Allergenicity Of Hydrolysates Research Articles

Enzymatic hydrolysis: A method in alleviating legume allergenicity

Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes – kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase® and Flavourzyme®. Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2 ± 7.7%, 87.1 ± 9.6% and 91.8 ± 7.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (p < 0.01). The release of histamine was decreased significantly when sensitized basophils were challenged with hydrolysates as compared to raw extracts. Significant reduction in the biopotency of hydrolysates was also observed in SPT where only 1/10 kidney bean-sensitive individuals, 2/6 black gram-sensitive individuals and 1/7 peanut-sensitive individuals were found positive to their respective hydrolysates. In conclusion, enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts.

Peptic hydrolysis of bovine beta‐lactoglobulin under microwave treatment reduces its allergenicity in an ex vivo murine allergy model

SummaryIn this study, the in vivo allergenicity of bovine beta‐lactoglobulin (BLG) in peptic whey protein hydrolysates generated during microwave and conventional heating treatments was assessed. The allergenicity of the hydrolysates was explored by studying the reaction of the murine jejunum from previously immunised Balb/c mice to treated BLG in an Ussing chamber. Intestinal anaphylactic reactions after stimulation of the gut‐associated immune system are a good indicator of potential in vivo allergenicity of whey hydrolysates. Fifty‐two per cent of BLG was hydrolysed by pepsin after only 3 min of microwave irradiation at 200 watts (W), yet it remained intact under conventional heating. Far‐ and near‐UV circular dichroism spectra indicated significant changes in BLG secondary and tertiary structures with microwave irradiation at 200 W. Pepsin whey protein hydrolysates obtained with microwave irradiation at 200 W for 3 min did not stimulate secretion of chloride in the Ussing chamber, as shown by the intensity of the short current values recorded (27.86 μA cm−2), compared to the conventional pepsin hydrolysates (68.21 μA cm−2). This demonstrates the low allergenicity of whey protein hydrolysates generated in this manner. These results confirm that microwave treatment combined with peptic hydrolysis could be applied to produce low allergenicity milk peptides.

The degree of whey hydrolysis does not uniformly affect in vitro basophil and T cell responses of cow's milk‐allergic patients

Several studies investigated whether hydrolysed proteins can induce tolerance to cow's milk (CM) in children at risk of developing CM allergy. Due to methodological problems and inconsistent findings, the evidence for a tolerogenic effect is limited. A major problem is that different hydrolysates may give different outcomes due to variations in their production and composition. The aim of the study was to investigate the effect of the degree of hydrolysis on the allergenicity and immunogenicity of whey hydrolysates. The hydrolysis of whey was stopped at different time-points between 1 and 60 min. In 18 CM allergic patients, the allergenicity of the hydrolysates was determined by immunoblot and the basophil activation test. To test immunogenicity, CM-specific T cell lines were generated. In most patients, increasing time of hydrolysis decreased IgE recognition and basophil activation. However, in five patients, hydrolysed proteins induced more basophil activation than non-hydrolysed proteins. The immunoblot data indicated that these patients recognized either a 25- to 30-kDa degradation product of casein or a 10-kDa degradation product of whey. Although T cell activation was decreased in all patients over time, half of them still showed a positive response to the proteins after 60 min of hydrolysis. Increasing the time of hydrolysis reduces both allergenicity and immunogenicity of whey hydrolysates in most but not all patients. This indicates that not the degree of hydrolysis is decisive but the presence and stability of IgE and T cell epitopes in the hydrolysate recognized by individual patients.

A Method of Reducing Allergenicity of Legume Proteins

S A T U R D A Y 67 A Method of Reducing Allergenicity of Legume Proteins Naveen Arora, Ramkrashan Kasera, Anand Singh, PhD, Shakuntala Lavasa, Komerla Nagendra; CSIRIGIB, Delhi, India, CSIR-Institute of Genomics and Integrative Biology, India, Institute of Genomics and Integrative Biology, DELHI, India, USLavasa Medical & Research Center, India, Bengaluru Allergy Centre, India. RATIONALE: Legumes are implicated in IgE mediated food allergy in different countries. The present study aimed to investigate the effect of enzymatic hydrolysis on allergenicityof kidney bean, black gramandpeanut. METHODS: The extracts were subjected to sequential enzymatic treatment by Alcalase and flavorzyme. The allergenicity of hydrolysates were determined by ELISA and SPT. Allergenic potential of legume proteins were assessed by ELISA inhibition and basophil histamine release. RESULTS: Hydrolysis reduced the number of IgE binding fractions of the three legumeskidney bean, black gram and peanut. Specific IgE binding was reduced 62%, 87% and 92% in the hyrolysates of kidney bean, black gram and peanut, respectively after 8 h of hydrolysis (p<0.01). Alcalase treatment alone increased the IC50 value of kidney bean, black gram and peanut to 4, 8.5 and 123 folds respectively. Sequential hydrolysis using both alcalase and flavourzyme increased the IC50 of the kidney bean, black gram and peanut to 25, 368 and 393 folds respectively further as compared to single enzyme treatment. Basophil histamine release showed reduced allergenicity of the hydrolyzed proteins. Significant reduction in the biopotency of hydrolyzed protein of kidney bean, black gram and peanut was observed in SPT where 6/13 kidney bean sensitive individuals, 8/13 black gram sensitive individuals and 9/15 peanut sensitive individuals were found SPT negative to their respective hydrolysates. CONCLUSIONS: Sequential hydrolysis by alcalase and flavourzyme reduced the allergenicity of legume proteins. The method has potential to reduce allergenicity of food extracts.