Fermentation and Functional Properties of Plant-Derived Limosilactobacillus fermentum for Dairy Applications

Abstract

Lactic acid bacteria (LAB) isolated from plant sources are gaining increasing attention due to their potential probiotic and postbiotic functionalities. In the present study, Limosilactobacillus fermentum isolated from Prunus padus (bird cherry) was evaluated for its physiological, functional, and technological attributes for application in fermented dairy products. The strain was isolated through anaerobic fermentation and identified using API 50 CHL and 16S rRNA sequencing. Its acid tolerance, antioxidant capacity, antibacterial effects, and hemolytic activity were assessed. The cell-free supernatant (CFS) was evaluated for thermal and pH stability. Fermentation trials were conducted using both mono- and co-culture combinations with the commercial yogurt starter strain YC-380. Physicochemical properties, viable cell counts, and viscosity were monitored throughout fermentation and refrigerated storage. The L. fermentum isolate exhibited strong acid resistance (48.28% viability at pH 2.0), non-hemolytic safety, and notable DPPH radical scavenging activity. Its CFS showed significant antibacterial activity against five Escherichia coli strains, which remained stable after heat treatment. Co-cultivation with YC-380 enhanced fermentation efficiency and improved yogurt viscosity (from 800 to 1200 CP) compared to YC-380 alone. During 24 days of cold storage, co-cultured samples maintained superior pH and microbial stability. Additionally, the moderate acidification profile and near-neutral pH of L. fermentum created favorable conditions for postbiotic compound production. These results indicate that L. fermentum derived from P. padus holds considerable promise as a functional adjunct culture in yogurt production. Its postbiotic potential, technological compatibility, and heat-stable bioactivity suggest valuable applications in the development of safe, stable, and health-promoting fermented dairy products.

1. Introduction

Natural resources have long served as a foundation for advancing human health, with their applications extending across various scientific and industrial fields. Nutraceuticals—such as dietary fibers, prebiotics, probiotics, polyunsaturated fatty acids, and antioxidants—have garnered increasing attention for their roles in promoting health and preventing disease [1]. Among these, lactic acid bacteria (LAB) are of particular interest due to their ability to produce lactic acid and bioactive compounds with beneficial health effects. LAB is widely used in agriculture, food fermentation, and clinical applications, contributing to food safety, sensory quality, and functional enhancement, thereby continuously driving research in this field. In recent years, probiotics—defined as live microorganisms that confer health benefits to the host when administered in adequate amounts—have become popular for their roles in supporting gut health, modulating the immune system, and preventing disease. In parallel, postbiotics—non-viable microbial cells, cell components, or metabolites—have emerged as promising functional ingredients due to their comparable health benefits, superior stability, and ease of incorporation into food matrices. While most probiotic research has focused on LAB strains isolated from dairy products and the human gut, increasing attention is being directed toward plant-derived LAB, which often exhibit superior acid tolerance and enhanced adhesion to intestinal epithelial cells compared to their dairy-origin counterparts [2,3,4]. Their natural origin and adaptive traits make them attractive candidates for developing next-generation probiotics and postbiotics tailored to modern functional food requirements. Simultaneously, there has been a growing interest in the use of traditional food plants within the nutraceutical and functional food industries. Plants rich in flavonoids, phenolic acids, and other antioxidant compounds are increasingly employed as natural alternatives to synthetic additives [5,6]. One such underutilized plant is Prunus padus (bird cherry), a member of the Rosaceae family, which is widely distributed across Europe and Asia—including Mongolia, Korea, and Japan—and has been traditionally used for both culinary and medicinal purposes [7]. The fruits of P. padus are known to contain a variety of bioactive compounds, including sugars, organic acids, flavonoids, and polyphenols such as caffeic acid, chlorogenic acid, gallic acid, quercetin, and catechin, all of which contribute to its strong antioxidant potential [5,8]. In Mongolia, these fruits are traditionally ground and mixed with milk cream for consumption and are also used as a folk remedy for ailments such as diarrhea. Despite its traditional use and rich phytochemical profile, P. padus remains largely unexplored with respect to its microbial ecology, and no studies to date have reported the isolation or characterization of LAB associated with this plant. To address this gap, the present study aimed to isolate and characterize a LAB strain derived from P. padus and to evaluate its probiotic and postbiotic potential. The strain’s functional properties were assessed through investigations of acid resistance, hemolytic activity, antibacterial activity, and fermentation performance in yogurt. By identifying a novel plant-derived LAB strain, this study contributes to the expanding field of plant-based probiotics and postbiotics and explores its potential application in the development of functional fermented dairy products.

2. Materials and Methods

2.1. Isolation and Identification of Lactic Acid Bacteria

Fresh P. padus cherries were harvested from a home garden in Ulaanbaatar, Mongolia, on 29 August 2023. The fruits were subjected to anaerobic fermentation at 37 °C for 6 weeks to enrich LAB. Following fermentation, the berries were homogenized with sterile distilled water, and the resulting suspension was spread onto De Man Rogosa and Sharpe (MRS) agar (Difco, Sparks, MD, USA) supplemented with 0.01% sodium azide (Sigma-Aldrich, St. Louis, MO, USA) [9]. The plates were incubated under both aerobic and anaerobic conditions at 37 ℃ until visible colonies developed. Colonies were selected based on morphological characteristics, such as shape and size, and subjected to Gram staining and microscopic examination for preliminary identification. Biochemical characterization was performed using the API 50 CHL kit (BioMérieux, Marcy-l’Étoile, France). Briefly, 100 µL of API 50 CHL medium inoculated with a single colony was dispensed into each capsule of the API strip and incubated at 37 ℃ for 48 h. Carbohydrate fermentation profiles were recorded based on color changes and interpreted using API Web (https://apiweb.biomerieux.com/, 30 November 2024) to determine strain identity. For molecular identification, 16S rRNA gene sequencing was outsourced to a commercial service provider (Solgent, Republic of Korea). The obtained sequence was compared to reference sequences in the NCBI database using BLAST 2.15.0 analysis. The isolated LAB was subsequently stored at −80 °C in 30% (v/v) glycerol after cultivation at 37 ℃ for 24 h under aerobic conditions for use in further experiments.

2.2. Preparation of Cell-Free Supernatant (CFS)

The isolated LAB was subcultured in MRS broth and incubated aerobically at 37 °C for 24 h. Following incubation, the culture was centrifuged at 4000 rpm for 20 min at 4 °C using a refrigerated centrifuge (Mega-17R, Hanil Science Industrial, Gimpo, Republic of Korea) to remove bacterial cells. The supernatant was collected and passed through a sterile 0.22 µm syringe filter (Advantec, Tokyo, Japan) to obtain a sterile CFS. The filtered CFS was aliquoted and stored at –20 °C until further use in antibacterial and cellular bioactivity assays.

2.3. Bacterial Strains and Culture Conditions

The LAB isolate, along with two commercial yogurt starter strains—Streptococcus thermophilus KCCM 40430 and Lactobacillus delbrueckii subsp. bulgaricus KCCM 35463—was obtained from the Korean Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea). A commercial yogurt starter culture, YC-380 (containing S. thermophilus and L. bulgaricus), was purchased from Chr. Hansen (Hørsholm, Denmark). All strains were subcultured in MRS broth at 37 ℃ for 24 h prior to experimentation.

Five pathogenic Escherichia coli strains (KCCM 11569, 11587, 11591, 11596, and 11600) were obtained from the Korea Microbial Conservation Center. These strains were cultured in Luria–Bertani (LB) broth (Difco, Sparks, MD, USA).

2.4. Hemolytic Activity

Hemolytic activity was evaluated according to the method described by Lkhagvasuren et al. [10]. The LAB isolate was pre-cultured in MRS broth at 37 °C for 24 h and streaked onto blood agar plates containing 5% defibrinated sheep blood (Kisan Biotech Co., Seoul, Republic of Korea). The plates were incubated aerobically at 37 °C for 48 h. Hemolytic activity was determined by examining zones of hemolysis around the colonies. Hemolysis was classified as α-hemolysis (partial, greenish discoloration), β-hemolysis (clear zone), or γ-hemolysis (no hemolysis) [11].

2.5. Acid Resistance

The acid tolerance of the LAB isolate was assessed based on the method described by Oh and Jung [12], with minor modifications. The strain was cultured in MRS broth at 37 °C for 12 h, after which the medium was adjusted to pH 2.0 using 1 N HCl to simulate gastric conditions. The acidified culture was incubated at 37 °C for an additional 2 h. After treatment, viable cell counts were determined using the standard plate count method on MRS agar. Serial dilutions were prepared, and 1 mL from each dilution was spread in triplicate onto MRS agar plates, followed by incubation at 37 °C for 48 h. The survival rate under acidic conditions was calculated using the following formula:

$$\mathrm{Survival} \mathrm{rate} (\%)=\left\{{\displaystyle \frac{\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} (\log \mathrm{C}\mathrm{F}\mathrm{U}/\mathrm{m}\mathrm{L}) \mathrm{s}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{d}}{\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} (\log \mathrm{C}\mathrm{F}\mathrm{U}/\mathrm{m}\mathrm{L}) \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{o}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}}}\right\}\times 100$$

(1)

2.6. Antioxidant Activity

The antioxidant activity of the CFS derived from the isolated LAB was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay, following the method developed by Jemil et al. [13]. Test samples included the CFS, 0.2 mM ascorbic acid (positive control), and MRS broth (negative control). Each sample was mixed with ethanol at a 1:1 (v/v) ratio. A 0.2 mM DPPH solution in ethanol was freshly prepared and added to the pre-treated samples in a 1:1 (v/v) ratio. The mixtures were incubated in the dark at room temperature for 30 min. Absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). A blank was prepared using a 1:1 (v/v) mixture of CFS and ethanol without DPPH. The DPPH radical scavenging activity was calculated using the following formula:

$$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} (\mathrm{\%})=\left\{{\displaystyle \frac{{(\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}+{\mathrm{A}}_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}-{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}})}{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\right\}\times 100$$

(2)

2.7. Antibacterial Activity of CFS Derived from the Isolated LAB

2.7.1. Paper Disk Diffusion Assay

The antibacterial activity of the CFS was evaluated against five E. coli strains (KCCM 11569, 11587, 11591, 11596, and 11600) using the paper disk diffusion method. LB agar (20 mL per plate) was sterilized at 125 ℃ for 20 min and cooled to approximately 50 ℃. Each pathogenic strain (1%, v/v) was inoculated into the cooled agar and poured into sterile Petri dishes. After solidification, sterile paper disks (Advantec, Tokyo, Japan) were loaded with 20 µL of CFS at concentrations of 100%, 50%, 25%, and 12.5% (v/v). The disks were gently placed on the surface of the inoculated agar and incubated at 37 ℃ for 3 h. Antibacterial activity was determined by measuring the diameter of the inhibition zones around each disk (mm).

2.7.2. 96 Well Plate Assay

The antibacterial activity of the cell-free supernatant (CFS) was further assessed using a 96-well microplate assay. The CFS was tested at concentrations of 20%, 10%, 5%, and 2.5% (v/v). After pre-culturing, 1% (v/v) of each Escherichia coli strain was inoculated into wells containing the respective concentrations of CFS diluted in LB broth. The plates were incubated at 37 ℃, and bacterial growth was monitored by measuring optical density (OD) at 655 nm using a microplate reader (Bio-Rad, Hercules, CA, USA) at 3 h intervals.

2.8. Measurement of pH Stability and Thermal Stability of CFS

The pH and thermal stability of the CFS were evaluated based on its antibacterial activity against five E. coli strains. For pH stability assessment, the pH of the CFS was adjusted to alkaline conditions using 1 N NaOH. The pH-adjusted CFS was added at 10% (v/v) to LB broth inoculated with 1% (v/v) of each E. coli strain. Bacterial growth was monitored by measuring OD at 655 nm at 3 h intervals during incubation at 37 °C for 24 h. For thermal stability assessment, aliquots of the CFS were subjected to heat treatment at four different conditions: 65 °C for 30 min, 75 °C for 15 min, 85 °C for 10 min, and 100 °C for 5 min. After cooling to room temperature, each heat-treated CFS was added at 10% (v/v) to LB broth inoculated with 1% (v/v) of each E. coli strain. Bacterial growth was monitored under the same conditions (OD 655 nm at 3 h intervals, 37 ℃ for 24 h).

2.9. Milk Fermentation

All LAB strains, including the isolated LAB, were subcultured twice in MRS broth at 37 °C to activate them prior to use in fermentation. For mono-strain fermentation, 2% (v/v) of each individual strain (S. thermophilus, L. bulgaricus, or the isolated LAB) was inoculated into 400 mL of autoclaved commercial milk in sterile 500 mL Erlenmeyer flasks. The cultures were incubated at 37 °C until fermentation was complete. For co-strain fermentation, the isolated LAB and YC-380 were mixed at a 1:1 (v/v) ratio and inoculated into 400 mL of sterilized milk at a final concentration of 2% (v/v). YC-380 alone served as the control. All fermentation mixtures were incubated at 37 °C, and pH was measured at 3 h intervals to monitor fermentation progress until the pH of the sample inoculated with the isolated LAB dropped below 4.6.

2.10. Measurement of pH, Titratable Acidity, and Viable Cell Counts

During fermentation, pH and titratable acidity were measured at 3 h intervals. pH was determined using a calibrated digital pH meter (Jenway 3510, Stone, Staffs, UK). Titratable acidity was measured according to AOAC methods. Specifically, 17.6 mL of fermented milk was diluted with an equal volume of distilled water (1:1, v/v) and titrated with 0.1 N NaOH to pH 8.3. Titratable acidity (%) was calculated using the following formula:

$$\mathrm{Titratable} \mathrm{acidity} (\%)=\left\{{\displaystyle \frac{\mathrm{N}\mathrm{a}\mathrm{O}\mathrm{H} \left(\mathrm{m}\mathrm{L}\right)\times 0.009}{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \left(\mathrm{g}\right)}} \right\}\times 100$$

(3)

Viable cell counts were determined every 12 h. Under aseptic conditions, 1 mL of each sample was homogenized with 9 mL of sterile 0.1% (w/v) peptone water and serially diluted in 10-fold steps. From each dilution, 1 mL was plated in triplicate on MRS agar using the pour plate method. Plates were incubated at 37 °C for 24 h, and colonies were counted. The results were expressed as colony-forming units (CFU)/mL and presented as the mean of three replicates.

After fermentation, all yogurt samples—including mono-strain (S. thermophilus, L. bulgaricus, isolated LAB), co-strain (isolated LAB and YC-380), and control (YC-380 only)—were stored at 4 ℃. Cold storage stability was evaluated by measuring pH, titratable acidity, and viable cell counts on days 6, 12, and 24.

2.11. Measurement of Viscosity

The viscosity of yogurt samples was measured after fermentation using an NDJ-8S digital rotational viscometer (WandJ Instrument Co., Ltd., Changzhou, China) equipped with spindle No. 4, operated at 30 rpm. A 50 mL aliquot of each sample was tested, and viscosity readings were recorded at 60 s intervals. Final viscosity was expressed as the average of three consecutive measurements to ensure reproducibility.

2.12. Statistical Analysis

All experimental data were analyzed using one-way analysis of variance (ANOVA), initially processed in Microsoft Excel (Office 365, Microsoft Corp., Redmond, WA, USA) and further validated using Minitab version 15 (Minitab Inc., State College, PA, USA). The results were expressed as mean ± standard deviation (SD) based on three independent replicates. Statistical significance was considered at * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results and Discussion

3.1. Isolation, Identification, and Characterization of LAB

Selected colonies were initially screened based on morphological characteristics, such as shape and size, followed by Gram staining and microscopic observation. The selected isolates exhibited rod-shaped morphology and were Gram-positive, consistent with typical LAB characteristics. Preliminary identification was conducted using the API 50 CHL system, which assesses carbohydrate fermentation profiles. The results of sugar utilization are presented in

Table 1. Carbon utilization of the isolated Limosilactobacillus fermentum using API 50CHL kit.

Carbohydrates	L. fermentum	Carbohydrates	L. fermentum
Control	−	Esculin	−
Glycerol	−	Salicin	−
Erythritol	−	D-Cellobiose	−
D-Arabinose	−	D-Maltose	+
L-Arabinose	−	D-Lactose	+
Ribose	−	D-Melibiose	+
D-Xylose	−	D-Sucrose	+
L-Xylose	−	D-Trehalose	+
D-Adonitol	−	Inulin	−
Methyl-β-D-Xylopyranoside	−	D-Melezitose	−
Galactose	+	D-Raffinose	+
Glucose	+	Starch	−
Fructose	+	Glycogen	−
Mannose	−	Xylitol	−
Sorbose	−	Gentiobiose	−
Rhamnose	−	D-Turanose	−
Dulcitol	−	D-Lyxose	−
Inositol	−	D-Tagatose	−
Mannitol	−	D-Fucose	−
Sorbitol	−	L-Fucose	−
Methyl-α-D-Mannopyranoside	−	D-Arabitol	−
Methyl-α-D-Glucopyranoside	−	L-Arabitol	−
N-Acetylglucosamine	−	Potassium Gluconate	−
Amygdalin	−	2-Ketogluconate	−
Arbutin	−	5-Ketogluconate	−

+, positive; −, negative.

. Based on the API database, the P. padus-derived isolate showed 90.3% identity with Limosilactobacillus fermentum. The API 50 CHL system is a widely used phenotypic tool that effectively differentiates among Lactobacillus species [14,15,16]. To confirm taxonomic identity, 16S rRNA gene sequencing was performed by Solgent (Daejeon, Republic of Korea). Sequence analysis revealed 99% similarity to L. fermentum strain CIP 102980, thereby confirming the isolate’s identity (Figure 1).

3.2. Hemolytic Activity

The safety of the isolated L. fermentum strain was evaluated by assessing its hemolytic activity on blood agar plates containing 5% defibrinated sheep blood. The strain demonstrated γ-hemolysis, indicated by the absence of clear or discolored zones around the colonies (Figure 2A). The γ-hemolytic profile observed in this study suggests that the strain does not exert cytotoxic effects on red blood cells. Similar results were observed in the control strain L. bulgaricus, which is recognized as safe (Figure 2B). According to FAO/WHO guidelines (2002), the absence of hemolytic activity is a critical safety criterion for probiotics, as hemolysis is considered a potential virulence factor that may compromise intestinal epithelial integrity and promote microbial translocation [17]. The non-hemolytic nature of the isolated strain supports its safety and suitability for use in food fermentation and probiotic applications.

3.3. Acid Resistance

The acid tolerance of the isolated L. fermentum strain was evaluated by adjusting the culture medium to pH 2.0 using 1 N HCl and incubating for 2 h. The initial viable cell count was 9.31 log CFU/mL. After 2 h of acid exposure, the viable count decreased to 4.42 log CFU/mL, corresponding to a survival rate of 48.28% (Figure 3). This result demonstrates that the L. fermentum strain exhibits notable intrinsic resistance to low pH, a trait not observed in the commercial probiotic strains tested in this study. All commercial strains showed complete loss of viability under the same conditions, in agreement with the findings reported by Pereira and Gibson [18]. The ability of the isolate to survive at pH 2.0 suggests that it can withstand conditions similar to those found in the human gastric environment, where pH typically ranges from 1.5 to 3.5 [19]. Furthermore, gastric pH often rises above 3.0 after food intake [20], indicating that the survival rate of the strain may be even higher under actual digestive conditions. Acid resistance is considered a key criterion for probiotic efficacy, as only strains capable of surviving the gastrointestinal tract (GIT) can exert functional benefits [21,22,23]. Martín et al. [24] reported that L. fermentum CECT5716, isolated from human breast milk, retained 30% viability after 80 min at pH 1.8. In comparison, the L. fermentum strain in this study maintained 42% viability after 120 min at pH 2.0, indicating superior acid tolerance. These results highlight the strain’s potential in probiotic applications. Additionally, Jeong et al. [25] reported that LAB derived from plant sources generally show enhanced acid resistance, a finding consistent with the present study, in which P. padus-derived L. fermentum demonstrated significantly higher acid survival. This supports the functional advantages of plant-origin LAB in probiotic development.

3.4. Antioxidant Activity of CFS

The CFS obtained from the isolated L. fermentum exhibited a DPPH radical scavenging activity of 59.87%, which was significantly higher (p < 0.05) than that of the negative control (MRS broth) (Figure 4). This result indicates a strong antioxidant capacity of the metabolites secreted by the strain. The observed activity is comparable to that of previously reported L. fermentum strains NS4 and JNU532, which showed approximately 60% DPPH scavenging activity in a study by Meng and Oh [26] on LAB isolated from kimchi. This consistency suggests that antioxidant potential may be a conserved feature among L. fermentum strains, regardless of their origin. The ability of probiotics to neutralize free radicals plays a crucial role in alleviating oxidative stress in the host. These findings support previous research on the antioxidative efficacy of LAB-derived postbiotic compounds and reinforce the potential application of L. fermentum as a functional strain for developing health-promoting fermented food products.

3.5. Antibacterial Activity and Stability of CFS

The antibacterial activity of the CFS derived from the isolated L. fermentum was evaluated using the paper disk diffusion method. As shown in Figure 5, the CFS at 100% and 50% (v/v) concentrations produced clear zones of inhibition against all five tested E. coli strains: KCCM 11569, 11587, 11591, 11596, and 11600. However, no inhibition zone was observed against the L. bulgaricus KCCM 35463 (control strain). These findings indicate that the CFS contains antibacterial compounds capable of suppressing the growth of pathogenic E. coli. To further validate the antibacterial potential of the CFS, a 96-well microplate assay was conducted using concentrations of 20%, 10%, 5%, and 2.5% (v/v). Bacterial growth was monitored for 24 h (Figure 6). At 10% concentration, E. coli KCCM 11596 and 11600 showed complete inhibition throughout the incubation period (Figure 6D,E). For KCCM 11569, inhibition was sustained for up to 15 h, whereas strains 11587 and 11591 exhibited growth suppression for approximately 9 h before resuming proliferation (Figure 6A–C). These results demonstrate a concentration-dependent antibacterial effect, with variable susceptibility among E. coli strains. The thermal stability of the antibacterial activity was assessed by subjecting the CFS to heat treatments at 65 ℃ for 30 min, 75 ℃ for 15 min, 85 °C for 10 min, and 100 °C for 5 min. Each treated sample (10%, v/v) was tested against the same panel of E. coli strains. As shown in Figure 7, no significant reduction in antibacterial activity was observed after any of the heat treatments, confirming that the active compounds in the CFS are heat-stable and retain efficacy under thermal stress. This supports their potential application in thermally processed food systems. To determine whether the antibacterial effect was due to organic acids or other non-pH-dependent compounds such as bacteriocins, the pH of the untreated CFS (initially pH 4.22) was adjusted to 7.0 using 1 N NaOH. The neutralized CFS was tested under the same conditions. As illustrated in Figure 8, no antibacterial activity was observed in the pH-adjusted CFS, whereas the unadjusted control retained strong inhibitory effects. This result strongly suggests that the antibacterial activity is primarily attributable to organic acids, likely including lactic acid, acetic acid, and phenyl lactic acid, which are ineffective under neutral or alkaline conditions. These findings align with previous studies. Cabo et al. [27] and Xin et al. [28] reported similar reductions in antibacterial activity following pH neutralization of LAB-derived CFS, despite retained heat stability. For instance, Lacticaseibacillus paracasei XLK 401 maintained antibacterial activity after heating at 100 ℃ for 60 min but lost efficacy at neutral pH, emphasizing the role of organic acids in microbial inhibition. Organic acids exert their antimicrobial effects by lowering environmental pH, disrupting microbial membrane potential, and interfering with critical enzymatic functions [29]. Bintsis [30] also emphasized their role in enhancing food safety and extending shelf life by inhibiting spoilage organisms. The high antibacterial activity, thermal stability, and pH sensitivity of the P. padus-derived L. fermentum CFS suggest its potential as an effective postbiotic candidate for use in food preservation and functional formulation. Beyond antimicrobial properties, postbiotic compounds such as organic acids and bacteriocins also contribute to gut microbiota balance and barrier function. Studies by Aguilar-Toalá et al. [31] and Sánchez et al. [32] demonstrated that postbiotics can enhance immune function, inhibit pathogen colonization, and promote gastrointestinal health. Taken together, these findings support the potential application of L. fermentum CFS from P. padus in the development of functional dairy products and broader applications in the food and health industries.

3.6. Changes in pH, Titratable Acidity, and Viable Cell Counts During Fermentation and Storage

The changes in pH, titratable acidity, and viable cell counts during a 60 h fermentation at 37 °C for mono-strain fermented milk are presented in Figure 9. Among the three strains tested, S. thermophilus exhibited the most rapid acidification, lowering the pH below 4.6 within the first 24 h. L. bulgaricus achieved a similar pH by 48 h, whereas L. fermentum showed delayed acidification, with minimal pH reduction until after 30 h. After 60 h, L. fermentum reached a final pH of 5.749, significantly higher than L. bulgaricus (4.164) and S. thermophilus (4.205) (Figure 9A). Consistent with these pH changes, titratable acidity increased progressively in the control strains, with S. thermophilus rising from 0.15% to 0.82% and L. bulgaricus from 0.14% to 0.81%. In contrast, L. fermentum showed a moderate increase, from 0.13% to 0.30%, indicating relatively limited acid production (Figure 9B). Viable cell counts measured at 12 h intervals are shown in Figure 9C. L. fermentum grew from 8.19 log CFU/mL at 12 h to 8.90 log CFU/mL at 48 h. In comparison, S. thermophilus and L. bulgaricus reached final counts of 9.47 and 9.74 log CFU/mL, respectively. These results suggest that L. fermentum maintains stable viability but exhibits weaker acidification capacity relative to traditional starter cultures. To evaluate cold storage behavior, yogurt samples fermented by each strain were stored at 6 °C for 24 days. As shown in Figure 9D–F, L. fermentum-fermented milk showed only a slight pH decrease and remained stable thereafter (Figure 9D). Titratable acidity decreased across all samples until day 6, followed by stabilization (Figure 9E). Viable cell counts remained consistent throughout storage, indicating high cell survival and product stability (Figure 9F). The fermentation performance of the commercial starter YC-380 and its co-culture with L. fermentum was also examined over 48 h (Figure 10). The co-culture resulted in a more pronounced pH drop, from 6.562 to 4.446, and a higher increase in titratable acidity (from 0.13% to 0.74%) compared to YC-380 alone (pH from 6.629 to 4.680; titratable acidity from 0.13% to 0.61%) (Figure 10A,B). This indicates that L. fermentum enhances lactose metabolism and lactic acid production, improving acidification efficiency. Viable cell counts were consistently higher in the co-culture. At 12 h, the co-culture reached 9.47 log CFU/mL versus 8.92 log CFU/mL for the control, and by 24 h, 11.10 log CFU/mL versus 10.16 log CFU/mL, respectively (Figure 10C). During 24-day storage, the co-culture maintained greater pH stability than YC-380 alone (Figure 10D). While titratable acidity declined in all samples by day 6, the co-culture maintained stable acidity thereafter, whereas YC-380 alone showed a gradual increase beginning after day 12 (Figure 10E). All samples retained viable counts above the 10⁷ CFU/g threshold recommended by Codex Stan 243–2003 (Figure 10F). Notably, microbial viability in the co-culture remained consistent, whereas YC-380 alone exhibited a gradual decline, suggesting a synergistic interaction between the two strains. These results indicate that L. fermentum enhances the fermentation performance of YC-380, improving acidification, microbial growth, and storage stability. In mono-strain fermentations, S. thermophilus and L. bulgaricus served as benchmark controls, as they are well-established reference cultures for evaluating LAB acidification performance [33,34,35,36]. While L. fermentum demonstrated slower acidification and lower final viability (8.90 log CFU/mL), these characteristics are consistent with the profile of postbiotic-producing strains. In postbiotics, functional efficacy is not necessarily dependent on the viability of live cells, as biological activity can persist through secreted metabolites. This offers advantages in formulation stability and shelf-life extension [37]. Unlike S. thermophilus and L. bulgaricus, which rapidly acidify milk, L. fermentum decreased pH from 6.589 to 5.749 over 60 h. This slower acidification is likely attributable to differences in sugar metabolism and enzymatic activity. However, co-culture with YC-380 accelerated pH reduction and titratable acidity increase, likely due to enhanced organic acid production, which improves casein coagulation near its isoelectric point and contributes to superior gel structure and texture [38,39]. Interestingly, the mild acidification observed in L. fermentum mono-cultures may support the generation of functional peptides. Proteolytic enzymes in LAB show peak activity in near-neutral environments (pH 5.5–7.5), facilitating the release of bioactive peptides from milk proteins [40,41]. These peptides are known to possess antioxidant, antimicrobial, and immunomodulatory properties [42,43,44,45]. This mechanism aligns with the study by Nami et al. [46], which demonstrated that postbiotics produced under pH-controlled fermentation (pH 6.0) showed enhanced immunological effects, including increased secretory IgA and resistance to Salmonella infection. These results highlight the importance of controlled acidification in postbiotic generation and further support the functional potential of L. fermentum in probiotic and postbiotic dairy formulations.

3.7. Viscosity Development During Storage and Functional Implications

Viscosity changes in the yogurt samples during 48 h of refrigerated storage are shown in Figure 11. All samples exhibited an increase in viscosity, though the extent of change varied by bacterial strain. The mono-strain fermented yogurt with L. fermentum showed an increase from 250 to 550 centipoise (cP), while samples fermented with L. bulgaricus and S. thermophilus increased from 1200 to 1350 cP and 1480 to 1700 cP, respectively. The commercial starter YC-380 displayed only a marginal viscosity increase (1680 to 1700 cP), whereas the co-culture of YC-380 and L. fermentum demonstrated a more pronounced rise, from 800 to 1200 cP. Viscosity is a key quality indicator in yogurt, reflecting its structural integrity, flow behavior, and mouthfeel. It is primarily influenced by the formation of protein networks through acid-induced casein aggregation, proteolysis, and extracellular polysaccharide (EPS) production [47,48]. Typically, lower pH promotes stronger gel formation, while higher pH leads to weaker curd structures and reduced viscosity [49]. The relatively high final pH of the L. fermentum mono-culture likely contributed to its lower viscosity compared to the reference strains. Nonetheless, L. fermentum significantly enhanced viscosity when co-cultured with YC-380, suggesting a synergistic effect—potentially driven by co-metabolism, increased EPS production, or enhanced protein–protein interactions. These findings are consistent with the results of Sahan et al. [50], who reported that viscosity can continue to rise during storage due to progressive gel network rearrangement. Collectively, the results indicate that while L. fermentum alone contributes modestly to yogurt viscosity, its combination with a commercial starter can markedly improve textural properties. This highlights the strain’s potential as both a functional and technological adjunct in fermented dairy product development.

4. Conclusions

In this study, a strain of L. fermentum was successfully isolated from P. padus and comprehensively characterized for its probiotic and postbiotic potential in functional food applications. The strain exhibited strong acid tolerance, maintaining over 48% viability at pH 2.0, surpassing several commercial probiotic references and demonstrating its potential for survival under gastric conditions. The cell-free supernatant (CFS) derived from the isolate displayed significant antioxidant activity, as evidenced by DPPH radical scavenging, and exerted broad-spectrum antibacterial effects against multiple pathogenic E. coli strains. Importantly, these antibacterial effects remained stable after thermal treatment, indicating the presence of heat-stable bioactive compounds suitable for application in thermally processed foods. The strain also exhibited γ-hemolytic activity, confirming its non-hemolytic and safe nature for food use. When co-cultured with the commercial starter YC-380, L. fermentum enhanced fermentation kinetics, increased microbial viability, and improved yogurt viscosity—likely due to synergistic metabolic interactions and EPS production. Furthermore, the moderate acidification pattern observed in mono-cultures of L. fermentum suggests a favorable environment for proteolytic activity and the release of functional peptides. This aligns with the objectives of postbiotic production, offering bioactivity without over-acidification, a common drawback in fermented dairy products. Overall, the findings of this study highlight the dual role of L. fermentum from P. padus as a safe, effective adjunct culture and a promising source of functional postbiotics for application in fermented milk systems. Future investigations should explore its regulatory effects on gut epithelial health and assess its scalability in industrial yogurt production aimed at combining health benefits with enhanced physicochemical quality.