Comprehensive phytochemical characterization and antibacterial evaluation of Zingiber officinale roscoe extract against selected foodborne pathogens: Implications for natural food preservation

Introduction

Foodborne illnesses continue to be a major global public health concern, causing millions of illnesses and large financial losses annually. The main cause of these disorders is microbial contamination of food, especially fresh produce, ready-to-eat items, and minimally processed meals. In tropical countries like India, warm, humid weather accelerates microbial growth, increasing the risk of food deterioration and the spread of dangerous pathogens (Abdul-Mutalib et al., 2015) [1] . E. coli, S. aureus and B. cereus are among the most commonly linked foodborne pathogens; they are linked to food poisoning, diarrhea, and serious consequences in susceptible groups (Kadariya et al., 2014; Yassen, 2016; Othman et al., 2020) [5,6,8,12] . The critical need for efficient and long-lasting natural antimicrobial drugs is highlighted by the rapid emergence of antimicrobial resistance among these pathogens, which has further hampered disease management and food safety policies (Tacconelli et al., 2018) [13] . While synthetic preservatives are still commonly employed, interest in plantderived bioactive chemicals as safer substitutes has increased due to growing consumer concerns about their possible negative health effects and the growing desire for clean-label products (Beristain-Bauza et al., 2019) [14] . Ginger (Zingiber officinale Roscoe), is a popular spice and traditional medicinal herb with a wide range of biological activity, including anti-inflammatory, antioxidant, and antibacterial qualities. Its antibacterial activity has been largely attributed to phenolic compounds such gingerols and shogaols as well as other sesquiterpenes (Karuppiah & Rajaram, 2012; Othman et al., 2020; Sulieman et al., 2024) [4,5,8,9] . Because ethanolic extracts may effectively extract both polar and somewhat non-polar bioactive components, they have consistently shown better antibacterial activity than aqueous preparations (Auta et al., 2011; Islam et al., 2014) [2,3] . Significant variances in the phytochemical content and antibacterial efficiency of ginger have been documented despite a great deal of research on the plant. These discrepancies are mostly caused by changes in harvest age, cultivar, cultivation practices, geographic origin, and extraction techniques. Furthermore, nothing is known about the antibacterial properties and phytochemical profile of Zingiber officinale grown in Salem, Tamil Nadu. To find locally accessible plant resources with promising uses for food safety, region-specific research is crucial. In this investigation, the antibacterial efficacy of an 80% ethanolic extract made from locally grown Zingiber officinale rhizomes against B. cereus, S. aureus and E. coli was assessed. Gas chromatography-mass spectrometry (GC-MS) and Fourier Transform Infrared (FTIR) spectroscopy were used to thoroughly evaluate the extract in order to determine its main bioactive components and functional groups. This study offers a thorough evaluation of the antibacterial potential of locally sourced ginger and advances the creation of natural antimicrobial agents for food safety and preservation applications by combining phytochemical profiling with agar well diffusion and Minimum Inhibitory Concentration (MIC) assays.

Materials and Methods

Plant Material Collection and Preparation

Zingiber officinale Roscoe's fresh rhizomes were gathered from fields in rural Salem, Tamil Nadu, India. After giving the rhizomes, a good wash under running water, they were rinsed with distilled water, peeled to remove the outer skin, and then cut into thin (2-3 mm) sections. For five to seven days, the slices were shade-dried at room temperature (25 to 28 °C). Before being extracted, the dried material was ground up using an electric grinder, put through a 40-mesh sieve, and kept in airtight amber glass containers at 4 °C (Sherifa et al., 2014; Adel & Prakash) [15] .

Preparation of Ethanolic Extract

A modified cold maceration approach was used to extract ethanol (Islam et al., 2014; Karuppiah & Rajaram, 2012) [3,4] . In short, 100 mL of 80% (v/v) analytical-grade ethanol (≥99.9%, Merck, India) was used to extract 10 g of the powdered rhizomes at a solid-to-solvent ratio of 1:10 (w/v). After 72 hours of agitation at 150 rpm in a shaking incubator at room temperature (25-28 °C), the mixture was filtered through Whatman No. 1 filter paper. The crude extract was made by concentrating the filtrate under low pressure at 40 °C using a rotary evaporator (Buchi R-300). After that, it was placed in sterile amber glass vials and stored at 4 °C for additional examination. The dry weight of the recovered crude extract in relation to the starting dry weight of the plant material was used to compute the extraction yield (%) (Mohammadi et al., 2020; Edo et al., 2024) [7,16] .

Phytochemical Characterization Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

A Shimadzu GCMS-QP2020 NX system with a Rtx-5MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) was utilized to describe the phytochemical profile of the 80% ethanolic extract. The carrier gas was 99.999% pure helium at a steady flow rate of 1.0 mL min⁻¹. An initial hold at 60 °C for two minutes, a ramp to 280 °C at 10 °C min⁻¹, and a final hold for ten minutes comprised the oven temperature program. The temperatures of the injector and interface were kept at 250 °C and 280 °C, respectively. One microliter of the material was injected in split mode (1:20). Over a scan range of m/z 40-650, mass spectra were obtained under electron ionization (70 eV). Compounds were identified using known fragmentation patterns and compared with the NIST 20 mass spectral library (≥90% similarity) (Sulieman et al., 2024; Gunasena et al., 2022; Abdullahi et al., 2020) [8,9,10] .

Fourier Transform Infrared (FTIR) Spectroscopy

The main functional groups in the extract were determined using FTIR spectroscopy (Bruker Alpha II FTIR). An FTIR spectrophotometer's Attenuated Total Reflectance (ATR) crystal was immediately coated with a tiny portion of the concentrated extract. 32 scans were used to record spectra in the 4000-400 cm⁻¹ region at a spectral resolution of 4 cm⁻¹. By comparing the measured absorption bands with conventional reference spectra for the distinctive phytochemical components of ginger, functional groups were given (Edo et al., 2024; Tabe et al., 2026) [7,11] .

Bacterial Strains

Based on their clinical and foodborne relevance, reference bacterial strains Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 11778) were chosen (Kadariya et al., 2014; Yassen, 2016; Othman et al., 2020) [5,6,8,12] . The strains were kept at 4 °C after being resurrected from glycerol stocks on nutrient agar.

Antibacterial Activity by Agar Well Diffusion Assay

A 100 mg/mL stock solution was created by reconstituting the crude extract in dimethyl sulfoxide (DMSO). In order to rule out any possible antibacterial impact of the solvent, the final concentration of DMSO in the test did not surpass 1% (v/v) (Auta et al., 2011; Islam et al., 2014) [2,3] . The extract solution was filtered through a 0.22 µm membrane filter to sterilize it before the experiment. The agar well diffusion method outlined by Bauer et al. (1966) was used to assess antibacterial activity. Bacterial suspensions calibrated to the 0.5 McFarland standard (about 1.5 × 10 8 CFU/mL) were uniformly injected into Mueller-Hinton (HiMedia Laboratories, India) agar plates. After aseptically punching 6 mm-diameter wells into the agar, 50, 75, and 100 µL of the extract solution were added to each well. Gentamicin solution (100 mg/mL; 100 µL per well) served as the positive control, and DMSO (100 µL per well) served as the negative control. Following a 24-hour incubation period at 37 °C, the inhibitory zone diameters were measured in millimeters (mm). Each experiment was carried out in triplicate, and the data were expressed using the mean± Standard Deviation (SD) (Karuppiah & Rajaram, 2012; Othman et al., 2020) [4,5,8] .

Determination of Minimum Inhibitory Concentration (MIC)

The broth dilution method was used to calculate the ethanolic ginger extract's Minimum Inhibitory Concentration (MIC) (Wiegand et al., 2008) [17] . To achieve final concentrations of 25, 50, and 100 mg/mL, a stock solution of the crude extract (100 mg/mL) was made in DMSO and diluted with sterile nutritional broth. The broth's final DMSO concentration was less than 1% (v/v), which has been shown to have no effect on bacterial growth inhibition (Auta et al., 2011) [2] . The corresponding extract concentrations were made in three sterile test tubes with 5 mL of nutritional broth. A tube holding nutritional broth with extract but without bacterial inoculation functioned as the negative (sterility) control, while another tube containing inoculated broth without extract served as the positive growth control. With the exception of the negative control, every tube was infected with a standardized bacterial culture that was adjusted to the 0.5 McFarland standard (about 1.5 × 10 8 CFU/mL) and incubated for 24 hours at 37 °C in an aerobic environment. Bacterial growth was assessed visually and using a UV-visible spectrophotometer to measure the optical density at 600 nm (OD 600 ) after incubation. Every experiment was conducted in triplicate, and the mean± Standard Deviation (SD) was used to express the results (Wiegand et al., 2008) [17] .

Results

Plant Material Processing and Extract Preparation

A dark reddish-brown, viscous crude extract with an extraction yield of 14.8% w/w was produced by cold macerating 295 g of fine powder with 80% ethanol. The extract was stable at 4 °C and had a strong, unique, pungent fragrance.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Based on comparison with the NIST 20 mass spectral library and published fragmentation data, a complex phytochemical profile including 42 compounds representing 97.2% of the total ion current was found by GC-MS analysis of the 80% ethanolic extract. Sesquiterpenes and phenolic ketones predominated in the extract. 6-gingerol (22.45%), αzingiberene (15.34%), 6-shogaol (9.67%), and βsesquiphellandrene (8.91%) were the main components. Significant bioactive potential was shown by the combined gingerol and shogaol content of 39.8%. Notes: ν = stretching; νas = asymmetric stretching; νs = symmetric stretching; δ = bending; γ = out-of-plane bending. Spectra were recorded in ATR mode (4000-400 cm⁻¹, 32 scans, 4 cm⁻¹ resolution).

Antibacterial Activity by Agar Well Diffusion Assay

Zingiber officinale's 80% ethanolic extract clearly showed concentration-dependent antibacterial activity against the foodborne pathogens that were tested. Zones of inhibition were seen after a 24-hour incubation period at 37 °C. The DMSO negative control showed no inhibitory zone. The inhibition zones grew as the extract concentration rose, indicating a concentration-dependent antibacterial activity against every strain of bacteria examined. At 100 µL, the extract generated the highest mean inhibition zone (18.3± 1.5 mm) against Staphylococcus aureus. Additionally, moderate antibacterial activity against Bacillus cereus and Escherichia coli was noted. The assay's validity was confirmed by the gentamicin positive control, which generated inhibition zones between 23 and 26 mm, while the DMSO negative control showed no inhibition zone.

Determination of Minimum Inhibitory Concentration (MIC)

The broth dilution method was used to calculate the minimum inhibitory concentration (MIC) of the 80% ethanolic ginger extract. Following a 24-hour incubation period, bacterial growth was evaluated both visually and by measuring optical density at 600 nm (OD 600 ). Bacterial growth was clearly inhibited by the extract in a dosedependent manner. The Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration exhibiting no discernible growth and OD 600 values similar to the sterility control (<0.1). While B. cereus required a higher dosage (MIC = 100 mg/mL) for total suppression, the extract showed considerable inhibitory activity against S. aureus and E. coli (MIC = 50 mg/mL). The concentrationdependent zones of inhibition seen in the agar well diffusion assay is in line with these findings.

Discussion

The 80% ethanolic extract of Zingiber officinale Roscoe, which was obtained from Salem, Tamil Nadu, showed concentration-dependent antibacterial activity against a few foodborne pathogens, according to the current study. The extraction yield of 14.8% (w/w) obtained by cold maceration, which is comparable to previous reports using similar ethanolic extraction systems, shows that 80% ethanol is an effective solvent for extracting both polar and moderately non-polar phytochemicals ( [3,4,7] . A highly varied phytochemical profile with 42 tentatively identified chemicals, accounting for 97.2% of the total ion current, was found by GC-MS analysis. Phenolic ketones and sesquiterpenes predominated in the extract, with the main ingredients being 6-gingerol (22.45%), α-zingiberene (15.34%), 6-shogaol (9.67%), and β-sesquiphellandrene (8.91%). The cultivar may be a rich source of these bioactive compounds because the combined gingerol and shogaol content (39.8%) was similar to or higher than values reported for ginger grown in several other geographical regions (Sulieman et al., 2024; Gunasena et al., 2022; Abdullahi et al., 2020) [8,9,10] . However, cultivar, environmental factors, cultivation techniques, harvest maturity, and post-harvest processing probably have an impact on this variation. These results were corroborated by FTIR analysis, which revealed hydroxyl, carbonyl, and aromatic functional groups frequently linked to the main bioactive components of ginger. [4,5,6,8] . This difference has been attributed to the outer lipopolysaccharide membrane of Gram-negative bacteria, which may reduce the penetration of hydrophobic phytochemicals. This variance is expected due to differences in the plant source, extraction methods, phytochemical content, and bacterial strains, even if the inhibitory zones found in this investigation were moderate when compared to those found in other studies ( [4,5,7,8] . Because of its innate physiological characteristics, B. cereus can need a somewhat higher concentration. The gradual inhibition of bacterial growth with increasing extract content is indicated by the dosedependent decrease in OD 600 values. Gingerols and shogaols, which have been shown to have antimicrobial qualities, may mediate the observed antibacterial activity. The current work combines GC-MS, FTIR, agar well diffusion, and MIC analyses to thoroughly assess the antibacterial potential of locally grown Zingiber officinale from Salem, Tamil Nadu, in contrast to numerous earlier studies that concentrated mainly on antibacterial activity. The results point to the possibility of using ginger ethanolic extract as a natural antibacterial agent for food preservation. Before being used in practice, its efficacy should be confirmed in actual food systems under various processing and storage circumstances. In order to increase stability and antimicrobial efficacy, future research should assess its activity against isolates produced from food, look into potential synergistic interactions with other natural preservatives, and investigate cutting-edge delivery methods such nanoformulations. Overall, the current study shows that Zingiber officinale grown locally has a rich phytochemical composition and has encouraging antibacterial efficacy against significant foodborne pathogens. These findings provide scientific support for the potential use of bioactive components derived from ginger to create natural antibacterial agents for food safety purposes.

Conclusion

The ethanolic extract of locally grown Zingiber officinale Roscoe from Salem, Tamil Nadu, was shown in this study to have potential concentration-dependent antibacterial activity against Bacillus cereus, Staphylococcus aureus, and Escherichia coli. The phytochemical profile rich in bioactive components shown by GC-MS and FTIR analysis may be responsible for the reported antibacterial action. These results offer scientific backing for the historic usage of ginger as a natural antibacterial agent and point to its possible use as a sustainable substitute for artificial preservatives to enhance food safety. Further research in real food systems is required to verify its practical efficacy and explore complex formulations for enhanced stability and antibacterial action.

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