Microbial strains and cultures preparation

The lyophilized LAB strain (La. plantarum PTCC1745) was activated and transferred into De Man, Rogosa, and Sharpe (MRS) broth or agar (Oxoid, Milan, Italy). Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 25,923), Escherichia coli (NCTC12900) and Salmonella typhi morium (ATCC 14,028) were cultured in Tryptic Soy broth (TSB, Oxoid). Fresh microbial suspensions of La. plantarum, the pathogenic bacteria, and spoilage mold strains were prepared by sub-culturing in De Mann, Rogosa, and Sharpe (MRS; Ibresco, Karaj, Iran) broth, Brain Heart Infusion (BHI; Ibresco, Iran) broth, and Potato Dextrose (PDA; Ibresco, Iran) agar, respectively. Aspergillus niger (PTCC 5010) and Aspergillus flavus (PTCC 5004) spores were used as target molds. These suspensions were standardized using a visible-ultraviolet spectrophotometer (Mecasys Co., Ltd., Daejeon, Korea) at 600 nm.

Experimental media for La. plantarum

The experimental media used in this study were MRS broth, cheese whey, and low-fat UHT milk with a 1.5% fat content. Cheese whey was provided by the Pegah dairy factory located in Mashhad, Iran, and UHT milk was purchased from a local market. The whey was prepared by reducing the pH to 4.5 using 5 N HCl (Merck, Darmstadt, Germany), denaturing the proteins through autoclaving at 121 °C for 15 min, and removing the precipitates by centrifugation (2360×g for 15 min). (Sharafi et al. 2022).

Growth kinetics of La. plantarum in selected cultures

A 2% overnight anaerobic culture in MRS broth was used to inoculate the experimental media. The cultures were incubated anaerobically at 37 °C for 36 h. The logarithmic value of bacterial cell concentration for each sample at 24 h was determined through dilution and pour plate counting. The samples were incubated in MRS agar at 37 °C for 48 h under anaerobic conditions.

Preparation of postbiotics solutions

La. plantarum was cultured in MRS broth, cheese whey, and low-fat UHT milk and incubated at 37 ± 1 °C for 48 h. The bacterial suspensions obtained from these three media were divided into three separate groups. The first group was heat-killed in a water bath at 95 °C for 5 min. The second group was sonicated for 5 min, and the last group remained intact. Then, all three groups were centrifuged at 4200×g for 10 min at 4 °C and subsequently filter-sterilized through a 0.45-µm-pore filter. The supernatant was harvested and stored at 4 °C (İncili et al. 2022).

Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations

The micro-dilution assay method in broth was used to determine the minimum inhibitory concentrations (MICs) of postbiotics derived from La. plantarum cultured in MRS broth, cheese whey, and low-fat UHT milk. (Lin and Pan 2019). Seventy-five microliters of a diluted culture of pathogenic bacteria (106 CFU/mL) were added to each well of a 96-well polystyrene flat-bottomed microtitre plate. The postbiotics were serially diluted to concentrations of 10,000, 8,000, 6,000, 5,000, 4,000, 3,000, 2,500, 2,000, 1,000, 500, and 250 µg/mL. Subsequently, 75 µL of each dilution was added to the respective wells. The treatment and control wells, which contained only bacterial suspensions, were then incubated at 37 °C for 18–20 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of CFS (cell-free supernatant) that showed no observable growth of the tested organisms upon macroscopic examination. The MIC value was reported in micrograms per milliliter (µg/mL). Furthermore, the concentrations that completely suppressed visible growth of the bacterial pathogens were determined. Next, 50 µL of each culture broth was plated onto agar plates and incubated at 37 °C for 24 h. The minimum bactericidal concentration (MBC) was determined as the concentration at which no bacterial colonies were observed to develop on the agar surface (Bajpai et al. 2016).

Antibacterial activity of postbiotics

The antibacterial activity of the postbiotics prepared from La. plantarum was determined using the agar well-diffusion method (Koohestani et al. 2018). Lawn cultures of pathogenic strains (6 log10 CFU/mL) were prepared on Mueller Hinton (MH) agar (Ibresco, Iran) and allowed to stand for 5 min. Wells were then created in the agar using a sterile filter pipette tip, and 100 µL of the prepared La. plantarum postbiotic was added to each well against each of the tested pathogens. Negative control was prepared using the same medium (sterile MRS medium) that was used to dissolve the samples. The plates were incubated at 37 ± 1 °C for 24 h. The antibacterial activity was assessed by measuring the zones of inhibition, which included the diameter of the well (6 mm), against the tested bacteria. The experiments were conducted in triplicate.

Antifungal activity of postbiotics

The antifungal activity was measured using the agar well-diffusion method (Poornachandra Rao et al. 2019). Aspergillus niger and Aspergillus flavus spores were used as target molds. A total of 10 µL of the conidial suspension (106 spores/mL) was evenly spread on PDA plates, and allowed to settle for 5 min. Wells with a diameter of 6 mm were then created in the agar. Each well was filled with 100 µL of postbiotics derived from La. plantarum. The plates were incubated at 30 °C for 48 h, and clear inhibition zones were observed. As a control, sterile MRS broth was used. The experiments were conducted in triplicate.

Determination of particle size

Average particle size was measured using dynamic light scattering (DLS) at a fixed angle of 135°. The diameter of the particles was measured based on the function of the intensity of the scattered light assuming that they are spherical. All experiments were performed at 25 ± 2 °C in triplicate. Size Particles were reported by intensity.

Statistical analysis

The data study was analyzed by analysis of variance (ANOVA (with GraphPad Prism version 5.0 for Windows. Duncan’s test was used to assess significant differences (p < 0.05) between the groups. All treatments were conducted in three plicate.