Research Notes
Antimicrobial properties of Bacillus amyloliquefaciens: A beneficial
bacterium isolated from curd
J.R. Krishna1, S. Aswin2, S.B. Nikhila2, R.P. Ramya1, T.S. Swapna2, O. Veena2
1PG Department of Biotechnology, College of Arts and Science, Thiruvananthapuram,
2Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India
Corresponding author: O. Veena, Email: dr.veena@keralauniversity.ac.in
Journal of Experimental Biology and Zoological Studies. 2(2): p 173-7, Jul-Dec 2026.
Received: 31/05/2026; Revised: 22/06/2026; Accepted: 25/06/2026; Published: 05/07/2026
_____________________________________________________________________________________
Abstract
Curd, a traditional fermented dairy product, harbours a diverse bacterial community that
contributes to its nutritional and functional properties. The present study was undertaken to isolate
a beneficial bacterial strain, Bacillus amyloliquefaciens, from curd and to evaluate its antimicrobial
properties. The findings suggest that Bacillus amyloliquefaciens possesses significant antibacterial
and antifungal activities, indicating its potential application in food preservation and in the
development of natural antimicrobial agents.
Keywords: Antibacterial activity, antifungal activity, Bacillus amyloliquefaciens, Gram staining.
______________________________________________________________________________
Introduction
Indian curd is a traditional fermented dairy product that is rich in diverse beneficial microbes. In
addition to lactic acid bacteria, curd also contains diverse species of Bacillus that aid in
fermentation and provide health benefits.[1] These microbes produce acids, enzymes, and
antimicrobial compounds that enhance digestion, immunity, and ensure food safety.[2] In this
study, a resilient bacterial strain, Bacillus amyloliquefaciens, was isolated from curd having
significant antimicrobial properties against some common pathogenic and food-degrading bacteria
and fungi. Its ability to withstand extreme conditions in the digestive tract, along with its capability
to produce useful enzymes and antimicrobial compounds, highlights its potential in fermentation,
natural bio-preservation, and functional food applications.[3] Bacillus amyloliquefaciens is
recognized as an important microorganism in the fermentation industry and has numerous
promising applications in industry, medicine, and agriculture. Metabolic engineers have attempted
to utilize this bacterium as a cellular factory for the commercial production of enzymes, vitamins,
and a variety of other valuable biomolecules.[3]
Materials and Methods
Bacillus amyloliquefaciens
The study was conducted at the Department of Biotechnology, Thiruvananthapuram, using
previously characterized Bacillus amyloliquefaciens stock cultures that had been originally isolated
from curd and maintained in De Man–Rogosa–Sharpe (MRS) medium in the Departmental laboratory.
[4] The isolates had been molecularly characterized by 16S rRNA gene sequencing following
primer-based PCR amplification, and the resulting sequences were analysed using the Basic Local
Alignment Search Tool (BLAST).
Gram staining
Gram staining was done to ensure that the culture obtained was pure showing only gram-positive
bacilli. (Figure 1).[5]
Figure 1: Gram-stained cells of Bacillus amyloliquefaciens, viewed under light microscopy
(400x magnification)
Antimicrobial activity
The antibacterial activities of Bacillus amyloliquefaciens were assessed against common bacterial
pathogens, like Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and
Staphylococcus aureus, and the antifungal activity was studied against Aspergillus brasiliensis,
Aspergillus niger, Candida albicans, and Fusarium oxysporum, using the well diffusion method.[6]
The uninoculated broth served as the negative control, and 25 μL streptomycin (1 mg/mL) served
as the positive control for antibacterial assay, whereas 25 μL clotrimazole (1 mg/mL) was used as
the positive control for antifungal assay. After incubation at 37°C for 18–24 h, the zones of
inhibition (mm) were measured and compared with those of the negative and positive controls.
All experiments were conducted in three independent replicates.
Results
Isolation of bacteria
The Bacillus amyloliquefaciens strain was successfully isolated from the curd sample in MRS agar
plates.
Gram staining
Bacillus amyloliquefaciens was identified to be a gram-positive bacterium (Figure 1).
Antibacterial activity
Bacillus amyloliquefaciens showed noticeable antibacterial effect against Escherichia coli and
Serratia marcescens; but caused no inhibitory effect against Pseudomonas aeruginosa and
Staphylococcus aureus (Table 1, Figure 2).
Table 1: Antibacterial activity of Bacillus amyloliquefaciens showing zones of inhibition (mm)
against the bacterial species tested
Tests performed
Zone of inhibition (mm)
Escherichia
coli
Pseudomonas
aeruginosa
Serratia
marcescens
Staphylococcus
aureus
Control (positive)
12.00 ± 2.00
13.00 ± 1.50
11.00 ± 1.00
9.00 ± 1.50
Control (negative)
Nil
Nil
Nil
Nil
Bacillus amyloliquefaciens
9.00 ± 2.00
Nil
10.00 ±2.00
Nil
Each value represents Mean ± Standard deviations of three data sets. Since no zone of inhibition was observed for
the negative controls (value = 0), any statistical comparison between the control and test groups would be significant;
therefore, significance is not denoted in the data.
Figure 2: Antibacterial activity of Bacillus amyloliquefaciens against selected bacterial species,
demonstrated by zones of inhibition on agar well diffusion plates. The inhibition zones are visible,
indicating antibacterial activity against (a) Escherichia coli and (c) Serratia marcescens. Inhibition was
not observed against (b) Pseudomonas aeruginosa and (d) Staphylococcus aureus (MP: Bacillus
amyloliquefaciens isolate; C+: positive control; C-: negative control. Wells labelled CC, C1, and C2
belong to species that were not included in this study)
Figure 3: Antifungal activity of Bacillus amyloliquefaciens against selected fungal species, demonstrated
by zones of inhibition on agar well diffusion plates. The inhibition zones are visible against (a)
Aspergillus brasiliensis, (b) Aspergillus niger, (c) Candida albicans, and (d) Fusarium oxysporum. (MP:
Bacillus amyloliquefaciens isolate; C+: positive control; C-: negative control. Wells labelled CC, C1, and
C2 belong to species that were not included in this study)
Table 2: Antifungal activity of Bacillus amyloliquefaciens showing zones of inhibition (mm)
against the fungal species tested.
Tests performed
Zone of inhibition (mm)
Aspergillus
brasiliensis
Aspergillus
niger
Candida
albicans
Control (positive)
21.00 ± 3.00
25.00 ± 2.00
25.00 ± 2.00
Control (negative)
Nil
Nil
Nil
Bacillus amyloliquefaciens
32.00 ± 2.00
37.00 ± 4.00
37.00 ± 4.50
Each value represents Mean ± Standard deviations of three data sets. Since no zone of inhibition was observed for
the negative controls (value = 0), any statistical comparison between the control and test groups would be significant;
therefore, significance is not denoted in the data.
Antifungal activity
Marked antifungal property was shown by Bacillus amyloliquefaciens against the four fungal
strains tested viz., Aspergillus braseliensis, Aspergillus niger, Candida albicans and Fusarium
oxysporum (Table 2, Figure 3).
Discussion
Bacillus amyloliquefaciens is a non-pathogenic, spore-forming soil bacterium. It is closely related
to Bacillus subtilis; but differs from Bacillus subtilis in its ability to grow in 10% NaCl and in its
characteristic growth on potato plugs. Additional distinguishing features include an enhanced
capability for α-amylase production and the ability to ferment lactose with acid production.[7]
Bacillus amyloliquefaciens is widely employed in industrial fermentation and biopreservation.[3]
Industrially produced α -amylase from Bacillus amyloliquefaciens is used in starch liquefaction,
textile desizing, and baking. Its proteases are utilized in detergents and meat tenderization. [3,8] In
addition, the bacterium produces cellulases, xylanases, and phytases, which degrade anti-
nutritional factors and improve nutrient assimilation in animal feed.[3]
As a prolific producer of secondary metabolites, Bacillus amyloliquefaciens is considered an
important biocontrol agent in agriculture and a natural probiotic and preservative in feed and food
applications.[9,10] Owing to these antimicrobial properties, it is widely incorporated into
commercial biofungicides. When applied to crops or soil, the bacterium colonizes the rhizosphere,
directly suppressing soil-borne fungal pathogens such as Fusarium and Rhizoctonia.
Simultaneously, it releases volatile organic compounds, including acetoin, that stimulate the
plant’s induced systemic resistance (ISR).[11] Owing to the availability of diverse genetic tools,
promoters, and plasmid expression systems, it has become a valuable organism in synthetic
biology, metabolic engineering, protein expression, and genetic engineering. Despite these
advantages, Bacillus amyloliquefaciens remains less extensively studied than the Gram-negative
bacterium Escherichia coli.[3]
The present study reveals that Bacillus amyloliquefaciens possesses antibacterial properties against
Escherichia coli and Serratia marcescens, as well as antifungal properties against Aspergillus
brasiliensis, Aspergillus niger, Candida albicans, and Fusarium oxysporum. Recent studies have
demonstrated that specific strains, such as B. amyloliquefaciens BS4, exhibit strong antagonistic
activity against clinically significant Gram-negative pathogens, including Escherichia coli,
Salmonella enterica, Klebsiella pneumoniae, Shigella flexneri, and Pseudomonas aeruginosa.[12]
Natchiappan et al. (2023) also demonstrated that Bacillus amyloliquefaciens inhibits Escherichia
coli and exhibits antifungal activity against Candida species.[13] Furthermore, the study by
Kadaikunnan et al. (2015) showed that extracts of Bacillus amyloliquefaciens inhibited several
fungal species, including Aspergillus clavatus, Aspergillus fumigatus, Aspergillus niger,
Gibberella moniliformis, Aspergillus oryzae, and Curvularia lunata.[14] Its antimicrobial activity
encompasses antibacterial, antifungal, and antiviral effects.[9]
It is reported that Bacillus amyloliquefaciens secretes antimicrobial peptides and small molecules
such as bacilysin, which inhibit competing bacteria by disrupting cell wall synthesis or membrane
integrity.[9,15] Substantial research has highlighted the profound medical and biopharmaceutical
importance of Bacillus amyloliquefaciens. Central to its therapeutic potential is its ability to
synthesize a structurally diverse array of antimicrobial peptides (AMPs) and secondary
metabolites.[16] Among these, cyclic lipopeptides—specifically surfactins, iturins, and fengycins—
exhibit potent antibacterial and antifungal properties by disrupting pathogen cell membranes.
Consequently, the bacterium represents a promising resource for developing alternatives to
conventional antibiotics.
Beyond direct antimicrobial activity, Bacillus amyloliquefaciens exerts vital immunomodulatory
and metabolic effects. It is well-documented to possess strong anti-inflammatory actions, which
help mitigate systemic inflammation.[3,15] Furthermore, its dietary or clinical supplementation has
been correlated with metabolic benefits, including reduced insulin resistance, enhanced glucose
metabolism, and improved lipid profiles by lowering serum cholesterol and triglycerides.[3,17]
These combined anti-inflammatory cascades and metabolic optimizations also extend to
neuroprotection; clinical models suggest that Bacillus amyloliquefaciens has the potential to
improve neurological symptoms and accelerate recovery trajectories following ischemic stroke.[3]
Conclusion
In conclusion, Bacillus amyloliquefaciens is increasingly valued as a robust, multi-functional
probiotic. Due to its endospore-forming nature, it successfully survives the harsh conditions of the
gastric environment, such as low pH and bile salts, to deliver its therapeutic benefits to the host.[14]
Given its dual antibacterial and antifungal capabilities, together with its metabolic and anti-
inflammatory benefits, Bacillus amyloliquefaciens appears as a promising candidate for future
biomedical applications and as a potential alternative to conventional antibiotics.
Financial support and sponsorship
Nil.
Conflicts of Interest
There are no conflicts of interest.
References
1. Khan MN, Bashir S, Imran M. Probiotic characterization of Bacillus species strains isolated from an artisanal fermented milk
product Dahi. Folia Microbiologica 2023;68(5):757–69.
2. Yohannes KW, Wan Z, Yu Q, Li H, Wei X, Liu Y, et al. Prebiotic, probiotic, antimicrobial, and functional food applications
of Bacillus amyloliquefaciens. J Agric Food Chem 2020; 68(50): 14709–27.
3. Zalila-Kolsi I, Ben-Mahmoud A, Al-Barazie R. Bacillus amyloliquefaciens: Harnessing its potential for industrial, medical,
and agricultural applications—A comprehensive review. Microorganisms 2023; 11(9): 2215.
4. Süle J, Kõrösi T, Hucker A, Varga L. Evaluation of culture media for selective enumeration of bifidobacteria and lactic acid
bacteria. Braz J Microbiol 2014; 45(3): 1023–30.
5. Tripathi N, Zubair M, Sapra A. Gram Staining. [Updated 2025 Mar 28]. In: StatPearls [Internet]. Treasure Island (FL):
StatPearls Publishing; 2026 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562156/ [Last accessed on 2026
Feb 15].
6. Hossain T J. Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and
limitations. European Journal of Microbiology and Immunology 2024; 14(2): 97–115.
7. Welker NE, Campbell LL. Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis. J Bacteriol 1967; 94(4): 1124–
30.
8. Dai Y, Chen Y, Lin X, Zhang S. Recent applications and prospects of enzymes in quality and safety control of fermented
foods. Foods 2024; 13(23): 3804.
9. Saiyam D, Dubey A, Malla MA, Kumar A. Lipopeptides from Bacillus: unveiling biotechnological prospects—sources,
properties, and diverse applications. Braz J Microbiol 2024; 55: 281-95.
10. Tran C, Cock IE, Chen X, Feng Y. Antimicrobial Bacillus: Metabolites and their mode of action. Antibiotics 2022; 11(1): 88.
11. Chowdhury S P, Hartmann A, Gao X, Borriss R. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42
– a review. Front Microbiol 2015; 6: 780.
12. Palacios-Rodriguez AP, Espinoza-Culupú A, Durán Y, Sánchez-Rojas T. Antimicrobial activity of Bacillus amyloliquefaciens
BS4 against Gram-negative pathogenic bacteria. Antibiotics 2024; 13(4): 304.
13. Natchiappan S, Ramasamy S, Gokul P, Arumugam V C. Probiotic Bacillus amyloliquefaciens from milk sample of a native
cow ‘Kangeyam’ breed in forest fringe village of Coimbatore, Tamil Nadu and value addition thereof. Int J Clin Biochem Res
2023; 11(1): 19–26.
14. Kadaikunnan S, Rejiniemon T, Khaled JM, Alharbi NS, Mothana R. In-vitro antibacterial, antifungal, antioxidant and
functional properties of Bacillus amyloliquefaciens. Ann Clin Microbiol Antimicrob 2015; 14: 9.
15. Wang B, Zhou Y, Tang L, Zeng Z, Gong L, Wu Y, et al. Effects of Bacillus amyloliquefaciens instead of antibiotics on
growth performance, intestinal health, and intestinal microbiota of broilers. Front Vet Sci 2021; 8: 679368.
16. Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of
potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact 2009; 8: 63.
17. Ahmat M, Cheng J, Abbas Z, Cheng Q, Fan Z, Ahmad B, et al. Effects of Bacillus amyloliquefaciens LFB112 on growth
performance, carcass traits, immune, and serum biochemical response in broiler chickens. Antibiotics 2021; 10(11): 1427.
_____________________________________________________________________________________
© 2026 Journal of Experimental Biology and Zoological Studies
Published by UNIZOA