Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 1-7 e-ISSN 2685-3167 p-ISSN 2828-6685 Endophytics Bacteria from Pangi Fruit (Pangium edule REINW.
) and Its Potential as Producer of Antibacterial Compound Natalia Veronica Sambuaga1*.
Mokosuli Yermia Semuel2 .
Helen Joan Lawalata2 Biology Study Program.
FMIPAK.
Manado State University.
Tondano.
Indonesia Department of Biology.
FMIPAK.
Manado State University.
Tondano.
Indonesia *Corresponding author: talyasambuaga@gmail.
Accepted: 10 September 2024 .
Approved : 24 Oktober 2024
ABSTRAK
Antibakteri alami dapat berasal dari berbagai jenis tumbuhan, salah satunya tumbuhan Pangi (Pangium edule REINW.
Buah Pangi memiliki kandungan senyawa antioksidan dan antibakteri yang bisa menghambat pertumbuhan bakteri.
Penelitian ini bertujuan untuk mengetahui isolat bakteri endofit yang terdapat dalam Buah Pangi dan potensinya sebagai penghasil senyawa antibakteri terhadap bakteri Escherichia coli dan Staphylococcus aureus.
Penelitian ini menggunakan metode penelitian deskripsi eksploratif.
Hasil penelitian isolasi bakteri endofit dari Buah Pangi diperoleh tujuh isolat.
Setiap isolat mempunyai morfologi yang berbeda-beda baik dari bentuk, warna, tepian, dan elevasi koloni.
Berdasarkan hasil pengujian aktivitas antibakteri, setiap isolat bakteri endofit memperlihatkan diameter zona bening yang berbeda-beda terhadap E.
,75 mm - 4,5 m.
dan S.
mm - 5 m.
Isolat bakteri endofit yang berpotensi sebagai antibakteri dengan diameter zona bening terbesar yaitu isolat PA 2.
,5 m.
terhadap coli dan isolat PA 3.
terhadap bakteri S.
Berdasarkan hasil karakterisasi morfologi isolat PA 1.
1 memiliki kemiripan dengan genus Basillus sp.
, isolat PA 1.
PA 1.
3 PA
PA 3.
1 dan PA 3.
2 memiliki kemiripan dengan genus Paracoccus alcaliphilus dan isolat PA 1 memiliki kemiripan dengan genus Alcaligenes sp.
Kata Kunci : Pangium edule REINW.
Bakteri Endofit.
Antibakteri ABSTRACT Natural antibacterials can be derived from various types of plants, one of which is the Pangi plant (Pangium edule REINW.
Pangi fruit contains an antioxidant and antibacterial compound that can inhibit bacterial growth.
This study aims to determine the isolates of endophytic bacteria contained in Pangi fruit and its potential as a producer of antibacterial compounds against Escherichia coli and Staphylococcus aureus bacteria.
This study used exploratory description research method.
Results Isolation research of endophytic bacteria from Pangi fruit obtained seven isolates.
Each Isolate has a different morphology both from the shape, color, edges, and colony elevation.
Based on the results of antibacterial activity testing, each isolate of endophytic bacteria showed different clear zone diameters.
Endophytic bacteria showed different clear zone diameters against E.
75 mm Ae 4.
5 m.
and S.
mm Ae 5 m.
Endophytic bacterial isolates That have the potential as antibacterial with the largest clear zone diameter are Isolate PA 2.
5 m.
against c oli and isolate PA 3.
against the bacteria S.
Based on the results of morphological characterization of isolate PA 1.
1 has similarities with the genus Basillus sp.
isolates PA 1.
PA 1.
3 PA 2.
PA 3.
1 and PA 3.
2 have similarity with the genus Basillus sp.
2 has similarities with the genus Paracoccus alcaliphilus and isolate PA 2.
1 has similarities with the genus Alcaligenes sp.
Keywords : Pangium edule REINW.
Antibacterial.
Endophytic Bacteria Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 1-7
e-ISSN 2685-3167
p-ISSN 2828-6685
INTRODUCTION
RESEARCH METHODS
Antibacterials are chemical or biological compounds that can inhibit the growth and activity of bacteria.
Natural antibacterials can come from various types of plants, one of which is the Pangi plant.
Pangi fruit contain compound antioxidant and antibacterial that can hinder growth bacteria1,2.
Bacteria endophyte is bacteria used as alternative producer compound antibacterial, sized micro, can found on network plants, roots, stems, leaves and fruit3,4.
Exploration of potential endophytic mikrobes an alternative to obtain new antibiotic Antibacterial compounds can be obtained from endophytic bacteria so there is no need to extract plants because endophytic bacteria that live in plants have the potential to produce the same antibacterial compounds as their host plants6,7.
Bacteria endophyte life symbiotic mutualism with plants8, namely bacteria utilise nutrition from results metabolism plant9,10 .
Besides that, the plant it is attached to also gets profit from bacteria Bacteria This capable protect plant in oppose herbivores, insects or network pathogen as well as stimulate growth plant11.
Bacteria endophyte produce compound antibiotics, antimalarials and antifungal in scale big and potential to obtain component new bioactive with different conditions.
Some bacteria endophytic from plant drug own efficacy strong antibacterial12,13.
Endophytic bacteria can maintain the sustainability of medicinal plants, especially rare plant species, so that they are not exploited continuously which will eventually result in extinction14.
Previous antibacterial and antioxidant activities of pangi fruit extract, but until now there has been no report on endophytic bacteria from pangi fruit that have the potential to act as antibacterials.
Based on the information on the content of antibacterial compounds in Pangi Fruit, researchers are motivated to conduct research on endophytic bacterial isolates that have the potential to be antibacterial from Pangi Fruit with the title Endophytic Bacteria from Pangi Fruit (Pangium edule REINW.
) and Its Potential as a Producer of Antibacterial Compounds.
Isolation of Endophytic Bacteria Endophytic bacteria were isolated from Pangi fruit and the parts of the fruit used were the flesh attached to the skin, the middle flesh of the fruit and the flesh attached to the fruit Sample then washed using running water until clean.
Sample one by one soaked in 70% ethanol for 1 minute then soaked again use solution Sodium Hypochlorite (NaClO) then rinsed using 70% ethanol as much as three Samples that have been ready planted in Nutrint Agar media containing nystatin then incubated with temperature 37AC in condition Observation done every 1 x 24 hours.
Bacteria endophyte growth purified one by one and cultivated in slanted NA media.
Every isolate bacteria endophyte cultured on two slant NA media for stock culture and working Characterization of Endophytic Bacteria Macroscopic includes observation of the shape, texture, appearance, edges, surface and color of the colony.
Microscopic observation of bacterial cell morphology including gram staining, citrate test, and catalase test3.
Gram Staining One loop of endophytic bacterial isolate is scratched on the object glass then fixed above the Bunsen flame.
Crystal violet solution is dripped on the object glass then left for 1 minute then rinsed using running water.
Drop lugol solution then let stand for 1 minute then Drop alcohol let stand for 10 seconds then rinsed.
Drop safranin solution, let stand for 1 minute then rinsed.
Dry the object glass then observe using a microscope with a magnification of 1003.
Citrate Test A total of 5.
75 grams of Simmon Citrate Agar (SCA) media was dissolved in 250 ml of distilled water and then heated in a hoplate while stirring until homogeneous.
The media was sterilized for 15 minutes, at a temperature of 121AC and a pressure of 2 Atm.
Pour the media into a test tube then tilt it to 45A and let it stand until solid.
Inoculate the bacteria aseptically with an ose needle then scratch it on Biofarmasetikal Tropis e-ISSN 2685-3167 p-ISSN 2828-6685 (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 1-7 the surface of the slanted media.
Incubate for 24 hours at a temperature of 37AC.
Positive results are indicated by a change in the color of the media from green to blue16.
Isolat PA 2.
Isolat PA 2.
Catalase Test Isolate bacteria taken as much as 1 loop then scratched on the object glass then dripped H2 O2.
Positive results are indicated by the presence of air bubbles on the surface17.
Figure 2.
Endophytic Bacteria in the Middle of Pangi Fruit Antibacterial Activity Test Pure cultures of test bacteria (S.
and E.
coli ) were taken as much as 50 AAl and put into semi-solid media .
75%) agar as much as 10 ml then inoculated into solid MHA media and then leveled.
The wells were made using sterile tip tips.
Endophytic bacterial isolates as much as 50 AAl were put into the wells that had been made and then incubated for 24 hours at a temperature of 37AC.
Observe the clear zone around the wells18.
Isolat PA 3.
Isolat PA 3.
Figure 3.
Endophytic bacteria in the flesh of the fruit attached to the seeds of the Pangi fruit
RESULTS AND DISCUSSION
Endophytic bacteria can enter plant tissue and spread directly to all parts of the plant.
Endophytic bacteria can live in vascular vessels or in intercellular spaces, roots, stems, leaves and fruits3.
The number of endophytic bacteria in plants cannot be determined with certainty, but these bacteria can be detected by isolating them on agar media.
The agar media used to isolate endophytic bacteria in this study was Nutrient Agar (NA) media.
This media contains yeast extract, peptone.
NaCl, and agar.
Endophytic bacteria can grow on NA media because this media is complex and most likely has conditions similar to conditions in plants19.
Endophytic bacteria from Pangi Fruit began to show growth after being incubated for A 48 hours .
The incubation time for at least 2 days aims to ensure that the body is endophytic bacteria, not contaminant bacteria.
The addition of nystatin .
to the media aims to maximize the isolation results and prevent contamination from fungi.
The results of cell morphology analysis, all isolates showed positive results in the catalase test and citrate test.
While in the gram test, only isolate PA 1.
1 showed gram positive while the other six isolates showed gram The results of the morphology analysis obtained the alleged genus Basillus sp.
(PA 1.
Paracoccus alcaliphilus (PA 1.
The results of the isolation of endophytic bacteria from pangi fruit obtained three isolates from the fruit flesh attached to the skin, namely PA 1.
PA 1.
PA 1.
3 (Figure .
, two isolates from the middle fruit flesh, namely PA 2.
2 (Figure .
and two isolates from the fruit flesh attached to the fruit seeds, namely PA 3.
and PA 3.
2 (Figure .
The total number of endophytic bacteria obtained from pangi fruit was seven isolates.
Isolat PA1.
Isolat PA1.
Isolat PA1.
Figure 1.
Endophytic Bacteria in Fruit Flesh Attached to Pangi Fruit Skin Biofarmasetikal Tropis e-ISSN 2685-3167 p-ISSN 2828-6685 (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 1-7 3.
PA 2.
PA 3.
1 and PA 3.
and Alcaligenes sp.
(PA 2.
(Table .
Biochemmical characterization was carried out with catalase and citrate test.
Catalase test was carried out to determine the ability of bacteria to degrade hydrogen peroxide (H2O.
through enzyme production.
Positive results are indicated by the formation of oxygen bubbles indicating that the microorganism is able to produce catalase enzyme which converts hydrogen peroxide into water H2 and oxygen O2.
Citrate test was carried out to determine the ability of microorganisms to use citrate as the only carbon used.
Table 1.
Results of Endophytic Isolate Cell Morphology Gram Citric Catalase PA 1.
PA 1.
PA 1.
PA 2.
PA 3.
PA 3.
Alcaligenes Paracoccus Paracoccus Paracoccus Based on the results of colony morphology analysis, colony PA 1.
1 which is suspected to be the genus Bacillus sp.
punctiform shape .
, glossy colony appearance, entire edge shape, viscous colony texture, milky white color, and a raised surface .
he height looks a little but even on all parts of the surfac.
flat and convex.
Colonies PA 1.
PA 1.
PA 2.
PA 3.
1 and PA 3.
2 have punctiform and circular colony shapes, milky white and cream colors, viscous and butyrous textures .
ike butte.
, dull and glossy appearance, convex surface, umbonate .
n the middle it looks a little protrudin.
and flat (Table .
Bacterial Cell Morphology Isolate Code PA 2.
Suspected Genus Bacillus sp.
Paracoccus Paracoccus Table 2.
Results of Endophytic Bacterial Isolate Colony Morphology Colony Morphology Appearan Texture Edge Isolate Code Form PA 1.
Punctiform Viscous Glossy Entire Raised PA 1.
Circular Butyrous Dull Lobate Covex PA 1.
Circular Viscous Dull Entire Flat PA 2.
Circular Viscous Dull Entire Umbonate PA 2.
Punctiform Viscous Glossy Undulate Flat PA 3.
Circular Butyrous Dull Entire Umbonate PA 3.
Punctiform Butyrous Glossy Undulate Convex Morphological characterization aims to observe the morphology of colonies and morphology of bacterial cells that have passed the selection.
Endophytic bacterial colonies that grow have different characteristics, ranging from colony shape, color, edges, appearance, texture, and surface.
This is in accordance with Shore's statement and Sathisha .
20 that endophytic bacteria in a host plant generally consist of several genera and species.
The Surface Color Milky Milky Beige Milky Milky Beige Milky diversity of bacteria is influenced by plant conditions, especially soil.
In some cases, not all plants of the same genus and species have the same endophytic bacteria.
In some plants there are distinctive and specific endophytic bacteria that inhabit the plant.
Testing the antibacterial activity of endophytic bacterial isolates was carried out using the well diffusion method and measuring the inhibition zone against E.
coli and S.
Biofarmasetikal Tropis (The Tropical Journal of Biopharmaceutica.
2024, 7.
, 1-7 e-ISSN 2685-3167 p-ISSN 2828-6685 Seven isolates of endophytic bacteria that have the potential to be antibacterial against S.
aureus and E.
coli bacteria were obtained, namely PA 1.
PA 1.
PA 1.
PA 2.
PA 3.
PA 3.
2 (Table .
The seven endophytic bacterial isolates that have been identified based on morphological, physiological and biochemical characterization of the endophytic bacterial isolates were found to be three species that were harmonized according to Bergey's book.
Manual of Determinative Bacteriology Seventh Edition .
biochemical tests that have been carried out show that isolates PA 1.
PA 1.
PA 2.
1 and PA 3.
2 reacted positively to the citrate and catalase tests.
This shows that the genus Paracoccus alcaliphilus is able to produce the catalase enzyme and use citrate to reduce nitrate to free nitrogen.
Arlita .
23 has conducted a study on the identification of carotenoid pigments in marine symbiotic bacteria caulerpa cupressioddes .
Agardh.
The results obtained in the study showed that isolate cj.
from seaweed came from the genus Paracoccus Bacillus sp.
Isolate PA 1.
1 was identified as a Bacillus sp.
species because the colonies were in the form of bacilli and gram-positive.
Data from biochemical tests that have been carried out showed that isolate PA 1.
1 reacted positively to the catalase and citrate tests.
This shows that the Bacillus sp.
species is able to produce the catalase enzyme and use citrate to be able to reduce nitrate to free nitrogen.
Alcaligenes sp.
Isolate PA 2.
1 was identified as alcaligenes sp.
which has a bacillus and gramnegative colony form.
Data from biochemical tests that have been carried out show that isolate PA 2.
1 reacts positively to the catalase test and citrate test.
This indicates that the species Alcaligenes sp.
is able to produce the catalase enzyme and use citrate to reduce nutrate to free nitrogen.
Arlita .
23 has conducted a study on the identification of carotenoid pigments in symbiotic bacteria of the seaweed caulerpa cupressioddes .
Agardh.
The results obtained in the study indicate that isolate cj.
k from seaweed comes from the genus Alcaligenes sp.
Table 3.
Diameter of Inhibition Zone of Endophytic Bacterial Isolates against Test Bacteria E.
coli and S.
Endophyti c Bacterial Isolates PA 1.
PA 1.
PA 1.
PA 2.
PA 2.
PA 3.
PA 3.
Escherichia
Staphylococcus
CONCLUSION
Based on the results of the research that has been conducted, it can be concluded:
The results of the isolation of endophytic bacteria from Pangi fruit obtained seven isolates, namely PA 1.
PA 1.
PA 1.
PA 2.
PA 2.
PA 3.
1, and PA 3.
Endophytic bacterial isolates from Pangi fruit have antibacterial activity against Staphylococcus aureus bacteria with an inhibitory diameter of 0.
75 - 4.
5 mm and against Escherichia coli bacteria with an inhibitory diameter of 1 - 5 mm.
Fifi et al.
, 201722 have conducted research on the isolation and characterization of the morphology and physiology of endophytic Bacillus sp.
bacteria from oil palm plants (Elaeis guineensis Jac.
and obtained the results of twelve isolates that were successfully isolated in this study.
REFERENCES