Effects of enzymatic hydrolysis on the allergenicity of natural cow milk based on a BALB/c mouse model

摘要

Cow milk allergy is one of the most prevalent food allergiesworldwide, particularly in infants and children.To the best of our knowledge, minimal research existsconcerning the antigenicity of cow milk (CM). Thisstudy was performed to evaluate the allergenicity ofenzymatically hydrolyzed cow milk (HM) in a BALB/cmouse model. The mice were randomly divided into5 groups (n = 12/group), which were sensitized withphosphate-buffered saline, CM, and HM (Alcalase-,or Protamex-, or Flavorzyme-treated cow milk; NovoNordisk; AT, PT, FT, respectively), respectively, usingcholera toxin as adjuvant on d 0, 7, 14, 21. On d 28,the test mice were orally challenged with phosphatebufferedsaline, CM, and HM (AT, PT, or FT) alone.Anaphylactic symptoms were monitored in the mice.Antibody, cytokine, histamine, and mouse mast cellprotease-1 (mMCP-1) levels were measured usingenzyme-linked immunosorbent assays. In addition, thenumbers of T helper (Th)1 and Th2 cells, as well as theproportions of CD~4+CD25~+Foxp3~+ Treg cells, in mousespleens were detected using flow cytometry. Statisticalsignificance was determined by one-way ANOVA.The results revealed significant differences betweenCM- and HM-challenged mice. Among these, the clinicalscores of HM-challenged mice (AT, 1.50; PT, 2.00;FT, 1.92) were lower than those of CM-challenged mice(positive control, 2.83), but body weight and temperatureof HM-challenged mice were higher than those ofCM-challenged mice. In addition, significant reductionsof allergen-specific IgE, IgG, histamine, and mMCP-1were showed in HM-challenged mice, especially for histamine,ranging from 171.42 ng/mL to 214.94 ng/mL.Remarkable reductions of IL-4, IL-5, and IL-13 levels,as well as elevations of interferon-γ and IL-10 levels inthe spleens of HM-challenged mice were also detected.Moreover, the number of Th2 cells decreased in theHM-challenged mice, to 2.36% (AT), 1.79% (PT), and4.03% (FT), respectively, whereas the numbers of Th1cells (AT, 6.30%; PT, 6.70%; FT, 6.56%) and the proportionsof CD4~+CD25~+Foxp3~+Tregs (AT, 8.86%; PT,9.21%; FT, 9.16%) increased significantly. Our findingsindicate that exposure to HM was sufficient to inducea shift toward a Th1 response, thereby reducing potentialallergenicity. Importantly, these results will lay atheoretical foundation for the development of hypoallergenicCM products.