تولید بستنی مبتنی بر تخم‌مرغ فراسودمند حاوی ایمونوگلوبین Y بر علیه باکتری هلیکوباکتر پیلوری

نوع مقاله : مقاله پژوهشی

نویسندگان

• سحر صباحی ¹
• سید علی مرتضوی ¹
• محمدرضا نصیری ²
• آرش قزوینی ³
• فخری شهیدی ¹

¹ گروه صنایع غذایی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

² گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران

³ گروه میکروبیولوژی، دانشگاه علوم پزشکی مشهد، مشهد، ایران

https://doi.org/10.22067/ifstrj.v16i5.83058

چکیده

امروزه، مصرف‌کنندگان به‌دنبال غذاهایی هستند که باعث سلامتی شده و خطر ابتلا به بیماری‌ها را کاهش دهد. تخم‌مرغ به‌عنوان منبع بزرگی از ایمونوگلوبین Y است که نقش بسیار مهمی در ایمنی غیرفعال دارد. ازاین‌رو، این پژوهش با هدف ایجاد ایمنی غیرفعال خوراکی در برابر باکتری هلیکوباکتر پیلوری که شیوع بسیار وسیعی در جهان داشته و عامل اصلی زخم و سرطان معده است، صورت پذیرفت. بدین منظور، سلول‌های باکتری هلیکوباکتر پیلوری با استفاده از فرمالدهید غیرفعال و پس از ترکیب با ادجوانت فعال و غیرفعال فروند به 4 نقطه از سینه مرغ نژاد لگهورن سفید تزریق زیرجلدی شد. استخراج آنتی‌بادی IgY با استفاده از پلی‌اتیلین گلایکول و کیفیت و غلظت آن با استفاده از روش‌های SDS-PAGE و برادفورد موردبررسی قرار گرفت. از زرده تخم‌مرغ‌های حاوی IgY به‌منظور تولید بستنی تخم‌مرغی استفاده و محصول در مقایسه با بستنی وانیلی مورد ارزیابی حسی قرار گرفت. از تست الایزا غیرمستقیم به‌منظور بررسی فعالیت IgY استخراجی، همچنین فعالیت IgY در طی سه ماه پس از تولید استفاده شد. حضور دو باند 25 و 67 کیلودالتونی بر روی ژل نشان از کیفیت مناسب روش استخراج داشت و غلظت IgY استخراجی 46/11 میلی‌گرم بر میلی‌لیتر برآورد شد. آنالیز فعالیت IgY با روش الایزا نشان داد که بیشترین میزان این آنتی‌بادی در هفته دوم داشته و بعد از 3 ماه نگهداری بستنی تخم‌مرغی در دمای 18-، 94 درصد از فعالیت آنتی‌بادی حفظ شده است. بررسی ارزیابی حسی بین بستنی حاوی IgY و نمونه شاهد  نشان دهنده عدم تفاوت معنی داردر 5 پارامتر اصلی تست چند نقطه ای می باشد. بنابراین، با توجه به فعال بودن آنتی‌بادی IgY در آزمایش‌های الایزا، امکان اتصال IgY حاضر در بستنی تخم‌مرغی تولیدشده در این پژوهش با باکتری هلیکوباکتری پیلوری فراهم بوده و این محصول می‌تواند در پیشگیری از تکثیر باکتری هلیکوباکتری پیلوری مؤثر عمل کند.  

کلیدواژه‌ها

• ایمونوگلوبین Y
• ایمنی‌زایی غیرفعال
• هلیکوباکترپیلوری
• بستنی

عنوان مقاله [English]

Production of Beneficial Ice Cream Containing Immunoglobulin Y against Helicobacter pylori

نویسندگان [English]

• Sahar Sabahi ¹
• Seyed Ali Mortazavi ¹
• Mohammad Reza Nassiri ²
• Arash Ghazvini ³
• Fakhri Shahidi ¹

¹ Department of Food Industry, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

² Department of Animal Science and Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

³ Department of Microbiology, Faculty of Medical science, Mashhad University of Medical Sciences, Mashhad, Iran

چکیده [English]

Introduction: Immunoglobin Y (IgY) or egg yolk antibodies have captured attention as substitutes for antibiotics against the presence of disease-causing organisms (Thomsen et al., 2016; Li et al., 2015; Pereira et al., 2019). Compared to mammalian IgG, IgY has several advantages including high yield, cost-effectiveness, and convenience (Li et al., 2015). It has been reported that the use of IgY against Helicobacter pylori can be particularly useful for prevention and treatment of diseases (Najdi et al., 2016). The growth inhibitory effect of IgY antibody is one of its crucial features (Alfarouk et al., 2019), meaning that IgY antibody could be consumed daily with no health risks. Therefore, daily consumption of IgY requires a suitable host. The ice cream is the best choice for this proposed, because this product is maintained at -18 C and ice cream composition has positive effect on the function of IgY. Therefore, we develop a new product for the prevention and treatment of Helicobacter pylori.
 
Material and Methods: The Mueller-Hinton media was used for H. pylori growth. Then, 109 colonies of bacterium were inactivated by formaldehyde methods. The 3 white leghorn chickens (Animal Science Department of Ferdowsi University of Mashhad) of 16 weeks old were immunized by H. pylori and Freund’s complete adjuvant with an equal volume. The reminder immunization was done two weeks later and using of Freund’s incomplete adjuvant for stage 2. One week after the chickens received their last injection, their eggs were collected on a daily basis for eight weeks, labeled and kept at 4 °C (Pauly et al., 2011). Extraction and purification of IgY antibody from egg yolk was done using polyethylene glycol 6000 (PEG 6000) powder (Merck, Germany) according to Polson’s method (Pauly et al., 2011). The IgY production wasverified by the SDS-PAGE and Bradford methods. The specific activity of IgY antibody against H. pylori was examined by ELISA. The absorbance of each well was measured at 492 nm (Nasiri et al., 2016). The egg yolk ice cream preparation contains IgY was prepared according to the method of Herald et al (2008). For estimation of IgY shelf life in ice cream, the residual activity of IgY after freezing was measured by ELISA. The Sensory evaluation of the egg yolk ice creams was conducted by 5-point hedonic scale through surveys to assess consumers’ level of acceptance.
 
Results and Discussion: The purification of IgY using the SDS-PAGE method was verified correctly and the size of heavy and light chains of IgY was estimated about 65 and 27 kDa. The IgY yield in samples was 11.46 mg/ml, therefore, the high purity and yield of IgY, it could be effectively used as an ideal antibody in the food industry. The purification of IgY from egg yolk by PEG method is remarkably cost effective. The ELISA assay indicated binding of IgY to H. pylori antigen that was fixed on plates. Also, the results revealed significant differences between the control and treatment groups (P-value <0.001). The shelf life of IgY in ice cream showed that this product could be most certainly used as bio-food. ELISA analysis confirmed that IgY could remain effective in ice cream for 3 months. The activity of IgY during this period decreased about 6%, which is insignificant. The sensory features of ice cream using hedonic scale revealed that this product could be suitable for marketing, as no significant difference was found between egg yolk and vanilla ice creams. Ice cream is considered as the best candidate for production of IgY bio-foods, as it possesses the features needed to protect IgY and to increase efficiency of IgY activity. The optimum temperature for protection of ice cream was -18 °C that is utterly suitable for IgY protection. Furthermore, ice cream is consumed while cold and studies showed that IgY can remain active at temperatures of up to 80 °C and pasteurization process had no effect on IgY activity (Horie et al., 2004). Jaradat et al (2000) reported that, IgY activity was inhibited in presence of carbohydrates (sucrose, lactose and trehalose). Therefore, here, we used freeze dried egg yolk to protect IgY. Consequently, ice-cream is an ideal candidate for transferring antibodies into human body, as the particular composition of ice cream can properly protect IgY antibodies.

کلیدواژه‌ها [English]

• Immunoglobin Y
• Ice cream
• H. pylori
• Inactive Immune System

مراجع

Abdel-Aal, el-S. M., Akhtar, H., Zaheer, K. & Ali, R. (2013). Dietary sources of lutein and zeaxanthin carotenoids and their role in eye health. Nutrients, 9;5(4):1169-85.

Aitila, P., Mutyaba, M., Okeny, S., Ndawula, K. M., Kasule, R., Ssedyabane, F., Okongo, B., Onyuthi Apecu, R., Muwanguzi, E. & Oyet, C. (2019). Prevalence and Risk Factors of Helicobacter pylori Infection among Children Aged 1 to 15 Years at Holy Innocents Children's Hospital, Mbarara, South Western Uganda. Journal of Tropical Medicine, 7: 930-942.

Amroa, W.A., Qaisib, W. A. & Razema, F. A. (2017). Production and purification of IgY antibodies from chicken egg yolk. Journal of Genetic Engineering and Biotechnology, 16, 99-103.

Backert, S., Neddermann, M., Maubach, G., & Naumann, M. (2016). Pathogenesis of Helicobacter pylori infection. Helicobacter, 21, 19–25.

Blanchard, T. G., & Nedrud, J. G. (2006). Laboratory maintenance of helicobacter species. Current protocols in microbiology, Chapter 8, Unit8B.1.

Cai, Y. C., Guo, J., Chen, S. H., Tian, L. G., Steinmann, P., Chen, M. X., Li, H., Ai, L. & Chen, J. X. (2012). Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum. International Journal for Parasitology, 61(3), 385-90.

Chen, J.P. & Chang, M.C. (2003) Effect of Anti-Helicobacter pylori Urease Antibody (IgY) as a Food Ingredient on the Decrease of H. pylori in the Stomach of Humans Infected with H. pylori. Taiwanese Journal of Agricultural Chemistry and Food Science, 41, 408-414.

Czinn, S. J., & Blanchard, T. (2011). Vaccinating against Helicobacter pylori infection. Nature Reviews Gastroenterology & Hepatology, 8, 133–140.

Hong, K. S., Ki, M. R., Ullah, H. M. A., Lee, E. J., Kim, Y. D., Chung, M. J., Jeong, K. S. (2018). Preventive effect of anti-VacA egg yolk immunoglobulin (IgY) on Helicobacter pylori-infected mice. Vaccine, 36(3), 371–380.

Horie, K., Horie, N., Abdou, A.M., Yang, J.O., Yun, S.S., Chun, H.N., Park, C.K., Kim, M. and Hatta, H. (2004). Suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on Helicobacter pylori in humans. Journal of Dairy Science, 87, 4073-4079.

Ikemori, Y., Peralta, R. C., Kuroki, M., Yokoyama, H. & Kodama, Y. (1993). Research note: avidity of chicken yolk antibodies to enterotoxigenic Escherichia coli fimbriae. Poultry Science, 72, 2361 – 23655.

Jaradat, Z.W. & Marquardt, R. R. (2000). Studies on the Stability of Chicken IgY in Different Sugars, Complex Carbohydrates and Food Materials. Food and Agricultural Immunology. 12, 263–272.

Kabir, S. (2007). The current status of Helicobacter pylori vaccines: A review. Helicobacter, 12, 89–102.

Kovacs-Nolan, J., Phillips, M., Mine, Y. (2005). Advances in the value of eggs and egg components for human health. Journal of Agricultural and Food Chemistry, 53, 8421 - 31;

Larsson, A., Bålöw, R. M., Lindahl, T. L. & Forsberg, P. O. (1993). Chicken antibodies: taking advantage of evolution. Poultry Science, 72, 1807 – 1802.

Lemamy, G. J., Roger, P., Mani, J. C., Robert, M., Rochefort, H. & Brouillet, J. P. (1999). High-affinity antibodies from hen’s-egg yolks against human mannose-6-phosphate/insulin-like growth-factor-II receptor (M6P/IGFII-R): characterization and potential use in clinical cancer studies. International Journal of Cancer, 80, 896 – 902.

Leslie, G. A. & Clem, L. W. (1969). Phylogeny of immunoglobulin structure and function. Journal of Experimental Medicine, 130,1337-1345.

Liu, L. N., Jing, F.J., Cui, J., Fu, G. Y. & Wang, Z. Q. (2013). Detection of circulating antigen in serum of mice infected with Trichinella spiralis by an IgY-IgM mAb sandwich ELISA. Experimental Parasitology.133(2):150-5.

Loibner, M., Buzina, W. & Viertler, C. (2016). Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative. PLoS One, 11:1-233 15.

Matsunaga, Y., Wakatsuki, Y., Tabata, Y., Kawasaki, H., Usui, T., Yoshida, M. (2000). Oral immunization with size-purified microsphere beads as a vehicle selectively induces systemic tolerance and sensitization. Vaccine, 19:579 – 88.

Mensink, M. A., Frijlink, H. W., van der Voort Maarschalk, K. & Hinrichs, W. L. (2017). How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. European Journal of Pharmaceutics and Biopharmaceutics, 114:288-295.

Mony, T. J., Kwon, H. S., Won, M. K., Kang, Y. M., Lee, S. H., Kim, S. Y., Bae, D. Y.& Elahi, F. (2019). Anti-urease immunoglobulin (IgY) from egg yolk prevents Helicobacter pylori infection in a mouse model. Food and Agricultural Immunology, 30, 662–676.

Müller, S., Schubert, A., Zajac, J.& Dyck, T. (2015). Oelkrug, C. IgY antibodies in human nutrition for disease prevention. Nutrition Journal, 20, 14:109.

Najdi, S., Nikbakht Brujeni, G., Sheikhi, N. & Chakhkar, S. (2016). Development of anti-Helicobacter pylori immunoglobulins Y (IgYs) in quail. Iranian Journal of Veterinary Research, 17(2):106-110.

Narat, M. (2003). Production of antibodies in chickens. Food Technology and Biotechnology; 41, 259 – 67

Nilsson, E., Hanrieder, J., Bergquist, J. & Larsson, A. (2008). Proteomic characterization of IgY preparations purified with a water dilution method. Journal of Agricultural and Food Chemistry, 56:11638 - 11642;

Rahman, S., Van Nguyen, S., Icatlo, F. C., Umeda, K., Kodama, Y. (2013). Oral passive IgY-based immunotherapeutics: a novel solution for prevention and treatment of alimentary tract diseases. Human Vaccines & Immunotherapeutics, 9(5):1039-48.

Rehault-Godbert, S., Guyot, N. & Nys, Y. (2019). The Golden Egg: Nutritional Value, Bioactivities, and Emerging Benefits for Human Health. Nutrients. 22;11(3):684.

Russo, M., Nahori, M. A., Lefort, J., Gomes, E., de Castro Keller, A., Rodriguez, D. (2001). Suppression of asthma-like responses in different mouse strains by oral tolerance. American Journal of Respiratory Cell and Molecular Biology, 24:518 - 26;

Schade, R., Gutierrez Calzado, E. J., Sarmiento, R., Chacana, P., Porankiewicz-Asplund, J., Horacio Raúl, T. (2005). Chicken Egg Yolk Antibodies (IgY-technology): A Review of Progress in Production and Use in Research and Human and Veterinary Medicine. Alternatives to laboratory animals, 33, 129-54.

Stålberg, J. & Larsson, A. (2001). Extraction of IgY from egg yolk using a novel aqueous two-phase system and comparison with other extraction methods. Upsala Journal of Medical Sciences, (2):99-110.

Sutton, P., & Lee, A. (2011). Helicobacter pylori vaccines-the current status. Alimentary Pharmacology and Therapeutics, 14, 1107–1118.

Taylor, A. I., Gould, H. J., Sutton, B. J. & Calvert, R. A. (2008). Avian IgY binds to a monocyte receptor with IgG-like kinetics despite an IgE-like structure. Journal of Biological Chemistry, 283,16384 – 90.

Xu, Y., Li, X., Jin, L., Zhen, Y., Lu, Y., Li, S. (2011). Application of chicken egg yolk immunoglobulins in the control of terrestrial and aquatic animal diseases: a review. Biotechnology Advances, 29:860 – 868.

Yamane, T., Saito, Y., Takizawa, S., Goshima, H., Kodama, Y., Horie, N. & Kim, M. (2003). Development of Anti-Helicobacter pylori Urease IgY and Its Application for Food Product. Food Processing and Ingredients, 38, 70-78.

Yong, X., Tang, B., Li, B. S., Xie, R., Hu, C. J., Luo, G., Yang, S. M. (2015). Helocobacter pylori virulence factor CagA promotes tumorigenesis of gastric cancer via multiple signaling pathways. Cell Communication and Signaling, 30, 13-20.