Vibrio vulnificus is a gram-negative food-borne pathogen that is widespread in estuarine and coastal environments (Kim and Chun, 2021) and frequently contaminates oysters, crab, shrimp and other seafood products (Bonnin-Jusserand et al., 2017). Human infection with V. vulnificus is usually caused by exposure to the organism from eating raw or undercooked seafood or from wound contact with seawater (Correa Velez and Norman, 2021). While seafood products are loved by a wide range of consumers, seafood food safety problems brought about by V. vulnificus have also occurred from time to time. Compared to other foods, seafood products have special characteristics: first, it is highly susceptible to spoilage (Almeida et al., 2022), and second, consumers prefer the freshness of the original unprocessed flavor (Hicks, 2017). Therefore, effective microbiological control techniques for seafood safety are a challenge for the food industry today.

In recent years, various natural, environmentally friendly, and safe plant essential oils have received much attention for their good antimicrobial properties (Falleh et al., 2020). Citral is widely available and naturally occurs in the leaves and fruits of a variety of plants, has a lemon aroma and flavor, and is a key component of essential oils such as sorrel essential oil and lemongrass (Zielinska et al., 2018). The European Commission recognizes citral as a flavoring ingredient in food (Regulation EU 872/2012), and the U.S. Food and Drug Administration (FDA) has approved and recognized citral as a safe food ingredient (GRAS 182.60), and it is also allowed to be used as an aromatic essential oil in China as an edible flavor (GB 1886.191-2016 “Food Additives Citral”) (Kang et al., 2021). Previous studies have shown that citral has a good inhibitory effect on the growth of Staphylococcus aureus (Liao et al., 2023) and Escherichia coli (Orizano-Ponce et al., 2022). Also, the inhibitory effect of citral on Vibrio parahaemolyticus, a common species in seafood, was demonstrated (Cao et al., 2021). Citral has become a widely used food additive in the food industry due to its antimicrobial activity, pleasant fruit aroma and food safety for consumers (Sun et al., 2019).

The viable but non-culturable (VBNC) state is a special survival mode of certain bacteria, a special dormant state in which bacteria lose their ability to grow and reproduce on conventional media, but can recover under the right conditions and return to a culturable state (Gao et al., 2021). Existing food microbial decontamination techniques in the food industry have been found to induce bacteria to form a VBNC state, including high-pressure carbon dioxide (Yang et al., 2021), thermosonication (Liao et al., 2018), food preservatives (Yadav et al., 2022), chlorination (Chen et al., 2018), etc. At each stage of the food chain, VBNC status bacteria are easily overlooked by conventional detection methods (Ramamurthy et al., 2014). Furthermore, VBNC status bacteria are more resistant to unfavorable environments and are more likely to survive unfavorable environments in the food industry (Cai et al., 2021). In addition, VBNC state bacteria may be infectious and virulent and have the potential to recover under the right conditions (Dong et al., 2019). Therefore, the invisibility, high tolerance and recoverability of bacteria in VBNC state in food are potential threats to food safety. Citral is an emerging food inhibitor, and there is no study on whether it can induce Vibrio vulnificus in seafood to form a VBNC state. Only by clarifying this key issue can the inhibitory effect of citral on Vibrio be comprehensively evaluated for better application of citral in the food industry.

Based on the above factors, this study firstly evaluated whether citral treatment could induce the formation of VBNC state in V. vulnificus, and explored the physiological characteristics of V. vulnificus induced by citral and its recovery ability. Finally, the differences in gene transcript levels for citral-induced formation and recovery of V. vulnificus were analyzed with the help of transcriptomics in an attempt to reveal possible molecular mechanisms, and further validated by RT-qPCR.