Multifunctional lactic acid bacteria from sugarcane juice with probiotic properties for gut adaptation, health benefits, food preservation and quality enhancement

Abstract

Lactic acid bacteria (LAB) are promising probiotics and natural biopreservatives, yet non-dairy sources remain underexplored. We identified four LAB from sugarcane juice−Limosilactobacillus fermentum SCJ26, Lactiplantibacillus plantarum SCJ27, L. fermentum SCJ28 and Lactiplantibacillus pentosus SCJ29, evaluated through an integrated “safety-to-application” framework. Unlike single-endpoint assessments, we combined gut-adaptation (acid, bile, phenol tolerance; adhesion traits), functional bioactivities (antimicrobial, antifungal, antioxidant, anti-biofilm), safety (antibiotic susceptibility, absence of hemolysis and DNase), and technological properties (EPS production, milk coagulation, biopreservation). SCJ28 and SCJ29 showed the highest simulated gastrointestinal survival, while SCJ26 and SCJ29 displayed superior aggregation and hydrophobicity. Functional bioactivities were strain-specific: SCJ27 produced inhibition zones up to ~ 20 mm against E. coli; SCJ29 reduced E. coli biofilm by ~ 53% and SCJ27 reduced S. aureus biofilm by ~ 70%; SCJ29 reached ~ 86% DPPH scavenging. Filtered supernatants effectively reduced microbial loads in food matrices, with reductions of up to 3.8 log₁₀ in fish and 3.6 log₁₀ in tomato. The isolates produced exopolysaccharides and coagulated milk, underscoring technological potential. None of the isolates exhibited hemolytic or DNase activity, and their antibiotic susceptibility profiles were typical for LAB, but warrant genomic follow-up to ascertain resistance traits are intrinsic. Overall, the LAB strains exhibited multifunctionality, supporting both health and food applications.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40844-5.

Introduction

Probiotics, particularly lactic acid bacteria (LAB), have emerged as pivotal players for their roles in gut health, immune modulation, antimicrobial defense, and food preservation^1–3. These advantages have increased interest in identifying novel probiotic strains with broader functional repertoires, especially those that combine health-promoting, technological, and food biopreservation traits³. While dairy-derived strains are the most studied probiotics^4–7, their adaptability and performance can vary under different host and processing conditions⁸. This has motivated exploration of alternative sources, especially sugar-rich, plant-based environments. Fresh sugarcane juice abundant in fermentable sugars, vitamins, and phytochemicals, represents an underexplored reservoir for isolating novel LAB⁹. The unique selective pressures of this sugar-rich environment may yield strains with functional traits distinct from dairy-derived counterparts, such as enhanced fermentative capacity and stress tolerance. Unlike earlier reports that assessed only single probiotic endpoints, the present study provides an integrated “safety-to-application” profile of sugarcane-derived LAB. We linked gut-adaptation markers (acid, bile, and phenol tolerance; auto-aggregation, co-aggregation, and surface hydrophobicity) with functional bioactivities (antimicrobial, antifungal, anti-biofilm, antioxidant) and food biopreservation. The novelty lies both in the choice of source – fresh, locally consumed sugarcane juice – and in the combined evaluation of probiotic, safety, and technological traits within the same strains, an approach not commonly applied in earlier single-endpoint studies.

Importantly, while sugarcane-derived LAB may not be intrinsically superior to dairy- or other plant-derived strains, our objective was to survey an underexplored, sugar-rich non-dairy niche and to generate an integrated ‘safety-to-application’ dataset that has not previously been available for fresh sugarcane juice isolates. Although individual traits may overlap with those reported for LAB from other sources, the combined multifunctional profile encompassing gut adaptation, safety assessment, technological attributes, and preliminary real-food biopreservation, has not previously been reported for LAB isolated from sugarcane juice.

Beyond source selection, the probiotic potential of LAB, therefore, depends on their resilience and functionality under host-relevant stresses¹⁰. Resistance to acid, bile, and phenolic compounds determines survival during gastrointestinal transit, while aggregation and adhesion traits underpin colonization and pathogen exclusion¹¹. Together, these characteristics support persistence in the gut, barrier protection, and immune modulation, thereby forming the biological basis for evaluating novel LAB candidates¹². Safety evaluation is also essential, since some LAB may harbor transferable resistance genes or produce harmful enzymes. Standard phenotypic assessments include hemolysis, DNase activity, and antibiotic susceptibility¹³; however, genomic confirmation is increasingly recommended to distinguish intrinsic from potentially transferable resistance¹⁴. Additionally, investigating the technological attributes of LAB, such as their ability to coagulate milk and produce exopolysaccharides¹⁵, can provide insights into their multifunctionality and incorporation into food systems. Beyond gut adaptation, LAB also contribute broader health benefits and food-preservation potential. Their antimicrobial activity, mediated by organic acids, bacteriocins, and other metabolites, inhibits foodborne pathogens and spoilage organisms^14,16. By limiting microbial growth, LAB can extend the shelf life of perishable foods such as fish, fruits, and vegetables, thereby linking probiotic functionality with practical biopreservation applications¹⁷.

Therefore, this study aimed to isolate LAB from fresh sugarcane juice and comprehensively evaluate their probiotic, functional, and technological attributes. Specifically, we integrated assays of gut adaptation, safety, antimicrobial and antioxidant activities, and real-food preservation into a single framework. This holistic evaluation highlights sugarcane juice as a valuable source of multifunctional probiotic candidates with potential applications in health promotion and natural food preservation.

Materials and methods

Sample collection and LAB isolation

Samples were procured in sterile containers from street‑side sugarcane juice vendors in Chattogram city, Bangladesh, to represent locally consumed juice. In parallel, whole sugarcane sticks were collected, washed, and mechanically blended to prepare fresh juice under aseptic conditions. All sample collection and laboratory processing followed standard microbiological aseptic procedures to minimize external contamination. Serial dilutions up to 10^{− 6} were prepared in sterile saline and plated on MRS agar The MRS agar was supplemented with sodium azide (0.2 g/l) to suppress Gram-negative bacteria, bromocresol purple (0.12 g/l) as a pH indicator to facilitates visual detection of acid-producing LAB colonies, and 2% sucrose. Plates were incubated aerobically at 37 °C for 48 h. Distinct yellow-zone colonies (n = 30) were picked; 20 Gram-positive, catalase- and oxidase-negative isolates consistent with LAB were retained for further screening, and four isolates showing the strongest antagonistic activity were preserved in 20% glycerol at −80 °C¹⁸.

Preparation of cell-free supernatant (CFS)

To obtain filtered supernatant, LAB cultures were centrifuged at 10,200×g for 10 min at 4 °C (Kubota, model 6930), and the supernatant was collected. The supernatant was then passed through a 0.22 μm syringe filter to remove bacterial cells and is hereafter referred to as CFS¹⁹.

Screening and characterization of LAB isolates

Preliminary screening and characterization The isolated LAB strains were initially screened by Gram staining, catalase, and oxidase tests. Gram-positive, catalase-negative, and oxidase-negative isolates were selected for further biochemical characterization, including carbohydrate fermentation tests.

Secondary screening using well diffusion method of antimicrobial assay The antimicrobial activity of the selected isolates was assessed against Bacillus cereus, Staphylococcus aureus, Salmonella typhi, and Escherichia coli using agar well diffusion method as previously described²⁰. Sterile MRS broth was used as negative control to exclude carrier effects.

Biochemical characterization and identification of the LAB isolates The selected isolates were characterized following standard protocols, including examination of cell morphology, colony characteristics, and biochemical properties. Biochemical tests performed included catalase, oxidase, citrate utilization, methyl red (MR), Voges–Proskauer (VP), indole production, nitrate reduction, deep glucose agar fermentation, motility, gelatin liquefaction, casein hydrolysis, and starch hydrolysis. Molecular identification was performed by sequencing the 16 S rRNA gene with primers 27 F and 1492R²¹. The sequences were compared against the NCBI database via BLAST to confirm their identification with the accession numbers OQ657038 to OQ657041. The two L. fermentum isolates (SCJ26 and SCJ28) differed by several bases in their 16 S rRNA gene sequences and were designated as distinct strains.

Gastrointestinal tolerance and probiotic competence

Acid, bile salt and phenol resistance assays These stress-tolerance assays evaluate the ability of LAB to survive gastrointestinal transit and subsequent probiotic function. To assess acid tolerance, MRS broth was adjusted to pH 3.0 and 6.5 using 1 M HCl. Overnight LAB cultures (OD600 ~ 1.8–2.0.8.0) were inoculated into 10 mL MRS medium at each pH and incubated at 37 °C. For bile salt tolerance, MRS medium with and without 0.3% bile salt (bacteriology grade) were inoculated with 1% LAB culture and incubated at 37 °C. Phenol tolerance was tested by cultivating strains in MRS medium with 0.3% and 0.4% phenol for 24 h at 37 °C. Growth was monitored by measuring OD₆₀₀ at specific time points, and resistance was inferred using the following Eqs^10,22–26.

Auto-aggregation assay Auto-aggregation reflects the ability of strains to form clusters, a property linked to colonization and persistence in the gut. To determine auto-aggregation, the LAB isolates were cultured in MRS medium at 37 °C for 18 h, harvested by centrifugation at 5000 rpm for 20 min, washed 3 times with phosphate-buffered saline (PBS), and resuspended in PBS. The suspension was incubated at 37 °C, and absorbance was measured at 0, 2, 4, 6, 12, and 24 h. Auto-aggregation was measured using the following equation¹⁰.

where A₀ is the absorbance at 0 h, and A_x is the absorbance at subsequent time points.

Coaggregation assay Co-aggregation with pathogens indicates potential to inhibit harmful microbes by preventing their adhesion and colonization. For co-aggregation analysis, LAB isolates and indicator organisms (S. aureus, E. coli, S. typhi) were cultured separately in MRS and nutrient broth (NB), respectively. Cell suspension was prepared as in auto-aggregation assay, and equal volume of LAB and pathogen suspensions (0.5 McFarland each) were mixed and incubated at 37 °C without agitation. Absorbance was measured at 0, 2, 4, 6, 12, and 24 h. Coaggregation (%) was determined using the following equation.

where, Apro + Apat represent the absorbance at 0 h, and Amix represent the absorbance at subsequent time points¹⁰.

Measurement of cell surface hydrophobicity Cell surface hydrophobicity is used as an indicator of the ability of LAB to adhere to intestinal epithelial surfaces, a key factor for probiotic colonization. The cell surface hydrophobicity of the LAB isolates was evaluated using the microbial adhesion to solvents (MATS) method. LAB cells were harvested at 5000 rpm for 20 min, washed twice, and resuspended in PBS. Equal volumes of the cell suspension and xylene were mixed and incubated at 37 °C for 1 h. After phase separation, the absorbance of the aqueous phase was measured at 600 nm, and hydrophobicity was calculated using the following formula²⁷.

where A₀ is the initial absorbance, and A_x is the absorbance after incubation with the solvent.

Health-promoting functional assays

Determination of antifungal activity The antifungal assay was conducted using the poisoned food technique²⁰. Candida albicans ATCC 10,231 was cultured on potato dextrose agar at 25 °C for 5–6 days, and 6 mm agar-blocks were taken from the fungal growth. One milliliter of cell-free LAB supernatant was mixed with nutrient agar using the pour-plate method. A 6 mm well was created in the agar, and a fungal block was placed inside. The control plate lacked the supernatant. After 5 days of incubation at 25 °C, fungal growth was measured, and inhibition was calculated using the following equation.

Where C is the diameter of the fungal colony in the control, and T is the diameter in the treatment.

Determination of anti-biofilm activity Anti-biofilm capacity is important for disrupting pathogenic biofilms, which are often resistant to treatment and contribute to infections. The anti-biofilm activity was measured using the crystal violet biofilm staining assay²⁸ with slight modification. Overnight cultures of biofilm-forming S. aureus and E. coli were grown in LB broth at 37 °C and diluted to an initial absorbance of 0.05 at 600 nm. Pathogen cultures were then incubated with LAB culture supernatants at a 1:1 (v/v) for 24 h at 37 °C. Untreated pathogen cultures served as negative controls. After 24 h of incubation at 37 °C, non-adherent cells were removed by washing with sterile PBS, and adherent biofilms were fixed with methanol, stained with crystal violet, and washed with distilled water. The bound dye was resolubilized with glacial acetic acid, and absorbance at 590 nm was measured to quantify anti-biofilm activity using the following formula.

where A_sample is the absorbance of the sample, and A_control is the absorbance of the control.

Assessment of antioxidant activity by DPPH scavenging assay The DPPH free radical-scavenging activity of LAB CFS was determined according to Tarannum et al.¹⁵. Briefly, LAB CFS (1.0 mL) was mixed with 1.0 mL of 0.1 mM ethanolic DPPH solution. The mixture was vortexed and absorbance was measured at 517 nm after 30 min of incubation in the dark. Ascorbic acid was used as standard. The scavenging ability was calculated as.

where A_sample is the absorbance of the sample, A_blank is the absorbance of the blank, and A_control is the absorbance of the control.

Technological and food-application attributes

Milk coagulation assay Milk coagulation reflects the fermentative ability of LAB and indicates their potential use in dairy product development. To assess milk coagulation activity, 50 mL of milk was boiled for ten minutes. After cooling, 5 mL of the boiled milk was transferred into sterile test tubes and pre-incubated at 40 °C for 5 min. Subsequently, 0.5 mL of the LAB culture was added to each test tube, mixed thoroughly, and incubated at 37 °C for 24 h²⁹. Coagulation was confirmed through visual inspection.

Assessment of exopolysaccharide (EPS) production EPS production is evaluated because it contributes to the texture and stability of fermented foods, enhancing functional food quality. To assess EPS production ability, fresh cultures were grown on aniline blue agar medium and the plates were incubated at 37 °C for 48 h. Strains that formed deep blue colonies were considered positive¹⁵. S. aureus was used as a positive control in this assay.

Determination of biopreservation efficacy of LAB isolates This assay indicates the antimicrobial potential of LAB in food matrices, suggesting possible applications in shelf-life extension. The biopreservation efficacy of selected LAB isolates in fish and tomato samples was assessed following a method adapted from Sarika et al. with slight modifications¹⁷. Fresh tilapia (Oreochromis niloticus) was collected from a local pond, cleaned, beheaded, eviscerated, deboned, and filleted into 10 g portions. Tomatoes, purchased from a local market, were washed with sterile distilled water and cut into 10 g slices. Each fish fillet and tomato slice was placed in a sterile Petri dish and evenly sprayed with 2 mL of LAB supernatant. Control samples were treated with sterile water. Both treated and control samples were incubated at room temperature for 72 h. Afterward, samples were serially diluted up to 10^{− 8}, plated on nutrient agar, and incubated at 37 °C for 24 h. Colony counts were compared between treated and control samples to evaluate the reduction in microbial load, thereby determining the biopreservative potential of the LAB isolates.

Safety evaluations

Antibiotic sensitivity test Antibiotic susceptibility was tested by the disc diffusion method on MRS agar following Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, according to a previously described protocol²⁰ against a panel of antibiotics including Amoxycillin, Clindamycin, Azithromycin, Ciprofloxacin, Tetracycline, Ofloxacin, Chloramphenicol, Doxycycline hydrochloride, Ampicillin, Gentamycin, and Vancomycin. Respective discs were placed on the agar plates streaked with LAB isolates and the zones of inhibition were measured after incubation at 37 °C for 24 h.

Hemolytic assay Assessment of hemolysis excludes strains with harmful cytotoxic activity, a key safety requirement. Hemolytic activity was assessed by streaking LAB isolates on blood agar plates containing 5% (w/v) sheep blood and incubating at 37 °C for 48 h³⁰. The presence of clear zones around the colonies indicated β-hemolysis, partial clearing indicated α-hemolysis, and no change in the medium was considered γ-hemolysis. Positive controls were prepared using Staphylococcus aureus.

Deoxyribonuclease (DNase assay) DNase testing helps exclude strains with nuclease-mediated virulence potential. DNase activity was determined by culturing LAB isolates on DNase test agar plates (HiMedia, India). Plates were incubated at 37 °C for 48 h, and DNase production was indicated by a colorless zone around the colonies³¹.

Use of large language models

The author used large language models (e.g., ChatGPT, GPT-4o) during manuscript preparation to assist in preparing and refining the text forclarity, flow, and readability. Final content was reviewed and approved by the authors.

Statistical analysis

All experiments were performed in triplicate and the corresponding data are expressed as the mean value with the standard deviation (mean ± SD). Data were analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test to assess significant differences among strains and treatments. Statistical significance was set at p < 0.05. Letters in figures and tables indicate groups that differ significantly.

Results and discussion

Selection and characterization of lactic acid bacteria from sugarcane juice

From the 30 colonies initially isolated, 20 met phenotypic criteria consistent with LAB, and most exhibited antagonistic activity against the tested pathogens. Four isolates (SCJ26, SCJ27, SCJ28, and SCJ29) showed the strongest inhibition against all four pathogens and were therefore selected for detailed probiotic and functional characterization. 16 S rRNA gene sequencing identified the isolates as Limosilactobacillus fermentum (SCJ26, SCJ28), Lactiplantibacillus plantarum (SCJ27), and L. pentosus (SCJ29). Sequence comparison showed several nucleotide differences between SCJ26 and SCJ28, supporting their classification as distinct L. fermentum strains. The identification of L. fermentum and L. plantarum aligns with previous reports that has consistently reported these taxa in sugar-rich environments, where they contribute to the fermentation process and exhibit broad-spectrum antimicrobial activity^5,7,32–35. Subsequent characterization of these isolates involved a detailed examination of their cultural and biochemical features, providing a comprehensive profile of their phenotypic and biochemical properties (Supplementary Data 1).

The successful isolation and identification of these LAB strains from sugarcane juice not only underscores their potential as natural antimicrobial agents but also highlights their broader applications within the food and health industries. Their robust antimicrobial activity against common foodborne pathogens suggests that they could be valuable candidates for the development of biopreservatives or probiotic supplements.

Tolerance to simulated gastrointestinal stressors

Acid tolerance Acid tolerance of the selected LAB strains was evaluated by exposing the isolates to a low pH, simulating gastric juice conditions. L. pentosus SCJ29 showed the highest initial survival of 58.88% at 2 h, although it decreased to 25.30% at 4 h (Fig. 1). L. fermentum SCJ28 maintained a survival rate of 39.48% at 2 h, with a subsequent decline to 12.43% at 4 h. In contrast, SCJ26 and SCJ27, demonstrated lower tolerance, with survival rates ranging from 36.05% to 27.47% at 2 h, and decreasing further over time. The ability to withstand acidic conditions is essential for probiotics, as they must survive the acidic environment of the stomach to reach the intestines. The higher acid tolerance of SCJ29 and SCJ28 suggests that these strains are particularly well-suited for potential probiotic applications, as they are likely to survive gastric transit. Comparable survival rates have been documented for other LAB strains including L. plantarum and L. fermentum which are known for their acid tolerance^36,37. For example, an L. fermentum strain isolated from bird cherry retained 48.28% viability at pH 2.0³⁸, whereas L. fermentum ATCC 2327 maintained over 60% survival under the same condition³⁹.

Fig. 1.

Tolerance of LAB isolates to simulated gastrointestinal conditions. Survival was tested under acidic pH (3.0), bile salts (0.3%), and phenol (0.4%). The data are expressed as means ± SD from triplicate experiments. Different letters indicate statistically significant differences (p < 0.05).

Bile tolerance The ability of the isolates to survive in the presence of 0.3% bile salt was measured over a period of 4 h at 37 °C (Fig. 1). L. fermentum SCJ28 exhibited the highest initial survival rate of 62.83% at 2 h but it decreased to 31.01% at 4 h. Conversely, L. pentosus SCJ29 demonstrated a more stable bile tolerance, maintaining a higher survival rate of 46.69% at 2 h and 40.28% at 4 h. SCJ26 and SCJ27 exhibited moderate bile tolerance, with survival rates ranging from 20.55% to 22.81% at 2 h and from 29.15% to 32.18% at 4 h. The observed bile tolerance reflects the potential of these LAB strains to survive in the small intestine where bile is present⁴⁰. The varying bile tolerance is consistent with other studies where LAB strains exhibited differential survival in bile⁴¹. For comparison, Lactiplantibacillus plantarum MB685 isolated from fermented broccoli maintained 68–76% survival at 0.3% bile in vitro, demonstrating robust bile-adaptation in that strain⁴².

Phenol tolerance Phenol tolerance was assessed in the presence of 0.3% and 0.4% phenol concentrations. L. fermentum SCJ28 again showed the highest tolerance, with survival rates of 59.37% at 0.3% phenol and 14.23% at 0.4% phenol (Fig. 1). The other isolates, SCJ26, SCJ27, and SCJ29, displayed lower phenol tolerance, with survival rates ranging from 21.81% to 28.88% at 0.3% phenol and further reduced survival at the higher phenol concentration. Phenol tolerance is an important trait for probiotics, as phenolic compounds can be present in the gut due to dietary intake and microbial metabolism^43,44. The robust resistance of SCJ28 to phenol suggests that it may have an enhanced probiotic potential, particularly in environments where phenol is present, adding to its versatility as a probiotic candidate.

The resistance of these isolates to acid, bile, and phenol highlights their potential as resilient probiotics capable of surviving and functioning in harsh conditions, such as the gastrointestinal tract and industrial fermentation processes⁴⁵. Cell survival in these high-throughput assays was assessed by optical density^22–26. While optical density reflects overall cell density rather than absolute viability, it can provide a practical means of ranking strains and potentially identify the tolerant isolates for further study. The observed variations in stress tolerance profiles suggest that these LAB strains possess diverse mechanisms to cope with adverse environmental conditions, with SCJ28 emerging as particularly promising candidate for probiotic applications. SCJ29 showed moderate survival under combined gastrointestinal stresses, maintaining ~ 47–58% viability. While this is lower than some other strains, it still indicates that a considerable fraction of cells may remain viable for potential gut colonization. Given that partial survival may suffice for probiotic action⁴⁶, SCJ29 can also be considered a candidate of interest. It should be noted that tolerance to acid, bile, and phenol was assessed independently in this study. Future studies incorporating sequential or simultaneous exposure to acid, bile, and phenolic compounds under in vitro or in vivo conditions will be important to more comprehensively assess probiotic survivability.

Adhesion-related functional traits

Autoaggregation Aggregation and adhesion metrics for the four strains are summarized in Fig. 2. SCJ29 and SCJ26 exhibited the strongest autoaggregation, reaching approximately 64.6% and 40.7%, respectively, at 24 h (Fig. 2). SCJ27 exhibited moderate autoaggregation (59.42% at 24 h), while SCJ28 showed the lowest capacity, reaching only 28.45% at 24 h. The high autoaggregation observed in SCJ29 and SCJ26 suggests a greater ability to form biofilms and persist in the gastrointestinal tract, which are important characteristics for effective probiotics. Comparable values have been reported for other probiotic strains; for example, L. plantarum Dad-13 from fermented buffalo milk and L. plantarum Mut-7 from fermented cassava showed autoaggregation levels of 40.9% and 57.5%, respectively⁴⁷, while L. pentosus 6MMI from yogurt exhibited 42.4%⁴⁸.

Fig. 2.

Autoaggregation, surface hydrophobicity, and coaggregation abilities of lab strains isolated from sugarcane juice. Coaggregation was tested against Staphylococcus aureus, Escherichia coli, and Salmonella typhi. Data are presented as means ± SD from triplicate measurements. Different letters indicate statistically significant differences (p < 0.05).

Surface hydrophobicity SCJ26 exhibited the highest hydrophobicity (33.73%) (Fig. 2). In contrast, SCJ27, SCJ28, and SCJ29, showed lower values of approximately 10–11%. High surface hydrophobicity is often associated with enhanced adhesive to epithelial cells, a critical feature for effective colonization and probiotic function. The relatively high hydrophobicity of SCJ26 implies that it may have an enhanced ability to adhere to mucosal surfaces, which could facilitate its persistence in the gastrointestinal tract and interaction with host cells⁴⁹. This observation is consistent with previous reports where LAB strains with higher surface hydrophobicity demonstrated better adhesion to intestinal surfaces, thereby improving their probiotic efficacy⁵⁰.

Coaggregation SCJ26 consistently demonstrated the highest coaggregation across all pathogens (Fig. 2), particularly with S. aureus (57.39% at 24 h) and E. coli (52.61% at 24 h). SCJ29 showed strong coaggregation with S. aureus (45.11%) and E. coli (35.35%), followed by SCJ27, which displayed moderate coaggregation, particularly with S. aureus (43.31%). SCJ28, however, demonstrated the lowest coaggregation abilities across all pathogens, with values not exceeding 31.27%. Previous studies have reported a wide range of co-aggregation values for lactic acid bacteria, from as low as 2.8–7.7% for L. fermentum LFB1295 with E. coli and S. aureus⁵¹, to 33.5% with E. coli and 62.7% with S. aureus in L. plantarum LB5⁵², while L. pentosus 6MMI achieved 51.3% with Listeria monocytogenes⁵³. This suggests that coaggregation capacity can vary considerably among LAB strains depending on strain background and target organism. The strong coaggregation observed in SCJ26 highlights its potential to inhibit pathogenic bacteria by preventing their adhesion and colonization, a key factor in probiotic efficacy⁵⁴.

The hydrophobicity, autoaggregation, and coaggregation properties observed in SCJ26 and SCJ29 suggest that these strains may have superior colonization potential, which is vital for maintaining a stable probiotic population in the gastrointestinal tract. These properties not only facilitate the persistence of the probiotics but also enhance their ability to interact with and inhibit pathogenic bacteria⁵⁵. The hydrophobicity and aggregation properties of these LAB strains indicate considerable potential as probiotic candidates, with SCJ26, in particular, demonstrating robust performance across all assessed criteria. However, multifunctionality selection may involve trade-offs: for example, SCJ27 combined strong antimicrobial and anti-biofilm traits with lower gastrointestinal stress tolerance, illustrating that a single isolate may not be optimal across all metrics. These inter-strain differences support selecting strains for particular applications.

Health promoting activities of the lactic acid bacteria

Antibacterial activity The four selected strains showed strain-specific functional profiles (Table 1). SCJ27 produced the largest inhibition zone (19.8 mm against E. coli), while both SCJ27 and SCJ29 demonstrated broad antimicrobial activity with strong inhibition against several pathogens. In contrast, SCJ26 and SCJ28 exhibited moderate antibacterial activity. Such activity may be particularly valuablein preventing or mitigating infections caused by these pathogens. The strong inhibition of E. coli and S. aureus is particularly noteworthy, as these bacteria are common causes of foodborne illnesses and skin infections, respectively^56,57. The variability in inhibition zones suggests strain-dependent antimicrobial mechanisms, potentially involving the production of bacteriocins, organic acids, or other inhibitory metabolites⁵⁸. To confirm these mechanisms, future studies should (a) compare neutralized versus non-neutralized supernatants, (b) apply proteinase K treatment to test for proteinaceous bacteriocins, and (c) employ chromatographic or LC-MS approaches to identify active compounds.

Table 1.

Antimicrobial, antifungal, anti-biofilm, and antioxidant activities of LAB isolates from sugarcane juice. Values represent means ± standard deviations (SD) from triplicate experiments. Different superscript letters within a column indicate statistically significant differences (p < 0.05).

Isolates	SCJ26	SCJ27	SCJ28	SCJ29
Antimicrobial zone of inhibition (mm):
B. cereus	12.83 ± 0.42c	16.80 ± 0.61b	13.60 ± 0.10c	17.23 ± 0.35a
S. aureus	10.23 ± 0.21d	17.37 ± 0.21a	11.80 ± 0.20c	16.80 ± 0.20b
E. coli	13.10 ± 0.26c	19.80 ± 0.20a	11.70 ± 0.26d	15.83 ± 0.15b
S. typhi	10.17 ± 0.15b	12.40 ± 0.30a	N/M	10.93 ± 0.21b
Antifungal activity (%):
C. albicans	58.59 ± 0.80a	58.36 ± 0.40a	1.84 ± 0.40ns	58.59 ± 0.40a
Anti-biofilm activity (%):
E. coli	28.80 ± 1.33c	43.49 ± 2.34b	15.09 ± 1.18d	53.06 ± 1.33a
S. aureus	58.25 ± 1.98c	69.90 ± 1.43a	26.43 ± 1.87d	62.84 ± 1.95b
DPPH radical scavenging (%):
	79.94 ± 0.49c	83.04 ± 1.39b	65.06 ± 1.73d	86.15 ± 3.09a

Antifungal activity SCJ26, SCJ27, and SCJ29, all showed substantial antifungal activity against C. albicans, with at least 58% inhibition of the fungal growth, while SCJ28 showed minimal antifungal activity (1.84%) (Table 1). The effectiveness of these isolates against C. albicans further underscores their potential role in controlling fungal infections, which could be particularly beneficial in the context of immunocompromised individuals or in food preservation^59,60.

Anti-biofilm activity The anti-biofilm properties of the LAB strains were tested against E. coli and S. aureus. SCJ29 exhibited the highest anti-biofilm activity against E. coli (53.06%) and SCJ27 against S. aureus (69.90%) (Table 1). SCJ26 also demonstrated significant anti-biofilm activity, particularly against S. aureus (58.25%), while SCJ28 showed the lowest activity against both pathogens. The ability of LAB to disrupt biofilm formation is critical, as biofilms contribute to chronic infections and increased resistance to antimicrobial treatments⁶¹. The observed anti-biofilm activity suggests that these LAB strains could be valuable in preventing or managing biofilm-associated infections.

Antioxidant activity The isolates demonstrated strong antioxidant potential with SCJ29 exhibiting the highest activity (86.15%), followed by SCJ27 (83.05%) and SCJ26 (79.94%) (Table 1). SCJ28 showed moderate antioxidant activity (65.06%). The robust antioxidant activity observed in these strains suggests their potential role in combating oxidative stress, which is implicated in various chronic diseases⁶². The antioxidant capacity of LAB is often linked to the production of exopolysaccharides, peptides, and other bioactive compounds, which can neutralize free radicals and protect cells from oxidative damage⁶³. This finding aligns with other studies where LAB strains demonstrated significant antioxidant properties, further supporting their application in health-promoting functional foods⁶⁴.

These combined health-promoting activities underscore the potential of the LAB isolates from sugarcane juice as promising candidates for probiotic formulations, with potential applications in food preservation and the prevention of various infections.

Safety profile of the LAB isolates

Antibiotic susceptibility and resistance The antibiotic resistance profiles of the LAB isolates were evaluated against a panel of standard antibiotics commonly used in clinical setting, which revealed a varied responses to the tested antibiotics (Table 2). All isolates were sensitive to Chloramphenicol (C), Tetracycline (TE), Ampicillin (AMP), Clindamycin (CD), Doxycycline hydrochloride (DO), and Azithromycin (AZM). However, resistance to Ciprofloxacin (CIP) and Amoxycillin (AMX) was observed in the isolates, with SCJ29 also showing intermediate resistance to Vancomycin (V) and Gentamycin (GEN). This pattern of resistance may reflect intrinsic resistance mechanisms typical of LAB, which are often considered non-transferable and thus pose minimal risk of contributing to antibiotic resistance dissemination⁶⁵. However, phenotypic assays alone cannot determine whether resistance determinants are chromosomal (intrinsic) or carried on mobile elements (transferable). In previous studies, similar resistance patterns were observed in LAB isolated from other food sources, suggesting that the resistance traits observed in this study are consistent with those found in LAB generally regarded as safe⁶⁶. Moreover, the resistance observed in Ciprofloxacin aligns with previously documented intrinsic resistance patterns⁶⁷. However, given the regulatory importance of vancomycin resistance, the intermediate response of SCJ29 warrants caution and genomic screening to detect specific resistance genes and mobile genetic elements prior to application. The sensitivity of the isolates to a broad range of antibiotics suggests their safe application in probiotic formulations, yet, further investigation can ensure the safety and non-transferability of resistance genes.

Table 2.

Antibiotic susceptibility profile of LAB isolates from sugarcane juice. Susceptibility was evaluated against 11 antibiotics, with results interpreted as susceptible (S), intermediate (I), or resistant (R) following Clinical and Laboratory Standards Institute (CLSI) guidelines.

Isolates	C	CN	TE	CIP	AMP	CD	DO	OF	AZM	AMX	V
SCJ26	S	S	S	R	S	S	S	I	S	R	S
SCJ27	S	S	S	R	S	S	S	R	S	R	S
SCJ28	S	I	S	R	S	S	S	R	S	R	S
SCJ29	S	I	I	R	S	S	S	R	S	R	I

DNase and hemolytic activity None of the LAB isolates exhibited DNase or hemolytic activity under the assay conditions, which supports an absence of two common virulence-associated phenotypes^68,69. DNase activity contributes to DNA degradation and immune invasion⁷⁰, while hemolytic activity is linked to host tissue damage⁷¹. The lack of hemolysisis consistent with previous reports showing that food-derived LAB generallylack hemolytic capabilities, making them suitable candidates for probiotic applications⁷¹.

Hence, the LAB isolates demonstrated a suggestive safety profile, characterized by sensitivity to most tested antibiotics, absence of hemolytic activity, and lack of DNase activity. These results provide an important initial safety baseline, consistent with probiotic assessment guidance (e.g., FAO/WHO). Nevertheless, further studies into the genetic basis of antibiotic resistance and long-term safety assessments are warranted to confirm these findings and ensure their continued safe use in food systems. Importantly, antibiotic susceptibility here was phenotypic only and that genomic screening for resistance genes and mobile elements is required to definitively distinguish intrinsic from potentially transferable resistance; this limitation is also noted in the Conclusion and is recommended as a necessary follow-up prior to any industrial application.

Potential for food quality enhancement

Milk coagulation All LAB isolates showed milk coagulation activities though the time required varied among them, with SCJ27 and SCJ29 achieving coagulation within 12–15 h, whereas SCJ26 and SCJ28 required 24 h. This finding highlights the potential application of these LAB strains in dairy product fermentation, where efficient milk coagulation is essential for producing yogurt, cheese, and other fermented products. The faster coagulation times observed in SCJ27 and SCJ29 suggest higher enzymatic activity or increased production of coagulant substances, which could be advantageous for developing dairy products requiring quicker processing times. In industrial settings, rapid coagulation is often preferred to enhance production efficiency and ensure product consistency⁷².

EPS production ability EPS production, assessed using the aniline blue agar plates assay, revealed that SCJ27 and SCJ29 showed relatively strong while SCJ26 showed moderate indication for EPS-producing capabilities. The ability to produce EPS is particularly important for the texture and stability of fermented foods. EPS contributes to the viscosity, mouthfeel, and water-holding capacity of products, enhancing their sensory attributes and shelf stability⁷³. The EPS potential of these isolates suggests that they could be valuable in formulating foods with improved texture and extended shelf life⁷⁴. However, further analysis is needed to fully evaluate their EPS production and the specific roles of EPS in food systems.

Biopreservative efficacy in food matrices Application of LAB supernatants to tilapia fillets and tomato slices resulted in marked decreases in total viable counts after 72 h compared with controls (Fig. 3). In tilapia, the control group exhibited 7.82 Log10 CFU/mL, whereas treatment with LAB CFS significantly reduced microbial loads. SCJ27 showed the strongest effect, reducing counts to 3.98 Log10 CFU/mL, while SCJ26, SCJ28, and SCJ29 also achieved marked reductions (4.02, 4.53, and 4.01 log CFU/mL, respectively). In tomato, the control reached 6.68 Log10 CFU/mL, with SCJ27 again demonstrating the highest antimicrobial activity (3.06 Log10 CFU/mL), closely followed by SCJ29 (3.08 log CFU/mL). While SCJ26 and SCJ28 reduced counts to 3.99 and 4.00 log CFU/mL, respectively. These reductions indicate that the LAB secretions can effectively suppress spoilage organisms and may extend the shelf life of perishable foods. Their ability to control microbial growth highlights the potential of LAB as natural preservatives reducing reliance on chemical preservatives while enhancing food safety⁷⁵.

Fig. 3.

Biopreservation potential of LAB isolates in fish and tomato samples. The ability of the strains to reduce microbial loads was evaluated after 72 h. Data represent means are presented as means ± SD from triplicate experiments. Different letters indicate statistically significant differences (p < 0.05).

Complementing their biopreservative effects, the LAB isolates displayed attributes that contribute to natural and sustainable food processing solutions. Their milk coagulation ability, EPS production, and biopreservation activities highlight a multifunctional potential in dairy fermentation, food texture enhancement, and natural preservation aligning with the growing demand for sustainable functional foods with health-promoting benefits^72,76,77. Notably, the EPS production and biopreservation efficacy of SCJ27 and SCJ29 suggest broader industrial applications, including the development of probiotic foods and natural preservative systems.

The observed microbial reduction in fish and tomato is likely mediated by the combined effects of organic acids, bacteriocins, and possibly hydrogen peroxide, as reported for other LAB⁷⁸. However, the 72-hour ambient incubation employed here represents only an initial screening and does not substitute for controlled shelf-life or challenge studies. Thus, while promising, these findings should be considered preliminary. Follow-up work should include neutralized and protease-treated CFS controls, dose–response testing, refrigerated shelf-life trials, and sensory evaluation to validate the practical applicability of these strains.

To place our findings in context, we assembled a literature-based comparison of key probiotic attributes for representative LAB strains from dairy and plant sources (Supplementary Data 2). While many individual trait values of the sugarcane-derived isolates fall within ranges reported for established probiotic candidates, the comparison highlights that the combined presence of gut-adaptation markers, antimicrobial/antifungal/anti-biofilm activities, technological traits (EPS production and milk coagulation), and preliminary food-matrix biopreservation is what renders these isolates relevant for application-oriented selection. Taken together, the contribution of this study lies in the integrated, safety-to-application evaluation of lactic acid bacteria isolated from an under-explored, sugar-rich, non-dairy niche, rather than in asserting inherent niche-driven superiority.

Conclusion

Our study showed that sugarcane juice is a promising non-dairy source of multifunctional lactic acid bacteria: the selected isolates exhibited complementary gut-adaptation markers, strain-specific antimicrobial, antifungal, and anti-biofilm activities, antioxidant potential, and useful technological traits (EPS production, milk coagulation) that together support their candidacy for probiotic and natural biopreservation applications. Important limitations temper these findings: (i) antimicrobial agents were not biochemically characterized, (ii) phenotypic antibiotic profiles were not corroborated by genomic data to assess gene presence or mobility, and (iii) the food-matrix tests were preliminary screening experiments rather than controlled shelf-life studies. Future studies should include genomic safety screening for resistance determinants and mobile elements, biochemical and proteomic characterization of active antimicrobial factors, and controlled shelf-life and refrigerated challenge studies with sensory assessments. Within these bounds, the present integrated screening highlights sugarcane-derived LAB as candidates worthy of further development for safe probiotic use and natural food preservation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

JF conceived and supervised the study. SI, SA, and SP conducted the experiments. SI and JF performed the primary data analysis and interpretation. MSHB and SP contributed to manuscript drafting. FA served as co-supervisor and performed statistical analysis. TJH prepared and wrote the manuscript, and contributed to result interpretation. All authors read and approved the final version of the manuscript.

Funding

This study was funded by Research and Publication Cell, University of Chittagong.

Data availability

Sequence data that support the findings of this study have been deposited in the NCBI GenBank with the accession numbers OQ657038 to OQ657041.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study did not involve human or vertebrate animal subjects requiring ethical approval; fish samples were obtained post-mortem from a university pond.