Research Article

Evaluation of the Antibacterial Activity of Dayak Onion Extract (Eleutherine palmifolia L.) against Pathogenic Salmonella spp. and Escherichia coli

Muhammad Mar’ie Sirajuddin1,3, Rusman Rusman2, Widodo Widodo2, Edi Suryanto2*

1Graduate School, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No. 3 Bulaksumur, Yogyakarta, Indonesia; 2Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No. 3 Bulaksumur, Yogyakarta, Indonesia; 3Departement of Food Technology, Faculty of Industrial Technology, Universitas Ahmad Dahlan, Jl. Ringroad Selatan, Tamanan, Banguntapan, Bantul, Yogyakarta, Indonesia.

Received | March 08, 2025; Accepted | April 29, 2025; Published | June 05, 2025

*Correspondence | Edi Suryanto, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna No. 3 Bulaksumur, Yogyakarta, Indonesia; Email: [email protected]

Citation | Sirajuddin MM, Rusman R, Widodo W, Suryanto E (2025). Evaluation of the antibacterial activity of dayak onion extract (Eleutherine palmifolia L.) against pathogenic Salmonella spp. and Escherichia coli. Adv. Anim. Vet. Sci. 13(7): 1517-1524.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.7.1517.1524

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Foodborne diseases represent a major global threat to public health. These illnesses primarily manifest as infections or irritations of the gastrointestinal tract, caused by the consumption of food or beverages contaminated with pathogenic bacteria (Rahim et al., 2016). Toxins produced by pathogenic bacteria can lead to infections, damage tissues and cells, and multiply while destroying healthy cells in the human body. Several diseases resulting from pathogenic bacterial infections include pneumonia, tuberculosis, cholera, diarrhea, typhoid fever, and leptospirosis (Henkel et al., 2010).

In the European Union (EU), Salmonellosis is the second most commonly reported gastrointestinal illness, primarily caused by the consumption of food contaminated with Salmonella spp. In 2018, Salmonellosis was responsible for 50% of all reported foodborne outbreaks in the EU (Ehuwa et al., 2021). This disease remains a significant challenge in numerous countries, including in the United States. Salmonella spp. are responsible for approximately 20% of foodborne illnesses in the United States (O’Bryan et al., 2022). Food products derived from poultry, beef, and animal feed are documented as the most prevalent sources of Salmonella contamination (Ehuwa et al., 2021). To reduce the presence of pathogenic bacteria in food, effective measures must be taken to eliminate or inhibit both Gram-positive and Gram-negative toxigenic microorganisms. One promising approach is the use of food additives (Singh, 2018).

Food additives are substances added to food products to alter or enhance their characteristics. They serve various purposes, such as preserving freshness, preventing oxidation, enhancing flavor, improving appearance, and enriching the nutritional value of meat products (Zulkarnain et al., 2021). Food additives with antibacterial properties help maintain food safety and quality by inhibiting the growth of toxin-producing pathogenic bacteria (Quinto et al., 2019). Antibacterial food additives are generally classified into two types: synthetic and natural. Excessive use of synthetic additives may cause adverse effects such as allergic reactions, blood disorders, and liver toxicity (Apriyanti et al., 2016; Farid et al., 2023).

Researchers are increasingly exploring natural plant sources as potential alternatives to synthetic food additives. This interest is driven by the abundance of bioactive compounds in plants that exhibit antibacterial activity (Melgarejo et al., 2006; Negi, 2012). The Dayak onion, native to Kalimantan, is known for its ability to inhibit harmful bacteria (Yuliantari et al., 2017; Yusnita et al., 2018). Studies by Harlita and Asnani (2018) have shown that Dayak onions possess antimicrobial activity against several pathogenic bacteria, including Staphylococcus aureus, Bacillus cereus, Shigella sp., and Pseudomonas aeruginosa. The greatest inhibition was observed at an extract concentration of 10 mg/mL, with a minimum inhibitory concentration (MIC) of 2 mg/mL. Romadhon et al. (2023) Also reported that Dayak onion extract demonstrated antimicrobial effects against Escherichia coli and Staphylococcus aureus at a concentration of 2 mg/mL. The antibacterial efficacy of Dayak onions is linked to their rich content of various secondary metabolites, such as alkaloids, saponins, phenolics, flavonoids, and triterpenoids (Warsiti et al., 2019).

Makarewicz et al. (2021) stated that there is a relationship between polyphenols and the human gut microbiota, including interactions with both pathogenic and beneficial bacteria. As a natural antibacterial agent rich in polyphenols and secondary metabolites, its effects on both pathogenic and non-pathogenic bacteria must be evaluated to ensure it does not disrupt the diversity or balance of the gut microbiota.

The antibacterial properties of Dayak onion extract, attributed to its flavonoid and phenolic content, function analogously to synthetic antimicrobial agents. For instance, A Aziz et al. (2022) demonstrated that structural modifications of ciprofloxacin, such as N-4 piperazinyl derivatives, enhance its efficacy against Gram-positive bacteria like Staphylococcus aureus by improving membrane penetration and target binding. This principle aligns with the extract’s effectiveness against Salmonella spp. The study indicated that these derivatives achieve lower minimum inhibitory concentration (MIC) values (1.70 µg/mL) compared to unmodified ciprofloxacin (5.49 µg/mL), illustrating how specific chemical modifications can enhance antimicrobial activity. Similarly, the flavonoid-metal chelation in Dayak onion may mimic the metal-binding properties of synthetic quinolones, interfering with bacterial DNA gyrase (A Aziz et al., 2022). This interaction between natural compounds and synthetic design approaches highlights the potential of plant-derived metabolites as frameworks for developing new antimicrobial agents.

MATERIALS AND METHODS

Preparation of Dayak Onion Powder

Fresh Dayak onion was prepared following the method of Lee et al. (2016). The Dayak onions were from a garden in Godean District, Sleman Regency, and harvested at six months of age. The onions were separated from roots and stalks, washed under running water to remove dirt, cut into pieces of 1–2 cm, and prepared for drying. Drying was carried out based on the method of Gong et al. (2022), with modifications involving the use of a deep freezer and adjusted freeze-drying temperatures. A 500 g sample of Dayak onion was frozen at −80 °C for 24 hours using a deep freezer, then dried at −52 °C for 24 hours using a freeze dryer. Another sample was dried in an oven at 55 °C for 24 hours. The dried samples were ground for 20 minutes and filtered through a 60-mesh sieve.

Dayak Onion Extraction

The extraction of Dayak onion was conducted following the method of Munaeni et al. (2019). The Dayak onion powder was macerated in 96% ethanol at a ratio of 1:4 (w/v) for 24 hours at room temperature, with continuous stirring using a magnetic stirrer throughout the maceration process in the same container. The resulting maceration was filtered through Whatman filter paper number 41. The meserated extract was then evaporated using a rotary evaporator at 40°C for 1 h. The appliance was turned off, the boiling flask was removed, and thick filtrate sediment was collected using a spatula. The thick extract from the meseration was weighed and stored in a dry and tightly closed container at − 20°C for further analysis (Santoso et al., 2021).

Microbial Inhibition Assay

Bacterial inhibitory activity was tested using the Kirby-Bauer test method. The bacteria tested were gram-negative pathogenic bacteria, namely Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091, and gram-positive bacteria, namely Lactobacillus acidophilus ATCC 4356 and Lactobacillus sp. FNCC 0020. The Dayak onion extract was prepared at various concentrations to test its antibacterial activity. Each concentration was tested in six replicates. Tetracycline was used as the positive control, and sterile distilled water served as the negative control.The samples tested included KP = Tetracycline 11 ppm, KN = Negative Control, B1 = Dayak Onion Extract 1 mL/L, B2 = Dayak Onion Extract 3 mL/L, B3 = Dayak Onion Extract 5 mL/L, B4 = Dayak Onion Extract 7 mL/L, B5 = Dayak Onion Extract 9 mL/L, and B6 = Dayak Onion Extract 11 mL/L.

Rejuvenation of Bacterial Culture

Bacterial culture rejuvenation was carried out according to the procedure of Wahyudi et al. (2011). Five pure cultures of Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, Escherichia coli FNCC 0091, Lactobacillus acidophilus ATCC 4356, and Lactobacillus sp. FNCC 0020 were used. A sterile inoculating loop was used to transfer bacteria into a test tube containing 7 mL of nutrient agar solution. The tube was inoculated with MRSA and NA slanted media, and the mixture was then placed in an incubator at 37°C for 24 hours.

Minimum Inhibitory Concentration (MIC) Assay

The MIC test was conducted following the Idiawati et al. (2017), with slight modifications.The method is slightly modified by pouring media, extracts, or bacterial suspensions using a single micropipette. This study aimed to obtain the minimum concentration that inhibit bacterial growth. The pathogenic bacterial cultures used were Salmonella typhimurium ATCC 700720 and Escherichia coli FNCC 0091, which have been standardized using optical density, namely the 0.8 McFarland standard, which is equivalent to 1.5 × 108 CFU/mL. Sample groups were put together in a 96-well microplate: positive control group (KP), negative control group (KN), and Dayak Onion extract group.

Each well of the microplate was filled with Nutrient Broth (NB) medium and 100 µL of test solution. The solution of Dayak onion extract was added to the wells at a volume of 100 µL from column 1 to column 10. Column 1 contains Dayak onion extract with a concentration of 11 mL/L, then columns 2 to 10 are diluted by the dilution method. Distilled water was added to column 11 as a negative control, and tetracycline at a concentration of 11 mL/L as a positive control in column 12. Fifty microliters of the bacterial suspension was added with 50 µL of bacterial suspension and incubated in an incubator for 24 h at 37 °C (Sandasi et al., 2010).

Statistical Analysis

Statistical analysis was performed using a completely randomized design (CRD). The data were analyzed using one-way analysis of variance (ANOVA) to determine significant differences among treatments. When significant effects were found at the 5% probability level (P < 0.05), Duncan’s New Multiple Range Test (DMRT) was applied as a post hoc test to compare group means. All analyses were conducted using SPSS software, version 21 (Hummel et al., 2021; Simon et al., 2021).

RESULTS AND DISCUSSIONS

Statistical analysis revealed a significant reduction in pathogenic bacterial growth (P < 0.01) across various treatments (Table 2). These treatments involved Dayak onion extract at various concentrations: 1 mL/L (B1), 3 mL/L (B2), 5 mL/L (B3), 7 mL/L (B4), 9 mL/L (B5), and 11 mL/L (B6). The highest concentration of 11 mL/L (B6) showed the largest inhibition zone among the extract treatments, although it was still less effective than the positive control, tetracycline. Dayak onion extract effectively inhibited the growth of Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091. These outcomes align with those of Hidayah et al. (2021), who highlighted the potent antibacterial properties of Dayak onion extract in suppressing the activity of pathogenic bacteria, particularly Salmonella spp. A previous study has shown that onions contain bioactive components capable of effectively suppressing the proliferation of Salmonella spp. (Indu et al., 2006).

 

Table 1: Diameter of the inhibitory power of Dayak onion extract.

Treatment	Gram-Negative			Gram-Positive
	Escherichia coli FNCC 0091	Salmonella typhimurium ATCC 700720D-5	Salmonella Enteritidis ATCC 14030.	Lactobacillus acidophilus ATCC 4356	Lactobacillus. sp. FNCC 0020
KN	0.00d	0.00c	0.00d	0.00b	0.00b
KP	22.13a	17.59a	17.28a	22.76a	27.46a
B1	15.26c	15.36b	7.00c	0.00b	0.00b
B2	15.53c	15.26b	11.37bc	0.00b	2.96b
B3	15.36c	15.53b	10.37bc	0.00b	0.00b
B4	16.13c	16.13b	11.63bc	0.00b	5.43b
B5	17.53b	17.53a	12.73ab	0.00b	2.96b
B6	17.73b	17.73a	12.96ab	0.00b	0.00b
SEM	1.26	1.16	1.09	1.57	1.91b
P-value	0.001	0.001	0.001	0.001	0.001
95% CI	± 5.42	± 4.99	± 4.69	± 6.76	± 8.22

 

a, b, c, d, e Different superscripts in the same column indicate significant differences (P < 0.05). KN: Negative control; KP: Positive control (tetracycline 11 mL/L); B1: 1 mL/L; B2: 3 mL/L; B3: 5 mL/L; B4: 7 mL/L; B5: 9 mL/L; B6: 11 mL/L.

 

Elgueta et al. (2021) explored the mechanism behind this inhibition, demonstrating the bactericidal effect of hydroethanolic extract of red onion on Salmonella enteritidis. These onion extracts impede Salmonella enteritidis growth and affect its ability to form biofilms and attach to human intestinal cells. This suggests that onion extract interferes with Salmonella enteritidis virulence factors by hindering growth and colonization. The antibacterial activity of onions is linked to the presence of phenolic compounds and secondary metabolites with antimicrobial properties. These substances disrupt bacterial permeability, enzymatic activity, and other crucial processes, thereby inhibiting bacterial growth.

The antibacterial efficacy of the Dayak onion extract is likely attributed to its flavonoid and phenol content. This finding aligns with the study of Kamsu et al. (2019), who reported that the antimicrobial properties of plant extracts correlate with their phenol and flavonoid contents. Higher concentrations of these compounds in plant extracts are associated with increased antimicrobial activity. phenolic compounds play a significant role in the inhibitory effect of the onion extract. Santacruz and Medrano (2021) further supported this finding, reporting that phenol compounds exhibit inhibitory effects on Salmonella spp., with in vitro microbiological analysis showing significant antibacterial properties at 1% and 1.5% concentrations. Phenol compounds, especially those from sources rich in polyphenolic components, show promise in combating Salmonella typhimurium. These compounds inhibit bacteria by compromising membrane integrity or blocking specific enzymatic activities (Vallejo et al., 2020).

Additionally, research has shown that both pure phenolic compounds and phenolic extracts from Nordic berries demonstrate antimicrobial activity against intestinal bacterial species, including Salmonella spp. Puupponen‐Pimiä et al. (2005) has shown that flavonoids extracted from Dayak onion possess antibacterial properties. This finding is supported by multiple studies that highlight the various ways flavonoids combat bacteria, including damaging bacterial membranes, preventing biofilm formation, impeding cell envelope and nucleic acid synthesis, disrupting electron transport chains and ATP production, and creating antibacterial complexes with metals (Rofida et al., 2020).

The antibacterial properties of Dayak onion extract are also related to the content of secondary metabolites, such as flavonols, found in various plant species, which are known for their antimicrobial properties. Studies have revealed that flavonols, especially catechins and theaflavins, demonstrate antibacterial effects against common harmful bacteria, such as S. aureus, E. coli, and H. pylori (Sychrová et al., 2022). The chemical structure of flavonols contributes to their antibacterial activity, and hydrocarbonyl substitution significantly enhances their efficacy (Fei et al., 2014). Flavonols exert their antibacterial effects through several mechanisms, including transferring transduction energy to the bacterial cytoplasmic membrane, inhibiting bacterial movement, and disrupting essential intracellular processes necessary for bacterial survival (Taylor et al., 2005; Sa’adah et al., 2022).

The antibacterial efficacy of Dayak onion extract is primarily attributed to its substantial flavonoid and phenol content, which disrupts bacterial membranes and impedes essential enzymatic functions (Kamsu et al., 2019; Vallejo et al., 2020). These natural compounds may function similarly to synthetic metal complexes, as elucidated by Abdel-Mawgoud et al. (2017), where Fe(III), Cu(II), and Ni(II) complexes demonstrated enhanced antimicrobial effects through metal-ion coordination and disruption of electron transport. This suggests that flavonoids in Dayak onion may chelate metal ions within bacterial cells, thereby augmenting their inhibitory effects. The extract’s activity against Salmonella spp. is dose-dependent, corroborating this mechanism, as higher concentrations provide more phenolic ligands for metal binding, resulting in increased membrane disruption and oxidative stress in pathogens.

RESULTS AND DISCUSSION

Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) test is a commonly used technique to determine the lowest concentration of antibacterial agents that inhibit bacterial growth. This method plays a vital role in evaluating the effectiveness of Dayak onion extract in suppressing bacterial proliferation. The MIC test results were used to determine the minimum concentration of antibacterial compounds required to inhibit the growth of Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091. Sterile distilled water was used as the negative control (Table 3).

 

Table 2: Results of the turbidity test for MIC determination of Dayak onion extract against bacteria.

Concentrations (mL/L)	Escherichia coli FNCC 0091	Salmonella typhimurium ATCC 700720D-5	Salmonella enteritidis ATCC 14030	Lactobacillus sp., FNCC 0020	Lactobacillus acidophilus ATCC 4356
11 mL/L	-	-	-	-	+
5.5 mL/L	-	-	-	-	+
2.75 mL/L	-	-	-	-	+
1.375 mL/L	-	-	-	+	+
0.687 mL/L	-	+	+	+	+
0.343 mL/L	-	+	+	+	+
0.171 mL/L	+	+	+	+	+
0.085 mL/L	+	+	+	+	+
0.042 mL/L	+	+	+	+	+
0.021mL/L	+	+	+	+	+
Negative Control	+	+	+	+	+

 

Description: (+) = Bacterial growth present; (–) = No bacterial growth.

 

The findings indicate that onion extract inhibits the proliferation of Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091, as shown by clear zones at concentrations of 11 mL/L, 5.5 mL/L, 2.75 mL/L, and 1.37 mL/L for Lactobacillus sp FNCC 0020. Growth suppression was noted at concentrations of 11, 5.5, and 2.75 mL/L. The study identified 1.37 mL/L as the optimal extract concentration for bacterial inhibition, as it effectively suppresses Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091, without hindering the growth of non-pathogenic bacteria Lactobacillus sp FNCC 0020 and Lactobacillus acidophilus ATCC 4356. This observation aligns with Mangalisu et al. (2022), who stated that antibacterial agents should inhibit bacterial growth without affecting non-pathogenic bacteria. Additionally, Noda et al. (2018) supported this finding, suggesting that antimicrobial agents such as bacteriocins from Lactobacillus Brevis can target specific pathogens while maintaining the growth of non-pathogenic bacteria. Following this, the minimum bactericidal concentration test was performed by culturing the test results with concentrations of 11 mL/L, 5.5 mL/L, 2.75 mL/L, and 1.37 mL/L on solid media. The results of the minimum bactericidal concentration test are displayed in Figures 1.

 

The research shows that onion extract inhibits the growth of Salmonella enteritidis ATCC 14030, Salmonella typhimurium ATCC 700720D-5, and Escherichia coli FNCC 0091, as demonstrated by inhibition zones at concentrations of 11 mL/L, 5.5 mL/L, 2.75 mL/L, and 1.37 mL/L. Lactobacillus sp FNCC 0020 was inhibited at 11, 5.5, and 2.75 mL/L. This antibacterial effect is likely due to the extract’s high content of sulfur compounds and flavonoids, which are known for their antimicrobial properties (Esakki et al., 2024).

Variations in inhibition suggest species-dependent efficacy. The study identifies 1.37 mL/L as the optimal concentration for inhibiting pathogenic bacteria while allowing the growth of beneficial bacteria such as Lactobacillus sp FNCC 0020 and Lactobacillus acidophilus ATCC 4356 (Omoniyi and Yaqub, 2024). This finding supports Mangalisu et al. (2022), who argued that antibacterial agents should target harmful bacteria without affecting beneficial ones. The selective inhibition of pathogenic bacteria by the extract while preserving beneficial microorganisms indicates the potential for improving antimicrobial treatments.

This ability to distinguish between harmful and beneficial bacteria is crucial for maintaining microbial balance (Jahani-Sherafat et al., 2023). This specificity could be valuable in applications that require a balanced microbial ecosystem. Noda et al. (2018) also support this by demonstrating that bacteriocins from Lactobacillus Brevis can target specific pathogens while allowing non-pathogenic bacterial growth. The minimum bactericidal concentration test was conducted by culturing results with concentrations of 11 mL/L, 5.5 mL/L, 2.75 mL/L, and 1.37 mL/L on solid media, as shown in Figures 1.

CONCLUSIONS AND RECOMMENDATIONS

All treatments with Dayak onion extract at various concentrations inhibited the growth of pathogenic bacteria but did not affect Lactobacillus acidophilus ATCC 4356. The optimal concentration for inhibiting pathogenic bacteria, as determined by the MIC test, was 1.37 mL/L.

ACKNOWLEDGEMENTS

The author expresses his deepest gratitude to the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia for the Unggulan scholarship, which enabled him to complete his education smoothly. He also thanks Universitas Gadjah Mada for the grant Rekognisi Tugas Akhir, which enabled him to conduct his research smoothly, as well as the entire team of Promoters for their guidance and advice.

NOVELTY STATEMENTS

This study examines the antibacterial effectiveness of local Dayak onion extracts from Indonesia against pathogenic and non-pathogenic bacteria, as well as determining the minimum dose that can inhibit bacteria, so that it can be used as a reference for industry and future researchers. Researchers also found that Dayak onion extracts have good selectivity, as they can inhibit the growth of pathogenic bacteria but do not significantly inhibit non-pathogenic bacteria.

AUTHOR’S CONTRIBUTIONS

Muhammad Mar’ie Sirajuddin conducted the experiments, performed laboratory analyses, evaluated the data, and prepared the initial manuscript draft. Edi Suryanto supervised the experimental work and revised the manuscript. Rusman supervised the experimental work. Widodo contributed to the experimental design and revised the manuscript. All authors were involved in the review process and provided final approval of the manuscript.

Conflict of Interest

All authors declare that they have no competing interests.

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