Changes in Solubility, Allergenicity, and Digestibility of Cow's Milk Proteins in Baked Milk

Abstract

It has been suggested that patients with cow's milk allergy can ingest baked milk without adverse reactions, although this has not been characterized sufficiently. Here, we investigated the solubility, allergenicity, and digestibility of αs1-casein and β-lactoglobulin when skim milk (SM) was mixed with food ingredients, starch and gluten, and then baked. Samples were analyzed using competitive ELISA, SDS-PAGE, and immunoblot. The protein bands of cow's milk proteins (CMP) in baked SM with starch and gluten were shifted up. αs1-Casein and β-lactoglobulin in the PBS extract from the baked SM with gluten decreased compared with that in the baked SM with starch. αs1-Casein and β-lactoglobulin were detected as insoluble fractions. The allergenicity of CMP was significantly decreased by baking and when mixed with gluten. The digestibility of αs1-casein and β-lactoglobulin was unaffected by food ingredients, and insolubilized β-lactoglobulin was not digested and thus remained, especially in baked SM with gluten.

Introduction

Cow's milk (CM) allergy is one of the most common food allergies in children. Among causative foods of new-onset food allergies, CM accounts for 24.3% allergies in <1-year-olds and 10.1% in 1-year-olds. On the other hand, in causative foods of accidental ingestion, CM accounts for 32.8%, 34.3%, 36.3%, 30.6%, and 17.2% in <1-year-olds, 1-year-olds, 2–3-year-olds, 4–6-year-olds, and 7–19-year-olds, respectively (Ebisawa et al., 2017). Thus, the rate of new-onset CM allergy shows a decreasing trend with increasing age, while the onset of CM by accidental ingestion is persistent into puberty.

Patients with persistent CM allergy require strict dietary management. In some cases, the patients cannot ingest unheated CM, but can consume baked milk (BM), such as bread and muffin. Furthermore, several recent studies showed that introducing BM into the diet of patients who have CM allergy could accelerate tolerance in these patients (Esmaeilzadeh et al., 2018; Lambert et al., 2017; Nowak-Węgrzyn et al., 2018; Kim et al., 2011). Kim et al. (2011) reported that 39 of 65 patients (60%) who were tolerant of BM at baseline were tolerant of unheated CM; in contrast, among 23 patients who reacted to BM at baseline, only 2 (9%) patients tolerated unheated CM. One possible reason for why patients who cannot ingest CM tolerate BM is that allergenicity of BM, which is made from various food ingredients, is presumably low. The allergens could be involved in interaction with other components of BM.

Some studies showed that heating produces an interaction between proteins (Kato et al., 2000; Luo et al., 2016; Lambrecht et al., 2017). Several recent studies have also shown that the mixing of allergic food with wheat flour and subsequent heating can reduce the antigenicity of the allergens (Shin et al., 2013a; Kato et al., 2001). Kato et al. (2001) demonstrated that kneading of egg with wheat flour for less than 20 min, followed by heating, significantly decreased the antigenicity of ovomucoid, which is a major egg allergen. Notably, the reduction in allergenicity of egg white proteins is dependent on duration and temperature of the heat treatment (Shin et al., 2013b).

Almost all CM proteins (CMP) have been identified as an allergen. The major CM allergens are β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and casein (CN) components (αs1-, αs2- , β-, and κ-CN). All of these proteins, except αs1- and β-CN, have cysteine (Cys) residues. Therefore, it is considered that the Cys residue-containing αs2- and κ-CN interact among themselves or other proteins that have Cys residue(s) during food processing. In fact, κ-CN was shown to interact with β-LG upon baking CM (Jang and Swaisgood, 1990; Anema, 2008). Kato et al. (2004) suggested that the decrease in the antigenicity of β-LG was based on heat-induced polymerization through intermolecular SS bonds between β-LG and wheat proteins. On the other hand, no reports have shown the antigenicity of αs1-CN when mixed with wheat flour and then heated. Both β-LG and αs1-CN are major CM allergens recognized by human IgE (Shek et al., 2005). Additionally, Ito et al. (2012) demonstrated that high levels of CN-specific IgE antibodies are strongly associated with milk allergy in children and might be associated with prolonged allergy. Since αs1-CN, which has no Cys residue, is the main isoform of CN, it is essential to study the allergenicity of αs1-CN in BM. In another study, β-LG heated with lactose on a stainless steel tray at 120 °C for 60 min showed a higher molecular weight (Shinmoto et al., 2013), suggesting carbohydrate modification of the protein (i.e., glycation). In addition, the modified β-LG with lactose retained its immunogenicity. However, CMP bands disappeared by heating at temperatures above 140 °C (Shinmoto et al., 2015). As of yet, there are no studies on the allergenicity of αs1-CN and β-LG after glycation.

In this study, we attempted to analyze the solubility, allergenicity, and digestibility of αs1-CN and β-LG when CMP was mixed with food ingredients, such as commercial starch and gluten, and baked. The results from this study could provide a basis for the clinical application of BM in oral immunotherapy (OIT).

Materials and Methods

Materials    Commercially available skim milk (SM, MEGMILK SNOW BRAND Co., Ltd), starch (Oogikaneyasu Co., Ltd), and gluten (FUJIFILM Wako Pure Chemical Corporation) were purchased. The amounts of protein in starch and gluten were measured by the Kjeldahl method and were 0.1 g and 76.9 g, respectively, in 100 g of powder. Rabbit anti-αs1-CN and anti-β-LG antiserum were procured from Sigma- Aldrich, Inc. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and alkaline phosphatase (AP)-conjugated anti-human IgE were purchased from Bethyl Laboratories.

Antisera of patients with CM allergy    The antisera of four patients with CM allergy were obtained from Dr. Komei Ito (Aichi Children's Health and Medical Center). Antisera showed CMP, CN, α-LA, and β-LG-specific IgE (median; 100, 100, 47.85, and 36.53 UA/mL, respectively) and were pooled. The experimental design involving the sera was approved by the institutional ethical committee at Nagoya University of Arts and Sciences (#200). Informed consent was obtained from the parents of each patient for participation and use of stocked sera for research purposes.

Baked milk    SM with and without each food ingredient (starch and gluten) was mixed in dry weight ratios of 4:1. For the BM, 30 g of mixture and 17 mL of water were kneaded for 2 min and baked in an oven at 180 °C for 10 min (i.e., there were three groups: baked SM alone, baked SM with starch, and baked SM with gluten). Similarly, non-baked milk (NBM) was also prepared: non-baked SM alone, non-baked SM with starch, and non-baked SM with gluten. All samples were freeze-dried and then ground into powder. The amounts of protein in the samples were determined using Kjeldahl method. The amounts of CMP in samples were calculated from the theoretical value.

Protein extraction    The protein extraction was performed as shown in Fig. 1. Briefly, 300 mg of the samples was mixed with 10 mL of phosphate-buffered saline (PBS). Each mixture was rotated at 4 °C for 120 min and then centrifuged at 10 000 rpm for 10 min. The supernatant was separated and the precipitate was mixed with 10 mL of PBS. Two additional extractions with PBS were carried out. The resulting supernatants were pooled and the precipitate was mixed with 10 mL of SDS+urea solution containing 4% SDS and 6 mol/L urea. Two additional extractions were carried out using SDS+urea solution, followed by three rounds of extraction in 2-mercaptoethanol (2-ME) solution containing 4% SDS, 6 mol/L urea, and 5% 2-ME. The pooled supernatants in each case were adjusted to 30 mL with respective extraction buffer (PBS, SDS+urea solution, or 2-ME solution). In addition, total proteins from the original samples were extracted with 2-ME solution. All extracts were stored at −20 °C until further use. We consider that protein in the PBS extract is soluble and proteins in the SDS+urea solution extract and 2-ME solution extract are insoluble.

Fig. 1.

Flowchart of protein extraction. Each procedure was repeated three times in total. cfg, centrifugation; sup., supernatant; ppt., precipitate.

In vitro digestion In vitro    digestion was carried out as reported in previous studies (Yamada et al., 2006) with some modifications. For pepsin digestion, each freeze-dried sample (48 mg of CMP) was mixed with pepsin (1:40 w/w in total proteins) in 20 mL of 30 mM NaCl, with pH adjusted to 1.2 with HCl, and then incubated at 37 °C for 0, 30, 60, and 120 min. To stop the enzyme reaction, 4 mL of 160 mM Na₂CO₃ was added. The pepsin-digested products were centrifuged and the supernatants were stored at −20 °C until analyzed; the protein in the precipitates was extracted with 12 mL of 2-ME solution.

Competitive ELISA    The solubility of αs1-CN and β-LG in PBS extracts were measured by competitive ELISA (Gan and Patel, 2013). Briefly, 96-well flat-bottomed ELISA plates (Nunc-ImmunoTM Plate II, Thermo Fisher Scientific, Roskilde, Denmark) were coated with αs1-CN and β-LG (10 µg/mL in PBS). PBS extracts (serially diluted in PBS) were pre-incubated with the same volume of the rabbit anti-αs1-CN (1:5 000) or anti-β-LG (1:10 000) antiserum diluted in 1% BSA/PBS containing 0.05% Tween-20 (PBST) at 4 °C. After incubation, plates were washed with PBST and blocked with 1% BSA/PBST at 4 °C for 1 h. Subsequently, pre-incubated PBS extracts were added to the wells of the ELISA plates. After incubation, plates were washed with PBST. HRP-conjugated anti-rabbit IgG [dilution 1:10 000 (αs1-CN) and 1:20 000 (β-LG) in 1% BSA/PBST] was added and incubated at 37 °C for 1 h. The plates were then washed, and TMB microwell peroxidase substrate (KPL, Gaithersburg, MD, USA) was added, followed by incubation at room temperature for 10 min. The reaction was stopped by the addition of HCl and absorbance was read at 450 nm using a microplate reader.

To measure the allerginicity of αs1-CN, β-LG, and CMP in PBS extracts, we performed competitive ELISA. The pooled antisera of patients with CM allergy (1:10) as the primary antibody, samples as the inhibitor, and AP-conjugated anti-human IgE (1:100) as the secondary antibody were used. The immune reaction was induced by the addition of PNPP (Thermo Fisher Scientific), stopped by the addition of NaOH, and then absorbance was read at 405 nm. A low absorbance indicated high IgE binding ability.

SDS-PAGE    The extracted samples were separated by SDS-PAGE using a 15% polyacrylamide gel according to the method of Laemmli (1970). The samples were diluted 1:3 v/v in water (samples after digestion were not diluted) and then mixed with sample buffer, containing 5% SDS and 5% 2-ME. A 5 µL (10 µg of CMP) aliquot of each sample (18 µL aliquot for supernatant and 9 µL aliquot for precipitates) was loaded into each well. The protein bands were stained with Coomassie Brilliant Blue R-250 (CBB).

Immunoblotting    Immunoblotting was performed according to the method of Towbin et al. (1979). The gel sheets after SDS-PAGE were electrophoretically transferred to PVDF membranes. The membranes were blocked with 1% BSA/tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) and then incubated with rabbit anti-αs1-CN antiserum (dilution 1:2 000) and anti-β-LG antiserum (dilution 1:10 000) in 1% BSA/TBST at room temperature for 1 h. After the membranes were washed three times with TBST, they were incubated with HRP-conjugated anti-rabbit IgG [dilution 1:5 000 (αs1-CN) and 1:10 000 (β-LG) in 1% BSA/TBST]. After the membranes were washed two times with TBST and twice with TBS, the protein bands were reacted with EzWestBlue (AE-1490, ATTO) and visualized with ATTO Printgraph (Tokyo, Japan). All membranes were analyzed with the same antibody at the same time.

Results and Discussion

Effects of the presence of gluten and baking    We analyzed the total protein composition of the samples using SDS-PAGE (Fig. 2). There was no significant difference between the samples. However, the CMP bands, including of αs1-CN, β-LG, and α-LA, were shifted up and smeared in the case of baked SM with starch and gluten in comparison to non-baked SM with starch and gluten, which was likely the result of glycation (i.e., Maillard reaction) and consistent with a previous study (Shinmoto et al., 2015). In the previous study, the molecular weight of milk allergens in commercial CM and a mixture of β-LG and lactose changed upon baking on a stainless steel tray at 120 °C for 60 min. In addition, CMP was aggregated by baking at temperatures higher than 140 °C and could not be detected by SDS-PAGE. In our study, CMP could be detected by SDS-PAGE even though we baked samples at 180 °C for 10 min, likely because we reduced the baking time. This result suggests that not only baking temperature but also baking time influences glycation of CMP. Although SM contains lactose, bands of CMP in SM did not shift up upon baking. This indicates that there are key factors other than baking that contribute to the glycation of CMP. In addition, glycation was confirmed in baked gluten, suggesting that glycation could occur even if only small amount of carbohydrate was present.

Fig. 2.

Protein composition of non-baked and baked milk. N, non-baked milk; B, baked milk.

We performed competitive ELISA to examine the solubility of αs1-CN and β-LG in the PBS extracts from all samples (Fig. 3A and B). The detectable αs1-CN and β-LG extracted from the mixture were decreased in BM samples compared with that in NBM samples. In addition, the solubility of total CMP, αs1- CN, and β-LG in NBM and BM samples was determined by extracting proteins using SDS+urea solution and 2-ME solution, followed by SDS-PAGE and immunoblotting (Fig. 4). Protein bands in the BM samples were deeply stained with CBB compared with the NBM samples, suggesting that αs1-CN and β-LG had aggregated and become insoluble upon baking. Moreover, this insolubilization phenomenon was also confirmed in baked SM alone samples, which did not contain sufficient glycated CMP to shift up the protein bands. This result indicates that glycation by carbohydrate derived from other ingredients has an insignificant effect on insolubilization.

Fig. 3.

Competitive ELISA using anti-αs1-CN and anti-β-LG rabbit antisera. A and B show inhibition curve of αs1-CN and β-LG in the presence of different food ingredients. N, not baked; B, baked.

Fig. 4.

SDS+urea solution extract fraction and 2-ME solution extract fraction of each baked milk. N, non-baked; B, baked; 1, SDS+urea solution extract fraction; 2, 2-ME solution extract fraction. Proteins were stained by CBB (A). αs1- CN (B) and β-LG (C) were detected from each sample using anti-α s1-CN and β-LG rabbit antisera.

Next, we compared the effect of the ingredients on CMP. The αs1-CN in PBS extracts from baked SM with starch and gluten slightly decreased compared with that from baked SM alone (Fig. 3A). The inhibition curves of β-LG in PBS extracts from baked SM with starch were similar to those from baked SM alone (Fig. 3B). In contrast, β-LG in the PBS extract from the baked SM with gluten was decreased compared with that from baked SM with starch. These effects were greatest in the case of β-LG from baked SM with gluten. According to previous studies, the SH group of the Cys residue in β-LG forms SS bonds with gluten (Kato, 2004) and κ-CN (Jang and Swaisgood, 1990; Anema, 2008). In the current study, putative bands of κ-CN and α-LA were stained in the 2-ME solution extracts from the baked SM with gluten (Fig. 4A). These proteins also contain Cys residues and might form SS bonds with gluten. Thus, our findings suggest that κ-CN, β-LG, and α-LA in the baked SM with gluten sample might interact with gluten.

κ-CN exists on the outside of the CN micelle and is reported to interact with gluten. Thus, following the interaction of κ-CN and gluten, the αs1-CN, which is within the CN micelle, might also be insolubilized upon interaction with gluten. The results of immunoblotting using rabbit anti-αs1-CN antiserum showed a very thin band in the 2-ME solution extract from baked SM with gluten. These results support our hypothesis that αs1-CN is also insolubilized with indirect interaction with gluten. However, since αs1-CN was also detected from the baked SM extract, insolubilization of αs1-CN also occurs upon interaction with other proteins or themselves, and not only with gluten.

Our results suggest that αs1-CN is insolubilized by aggregating with other proteins, CMP or wheat proteins, even though it may not form SS bonds. However, αs1-CN in the SDS+urea solution extracts from NBM samples was detected as very thin bands, suggesting that the insolubilization phenomenon occurs even without baking.

Through immunoblotting, β-LG was detected in 2-ME solution extracts from the BM samples, with β-LG in the baked SM with gluten samples more strongly detected than that in baked SM with starch samples. Furthermore, β-LG was also detected in 2-ME solution extract from non-baked SM with gluten samples, and in the SDS+urea solution extract from SM samples, irrespective of baking. These results indicate that β-LG forms SS bonds with other proteins, especially gluten, similar to a previous study (Kato, 2004; Jang and Swaisgood, 1990; Anema, 2008). However, we consider that the amount of β-LG in the 2-ME solution extract from non-baked SM with gluten sample and in the SDS+urea solution extract from SM sample were low because bands of β-LG were not detected in CBB (Fig. 4A).

Next, we evaluated the effects of different variations of the ratio of SM to food ingredients, such as dry weight ratios of 0.5:1, 1:1, and 2:1 (Fig. 5A and B). The solubility of αs1-CN and β-LG in samples with a low percentage of SM was decreased (as the ratio of food ingredients increased). Based on these results, we hypothesize that as the ratio of starch increases, the aggregation of proteins becomes difficult, since total protein in the mixture decreases. However, the results of β-LG from baked SM with starch were positively correlated with the amounts of protein. A limitation of this study is that we did not evaluate other ingredients. Nonetheless, the ratio of CMP and gluten in general BM for practical applications, such as bread and muffin, is 0.5:1, and β-LG shows insolubilization even when the ratio of CMP and gluten is 4:1. Therefore, we can expect that β-LG in most BM is in a state of insolubilization.

Fig. 5.

Competitive ELISA in samples with a varying ratio of SM to food ingredients. A and B show inhibition curves of αs1-CN (A) and β-LG (B) upon varying the ratio of SM to food matrix. Upper panels show inhibition curve. N, not baked; B, baked. The dry weight ratios of SM:food ingredients of 0.5:1, 1:1, 2:1, and 4:1 are shown as 0.5, 1, 2, and 4, respectively.

Allergenicity of CMP, αs1-CN, and β-LG in BM    CMP, αs1-CN, and β-LG in all samples were analyzed using human IgE in order to understand the change in allergenicity of CMP by baking. The allergenicity of αs1-CN and β-LG in the SM alone sample, which does not sufficiently induce glycation of CMP to shift up the protein bands (Fig. 2), were decreased upon baking (Fig. 6). Therefore, it is suggested that glycation might not affect their allergenicity.

Fig. 6.

Competitive ELISA using pooled antisera of patients with CM allergy. SM, skim milk; N, non-baked; B, baked. Inhibition ratios to (A) CMP, (B) αs1-CN, and (C) β-LG.

The conformation of αs1-CN is not affected by heating. In our study, allergenicity of αs1-CN was decreased by mixing with ingredients and baking; however, the difference associated with ingredients by baking was little (Fig. 6B). This result indicates that the reduced allergenicity of αs1-CN in BM can be attributed to its aggregation with other proteins in addition to its interaction with gluten.

The allergenicity of β-LG was especially decreased by mixing with gluten and baking (Fig. 6C). Morgan et al. (1999) reported that glycation-mediated structural changes in β-LG were confined to localized region and had little consequence on the binding ability of mouse monoclonal IgG antibodies. On the other hand, Ehn et al. (2004) reported that β-LG denatured at 70 °C and the binding ability of IgE decreased at 74 °C. Additionally, it seems that denatured β-LG at 70 °C is different from the denaturation by the interaction with gluten. It is unclear whether these interactions are specific to gluten or can be induced by other food proteins. Further studies are needed to evaluate the effect of other food matrixes.

We also analyzed allergenicity of whole CMP. As a result, the allergenicity of CMP was found to be decreased by baking, and the change was remarkable in the mixture with gluten (Fig. 6A). We presumed that the allergenic proteins other than αs1-CN and β-LG may also interact with gluten. In contrast, the allergenicity of CMP in baked SM with starch remained similar to that observed in baked SM alone. Therefore, the reduced allergenicity of CMP in BM can be attributed to its mixing with gluten.

Digestibility of allergens in BM    To examine the change in digestibility of CMP, including αs1-CN and β-LG, in samples by baking, digested samples were analyzed by SDS-PAGE and immunoblotting. In SM samples, αs1-CN (which appeared as 33.6 kDa) was not detected after digestion (Fig. 7 and Fig. S1, lane 2–4 and 6–8). In non-baked SM with starch samples, the proteolytic fragments of αs1-CN were detected after 30 min of digestion (Fig. 7A, lane 2). Furthermore, in non-baked SM with gluten samples, the proteolytic fragments of αs1-CN were detected after 120 min of digestion (lane 4). Notably, αs1-CN was not detected in BM samples after 30 min of digestion (lane 6–8). In the precipitate after digestion of samples (Fig. S1B), very little staining was observed in the mixture with starch and gluten. In addition, αs1-CNwasnotdetected by immunoblotting (Fig. 7B). These findings suggest that αs1-CN in BM samples is completely resolved by digestion.

Fig. 7.

Digestibility of αs1-CN in samples after pepsin digestion. The pepsin digestive products were separated as supernatants and precipitates. Proteins in the precipitates after digestion were extracted with 2-ME solution, and those in supernatants and 2-ME solution extracts were analyzed by immunoblot using anti-αs1-CN antibody. Lanes 1, 2, 3, and 4 show non-baked samples after 0, 30, 60, and 120 min of digestion, respectively. Lanes 5, 6, 7, and 8 show baked samples after 0, 30, 60, and 120 min of digestion, respectively. αs1-CN in supernatants (A) and precipitate (B) were detected from each sample. Arrow indicates the proteolytic fragments of αs1-CN.

Fig. S1.

Digestibility of samples after pepsin digestion

The pepsin digestive products were separated as supernatants and precipitates, and then proteins in supernatants were analyzed by SDS-PAGE. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 show non-baked samples after 0, 30, 60, and 120 min of digestion, respectively. Lanes 5, 6, 7, and 8 show baked samples after 0, 30, 60, and 120 min of digestion, respectively. Proteins in supernatants (A) and precipitate (B) were stained by CBB.

In the case of β-LG in SM samples, intact β-LG was weakly detected after 30 min of digestion, and some proteolytic fragments were detected in a low molecular weight region (Fig. 8A and Fig. S1, lane 2–4 and 6–8). While the banding patterns before and after digestion were decidedly different, the banding pattern after digestion did not change even after digestion up to 120 min. The digestibility of β-LG in supernatant from non-baked SM with starch and gluten samples was similar to that from non-baked SM alone samples (lane 2–4). In the precipitate after digestion (Fig. 8B), although β-LG from non-baked SM with starch and gluten samples was detected after 30 min of digestion (lane 2), the staining intensity of β-LG was weak during digestion, and was undetectable after 120 min of digestion (lane 4).

Fig. 8.

Digestibility of β-LG in samples after pepsin digestion. The pepsin digestive products were separated as supernatants and precipitates. Proteins in the precipitates after digestion were extracted with 2-ME solution, and those in supernatants and 2-ME solution extracts were analyzed by immunoblot using anti-β-CN antibody. Lanes 1, 2, 3, and 4 show non-baked samples after 0, 30, 60, and 120 min of digestion, respectively. Lanes 5, 6, 7, and 8 show baked samples after 0, 30, 60, and 120 min of digestion, respectively. β-LG in supernatants (A) and precipitate (B) were detected from each sample.

In the supernatant of digested BM samples (Fig. 8A), smeared α-LG bands were clearly detected after 30 min of digestion (lane 6), and detected up to 120 min of digestion (lane 8). Our results showed that digestibility of β-LG may be unaffected by the ingredients. Furthermore, β-LG was detected before digestion, but not after digestion, in the baked SM with starch sample (Fig. 8B, lane 6–8). In contrast, β-LG from the baked SM with gluten sample could be detected after as well as before digestion (lane 6–8). These results suggest that insolubilization of β-LG impacts digestibility. Additional studies are needed to confirm whether insolubilized β-LG is absorbed in some way or is excreted.

β-LG is well known as an indigestible protein. Commercial CM is thermally treated by pasteurization. Sakai et al. (1998) reported that β-LG in low temperature long time-treated (LTLT, at 62–65 °C for 30 min) milk and high temperature short time-treated (HTST, at 72 °C for 15 s) milk was not digestible by pepsin and pancreatin. However, β-LG in ultra-high temperature (UHT, at 120–150 °C for 1–5 s)-treated milk was digestible. In addition, β-LG in boiled (for 15 min) LTLT milk was digestible. These results suggest that the digestibility of β-LG is affected by the heating process. In this study, β-LG was hard to digest even after baking at 180 °C for 10 min; these contrasting results might be due to the differences in the protocol of the digestion experiment. Sakai et al. (1998) performed sequential pepsin and pancreatin digestion for 120 min. Additionally, we used SM powder instead of commercial CM, and CMP in SM might resist digestion.

Conclusion

In this study, we analyzed the solubility, allergenicity, and digestibility of αs1-CN and β-LG when CMP was mixed with food ingredients and baked. The allergenicity of CMP, including αs1-CN and β-LG, decreased by baking due to protein aggregation, especially with gluten. Further, although baking promotes glycation of protein, aggregation among proteins might affect the allergenicity of them more than glycation. The insolubilized allergens do not re-solubilize after digestion by pepsin, although it is unclear whether the insolubilized allergens get absorbed. Together, our findings indicate that the risk of allergy may be decreased by BM consumption instead of CM for a patient with CM allergy, since allergen in BM may not exceed the symptom-inducing dose found in CM. Therefore, BM might be suitable for ingestion in patients with severe allergy, and might have potential application in initial OIT.

Acknowledgments    We thank Dr. Komei Ito (Aichi Children's Health and Medical Center) for providing the antisera of patients with CM allergy.

This work was supported by JSPS KAKENHI Grant Number JP17K00831.

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