Antimicrobial and Antioxidant Properties of Hawthorn Vinegar

Abstract

This study investigated the antimicrobial and antioxidant properties of hawthorn vinegar. The antimicrobial activity was evaluated against Staphylococcus aureus, Salmonella, Saccharomyces cerevisiae, and Bacillus subtilis using the filter paper disc method. Antioxidant capacity was assessed through DPPH, hydroxyl, and superoxide anion radical scavenging assays. The results show that hawthorn vinegar exhibited inhibitory effects against all tested microorganisms, with the most potent activity against Salmonella. The vinegar extract demonstrated considerable antioxidant potential, with maximum scavenging rates of 71%, 82.2%, and 81.3% for DPPH, hydroxyl, and superoxide anion radicals, respectively. These findings suggest that hawthorn vinegar possesses notable antimicrobial and antioxidant properties, warranting further investigation for potential applications in food preservation and health promotion.

1. Introduction

Hawthorn (Crataegus sp.) is a medicinal plant with a long history of use in traditional medicine and culinary applications [1]. The fruit, leaves, and flowers of hawthorn are rich in bioactive compounds, including polyphenols, flavonoids, terpenoids, organic acids, and vitamins. These compounds contribute to hawthorn’s potential health benefits, such as its anti-inflammatory, antioxidant, and immune-modulating effects, as well as its ability to promote overall health [2,3,4]. In recent years, there has been growing interest in the development of functional foods that provide health benefits beyond basic nutrition. As a separate but related line of inquiry, hawthorn has attracted attention from researchers due to its rich phytochemical profile and potential health-promoting properties [2,3,4].

Vinegar, a fermented food product, has long been valued not only for its culinary uses but also for its potential health effects [5]. The primary functions of vinegar are linked to its acetic acid, which exhibits anti-glycemic and antidiabetic properties, and its polyphenols, which act as antioxidants [6]. But other compounds present in the raw materials or formed during fermentation also contribute to its properties. Hawthorn vinegar, as an emerging functional beverage, combines the potential health benefits of hawthorn with the functional characteristics of vinegar and has garnered significant research interest in recent years [2,3,4].

Hawthorn is rich in various bioactive substances, primarily including polyphenolic compounds (such as chlorogenic acid, procyanidin B2, and epicatechin), flavonoids (proanthocyanidins, luteolin, quercetin, and rutin), pentacyclic triterpenoids (ursolic acid, hawthornic acid, and oleanolic acid), and organic acids (like malic acid and citric acid). These active components endow hawthorn and its processed products (such as hawthorn vinegar) with multiple biological activities, including antioxidant, anti-inflammatory, lipid-regulating, and cardiovascular protective effects [7,8]. Multiple functional components have been identified in hawthorn vinegar, such as organic acids, phenolic and flavonoid compounds, and bioactive volatiles [9]. The composition and content of these compounds may be influenced by processing methods. For instance, ultrasound treatment has been reported to enhance the total phenolic and flavonoid contents as well as DPPH and CUPRAC antioxidant activities in hawthorn vinegar [4].

Previous studies have demonstrated the antioxidant properties, immunomodulatory roles, and metabolic functions of hawthorn vinegar [2,3,4,5]. Antioxidants are compounds that can neutralize harmful free radicals in the body, potentially reducing oxidative stress and associated health risks [10]. The antioxidant capacity of various fruit vinegars has been reported, often correlating with their polyphenol content [11,12]. The specific mechanisms of action and the relative contributions of different antioxidant assays have not been comprehensively analyzed. Some research has also explored its antimicrobial activities and its impact on the quality parameters of meat products [9]. Antimicrobial activity is a crucial characteristic for food preservation and safety. The ability of vinegar to inhibit microbial growth has been reported to be linked with acetic acid playing a primary role in this effect [13]. However, the comparative efficacy of hawthorn vinegar against different types of microorganisms (Gram-positive bacteria, Gram-negative bacteria, and fungi) under standardized conditions remains to be fully elucidated.

The present study aims to evaluate the antimicrobial activity of hawthorn vinegar against a diverse panel of microorganisms, including Staphylococcus aureus, Salmonella, Saccharomyces cerevisiae, and Bacillus subtilis, using standardized methods. It also seeks to assess the antioxidant capacity of hawthorn vinegar through multiple free radical scavenging assays (DPPH, hydroxyl, and superoxide anion) to provide a comprehensive profile of its antioxidant mechanisms. By addressing these objectives, this study aims to provide a more comprehensive understanding of the functional properties of hawthorn vinegar. The findings may have implications for its potential applications in food preservation and as a functional food ingredient, contributing to the broader field of functional food research and development.

2. Materials and Methods

2.1. Hawthorn Vinegar and Extract Preparation

Commercial hawthorn vinegar (Ruitai Hawthorn Vinegar, Yuncheng, China) was used for all experiments. According to the manufacturer’s instructions, the hawthorn vinegar was produced using Chinese hawthorn fruit (Crataegus pinnatifida Bunge) and water as raw materials. The total acidity of the product is ≥3.5 g/100 mL. For the antioxidant assays, a vinegar extract was prepared. Briefly, 150 mL of hawthorn vinegar (100%) was concentrated under vacuum and then extracted with 150 mL ethyl acetate. The organic layer was separated and combined with 150 mL anhydrous ethanol. This mixture was centrifuged, and the supernatant was filtered and evaporated. The resulting extract was dissolved in 150 mL distilled water to obtain the final sample solution.

2.2. Antimicrobial Assay

The antimicrobial activity was evaluated using the filter paper disc method [14,15] with modifications against Staphylococcus aureus (CICC 21600), Salmonella enterica serovar Enteritidis (CICC 21482), Saccharomyces cerevisiae (CICC 31362), and Bacillus subtilis (CICC 10732). All microorganisms used in this study were obtained from the China Center of Industrial Culture Collection (CICC) and maintained in our laboratory. Hawthorn vinegar was used at full strength (100%) and diluted with distilled water to prepare solutions of 50% and 33.3% (v/v) for antimicrobial assays. Sterile filter paper discs (7.5 mm in diameter, modified from the 6 mm discs described in the previous method [14]) were impregnated with these solutions for 2 h. Distilled water served as a negative control. Microbial suspensions were prepared and spread on appropriate agar plates. Specifically, S. aureus and S. enterica were cultured on Luria Agar (LA) at 37 °C, S. cerevisiae was grown on Yeast Extract Peptone Dextrose (YPD) agar at 28 °C, and B. subtilis was cultured on Nutrient Agar (NA) at 37 °C. The impregnated discs were placed on the inoculated agar surfaces. Plates were incubated at 37 °C for 24 h (bacteria) or at 28 °C for 48 h (Saccharomyces cerevisiae). The inhibition zone diameters were measured using the Kirby–Bauer disc diffusion susceptibility test protocol [14]. The zone size was determined by measuring the entire diameter of the inhibition zone, including the 7.5 mm filter paper disc. Inhibition zone diameters were measured in triplicate for each concentration and microorganism.

2.3. Antioxidant Assays

2.3.1. DPPH Radical Scavenging Assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was determined [16]. Briefly, 2 mL of 0.2 mmol/L DPPH solution in ethanol was mixed with different volumes (0.2–1.4 mL) of the vinegar extract. The mixture was adjusted to 10 mL with anhydrous ethanol and incubated in the dark for 30 min. Absorbance was measured at 490 nm. The DPPH radical scavenging rate (%) was calculated as [1 − (A_sample − A_{sample-ethanol})/A_DPPH-ethanol] × 100%, where A_sample is the absorbance of the reaction mixture containing the sample and DPPH, A_{sample-ethanol} is the absorbance of the sample solution without DPPH, and A_DPPH-ethanol is the absorbance of the reaction system containing all reagents except the vinegar extract, with an equivalent volume of distilled water used in place of the sample.

2.3.2. Hydroxyl Radical Scavenging Assay

The hydroxyl radical scavenging activity was assessed [17]. The reaction mixture contained 2 mL FeSO₄ (9 mmol/L), 2 mL salicylic acid–ethanol (9 mmol/L), different volumes of vinegar extract (0.2–1.4 mL), and 2 mL H₂O₂ (9.9 mmol/L). The volume was adjusted to 10 mL with distilled water. After incubation at 37 °C for 30 min, absorbance was measured at 510 nm. The hydroxyl radical scavenging rate (%) was determined using the formula [1 − (A_sample − A_sample-H2O2)/A_blank] × 100%, where A_sample represents the absorbance of the complete reaction system, A_sample-H2O2 is the absorbance of the reaction system without H₂O₂, and A_blank is the absorbance of the reaction system containing all reagents except the vinegar extract, with an equivalent volume of distilled water used in place of the sample.

2.3.3. Superoxide Anion Radical Scavenging Assay

The superoxide anion radical scavenging activity was determined [18]. The reaction mixture contained 6 mL Tris-HCl buffer (pH 8.2, 10 mmol/L), different volumes of vinegar extract (0.2–1.4 mL), and distilled water to make up 9.7 mL. After pre-incubation at 25 °C for 20 min, 0.3 mL pyrogallol (3 mmol/L in 10 mmol/L HCl) was added. Absorbance at 380 nm was measured after 3 min. The superoxide anion scavenging rate (%) was calculated as (A_control − A_sample)/A_control × 100%, where A_control is the absorbance of the reaction system containing all reagents except the vinegar extract, with an equivalent volume of distilled water used in place of the sample, and A_sample is the absorbance of the complete reaction system with the vinegar extract.

2.4. Statistical Analysis

All experiments were performed in triplicate. Results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software (version 10.0). The flowchart for experimental design was created using the Figdraw tool.

3. Results and Discussion

3.1. Antimicrobial Activity of Hawthorn Vinegar

The experimental procedure for assessing the antimicrobial activity of hawthorn vinegar against four microbial strains is illustrated in Figure 1, and the corresponding results are shown in Figure 2. Hawthorn vinegar exhibited inhibitory effects against all tested microorganisms, with varying degrees of effectiveness depending on the concentration and the specific microorganism.

The most potent antimicrobial activity was observed against Salmonella, with inhibition zone diameters of 29 ± 2.0 mm, 23 ± 2.7 mm, and 18 ± 3.5 mm for vinegar concentrations of 100%, 50%, and 33.3%, respectively (Figure 2B). Saccharomyces cerevisiae and B. subtilis showed less sensitivity, with similar inhibition patterns (100%: 24 ± 6.6 mm and 23 ± 3.6 mm; 50%: 17 ± 1.0 mm and 18 ± 1.7 mm; 33.3%: 11 ± 1.0 mm and 13 ± 1.7 mm, respectively) (Figure 2C,D). S. aureus exhibited the least sensitivity, with inhibition zones of 22 ± 2.0 mm, 16 ± 2.7 mm, and 12 ± 2.0 mm for the three concentrations (Figure 2A).

These results suggest that hawthorn vinegar possesses broad-spectrum antimicrobial activity. While this activity is likely primarily attributed to its acetic acid content, a well-known antimicrobial agent, other bioactive compounds present in hawthorn fruit may also contribute to this effect such as flavonoids and phenolic compounds [19]. The observed concentration-dependent effect, where the antimicrobial potency increased with higher vinegar concentrations, is consistent with this hypothesis. However, further research is needed to elucidate the specific contributions of acetic acid and these other compounds to the overall antimicrobial activity of hawthorn vinegar.

The more potent inhibitory effect against Salmonella is particularly noteworthy given the importance of this pathogen in food safety [20]. This suggests that hawthorn vinegar could potentially be used as a natural preservative in food products, especially those susceptible to Salmonella contamination.

The relatively lower sensitivity of S. aureus to hawthorn vinegar, compared to the other tested microorganisms, is an interesting observation. This difference in susceptibility among microbial species could be due to variations in the cell wall structure or other resistance mechanisms [21,22] and warrants further investigation.

Compared to previous research that tested Listeria monocytogenes, Escherichia coli, and Salmonella [9], our study expanded the scope of antimicrobial activity of hawthorn vinegar by including S. aureus, S. cerevisiae, and B. subtilis. The inclusion of yeast and spore-forming bacteria provides insights into hawthorn vinegar’s broader antimicrobial spectrum. Interestingly, our study found the most potent inhibition against Salmonella, while Karatepe et al. reported more uniform inhibition across tested pathogens [9]. This difference may reflect variations in vinegar composition or bacterial strain sensitivities. Notably, Karatepe et al. determined specific MIC values, which could be a valuable addition to our future studies.

3.2. Antioxidant Activity of Hawthorn Vinegar

The antioxidant capacity of hawthorn vinegar extract was evaluated using three different radical scavenging assays. The results are summarized in Figure 3.

DPPH Radical Scavenging (Figure 3B): The hawthorn vinegar extract demonstrated dose-dependent DPPH radical scavenging activity. The scavenging rate increased from 20% at 0.2 mL to a maximum of 71% at 1.4 mL of extract. This indicates a potent ability to donate hydrogen atoms or electrons, neutralizing the DPPH radical [23].

Hydroxyl Radical Scavenging (Figure 3C): The extract exhibited potent hydroxyl radical scavenging activity, with the scavenging rate ranging from 30% at 0.2 mL to 82.2% at 1.4 mL. This high scavenging ability is particularly significant given the highly reactive and damaging nature of hydroxyl radicals in biological systems [24].

Superoxide Anion Radical Scavenging (Figure 3D): Hawthorn vinegar extract also showed considerable superoxide anion radical scavenging activity, with rates increasing from 25% at 0.2 mL to 81.3% at 1.4 mL. This suggests that the extract contains compounds capable of inhibiting the formation of superoxide radicals or promoting their decomposition [25].

The potent antioxidant activity observed across all three assays indicates that hawthorn vinegar contains a variety of antioxidant compounds. These likely include polyphenols, flavonoids, and organic acids, which are known to be present in hawthorn fruit and may be retained or even concentrated during the vinegar production process [1,4,19].

The dose-dependent nature of antioxidant activity suggests that higher concentrations of hawthorn vinegar or its extract could provide greater protective effects against oxidative stress. However, it is important to note that in vitro antioxidant activity does not always directly translate to in vivo effects, and further studies would be needed to confirm the potential health benefits. In fact, an in vivo study using rats demonstrated that hawthorn vinegar exhibits significant antioxidant capacity, which can be enhanced by ultrasound treatment [4]. These findings complement our in vitro results, and both studies highlight hawthorn vinegar’s potential as a natural antioxidant source.

The particularly high scavenging rates for hydroxyl and superoxide anion radicals (82.2% and 81.3%, respectively, at the highest concentration) are significant findings. Hydroxyl radicals are highly reactive and can cause severe damage to cellular components, while superoxide anions are precursors to other reactive oxygen species [26]. The ability of hawthorn vinegar extract to effectively scavenge these radicals suggests that it could potentially play a role in preventing oxidative damage in biological systems.

It is worth noting that the mechanisms underlying the observed antimicrobial and antioxidant effects were not investigated in this study, and the specific compounds responsible for these activities were not identified. Additionally, the potential effects of the vinegar production process on the bioactive compounds in hawthorn fruit remain to be elucidated. These areas represent important directions for future research.

Further studies should focus on identifying and quantifying the bioactive compounds in hawthorn vinegar, investigating the mechanisms of action, and exploring potential synergistic effects among different components. In vivo studies will also be crucial to determine the bioavailability and physiological effects of these compounds.

4. Conclusions

This study provides evidence for the antimicrobial and antioxidant properties of hawthorn vinegar. The vinegar demonstrated broad-spectrum antimicrobial activity, with particularly potent effects against Salmonella. The vinegar also exhibited significant antioxidant capacity, effectively scavenging DPPH, hydroxyl, and superoxide anion radicals. These findings suggest potential applications for hawthorn vinegar in food preservation and as a source of natural antioxidants.