Inactivation of Morganella morganii by high hydrostatic pressure combined with lemon essential oil

Abstract The inactivation and damage of histamine‐forming bacterium, Morganella morganii, in phosphate buffer and tuna meat slurry by high hydrostatic pressure (HHP) alone or in combination with 0.2% lemon essential oil (LEO) treatments were studied using viability measurement and scanning electron microscopy (SEM). HHP alone or in combination with LEO treatments showed first‐order destruction kinetics to M. morganii during pressure holding period. The D values of M. morganii (200 to 600 MPa) in phosphate buffer ranged from 16.4 to 0.08 min, whereas those in tuna meat slurry ranged from 51.0 to 0.10 min, respectively. M. morganii in tuna meat slurry had higher D values and were more resistant to HHP treatments than in phosphate buffer. In addition, the D values of HHP in combination with LEO treatment were lower than those of HHP treatment alone at <400 MPa of pressure, indicating that it is more effective to inactivate M. morganii under the same pressure. The results showed the M. morganii at HHP in combination with LEO treatment was more susceptible to pressure treatment alone. HHP with or without LEO treatments can be used to inactivate M. morganii by causing disruption to bacterial cell membrane and cell wall as demonstrated by SEM micrographs.

High hydrostatic pressure (HHP) is a nonthermal technology for food pasteurization and preservation (Wang et al., 2016). In commercial setting, HHP was used at a pressure above 300 MPa to kill spoilage and pathogenic microorganisms for shelf-life extension and safety improvement of jams, fruit juices, guacamole, meats, dairy and egg products and seafood (Considine, Kelly, Fitzgerald, Hill, & Sleator, 2008;Phuasate & Su, 2015). The usage of HHP treatment to preserve the freshness of food was also shown to not affect some of the food quality characteristics such as the color, natural flavor, and nutrients (Phuasate & Su, 2015;Singh & Ramaswamy, 2013). HHP treatment was reported to be capable of killing Listeria monocytogenes, Escherichia coli, and Vibrio parahaemolyticus through morphological damages to both the internal and external structures (Ramaswamy, Zaman, & Smith, 2008;Wang, Huang, Hsu, Shyu, & Yang, 2013). A treatment at a pressure of >300 MPa can cause irreversible denaturation of enzymes and proteins to affect the integrity of the cell membrane, lower protein biosynthesis, and inhibit protein repairs, and ultimately resulting in cell membrane rupture, excretion of internal substances, and bacterial death (Huang, Lung, Yang, & Wang, 2014;Wang et al., 2013).
Therefore, it can act as a natural preservative for improving food safety and shelf life. In addition, lemon juice and lemon fruit are extensively used as flavoring ingredients in a wide variety of foods.
These ingredients are commonly added to fishes consumed raw and after cooking, especially in Asia (Lin et al., 2010). Thus, lemon aroma is well accepted for fish and the addition of LEO could be positively applied also on seafood products.
A hurdle technology is combining two or more physical or chemical preservations to inactivate spoilage and pathogenic microorganisms in foods, to lower level of chemicals (Chien et al., 2017).
Recently, the inactivation effect of HHP treatment on M. morganii was observed using viability counting (Lee et al., 2020). Since only limited information was available on the inactivation effect and morphological damage of M. morganii by HHP alone and in combination with LEO treatments, the aims of this study were to find out the inactivation kinetics of HHP alone and in combination with LEO processing on M. morganii in 0.1 M phosphate buffer (pH 6.8) and tuna meat slurry, and to evaluate whether morphological damages occurred in HHP-treated HFB cells.

| Bacterial culture and lemon essential oil preparation
Stock culture of M. morganii isolated from albacore tuna was kindly provided by Dr. S. H. Kim (Kim et al., 2001). It was maintained in our laboratory on Trypticase Soy Agar (Difco Becton-Dickinson Co) at 4°C. The LEO was prepared from lemon peels according to our previous method (Lin et al., 2010). Briefly, the lemon peels (C. lemon L.) were diced into 1 × 1 cm pieces and stored at −20°C before extraction. The peel pieces were vacuum-freeze dried and then ground into powder. One hundred gram of powder was placed into the supercritical CO 2 extractor, designed by Dr. Shane-Rong Sheu at Far East University, Tainan, Taiwan. The extraction parameters are as follows: 1.5 L capacity, temperature, 323 K; pressure, 10 MPa; flow rate of CO 2 , 3.5 kg/h; time, 90 min. Components of the extracted essential oil were analyzed according to the previous method (Lin et al., 2010) using a gas chromatograph. The major compositions of lemon essential oil were limonene (80.5%), γ-terpinene (6.4%), β-pipene (6.0%), and myrcene (3.5%) (data not shown).

| Preparation of M. morganii in phosphate buffer and tuna meat slurry
One loopful of M. morganii was inoculated into Trypticase Soy Broth (TSB) tube (5 ml) and incubated at 35°C for 12 hr; then, 100 μL aliquot of the bacterial culture was added to 100 ml sterile TSB medium at 35°C for 24 hr. The cultured broth was centrifuged at 8,000 x g for 15 min at 4°C, and the bacterial pellet was washed and re-suspended in 0.1 M phosphate buffer (pH 6.8). The bacterial suspension was then adjusted to a concentration of 10 9 CFU/mL. Fresh tuna flesh was purchased from a local market in Kaohsiung City, Taiwan, and transported in ice to the laboratory immediately.
After washing with a 75% ethanol solution for 1 min and rinsing with sterile water, the flesh was ground to mince in a sterile food homogenizer. The fish mince was then blended with 0.1% peptone water (1:4, w/w) for 2 min in a blender (Omni International, Waterbury, CT, USA). Both the sterile phosphate buffer (0.1 M, pH 6.8, 99 ml) and the tuna meat slurry (99 ml) were inoculated with 1 ml of M. morganii inoculum (10 9 CFU/mL) to get a final bacterial population of 10 7 CFU/mL. In LEO treatments, the phosphate buffer or tuna meat slurry was added and mixed with LEO solution to get at 0.2% LEO concentration before M. morganii inoculation. The test samples were added to sterile vacuum bags in 10 ml portions, vacuum packaged and heat-sealed, and then subject to HHP treatments immediately.

| High hydrostatic pressure treatment
Test bags in triplicate were treated with a laboratory model of high pressure processing system (BaoTou KeFa, High Pressure Technology Co. Ltd) at 200 to 600 MPa for 0 to 15 min at room temperature (25°C). This high pressure processing system having a 6.2-L chamber can be operated at up to 600 MPa at a pressure increase rate of approximately 300 MPa/min and the release times of less than 20 s at all pressures. Water was used as a pressure transmission medium in this study, and the reported pressurization times did not include the time for pressure increase or release. An untreated bag placed in ice water at ambient pressure (0.1 MPa) served as a control. Samples subject to pressure treatment were set in ice water and immediately processed for bacterial counting and SEM analyses.

| Enumeration of M. morganii surviving cells and decimal reduction time
The HHP-treated, HHP in combination with LEO-treated and nontreated bacterial suspensions in phosphate buffer or fish slurry were 10-fold serially diluted in sterile phosphate buffer (0.1 M, pH 6.8). With regard to pour plate counting, aliquots (1.0 ml) of the diluents were mixed in petri dishes with 15 ml TSA (Difco) at 45-50°C. After the agar medium was solidified in a laminar flow hood, the plates were transferred to an incubator and incubated at 30°C for 24-48 hr. Bacterial colonies numbers on the plates were counted. The detection limit of bacterial count was 1.0 log CFU/mL. Data from triplicate samples were presented as mean ± standard deviation.
The linear first-order reaction was used as follows to determine the pressure destruction kinetics of M. morganii during the pressure- The Zp is the increase of pressure needed to change the D value by 90%.

| Scanning electron microscopy (SEM) analysis
Morganella morganii cells in 0.1 M phosphate buffer (pH 6.8) were harvested from pressure-treated (500 MPa for 10 min), LEO pressure-treated (500 MPa for 10 min), and nontreated suspensions via centrifugation at 5,000 rpm for 20 min. After two washes with phosphate buffer, the pellets were re-suspended in 1 ml of phosphate buffer and then filtered through Millipore membranes    to pressure changes; therefore, the destruction rate of HHP treatments alone is more sensitive to changes in pressure than HHP in combination with LEO treatments.  to pressure changes; therefore, the destruction rate of HHP treatments alone is more sensitive to changes in pressure than HHP in combination with LEO treatments.

| Inactivation kinetics of HHP treatment on M. morganii in tuna meat slurry
The combination of HHP and carvacrol was found to inactivate L. monocytogenes (Karatzas, Kets, Smid, & Bennik, 2001 Many intrinsic and environmental parameters, especially the nature of the suspension medium, influence the resistance of microorganisms to pressure treatment. Simpson and Gilmour (1997) reported that bacteria existing in nutrient-rich media had great survival ability to high pressure treatment because the media contained nutrients that are essential for repairing or substances that may provide protection against damage. Microorganisms in food systems were more resistant to HHP treatment than in buffer solution, while such resistance ability to pressure treatment increased as the water activity decreased (Cheftel & Culioli, 1997). Fish matrix was reported to have higher protective effect to spoilage bacteria than the phosphate buffer at pressures below 550 MPa (Panagou et al., 2006). Patterson (2005 also stated that some food constituents such as lipids, proteins, carbohydrates, and salt can have a protective effect for the microbial cells. Therefore, the M. morganii cell in tuna meat slurry are more protected against HHP treatment due to protein and lipid contents.

| CON CLUS IONS
This study, aiming of investigating the inactivation of M. morganii using HHP alone and with LEO treatments, showed that HHP can be applied to inactivate histamine-forming bacterium M. morganii by damaging cell wall and cell membrane. The results showed that M. morganii in tuna meat slurry were more resistant to HHP treatment than in phosphate buffer. With LEO treatment, M. morganii was more susceptible to pressure treatment than HHP treatment alone.

ACK N OWLED G M ENTS
The study was supported by the Ministry of Science and Technology, R.O.C. (Contract No. MOST 106-2221-E-022-017-MY3).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest in the publication of this article.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.