Assessment Of Allergenicity Of Proteins Derived From Different Kinds Of Milk

Milk allergy is a common disease occurred in infants and children with a ratio of about2.5%in the first three years after birth. As for infants who can not access to breast milk, infant formula is a very important breast-milk substitute. Currently, most infant formula, especially formula for premature infants are developed using cow’s milk protein-based materials. The cow’s milk proteins consist of casein, a-lactalbumin, β-lactoglobulin, etc., which are very important potential allergens for infants, especially premature infants and makes the allergy problem be more prominent. Therefore, the research and development to the milk-based materials that are suitable for hypoallergenic infant formula have attracted great concern of medical profession and nutrition scholars. Although the hydrolysis, high pressure, heating, methylation and other methods can be used to reduce the allergenicity of milk, these treatment methods will result in that the nutrients in milk are destroyed and the taste is greatly reduced even with bitterness. If we can find out good protein materials that are with low allergenicity without the need of treatment or only need of slight treatment, it will be very beneficial for successful development of hypoallergenic infant formula. Although the common protein material for infant formula is cow’s milk, it is generally believed that the non-dairy mammal milk, including goat’s milk, sheep’s milk, mare’s milk and donkey milk, provide an acceptable alternative materials for the development of infant formula for babies suffering from cow’s milk protein allergy. Existing relevant studies have shown that the mare’s milk and goat’s milk are closer to breast milk than cow’s milk, and therefore we try to analyze whether the mare’s milk or goat’s milk are more suitable for the development of hypoallergenic infant formula than cow’s milk from the point of view of immunological analysis to provide theoretical basis for the development of hypoallergenic infant formula as well as infant formula for premature infant.In this experiment, Balb/c mice were used to establish animal model to compare the effects of cow’s milk, goat’s milk and mare’s milk on the mice body weight and intestinal vascular permeability. MTS and ELISA methods were used to detect cell proliferation, IgE and IgG1in serum and intestinal juice as well as histamine secretion levels in plasma. The effects of cow’s milk, goat’s milk and mare’s milk on the intestine and lung morphology were observed by preparing paraffin sections and conducting HE staining. Real-time PCR, ELISA and flow cytometry methods are used to detect the differentiation of helper T cells in T lymphocytes from molecule and protein levels. The results are shown as below: The results of cell proliferation showed that in the absence of ConA, the SI of B-lg was significantly higher than that of cow’s milk, goat’s milk, mare’s milk and the control (P<0.01). In the presence of ConA, the SI of β-lg was significantly higher than that of cow’s milk, goat’s milk, mare’s milk and the control, but the SI in the presence of ConA was higher than that in the absence of Con A, indicating that under the induction of ConA, the stimulation of B-1g, cow’s milk, goat’s milk and mare’s milk on spleen lymphocytes were enhanced.ELISA method was used to detect the levels of IgE in serum and small intestine juice. The results showed that the IgE levels in serum of mice stimulated by B-lg and cow’s milk were significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01), and there was no significant difference between the groups stimulated by goat milk and mare’s milk (P>0.05). The IgE levels in small intestinal juice of mice stimulated by B-lg was significantly higher than that stimulated by cow’s milk, goat’s milk, mare’s milk and PBS (P<0.01). The IgE level in small intestinal juice of mice stimulated by cow’s milk was significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01). There was no significant difference between the groups stimulated by goat’s milk, mare’s milk as well as PBS (P>0.05). In addition, we also detected the levels of IgG1in serum and small intestine juice. The results indicated that the IgG1levels in serum of mice stimulated by B-lg and cow’s milk were significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.05), and there was no significant difference between the groups stimulated by goat milk and mare’s milk (P>0.05). The IgG1level in small intestinal juice of mice stimulated by B-lg was significantly higher than that stimulated by cow’s milk, goat’s milk, mare’s milk and PBS (P<0.01). The IgG1levels in small intestinal juice of mice stimulated by cow’s milk was significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01). There was no significant difference between the groups stimulated by goat’s milk, mare’s milk as well as PBS (P>0.05).ELISA kits were used to detect the secretions of four cytokines of IL-4, IL-10, IL-17and IFN-y in serum and spleen cells supernatants of mice. The results showed the IL-4level in serum of mice stimulated by B-lg was significantly higher than that stimulated by cow’s milk, goat’s milk, mare’s milk as well as PBS (P<0.01). The IL-4level in serum of mice stimulated by cow’s milk was significantly higher than that stimulated by goat’s milk mare’s milk and PBS (P<0.01) and there was no significant difference between the groups stimulated by goat’s milk, mare’s milk as well as PBS. The IL-10levels in serum of mice stimulated by β-lg and cow’s milk were significantly lower than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01) and there was no significant difference between the groups stimulated by goat’s milk, mare’s milk and the Control. The IL-17levels in serum of mice stimulated by B-lg and cow’s milk were significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01) and there was no significant difference between the groups stimulated by goat’s milk, mare’s milk as well as PBS (P>0.05). The IFN-y levels in serum of mice stimulated by B-lg and cow’s milk were significantly lower than that of goat’s milk, mare’s milk and the Control (P<0.001), and there was no significant difference between the groups stimulated by goat’s milk, mare’s milk and PBS (P>0.05).ELISA kit was also used to detect the levels of histamine in plasma and the results showed that the level of histamine in plasma of mice stimulated by B-lg was significantly higher than that in cow’s milk, goat’s milk, mare’s milk and PBS (P<0.05). The level of histamine in plasma of mice stimulated by cow’s milk was significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01). but no significant differences between the goat’s milk group, mare’s milk and control groups (P>0.05). There was no significant difference between the groups stimulated by goat’s milk, mare’s milk and PBS.Histological biopsy results showed that, compared with the control group, the number of goblet cells was increased in intestinal gland of mice stimulated by β-1g and cow’s milk, indicating accompanied by catarrhal enteritis and small amount of shedding and necrosis of epithelial cells. The number of goblet cells was increased in intestinal gland of mice stimulated by mare’s milk and goat’s milk, but the intestinal villi was under normal condition and there was no increase in goblet cells as well as intestinal epithelial cell shedding and necrosis. Compared with the control group, the lung of mice stimulated by β-1g, mare’s milk and cow’s milk showed pulmonary alveoli wall incrassation, accompanied by capillary, arterial and venous congestion, congestion, interstitial pneumonia, alveolar collapse, etc. However, the lung of mice stimulated by goat’s milk was under normal group, occasionally with a small amount of congestion. The results of vascular permeability showed that the vascular permeability of intestinal wall of mice stimulated by β-1g and cow’s milk was significantly higher than that stimulated by goat’s milk, mare’s milk and PBS (P<0.01) and there was no significant difference between the groups stimulated by goat’s milk, mare’s milk and PBS (P>0.05).To further explore whether the cow’s milk allergy was related to Thl, Th2, Thl7and Treg cells imbalance, the RNA of spleen stimulated by different milk samples was extracted and real-time PCR was used to detect the expression of cytokines secreted by helper T cells after reverse transcription as well as nuclear transcription factors. The results showed that the IL-4and IL-17mRNA expression levels of spleen coming from mice stimulated by B-lg and cow’s milk were significantly higher than that stimulated by goat’s milk and mare’s milk. The IFN-y and IL-10mRNA expression levels of spleen coming from mice stimulated by goat’s milk and mare’s milk were significantly higher than that stimulated by cow’s milk. The representative nuclear transcription factors GATA-3, Foxp3, RORyt and T-bet expression levels of helper T cells were similar to their corresponding representative cytokines.The levels of four representative cytokines of helper T cells within spleen were detected using flow cytometry method. The results showed that the number of spleen cells expressing IL-4and IL-17of mice stimulated by cow’s milk was significantly higher than that stimulated by goat’s milk, mare’s milk and PBS. There was no significant difference among the number of spleen cells expressing IL-4and IL-17of mice stimulated by goat’s milk and mare’s milk. The number of speen cells expressing IFN-γ and IL-10of mice stimulated by cow’s milk was significantly lower than that stimulated by goat’s milk and mare’s milk and there was no significant difference between the number of speen cells expressing IFN-γ and IL-10of mice stimulated by goat’s milk and mare’s milk. These results were basically consistent with the results of IL-4, IL-10, IL-17and IFN-y in sera and culture supernatants. The flow cytometry method was also used to detect the ratio of CD4+and CD8+T cells in speen, and the results showed that among all groups, the proportion of CD3+CD4+T cells was higher than CD3+CD8+T cells, and there was no significant differences among all groups (P>0.05).In conclusion, Balb/c mice were used to successfully establish allergy animal model and immunological methods were used to compare the sensitization differences between cow’s milk, mare’s milk and goat’s milk. The results confirm that the sensitization of mare’s milk and goat’s milk is lower than cow’s milk, so they are more suitable to produce hypoallergenic infant formula or premature infant formula. These results provide a theoretical basis for the development of hypoallergenic infant formula or premature infant formula.