Production of Beneficial Ice Cream Containing Immunoglobulin Y against Helicobacter pylori

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.

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.

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.