Chemical Compositions and Antibacterial Activities of Litsea cubeba Essential Oil and Its Distillates Prepared by Vacuum Fractional Distillation and Molecular Distillation

Abstract

Litsea cubeba essential oil (LCEO) has various bioactivities and wide applications. However, most reported LCEOs are directly extracted from plants, and studies on further processing of LCEO to enrich bioactive components using modern separation techniques are scarce. In this study, LCEO was extracted by hydrodistillation and further processed via vacuum fractional distillation (VFD) and molecular distillation (MD). The chemical compositions of LCEO and seven distillates were analyzed, and the activities of the EOs and eight individual constituents against seven bacteria were tested. Distillates VFD3 and MDH2 showed the best activity against Escherichia coli, Staphylococcus aureus, and Agrobacterium rhizogenes. VFD3 exerted antibacterial action against A. rhizogenes by inhibiting biofilms, damaging the cell membrane and cell wall, and perturbing metabolic pathways. VFD and MD are effective processing methods for changing the chemical composition and enhancing the bioactivity of LCEO, which might be used to improve the quality and extend the applications of LCEO and other EOs.

1. Introduction

Litsea cubeba (Lour.) Pers. (L. cubeba) is an important natural woody plant that is widely distributed in Southeast Asia, China, and Japan.^1,2 The extract of L. cubeba is rich in various natural components that are widely used in the food, medicine, and cosmetics industries.^3,4 The essential oil (EO) of L. cubeba has received much attention because of its wide range of bioactivities.⁵ The chemical compositions of EOs extracted from different parts of L. cubeba are quite different, and the highest yield of EO is from the fruit.^6,7 The origin, extraction, and processing methods of LCEO affect its components and yield.^7,8 China is a major producer of LCEO in the world, with an annual output of 1500–2000 tonnes.⁸ Chinese customs have reported that from 2019 to 2023, the total export volume of LCEO reached 1682 tonnes.⁹ Therefore, conducting research on LCEO can help researchers better utilize the advantages of high LCEO production in China.

LCEO is mainly composed of oxygenated monoterpenes, monoterpenes, and sesquiterpenes. Citral, an oxygenated monoterpene, is a major component of LCEO.¹⁰ Zhao et al. used the hydrodistillation (HD) method to extract LCEO from L. cubeba fruit collected in Hangzhou City, China, and reported that geranial (α-citral) and neral (β-citral) accounted for 41.09 and 34.70%, respectively, of the total EO.¹⁰ LCEO has antioxidant, anticancer, antimicrobial, and anthelmintic activities, which are affected by its chemical composition.⁴ Citral exhibits bacteriostatic activity against methicillin-resistant Staphylococcus aureus (MRSA), and the activity is antagonistically modulated by the accompanying components.¹¹ Therefore, the key to improving the application of LCEO and enhancing its biological activity is determining how to better enrich its active components. LCEO samples used in most reported studies were extracted using the HD method without further processing.¹² In this study, LCEO was extracted from the fruits of L. cubeba by HD, and then, vacuum fractional distillation (VFD) and molecular distillation (MD) were performed to further fractionate the LCEO and enrich the bioactive components. Distillation is the most important technique for extracting and separating fluid mixtures in the process industry. The main methods used to extract EO from the plant material include HD, cold pressing, supercritical fluid extraction, etc.⁴ HD is the most common method used to extract EO without organic solvents. The process involves immersing the plant material in a water-filled container and heating it to a boil. The resulting water–oil vapor is condensed, and the oil and water phases are separated.¹³ The main distillation methods used to process EO into different fractions include normal atmospheric distillation, vacuum distillation (VD), fractional distillation (FD), VFD, and MD. VFD is a physical separation process for liquid mixtures based on differences in the boiling points of the components in the mixture. This decreases the boiling temperatures of the compounds under vacuum conditions. It is an efficient process that takes advantage of the preservation of thermally sensitive compounds.¹⁴ MD is a specialized high-vacuum technique used to separate thermally unstable and high-boiling compounds based on different mean free path lengths of the molecules at different pressures and temperatures.¹⁵

The application of VFD to separate the constituents of EO has rarely been reported. Do et al. separated lemongrass EO by VFD to obtain fractions containing high citral contents.¹⁶ Silvestre et al. used VFD to fractionate rosemary (Rosmarinus officinalis L.) EO and green mandarin (Citrus deliciosa Tenore) EO; they reported that VFD effectively separated compounds belonging to different chemical classes.^14,17 However, bioactivity tests of these fractions have not been reported. In our study, LCEO was separated by VFD into a low-boiling-point fraction as VFD1, a medium-boiling-point fraction as VFD2, and a high-boiling-point fraction as VFD3. Next, double MD was conducted to fractionate LCEO.¹⁸ By conducting the first MD, the light phase fraction and heavy phase fraction were obtained and expressed as MDL1 and MDH1, respectively. MDH1 was used as the feed during the second MD. After distillation, the light phase fraction and heavy phase fraction were expressed as MDL2 and MDH2, respectively. Some studies have used only LCEO samples prepared via HD; we further processed LCEO by VFD and MD to obtain different fractions and compared the chemical compositions and antibacterial activities of the fractions.

The most common pathogenic bacteria responsible for foodborne outbreaks around the world are Escherichia coli (E. coli), Legionella spp., Listeria monocytogenes, Bacillus cereus, Salmonella spp., Shigella spp., and S. aureus. They cause considerable damage to the food industry and threaten human health.^19,20 According to the World Health Organization, an estimated 600 million people fall ill, and 0.42 million people (including 0.125 million children) die due to the consumption of unsafe food each year.²¹ Foodborne pathogens such as E. coli and S. aureus greatly contribute to foodborne illness. This infection may cause significant and even life-threatening health risks.²⁰ Some bacteria not only affect humans but also threaten plant growth. Agrobacterium is a soil-borne Gram-negative plant pathogen that transfers tumor-inducing or root-inducing plasmids into plant cells and causes crown gall and hairy root diseases; these diseases are incurable, and infected plants remain for their entire lives and continuously release opines.^22,23Agrobacterium mainly affects dicotyledonous plants (apples and roses), grape vines, shade trees, herbaceous perennials, and even hydroponically grown tomatoes and cucumbers. It interferes with plant metabolism and decreases nutrient accumulation and fruit production, resulting in large economic losses.²⁴⁻²⁶ The disease was first reported in the United States in the 1870s and was subsequently reported in South America, Asia, and European countries.²⁷ Therefore, effective control of foodborne and plant pathogens is important for human health and agricultural practices. Antibiotics are widely used in human medicine and agriculture, and misusing these drugs can lead to the development of multidrug resistance, which increases the risk of treating infections caused by pathogenic bacteria and poses a hazard to human and plant health.^20,28 To reverse the threat posed by antibiotics, the search for natural plant-derived agents to control foodborne and plant diseases is inevitable. EOs are potential sources of novel natural bacteriostatic agents.^1,4 LCEO, which is generally regarded as safe (GRAS) and capable of reducing risks to the environment and human health, has various biological activities, especially antimicrobial activities.²⁹ Its use as a natural antimicrobial agent has been applied in areas such as fruit, vegetable, and meat preservation. Dai et al. added LCEO to different vegetable juices, and the bacterial inhibition rate remained above 99.9% for 4 days.¹ Su et al. reported that after cherry tomatoes were treated with an LCEO nanoemulsion for 9 min, the Salmonella count decreased by 6.50 ± 0.20 log CFU/mL.³⁰ Kunová et al. reported that the number of coliform bacteria in deer meat decreased from 1.02 log CFU/mL to 0 under 1% LCEO treatment.³ Thielmann et al. demonstrated that LCEO has high antibacterial activity against S. aureus and E. coli and has been used to preserve strawberries.³¹ Our study, in which processed LCEO was used to examine its activity against foodborne pathogens, is an extension of this field. We also studied the inhibitory activity of LCEO and seven distillates against Agrobacterium, which has not been previously reported. Some studies have shown that EOs can exert bacteriostatic effects by inhibiting biofilm formation and causing irreversible damage to cell membranes and cell morphology.^1,32,33 We also assessed the mechanism by which the most active fraction, VFD3, inhibits Agrobacterium rhizogenes (A. rhizogenes) based on biofilm inhibition, ROS levels, AKP activity, and cell morphology. Metabolomics is a powerful tool for evaluating secondary metabolites and antibacterial mechanisms and has been widely used in various fields.³³ Hu et al. reported that the inhibitory activity of LCEO on MRSA was related to the disruption of cell membranes, the inhibition of the hexose monophosphate pathway, and the activity of key enzymes.³⁴ Chen et al. showed that LCEO significantly affected Cutibacterium acnes by altering 86 metabolites and perturbing 34 metabolic pathways.³⁵ To elucidate its bacteriostatic mechanism, we used UPLC-QTOF-MS-based untargeted metabolomics and UPLC-QQQ-MS-based targeted metabolomics as the standard methods to conduct qualitative and quantitative analysis of the effects of VFD3 on metabolites of A. rhizogenes.³⁶ Our study may help optimize the processing of EOs, enrich their active compounds, improve their bioactivities, and explore the application of EOs as natural antibacterial agents.

2. Materials and Methods

2.1. Materials

Fruits of L. cubeba (Lour.) Pers. were collected in Anshun City, Guizhou Province, China, in mid-June 2022 and were dried in the shade at room temperature. Other materials including the bacterial and authentic compounds used are shown in Supporting Information 1.1.

2.2. LCEO Extraction and Processing

2.2.1. LCEO Extraction by HD

L. cubeba fruits (5.0 kg) were placed in a Clevenger-type distillation device, and 15.0 L of H₂O was added. HD was performed for 12 h. The upper LCEO layer was collected and dried with sodium sulfate (Na₂SO₄). After filtration, LCEO was weighed and stored in a refrigerator at 4 °C before further processing.

2.2.2. Vacuum Fractional Distillation of LCEO

LCEO (231.40 g) was added to a distillation flask, and a fractionating column was attached to the flask. The temperature of the cooling water was 3 °C. When the pressure stabilized at 0.4 kPa, the heating was turned on and the VFD process started. The distilled fractions were collected. Three distillates, VFD1, VFD2, and VFD3, were collected at boiling temperatures ranging from 38 to 44, 44 to 76, and 76 to 97 °C, respectively.

2.2.3. Molecular Distillation of LCEO

The double MD process was conducted with a 2 inch. Pope Wiped-Film Molecular Evaporator (Pope Scientific Inc., Saukville, WI, USA). In the first stage, the distillation temperature, cooling temperature, vacuum pressure, wiper speed, and feed rate were set at 55 °C, 3 °C, 0.6 kPa, 350 rpm, and 4 mL/min, respectively. The light and heavy phase fractions were expressed as MDL1 and MDH1, respectively. MDH1 was used as the feed at the second MD stage, and similar conditions were set at 65 °C, 3 °C, 0.5 kPa, 400 rpm, and 4 mL/min. After distillation, the light phase fraction and heavy phase fraction were expressed as MDL2 and MDH2, respectively.

2.3. Gas Chromatography and Mass Spectrometry (GC-MS) Characterization of EOs

GC-MS was performed to analyze and identify the compositions of LCEO and seven distillates, according to Peng et al., with minor modifications.³⁷ The experimental details are given in Supporting Information 1.2. The constituents of the EOs were identified by comparing their mass spectra with the National Institute of Standards and Technology data library (2010 version) and the retention index (RI). The RI was measured by injecting C8–C20 n-alkanes with n-hexane-diluted EO via GC-MS under the same conditions described above. The major compounds were also identified by comparing the mass spectra and RI values with those of commercially available authentic compounds injected into the GC-MS apparatus.

2.4. Antibacterial Activity of LCEO, Seven Distillates, and Eight Individual Constituents

2.4.1. Preparation of Bacterial Cultures

Four foodborne pathogens, S. aureus, E. coli, Pseudomonas aeruginosa (P. aeruginosa), and Salmonella typhimurium (S. typhimurium), two phytopathogens, Agrobacterium tumefaciens (A. tumefaciens) and A. rhizogenes, and Bacillus subtilis (B. subtilis, a foundational model organism in microbiology research³⁸) were used to determine the antimicrobial activity of EOs. Foodborne bacteria and B. subtilis were activated on nutrient agar (NA) overnight at 37 °C. Agrobacterium was activated on yeast extract peptone (YEP) broth medium at 28 °C. Bacteria were diluted and used to determine the inhibition zone diameter (IZD) by using the filter paper diffusion method and the minimum inhibitory concentration (MIC). To avoid the growth of other bacteria, rifampicin (25 μg/mL) was added to the growth medium of A. tumefaciens, and streptomycin sulfate (50 μg/mL) was added to the growth medium of A. rhizogenes.

2.4.2. Determination of the Inhibition Zone Diameter

The filter paper diffusion method was used to evaluate the inhibitory activity of LCEO and the distillates on four foodborne pathogens and Bacillus subtilis (B. subtilis), as described by Hao et al.³⁹ In total, 150 μL of each pathogen suspension was spread on a solid medium in a Petri dish with a diameter of 90 mm. The IZD of Agrobacterium was determined using the method described by Long et al.⁴⁰ The YEP medium was melted to a certain temperature, after which Agrobacterium was added, vortexed, mixed, and poured into a 90 mm diameter Petri dish. Next, 5 μL of EO was added to the center of the filter paper (6 mm in diameter) and then incubated at 37 °C for 24 h (four foodborne pathogens and B. subtilis) or at 28 °C for 36 h (Agrobacterium). Ampicillin (100 μg/mL) was used as a positive control. The IZD was measured with a caliper. Each bacterium was measured three times in parallel.

2.4.3. Determination of the Minimum Inhibitory Concentrations

The MICs of all EOs were determined by the broth microdilution method.¹⁷ EOs were dissolved in Tween 80 at 1% v/v and diluted in a 2-fold gradient in nutrient broth (NB) or YEP. Each well consisted of 100 μL of each sample at each concentration, 80 μL of NB or YEP, and 20 μL of bacterial suspension. Ampicillin was used as the positive control, and bacteria enriched with only NB or liquid YEP or with Tween 80 (1% v/v) without EO were used as the negative control. After incubation at 37 °C for 24 h or at 28 °C for 36 h, 10 μL of 20 mg/mL 2,3,5-triphenyltetrazolium chloride (TTC) was added to each well and incubated at 37 or 28 °C for 30 min. The MIC was the minimum EO concentration of the wells that did not turn red. Each set of experiments was repeated three times.

2.5. Antibacterial Mechanism of VFD3 against A. rhizogenes

The antibacterial mechanism of VFD3 on A. rhizogenes was investigated by measuring the biofilm inhibition rate,³³ accumulation of ROS,⁴¹ AKP activity,⁴¹ and SEM images of the cell morphology.²⁹ The experimental details are shown in Supporting Information 1.3.

2.6. Analysis of Metabolites of A. rhizogenes

2.6.1. Sample Preparation

Suspensions of A. rhizogenes from the VFD3 (at a concentration of MIC)-treated group and the control group (CK) were collected by centrifugation at 6000 r/min for 5 min at 4 °C.³² The samples were washed twice with 2 mL of phosphate-buffered saline (0.01 M, pH 7.0), which was rapidly placed in liquid nitrogen for 5 min and then on dry ice for 5 min. The samples were thawed on ice and vortexed for 2 min. The thawed samples were submerged and completely suspended in 80% methanol. The supernatant was obtained by vortexing three times at intervals of 5 min and centrifuged twice at 12,000 rpm and 4 °C. An aliquot of the supernatant from each sample was pipetted and mixed into a linear tube of the corresponding injection vial for LC-MS analysis. Three replicates were maintained for the VFD3 treatment group and the control group.

2.6.2. Untargeted Analysis of the VFD3 Treatment Group by UPLC-QTOF-MS

The metabolites of A. rhizogenes after VFD3 treatment were determined by a nontargeted method based on UPLC-QTOF-MS. The experimental details are shown in Supporting Information 1.4.1.

2.6.3. Target Analysis of the VFD3 Group by UPLC-QQQ-MS

The UPLC-QQQ-MS technique was used to quantitatively analyze the metabolites. The experimental details are shown in Supporting Information 1.4.2.

2.6.4. Screening of Differential Metabolites

The metabolites of the VFD3 and CK groups were screened by a combination of variable importance projection (VIP) derived from orthogonal partial least-squares-discriminant analysis (OPLS-DA) and the p-values from Student’s t tests. The screening criteria for differential metabolites (DMs) were VIP > 1 and p < 0.05. The VIP value indicates the strength of the effect of intergroup differences in the corresponding metabolite on the classification of each group of samples. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze the metabolic pathways.

2.7. Statistical Analysis

All experiments were repeated three times. Except for the MIC values, the results were expressed as the mean ± the standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey’s post hoc tests were performed using IBM SPSS Statistics 27 software, and p < 0.05 was considered to indicate a statistically significant difference.

3. Results and Discussion

3.1. Yields and Chemical Compositions of LCEOs

The extraction yield of LCEO by HD may be affected by factors such as origin and extraction conditions, and the oil yield from fresh fruit is 3–6%.⁶ In this study, LCEO was extracted by HD with a yield of 3.5%. After the VFD of LCEO, fractions VFD1, VFD2, and VFD3 were obtained in yields of 24.36, 37.45, and 26.88%, respectively. After the MD of LCEO, fractions MDL1 and MDH1 were obtained in yields of 20.47 and 19.32%, respectively. After the second MD using MDH1 as the feed, MDL2 and MDH2 were collected in 9.22 and 48.44% yields, respectively.

The chemical compositions and relative contents of LCEO and seven fractions are shown in Table 1. In total, 36 compounds were detected in all EOs by GC-MS. The main components of the LCEO were β-citral (14.69%), citronellal (14.00%), limonene (11.40%), linalool (9.25%), α-citral (7.56%), and 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde (7.18%). The diversity of the chemical composition and content of the LCEO is related to climate conditions, soil types, and other factors.⁴² Gogoi et al. extracted the LCEO by HD in Assam, India, and reported that the relatively high-content compounds were sulcatone (30.9%), limonene (23.14%), β-citral (14.00%), α-citronellol (6.52%), linalool (2.26%), and caryophyllene (1.73%). α-Citral was not detected.⁴³ Sattayakhom et al. identified 25 compounds of the LCEO produced in Nonthaburi, Thailand, and reported that α-citral (33.36%) and β-citral (26.78%) were the predominant components.⁴⁴ The total ion chromatograms are shown in Figure S1, with the top 10 high-content compounds in each EO labeled with numbers, with each number corresponding to the serial number of the compound in Table 1.

Table 1. Chemical Compositions of LCEO and Seven Distillates Identified by GC-MS^a.

				composition (%)
no.	RIexp.	RIlit.	compounds	LCEO	VFD1	VFD2	VFD3	MDL1	MDH1	MDL2	MDH2
1	927	927	α-thujene	0.16 ± 0.02 b	0.56 ± 0.03 a	—	—	0.44 ± 0.12 a	—	0.21 ± 0.01 b	—
2	933	929	α-pinene	4.51 ± 0.20 d	13.02 ± 0.69 b	1.01 ± 0.14 ef	0.17 ± 0.08 g	16.07 ± 0.21 a	1.37 ± 0.01 e	6.36 ± 0.23 c	0.31 ± 0.01 fg
3	947	951	camphene	0.99 ± 0.03 d	3.21 ± 0.06 b	0.30 ± 0.04 e	—	3.39 ± 0.03 a	0.35 ± 0.00 e	1.60 ± 0.05 c	—
4	974	970	sabinene	5.82 ± 0.16 d	14.99 ± 0.12 b	2.67 ± 0.23 e	—	16. 44 ± 0.36 a	2.25 ± 0.09 e	10.16 ± 0.30 c	0.70 ± 0.05 f
5	977	972	β-pinene	2.23 ± 0.19 c	8.19 ± 0.73 a	1.75 ± 0.38 c	—	8.86 ± 0.60 a	1.79 ± 0.08 c	6.82 ± 0.17 b	0.59 ± 0.01 d
6	991	988	sulcatone	3.88 ± 0.05 c	8.26 ± 0.33 a	5.19 ± 0.25 b	—	8.29 ± 0.98 a	2.16 ± 0.40 d	9.19 ± 0.29 a	0.97 ± 0.45 de
7	994	992	β-myrcene	3.00 ± 0.16 c	7.95 ± 0.28 a	2.97 ± 0.80 c	—	6.48 ± 0.82 b	2.53 ± 0.20 cd	6.58 ± 0.46 b	1.32 ± 0.12 d
8	1006	1006	α-phellandrene	0.26 ± 0.01 b	0.51 ± 0.04 a	—	—	0.17 ± 0.01 c	—	—	—
9	1027	1030	limonene	11.40 ± 0.33 d	27.45 ± 0.49 a	13.18 ± 1.44 c	0.19 ± 0.03 g	23.22 ± 0.31 b	7.10 ± 0.17 e	24.53 ± 0.03 b	3.44 ± 0.26 f
10	1029	1023	eucalyptol	1.45 ± 0.05 d	—	—	—	4.03 ± 0.02 b	2.23 ± 0.14 c	5.25 ± 0.22 a	1.38 ± 0.06 d
11	1040	1039	trans-β-ocimene	0.17 ± 0.01 c	0.44 ± 0.04 a	0.27 ± 0.02 bc	—	0.28 ± 0.03 b	—	0.36 ± 0.10 ab	—
12	1050	1050	α-ocimene	0.18 ± 0.01 d	0.44 ± 0.05 a	0.26 ± 0.01 c	—	0.27 ± 0.01 c	—	0.36 ± 0.00 b	—
13	1060	1060	γ-terpinene	0.19 ± 0.01 d	0.45 ± 0.05 a	0.27 ± 0.01 c	—	0.31 ± 0.02 bc	0.14 ± 0.03 d	0.35 ± 0.02 b	—
14	1071	1074	cis-sabinene hydrate	0.10 ± 0.00 d	—	0.24 ± 0.01 a	—	0.08 ± 0.02 d	0.13 ± 0.01 c	0.18 ± 0.01 b	—
15	1089	1089	terpinolene	0.38 ± 0.01 c	0.72 ± 0.08 a	0.61 ± 0.10 ab	—	0.41 ± 0.03 c	—	0.57 ± 0.04 b	—
16	1108	1102	linalool	9.25 ± 0.04 c	4.99 ± 0.33 e	20.05 ± 0.62 a	6.18 ± 0.46 d	4.21 ± 0.06 e	12.54 ± 0.26 b	10.06 ± 0.17 c	13.02 ± 0.70 b
17	1112	1116	α-cyclocitral	0.31 ± 0.04 b	0.63 ± 0.00 a	—	—	0.16 ± 0.04 bc	0.51 ± 0.10 a	0.30 ± 0.10 b	0.53 ± 0.07 a
18	1125	1123	trans-p-mentha-2,8-dienol	0.19 ± 0.02 b	—	0.30 ± 0.05 a	—	—	0.16 ± 0.02 b	—	—
19	1140	1139	trans-limonene oxide	0.29 ± 0.07 b	0.18 ± 0.02 bc	0.57 ± 0.02 a	—	0.09 ± 0.05 cd	—	0.25 ± 0.10 b	—
20	1144	1143	camphor	0.15 ± 0.00 b	—	0.37 ± 0.01 a	—	0.06 ± 0.01 c	0.21 ± 0.02 b	0.16 ± 0.07 b	0.18 ± 0.04 b
21	1146	1152	isopulegol	0.54 ± 0.08 b	—	—	—	0.07 ± 0.02 c	0.71 ± 0.03 ab	0.18 ± 0.07 c	0.91 ± 0.20 a
22	1151	1146	β-pinene oxide	0.36 ± 0.07 bc	—	0.45 ± 0.11 b	1.32 ± 0.04 a	—	0.24 ± 0.04 cd	0.15 ± 0.03 d	—
23	1159	1155	citronellal	14.00 ± 1.00 d	3.26 ± 0.32 f	14.04 ± 0.51 d	16.23 ± 1.23 c	2.24 ± 0.09 f	18.40 ± 0.42 b	5.35 ± 0.15 e	21.32 ± 1.11 a
24	1169	1170	cis-verbenol	4.30 ± 0.40 bc	0.20 ± 0.04 e	1.65 ± 0.10 d	8.14 ± 0.45 a	0.12 ± 0.02 e	3.99 ± 0.10 c	0.29 ± 0.16 e	4.91 ± 0.26 b
25	1181	1178	terpinen-4-ol	1.46 ± 0.03 cd	0.51 ± 0.08 e	2.68 ± 0.50 a	1.76 ± 0.30 bc	0.36 ± 0.00 e	1.57 ± 0.03 bc	0.88 ± 0.03 de	2.15 ± 0.27 ab
26	1188	1171	1,3,4-trimethyl-3-cyclo-hexene-1-carboxaldehyde	7.18 ± 0.58 bc	0.52 ± 0.11 e	3.71 ± 0.11 d	10.48 ± 0.83 a	0.20 ± 0.11 e	6.38 ± 0.35 c	0.67 ± 0.03 e	7.66 ± 0.31 b
27	1196	1191	α-terpineol	1.44 ± 0.03 b	-	2.15 ± 0.75 ab	2.47 ± 0.11 a	0.11 ± 0.02 c	1.41 ± 0.08 b	0.35 ± 0.13 c	1.88 ± 0.05 ab
28	1249	1249	β-citral	14.69 ± 1.12 e	2.39 ± 0.30 g	17.01 ± 1.15 d	30.03 ± 1.38 a	2.07 ± 0.18 g	19.80 ± 0.14 c	5.86 ± 0.31 f	22.64 ± 0.66 b
29	1278	1273	α-citral	7.56 ± 0.64 c	0.53 ± 0.04 d	6.06 ± 0.86 c	17.04 ± 1.07 a	0.35 ± 0.12 d	9.83 ± 0.27 b	1.33 ± 0.39 d	10.82 ± 0.53 b
30	1373	1375	copaene	0.53 ± 0.03 c	—	0.68 ± 0.13 bc	1.00 ± 0.09 a	0.14 ± 0.19 d	0.71 ± 0.04 bc	0.20 ± 0.02 d	0.83 ± 0.05 ab
31	1427	1428	β-caryophyllene	0.32 ± 0.03 bc	—	0.24 ± 0.05 c	0.71 ± 0.08 a	—	0.42 ± 0.02 b	—	0.84 ± 0.10 a
32	1465	1465	α-caryophyllene	0.17 ± 0.00 d	—	—	0.39 ± 0.02 a	—	0.24 ± 0.02 c	—	0.29 ± 0.03 b
33	1471	1473	β-farnesene	0.15 ± 0.01 c	—	—	0.37 ± 0.06 a	—	0.24 ± 0.02 b	—	0.28 ± 0.03 b
34	1498	1482	γ-elemene	0.23 ± 0.16 bc	—	—	0.61 ± 0.22 a	—	0.44 ± 0.02 ab	—	0.52 ± 0.05 a
35	1514	1511	β-bisabolene	0.23 ± 0.07 b	—	—	0.36 ± 0.06 ab	—	0.28 ± 0.03 ab	—	0.41 ± 0.11 a
36	1591	1583	caryophyllene oxide	0.73 ± 0.07 c	—	—	0.76 ± 0.09 c	—	0.93 ± 0.04 b	—	1.11 ± 0.12 a
total				98.82	99.40	98.67	98.19	98.92	99.07	98.54	99.01
monoterpene hydrocarbons				29.30	77.92	23.28	0.35	76.34	15.53	57.89	6.36
oxygenated monoterpenes				56.10	12.69	65.56	83.17	13.95	71.72	30.58	79.75
sesquiterpene hydrocarbons				1.63	0.00	0.92	3.43	0.14	2.33	0.20	3.17
oxygenated sesquiterpenes				0.73	0.00	0.00	0.76	0.00	0.93	0.00	1.11
others				11.07	8.78	8.90	10.48	8.50	8.55	9.86	8.63

^aRI_exp., experimental retention index on an HP-5MS column. RI_lit., literature retention index. “—”: not detected. The results are expressed as the mean area percentage (%) ± SD of three independent injections. The mean values in the same row followed by different superscript letters are significantly different (p < 0.05), according to ANOVA followed by Tukey’s post hoc test.

The effects of the VFD and MD on the individual compounds in the eight EOs were illustrated by OPLS-DA (Figure 1). The contribution of compounds with VIP > 1 to the differences between EOs was significant. We identified 13 differential compounds (marked in red in Figure 1), including β-citral, linalool, 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde, etc. OPLS-DA can efficiently separate EOs into different areas of the coordinate chart. β-Citral, 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde, and α-citral were enriched in the VFD3 area in quadrant 1. Linalool and citronellal were the main components of VFD2 and MDH2 in the fourth quadrant, respectively. α-Pinene, sabinene, and β-pinene were mainly concentrated in the same quadrant as MDL1 and VFD1 at close distances, indicating that these compounds were similar. The seven distillates were a certain distance from the LCEO (Figure 1), indicating that VFD and MD could effectively separate the compounds in the LCEO. The relative contents of β-citral and α-citral increased from 14.69 and 7.56%, respectively, in LCEO to 30.03 and 17.04%, respectively, in VFD3, and to 22.64 and 10.82%, respectively, in MDH2.

Figure 1.

OPLS-DA score scatter plot of eight EOs and individual constituents.

The 36 compounds were categorized into five groups, and the compounds with the most significant effects on the relative contents of VFD and MD included oxygenated compounds, sesquiterpenes, and monoterpenes. Regarding the VFD fractions, the relative content of monoterpene hydrocarbons decreased considerably from 77.92% in VFD1 to 23.28% in VFD2, and only 0.35% was found in VFD3. However, the relative content of oxygenated monoterpenes increased considerably: 12.69% in VFD1, 65.56% in VFD2, and 83.17% in VFD3. In the MD process, compared to that in the first distillation, the monoterpene content of the light phase fractions decreased in the second distillation, from 76.34% in MDL1 to 57.89% in MDL2. However, the percentage of oxygenated monoterpenes increased from 71.72% in MDH1 to 79.75% in MDH2. Compared to those in LCEO, oxygenated compounds in VFD3 and MDH2 increased by 27.07 and 23.65%, respectively, and monoterpenes decreased by 28.95 and 22.94%, respectively. The contents of sesquiterpenes in VFD3 and MDH2 were about twice as high as those in LCEO. Overall, the VFD and MD processes greatly increased the relative contents of oxygenated monoterpenes and decreased the relative contents of monoterpene hydrocarbons in VFD3 and MDH2. These results indicated that the VFD and MD changed the composition and relative content of the LCEO, which could be used to enrich the active compounds for other EOs.

3.2. Analysis of the Bacteriostatic Activity of LCEO, Seven Distillates, and Eight Individual Constituents

The activity of the EOs and individual constituents against seven bacteria was determined by the filter paper diffusion method and the broth microdilution method. Based on the criteria defined in another study,⁴⁵ bacterial sensitivity to EOs can be classified as follows: IZD ≤ 8.0 mm, insensitive; IZD between 8.0–14.0 mm, moderately sensitive; IZD between 14.0–20.0 mm, sensitive; and IZD ≥ 20.0 mm, extremely sensitive.

As shown in Tables 2 and 3, eight EOs displayed different antibacterial activities. The activities of LCEO and the three VFD fractions were ranked as follows: VFD3 > VFD2 > LCEO > VFD1. The order of antibacterial activity of the MD distillates was MDH2 > MDH1 > MDL2 > MDL1. B. subtilis and S. aureus were extremely sensitive to VFD2, VFD3, and MDH2, with IZD values greater than 20.00 mm and MIC values ranging from 0.20 to 1.56 μL/mL. Most studies on the bacteriostatic activity of LCEO have focused on foodborne pathogens.¹ Nguyen et al. studied the bacteriostatic activity of LCEO extracted by HD in Vietnam and reported MIC values ranging from 2800 to 5500 μg/mL against B. subtilis, S. aureus, and E. coli.⁴⁶ The activity of the LCEO in that study was close to that of the LCEO found in this study; however, it was weaker than VFD3 and MDH2. Ambrosio et al. assessed the activity of 28 types of EOs against S. aureus and B. subtilis and reported that orange EO had the best activity, with IZDs of 28.40 and 27.90 mm, respectively, whereas the IZDs of VFD3 were greater than 90.00 mm for both bacteria.⁴⁵ Miladinović et al. reported the inhibitory activities of six EOs against S. aureus and E. coli, with MICs ranging from 1326 to 5375 μg/mL, which were comparable to the MICs of VFD3 and MDH2 (0.78–1.56 μL/mL).⁴⁷ In this study, the bacteriostatic effects of eight EOs against A. tumefaciens and A. rhizogenes were determined. Hsouna et al. reported that peppermint (Mentha piperita L.) EO had good antibacterial activities against the phytopathogenic bacteria A. tumefaciens, with an MIC of 12.50 mg/mL,⁴⁸ which was weaker than those of LCEO, VFD2, VFD3, and MDH2, with MICs of 1.56–3.13 μL/mL. Like foodborne pathogens, VFD3 also showed the best bacteriostatic activity against both plant pathogens, with the same MIC of 1.56 μL/mL. Lee et al. investigated 50 EOs and reported that cinnamon bark (Cinnamomum verum) EO had the best activity against A. tumefaciens, with an MIC of 250 μg/mL, which was better than those of VFD3 and MDH2.²⁸ VFD2 and MDH2 also exhibited good bacteriostatic activity against the two plant pathogens, with MICs of 3.13 μL/mL for A. rhizogenes and 1.56 μL/mL for A. tumefaciens.

Table 2. Inhibition Zone Diameter (IZD, mm) of EOs and Individual Constituents.

bacterial	B. subtilis	S. aureus	E. coli	P. aeruginosa	S. typhimurium	A. rhizogenes	A. tumefaciens
EOs
LCEO	>90.00 a	16.08 ± 0.03 g	8.11 ± 0.09 e	7.68 ± 0.18 a	7.94 ± 0.27 e	8.35 ± 0.08 i	14.68 ± 0.06 d
VFD1	12.99 ± 0.30 g	12.98 ± 0.16 i	6.00 ± 0.00 i	6.00 ± 0.00 e	7.25 ± 0.11 f	11.25 ± 0.04 e	7.50 ± 0.08 j
VFD2	>90.00 a	34.85 ± 0.08 b	11.74 ± 0.20 b	6.57 ± 0.29 c	10.24 ± 0.09 b	17.69 ± 0.13 b	13.67 ± 0.13 e
VFD3	>90.00 a	>90.00 a	13.41 ± 0.09 a	6.83 ± 0.02 b	11.50 ± 0.23 a	21.53 ± 0.11 a	21.48 ± 0.11 a
MDL1	13.64 ± 0.05 f	13.90 ± 0.07 h	6.54 ± 0.04 g	6.00 ± 0.00 e	6.30 ± 0.04 h	7.18 ± 0.01 l	7.50 ± 0.04 j
MDH1	90.00 ± 0.00 a	17.46 ± 0.36 f	8.01 ± 0.01 e	6.14 ± 0.01 de	6.99 ± 0.03 fg	9.49 ± 0.11 g	12.52 ± 0.22 f
MDL2	21.42 ± 0.08 c	17.33 ± 0.09 f	7.67 ± 0.02 f	6.09 ± 0.01 de	6.83 ± 0.03 g	10.85 ± 0.07 f	7.76 ± 0.01 j
MDH2	>90.00 a	20.46 ± 0.08 e	9.43 ± 0.09 d	6.28 ± 0.03 d	7.18 ± 0.01 f	16.14 ± 0.10 d	18.65 ± 0.05 c
citral	>90.00 a	32.24 ± 0.08 c	6.00 ± 0.00 i	6.22 ± 0.04 de	8.59 ± 0.03 c	16.70 ± 0.09 c	19.57 ± 0.13 b
citronellal	>90.00 a	10.67 ± 0.16 j	6.00 ± 0.00 i	6.00 ± 0.00 e	6.00 ± 0.00 i	7.79 ± 0.05 j	8.75 ± 0.20 i
comp. 26a	>90.00 a	7.23 ± 0.06 m	6.00 ± 0.00 i	6.00 ± 0.00 e	6.00 ± 0.00 i	6.56 ± 0.08 m	6.59 ± 0.15 k
linalool	9.22 ± 0.09 h	9.35 ± 0.07 k	11.20 ± 0.02 c	6.00 ± 0.00 e	8.28 ± 0.07 d	9.38 ± 0.07 g	12.48 ± 0.01 f
α-terpineol	15.20 ± 0.04 e	10.43 ± 0.09 j	11.81 ± 0.07 b	6.00 ± 0.00 e	8.39 ± 0.01 cd	7.43 ± 0.14 k	12.21 ± 0.07 g
terpinen-4-ol	16.29 ± 0.11 d	8.40 ± 0.07 l	13.28 ± 0.09 a	6.00 ± 0.00 e	11.36 ± 0.06 a	8.61 ± 0.17 h	10.15 ± 0.06 h
β-pinene oxide	7.50 ± 0.02 i	7.12 ± 0.03 m	6.28 ± 0.05 d	6.00 ± 0.00 e	6.00 ± 0.00 i	6.27 ± 0.02 n	6.27 ± 0.07 l
caryophyllene oxide	7.30 ± 0.02 i	6.53 ± 0.07n	6.00 ± 0.00 i	6.00 ± 0.00 e	6.00 ± 0.00 i	6.00 ± 0.00°	6.00 ± 0.00 m
ampicillin	22.15 ± 0.28 b	28.06 ± 0.18 d	6.00 ± 0.00 h	6.00 ± 0.00 e	8.37 ± 0.23 cd	11.46 ± 0.10 e	12.06 ± 0.10 g

^aComp. 26 indicates 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde and is shown in Table 1. The IZD values are presented as the mean ± SD for at least three experiments in mm, including a 6.0 mm disk diameter. The mean values in the same column followed by different superscript letters were significantly different (p < 0.05), according to ANOVA followed by Tukey’s post hoc test.

Table 3. Minimum Inhibitory Concentration (MIC, μL/mL) of EOs and Individual Constituents against Seven Bacterial Strains.

bacteria	B. subtilis	S. aureus	E. coli	P. aeruginosa	S. typhimurium	A. rhizogenes	A. tumefaciens
EOs
LCEO	0.78	6.25	6.25	200.00	12.5	6.25	3.13
VFD1	3.13	12.50	100.00	200.00	100.00	25.00	12.50
VFD2	0.20	1.56	6.25	200.00	3.13	3.13	1.56
VFD3	0.39	0.78	1.56	100.00	0.78	1.56	1.56
MDL1	3.13	1.56	12.50	100.00	100.00	25.00	12.50
MDH1	0.39	1.56	1.56	50.00	3.13	6.25	6.25
MDL2	1.56	3.13	3.13	50.00	6.25	25.00	12.50
MDH2	0.39	0.78	1.56	50.00	1.56	3.13	1.56
citral	0.39	0.39	0.78	400.00	6.25	1.56	1.56
citronellal	0.39	0.78	3.13	400.00	6.25	12.50	12.50
comp. 26a	0.39	6.25	6.25	200.00	12.50	3.13	3.13
linalool	3.13	3.13	3.13	400.00	6.25	3.13	3.13
α-terpineol	3.13	0.78	0.78	400.00	0.78	3.13	3.13
terpinen-4-ol	0.78	1.56	1.56	400.00	0.78	3.13	3.13
β-pinene oxide	12.5	6.25	3.13	400.00	12.50	3.13	3.13
caryophyllene oxide	12.5	6.25	3.13	400.00	12.50	12.50	12.50
ampicillin (μg/mL)	25.00	0.10	3.13	800.00	1.56	250.00	625.00

^aComp. 26 indicates 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde and is shown in Table 1.

Among the single constituents of VFD3 and MDH2, which had the best inhibitory activity against most bacteria, citral (47.07, 33.46%), citronellal (16.23, 21.32%), 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde (10.48, 7.66%), linalool (6.18, 13.02%), α-terpineol (2.47, 1.88%), terpinen-4-ol (1.76, 2.15%), and caryophyllene oxide (0.76, 1.11%) were the common constituents in both distillates with the highest contents. To explore the contributions of individual constituents to EOs, the bacteriostatic activities of the above seven constituents and β-pinene oxide, which had relatively high contents in VFD3, were tested. Like the eight EOs, the above eight compounds inhibited all bacteria except P. aeruginosa with MICs ranging from 0.39 to 12.50 μL/mL, as shown in Table 3. Oxygenated monoterpenes, such as citral, linalool, α-terpineol, terpinen-4-ol, and β-pinene oxide, displayed strong activity against A. rhizogenes and A. tumefaciens, with MICs ranging from 1.56 to 3.13 μL/mL. However, caryophyllene oxide, as the only representative oxygenated sesquiterpene in the EOs tested, showed the weakest activity among the eight constituents tested, with an MIC of 12.5 μL/mL against the above two plant pathogens. Rasoul et al. reported that linalool displayed bacteriostatic activity against A. tumefaciens, with an MIC of 2500 mg/L.⁴⁹ Kotan et al. reported that citronellal, linalool, and α-terpineol had antibacterial effects on A. tumefaciens, with IZD values ranging from 7 to 8 mm.⁵⁰ In our study, linalool (20.05% in VFD2) had an MIC of 3.13 μL/mL against A. tumefaciens, with less inhibitory activity than VFD2 (MIC of 1.56 μL/mL), suggesting that the inhibitory effect may be a result of the combined action of several compounds rather than a single compound. The results of the bacteriostatic activity of eight individual compounds further proved that the VFD and MD were efficient methods for enriching bioactive constituents.

Spearman correlation analysis was performed to reveal the relationships between the individual compounds and the bacteriostatic activity of the eight EOs to identify highly active compounds. The clustered heat map (Figure 2) revealed a positive or negative correlation between the bacteriostatic activities (MICs) of the different compounds. β-Citral, citronellal, linalool, 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde, α-citral, α-terpineol, and terpinen-4-ol were negatively correlated with the MICs against different bacteria, and these compounds had relatively high contents in VFD3 and MDH2, which presented the best bacteriostatic activity. Citral may play an important role in the bacteriostatic effects of VFD3 (47.07%) and MDH2 (33.46%) against B. subtilis, S. aureus, and E. coli, with MICs of 0.39–0.78 μL/mL. Guo et al. reported that citral, terpineol, and linalool had good antibacterial activity against B. subtilis, S. aureus, and E. coli, with MICs ranging from 0.48 to 15.84 μL/mL.⁵¹ Similar findings were reported in this study, with MICs ranging from 0.39 to 3.13 μL/mL for the three constituents against the three bacteria. Among the five classes of compounds, oxygenated monoterpenes presented significant negative correlations with all bacteria except P. aeruginosa. Monoterpenes were significantly positively correlated with seven species of bacteria. Other studies have shown that some oxygenated compounds in EOs have better antibacterial activity than terpenes.⁵² Sidorova et al. reported that the two monoterpenes limonene and α-pinene did not inhibit A. tumefaciens even at relatively high concentrations (400 μmol and 600 μmol); however, 2-octanone and isoamyl alcohol strongly inhibited A. tumefaciens.⁵³ However, the contents of monoterpene hydrocarbons in VFD1 (77.92%) and MDL1 (76.34%) were significantly greater than those in VFD3 (0.35%) and MDH2 (6.36%). The contents of oxygenated monoterpenes in VFD3 and MDH2 were 10 times and 6 times greater than those in VFD1 and MDL1, respectively (Table 1), suggesting that oxygenated monoterpenes may be the dominant factor facilitating the inhibitory effects of VFD3 and MDH2 on the foodborne pathogens and plant pathogens tested. As the antibacterial activities of VFD3, VFD2, and MDH2 were better than those of LCEO, we concluded that VFD and MD are extremely efficient methods to fractionate LCEO and may be used for other EOs and practical applications. This study revealed that some LCEO distillates, such as VFD3 and MDH2, and individual constituents not only effectively inhibited foodborne pathogens but also had good antibacterial effects on plant pathogens. Considering the few studies on LCEO and individual compounds targeting A. rhizogenes and A. tumefaciens, this study provided a theoretical basis for the use of LCEO distillates and active individual compounds as plant-derived bacteriostatic agents to control plant diseases in agriculture. Qiu et al. reported that LCEO is a new promising natural cosmetic ingredient.⁵⁴ Chen et al. reported that citral-rich LCEO may help treat inflammatory diseases.⁵⁵ Therefore, the results of this study may deepen our understanding of the bioactivity of LCEO and its distillates and broaden its application prospects in the medical and cosmetic industries.

Figure 2.

Spearman correlation analysis. The correlation values showed positive (above zero) and negative (below zero) associations between chemical compositions, eight EOs and their antibacterial activity. “*”, “**”, and “***” indicate a significant difference at the p < 0.05, p < 0.01, and p < 0.001 level, respectively.

3.3. Analysis of Antibacterial Mechanism of VFD3 against A. rhizogenes

The growth and reproduction of pathogens are tightly dependent on biofilm formation. The hydrophobic components of EOs might permeate the cell membrane to affect biofilm formation.⁴¹ The effects of different concentrations of VFD3 on biofilm formation were investigated by a crystal violet staining assay. The effect of inhibiting biofilms was more prominent with increasing concentrations of VFD3 (Figure 3A), and the inhibition rates at 1 MIC and 2 MIC reached 58.58 and 82.46%, respectively.

Figure 3.

Effects of different concentrations of VFD3 on biofilm activity, AKP activity, and ROS levels in A. rhizogenes. (A) Biofilm inhibition rate at different concentrations of VFD3; MIC = 1.56 μL/mL; CK was the blank control. (B) Effects of different concentrations of VFD3 on ROS levels. (C) Effects of different concentrations of VFD3 on AKP activity. Different lowercase letters above the columns within (A–C) indicate significant statistical differences at p < 0.05.

Oxidative stress leads to the accumulation of high levels of ROS, disrupting bacterial cell membranes and inhibiting their growth.⁴¹ EOs kill bacteria through the accumulation of intracellular ROS. A DCFH-DA fluorescent probe was used to detect intracellular ROS production. The level of ROS accumulation was positively correlated to the concentration of VFD3 (Figure 3B). After treatment of A. rhizogenes with VFD3 at 1–4 MICs, the ROS level was 5–7 times greater than that in the CK group.

Alkaline phosphatase (AKP) is considered to be a cell wall damage marker, and a leakage assay revealed that the permeability of the cell wall increased after treatment with VFD3. AKP activity increased in a concentration-dependent manner and was about 4-fold greater than that of the control group after treatment with 4 MIC VDF3 (Figure 3C), suggesting that VFD3 damaged the cell wall, which allowed the leakage of AKP into the cell culture medium.

SEM analysis was performed to assess the effect of VFD3 on the morphology of A. rhizogenes cells (Figure S2). Untreated cells maintained a normal and intact appearance with smooth surfaces (Figure S2A), whereas VFD3-treated cells became irregular, with collapsed or shrunken cell surfaces (Figure S2B,C). These findings suggested that VFD3 can alter the morphology of A. rhizogenes cells, which might affect the integrity of the cell membrane and the cell wall.

To summarize, VFD3 might exert its antibacterial effect by inhibiting cell biofilm formation, inducing the accumulation of ROS, increasing extracellular AKP activity, and altering the cell morphology, leading to disruption of the cell membrane and cell wall integrity.

3.4. Analysis of Metabolomics Data

3.4.1. Effect of VFD3 on the Metabolism of A. rhizogenes

To assess the effects of VFD3 on A. rhizogenes, a combination of untargeted and targeted UPLC-MS/MS techniques was used to quantitatively and qualitatively analyze the metabolites. OPLS-DA was used to analyze differences in metabolites between the VFD3 and CK groups (Figure 4A). The DMs of A. rhizogenes in the VFD3 group were visualized by using a volcano plot (Figure 4B). In total, 326 DMs were screened, of which 133 DMs were significantly upregulated and 193 DMs were downregulated. The scatter plot (Figure S3) and clustered heat map (Figure S4) revealed large differences in the DMs between the VFD3 and CK groups. Among them, the DMs with significant changes were found to be mainly amino acids and their metabolites, which were significantly lower than those in the CK group. Our results agreed with those of Chen et al., who reported that almost all amino acids were significantly reduced in the cells of Cutibacterium acnes after LCEO treatment.⁵⁶

Figure 4.

Metabolomic analysis of A. rhizogene with and without VFD3 treatment. (A) OPLS-DA score plot. (B) Volcano plot. The red and green dots represent increases and decreases in metabolite abundance, respectively, compared with those in the CK group.

3.4.2. Metabolic Pathway Analysis of A. rhizogene with VFD3 Treatment

Pathway analysis of the 326 DMs screened via the KEGG database yielded 64 metabolic pathways (Figure S5). According to the hypergeometric test p-value, the top 20 ranked pathways are presented from the smallest to largest, and the results are shown in the enrichment analysis bubble (Figure 5). After VFD3 treatment, metabolites in the bacterial secretion system pathway, which was related to membrane transport, were the most highly enriched and upregulated.⁵⁷ Guillín et al. reported that the main DMs were involved in the pathways of aminoacyl-tRNA biosynthesis, glutathione metabolism, and amino acid metabolism.³³ In this study, DMs were significantly enriched in the pathways of glutathione metabolism, glycerophospholipid metabolism, and peptidoglycan biosynthesis. The first two pathways were downregulated in expression. In the glutathione metabolic pathway, oxidized glutathione, cysteinylglycine, and the intermediate 5-oxoproline were significantly downregulated, resulting in blocked transport. A similar mode of action was reported by Zhao et al., who reported that glycerophospholipid metabolism was significantly altered by menthone treatment of MRSA. The structure of the cell membrane was disrupted by interference with lipid synthesis, thus exerting an antibacterial effect.⁵⁸

Figure 5.

Differential metabolite pathway enrichment maps of A. rhizogene in response to VFD3 treatment.

We found that in the VFD3-treated group, uridine-5′-diphospho-N-acetylgalactosamine-disodium-salt and UDP-N-acetylmuramic acid, which are related to peptidoglycan (PG), were significantly downregulated. PG plays a crucial role in maintaining the morphological, structural, and functional integrity of bacterial cells, and impaired PG synthesis leads to severe growth defects, ultimately leading to cell death.^59,60 Therefore, the significant downregulation of the PG biosynthetic pathway may also be a reason for the bacteriostatic effect of VFD3.

These results suggested that VFD3 treatment may affect A. rhizogenes by interfering with the pathways of glutathione metabolism, glycerophospholipid metabolism, and PG biosynthesis, resulting in bacteriostatic effects.

To summarize, two separation technologies, VFD and MD, were used to process LCEO, and the chemical composition and antibacterial activity of the seven distillates were significantly varied. Some active components, such as citral, were strongly enriched in the VFD3 and MDH2 distillates, resulting in better bacteriostatic effects on some foodborne pathogens, such as E. coli and S. aureus. VFD3 and MDH2 had strong antibacterial activity against two strains of plant pathogens, A. rhizogenes, and A. tumefaciens, which have not been previously reported. VFD and MD techniques played an important role in enhancing the content of the active ingredients and the antibacterial activity of LCEO while retaining heat-sensitive components under high-vacuum and low-temperature conditions. The antibacterial mechanism of VFD3 on A. rhizogenes was investigated by measuring the biofilm inhibition rate, accumulation of ROS, AKP activity, and SEM images of cell morphology. Additionally, the UPLC-MS/MS technique was used to qualitatively and quantitatively analyze the metabolites. The effect of VFD3 might involve damage to the cell membrane and cell wall integrity and interference with metabolic pathways. The results of this study may provide a reference for EO processing and separation research to increase the bioactivity and improve the application potential of LCEO and other EOs.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c11955.

• Materials, experimental details of GC-MS characterization of EOs, antibacterial mechanism of VFD3 against A. rhizogenes, metabolite analysis, abbreviations used, and Figures S1–S5 (PDF)

The authors declare no competing financial interest.