Vinegar is produced by bacteria and yeast metabolism through the fermentation of carbohydrates ⁷. A wide range of raw materials, including cereals (rice, barley, and millet etc.), fruits (apples, grapes, and coconuts etc.), vegetables (onion etc.), animal derived foods (honey and whey), sugar cane and roots can be used to produce vinegar ^3,8. The production of traditional vinegar involves a two-stage fermentation process.

In the initial stage, carbohydrates are converted to alcohols by the metabolic actions of yeass, particularly those belonging to the Saccharomyces species. Subsequently, the alcoholic compounds are degraded to carboxylic acids, including acetic acid. The second stage of fermentation is facilitated by the involvement of various bacteria belonging to the genera of Acetobacter, Gluconobacter, and Gluconacetobacter ⁹.

Due to producing through the fermentation carbonhydrates, vinegars contain a variety of bioactive compounds, including organic acids, polyphenols, and volatiles ⁷. However, the amount and variety of bioactive compounds may vary depending on the raw material and the fermentation process, including temperature, duration, and microorganisms. ^6,10. It is well documented that vinegars exhibit a variety of bioactive properties. In this manner, a number of bioactive properties have been identified in vinegars, including antimicrobial, anti-inflammatory, antidiabetic, anticarcinogenic, antioxidative, kidney stone-prevention, blood sugar-lowering, and immune-stimulating effects ^3,10-12. Additionally, vinegar has been shown to promote digestion and facilitate weight loss ^1,3,13-16.

The contamination of foods by pathogenic microorganisms (mainly bacteria) may result in the occurrence of foodborne diseases and outbreaks. In this context, the European Food Safety Authority (EFSA, 2022) have reported that the most prevalent foodborne pathogens include Salmonella spp., Shiga-toxigenic Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus ¹⁷. Furthermore, certain fungal strains, including Aspergillus spp., are not only pathogenic to humans by producing mycotoxins, but also contribute to the deterioration of a great variety of foods (18). Consequently, further investigation is still required to identify effective decontamination approaches and/or preservatives that can be employed to prevent and control these foodborne zoonoses and spoilage microorganisms. ^2,8,19.

As previously stated, numerous studies have demonstrated the presence of a diverse array of bioactive compounds and beneficial properties commonly associated with vinegars. However, the complexity of the raw materials and the extensive range of fermenting microorganisms suggest that there is a significant scope for further investigation into the bioactive potential of vinegars ¹⁶. Therefore, further characterization and testing the antimicrobial activity of vinegars produced from different raw materials are still required. In the light of this aforementioned information, the aim of the current research is to identify the bioactive compoounds of vinegars derived from different raw materials, including date, fig, mulberry, black mulberry, and pomegranate. Additionally, it was aimed to evaluate the antimicrobial activity of these vinegars against foodborne microorganisms, including Escherichia coli O157:H7, Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, Staphylococcus aureus, and Aspergillus parasiticus.

Methods
Characterization of Vinegars: The organic acids, volatiles, pH, TPC, individual phenolic and flavonoid compounds of the vinegars were determined by the method outlined in our previously published research ⁸. The organic acids were determined by using a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC, Shimadzu LC 20AD, Japan). The identification of volatiles was conducted through the using of Gas Chromatography-Mass Spectrometry. For the determination of TPC, Folin-Ciocalteu’s assay was used, and the results were expressed as mg gallic acid equivalent GAE/L. Individual phenolic compounds in the vinegars were determined by HPLC (Shimadzu LC 20AD Corporation, Kyoto, Japan), and the results are given as mg/L.

Determination of in vitro Antimicrobial Activities of Vinegars: The in vitro antimicrobial activity of the vinegars were evaluated against Escherichia coli O157:H7 ATCC 43895, Escherichia coli ATCC 35218, Listeria monocytogenes ATCC 13932, Salmonella Typhimurium ATCC 14028, Salmonella Enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923, and Aspergillus parasiticus NRRL 2999. The diameter of inhibition zones, MIC, MBC, MFC values of the vinegars were determined against tested strains. Prior to conducting in vitro antimicrobial tests, the vinegar samples were filtered through a 0.22 μm pore size membrane to remove the initial microbial load. The disc diffusion method was used to determination of the diamater of inhibition zones. For this purpose, the tested microorganisms were separately inoculated onto Mueller-Hinton agar at a level of 6.0 log10. Following inoculation, the plates were incubated at ambient temperature for 10 minutes to allow for microbial attachment to the agar surface. Subsequently, 20 μL of the filtered vinegar samples were embedded in sterile empty filter papers, which were then placed on the plates. The plates were then incubated at 37 °C for 24 h. Afterwards, the diamater of inhibition zones were measured with digital callipers ²⁰. The MIC values of the vinegars were determined using the broth dilution method. The two-fold dilutions starting from 50% concentration of vinegars in Nutrient broth were prepared, and then 100 μL inoculum of each pathogenic bacteria (6.0 log10) was separately added to the tubes. Then, the inoculated tubes were kept at 35 ± 1 °C for 24 h, and the lowest concentration of the vinegars with no visible bacterial growth was accepted as MIC values ²¹. The MBC and MFC value was subsequently determined following the completion of the MIC assay. A 10-μL aliquot from each of the tubes containing concentrations of 2 × MIC, 4 × MIC, and 8 × MIC was spread onto MHA plates. After 24 h of incubation at 37°C, the minimum concentration that completely inactivated the microorganisms was determined as the MBC and MFC values ²².

Statistical Analyses: In the present study, the data were obtained from three independent replicates. Three different samples from each vinegar were employed in each replicate. Following preliminary statistical evaluation, it was ascertained that the data obtained satisfied the parametric test assumptions. Consequently, analysis of variance (ANOVA) and post-hoc Tukey's test was employed to ascertain the differences between the vinegars. The statistical analyses were conducted using the SPSS software, version 21.0. All data sets were presented as mean ± standard deviation, and a p -value of 0.05 was considered statistically significant.

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Table 1: Organic acids present in the vinegars (Mean ± S.D.)

As illustrated in Figure 1, a total of 7 carboxylic acids, 21 alcohols, 19 esters, 8 ketones, 6 phenyl and phenol compounds, 5 terpenes, and 8 miscellaneous compounds were identified in the volatile fractions of the vinegars. Among the carboxylic acids, acetic acid was the most abundant compound in all vinegars. The black mulberry vinegar had the highest carboxylic acid concentration (955.77 peak area) among the vinegars used in the study. On the other hand, alcohols comprised the highest peak area in the volatile fractions of the date and fig. The ester compounds were identified in black mulberry vinegar, with a concentration of 1046.93 peak area. Additionally, these compounds were identified as the most prevalent volatiles in the black mulberry vinegar. The study revealed that ketones, phenyl and phenol compounds, as well as acids and esters, exhibited higher peak areas in the black mulberry vinegar in comparison to the other samples.

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Figure 1: The hierarchical clustering analysis with a heatmap presentation of the volatiles identified in the vinegar samples used in the study

It was found that the pH values of date, fig, mulberry, black mulberry, and pomegranate vinegars were found to be 3.58, 3.74, 3.68, 3.27, and 3.59 respectively (Figure 2). TPC values of the vinegar samples were found to range between 96.74 and 982.61 mg GAE/L. The differences between the vinegars were found to be significant in terms of TPC, as shown in Figure 3 (p≤0.05). In the current study, a total of 20 different phenolic and flavonoid compounds were identified in the date, 17 in the fig and mulberry, 21 in the black mulberry, and 13 in the pomegranate vinegars, as shown in Table 2. Caftaric acid was identified as the most prevalent phenolic compound in the date, mulberry, and black mulberry vinegars, with concentrations of 3.83, 12.88, and 34.00 mg/L, respectively. Additionally, procyanidin B2 was identified as the most prominent polyphenol in fig and pomegranate vinegars, with concentrations of 4.38 and 4.25 mg/L, respectively. The characterization analysis revealed that the black mulberry vinegar had the highest concentrations of organic acids, phenolic and flavonoid compounds, phenyl and phenols, ketones, esters, and acids in comparison to the other vinegars.

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Figure 2: The pH values of the vinegars (Mean ± S.D.). A-C: The mean values with different letters are significantly different (p<0.05)

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Figure 3: The total phenolic content (TPC) values of the vinegars (Mean ± S.D.). A-D: The mean values with different letters are significantly different (p<0.05).

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Table 2: Individual phenolic and flavonoid compunds present in the vinegars (Mean ± S.D.)

The MIC, MBC, and MFC values, and the diameter of inhibition zones are shown in Table 3. It was found that the MIC values of the vinegars were found to range between 6.25 to 20.83% against the tested strains. Furthermore, the MBC and MFC values displayed a range of 6.25% to 50%. The black mulberry had the lowest MIC, MBC, and MFC values, and displayed the highest inhibition zones against the tested pathogens compared to the other vinegars (p˂0.05). The in vitro antimicrobial activity tests showed that the pomegranate vinegar had the highest MIC, MBC, MFC, and the lowest diamater of inhibition zones compared to other vinegar samples.

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Table 3: Minimum inhibitory concentration (%), minimum bactericidal concentration (%), and the diameter of inhibition zones (mm) of the vinegar against different pathogenic bacteria (Mean ± S.D.)

In this study, a total of five organic acids were identified in the vinegars, with significant differences in their concentration between the samples. Similarly, other studies have documented comparable variations in the organic acid composition of the vinegars ^3,5,24.

It is well known that volatile compounds exert a significant influence on the sensory attributes of foods and consumer acceptance ²⁵. As mentioned above, the level of these volatiles is primarily depends on the raw material used in the vinegar production and the fermantation conditions. In line with this information, the results of this study demonstrated that the vinegars comprised a considerable quantity of volatiles. In addition, alcohols and esters, which are produced through the fermentation of sugars by microorganisms, are known for display a diverse array of bioactivities. These include antimicrobial, anticancer, and antitumour properties ⁸. A variety of volatile compounds have been identified in a number of vinegars produced by different fruits, including black mulberry, white mulberry, hawthorn, grapes, apple etc. The studies have revealed that the vinegar produced from a given fruit can exhibit a distinctive profile of volatile compounds, which may be influenced by factors such as the variety of fruit, the processing method, and the fermentation conditions ^{8,16,19,25-27}. These pieces of information can explain the differences in the levels of the bioactive compounds and bioactivies of the vinegars used in the current study.

The pH values of date, fig, mulberry, black mulberry, and pomegranate vinegars were found between 3.27 and 3.74. Consistently, in a study conducted by Silva et al. ^28, the pH values of the six different vinegar samples were found to range between 2.83 and 3.49. Furthermore, Kara et al. ⁶ reported that the pH values of the 11 vinegars were found to be between 2.37 and 4.47. As shown in Figure 2, the black mulberry vinegar had the lowest pH (3.27±0.02). Contrarily, Kara et al. ^5, reported that the four apple vinegars had the lowest pH (3.6). In addition, in a study conducted by Yıldız et al. (9), the pH value of whey vinegar was found to be 4.55. As demonstrated by the literature reviewed above, there are considerable variations in the pH values of the various vinegars. The possible reasons for this phenomenon are thought to be attributable to the type of fruit used in vinegar production or the method practised in vinegar production process ¹⁹.

As shown in Figure 3, significant differences were found in the levels of TPC among the vinegars, and the highest TPC was found in the black mulberrry (P ≤ 0.05). The results revealed that the vinegars varied significantly in terms of TPC, and this finding is consistent with the existing literature ^6,28. It is well known that phenolics and flavonoids have antimicrobial and antioxidant properties. Furthermore, flavonoids have been demonstrated to exhibit a variety of bioactive effects, including anti-inflammatory, anti-thrombotic, and anti-mutagenic activities. ⁸. These compounds have also affect on the sensory characteristics of vinegars ¹⁶. The phenolic compounds present in vinegar are mainly derived from the raw materials used in its production. Furthermore, it has been demonstrated that the total polyphenol concentration in vinegar increases in line with the duration of the ripening process ^3,10,29,30. In the current study, a total of 20 different phenolic and flavonoid compounds were identified in the date, 17 in the fig and mulberry, 21 in the black mulberry, and 13 in the pomegranate vinegars (Table 2). The reasons for the differences in the variety and quantity of polyphenols identified in the various vinegar samples may be associated with the aforementioned factors.

The MIC, MBC, and MFC values, as well as the diameter of inhibition zones of the vinegars, were found to range between 1.56 and 20.83%, 6.25 and 50%, and 8.22 and 19.81 mm, respectively, against the tested microorganisms. In contrast, Sengün et al. ³¹ reported that the MIC values of the different vinegars were found to range betwee 3.12 to 12.5% against certain foodborne pathogens. In another study, Kahraman et al. ¹⁹ found that the MIC values of apple and grape vinegars against eleven pathogens were ranged from 6.25% to12.5%, and the MBC values were ranged from 6.25 to 25%. In addition, it was found that the MIC values of the vinegars were recorded as 1.95 and 500 μL/mL against S. aureus and C. Albicans, respectively ⁶. However, they also reported that the Aspergillus parasiticus exhibited resistance against all of the vinegars ⁶. Yıldız et al. ⁹ reported that the MIC and MBC values were found to be 6.25 and 50 μL/mL against seven pathogenic microorganisms. On the other hand, Kara et al. ⁵ tested the antimicrobial effect of the four different apple vinegars against five pathogenic microorganisms, and the MIC values were found between 1.95 and 500 μL/mL, and the MBC values were ranged from 3.91 and 500 μL/mL.

In our previous study, the diameters of inhibition zones of the hawthorn vinegar were ranged from 9.22 to 11.43 mm against 12 pathogenic bacteria ⁸. In different studies, the diamater of inhibition zones of the various vinegars were foudn to be range between 6.30 to 40.17 mm ^5,6,9,28. It is believed that the differences between the studies are probably due to the pH value of the vinegars, the level of bioactive compounds that contribute to the antimicrobial activity (organic acids, volatiles, and polyphenols, etc.), and the susceptibility of the tested microorganisms.

The characterization analyses of the vinegars revealed that all vinegars used in the study contain a wide range of bioactive compounds, including organic acids, individual phenolic and flavonoid compounds, and volatiles. Nevertheless, the quantities of these bioactive compounds exhibited considerable variation between the vinegars. Consequently, the vinegars exhibited a diverse spectrum of antimicrobial activity against the tested microorganisms. Among the vinegars, black mulberry exhibited highest antimicrobial activity in comparion to the other vinegars. The result of the current research indicate that the vinegars displayed a considerable potential in terms of in vitro antimicrobial activity. Although they have a strong antimicrobial effect, their use in foods should take into account that they may have a pungent taste and aroma specific to the fruit from which they are derived. In conclusion, vinegars can be employed as a natural preservative to enhance the microbiological quality and safety of foods.

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