Skip to main content
NCBI home page
Current Research in Food Science logoLink to Current Research in Food Science
. 2025 Aug 6;11:101164. doi: 10.1016/j.crfs.2025.101164

Phenotypic and transcriptomic analysis reveals the antibiofilm mechanism of Litsea cubeba essential oil against Bacillus cereus and its application in pork preservation

Haixia Liao a,1, Sisi Liu b,1,∗∗, Wei Hu b,c, Yanna Zhao d, Zhihong Xiao b,e, Yingzi Ma a,∗, Changzhu Li b,c,∗∗∗
PMCID: PMC12398906  PMID: 40896510

Abstract

Bacillus cereus is a common foodborne pathogen that can result in diarrhea and vomiting. It can easily exist in food production environments and form biofilms that are difficult to remove. This study investigated the inhibitory effect and mechanism of Litsea cubeba essential oil (LC-EO) against B. cereus biofilm based on phenotype and transcriptomics, as well as the application in pork preservation. The results showed the MIC and MBIC were 0.125 mg/mL and 0.250 mg/mL, respectively. After LC-EO treatment, the extracellular DNA (eDNA) decreased significantly, the swimming motility was almost completely inhibited. Additionally, LC-EO interferes with AI-2/LuxS QS system by inhibiting key enzyme genes (metC, metK, metQ) in AI-2 signaling molecule synthesis pathway and the key genes (lsrF, lsrK) in the downstream signal transduction pathway. Moreover, molecular docking studies have shown that citral can target five key enzymes (LuxS, metC, metK, LsrF and LsrK). qRT-PCR results also showed that LC-EO significantly inhibited flagellar motility related genes (fla, fliY), QS related genes (codY, SinR, SinI, plcR) and ten virulence-related genes. Finally, the experiment of LC-EO in pork preservation showed that LC-EO can be used to extend the shelf life of pork.

Keywords: Bacillus cereus, Litsea cubeba essential oil, Antibiofilm, Mechanism, Pork preservation

Graphical abstract

Image 1

Open in a new tab

Highlights

  • •

    Exploring antibiofilm mechanism by phenotype, transcriptome and molecular docking.

  • •

    LC-EO can inhibit the enzyme genes luxS and metC/K/Q in the AI-2 synthesis pathway.

  • •

    LC-EO also inhibited gene lsrF and lsrK in the signal transduction pathway of AI-2.

  • •

    LC-EO target key enzymes LuxS, MetC, MetK, LsrF and LsrK in the AI-2/LuxS QS system.

1. Introduction

As a foodborne gram-positive pathogen, B. cereus is widely distributed in soil, water source and food (Haque et al., 2021). It can produce a series of virulence factors and cause gastrointestinal diseases with different symptoms after entering the human body, including diarrhea and vomiting (Jovanovic et al., 2021). Bacterial biofilm is associated with most foodborne diseases and are key to persistent contamination caused by foodborne pathogenic bacteria (Abebe, 2020). Biofilm is a community of microbial cells, usually wrapped in a self-produced extracellular polymeric substance (EPS) composed of polysaccharides, proteins and extracellular DNA (eDNA), which is a survival mode for many bacteria in a self-protective state (Sauer et al., 2022). As a dynamic system with complex structure, biofilm formation is a continuous dynamic process, which usually includes four stages: bacterial adhesion, microcolony formation, biofilm maturation and diffusion (Rumbaugh and Sauer, 2020).

Bacteria can sense some environmental signals and trigger a density-dependent communication system called quorum sensing (QS) (Mukherjee and Bassler, 2019), accumulate signaling molecules, and when the concentration of signaling molecules reaches a minimum threshold, they bind to receptor proteins, thereby activating the expression of genes associated with biofilm formation and producing a large number of EPS, which can promote the formation of microcolonies and thicken the biofilm structure (Paluch et al., 2020). However, the normal operation of QS system requires the participation of signal molecules, and different types of bacteria secrete different signal molecules. For example, AHL secreted by gram-negative bacteria (Liu et al., 2022, Liu et al., 2022), AIP secreted by gram-positive bacteria (McBrayer et al., 2020), and universal signaling molecule AI-2 (Deng et al., 2022), which is involved in regulating biofilm formation of both gram-negative and gram-positive bacteria.

AI-2 is an important signaling molecule in AI-2/LuxS quorum sensing system, and its synthesis is closely related to methionine cycle. Methionine participates in the methylation reaction of bacteria by converting to S-adenosylmethionine (SAM), and SAM is converted to S-adenosylhomocysteine (SAH) after the methylation. Then, S-ribosylhomocysteine (SRH) is catalyzed by S-adenosylhomocysteine (Pfs) to produce S-ribosylhomocysteine. LuxS catalyzed SRH to produce homocysteine and AI-2 signaling precursor 4,5-dihydroxy, 2,3-pentanedione (DPD), and DPD generated AI-2 signaling molecules through its own cyclization rearrangement (Wang et al., 2019). The homocysteine reacts to form a new SAM and enters a new cycle. When AI-2 concentration accumulates to a threshold, the downstream regulatory network is activated by binding to specific receptors such as Lsr protein (Gatta et al., 2020), thereby regulating gene expression of key phenotypes including biofilm formation, virulence factor secretion, and motility.

Litsea cubeba essential oil (LC-EO) are volatile secondary metabolites mainly extracted from the fruit of L. cubeba, which contains monoterpene and sesquiterpene compounds such as citral and limonene (Thielmann and Muranyi, 2019), and LC-EO exhibits excellent antibacterial activity, with citral deemed to be the main cause of the antimicrobial activity because of its “antimicrobial impact” (AI) values (Liu et al., 2024). It has a good inhibition effect on bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) (Hu et al., 2019), Vibrio parahaemolyticus (Li et al., 2022, Li et al., 2022), Escherichia coli (Mei et al., 2020), and Salmonella typhimurium (Wang et al., 2025).and fungi such as Aspergillus niger (Zhang et al., 2025). Although there are many reports on the antibacterial activity of LC-EO, relatively few reports on the antibiofilm activity of LC-EO, especially on the mechanisms related to antibiofilm or antibacterial, and most of them are limited to the aspect of microbial physiology (Bai et al., 2021). At present, the antibacterial and antibiofilm activity of LC-EO against B. cereus and the underlying mechanisms remain unclear.

In this study, we selected the model strain B. cereus ATCC14579 and the LC-EO which we had previously characterized for chemical composition (Huang et al., 2023) as the research objects. Firstly, the inhibitory effect of LC-EO on B. cereus biofilm and its mechanism were investigated from the aspects of motility, EPS and biofilm structure. Secondly, transcriptomics, molecular docking and molecular biology techniques were used to study the effects of LC-EO on the synthesis and downstream transduction of AI-2 signaling molecules in AI-2/LuxS QS system at the molecular level, so as to reveal the potential mechanism of LC-EO mediating AI-2/LuxS QS system to inhibit biofilm formation. In addition, qRT-PCR was used to study the effects of LC-EO on the expression levels of motility related genes and virulence genes during the biofilm formation of B. cereus. Finally, the application potential of LC-EO as a natural antibacterial agent in pork preservation was discussed.

2. Materials and methods

2.1. Strains and reagents

B. cereus ATCC 14579 and Vibrio harveyi BB 170 were purchased from the American Type Culture Collection (ATCC) (Manassas, USA). Litsea cubeba essential oil (LC-EO) was purchased from Yongzhou Samshiang Flavours & Fragrances Co., LTD. (Yongzhou, China), Luria–Bertani (LB) broth, Tryptone Soy Broth (TSB) and tryptic soy agar (TSA) medium were purchased from Hangzhou Baisi Biotechnology Co., Ltd. (Hangzhou, China), Autoinducer Bioassay (AB) Medium was purchased from Shandong Tuopu Biol-Engineering Co., LTD. (Zhaoyuan, China).

2.2. Antibacterial activity assays

The MIC and MBC of LC-EO against B. cereus ATCC 14579 were determined by double dilution method. Firstly, LC-EO was dissolved in TSB with the help of a vortex shaker. 200 μL of B. cereus (1 × 106 CFU/mL) was inoculated into each well of 24-well plate, and 2 mL of TSB medium containing different concentrations of LC-EO was added to make the final concentrations of LC-EO to be 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mg/mL, respectively. The MBIC and MBEC were determined by Andersen et al. (2024) and Gao et al. (2024).

2.3. Microstructure of B. cereus biofilm under LC-EO treatment at MBIC

The cell slides were immersed in a 24-well plate containing B. cereus ATCC 14579 bacterial solutions so that the biofilm formed could attach to the slides. After incubation for 24 h at 37 °C with or without LC-EO, the slides were removed and carefully washed twice with 0.01M PBS to remove planktonic bacteria, followed by 2.5 % glutaraldehyde (pre-cooled at 4 °C) and fixed at 4 °C. After the fixative was removed, different volume ratios of ethanol (30 %, 50 %, 70 %, 80 %, 95 % and 99.9 %) were added for gradient elution for 10 min, followed by freeze-drying (Gao et al., 2025). Finally, it was sprayed with gold and observed by SEM (TESCAN MIRA4, Brno, Czech) at a voltage of 5 kV and a magnification of × 3, 000 to × 10, 000.

2.4. Effect of LC-EO on early motility and initial adhesion of B. cereus

TSA plates suitable for observing the motility of bacteria swimming (0.3 % agar), swarming (0.5 % agar) and twitching (1.0 % agar) were prepared 24 h before the experiment (Badal et al., 2021). Then, 5 μL of TSA medium with LC-EO added (1 × 106 CFU/mL) was inoculated in the center of the above three TSA plates, and incubated at 37 °C. Observations were made at 24 h and 48 h, and diameters were measured by cross crossing method. The data analysis is presented by radial bar graph using Origin 2021 (OriginLab, New York, USA).

100 μL of bacterial suspension (1 × 106 CFU/mL) was added to each well of a 24-well plate, and then 1 mL of TSB medium containing LC-EO was added to the wells to make the final concentration of LC-EO in the wells was 1/2 MBIC, MBIC, and 2 MBIC, respectively, with no LC-EO added as a control. After incubating at 37 °C for 8 h to make initial adhesion occur, the initial adhesion ability of bacteria was determined according to the method described by Liu et al. (2022), and the calculation formula is as follows:

Relative adhesion rate (%) = (OD570 nm treatment/OD570 nm control) × 100 %

2.5. Effect of LC-EO on the extracellular polymeric substance (EPS) and the bacterial metabolism in biofilms of B. cereus

The extracellular polysaccharide content of B. cereus biofilm was determined by phenol-sulfuric acid method (Hooshdar et al., 2020). Extracellular protein content was determined by Coomassie brilliant blue method (Cui et al., 2020). Extracellular DNA content was extracted by bacterial genome DNA rapid extraction kit (Aidlab, Beijing, China) and then determined by a microplate reader (Epoch BioTek, Winooski, USA).

The cell slides were placed in the 24-well plate, then the bacterial solution (1 × 106 CFU/mL) and TSB medium were added. After the biofilm was mature and attached to the slide, the supernatant was discarded. TSB medium containing different concentrations of LC-EO (2MBIC, MBIC, 1/2MBIC, 0) was used to continue the culture of the biofilm. After 4 h, the slide attached with the biofilm was transferred to a 10 mL centrifuge tube containing 1 mL sterile water, then sonicated for 5 min, vortexed and resuspended the biofilm. Resazurin staining solution was added to the bacterial suspension at a volume ratio of 10 %, and avoid light incubated at 37 °C and 180 rpm for 2 h in a shaking table, and then was centrifuged at 10000 rpm for 4 min, and the supernatant was transferred to a 96-well black microplate for detection. The excitation and emission light wavelength were set to 560 nm and 590 nm respectively.

2.6. Transcriptomic analysis

2.6.1. RNA extraction and sequencing

The bacterial solution (1 × 106 CFU/mL) was added with LC-EO, so that the concentration of LC-EO in the mixture was 1/4 MBIC, and the solution without LC-EO was used as a control. The above two cultures were incubated at 37 °C with shaking at 180 rpm for 12 h and 24 h, respectively. Then, the cells were collected by centrifugation at 10000 rpm and 4 °C for 10 min and quick-frozen in liquid nitrogen. Finally, the cells were stored at −80 °C. Total RNA was extracted by TruSeq™ Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA), and the sequencing was performed using Illumina's NovaSeqXPlus platform at Shanghai Meiji Biotechnology Co., LTD. (Shanghai, China).

2.6.2. Transcriptomic data analysis

Firstly, the clean reads were obtained by removing the reads with low-quality. And the gene expression levels were established using the RSEM method, the differentially expressed genes (DEGs) were calculated by the DEseq2 software based on the combined criteria of |log2 (fold change)| > 2 and p < 0.05. At the same time, the Venn and Volcano diagram of DEGs were analyzed. Finally, the DEGs were enriched by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (p < 0.05).

2.7. qRT-PCR validations of DEGs

In this experiment, B. cereus LuxS/AI-2 QS system (lsrF, lsrK, metC, metK and metQ), biofilm formation (fla, fliY, codY, SinR, SinI, plcR) and toxin related genes (hblA, hblC, hblD, cesB, nheA, nheB, nheC, entFM, bceT and cytK) were selected, Specific primers (Table S1) were designed using Primer Premier 6.0 software, and all primers were synthesized by Beijing Tsingke Biotech Co., Ltd., and 16s rRNA gene was used as the reference gene (Lee et al., 2021). Total RNA was extracted using the EASYSpin Plus bacterial RNA quick extract kit (Aidlab, Beijing, China), followed by reverse transcription to obtain cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). The reaction system was configured according to the instructions of the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and the qRT-PCR experiment was performed on the QuantStudio™ 7 Flex RealTime PCR system (Applied Biosystems®, USA). After Ct values were obtained, the 2−ΔΔCt algorithm was used to calculate the relative expression.

2.8. Effect of LC-EO on AI-2 signaling molecules in B. cereus

V. harveyi BB170 was cultured with LB broth medium to OD600 nm = 1.0 (9.2 × 108 CFU/mL, Jing et al., 2021), and 1 mL of the above V. harveyi BB170 was diluted with AB medium at 1:1000 ratio (Zhao et al., 2021). B. cereus ATCC 14579 (1 × 106 CFU/mL) was cultured at 37 °C in TSB medium containing LC-EO at final concentrations of 2MBIC, MBIC, and 1/2MBIC, respectively. The sample without treatment with LC-EO was used as a control. The supernatants of 12 h and 24 h culture was obtained by centrifugation at 4 °C and 12000 rpm for 10 min, and filtered into a 2 mL centrifuge tube with a 0.22 μm sterile filter membrane. Then 200 μl of the filtrate was diluted with the above diluted V. harveyi BB170 bacteria solution (volume ratio: 1:10), and were incubated for 5 h at 30 °C with shaking at 125 rpm. Then 200 μL of the shaking culture was added to a black-bottom 96-well plate for bioluminescence assay using a microplate reader.

2.9. Molecular docking study

According to the transcriptome data of B. cereus measured in this study, the amino acid sequence of the target protein was obtained and submitted to the online platform Swiss model (https://www.swissmodel.expasy.org/) for homologous protein template search. Then the protein molecules with high amino acid sequence coverage and similarity (not less than 30 %) are selected as homologous templates to construct a 3D model of protein molecules and the rationality of the model is verified by Saves 6.0 (https://saves.mbi.ucla.edu/).The PDB file of protein meeting the quality requirements of molecular docking was downloaded, and PyMOL 2.2.0 software was used to remove water, phosphate and other unrelated ions in the protein.

The 3D structure sdf files of citral molecule were downloaded from Pubchem database and converted to PDB format files using Openbabel 2.4.1 software. The protein molecules and small molecule ligands were preprocessed using AutoDockTools 1.5.6 software before docking. And the pre-processed target proteins and small molecule ligands were converted into pdbqt format files, AutoDock Vina software was run for Autogrid and Docking operation, PyMOL 2.2.0 software was used to open the docking complex after docking, and 3D interaction diagram was exported. Finally, the two-dimensional structure of protein-ligand interaction is visualized using LigPlus software.

2.10. Application of LC-EO in fresh pork storage

This assay was performed as previously reported by Li et al. (2022). Fresh pork was purchased from local supermarkets (Changsha, China), and shipped to the laboratory wrapped in ice. The pork was cut into small pieces of almost the same size, shape and thickness, each weighing 10 g, then all pork samples were rinsed twice with sterile water and UV-irradiated for 20min on both sides, respectively. Then the pork pieces were randomly divided into three groups. The control group was not further treated, and the other two groups were immersed in B. cereus suspension (1 × 106 CFU/mL) for 2 min to ensure full contact, and then were immersed in LC-EO with MIC and 2MIC for 2 min respectively after drying for 10 min in a sterile environment. Finally, all pork pieces were placed in petri dishes at 25 °C, and the quality of pork was observed at 0, 2 and 4 days, respectively.

2.11. Statistical analysis

All experiments were performed in triplicate. The data were analyzed by SPSS 20.0 software, and p < 0.05 was considered as significant difference. "∗" p < 0.05; "∗∗" p < 0.01; "∗∗∗" p < 0.001.

3. Results and discussion

3.1. Antibacterial and antibiofilm effects of LC-EO on B. cereus

The results were similar to garlic essential oil (Jin et al., 2021), Thymus vulgaris essential oil (Sateriale et al., 2023) and clove essential oil (Li et al., 2022), LC-EO had a significant inhibitory effect on the B. cereus and its biofilm. It was shown that the MIC, MBC, MBIC and MBEC of LC-EO against B. cereus ATCC 14579 was 0.125 mg/mL (Fig. 1A), 0.5 mg/mL (Figs. 1B), 0.25 mg/mL (Fig. 1C) and 1.0 mg/mL (Fig. 1D), respectively.

Fig. 1.

Fig. 1

Open in a new tab

Mic, MBC, MBIC and MBEC of LC-EO against B. cereus ATCC14579.

The SEM results more directly showed the change of the integrity of B. cereus biofilm. Without LC-EO, a large number of B. cereus cell could be observed to aggregate on the surface of the cell slide after 24 h of culture, and adhere to each other, showing a preliminary three-dimensional structure (Fig. 2A and B). After 48 h, a thick and dense three-dimensional biofilm structure was formed, with a large number of adhesive substances on the surface of the bacteria, and the bacteria were embedded in the extracellular polymeric substance (Fig. 2C and D.). When the MBIC concentration of LC-EO was added (Fig. 2E–H), a large number of B. cereus cell structures were destroyed, the intracellular matter of the bacteria leaked out, and the number of bacteria was greatly reduced. As a result, the three-dimensional structure of the biofilm was destroyed, the dense structure became sparse, and the coverage area and thickness of the biofilm were reduced. Most of the bacteria were scattered and isolated, and there was almost no extracellular polymeric substance in the intercellular space, which is similar with the results of other essential oils studies (Li et al., 2025). It is worth noting that after treatment with LC-EO for 48 h, the destructive effect of LC-EO on B. cereus was not significantly enhanced, suggesting that LC-EO has played a role within 24 h and has a rapid effect.

Fig. 2.

Fig. 2

Open in a new tab

SEM images of B. cereus ATCC 14579 biofilms cultured for 24h and 48h in the absence (CK: 0 mg/mL) and presence (MBIC: 0.25 mg/mL) of LC-EO, respectively.

3.2. Effect of LC-EO on early motility and initial adhesion of B. cereus

In the early stage of biofilm formation, the adhesion of bacteria is mainly depended on, and the initial adhesion during biofilm formation is closely related to the early motility of bacteria (Alotaibi and Bukhari, 2021). B. cereus can form approximately round colonies on agar medium, which are waxy white in color. With the increase of LC-EO concentration in the medium, the growth range of B. cereus in the plate is significantly reduced, which shows the swimming, swarming and twitching motility of B. cereus were inhibited. Fig. 3A and B and C shows that B. cereus has the strongest swimming motility, and LC-EO also has the most significant inhibitory effect on B. cereus swimming motility, followed by swarming. Moreover, this study also analyzed the effect of LC-EO on the initial adhesion of biofilm-forming B. cereus. 1/2 MBIC LC-EO can inhibit the initial adhesion of biofilm-forming B. cereus, and MBIC LC-EO has a stronger inhibition effect on the initial adhesion (Fig. 3D). Compared with CK, the adhesion rate significant decreases by 77.93 %, and when treated with 2MBIC, the adhesion rate even significant decreases to 10.52 %. This is consistent with the results of biofilm formation measured by crystal violet staining.

Fig. 3.

Fig. 3

Open in a new tab

Effect of LC-EO on three motility (A, B and C) and initial adhesion (D) of B. cereus. The scale is 1.0 cm. Swimming (line 1), swarming (line 2) and twitching (line 3).

3.3. Effects of LC-EO on EPS and bacterial metabolism in B. cereus biofilms

EPS of bacterial biofilm is usually composed of protein, polysaccharide, extracellular DNA (eDNA), etc. Extracellular proteins adhere to extracellular polysaccharide and jointly promote the formation of three-dimensional structure of biofilm (Pan et al., 2022). The results showed the inhibitory effect of LC-EO on extracellular polysaccharide (Fig. 4A) and extracellular protein (Fig. 4B) was not obvious, but the eDNA was very significant (Jannat et al., 2023; Gao et al., 2024), when B. cereus was treated with 1/2MBIC and MBIC concentrations of LC-EO for 48 h respectively, the eDNA content decreased significantly from 3.31 to 2.28 and 1.47 μg/ml (Fig. 4C), respectively. The reason maybe that citral, a key component of LC-EO, can form a chimera with B. cereus DNA to change the DNA structure, resulting in a significant decrease in eDNA content, and change the distribution of extracellular polysaccharide and extracellular protein in B. cereus biofilm, but did not have a significant effect on their content.

Fig. 4.

Fig. 4

Open in a new tab

Effects of LC-EO on extracellular polysaccharide (A), extracellular protein (B), eDNA (C) and bacterial metabolism (D) in B. cereus biofilm.

The test results of bacterial metabolic capacity (Fig. 4D) indicated that with the increase of LC-EO treatment concentration, the bacterial metabolic activity in the biofilm showed a continuous downward trend. After 1/2 MBIC LC-EO treatment, the bacterial metabolic activity in the B. cereus biofilm dropped to 78.53 %. After the concentration of LC-EO was increased to MBIC, the metabolic activity rapidly decreased to 9.21 %. Subsequently, when the concentration of LC-EO was increased to 2 MBIC, the rate of decline slowed down. The reason maybe was that when the concentration of LC-EO was MBIC, most of the bacteria in the biofilm had become inactivated or died, and due to the marginal effect, when the treatment concentration of LC-EO was further increased, there was no significant change in the downward trend of bacterial metabolic activity, which was basically consistent with the SEM results, and further revealed the mechanism of LC-EO inhibiting B. cereus biofilm (Hu et al., 2019).

3.4. Transcriptomic analysis of LC-EO against B. cereus biofilm

3.4.1. DEG analysis

As shown in Fig. 5A and B, compared with B. cereus without LC-EO, there were 1076 DEGs in B. cereus treated with 1/4MBIC concentration of LC-EO for 12 h, among which 518 DEGs were up-regulated and 558 DEGs were down-regulated. And there were 409 DEGs in B. cereus treated with LC-EO for 24 h, of which 185 were up-regulated and 224 were down-regulated. As shown in Fig. 5C and D, there were 35 DEGs that were significantly up-regulated in B. cereus after treatment with 1/4 MBIC concentration of LC-EO both for 12 h and 24 h, and 72 DEGs were significantly down-regulated both for 12 h and 24 h, and the results imply that within 24 h after treatment with 1/4 MBIC of B. cereus essential oil, 35 DEGs were significantly and continuously up-regulated and 73 DEGs were known to be significantly and continuously down-regulated.

Fig. 5.

Fig. 5

Open in a new tab

Volcano map of DEGs of B. cereus treated with 1/4 MBIC concentration of LC-EO for 12 h (A) and 24 h (B), Venn diagram of continuously significantly up-regulated DEGs (C) and continuously significantly down-regulated DEGs (D).

3.4.2. GO and KEGG enrichment analysis

GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for DEGs. The enriched GO terms were sorted according to their significance, and the first 5 GO terms in the categories of Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) were selected (Fig. 6A), respectively. The results showed that in the BP category, DEGs mainly focus on small molecule metabolic process, organonitrogen compound metabolic process and pyrimidine-containing compound term. In CC category, it is mainly concentrated in ribosomal subunit, cytosolic large ribosomal subunit and ribonucleoprotein complex term. In MF category, it is mainly enriched in rRNA binding, oxidoreductase activity and metal ion binding term. The KEGG pathway enrichment analysis results (Fig. 6B) showed that most of the DEGs were enriched in amino acid metabolism, carbohydrate metabolism, energy metabolism, membrane transport, signal transduction pathways.

Fig. 6.

Fig. 6

Open in a new tab

GO enrichment analysis of DEGs (A) and KEGG enrichment analysis of DEGs (B).

3.5. qRT-PCR validation of DEGs related to AI-2/LuxS QS system, virulence and motility of B. cereus

The QS is closely related to biofilm formation, and AI-2 signaling molecule can promote biofilm formation during the 24 h growth period of bacteria (Lee et al., 2021). AI-2 is a by-product of methionine cycling pathway (Zhang et al., 2020), so AI-2 synthesis is closely related to methionine metabolism. The cystathionine β-lyase encoded by the metC gene is a key enzyme in the methionine biosynthesis pathway, responsible for catalyzing the decomposition of cystathionine into homocysteine, pyruvate and ammonia, and finally into methionine (Gai et al., 2021). The metK gene encodes S-adenosylmethionine synthetase, responsible for converting methionine to S-adenosylmethionine (SAM) (Yadav et al., 2022), while metQ is a methionine binding protein, responsible for recognizing and binding methionine in the environment (Pei et al., 2020), assisting in its transmembrane transport.

The qRT-PCR results (Fig. 7A–B) showed that only metC gene was significantly down-regulated in B. cereus after treatment with 1/4MBIC concentration of LC-EO for 12 h, while metC, metK and metQ genes were significantly down-regulated for 24 h, especially metQ. Interestingly, when treated with 1/4 MBIC LC-EO for 12 h, the luxS gene was significantly up-regulated. This phenomenon may be related to the bacterial stress response mechanism, and the active components of LC-EO in the early stage may activate the bacterial oxidative stress pathway and induce the transient upregulation of luxS gene to enhance the AI-2 mediated group cooperation against environmental stress. With the extension of treatment time to 24 h, the expression of luxS gene decreased significantly and was significantly inhibited. These results suggest that LC-EO may interfere with methionine anabolic pathway by inhibiting the expression of metC, metK, metQ and luxS genes, thus affecting AI-2 production.

Fig. 7.

Fig. 7

Open in a new tab

Effects of LC-EO on AI-2 synthesis and signal transduction related genes (A and B), virulence-related genes (C), motility-related genes (D) and the content of AI-2 (E) of B. cereus.

Meanwhile, the expression of lsrF and lsrK was also significantly inhibited by the LC-EO after 24 h treatment. LsrF and LsrK are the core components of the AI-2/LuxS QS system, responsible for sensing and metabolizing AI-2 signaling molecules. The lsrK encoding LsrK is an AI-2 kinase that phosphorylates extracellular AI-2 and tranships it into the intracellular and initiates the signaling cascade reaction (Zuo et al., 2019). The LsrF encoded by lsrF is involved in the further metabolism of phosphorylated AI-2 and its transformation into an intermediate product of bacterial central metabolism (Marques et al., 2014). Previous studies by Li et al. (2007) confirmed that the deletion of lsrK or lsrR can significantly affect the biofilm structure of E. coli and the expression of related genes, and reduce the secretion of virulence factors. This mechanism may be applicable to B. cereus, therefore, it is speculated that LC-EO can inhibit the expression of lsrF and lsrK genes of B. cereus, thus interfering with the intraspecific communication mediated by AI-2 signaling molecules and weakening the biofilm formation of B. cereus.

Moreover, we further analyzed the effects of LC-EO on the expression of several common virulence-related genes in B. cereus by qRT-PCR. As shown in Fig. 7C, after B. cereus was cultured in TSB medium with a final concentration of 1/4 MBIC of LC-EO for 24 h, the expression levels of several major enterotoxin genes and vomitoxin genes were significantly down-regulated. Fig. 7D shows the effects of LC-EO on flagellar motility genes (fla, fliY) and other biofilm-forming genes (codY, SinR, SinI and plcR) of B. cereus. The early motility of bacteria is mainly mediated by flagella and fimbriae, and swimming motility is mainly mediated by flagella (Grognot and Taute, 2021). qRT-PCR results showed that LC-EO significantly inhibited the expression of flagellar motility genes fla and fliY in B. cereus, which was consistent with the results of this study that LC-EO significantly inhibited the swimming motility of B. cereus.

3.6. Effect of LC-EO on the content of AI-2 signaling molecules

V. harveyi BB170 is a commonly used indicator strain for detecting AI-2 signaling molecules, and it can sense AI-2 signaling molecules and produce light, and the concentration of AI-2 was analyzed by detecting the luminescence intensity. In order to verify the effects of LC-EO on the AI-2 signaling molecules of B. cereus, we used V. harveyi BB170 sensor strain bioassay to determine the AI-2 content of B. cereus with LC-EO treatment for 12 h and 24 h, respectively. The results showed that the content of AI-2 signaling molecules in the culture medium of B. cereus decreased significantly after treatment with MBIC concentration of LC-EO (Fig. 7E). This result suggests that LC-EO inhibits the synthesis of AI-2 signaling molecules of B. cereus, interferes with intercellular communication, and inhibits the QS of B. cereus, thus inhibiting the formation of its biofilm. It is interesting that there are also studies showing that the addition of essential oil components has no significant effect on the content of B. cereus AI-2 signaling molecules (Zhao et al., 2021).

3.7. Interaction between citral and metC/K, lsrF/lsrK and LuxS protein in AI-2/LuxS QS system

The molecular docking results showed that the citral could bind to the key proteins (MetC, MetK, LsrF, LsrK and LuxS) in the AI-2 signaling molecule synthesis and transduction pathway and form a stable complex. The binding energies were −5.2 kcal/mol, −4.7 kcal/mol, −5.7 kcal/mol, −5.9 kcal/mol and −5.6 kcal/mol, respectively, the larger the absolute value of binding energy indicates the more stable binding, and the binding ability is good when the binding energy is lower than −1.2 kcal/mol. The binding energy between citral and LsrK protein was the lowest among the five target proteins (−5.9 kcal/mol), suggesting that citral may preferentially act on the key kinase LsrK in the AI-2 signal transduction pathway to block the phosphorylation and activation process of AI-2 signal. It is worth noting that the binding stability of citral to LuxS and LsrF was close to that of LsrK, indicating that citral may achieve comprehensive interference of AI-2 signaling pathway by simultaneously inhibiting multiple key enzymes in AI-/LuxS QS. This is highly consistent with the downregulation of gene expression observed in qRT-PCR experiments.

Specifically, citral forms hydrogen bonds of 3.0, 2.8, and 3.5 Å with Lys43, Tyr44, and Glu55 of MetC (Fig. 8A), respectively, and a single hydrogen bond of 3.4 Å with Tyr372 of MetK (Fig. 8B). Thus, the citral disrupts the substrate binding pocket of MetC and MetK. It affects the substrate supply of LuxS enzyme, and then affects the synthesis of AI-2 signaling molecules. The citral also formed single hydrogen bonds with Lys172 (3.0 Å) and Arg74 (3.1 Å) and double hydrogen bonds with Asp24 (3.4 and 3.5 Å) of LsrF (Fig. 8C), and formed double hydrogen bonds with Arg308 (3.2 and 2.9 Å) and single hydrogen bonds with Tyr330 (3.0 Å) of LsrK (Fig. 8D), suggesting that citral may inhibit ATP binding required for AI-2 phosphorylation. Additionally, Citral forms a double hydrogen bond with Arg65 of LuxS (3.0 and 3.1 Å) (Fig. 8E), thereby blocking AI-2 synthesis.

Fig. 8.

Fig. 8

Open in a new tab

Molecular docking of citral with MetC, MetK, LsrF, LsrK and LuxS proteins.

3.8. Effect of LC-EO on storage quality of fresh pork

Meat spoilage is mainly caused by many microorganisms including B. cereus. In order to verify the inhibition ability of LC-EO against B. cereus in fresh tissues, we carried out a study on the application of LC-EO in pork storage. As shown in Fig. 9, the sensory quality of pork was sharply decreased after being stored at 25 °C for 4 d. A large number of microorganisms proliferated on the surface of the pork in CK and produced mucus material, accompanied by a pungent odor. Compared with CK, the pork in 2MIC group was still a bright pink (red) color on the second day and slightly sticky on the fourth day, and the quality of pork in MIC group was also significantly better than CK. The results imply that LC-EO has a good ability to against B. cereus, which can delay the contamination of pork and extend the shelf life of pork.

Fig. 9.

Fig. 9

Open in a new tab

Effects of LC-EO on storage quality of fresh pork.

The experiment of LC-EO applied in pork to inhibit B. cereus showed that LC-EO could significantly inhibit the damage of B. cereus to pork quality and prolong the shelf life of pork stored at room temperature 25 °C for 36–48 h. It is consistent with the results of pork storage studies with other essential oils such as oregano (Khruengsai et al., 2024) and clove essential oil (Li et al., 2022), which can prolong the shelf life of pork (Rasheed et al., 2024; Sobhy et al., 2025). Furthermore, the delay in the color change process of pork during storage may be attributed to the inhibitory effect of citral on myoglobin oxidation (Pateiro et al., 2018; Cui et al., 2025). Currently, the most commonly used method for pork preservation is freezing, usually without preservatives. Our results suggest that LC-EO has potential that can be used as a natural preservative or edible membrane component for pork preservation.

4. Conclusions

LC-EO has broad-spectrum antibacterial and antibiofilm activities, but its antibiofilm effects on gram-positive bacteria are less studied, and the mechanism of action is still unclear. Therefore, this study focused on the inhibition mechanism of LC-EO on biofilm and its application potential in meat preservation by taking the common gram-positive bacteria B. cereus as the research object. Studies have shown that LC-EO can significantly inhibit B. cereus biofilm formation in phenotype. Specifically, after treatment with LC-EO, the three-dimensional structure of B. cereus biofilm is damaged, the content of eDNA is reduced to different degrees, the amount of biofilm is significantly reduced, and the swimming, twitching and swarming motility were inhibited to varying degrees.

At the molecular level, on the one hand, LC-EO can inhibit the formation of B. cereus biofilm by influencing the AI-2/LuxS QS system. The experimental results showed that LC-EO interfered with AI-2/LuxS QS system by inhibiting the key enzyme genes (metC, metK, metQ) in AI-2 signaling molecule synthesis pathway and the expression of key genes (lsrF, lsrK) in the downstream signal transduction pathway of AI-2 molecule. At the same time, we used molecular docking technology to analyze whether citral, the main active component of LC-EO, can target key enzymes in AI-2 signaling molecule synthesis and signal transduction pathway, and the results showed that citral can interact with five key enzymes such as LuxS, metC, metK, LsrF and LsrK through intermolecular interaction forces such as hydrogen bonding. On the other hand, it was found that LC-EO could inhibit the expression of B. cereus virulence genes (nheA/B/C, hblA/B/C, entFM, bceT, cytK, cesB), flagellar motility-related genes (fla and fliY), and other biofilm-forming related genes (codY, SinR, SinI, plcR). Finally, the application potential of LC-EO as a bacteriostatic agent in pork preservation was explored, and the results showed that LC-EO could maintain the freshness and flavor of pork to a certain extent.

CRediT authorship contribution statement

Haixia Liao: Data curation, Investigation, Methodology, Visualization, Writing – original draft. Sisi Liu: Conceptualization, Data curation, Methodology, Visualization, Writing – original draft. Wei Hu: Data curation, Methodology. Yanna Zhao: Methodology, Formal analysis. Zhihong Xiao: Resources, Supervision. Yingzi Ma: Project administration, Resources, Supervision, Writing – review & editing. Changzhu Li: Project administration, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare no conflict of interest regarding the presented data in the manuscript.

Acknowledgement

This work was supported by the Special Project for the Construction of Chenzhou National Sustainable Development Agenda Innovation Demonstration Zone (No. 2022sfq53), Construction of State Key Laboratory of Woody Oil Resources (No. 2024PT0001), Changsha City Major Science and Technology Projects (No. kq2301004) and Hunan Forestry Science and Technology Innovation Fund (No. XLK202404).

Footnotes

Appendix B

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crfs.2025.101164.

Contributor Information

Sisi Liu, Email: liusisi274@126.com.

Yingzi Ma, Email: ma_yingzi@163.com.

Changzhu Li, Email: lichangzhu2013@aliyun.com.

Appendix B. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (19.5KB, docx)

Data availability

Data will be made available on request.

References

  1. Abebe G.M. The role of bacterial biofilm in antibiotic resistance and food contamination. Int. J. microbiol. 2020;2020(1) doi: 10.1155/2020/1705814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alotaibi G.F., Bukhari M.A. Factors influencing bacterial biofilm formation and development. Am. J. Biomed. Sci. Res. 2021;12(6):617–626. doi: 10.34297/AJBSR.2021.12.001820. [DOI] [Google Scholar]
  3. Andersen J.B., Rybtke M., Tolker-Nielsen T. The dynamics of biofilm development and dispersal should be taken into account when quantifying biofilm via the crystal violet microtiter plate assay. Biofilm. 2024;8 doi: 10.1016/j.bioflm.2024.100207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Badal D., Jayarani A.V., Kollaran M.A., Prakash D., P M., Singh V. Foraging signals promote swarming in starving Pseudomonas aeruginosa. mBio. 2021;12(5):10–1128. doi: 10.1128/mbio.02033-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bai M., Li C.Z., Cui H.Y., Lin L. Preparation of self-assembling Litsea cubeba essential oil/diphenylalanine peptide micro/nanotubes with enhanced antibacterial properties against Staphylococcus aureus biofilm. LWT--Food Sci. Technol. 2021;146:9. doi: 10.1016/j.lwt.2021.111394. [DOI] [Google Scholar]
  6. Cui H., Chen Y., Aziz T., Al-Asmari F., Alwethaynani M.S., Shi C., Lin L. Antibacterial mechanisms of diacetyl on Listeria monocytogenes and its application in Inner Mongolian cheese preservation via gelatin-based edible films. Food Control. 2025;168 doi: 10.1016/j.foodcont.2024.110920. [DOI] [Google Scholar]
  7. Cui H., Zhang C., Li C., Lin L. Inhibition of Escherichia coli O157: H7 biofilm on vegetable surface by solid liposomes of clove oil. LWT. 2020;117 doi: 10.1016/j.lwt.2019.108656. [DOI] [Google Scholar]
  8. Deng Z., Hou K., Valencak T.G., Luo X.M., Liu J., Wang H. AI-2/LuxS quorum sensing system promotes biofilm formation of Lactobacillus rhamnosus GG and enhances the resistance to enterotoxigenic Escherichia coli in germ-free zebrafish. Microbiol. Spectr. 2022;10(4) doi: 10.1128/spectrum.00610-22. 22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gai Y., Li L., Ma H., Riely B.K., Liu B., Li H. Critical role of MetR/MetB/MetC/MetX in cysteine and methionine metabolism, fungal development, and virulence of Alternaria alternata. Appl. Environ. Microbiol. 2021;87(4) doi: 10.1128/AEM.01911-20. 20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao Y., Li C., Tu J., Xiao J., Xiao Z., Ma Y., Liu S. Mechanism and application of camellia saponins against Bacillus cereus biofilm formation on different food contact surfaces. Food Control. 2025;168 doi: 10.1016/j.foodcont.2024.110903. [DOI] [Google Scholar]
  11. Gao Y.D., Liu S.S., Ma Y.Z., Li C.Z., Xiao Z.H., Nie S.L., Tu J. Effects of camellia saponins on biofilm formation and virulence factor genes of Bacillus cereus. LWT--Food Sci. Technol. 2024;198:11. doi: 10.1016/j.lwt.2024.116023. [DOI] [Google Scholar]
  12. Gatta V., Tomašič T., Ilaš J., Zidar N., Peterlin Mašič L., Barančoková M., et al. A new cell‐based AI‐2‐Mediated quorum sensing interference assay in screening of lsrk-targeted inhibitors. Chembiochem. 2020;21(13):1918–1922. doi: 10.1002/cbic.201900773. [DOI] [PubMed] [Google Scholar]
  13. Grognot M., Taute K.M. More than propellers: how flagella shape bacterial motility behaviors. Curr. Opin. Microbiol. 2021;61:73–81. doi: 10.1016/j.mib.2021.02.005. [DOI] [PubMed] [Google Scholar]
  14. Haque M.A., Quan H., Zuo Z., Khan A., Siddique N., He C. Pathogenicity of feed-borne Bacillus cereus and its implication on food safety. Agrobiol. Rec. 2021;3:1–16. doi: 10.47278/journal.abr/2020.015. [DOI] [Google Scholar]
  15. Hooshdar P., Kermanshahi R.K., Ghadam P., Khosravi-Darani K. A review on production of exopolysaccharide and biofilm in probiotics like lactobacilli and methods of analysis. Biointerface Res. Appl. Chem. 2020;10:6058–6075. doi: 10.33263/BRIAC105.60586075. [DOI] [Google Scholar]
  16. Hu W., Li C., Dai J., Cui H., Lin L. Antibacterial activity and mechanism of Litsea cubeba essential oil against methicillin-resistant Staphylococcus aureus (MRSA) Ind. Crop. Prod. 2019;130:34–41. doi: 10.1016/j.indcrop.2018.12.078. [DOI] [Google Scholar]
  17. Huang J., Liu S., Liu R., Yi Y., Li C., Xiao Z., Tu J., Xiao J. Mechanisms of Litsea cubeba essential oil in the control of Colletotrichum scovillei in pepper (Capsicum annuum L.): cell membrane/wall perspective. Physiol. Mol. Plant Pathol. 2023;127 doi: 10.1016/j.pmpp.2023.102103. [DOI] [Google Scholar]
  18. Jannat B., Alizadeh A.M., Farshi P., Dadgarnejad M., Hosseini H., Hashempour-Baltork F., Jafari S.M. Anti-biofilm activity of essential oils in fruit and vegetable: a systematic review. Food Control. 2023;152:10. doi: 10.1016/j.foodcont.2023.109875. [DOI] [Google Scholar]
  19. Jin Z.K., Li L., Zheng Y., An P.P. Diallyl disulfide, the antibacterial component of garlic essential oil, inhibits the toxicity of Bacillus cereus ATCC 14579 at sub-inhibitory concentrations. Food Control. 2021;126:8. doi: 10.1016/j.foodcont.2021.108090. [DOI] [Google Scholar]
  20. Jing Y., Zuo J., Phouthapane V., Chen Z., Han X. An optimized method for detecting AI-2 signal molecule by a bioassay with Vibrio harveyi BB170. Microbiology. 2021;90(3):383–391. doi: 10.1134/S0026261721030048. [DOI] [Google Scholar]
  21. Jovanovic J., Ornelis V.F., Madder A., Rajkovic A. Bacillus cereus food intoxication and toxicoinfection. Compr. Rev. Food Sci. Food Saf. 2021;20(4):3719–3761. doi: 10.1111/1541-4337.12785. [DOI] [PubMed] [Google Scholar]
  22. Khruengsai S., Phoopanasaeng P., Sripahco T., Soykeabkaew N., Pripdeevech P. Application of chitosan films incorporated with Zanthoxylum limonella essential oil for extending shelf life of pork. Int. J. Biol. Macromol. 2024;262 doi: 10.1016/j.ijbiomac.2024.129703. [DOI] [PubMed] [Google Scholar]
  23. Lee N., Kim M.D., Lim M.C. Autoinducer-2 could affect biofilm formation by food-derived Bacillus cereus. Indian J. Microbiol. 2021;61:66–73. doi: 10.1007/s12088-020-00918-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li A., Shi C., Qian S., Wang Z., Zhao S., Liu Y., Xue Z. Evaluation of antibiotic combination of Litsea cubeba essential oil on Vibrio parahaemolyticus inhibition mechanism and anti-biofilm ability. Microb. Pathog. 2022;168 doi: 10.1016/j.micpath.2022.105574. [DOI] [PubMed] [Google Scholar]
  25. Li H., Li J., Hua Z., Aziz T., Khojah E., Cui H., Lin L. Transcriptomic combined with molecular dynamics simulation analysis of the inhibitory mechanism of clove essential oil against Staphylococcus aureus biofilm and its application on surface of food contact materials. Food Bioprod. Process. 2025;150:131–140. doi: 10.1016/j.fbp.2025.01.009. [DOI] [Google Scholar]
  26. Li J., Attila C., Wang L., Wood T.K., Valdes J.J., Bentley W.E. Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects on small RNA and biofilm architecture. J. Bacteriol. 2007;189(16):6011–6020. doi: 10.1128/jb.00014-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Li J., Li C., Shi C., Aliakbarlu J., Cui H., Lin L. Antibacterial mechanisms of clove essential oil against Staphylococcus aureus and its application in pork. Int. J. Food Microbiol. 2022;380 doi: 10.1016/j.ijfoodmicro.2022.109864. [DOI] [PubMed] [Google Scholar]
  28. Liu L., Zeng X., Zheng J., Zou Y., Qiu S., Dai Y. AHL-mediated quorum sensing to regulate bacterial substance and energy metabolism: a review. Microbiol. Res. 2022;262 doi: 10.1016/j.micres.2022.127102. [DOI] [PubMed] [Google Scholar]
  29. Liu Y., Bu Y., Li J., Liu Y., Liu A., Gong P., Liu T., Zhang L., Wang S., Yi H. Inhibition activity of plantaricin Q7 produced by Lactobacillus plantarum Q7 against Listeria monocytogenes and its biofilm. Fermentation. 2022;8(2):75. doi: 10.3390/fermentation8020075. [DOI] [Google Scholar]
  30. Liu Y., Ren H., Li K. Litsea cubeba essential oil: extraction, chemical composition, antioxidant and antimicrobial properties, and applications in the food industry. J. Food Sci. 2024;89(8):4583–4603. doi: 10.1111/1750-3841.17236. [DOI] [PubMed] [Google Scholar]
  31. Marques J.C., Oh I.K., Ly D.C., Lamosa P., Ventura M.R., Miller S.T., Xavier K.B. LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2. Proc. Natl. Acad. Sci. 2014;111(39):14235–14240. doi: 10.1073/pnas.1408691111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McBrayer D.N., Cameron C.D., Tal-Gan Y. Development and utilization of peptide-based quorum sensing modulators in Gram-positive bacteria. Org. Biomol. Chem. 2020;18(37):7273–7290. doi: 10.1039/D0OB01421D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mei C., Wang X., Chen Y., Wang Y., Yao F., Li Z., Gu Q., Song D. Antibacterial activity and mechanism of Litsea cubeba essential oil against food contamination by Escherichia coli and Salmonella enterica. J. Food Saf. 2020;40(4) doi: 10.1111/jfs.12809. [DOI] [Google Scholar]
  34. Mukherjee S., Bassler B.L. Bacterial quorum sensing in complex and dynamically changing environments. Nat. Rev. Microbiol. 2019;17(6):371–382. doi: 10.1038/s41579-019-0186-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Paluch E., Rewak-Soroczyńska J., Jędrusik I., Mazurkiewicz E., Jermakow K.J.A.M. Prevention of biofilm formation by quorum quenching. Appl. Microbiol. Biotechnol. 2020;104:1871–1881. doi: 10.1007/s00253-020-10349-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pan M., Li H., Han X., Ma W., Li X., Guo Q., Yang B., Ding C., Ma Y. Effects of hydrodynamic conditions on the composition, spatiotemporal distribution of different extracellular polymeric substances and the architecture of biofilms. Chemosphere. 2022;307 doi: 10.1016/j.chemosphere.2022.135965. [DOI] [PubMed] [Google Scholar]
  37. Pateiro M., Barba F.J., Domínguez R., Sant'Ana A.S., Khaneghah A.M., Gavahian M., Gómez B., Lorenzo J.M. Essential oils as natural additives to prevent oxidation reactions in meat and meat products: a review. Food Res. Int. 2018;113:156–166. doi: 10.1016/j.foodres.2018.07.014. [DOI] [PubMed] [Google Scholar]
  38. Pei X., Liu J., Liu M., Zhou H., Wang X., Fan H. Quantitative proteomics revealed modulation of macrophages by MetQ gene of Streptococcus suis serotype 2. AMB Express. 2020;10:1–12. doi: 10.1186/s13568-020-01131-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rasheed H.A., Rehman A., Karim A., Al-Asmari F., Cui H., Lin L. A comprehensive insight into plant-derived extracts/bioactives: exploring their antimicrobial mechanisms and potential for high-performance food applications. Food Biosci. 2024;59 doi: 10.1016/j.fbio.2024.104035. [DOI] [Google Scholar]
  40. Rumbaugh K.P., Sauer K. Biofilm dispersion. Nat. Rev. Microbiol. 2020;18(10):571–586. doi: 10.1038/s41579-020-0385-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sateriale D., Forgione G., De Cristofaro G.A., Pagliuca C., Colicchio R., Salvatore P., Paolucci M., Pagliarulo C. Antibacterial and antibiofilm efficacy of thyme (Thymus vulgaris L.) essential oil against foodborne illness pathogens, Salmonella enterica subsp. enterica serovar typhimurium and Bacillus cereus. Antibiotics. 2023;12(3):485. doi: 10.3390/antibiotics12030485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sauer K., Stoodley P., Goeres D.M., Hall-Stoodley L., Burmølle M., Stewart P.S., Bjarnsholt T. The biofilm life cycle: expanding the conceptual model of biofilm formation. Nat. Rev. Microbiol. 2022;20(10):608–620. doi: 10.1038/s41579-022-00767-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sobhy M., Abdelkarim E.A., Hussein M.A., Aziz T., Al-Asmari F., Alabbosh K.F., et al. Essential oils as antibacterials against multidrug-resistant foodborne pathogens: mechanisms, recent advances, and legal considerations. Food Biosci. 2025 doi: 10.1016/j.fbio.2025.105937. [DOI] [Google Scholar]
  44. Thielmann J., Muranyi P. Review on the chemical composition of Litsea cubeba essential oils and the bioactivity of its major constituents citral and limonene. J. Essent. Oil Res. 2019;31(5):361–378. doi: 10.1080/10412905.2019.1611671. [DOI] [Google Scholar]
  45. Wang C., Chen X., Liu M., Tang X., Li Y., Zhan Y., Hao Z. Antibacterial activity and mechanism of Litsea cubeba essential oil against Salmonella typhimurium. Plants. 2025;14(9):1343. doi: 10.3390/plants14091343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wang Y., Liu B., Grenier D., Yi L. Regulatory mechanisms of the LuxS/AI-2 system and bacterial resistance. Antimicrob. Agents Chemother. 2019;63(10):10–1128. doi: 10.1128/aac.01186-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yadav J., Das S., Singh S., Jyoti A., Srivastava V.K., Sharma V., Kumar S., Kumar S., Kaushik S. Deciphering the role of S-adenosyl homocysteine nucleosidase in quorum sensing mediated biofilm formation. Curr. Protein Pept. Sci. 2022;23(4):211–225. doi: 10.2174/1389203723666220519152507. [DOI] [PubMed] [Google Scholar]
  48. Zhang J., Tao R., Wang Y., Jiang J., Chen Y. Enzyme-assisted deep eutectic solvent extraction of Litsea cubeba essential oil and its Anti-Aspergillus flavus activity. LWT. 2025;217 doi: 10.1016/j.lwt.2024.117292. [DOI] [Google Scholar]
  49. Zhang L., Li S., Liu X., Wang Z., Jiang M., Wang R., Xie L., Liu Q., Xie X., Shang D., Li M., Wei Z., Wang Y., Fan C., Luo Z., Shen X. Sensing of autoinducer-2 by functionally distinct receptors in prokaryotes. Nat. Commun. 2020;11(1):5371. doi: 10.1038/s41467-020-19243-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhao L., Duan F., Gong M., Tian X., Guo Y., Jia L., et al. (+)-Terpinen-4-ol inhibits Bacillus cereus biofilm formation by upregulating the interspecies quorum sensing signals diketopiperazines and diffusing signaling factors. J. Agric. Food Chem. 2021;69(11):3496–3510. doi: 10.1021/acs.jafc.0c07826. [DOI] [PubMed] [Google Scholar]
  51. Zuo J., Yin H., Hu J., Miao J., Chen Z., Qi K., Wang Z., Gong J., Phouthapane V., Jiang W., Mi R., Huang Y., Wang C., Han X. Lsr operon is associated with AI-2 transfer and pathogenicity in avian pathogenic Escherichia coli. Vet. Res. 2019;50:1–15. doi: 10.1186/s13567-019-0725-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.docx (19.5KB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Current Research in Food Science are provided here courtesy of Elsevier

RESOURCES