Shahid Beheshti University of Medical Sciences & Iranian Probiotic and Functional Foods Society
Doi
Abstract
Background and Objective: Cheese is one of the major dairy products with high nutritional value, but its susceptibility to microbial growth and early spoilage remains a challenge for the dairy industry. While chemical additives are widely applied to control microbial contamination, increasing awareness of the potential hazards of synthetic preservatives has led to a growing demand for natural alternatives. This study was designed to evaluate the antimicrobial potential of postbiotics derived from lactiplantibacillus plantarum and Lacticaseibacillus casei against common spoilage and pathogenic microorganisms in cheese, as well as to investigate their impact on the microbiological and chemical properties of cheese during storage.
Material and Methods: Postbiotics were extracted from cultures of L. plantarum and L. casei and their antimicrobial activities were tested using standard microbiological assays against Gram-positive and Gram-negative bacteria. To assess practical application, the postbiotics were applied as coatings on cheese samples either alone or in combination with whey protein concentrate (WPC). Microbiological counts, chemical parameters, and sensory evaluation were performed throughout storage. Data were analyzed to determine the comparative effectiveness of treatments.
Results and Conclusion: The findings showed that the postbiotic derived from L. plantarum demonstrated stronger antimicrobial effects, particularly against Gram-positive bacteria, compared to that from L. casei. However, when combined with WPC, the antimicrobial activity of both postbiotics declined. Despite this limitation, postbiotics applied alone significantly reduced microbial counts during storage without altering the main chemical properties of the cheese. Sensory evaluation confirmed the overall acceptability of postbiotic and postbiotic-WPC treated samples. In conclusion, postbiotics can serve as promising natural antimicrobial agents in cheese preservation, though further optimization is required to enhance their activity when combined with protein-based carriers such as WPC.
Keywords: Antimicrobial activity, Cell-free supernatant, Cheese, Lactic acid bacteria, Postbiotic
Introduction
The food industry consistently faces substantial challenges from pathogenic and spoilage microorganisms, which are major drivers of foodborne diseases (FBDs), product quality degradation, and significant economic losses [1]. Globally, FBDs remain a pressing public health issue, with more than 600 million cases and nearly 420,000 deaths reported annually due to contaminated food and water [2,3]. These statistics have reinforced the urgency of strengthening food safety measures and minimizing contamination throughout production, processing, and storage stages [4,5]. At the same time, consumer concerns over the potential health risks associated with chemical preservatives, alongside the increasing demand for minimally processed foods, have fueled interest in natural preservation strategies [6,7]. Such approaches not only lessen dependence on artificial additives but also align with the growing trend toward clean-label products.
In this regard, biological protection has emerged as a promising strategy, harnessing beneficial microorganisms and their antimicrobial metabolites to suppress the proliferation of spoilage and pathogenic organisms [4]. Probiotics, especially lactic acid bacteria (LAB), have drawn considerable attention for their ability to generate bioactive substances such as organic acids, hydrogen peroxide, diacetyl, and bacteriocins [8]. Among these, LAB species such as Lactobacillus and Bifidobacterium are generally recognized as safe (GRAS) and have been widely employed for decades in the fermentation of diverse foods, including dairy products, cereals, and vegetables [9]. More recently, growing attention has been directed toward postbiotics, defined as non-viable microbial cells, cellular components, or metabolites that provide functional health benefits [10]. Compared with probiotics, postbiotics present notable practical advantages: they are independent of cell viability, exhibit greater stability under processing and storage conditions, and pose a lower risk of antibiotic resistance or incompatibility with food matrices. Additionally, postbiotics retain antimicrobial activity across a wide range of pH and temperature conditions, are capable of disrupting pathogenic biofilms, and can neutralize harmful contaminants such as pesticides and mycotoxins. In food systems, they have been investigated both as direct additives and as functional components of active packaging technologies, thereby overcoming limitations associated with the use of live microbial cultures [4].
Cheese provides a particularly critical application, as it is highly vulnerable to microbial contamination during both processing and storage. Among potential threats, Listeria monocytogenes is of significant concern due to its ability to withstand stress conditions and its high fatality rate in human infections [11,12]. Epidemiological evidence has repeatedly linked outbreaks of listeriosis to cheeses manufactured from raw or inadequately pasteurized milk [13], underscoring the urgent demand for innovative, safe, and effective antimicrobial approaches in dairy preservation.
Recent investigations have demonstrated that postbiotics and bacteriocin-like compounds derived from lactic acid bacteria (LAB) are capable of inhibiting pathogenic bacteria across diverse food matrices, including meat, seafood, and dairy products [14–19].
In parallel, whey protein, a major by-product of cheese production, has attracted considerable interest as a functional material for edible coatings and packaging, owing to its excellent barrier properties and strong film-forming capacity [11]. Incorporating antimicrobial postbiotics into whey protein systems may therefore offer dual advantages: enhancing microbial safety and prolonging shelf life while preserving desirable sensory characteristics.
Although evidence supporting postbiotics as natural preservatives is growing, relatively few studies have systematically assessed their performance in real cheese systems, particularly when combined with whey protein–based coatings. This gap in knowledge constrains a comprehensive understanding of their effectiveness under practical conditions.
The present study aims to bridge this gap by examining the antimicrobial activity of postbiotics derived from lactiplantibacillus plantarum and Lacticaseibacillus casei against key dairy pathogens, while also evaluating the microbial and chemical quality of cheese coated with whey protein concentrate (WPC) enriched with these postbiotics. This integrated strategy offers a novel, effective, and sustainable approach to improving food safety and preservation in dairy products.
Materials and Methods
2.1 Study design
This descriptive-analytical study was conducted between 22 November 2022 and 15 September 2023. The study protocol was reviewed and approved by the Medical Ethics Committee of Gonabad University of Medical Sciences (IR.GMU.REC.1401.073).
2.2 Bacterial strains
Three lactiplantibacillus plantarum strains were isolated from traditional Iranian cheeses [20]. In addition, one Lacticaseibacillus casei strain (1608 PTCC, IBRC of Iran), Listeria monocytogenes (7644 ATCC), Escherichia coli (1338 PTCC), and Staphylococcus aureus (1431 PTCC) were obtained from the Laboratory of Specialization in Nutrition, Mashhad University of Medical Sciences.
2.3 Postbiotic preparation
Each lactic acid bacterial strain was cultured separately in MRS broth medium and incubated under anaerobic conditions at 37 °C for 24 hours. The cultures were then centrifuged at 6000 rpm for 10 minutes at 4 °C. The resulting cell-free supernatants were filtered through a 0.4 µm membrane filter and subsequently freeze-dried (freezing temperature −83 °C, pump pressure 0.0026 mBar, storage temperature −60 °C) for use in subsequent experiments [21].
2.4 Chemical analysis of cell-free supernatants of Lactobacillus sp.
The chemical compounds of postbiotics were identified following the method described by Ryan et al. (2009), with minor modifications. For derivatization, 1 mL of the supernatant was mixed with 10 mL of absolute ethanol and 15 drops of sulfuric acid (97%), and the mixture was stirred at 80 °C for one hour. After cooling, 20 mL of distilled water was added, and extraction was performed five times with 50 mL of dichloromethane, collecting the lower phase each time. The pooled extracts were combined with 50 g of sodium sulfate and passed through filter paper. The solvent was then removed using a vacuum evaporator at 50 °C, and the remaining residue was injected into the gas chromatography–mass spectrometry (GC–MS) system.
The chemical composition of the derivatized postbiotics was analyzed using a GC instrument (Agilent HP-6890, Agilent Technologies, Palo Alto, CA, USA) operated with Agilent GC/MS Mass Hunter Acquisition software. The GC system was equipped with an Agilent HP-5ms column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed to increase from 110 °C to 240 °C at a rate of 4 °C/min with no hold time. A 10 μL sample was injected with a 5:1 split ratio [22].
2.5 Antimicrobial Activity In-vitro
2.5.1 Agar-well diffusion
Three pathogens (L. monocytogenes, E. coli, and S. aureus) were inoculated separately at a concentration of 5 log10 CFU/mL onto the surface of Muller Hinton Agar (MHA) plates. Wells with an 8 mm diameter were then created in the agar, and 100 µL of postbiotic suspensions at concentrations of 5%, 10%, and 20% were added to the wells. Plates were incubated aerobically at 37 °C for 24–48 hours. Nisin (625 IU/mL) and sterile distilled water served as the positive and negative controls, respectively. Antimicrobial activity was expressed as the mean diameter (mm) of inhibition zones, measured as the clear areas surrounding the wells. Each assay was performed in triplicate [23].
2.5.2 Determination of minimum inhibitory concentra-tion (MIC) and minimum bactericidal concentration (MBC) by the microdilution method
MIC and MBC values were determined according to the Clinical and Laboratory Standards Institute (CLSI, 2017) guidelines. After 24 hours of aerobic incubation at 37 °C, the wells were examined for turbidity. To determine the MBC, 10 µL samples from wells without turbidity (corresponding to MIC and higher concentrations) were streaked onto MHA plates in triplicate and incubated under the same conditions [24].
2.5.3 Anti-listeria activity of coatings containing postbiotics
To prepare the coating solution, 50 mL of deionized water was heated to 90 °C in a bain-marie, and 4.34 g of whey protein concentrate (WPC; protein 81.2%, lactose 7.4%, fat 6%, moisture 5%, ash 4%, pH 6.1; Alinda, Greece) was fully dissolved. The solution was maintained at this temperature for 45–60 minutes, during which 2.71 g of glycerol and 0.081 g of Tween 80 were added. After cooling, cell-free supernatants (CFS) were incorporated at concentrations corresponding to MIC and MBC [25]. The anti-listeria activity of coatings containing postbiotics was then evaluated using the agar-well diffusion method. CFS without coating, at equivalent concentrations, was included as a positive control [26].
2.6 Inoculation and coating of cheese samples
A pasteurized traditional cheese, commercially available in local markets, was selected for this study. The cheeses analyzed were pasteurized varieties inoculated with a fungal starter culture. Four treatment groups were prepared to evaluate microbial and chemical characteristics over storage on days 0, 1, 2, 4, 6, 8, and 10 (Table 1).
Cheese pieces of approximately 10 g (3 × 3 × 1 cm) were inoculated with L. monocytogenes at a level of 1 × 10⁵ CFU/g by spreading 1 mL of an appropriately diluted suspension onto the surface [18]. Samples were then allowed to stabilize to ensure bacterial adherence. The treatments were as follows:
Inoculated cheese without further treatment,
Cheese immersed in CFS solution at the designated concentration,
Cheese coated with WPC solution without CFS, and
Cheese coated with WPC solution containing CFS (immersion for 4–5 minutes).
All samples were stored at 4 °C until further analysis.
2.7 Microbial analyses in-situ
monocytogenes, total viable microorganisms, molds, and yeasts were enumerated using Palcam Agar, PCA, and SDA media, respectively. Palcam and PCA plates were incubated at 37 °C for 48 hours, while SDA plates were incubated at 25 °C for 3–5 days [26]. The enumeration of molds and yeasts was carried out according to the Iranian National Standard No. 2406: Microbiology of milk and milk products — Specifications and test methods [27].
2.8 Chemical analyses
The pH of cheese samples was measured using a pre-calibrated pH meter, and moisture content was determined by the gravimetric method on the designated sampling days [17,21].
2.9 Sensory analyses
Cheese slices coated with cell-free supernatants, free of L. monocytogenes, were evaluated for taste, color, aroma, texture, and overall acceptability by a panel of 10 semi-trained assessors using a 5-point hedonic scale [15]. All participants were adults above the legal age and voluntarily provided written informed consent in compliance with ethical standards for human subject research. Evaluations were conducted under identical environmental and temporal conditions to ensure consistency.
2.10 Statistical analyses
Statistical analyses were performed using SPSS software version 26. Mean values from three independent replicates were compared between two groups using the independent t-test, while comparisons among more than two independent groups were carried out using one-way ANOVA. Changes in data trends over the 10-day storage period were analyzed using one-way repeated measures ANOVA. A p-value of <0.05 was considered statistically significant.
Results and Discussion
3.1 Antimicrobial activity of postbiotics
Postbiotics derived from both Lactobacillus species demonstrated inhibitory effects against the three tested pathogens, with higher postbiotic concentrations corresponding to stronger antimicrobial activity (Fig. 1).
Fig. 1. Antimicrobial activity of postbiotics against L. monocytogenes evaluated by the agar-well diffusion method.
The greatest inhibition was observed with the postbiotic from L. plantarum at a 20% concentration against L. monocytogenes, producing an inhibition zone of 30.67 ± 0.57 mm. In contrast, the weakest inhibition was observed with the postbiotic from L. casei at a 5% concentration against S. aureus, yielding an inhibition zone of 8.63 ± 0.55 mm. Across all concentrations, the postbiotic of L. plantarum exhibited significantly stronger inhibitory activity against L. monocytogenes and S. aureus compared to that of L. casei (p < 0.05) (Tables 2–4). For E. coli, no significant differences were detected between the two postbiotics except at the 10% concentration (Table 3). These findings suggest that postbiotics from L. plantarum are more effective against Gram-positive pathogens than those from L. casei.
Overall, the results indicate that L. plantarum postbiotics exert stronger antimicrobial effects against Gram-positive bacteria, with the most consistent reductions achieved through the “CFS only” treatment rather than the CFS–WPC combination. The maximum reduction compared to control (~0.80 log CFU/g for L. monocytogenes at day 2) declined over subsequent storage days, while pH, moisture, and sensory acceptability remained unaffected. This strain- and target-dependent pattern is consistent with previous evidence showing that LAB-derived cell-free supernatants inhibit Gram-positive pathogens primarily through organic acids and bacteriocin-like metabolites, mechanisms that involve pH reduction and disruption of microbial membranes [4,6,9,28–30]. Arena et al. also reported strong anti-pathogen activity of L. plantarum supernatants, with acidification identified as a major contributing factor [31,32]. The reduced inhibitory effect observed when CFS was incorporated into a whey protein carrier is consistent with the well-documented “matrix effects” described in the literature. Previous studies have shown that interactions between proteins and bioactive metabolites, along with the barrier properties of protein films, can delay the release and reduce the bioavailability of antimicrobial compounds [11,26,33–36]. Similar patterns of initial but transient inhibition, followed by partial recovery of pathogen populations, have been reported in fresh cheese, meat, and fish products treated with CFS- or bacteriocin-based films [15–17,19].
In agreement with these findings, the present study demonstrated that chemical attributes (pH and moisture) and sensory acceptance were not adversely affected, supporting the feasibility of integrating postbiotics into dairy preservation systems. However, further optimization of carrier composition and release kinetics is required to maximize antimicrobial effectiveness [37].
The MIC and MBC values of L. plantarum postbiotics against the three tested pathogens were determined as 31.25 mg/mL and 62.5 mg/mL, respectively, for Gram-positive bacteria, and 125 mg/mL for E. coli. These results highlight a greater inhibitory effect against Gram-positive bacteria at lower concentrations (Table 5).
The postbiotic of L. casei exhibited a comparable inhibitory effect against L. monocytogenes to that of L. plantarum, except at the 5% concentration, where a difference was observed in comparison with E. coli (Table 6). No significant differences were detected in the inhibition of S. aureus and E. coli (p > 0.05) (Table 7).
The MIC of L. casei postbiotics against L. monocytogenes was higher (62.5 mg/mL) than that observed for L. plantarum, although both species showed identical MBC values. For E. coli, the MBC of L. casei postbiotics was lower than that of L. plantarum. In contrast, for S. aureus, the MIC and MBC values were the same for both postbiotics.
Arrioja et al. (2020) similarly reported that CFS derived from L. plantarum exhibited stronger inhibitory activity against most pathogens compared with CFS from L. casei [33]. Arena et al. (2016) further demonstrated variability in inhibition zones and MICs among different L. plantarum strains against various pathogens, with generally greater effects observed against Gram-positive bacteria [31]. Consistent with the present findings, Tenea and Barrigas (2018) showed that bacteriocin-containing supernatants from L. plantarum (Cys5-4) exhibited variable inhibitory effects against two E. coli strains [7]. Koohestani et al. (2018) also reported that CFS from L. casei 431 produced an inhibition zone of 13 mm against S. aureus, which closely aligns with the current results [23]. In contrast, Yordshahi et al. (2020) documented smaller inhibition zones for L. plantarum postbiotics against L. monocyte-genes compared with those observed in this study [21].
The antimicrobial activity of lactic acid bacteria has been attributed to a range of metabolites, including organic acids, polyamines, proteases, and bacteriocins. The effectiveness of postbiotics depends on multiple factors such as bacterial strain, metabolite composition and concentration, preparation method, and pathogen type. Numerous studies have confirmed that Gram-positive bacteria are generally more susceptible to the antagonistic compounds in postbiotics than Gram-negative bacteria, consistent with the findings of the present work [38].
Results from the agar-well diffusion assays indicated that postbiotics incorporated into WPC coating solutions at MBC concentrations generally exhibited reduced inhibition against L. monocytogenes compared to postbiotics applied alone (Table 8). However, the combination of L. plantarum postbiotics with WPC coatings produced greater inhibition than L. casei postbiotics at equivalent concentrations.
Based on the in-vitro assays, the L. plantarum postbiotic at twice the MBC concentration was identified as the most effective formulation and was subsequently selected for testing in the food model.
3.2 Identification of chemical compounds of extracted CFS
The chemical compounds identified in the postbiotics derived from L. plantarum and L. casei are presented in their respective chromatograms (Figs. 2 and 3).
Sezen Özcelik et al. (2016) reported that LAB strains are particularly efficient producers of succinic acid, especially when cultivated in MRS broth. Succinic acid serves as a key intermediate in the Krebs cycle and a common fermentation byproduct, reflecting the strong metabolic capacity of LABs. The quantity and composition of organic acids produced by LABs vary considerably across strains and culture media, with pH and temperature exerting significant influence [28]. Similarly, Iqbal Hossain et al. (2021) identified nine distinct organic acids, including succinic acid, in several LAB strains such as L. plantarum, highlighting the diverse metabolite production potential of these bacteria [29]. In the present study, the derivatization technique applied proved particularly effective in detecting compounds such as esters and alkanes.
Shehata et al. demonstrated that LABs synthesize antifungal metabolites that differ across strains, with organic acids and hydrogen peroxide serving as the primary contributors to antifungal activity. Their study identified compounds such as pentadecane and 2,4-di-tert-butylphenol, both known to inhibit foodborne pathogens and fungi. Additionally, antimicrobial compounds including 6-octadecenoic acid methyl ester and hexadecanoic acid methyl ester—also de
