Digestibility and allergenicity assessment of enzymatically crosslinked β‐casein

Abstract

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of beta-casein (CN). beta-CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to beta-CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS-PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the beta-CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of beta-CN.

Green tea catechins of food supplements facilitate pepsin digestion of major food allergens, but hampers their digestion if oxidized by phenol oxidase

“…Various in vitro digestion models have been developed to assess the stability of food allergens during digestion, but systematic evaluation of the stability of food allergens that are active via the gastrointestinal tract have been currently tested in traditional pepsin digestibility models (Ladics, 2008;Thomas et al, 2004). In addition, more evidence is building up on the influence of food processing on pepsin digestion and allergenicity of proteins (Mills & Mackie, 2008;Monogioudi et al, 2011;Roth-Walter et al, 2008;Stanic et al, 2010). The effect of some phenolic compounds, resveratrol, catechin, epigallocatechin-3-gallate, quercetin and phenolics-rich beverages (red wine and green tea) on pepsin activity has previously been described.…”

Enzymatic and Chemical Modifications of Food Allergens

“…In the study of Monogioudi et al (Monogioudi et al, 2011) -casein was cross-linked of by tyrosinase and demonstrated that enzymatically cross-linked b-casein was stable under acidic conditions and was more resistant to pepsin digestion when compared to non cross-linked b-casein. In the study of Stanic et al (Stanic et al, 2010) tyrosinase treated casein showed mitigated IgE binding reactivity, compared with the untreated CN, only in the presence of caffeic acid as mediator in BAT.…”

“…Enzymatic processing of BLG decrease the bi-phasal pepsin-pancreatin digestibility of the monomeric and crosslinked protein, thus decreasing its nutritional value. Stanic et al (Stanic et al, 2010) crosslinked -casein (CN) by laccase and caffeic acid and demonstrated that crosslinking was not very efficient, leaving mostly monomeric CN modified by caffeic acid. Regardless to that ability of crosslinked CNs to activate basophils was significantly reduced in seven patients and reduced inhibition potential is possibly due to hindering of epitopes by monomer modification.…”

Methods of Protein Digestive Stability Assay - State of the Art

“…One of the simplest models of protein digestion is the use of the purified major enzymes of stomach (pepsin) and pancreas (trypsin and chymotrypsin) (Horii et al 2009;Huang et al 2010;Kamata et al 1982;Nielsen et al 1988;Stanic et al 2010;Yagami et al 2000), implemented either separately of subsequently. The products of the digestion are usually analyzed using SDS-PAGE with the separation range from 3 to 20-30 kDa (Stanic et al 2010;Yu et al 2011) or reverse-phase HPLC (Bublin et al 2008).…”

“…The products of the digestion are usually analyzed using SDS-PAGE with the separation range from 3 to 20-30 kDa (Stanic et al 2010;Yu et al 2011) or reverse-phase HPLC (Bublin et al 2008). In rare cases, mass spectrometry is also employed to characterize the digestion products (Dupont et al 2010).…”