Document Type : Original Research Paper


1 Department of Food Hygiene, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies (AUSMT), Amol, Iran

2 Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Faculty of Engineering Modern Technologies, Amol University of Special Modern Technologies (AUSMT), Amol, Iran


This research aimed to assess the effect of biodegradable coating chitosan nano-gel/emulsion loaded by Bunium persicum essential oil and nisin on E. coli O157:H7 in rainbow trout fillet during 12 days at refrigeration (4˚C). Trout fillet Sample was divided into 6 groups after inoculation of bacteria (E. coli O157:H7), including control (without any coating), coated with chitosan 2% and other groups including nano-emulsion chitosan 2%, Nano-emulsion of chitosan containing Bunium persicum essential oil (0.5%), Nano-gel of chitosan containing nisin (200 IU/g) and Nano-gel/emulsion of chitosan containing Bunium persicum essential oil (0.5%) and nisin (200 IU/g). The samples were stored at the cool condition, and the bacterial count was performed on days: 0, 1, 2, 4, 8, and 12. The mean number of the bacterial count was significantly different among treatment (p<0.001). The most significant inhibitory effect on the growth of E.coli O157:H7 was observed in chitosan Nano-emulsion coating containing Bunium persicum essential oil (0.5%) and nisin (200 IU/g). According to this study, it was concluded that the use of Nano-gel/emulsion of chitosan coating Bunium persicum essential oil and nisin could be effective on the decrease of E.coli O157:H7 growth in food.



Seafood, especially fish, one of the essential meats for human health, has high nutritional value and rich in omega-3 polyunsaturated fatty acids, protein, and minerals [1, 2]. Thus, it is sensitive to contamination by foodborne pathogens such as E. coli O157:H7 that could cause diverse forms of the disease that leads to severe complications, so the need for a precise control way to decrease bacterial growth is necessary [3-5]. This bacterial is liable for many outbreaks involving various types of food products due to cross-contamination particularly by post-processing contamination [6].

The recent studies are looking for; naturally, preservatives mainly medicinal plants have increased because people are more conscious about the side effects of chemical preservatives on health and prolong the shelf life of food products; on another hand, the environment and its health are essential to humanity. In this regard, the use of edible biodegradable coatings and anti-microbial agents like chitosan and essential oil respectively is considerable [7-9]. Essential oils (EOs) that can be extracted from plants contain various compounds, such as flavonoids and phenolic acids, with antimicrobial nature against food-borne pathogens [1, 10-12].

Bunium persicum (BPEO) is a widely used spice in food preservative can be grown in different regions of Asia such as Afghanistan, Iran, Pakistan and India [13]. This essential oil has to potent anti-bacterial and oxidant effects due to the high level of oxygenated mainly ρ-cymene, monoterpenes, limonene and γ-Terpinene [14]. Other natural antimicrobial agents, for example, nisin, that known as generally recognized as safe (GRAS), recently they have been of interest [15-17].

Nisin is produced by Lactococcus lactis spp. lactis and is the only bacteriocin with Food and Drug Administration (FDA) approved GRAS status for use in food products as a preservative in foods [18]. Reportedly nisin is a durable substance to eliminate gram-positive bacteria and, to a lesser extent, in gram-negative [15, 19].

Nano-emulsion is one of the most exciting applications for food products because they can act as delivery systems for lipophilic compounds, such as flavors, drugs, nutraceuticals, antioxidants, and antimicrobial agents[9, 20]. Also, Nano-emulsion of coating solutions containing antimicrobial agents indicates higher anti-bacterial activity compared with course emulsions [21, 22]. Various techniques can provide nano-emulsions, for example, low and high-energy. The high-energy methods in this regard are ultrasonic emulsification which could be effectively applied to prepare Nano-emulsions with small droplet diameters and also low size distributions [23-26]. Nano-emulsions are very stable, and some reports have used an electrical field method to separate them from each other [27, 28].

Several studies were concentrating on the shelf life and safety of food with the application of natural anti-microbial such as edible coating [1, 29-31]. The use of chitosan-based edible coatings reported in previous studies in many kinds of food; although, there have been few studies in the use of nano-chitosan solution with antimicrobial agents in kinds of seafood [32-36]. This research aimed to assess the effect of biodegradable coating nano-gel/emulsion solutions with nisin and Bunium persicum essential oil in rainbow trout fillet.


Experimental Materials

The essential oil was purchased from medicinal plants, Karaj, Iran. Chitosan with Low molecular weight (LMW; 1.03×۱۰5) was also optioned from Sigma-Aldrich Company (St. Louis, MO, USA). Nisin and other Experimental Materials with analytical grade were prepared from the Sigma. The pathogen bacteria (NCTC 12900) was obtained from the department of food hygiene, faculty of veterinary medicine, Amol University of Special Modern Technologies, Amol, Iran.

Preparation of bacteria and chitosan coatings

E. coli O157:H7 was cultured in 9 mL of brain heart infusion (BHI) broth, then incubated at 37°C for 24 and 18 hours at 37°C [37]. To prepare 0.5 McFarland turbidity standard (containing 1.5 × 10CFU/mL) and diluted (1:10) to density of 1.5 × 10CFU/mL [37]. To prepare chitosan solution 2% (w/v), 2g of chitosan in 1% (v/v) acetic acid thus blended with 90ml of distilled water and mixed by magnetic stirrer at 40C. Then glycerol (0.75ml/gr) was added to the chitosan solution as a plasticizer and stirred for 10 min [38].

Preparation of edible biodegradable coating

At this stage, the chitosan (2% w/v) was prepared in sterile distilled water with acetic acid (1%, w/v), and glycerol, then the solutions stirred for 15 min in order to obtain explicit solutions. Following that, nisin (50 mg) was dissolved in hydrochloric acid (0.02 mol/L) to prepare a stock solution of nisin. The BPEO (0.5% V/W) and nisin (200 IU/g) were mixed in the chitosan solutions. At this stage, tween 80 (0.2% w BPEO), as an emulsifier, added to chitosan solutions to become uniform, stable, and transparent emulsion. Then coating solutions were subjected to ultraturrax for 3-5 min at 3000 rpm, then ultrasonic sonicator (60 °C, pulse; 45s and rest; 15s) for six min [39]. Here the transparent Nano-emulsion was prepared (Fig.1) and finally, Particle size was measured by DLS device (Nanophox Sympatec GmbH, Clausthal, Germany) as well.

Preparation of trout fillets and inoculation of the bacterial

Fresh rainbow trout fish were purchased from a local fish farm at Amol (Mazandaran, Iran), and transferred to the laboratory. Then the fillets were washed and slimed and dried. The fillets were cut to pieces with 10g weight then burnt to exterminate the surface microorganisms. E. coli O157:H7 were inoculated (using adjustable volume micropipettes) on each side of separate

Preparation of treatments

The samples that inoculated with E. coli O157:H7 were divided into six groups (Table 1) and then were treated by immersing in Nano-gel/emulsion (Fig. 2) for 1 minute, drained for 15 minutes and stored at 4 ± 1°C for 12 days to be analyzed at 7-day intervals: 0, 1, 2, 4, 6, 8 and 12 [1].

Enumeration of bacteria

Firstly, the fillets (10 g) were brought to a final volume of 90 mL with 0.1% sterile peptone water and then homogenized by a stomacher (Seward Medical, London, UK) for 3 minutes. Then, serial dilutions of homogenates were plated on Sorbitol MacConkey)SMAC) Agar for enumeration of bacteriaafter incubation at (35-37°C for 24 h) [40].

Statistical analysis

The procedure of changes in the logarithmic bacterial count was analyzed through Repeated measure ANOVA in 12 days periods of time, and the double-two comparison of groups was done by Bonferroni post hoc test. The SPSS ver. 21 software was employed for Statistical analysis, and a P-value of less than 0.05 is considered as significant.


Nano-gel/emulsion characterization

As can be seen in Table 2 and Fig. 3, the mean droplet size and PDI also decreased after the preparation of different Nano-gel/emulsion solutions. The mean droplet size decreased in the Nano-chitosan solution in comparison with that of the chitosan solution. The largest particle size was observed in the chitosan solution (3159 nm), and the lowest was observed in Nano-gel/emulsion of chitosan solution with nisin and BPEO (242.4 nm). Other researchers fabricated Nano-emulsion by the incorporation of EOs and antibacterial agents in the biopolymer, such as alginate solutions [41] and chitosan [42] before the sonication process. All of these researchers reported droplet sizes smaller than 1000 nm than that obtained in this research (the lowest was 242.4 nm). As can be seen, the PDI also decreased after Nano-gel/emulsion fabrication. The PDI obtained in this research was less than 0.550 in different groups. Noori et al(2018), observed that the PDI was recorded as 0.584 for GEO emulsion and decreased to 0.222 when conventional Nano-emulsion was prepared. Also, some other researchers reported that the PDI decreased after sonication (in all of them, PDI less than 0.600 reported) [43, 44]. The lower PDI of Nano-emulsion approved the efficiency of the ultra-sonication method information of a uniform size distributed Nano-emulsion [43].


Fig. 4 and Table 3 represent the effect of the treatments on the growth of E. coli O157:H7 during the 12 days of storage. The initial count of E. coli O157:H7 was 6.55 ± 0.11 log CFU/g, which decreased during the storage in all samples especially for Nano-emulsion of chitosan containing 0.5% (w/v) BPEO samples (4.19 ± 0.18) and Nano-emulsion of chitosan containing 0.5% (w/v) BPEO and nisin 200IU/g (3.20 ± 0.12 ) that similar result was obtained in previous studies [37]. The reduction of the bacterial count was considerable in the Nano-gel/emulsion chitosan+ Nisin+ BPEO due to the significant impact of the Nano-chitosan solution and essential oil and Nisin [45]. Also, in the control group during 12 days, the bacterial count was decreased from 6.55 ± 0.11 to 5.19 ± 0.18 because this bacterial is mesophilic [46]. A comparison of the chitosan and Nano-chitosan treatments showed a higher decrease in bacterial count in Nano-chitosan treatment than chitosan treated samples. Therefore, the use of combinational antimicrobial agents is more effective against microbial growth than their individual use [15, 37].

The mean reduction rate of E. coli O157:Hcount in different treatments is shown in Table 4 when treatment was compared to each other. The maximum reduction rate was related to Nano-gel/emulsion chitosan+ Nisin+ BPEO (1.18 log CFU/g) and Nano-chitosan+ BPEO (0.86 log CFU/g), when they were compared to Samples without any coating (CON). Also, in Table 4 showed that the use of combinational antimicrobial agents is more effective against microbial growth than their individual use [15, 37]. Several previous studies (Raeisi et al., 2016; Shahbazi et al., 2015) confirmed the above finding; nevertheless, they may have synergistic, antagonistic, or additive effects according to the type of antimicrobial agent and microorganism.


According to the results from the present research, the use of Nano-gel/emulsion of chitosan solution with nisin and BPEO (Nano ch+nisin + BPEO) has a potential anti-microbial effect against foodborne pathogens like as E. coli O157:H7 in rainbow trout fillet at 4°C. Also, the treatments had an acceptable effect on high doses of this foodborne pathogen and could effectively accelerate its reduction rate in contaminated rainbow trout fillets stored in at 4°C. According to these results, given the preference of producer and consumer for using natural food additives, we recommend the administration of Nano-gel/emulsion of chitosan enriched with BPEO and nisin in rainbow trout fillet to increase its safety against the pathogenic bacteria.


This research work has been supported by a research grant from the Amol University of Special Modern Technologies, Amol, Iran.


There is no conflict of interest in this study.



1. Sharifi F, Khanzadi S, Hashemi M, Azizzadeh M. Control of Listeria Monocytogenes and Escherichia coli O157:H7 Inoculated on Fish Fillets Using Alginate Coating Containing Lactoperoxidase System and Zataria multiflora Boiss Essential Oil. Journal of Aquatic Food Product Technology. 2017;26(9):1014-21.

2. Mofidian R, Barati A, Jahanshahi M, Shahavi MH. Optimization on thermal treatment synthesis of lactoferrin nanoparticles via Taguchi design method. SN Applied Sciences. 2019;1(11).

3. Hyldgaard M, Mygind T, Meyer RL. Essential Oils in Food Preservation: Mode of Action, Synergies, and Interactions with Food Matrix Components. Frontiers in Microbiology. 2012;3.

4. Ghorbanpour, M., M. Moghimi, and S. Lotfiman, Silica-supported copper oxide nanoleaf with antimicrobial activity against Escherichia coli. Journal of Water and Environmental Nanotechnology, 2017. 2(2): p. 112-117.

5. Sheikhaghaiy, T. and B. Golestani Eimani, Investigation of synergistic effect of cuo nanoparticles and nisin on genomic of Escherichia coli bacteria. Journal of Water and Environmental Nanotechnology, 2018. 3(4): p. 355-367.

6. Djenane D, Yangüela J, Amrouche T, Boubrit S, Boussad N, Roncalés P. Chemical composition and antimicrobial effects of essential oils of Eucalyptus globulus, Myrtus communis and Satureja hortensis against Escherichia coli O157:H7 and Staphylococcus aureus in minced beef. Food Science and Technology International. 2011;17(6):505-15.

7. Raeisi M, Tabaraei A, Hashemi M, Behnampour N. Effect of sodium alginate coating incorporated with nisin, Cinnamomum zeylanicum , and rosemary essential oils on microbial quality of chicken meat and fate of Listeria monocytogenes during refrigeration. International Journal of Food Microbiology. 2016;238:139-45.

8. Ebrahimpour, M., et al., Nanotechnology in Process Biotechnology: Recovery and Purification of Nanoparticulate Bioproducts Using Expanded Bed Adsorption. Dynamic Biochemistry, Process Biotechnology and Molecular Biology, 2009. 3(2): p. 57-60.

9. Jahanshahi M, Najafpour G, Ebrahimpour M, Hajizadeh S, Shahavi MH. Evaluation of hydrodynamic parameters of fluidized bed adsorption on purification of nano-bioproducts. physica status solidi (c). 2009;6(10):2199-206.

10. Shahavi, M.H., et al., Preparation of nanoemulsion clove oil in water as a green nano pesticide, in 9th International conference on natural sciences and technologies. 2014: Kalmar, Sweden.

11. Shahavi, M.H., et al., Clove Oil Nanoemulsion as an Eco-Friendly Pesticide: Effect of Sonication Time on Droplet Size, in Asia Nano Forum Conference 2015 (ANFC 2015). 2015: Kish Island, Iran.

12. Rad AS, Samipour V, Movaghgharnezhad S, Mirabi A, Shahavi MH, Moghadas BK. X12N12 (X = Al, B) clusters for protection of vitamin C; molecular modeling investigation. Surfaces and Interfaces. 2019;15:30-7.

13. Aminzare M, Amiri E, Abbasi Z, Hassanzad Azar H, Hashemi M. Evaluation of In Vitro Antioxidant Characteristics of Corn Starch Bioactive Films Incorporated With Bunium Persicum and Zataria Multiflora Essential Oils. SSRN Electronic Journal. 2017.

14. Agah, S., et al., Cumin extract for symptom control in patients with irritable bowel syndrome: a case series. Middle East journal of digestive diseases, 2013. 5(4): p. 217.

15. Shahbazi Y, Shavisi N, Mohebi E. Effects ofZiziphora clinopodioidesEssential Oil and Nisin, Both Separately and in Combination, to Extend Shelf Life and ControlEscherichia coli O157:H7 andStaphylococcus aureusin Raw Beef Patty during Refrigerated Storage. Journal of Food Safety. 2015;36(2):227-36.

16. Yoon JI, Bajpai VK, Kang SC. Synergistic effect of nisin and cone essential oil of Metasequoia glyptostroboides Miki ex Hu against Listeria monocytogenes in milk samples. Food and Chemical Toxicology. 2011;49(1):109-14.

17. Taheri ES, Jahanshahi M, Hamed Mosavian MT, Shahavi MH. Investigation of hydrodynamic parameters in a novel expanded bed configuration: local axial dispersion characterization and an empirical correlation study. Brazilian Journal of Chemical Engineering. 2012;29(4):725-39.

18. Economou T, Pournis N, Ntzimani A, Savvaidis IN. Nisin–EDTA treatments and modified atmosphere packaging to increase fresh chicken meat shelf-life. Food Chemistry. 2009;114(4):1470-6.

19. Gao M, Feng L, Jiang T, Zhu J, Fu L, Yuan D, et al. The use of rosemary extract in combination with nisin to extend the shelf life of pompano (Trachinotus ovatus) fillet during chilled storage. Food Control. 2014;37:1-8.

20. Weiss J, Decker EA, McClements DJ, Kristbergsson K, Helgason T, Awad T. Solid Lipid Nanoparticles as Delivery Systems for Bioactive Food Components. Food Biophysics. 2008;3(2):146-54.

21. Oh YA, Oh YJ, Song AY, Won JS, Song KB, Min SC. Comparison of effectiveness of edible coatings using emulsions containing lemongrass oil of different size droplets on grape berry safety and preservation. LWT. 2017;75:742-50.

22. Ebrahimpour, M., et al., Nanotechnology in Process Biotechnology: Recovery and Purification of Nanoparticulate Bioproducts Using Expanded Bed Adsorption. Dynamic Biochemistry, Process Biotechnology and Molecular Biology, 2009. 3: p. 57-60.

23. Salvia-Trujillo L, Soliva-Fortuny R, Rojas-Graü MA, McClements DJ, Martín-Belloso O. Edible Nanoemulsions as Carriers of Active Ingredients: A Review. Annual Review of Food Science and Technology. 2017;8(1):439-66.

24. Hamed Mashhadzadeh A, Fathalian M, Ghorbanzadeh Ahangari M, Shahavi MH. DFT study of Ni, Cu, Cd and Ag heavy metal atom adsorption onto the surface of the zinc-oxide nanotube and zinc-oxide graphene-like structure. Materials Chemistry and Physics. 2018;220:366-73.

25. Shahavi MH, Hosseini M, Jahanshahi M, Meyer RL, Darzi GN. Clove oil nanoemulsion as an effective antibacterial agent: Taguchi optimization method. Desalination and Water Treatment. 2015;57(39):18379-90.

26. Shahavi MH, Hosseini M, Jahanshahi M, Meyer RL, Darzi GN. Evaluation of critical parameters for preparation of stable clove oil nanoemulsion. Arabian Journal of Chemistry. 2019;12(8):3225-30.

27. Hosseini M, Shahavi MH. Electrostatic Enhancement of Coalescence of Oil Droplets (in Nanometer Scale) in Water Emulsion. Chinese Journal of Chemical Engineering. 2012;20(4):654-8.

28. Hosseini, M., M.H. Shahavi, and A. Yakhkeshi, AC & DC-currents for separation of nano-particles by external electric field. Asian Journal of Chemistry, 2012. 24(1): p. 181-184.

29. Pérez Quiñones J, Brüggemann O, Kjems J, Shahavi MH, Peniche Covas C. Novel Brassinosteroid-Modified Polyethylene Glycol Micelles for Controlled Release of Agrochemicals. Journal of Agricultural and Food Chemistry. 2018;66(7):1612-9.

30. Shahavi, M.H., et al., Optimization of encapsulated clove oil particle size with biodegradable shell using design expert methodology. Pakistan J Biotechnol, 2015. 12: p. 149-160.

31. Niksefat Abatari M, Sarmasti Emami MR, Jahanshahi M, Shahavi MH. Superporous pellicular κ-Carrageenan–Nickel composite beads; morphological, physical and hydrodynamics evaluation for expanded bed adsorption application. Chemical Engineering Research and Design. 2017;125:291-305.

32. Yuan G, Chen X, Li D. Chitosan films and coatings containing essential oils: The antioxidant and antimicrobial activity, and application in food systems. Food Research International. 2016;89:117-28.

33. Vieira JM, Flores-López ML, de Rodríguez DJ, Sousa MC, Vicente AA, Martins JT. Effect of chitosan– Aloe vera coating on postharvest quality of blueberry ( Vaccinium corymbosum ) fruit. Postharvest Biology and Technology. 2016;116:88-97.

34. Soleymani Lashkenrai A, Najafi M, Peyravi M, Jahanshahi M, Mosavian MTH, Amiri A, et al. Direct filtration procedure to attain antibacterial TFC membrane: A facile developing route of membrane surface properties and fouling resistance. Chemical Engineering Research and Design. 2019;149:158-68.

35. Shahavi, M.H., et al., Expanded bed adsorption of biomolecules by NBG contactor: Experimental and mathematical investigation. World Applied Sciences Journal, 2011. 13(2): p. 181-187.

36. Yadav, B., et al., Electrical Behaviour of Chitosan-Silver Nanocomposite in Presence of Water Vapour. Journal of Water and Environmental Nanotechnology, 2017. 2(2): p. 71-79.

37. Ehsani A, Hashemi M, Naghibi SS, Mohammadi S, Khalili Sadaghiani S. Properties ofBunium PersicumEssential Oil and its Application in Iranian White Cheese AgainstListeria MonocytogenesandEscherichia Coli O157:H7. Journal of Food Safety. 2016;36(4):563-70.

38. Ojagh SM, Rezaei M, Razavi SH, Hosseini SMH. Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chemistry. 2010;120(1):193-8.

39. Ghosh V, Mukherjee A, Chandrasekaran N. Ultrasonic emulsification of food-grade nanoemulsion formulation and evaluation of its bactericidal activity. Ultrasonics Sonochemistry. 2013;20(1):338-44.

40. Shin JH, Chang S, Kang DH. Application of antimicrobial ice for reduction of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes) on the surface of fish. Journal of Applied Microbiology. 2004;97(5):916-22.

41. Alparslan Y, Baygar T. Effect of Chitosan Film Coating Combined with Orange Peel Essential Oil on the Shelf Life of Deepwater Pink Shrimp. Food and Bioprocess Technology. 2017;10(5):842-53.

42. Wu C, Wang L, Hu Y, Chen S, Liu D, Ye X. Edible coating from citrus essential oil-loaded nanoemulsions: physicochemical characterization and preservation performance. RSC Advances. 2016;6(25):20892-900.

43. Noori S, Zeynali F, Almasi H. Antimicrobial and antioxidant efficiency of nanoemulsion-based edible coating containing ginger (Zingiber officinale) essential oil and its effect on safety and quality attributes of chicken breast fillets. Food Control. 2018;84:312-20.

44. Hosseinnia M, Khaledabad MA, Almasi H. Optimization of Ziziphora clinopodiodes essential oil microencapsulation by whey protein isolate and pectin: A comparative study. International Journal of Biological Macromolecules. 2017;101:958-66.

45. Khanjari, A., et al., Evaluation of the antimicrobial effect of chitosan and whey proteins isolate films containing free and nanoliposomal garlic essential oils against Listeria monocytegenes, E. coli O157: H7 and Staphylococcus aureus. Iranian Journal of Medical Microbiology, 2016. 10(5): p. 45-51.

46. Elliot RM, McLay JC, Kennedy MJ, Simmonds RS. Inhibition of foodborne bacteria by the lactoperoxidase system in a beef cube system. International Journal of Food Microbiology. 2004;91(1):73-81.