Intek Akuakultur.
Volume 6.
Nomor 1.
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E-ISSN 2579-6291.
Halaman 103-111 The condition of pH 2 was able to affect the survival rate of probiotics, while bile salts were also able to affect the mortality of probiotics (El-Saadony et , 2.
Bacteria have 4 growth phases, namely: lag phase, exponential phase, stationary phase and death phase.
The lag .
phase for isolates A and B was at 0-4 hours, isolate C at 0-14 hours, isolate I at 0-8 hours, isolate M at 0-6 hours, while isolate O was at 0-16 hours.
In the adaptive phase, there is slow growth influenced by bacterial activity in the process of adjusting to environmental conditions such as pH, temperature, and nutrition conditions (Mardanela, 2.
Cumulatively, the lag phase is an adaptive response to stress and damage, bacteria have a shorter time to adapt, depending on their tolerance to stress (Bertrand, 2.
The exponential phase is a phase of increasing microbial activity that changes shape and also increases the number of cells to the maximum speed so that a curve is obtained in an exponential phase.
The exponential phase for isolates A and B was at 4-24 hours, isolate C at 14-24 hours, isolate I at 8-24 hours, isolate M at 6-24 hours, while isolate O was at 16-24 hours.
The bacterial growth curve in this study aims to determine the logarithmic phase to determine the harvest time.
The number of cells in the logarithmic phase is the largest number of other phases.
CONCLUSION
There are six isolates of Bacillus sp.
, which has the potential for probiotics in the digestive tract of silver pompano, which can produce protease, lipase, and amylase enzymes and survive in acidic and alkaline conditions.
REFERENCES