According to the official definition made by the International Scientific Association for Probiotics and Prebiotics (ISAPP) in 2021, a postbiotic is defined as a ''preparation of inanimate microorganisms and/or their components that confers a health benefit on the host'' ^7,10,11. Postbiotics contain many bioactive compounds, such as organic acids, bacteriocins, exopolysaccharides, peptidoglycans, carboxylic acids, free fatty acids, amino acids, phenolics, and volatile compounds ^12-14. Owing to these metabolites in their structure, postbiotics possess a very broad spectrum of biological activity, including antimicrobial and antioxidant properties as well as immunomodulatory, anti-inflammatory, anti-tumor, anti-allergic, antidiabetic, and antihypertensive effects ^12,13,15,16.

Postbiotics exert their antimicrobial effect by disrupting the cell membrane (bacteriocins, free fatty acids), lowering the environmental pH (organic acids), damaging pathogen DNA and protein structures as a result of triggering oxidative stress (hydrogen peroxide, reuterin, etc.), inhibiting biofilm formation (exopolysaccharides), suppressing the virulence genes of pathogens, and preventing the spread of antibiotic resistance genes ^7,12,17. Thanks to these versatile properties, postbiotics are accepted as a ''clean-label''-compatible strategic component that protects public health by preventing both microbiological and chemical spoilage in foods, while also functioning as a natural biopreservative that extends shelf life ^17-19. The concentration of postbiotics is a critical technological process that directly increases the amount of bioactive components they contain and, consequently, their antimicrobial efficacy. Studies have revealed that concentrating postbiotics by removing their liquid portion synergistically multiplies their inhibitory power against food pathogens ^4,20,21.

Reports from the Centers for Disease Control and Prevention (CDC) and the European Food Safety Authority (EFSA) emphasize that foods, particularly meat and meat products, are at risk of serious pathogen contamination that threatens public health ^22,23. These pathogens can become contaminated at various stages of the food chain, such as production, processing, storage, and distribution, and may cause serious zoonotic diseases and foodborne outbreaks. E. coli are gram-negative (-), rod-shaped, facultative anaerobic microorganisms. Non-pathogenic E. coli bacteria are found as normal flora in the human digestive system and are used as reference strains in studies ²⁴. However, pathogenic E. coli strains cause serious public health problems such as diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome and neonatal meningitis ^13,25. On the other hand, Staphylococcus aureus poses a constant threat to food safety with the disinfectant-resistant biofilm structures it forms on food-processing surfaces, while methicillin-resistant S. aureus (MRSA) strains are considered among the most clinically risky pathogens ^12,20.

Within the scope of combating these microbial threats, EFSA carries out strain-based risk assessments through the Qualified Presumption of Safety (QPS) approach to ensure the safety of microorganisms and emphasizes the development of natural biopreservative strategies, such as postbiotics, as a vital priority ¹². The reports of these international organizations reveal that the control of pathogens and the development of natural preservation methods, such as postbiotics, are of vital importance for global food safety strategies ²³.

In light of this information, the aim of this study was to determine the in vitro antimicrobial activity against E. coli and S. aureus, as well as the pH and titratable acidity parameters, of non-concentrated (1×) and differently concentrated (10×, 12.5×, and 15×) postbiotics obtained using the Lactiplantibacillus plantarum ATCC 14917 strain.

In the study, the Lactiplantibacillus plantarum ATCC 14917 strain and the S. aureus (MRSA) ATCC 33591 and 43300 and E. coli ATCC 35218 and 25922 strains present in our department's culture collection were used.

Postbiotic Production, Concentration by Evaporation, and Formation of the Groups: For postbiotic production, the Lactiplantibacillus plantarum ATCC 14917 strain was incubated in MRS Broth at 37°C for 48 hours, then centrifuged at 4200 ×g for 10 minutes to separate the bacteria from the supernatant. The supernatant was then filtered using a 0.22 μm syringe filter to obtain the postbiotic. Concentration of the obtained postbiotics was performed with minor modifications to the method used by Sezen et al. ⁴. For this purpose, 10-, 12.5-, and 15-fold concentrated postbiotics were obtained using a rotary evaporator (Buchi Interface I-300) (at 50°C, 42 mbar pressure, and 250 rpm) ^4,13. The concentration of the postbiotics was determined based on dry matter. The degree of concentration was calculated by considering the ratio between the initial weight and the final weight obtained. The groups were divided into four: 1× (non-concentrated), 10× (10-fold concentrated postbiotic), 12.5× (12.5-fold concentrated postbiotic), and 15× (15-fold concentrated postbiotic) (Figure 1).

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Figure 1: Postbiotics obtained from L. plantarum (1×, 10×, 12.5×, and 15×). 1×: non-concentrated, 10×: 10-fold concentrated, 12.5×: 12.5-fold concentrated, 15×: 15-fold concentrated postbiotic.

Determination of pH and Titratable Acidity: The pH values of the postbiotics were measured using a digital pH meter (HI 11310, Hanna Instruments, USA). The amount of titratable acidity was determined using the method described by Serter et al. ²⁶. Briefly, after adding a few drops of phenolphthalein indicator to 10 mL of postbiotic, titration was performed with 0.25 N NaOH (Merck, Emplura, Darmstadt, Germany) until a pink color developed. To determine the amount of NaOH consumed for 100 mL of postbiotic, the amount of 0.25 N NaOH consumed was multiplied by 10. The result obtained was multiplied by a coefficient of 0.0225 to express it in terms of % g lactic acid.

In vitro antimicrobial activity: The in vitro antimicrobial activity of the postbiotics against S. aureus (MRSA) ATCC 33591 and 43300 and E. coli ATCC 35218 and 25922 strains was determined using the agar spot test. To propagate the bacteria, 100 μL from each culture (from stock cultures stored at -18°C in 20% glycerol (v/v) and Tryptic Soy Broth) was added to 9.9 mL of TSB and kept at 37°C for 24 hours, then centrifuged at 4200 ×g for 10 minutes. The absorbance values of the pellets were adjusted to 1.0 at 600 nm. Accordingly, a stock inoculum value of 8.0 log10 cfu/mL was obtained. Ten-fold dilutions were prepared to obtain a bacterial count of 6.0 log10 cfu/mL for use in the in vitro antimicrobial activity tests. Then, 100 μL of bacterial culture from the appropriate prepared dilution was spread onto the surface of previously prepared Mueller-Hinton agar (15 mL), and the plates were kept at ambient temperature for 10 minutes to allow the bacteria to adhere to the surface. Subsequently, 20 μL of postbiotic was absorbed into the Petri dishes, and after 24 hours of incubation at 37°C, the diameters of the clear zones were measured using a digital micrometer ²⁷.

Statistical Analysis: When performing the statistical analyses, the data were analyzed using the SPSS program (IBM SPSS, version 21.0, IBM Corporation, USA). After the obtained data were transformed logarithmically, all values were presented as mean ± standard deviation. The data were first analyzed for normality. Analysis of variance (ANOVA) was used for normally distributed data, and the post hoc Tukey test was applied at a 5% confidence level (p<0.05); the Kruskal-Wallis analysis was applied to abnormally distributed data ²⁸.

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Table 1: pH and titratable acidity content of concentrated postbiotics obtained from L. plantarum. Values with different superscripts (a-c) in the same column are statistically significant (p<0.05). 1×: non-concentrated, 10×: 10-fold concentrated, 12.5×: 12.5-fold concentrated, 15×: 15-fold concentrated postbiotic.

The inhibition zone diameters and images against the pathogenic bacteria used in the study are presented (Table 2) (Figure 2). When the in vitro antimicrobial analysis results are examined, it was determined that as the concentration ratio of the postbiotics (1×, 10×, 12.5×, and 15×) increased, a higher inhibition zone was formed against the specified pathogens (p<0.05). The highest inhibition zone on E. coli ATCC 25922 and 35218 was determined to be in the 15× group, at 9.44 and 10.18 mm, respectively. Similarly, on S. aureus ATCC 43300 and 33591, it was observed that 15× provided the highest inhibition zone diameters, at 10.79 and 10.67 mm (p<0.05).

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Table 2: Inhibition zone diameters (mm) of concentrated postbiotics obtained from L. plantarum. Values with different superscripts (a-d) in the same row are statistically significant (p<0.05). 1×: non-concentrated, 10×: 10-fold concentrated, 12.5×: 12.5-fold concentrated, 15×: 15-fold concentrated postbiotic.

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Figure 2: Inhibition zone diameter images (mm) of 1×, 10×, 12.5×, and 15× concentrated postbiotics obtained from L. plantarum. 1×: non-concentrated, 10×: 10-fold concentrated, 12.5×: 12.5-fold concentrated, 15×: 15-fold concentrated postbiotic.

Titratable acidity (TA) is a measure of the total amount of acid in a postbiotic solution and is generally expressed in terms of % lactic acid equivalent (LAE) in the characterization of postbiotics. This parameter, together with the pH value of the postbiotic, is considered one of the fundamental factors determining the inhibitory power against pathogenic microorganisms ^25,28. The amount of titratable acidity can vary significantly depending on the bacterial strain used and the production method of the postbiotic. In the present study, titratable acidity increased as the concentration ratio increased, supporting the pH results (p<0.05) (Table 1). The highest titratable acidity value was determined to be at 15×, at 14.38 % l.a. (p<0.05). Similar to the findings of the study, in a study conducted by Sezen et al. ^4, it was determined that when postbiotics were concentrated 2× and 4×, the titratable acid amounts increased significantly (while the acidity in a non-concentrated postbiotic was between 1.97-2.19%, this ratio was between 6.78-7.08% in the 4× concentrated form). Furthermore, in a previous study conducted using Pediococcus acidilactici, the acidity level of the whole-cell postbiotic (WCP) (1.44% LAE) was found to be higher than that of the cell-free supernatant (CFS) (1.27% LAE), and it is thought that the reason for this is the release of intracellular organic acids into the medium due to the disintegration (lysis) of bacterial cells during autoclaving ²⁹. In another study, it was determined that the CFS obtained from a Latilactobacillus sakei/Staphylococcus xylosus (LS) mixture exhibited higher titratable acidity and lower pH compared to that obtained from the Pediococcus acidilactici (PA) strain alone ²⁵. Postbiotics exhibit broad-spectrum and potent antimicrobial activity against foodborne pathogenic bacteria under in vitro conditions. This effect occurs through the synergistic interaction of the organic acids, bacteriocins, hydrogen peroxide, functional peptides, and volatile components contained in postbiotics ^12,15.

In the present study, it was observed that the inhibition zone diameters also increased proportionally as the concentration ratio increased compared to the non-concentrated postbiotic on the pathogenic microorganisms used (Table 2). Similarly, in a study conducted by Sezen et al. ⁴ using L. plantarum, L. curvatus, and L. sakei cultures, it was determined that the inhibition zone diameter increased as the concentration ratio (2×, 4×) increased. In another study ^15, L. plantarum postbiotic was used, and zone diameters of 20.21 mm on E. coli O157:H7 and 22.56 mm on S. aureus were obtained. In a previous study conducted using Pediococcus acidilactici postbiotic on some pathogenic microorganisms (E. coli O103 and S. aureus), inhibition zone diameters ranging from 8.22 to 10.49 mm were obtained ²⁹. The inhibition zone diameters obtained in studies vary depending on the bacterial strain used, the amount of postbiotic used, the application form within the food matrix (liquid, powder, or film), the formulation, and the concentration process ¹³.

In conclusion, it was determined that the 10×, 12.5×, and 15× concentrated postbiotics obtained from the Lactiplantibacillus plantarum culture formed inhibition zones ranging from 8.05 to 10.79 mm against E. coli and S. aureus strains. Additionally, the pH values of the 10×, 12.5×, and 15× concentrated postbiotics ranged from 4.44 to 4.97, and the titratable acidity values ranged from 1.42 to 14.38 % g lactic acid. In line with the findings obtained, it was determined that as the concentration ratio increased, the postbiotic formed a higher inhibition zone against the specified bacteria, the pH value decreased significantly, and the titratable acidity ratio increased (p<0.05). In short, it is thought that postbiotics, owing to the organic acids, bacteriocins, and other bioactive components they contain, will contribute to enhancing microbiological and chemical quality by both preserving food safety and extending shelf life and by exhibiting antimicrobial effects on major pathogens.

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