Microbial transglutaminase alters the immunogenic potential and cross-reactivity of horse and cow milk proteins

Abstract

Horse milk is a valuable raw material and a very attractive alternative for scientific research to address the issue of cow milk (CM) allergy due to its protein profile. A decrease in immunoreactive properties can be achieved by thermal, enzymatic, and hydrolytic processing. Therefore, the aim of this study was to explore the possibility of reducing the immunoreactivity of horse milk proteins by microbial transglutaminase (TG) polymerization. To determine how TG linking alters immunoreactivity under simulated digestion of the examined milk, analyses were performed before, during, and after digestion. The dose-dependent (1, 10, and 100 U) effects of microbial TG on horse and cow milk were analyzed. A consecutive 3-stage digestion was simulated with salivary, gastric, and intestinal fluids. The effects of digestion were analyzed by SDS-PAGE, particle size analysis, and size-exclusion chromatography. Immunoreactivity was assessed using competitive ELISA (β-lactoglobulin and α-casein) and immunodot (sera from 7 patients aged 3 to 13 years who are allergic to CM proteins). Horse milk contained almost half of the amount of total proteins in CM. The dose 1 U/g of total milk protein changed the immunoreactivity of both cow and horse milk. With increasing TG doses, α-casein immunoreactivity increased, and β-lactoglobulin decreased. After total digestion, horse milk was characterized by 2.4-fold lower average IgE and 4.8-fold lower IgG reactivity than CM. We found that TG alters the IgE and IgG reactivity of CM after in vitro digestion. Horse milk was less reactive to IgE and IgG than was CM, with animal and patient sera.The effect of TG on immunoreactivity depends on enzyme quantity and milk protein type. The diet based on modified horse milk proteins could be an alternative for some patients with CM protein allergy; however, confirmation through clinical trials is needed.

IgE-Binding and Immunostimulating Properties of Enzymatic Crosslinked Milk Proteins as Influenced by Food Matrix and Digestibility

“…This is consistent with the data obtained when specific inhibition ELISA was performed on CN and WP samples ( Figure 2 c,d). Different results were found by Fotschki et al, who showed that polymerization with TG decreased β-LG immunoreactivity and increased IgE binding of α-CN [ 20 ]. However, they used polyclonal rabbit antibodies, obtained from rabbits immunized against commercial purified allergens instead of human sera from naturally sensitized allergic patients.…”

“…WP, due to their compact globular structures, cross-linked less efficiently [ 17 ]. Although there is no specific legislation about minimum or maximum quantities of TG allowed to be added to food products in the European Union or in the United States, most studies that addressed the use of TG in dairy foods use a maximum of 100 U/g of protein [ 19 , 20 ]. We evaluated the use of 1000 U/g of protein to assess whether higher enzyme activity could result in higher crosslinking efficiency, but although polymerization increased slightly compared to 100 U/g, it was not enough to convert all monomeric proteins into polymers.…”

“…However, after treatment with TG, IgE binding to WP increased ( Figure 2 a), probably due to the exposure of hidden epitopes due to conformational changes in the protein [ 21 ]. Other allergenic proteins or extracts, such as ovalbumin [ 22 ], Ara h 1 [ 23 ], tropomyosin [ 24 ], β-LG [ 20 ], and wheat flour [ 25 ], have shown a reduced allergenicity after cross-linking with TG. However, peanut [ 26 ] and wheat [ 27 ] proteins showed higher IgE binding capacity after cross-linking with TG.…”

Reducing the potential allergenicity of amandin through binding to (−)-epigallocatechin gallate

“…The antigenic epitope of amandin was destroyed or buried, and its potential allergenicity was reduced. Structural and conformational changes caused by processes such as food processing alter the immunoreactivity of proteins ( Fotschki et al, 2020 ). The reduced potential allergenicity of amandin might be mainly due to the absence of disulfide bonds, decreased solubility such as formation of protein–polyphenol insoluble conjugates, and burial of antigenic epitopes ( Chhabra et al, 2017 ).…”

Results of the complex evaluation of bryndza cheese and the byproduct milk whey with the use of enzyme MTG and its activity degree during storage

“…Трансглутаминазу иногда называют «природным клеем», так как она участвует в процессах: свёртываемости крови, синтезе кожи, модификации клеточного матрикса и других процессах. В 1989 году японскими учеными была получена трансглутаминаза из штамма почвенных бактерий Streptoverticillium mobaraense, которые в больших количествах синтезируют легко очищаемую ТГ [11,12,13,14].…”