تولید نانولیپوزوم‌های حامل نایسین با پوشش کیتوزان و ارزیابی خواص فیزیکی و ضدباکتریایی محصول علیه Bacillus cereus و Staphylococcus aureus

نوع مقاله : مقاله کوتاه

نویسندگان

• سهیل ریحانی پول ¹
• سکینه یگانه ²
• رضا صفری ³

¹ گروه فراوری محصولات شیلاتی، دانشکده شیلات و محیط زیست، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران.

² گروه شیلات، دانشکده علوم دامی و شیلات، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران.

³ پژوهشکده اکولوژی دریای خزر، ساری، ایران.

https://doi.org/10.22067/ifstrj.2021.72715.1097

چکیده

از آنجا که فعالیت ضدباکتریایی نایسین در واکنش با پروتئین‌ها، قندها و لیپیدها کاهش می‌یابد، نانوریزپوشانی این نگهدارنده جهت حفاظت از خواص ذاتی آن راه حل مناسبی به‌نظر می‌رسد. هدف تحقیق حاضر تولید نانولیپوزوم‌های حامل نایسین با (و بدون) پوشش کیتوزان و ارزیابی خواص فیزیکی و ضدباکتریایی محصول بود. به همین منظور چهار تیمار شامل NN (نانولیپوزوم‌های حامل نایسین)، NN-CH (0.05)، NN-CH (0.1) و NN-CH (0.5) (نانولیپوزوم‌های حامل نایسین به‌ترتیب با پوشش‌های 05/0، 1/0 و 5/0 درصد کیتوزان) طراحی و مورد آزمون‌های مختلف قرار گرفتند. نتایج نشان داد میانگین سایز ذرات در تیمارهای مختلف از حدود 110 تا 327 نانومتر متغیر است؛ ضمن اینکه با افزایش میزان کیتوزان در پوشش، ذرات بزرگتر شدند (05/0>P). شاخص توزیع اندازه ذره‌ای در تیمارها کمتر از 3/0 ثبت شد و ارتباطی با میزان کیتوزان در پوشش نداشت. با افزایش میزان کیتوزان اطراف نانولیپوزوم‌ها، پتانسیل زتا به صورت معنی‌داری افزایش (05/0>P) و از 34/55- در تیمار NN به 14/53 میلی‌ولت در تیمار NN-CH (0.5) ارتقا یافت. همچنین راندمان ریزپوشانی نیز به صورت معنی‌داری بیشتر شد و از 19/32 در تیمار NN به 14/75 درصد در تیمار NN-CH (0.5) تغییر کرد (05/0>P). مطابق نتایج فعالیت ضدباکتریایی نایسین در دو روش انتشار در محیط کشت آگار و رقیق­سازی در لوله، با نانوریزپوشانی نایسین با (و بدون) پوشش کیتوزان، فعالیت ضدباکتریایی آن بیشتر شد (05/0>P). همچنین با افزایش غلظت کیتوزان، فعالیت ضدباکتریایی نانولیپوزوم‌های حامل افزایش یافت و بیشترین حد آن در تیمار NN-CH (0.5) ثبت گردید (05/0>P). قطر هاله عدم رشد باسیلوس سرئوس در برابر تیمارهای تحقیق (با پنج غلظت 5/2 تا 25 میکروگرم بر میلی‌لیتر) از حدود 5/4 تا 5/17 میلی‌متر متغیر بود. این میزان برای استافیلوکوکوس اورئوس حدود 1/2 تا 5/26 میلی‌متر ثبت شد. حداقل غلظت مهارکنندگی (MIC) و حداقل غلظت کشندگی (MBC) تیمارهای تحقیق برای باسیلوس سرئوس به‌ترتیب در بازه 100 تا 400  و 200 تا 500 میکروگرم بر میلی‌لیتر قرار داشت. این دو غلظت برای استافیلوکوکوس اورئوس به‌ترتیب 50 تا 200 و 100 تا 400 میکروگرم بر میلی‌لیتر ثبت گردید. مطابق یافته‌ها، نانوریزپوشانی نایسین در قالب نانولیپوزوم‌های حامل با پوشش کیتوزان موجب ارتقای خواص فیزیکی و ضدباکتریایی آن می‌شود.

کلیدواژه‌ها

• نایسین
• نانولیپوزوم
• کیتوزان
• خواص فیزیکی
• فعالیت ضدباکتریایی

عنوان مقاله [English]

Production of nanoliposomes carrying nisin with chitosan coating and evaluation of physical and antibacterial properties of the product against Bacillus cereus and Staphylococcus aureus

نویسندگان [English]

• Soheyl Reyhani Poul ¹
• Sakineh Yeganeh ²
• Reza Safari ³

¹ Department of Processing of Fishery Products, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.

² Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agricultural Science and Natural Resources University, Sari, Iran.

³ Caspian Sea Ecology Research Institute, Sari, Iran.

چکیده [English]

[1]Introduction: Nisin is one of the antimicrobial substances that is used today as a preservative in various foodstuffs. It is a bacteriocin comprised of 34 amino acids and a molecular weight of 3.5 Da. With all the benefits of nisin, there are barriers to its use in dairy and protein rich products. One of these barriers is the combination of nisin with fats, proteins and sugars and the consequent reduction of its antibacterial activity. In the food science and industry, the use of the technique of encapsulation and production of liposome is the best possible solution in such cases. Also, by adding an antimicrobial agent such as chitosan to the coating of nanoliposomes, the antibacterial activity of the product may be increased. The aim of the present research was to produce nanoliposomes carrying nisin with (and without) chitosan coating and to evaluate the physical and antibacterial properties against two gram-positive bacteria, Bacillus cereus and Staphylococcus aureus.
 
Materials and Methods: In this study, four treatments of nanoliposomes carrying nisin (NN), nanoliposomes carrying nisin coated with chitosan 0.05% ((NN-CH (0.05)), nanoliposomes carrying nisin coated with chitosan 0.1% (NN-CH (0.1)) and nanoliposomes carrying nisin coated with chitosan 0.5% (NN-CH (0.5)) were prepared and examined in terms of physical properties (average particle size, particle dispersity index, zeta potential and encapsulation efficiency) and antibacterial activity (against two gram-positive bacteria, Bacillus cereus and Staphylococcus aureus with two diffusion methods in agar medium and microdilution test). This research was conducted in a completely randomized design and SPSS and EXCEL softwares were used for statistical analysis and drawing of diagram, respectively. Data were analyzed by one-way analysis of variance and the difference between the means was evaluated by Duncan's test at 95% confidence level.
 
Results and Discussion: The results showed that the average particle sizein different treatments with each other are significantly different (P<0.05) and vary from about 110 to 327nm; Also as the amount of chitosan in the coating increased, the particle size increased (P<0.05). This indicates the successful binding of chitosan to the surface of the nanoliposome, which results in the formation of a layer around the nanoliposome and an increase in particle size. Particle dispersity index was recorded less than 0.3 in all treatments and was not related to the amount of chitosan in the coating. With increasing the amount of chitosan in the coating of nanoliposomes, zeta potential increased significantly (P<0.05). This index changed from -55.34 in NN treatment to 53.14 mV in NN-CH (0.5) treatment. In fact, chitosan as a cationic polysaccharide changes the potential to positive values. As the amount of chitosan in coating of nanoliposomes increased, the encapsulation efficiency increased significantly in the treatments (P<0.05); this index increased from 32.19% in NN treatment to 75.14% in NN-CH (0.5) treatment. The results of the antibacterial activity of nisin in two methods of diffusion in agar medium and microdilution test showed that its antibacterial activity increased with nanoencapsulation of nisin with (and without) chitosan coating (p<0.05). Also, with the increase in chitosan concentration, the antibacterial activity of carrier nanoliposomes increased and the highest antibacterial activity was recorded in NN-CH (0.5) treatment (p<0.05). The diameter of the non-growth halo of Bacillus cereus against the research treatments (with five concentrations of 2.5 to 25 μg/ml) varied from about 4.5 to 17.5 mm. This amount for Staphylococcus aureus was recorded from 2.1 to 26.5 mm. By increasing the concentration of nisin and carrier nanoliposomes, the diameter of the halo of non-growth of both bacteria increased significantly (p<0.05). But an exception was recorded in this case; The diameter of the non-growth halo for Staphylococcus aureus in two concentrations of 2.5 and 5 μg/ml of treatments was the same and had no significant difference (p>0.05). The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the examined treatments for Bacillus cereus were in the range of 100 to 400 and 200 to 500 μg/ml, respectively. These two concentrations for Staphylococcus aureus were recorded as 50 to 200 and 100 to 400 μg/ml respectively. Based on the values of diameter of non-growth halo, MIC and MBC it can be claimed that Bacillus cereus is more resistant to the examined treatments than Staphylococcus aureus.
Nanoencapsulation of nisin in the form of carrier nanoliposomes with chitosan coating is a suitable solution to improve its physical and antibacterial properties. In such a way that by increasing the concentration of chitosan in the coating, both of the aforementioned properties improved significantly. Nanoliposomes carrying nisin with (and without) chitosan coating have the ability to inhibit the growth and killing Bacillus cereus and Staphylococcus aureus bacteria. The antibacterial activity increases with the increase in nisin and carrier nanoliposomes concentrations. The value of non-growth halo, minimum inhibitory concentration and minimum bactericidal concentration confirm that Bacillus cereus is more resistant to nisin and its carrier nanoliposomes than Staphylococcus aureus.
 

کلیدواژه‌ها [English]

• Nisin
• Nanoliposome
• Chitosan
• Physical properties
• Antibacterial activity

مراجع

1. Alboghbeish, H., and Khodanazary, A. (2017). Comparative effects of chitosan and nanochitosan coatings enriched with green tea (Camellia sinensis) extract on quality of Costal trevally fish (Carangoides coeruleopinnatus) during refrigerated storage. Iranian Scientific Fisheries Journal, 26 (5), 95-109. [In Persian].
2. Bang, S. H., Hwang, I. C., Yu, Y. M., Kwon, H. R., Kim, D. H., and Park, H. J. (2011). Influence of chitosan coating on the liposomal surface on physicochemical properties and the release profile of nanocarrier systems. Journal of Microencapsulation, 28(7), 595-604.
3. Begde, D., Bundale, S., Mashitha, P., Rudra, J., Nashikkar, N., and Upadhyay, A. (2011). Immunomodulatory efficacy of nisin, a bacterial lantibiotic peptide. Journal of Peptide Science, 17(6), 438-444. https://doi.org/10.1002/psc.1341
4. Benech, R. O., Kheadr, E. E., Laridi, R., Lacroix, C., and Fliss, I. (2002). Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture. Applied and Environmental Microbiology, 68(8), 3683-3690.
5. Cheikhyoussef, A., Pogori, N., Chen, W., and Zhang, H. (2008). Antimicrobial proteinaceous compounds obtained from bifidobacteria: From production to their application. International Journal of Food Microbiology, 125(3), 215-222. https://doi.org/10.1016/j.ijfoodmicro.2008.03.012
6. Chollet, E., Sebti, I., Martial-Gros, A., and Degraeve, P. (2008). Nisin preliminary study as a potential preservative for sliced ripened cheese: NaCl, fat and enzymes influence on nisin concentration and its antimicrobial activity. Food Control, 19(10), 982-989. https://doi.org/10.1016/j.foodcont.2007.10.005
7. Da Rosa Zavareze, E., Telles, A. C., El Halal, S. L. M., da Rocha, M., Colussi, R., de Assis, L. M., ... and Prentice-Hernández, C. (2014). Production and characterization of encapsulated antioxidative protein hydrolysates from Whitemouth croaker (Micropogonias furnieri) muscle and byproduct. LWT-Food Science and Technology, 59(2), 841-848.
8. Da Silva Malheiros, P., Daroit, D. J., and Brandelli, A. (2010). Food applications of liposome-encapsulated antimicrobial peptides. Trends in Food Science & Technology, 21(6), 284- 292. https://doi.org/10.1016/j.tifs.2010.03.003
9. Drusch, S., Serfert, Y., Berger, A., Shaikh, M. Q., Rätzke, K., Zaporojtchenko, V., and Schwarz, K. (2012). New insights into the microencapsulation properties of sodium caseinate and hydrolyzed casein. Food Hydrocolloids, 27(2), 332-338. https://doi.org/10.1016/j.foodhyd.2011.10.001
10. Fang, Z., and Bhandari, B. (2010). Encapsulation of polyphenols–a review. Trends in Food Science & Technology, 21(10), 510-523. https://doi.org/10.1016/j.tifs.2010.08.003
11. Ghorbanzadeh, T., Jafari, S. M., Akhavan, S., and Hadavi, R. (2017). Nano-encapsulation of fish oil in nano-liposomes and its application in fortification of yogurt. Food Chemistry, 216, 146-152. https://doi.org/10.1016/j.foodchem.2016.08.022
12. Hamidi, M., Mousavi Nasab, D., Ahmadi, N., Basati, Gh., Aolad, G., Salimian, J., and Zargar, M. (2012). Synthesis of antimicrobial peptides in bacteria. Scientific Journal of Ilam University of Medical Sciences, 20 (4), 158-170. [In Persian].
13. Hasani, Sh., Shahidi, M., and Ojagh. M., (2018). The production and evaluation of nanoliposomes containing bioactive peptides derived from fish wastes using the alkalase enzyme. Research and Innovation in Food Science and Industry, 8 (1), 31-44. [In Persian].
14. Henriksen, I., Smistad, G., and Karlsen, J. (1994). Interactions between liposomes and chitosan. International journal of pharmaceutics, 101(3), 227-236. https://doi.org/10.1016/0378-5173(94)90218-6
15. Hsieh, Y. F., Chen, T. L., Wang, Y. T., Chang, J. H., and Chang, H. M. (2002). Properties of liposomes prepared with various lipids. Journal of Food Science, 67(8), 2808-2813.
16. Laridi, R., Kheadr, E. E., Benech, R. O., Vuillemard, J. C., Lacroix, C., and Fliss, I. (2003). Liposome encapsulated nisin Z: optimization, stability and release during milk fermentation. International Dairy Journal, 13(4), 325-336. https://doi.org/10.1016/S0958-6946(02)00194-2
17. Li, Z., Paulson, A. T., and Gill, T. A. (2015). Encapsulation of bioactive salmon protein hydrolysates with chitosan-coated liposomes. Journal of Functional Foods, 19, 733-743. https://doi.org/10.1016/j.jff.2015.09.058
18. Liu, W., Ye, A., Liu, W., Liu, C., Han, J., and Singh, H. (2015). Behaviour of liposomes loaded with bovine serum albumin during in vitro digestion. Food Chemistry, 175, 16-24. https://doi.org/10.1016/j.foodchem.2014.11.108
19. Mosquera, M., Giménez, B., da Silva, I. M., Boelter, J. F., Montero, P., Gómez-Guillén, M. C., and Brandelli, A. (2014). Nanoencapsulation of an active peptidic fraction from sea bream scales collagen. Food Chemistry, 156, 144-150. https://doi.org/10.1016/j.foodchem.2014.02.011
20. Mozafari, M. R., Flanagan, J., Matia‐Merino, L., Awati, A., Omri, A., Suntres, Z. E., and Singh, H. (2006). Recent trends in the lipid‐based nanoencapsulation of antioxidants and their role in foods. Journal of the Science of Food and Agriculture, 86(13), 2038-2045. https://doi.org/10.1002/jsfa.2576
21. No, H. K., Lee, S. H., Park, N. Y., and Meyers, S. P. (2003). Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process. Journal of Agricultural and food Chemistry, 51(26), 7659-7663.
22. Nowzari, F., Shábanpour, B., and Ojagh, S. M. (2013). Comparison of chitosan–gelatin composite and bilayer coating and film effect on the quality of refrigerated rainbow trout. Food Chemistry, 141(3), 1667-1672. https://doi.org/10.1016/j.foodchem.2013.03.022
23. Ojagh, S. M., Rezaei, M., Razavi, S. H., and Hosseini, S. M. H. 2010. Effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. Food Chemistry, 120(1), 193-198. https://doi.org/10.1016/j.foodchem.2009.10.006
24. Paomephan, P., Assavanig, A., Chaturongakul, S., Cady, N. C., Bergkvist, M., and Niamsiri, N. (2018). Insight into the antibacterial property of chitosan nanoparticles against Escherichia coli and Salmonella Typhimurium and their application as vegetable wash disinfectant. Food Control, 86, 294-301. https://doi.org/10.1016/j.foodcont.2017.09.021
25. Ramezanzade, L., Hosseini, S. F., and Nikkhah, M. (2017). Biopolymer-coated nanoliposomes as carriers of rainbow trout skin-derived antioxidant peptides. Food Chemistry, 234, 220-229. https://doi.org/10.1016/j.foodchem.2017.04.177
26. Rasti, B., Jinap, S., Mozafari, M. R., and Yazid, A. M. (2012). Comparative study of the oxidative and physical stability of liposomal and nanoliposomal polyunsaturated fatty acids prepared with conventional and Mozafari methods. Food Chemistry, 135(4), 2761-2770. https://doi.org/10.1016/j.foodchem.2012.07.016
27. Reyhani Poul, S., and Jafarpour, A. (2020). Effect of edible active film of chitosan containing fish protein hydrolysate (FPH) on chemical and microbial properties of rainbow trout (Oncorhynchus mykiss) fillets during the refrigerated storage. Iranian Food Science and Technology Research Journal, 16 (4), 493-505. [In Persian].
28. Romero-Pérez, A., García-García, E., Zavaleta-Mancera, A., Ramírez-Bribiesca, J. E., Revilla-Vázquez, A., Hernández-Calva, L. M., ... and Cruz-Monterrosa, R. G. (2010). Designing and evaluation of sodium selenite nanoparticles in vitro to improve selenium absorption in ruminants. Veterinary Research Communications, 34(1), 71-79. https://doi.org/10.1007/s11259-009-9335-z
29. Sadeghian, Y., Sadeghi, A.R., Ghorbani, M., Alami, M., and Joshaghani, H. (2019). Investigation of the physical, chemical and antioxidant properties of nanoliposomes loaded with quinoa seed hydrolyzed proteins. Iranian Journal of Nutrition Sciences & Food Technology, 15 (2), 71-82. [In Persian].
30. Safari, R., Raftani Amiri, Z., and Esmaeilzadeh Kenari, R. (2018). Optimizing the extraction of phycocyanin pigment from Spirulina platensis algae and investigating the qualitative properties of the encapsulated pigment. PhD thesis, Sari Agricultural Sciences and Natural Resources University. [In Persian].
31. Sarada, R. M. G. P., Pillai, M. G., and Ravishankar, G. A. (1999). Phycocyanin from Spirulina sp: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin. Process Biochemistry, 34(8), 795-801. https://doi.org/10.1016/S0032-9592(98)00153-8
32. Sitohy, M., Osman, A., Ghany, A. G. A., and Salama, A. (2015). Antibacterial phycocyanin from Anabaena oryzae SOS13. International Journal of Applied Research in Natural Products, 8(4), 27-36.
33. Tan, C., Xia, S., Xue, J., Xie, J., Feng, B., and Zhang, X. (2013). Liposomes as vehicles for lutein: preparation, stability, liposomal membrane dynamics, and structure. Journal of Agricultural and Food Chemistry, 61(34), 8175-8184.
34. Wald, M., Schwarz, K., Rehbein, H., Bußmann, B., and Beermann, C. (2016). Detection of antibacterial activity of an enzymatic hydrolysate generated by processing rainbow trout by-products with trout pepsin. Food Chemistry, 205, 221-228. https://doi.org/10.1016/j.foodchem.2016.03.002
35. Watanabe, N., Kamei, S., Ohkubo, A., Yamanaka, M., Ohsawa, S., Makino, K., and Tokuda, K. (1986). Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer. Clinical Chemistry, 32(8), 1551-1554.
36. Xia, S., Xu, S., and Zhang, X. (2006). Optimization in the preparation of coenzyme Q10 nanoliposomes. Journal of Agricultural and Food Chemistry, 54(17), 6358-6366.
37. Yeganeh, S., and Reyhani Poul. (2021). Nanoencapsulation of bioactive peptides from shrimp wastes enzymatic hydrolysis with combined coating of nanoliposome -chitosan and evaluation of antibacterial, antioxidant and antihypertensive activity of the product. Iranian Scientific Fisheries Journal, 30 (6), 83-95. [In Persian].
38. Zaerzadeh, E., Mortazavi, A., Jafari, M., Afsharnejad, S., Tabatabaii, F., and Nassiri, M. (2011). Antibacterial effect of nanoencapsulated nisin in liposoms in contrast to free nisin in control of Listeria monocytogenes in iranian feta cheese (UF). Iranian Food Science and Technology Research Journal, 7 (3), 191-199. [In Persian].