Food safety is a worldwide concern. Microbial contamination ranks first among various factors that affect food safety^[1]. It has been reported that in 2005 alone that 1.8 million people died from diarrheal diseases,a great proportion of which can be attributed to contamination of food and drinking water^[2]. The prevention and control of pathogens pollution for reducing the incidence of food-borne diseases and ensuring the safety and quality of food have received wide attention.

Citrus oil is generally recognized as safe (GRAS) and has been found as inhibitory in both direct oil and vapour form against a range of both gram-positive and gram-negative bacteria^[3-4]. Although the studies have made some achievements,but there are some aspects remaining to be solved or improved,for example,it is still not clear that which component or component combination presents antibacterial activity because essential oil is a mixture of many compounds,and research on the quantitative data (minimal inhibitory concentration MIC and minimal bactericide concentration MBC) are far from enough^[5-7]. Another problem in the application of essential oil is its hydrophobic property,which makes appropriate solvent selection important for the antibacterial activity.

In this paper,the antibacterial effects of limonene,major component of citrus oil,under different solvents on food-borne pathogens were studied. The results would accumulate certain qualitative and quantitative data for limonene antibacterial property,as well as provide new methods for the management of food-borne pathogens.

1 Materials and methods 1.1 Limonene and emulsification

Limonene (purity of 95%) was procured from Damas-beta Company egg yolk lecithin was obtained from Institute for the Control of Agrochemicals,Ministry of Agriculture,China. Limonene was emulsified to improve its solubility. Emulsification method was according to the previously reported methods^[8] with slight changes. Optimized selection of limonene emulsion was conducted by changing the content of emulsifier and ultrasonic processing time. Egg yolk lecithin was used as the emulsifier. Initially,the organic phase (limonene + egg yolk lecithin) was stirred magnetically (Shanghai Weicheng Instrument Co.,Ltd,Shanghai,China) for 50 min. Then,the aqueous phase (distilled water) was added to the organic phase,at a ratio of 4 g aqueous phase with 1 g organic phase. Then they were agitated for 20 min on a magnetic stirrer. After that,the solution underwent sonification using an ultrasound with 70% amplitude (Kunshan Ultrasonic Instrument Co.,Ltd.,Suzhou,China). Two groups with three treatments each were set up as follows:

Group Ⅰ: changing the content of emulsifier. The mass ratio of egg yolk lecithin and limonene was 25∶1,25∶2,25∶3,respectively;

Group Ⅱ: changing the ultrasonic processing time for 8,12,16 min,respectively.

Emulsification conditions were recorded at the moment and 24 h later. The optimized limonene emulsion was obtained for the following antibacterial test,with emulsion with only egg yolk lecithin under the same method as the control emulsion. The emulsions were stored at 4 ℃ before use.

1.2 Food-borne pathogens and growth conditions

The strains of Yersinia enterocolitica (GIM1.265),Staphylococcus aureus (GIM1.221) and Listeria monocytogenes (GIM1.347) were purchased from Microbial Culture Collection Center of Guangdong Institute of Microbiology (Guangdong,China).

During this investigation,the culture was maintained in cryovials at -80 ℃. Broth subcultures were prepared by inoculating,with one single colony from a plate,a test tube containing 5 mL of sterile broth subcultures (Y. enterocolitica and S. aureus using nutrient broth,NB; L. monocytogenes using tryptic soy broth with 0.6% yeast extract added,TSBYE) (Haibo Biological Technology Co.,Ltd.,Qingdao,China). After inoculation,the tubes were incubated overnight at 28 ℃ (Y. enterocolitica) or 37 ℃ (S. aureus and L. monocytogenes). With these subcultures,250 mL Erlenmeyer flasks containing 50 mL of NB or TSBYE were inoculated to a final concentration of 10⁶-10⁷ cells/mL. These flasks were incubated under agitation (250 r/min,Tianxiangfeiyu Instrument Equipment Co.,Ltd.,Beijing,China) at the appropriate temperature (see above) until the stationary growth phase was reached.

1.3 Antimicrobial assay (disk diffusion assay)

The antimicrobial activity against three species of food-borne pathogens of limonene solution [limonene acetone solution (50,500,5 000 μL/L) and limonene emulsion (the optimize limonene emulsion obtained in 1.1)] was tested using the agar diffusion method. Filter paper disks (What-man No.1,6 mm diameter) containing 15 μL limonene solution (see above) were applied to the surface of agar plates (Y. enterocolitica and S. aureus using nutrient agar,NA; L. monocytogenes using tryptic soy agar with 0.6% yeast extract added,TSAYE) (Haibo Biological Technology Co.,Ltd.,Qingdao,China) that were previously seeded by spreading one sterile hyssop impregnated with a stationary phase culture. The plates were incubated overnight at the appropriate temperature (see above),and the diameter of inhibition zone was measured. Filter paper disks under the same method were prepared as negative controls (without limonene) and positive controls (ofloxacin,commercially available eye drops).

1.4 Determination of minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC)

MIC and MBC of limonene against the same food-borne pathogen were determined using the tube dilution method. The highest and the lowest concentrations of limonene acetone solution tested were 50 μL/L and 0.4 μL/L. The optimized limonene emulsion was half times diluted into 8 concentrations. Solutions under the same method were prepared as negative controls (without food-borne pathogens) and positive controls (without limonene). After a 24 h incubation at the appropriate temperature in a shaking thermostatic bath (Tianxiangfeiyu Instrument Equipment Co.,Ltd.,Beijing,China),the MIC was checked. The MIC was the lowest concentration of limonene at which bacteria failed to grow,so no visible changes were detected in the broth medium. In order to evaluate MBC,after 24 h incubation at the appropriate temperature (see above) in the shaking thermostatic bath,100 μL of each tube in which microbial growth was not observed was spread plated in NA or TSAYE. Plates were incubated at the appropriate temperature (see above) for 24 h. The MBC was the lowest concentration at which bacteria failed to grow in broth subcultures and the subsequent agar subcultures.

1.5 Data analysis

All experiments were carried out in triplicate. Data of limonene emulsification and determination of MIC and MBC experiments were directly obtained through measurement or observation. Data of antimicrobial assay were average diameters.

2 Results 2.1 Limonene emulsion under different methods

Optimized selection of limonene emulsion was conducted by changing the content of emulsifier and ultrasonic processing time (Table 1). Under the same ultrasonic processing time,the instant emulsification effect was good when the mass ratio of limonene and egg yolk lecithin (emulsifier) was 25∶1,while limonene floated on the top after 24 h. Emulsification stability improved when the mass ratio of limonene and emulsifier was 25∶2 and 25∶3. When the mass ratio of limonene and emulsifier was fixed at 25∶1,the extension of ultrasonic processing time could promote the emulsification of limonene. Good limonene emulsion was got when the ultrasonic processing time was 8 or 10 min. Therefore,best limonene emulsion was got when the mass ratio of limonene and egg yolk lecithin (emulsifier) was 25∶1,and the ultrasonic processing time was 8 min,which obtained good emulsification effect with the least amount of emulsifier and time.

2.2 Antibacterial effect of limonene 2.2.1 Antibacterial effect of limonene on Y. enterocolitica

The antibacterial effect of limonene acetone solution and limonene emulsion on Y. enterocolitica was presented in Table 2. The results of the agar diffusion test showed that all treatments except the control emulsion showed good activity. The best efficiency one was 500 μL/L limonene acetone solution,with the inhibition zone diameter of 32.2 mm,followed by limonene acetone solution at the concentration of 5 000 μL/L,with the inhibition zone diameter of 27.4 mm,and the efficiency of limonene acetone solution at the concentration of 50 μL/L was similar to that of limonene emulsion. The tube dilution test showed that limonene was effective on Y. enterocolitica. Limonene acetone solution showed higher antimicrobial activity with lower values of MIC and MBC than limonene emulsion. It may be caused by the organic solvent,which had antibacterial activity itself and could improve the efficiency of limonene by simultaneously increasing the dissolution and diffusion.

2.2.2 Antibacterial effect of limonene on S. aureus

Results shown in Table 3 indicated that the most effective treatment to S. aureus was ofloxacin (positive control),followed by limonene acetone solution when the concentration was 50 μL/L,with the inhibition zone diameter of 30.3 mm,and the efficiency of other treatments except the emulsion control was similar. The tube dilution test showed that both limonene acetone solution and limonene emulsion were effective on S. aureus,and greater antimicrobial activity was obtained in the former one than in the latter one.

2.2.3 Antibacterial effect of limonene on L. monocytogenes

The diameters inhibition zone of limonene acetone solution and acetone (solvent control) against L. monocytogenes was similar,while there was no inhibition zone of limonene emulsion,which indicated that it was acetone but not limonene had the activity against L. monocytogenes. The tube dilution test confirmed the same conclusion (Table 4).

3 Discussion

The major problem in the application of essential oil’s antibacterial properties is their hydrophobic nature which makes them insoluble in water-based media. This may be overcome with the use of emulsifiers such as Tween 20 or 80 alone or in combination with acetone,polyethylene glycol or ethanol^[9]. This paper studied the solubility of limonene under different emulsifying methods. Best limonene emulsion was got when the ratio of limonene and egg yolk lecithin (emulsifier) was 25∶1,and the ultrasonic processing time was 8 min,which obtained good emulsification effect with the least amount of emulsifier and time. Green emulsified solution,instead of harmful organic solvents,will be conducive to better development and utilization of limonene,and provide a feasible way for the application of plant essential oil in food.

With the consumer trends for natural alternatives to chemical-based bactericides,citrus oils may provide a solution for both food industry and consumers,as their components are GRAS^{[3, 10]}. Citrus oils make up the largest sector of the world production of essential oils. The major chemical component of citrus oils is limonene,ranging from 32% to 98%^[10],which provide rich resources for a broad prospect of development and utilization of limonene. Since first noted in 1949,the antimicrobial properties of citrus oils and their major components were proved in many plant pathogenic bacteria and food-borne pathogenic bacteria,such as Penicillium digitatum,Aspergillus flavus,Aspergillus parasiticus,Bacillus subtilis,Salmonella enterica subsp enterica,Escherichia coli,Bacillus cereus,Pseudomonas aeruginosa,L. monocytogenes,and S. aureus^{[5, 11-17]}. In this paper,research showed that limonene was effective against Y. enterocolitica and S. aureus,but not against L. monocytogenes. The activity of limonene against gram-negative bacteria was greater than that against gram-positive bacteria,which was not agree with previous researches. Some studies have found gram-positive bacteria to be more sensitive to essential oils than gram-negative bacteria,which may be due to the relatively impermeable outer membrane that surrounds gram-negative bacteria^{[9, 18-19]}. This may be related to the different targets of action. Previous studies regarded that considering the large number of chemical compounds present in essential oils,it was most likely that their antibacterial activity is not attributable to one specific mechanism but that there are several targets in the cell^[20-21]. The mechanism needs further researches.

The antimicrobial properties of essential oils lend themselves to be used in food as potential food preservatives. Considering their potential,studies involving citrus oils have not been well documented. For essential oils have an antimicrobial application in food,they must not only be safe for consumption but also reduce the initial microbial load during production to extend the foodstuffs shelf-life^[11]. High doses of essential oils have been shown to have a cytotoxic and increase the damage to the cell population,though low doses cause no damage over a short period^[22],which emphasizes the necessity to find a balance between the risk of food poisoning and the dosage of essential oils required to have an effect. Therefore,it is very necessary to maximize the role of plant essential oil with minimum concentration^[4]. There was no linear or logarithmic relationship between the radius of inhibition and the concentration of limonene acetone solution applied in the agar diffusion method,which was consistent with previous research conclusions^[23]. It is important to study quantitative data such as MIC,MBC for the application of the essential oil in food. Previous studies have reported that MICs of 12 800 mg/L for lemon and orange oils and 6 400 mg/L for lime oil were required against several food-borne pathogens^[14-15]. However,these high MICs would be unacceptable for the application in food products due to the possible changes in organoleptic properties especially aroma. In our study,MBC of limonene emulsion against Y. enterocolitica and S. aureus was 6 009.6 and 12 019.3 μL/L,respectively. Whether these values are too high for limonene to be applied to food products requires further evaluation. In order to solve this problem,some scholars put forward some methods combined with other physical methods,such as heat treatment,high pressure slurry^{[8, 24-25]},to improve the antibacterial activity for reducing the essential oil content in food to an acceptable extent.

4 Conclusions

Best limonene emulsion was got with the least amount of emulsifier and time,which will be helpful for better development and utilization of limonene. Limonene acetone solution showed higher antibacterial activity against three kinds of food-borne pathogens than limonene emulsion. Among the three selected food-borne pathogens,Y. enterocolitica was the most sensitive one to limonene,followed by S. aureus and L. monocytogenes. It is feasible to use limonene emulsion in food industry as food preservative for its antibacterial property and safety.