Exploring Human Milk Dynamics: Interindividual Variation in Milk Proteome, Peptidome, and Metabolome.

Human milk is a dynamic biofluid, and its detailed composition receives increasing attention. While most studies focus on changes over time or differences between maternal characteristics, interindividual variation receives little attention. Nevertheless, a comprehensive insight into this can help interpret human milk studies and help human milk banks provide targeted milk for recipients. This study aimed to map interindividual variation in the human milk proteome, peptidome, and metabolome and to investigate possible explanations for this variation. A set of 286 milk samples was collected from 29 mothers in the third month postpartum. Samples were pooled per mother, and proteins, peptides, and metabolites were analyzed. A substantial coefficient of variation (>100%) was observed for 4.6% and 36.2% of the proteins and peptides, respectively. In addition, using weighted correlation network analysis (WGCNA), 5 protein and 11 peptide clusters were obtained, showing distinct characteristics. With this, several associations were found between the different data sets and with specific sample characteristics. This study provides insight into the dynamics of human milk protein, peptide, and metabolite composition. In addition, it will support future studies that evaluate the effect size of a parameter of interest by enabling a comparison with natural variability.

Nutritional content, protein quantity, protein quality and carbon footprint of plant-based drinks and semi-skimmed milk in the Netherlands and Europe.

OBJECTIVE: To compare the nutritional composition of bovine milk and several plant-based drinks with a focus on protein and essential amino acid content and to determine the ratio of essential amino acids to greenhouse gas emission. DESIGN: Nutritional information on the label was extracted for semi-skimmed milk, soy, oat, almond, coconut and rice drink from the Innova database between January 2017 and March 2020 for the Netherlands, Belgium, Germany, Spain, Italy, and Sweden. Protein and amino acids were measured and carbon footprint was calculated for a selection of Dutch products. Protein quality was determined by calculating the contribution to the WHO essential amino acids requirements. SETTING: The bovine milk and plant-based drinks market in Netherlands, Belgium, Germany, Spain, Italy, and Sweden. PARTICIPATING PRODUCTS: Semi-skimmed bovine milk and soy-, oat-, almond-, coconut- and rice drink. RESULTS: Nutritional label information was collected for 399 products. Milk naturally contains many micronutrients, e.g. vitamin B2, B12, and calcium. Approximately 50% of the regular plant-based drinks was fortified with calcium, whereas the organic plant-based drinks were mostly unfortified. Protein quantity and quality were highest in milk. Soy drink had the best protein quality to carbon footprint ratio and milk came second. CONCLUSIONS: The nutrition - climate change balance presented in this study, is in line with previous literature, which shows that semi-skimmed bovine milk and fortified soy drink deserve a place in a sustainable diet.

How processing may affect milk protein digestion and overall physiological outcomes: A systematic review.

Dairy is one of the main sources for high quality protein in the human diet. Processing may, however, cause denaturation, aggregation, and chemical modifications of its amino acids, which may impact protein quality. This systematic review covers the effect of milk protein modifications as a result of heating, on protein digestion and its physiological impact. A total of 5363 records were retrieved through the Scopus database of which a total of 102 were included. Although the degree of modification highly depends on the exact processing conditions, heating of milk proteins can modify several amino acids. In vitro and animal studies demonstrate that glycation decreases protein digestibility, and hinders amino acid availability, especially for lysine. Other chemical modifications, including oxidation, racemization, dephosphorylation and cross-linking, are less well studied, but may also impact protein digestion, which may result in decreased amino acid bioavailability and functionality. On the other hand, protein denaturation does not affect overall digestibility, but can facilitate gastric hydrolysis, especially of ß-lactoglobulin. Protein denaturation can also alter gastric emptying of the protein, consequently affecting digestive kinetics that can eventually result in different post-prandial plasma amino acid appearance. Apart from processing, the kinetics of protein digestion depend on the matrix in which the protein is heated. Altogether, protein modifications may be considered indicative for processing severity. Controlling dairy processing conditions can thus be a powerful way to preserve protein quality or to steer gastrointestinal digestion kinetics and subsequent release of amino acids. Related physiological consequences mainly point towards amino acid bioavailability and immunological consequences.

Binding of CML-Modified as Well as Heat-Glycated ß-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity.

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, ß-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to ß-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of ß-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified ß-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

Endogenous human milk peptide release is greater after preterm birth than term birth.

BACKGROUND: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile. OBJECTIVE: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely. METHODS: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d). RESULTS: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. CONCLUSION: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Short communication: Practical issues in implementing volatile metabolite analysis for identifying mastitis pathogens.

Several parameters for improving volatile metabolite analysis using headspace gas chromatography-mass spectrometry (GC-MS) analysis of volatile metabolites were evaluated in the framework of identification of mastitis-causing pathogens. Previous research showed that the results of such volatile metabolites analysis were comparable with those based on bacteriological culturing. The aim of this study was to evaluate the effect of several method changes on the applicability and potential implementation of this method in practice. The use of a relatively polar column is advantageous, resulting in a faster and less complex chromatographic setup with a higher resolving power yielding higher-quality data. Before volatile metabolite analysis is applied, a minimum incubation of 8h is advised, as reducing incubation time leads to less reliable pathogen identification. Application of GC-MS remained favorable compared with regular gas chromatography. The complexity and cost of a GC-MS system are such that this limits the application of the method in practice for identification of mastitis-causing pathogens.

Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides.

Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides - many of which are homologous with known antimicrobial and immunomodulatory peptides - and nearly 50 oligosaccharide compositions (not including structural isomers). As these endogenous peptides and oligosaccharides have low-molecular weight and whey permeate is currently an under-utilized product stream of the dairy industry, we hypothesized that whey permeate may serve as an inexpensive source of naturally occurring functional peptides and oligosaccharides. Laboratory fractionation of endogenous peptides and oligosaccharides from bovine colostrum sweet whey was expanded to pilot-scale. The membrane fractionation methodology used was similar to the methods commonly used industrially to produce whey protein concentrate and whey permeate. Pilot-scale fractionation was compared to laboratory-scale fractionation with regard to the identified peptides and oligosaccharide compositions. Results were interpreted on the basis of whether industrial whey permeate could eventually serve as a source of functional peptides and oligosaccharides. The majority (96%) of peptide sequences and the majority (96%) of oligosaccharide compositions found in the laboratory-scale process were mirrored in the pilot-scale process. Moreover, the pilot-scale process recovered an additional 33 peptides and 1 oligosaccharide not identified from the laboratory-scale extraction. Both laboratory- and pilot-scale processes yielded peptides deriving primarily from the protein ß-casein. The similarity of the laboratory-and pilot-scale's resulting peptide and oligosaccharide profiles demonstrates that whey permeate can serve as an industrial-scale source of bovine milk peptides and oligosaccharides.

Glycation of goat milk with different casein-to-whey protein ratios and its effects on simulated infant digestion.

This research compared the effects of dry heating on the digestion of goat milk proteins with different casein-to-whey ratios (40% casein, C40 and 80% casein, C80). The glycation markers of heated samples were determined by LC-MS. Heating at 60 °C for 8 h induced early glycation while heating at 60 °C for 72 h induced advanced glycation. Unheated C80 samples showed a higher digestibility than unheated C40 samples, which may be due to their higher protein solubility. After dry heating for 72 h, no significant difference in digestibility was observed between C80 and C40 samples. Heating for 72 h decreased the digestibility of C40 samples compared to unheated samples, probably due to glycation, while protein aggregation was the main reason for the reduced digestibility of heated C80 samples. Overall, this study showed that dry heating for 72 h induced a lower digestibility of C80 and C40 samples, although with different underlying mechanisms.

sRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion.

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

Network analysis of the proteome and peptidome sheds light on human milk as a biological system.

Proteins and peptides found in human milk have bioactive potential to benefit the newborn and support healthy development. Research has been carried out on the health benefits of proteins and peptides, but many questions still need to be answered about the nature of these components, how they are formed, and how they end up in the milk. This study explored and elucidated the complexity of the human milk proteome and peptidome. Proteins and peptides were analyzed with non-targeted nanoLC-Orbitrap-MS/MS in a selection of 297 milk samples from the CHILD Cohort Study. Protein and peptide abundances were determined, and a network was inferred using Gaussian graphical modeling (GGM), allowing an investigation of direct associations. This study showed that signatures of (1) specific mechanisms of transport of different groups of proteins, (2) proteolytic degradation by proteases and aminopeptidases, and (3) coagulation and complement activation are present in human milk. These results show the value of an integrated approach in evaluating large-scale omics data sets and provide valuable information for studies that aim to associate protein or peptide profiles from biofluids such as milk with specific physiological characteristics.

Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Heating affects protein digestion of skimmed goat milk under simulated infant conditions.

The objective of this research was to analyse the effects of heating on digestion of skimmed goat milk proteins. Most previous goat milk digestion studies evaluated the digestion only based on the supernatant. In this study, digestion of skimmed goat milk was studied in both supernatant and gastric clot. The results indicated that, compared to mild temperature heated samples (≤75 °C), samples heated at ≥80 °C showed more extensive gastric clot formation with a higher protein digestion rate, but also resulted in a larger amount of undigested whey proteins due to its severe aggregation. For the peptidome, ß-casein was the major source of bioactive peptides. The samples heated at 65 °C showed higher bioactive peptide abundances, whereas at temperatures higher than 75 °C, it was reduced due to cleavage into smaller peptides. Overall, this study showed that different heating temperatures induced different whey protein denaturation degrees, which affected their digestion in skimmed goat milk..

Maternal diet and human milk composition: an updated systematic review.

Context: Exclusive breastfeeding for 6 months after birth provides infants with the best start for life. A review by Bravi et al. summarized the importance of maternal diet as a determinant of human milk composition based on data up to 2015, but evidence on nutrient intake level was limited. Objective: We updated the review by Bravi et al., critically assessed differences in study designs and sampling methods, and graphically visualized trends and associations. Data sources: PubMed was systematically searched for articles published between January 2015 and March 2021. Data extraction: Article screening, selection, and data extraction was done by two independent researchers, including a risk of bias assessment based on 11 criteria. Articles were eligible when including: quantitative information, commonly used effect estimates, healthy mother-infant dyads. Results: Twenty seven observational and five intervention studies were identified (n = 7,138) and combined with results of Bravi et al. Fatty acids were still the most studied human milk components in relation to maternal diet (n = 17 studies) with maternal fish intake being predominantly positively associated with milk ALA (r = 0.28-0.42), DHA (r = 0.24-0.46), and EPA (r = 0.25-0.28) content. PUFAs from diet were generally positively correlated with their concentrations in milk, while SFA intake was negatively associated with several fatty acids in milk. Studies on associations with maternal diet and milk carbohydrates, proteins, vitamins and minerals were limited in number and varied in methods and results. Conclusion: This updated review shows that evidence on the association between maternal diet and human milk fatty acids is rapidly increasing, but still diversified in methodology and results. Further studies, preferably intervention studies, assessing diet and milk carbohydrates, proteins, vitamins and minerals are needed to be able draw conclusions on the importance of maternal diet for human milk composition as a whole.

Corrigendum: The human milk proteome and allergy of mother and child: exploring associations with protein abundances and protein network connectivity.

[This corrects the article DOI: 10.3389/fimmu.2022.977470.].

Tolerance Induction in Cow's Milk Allergic Children by Heated Cow's Milk Protein: The iAGE Follow-Up Study.

Accelerating the induction of tolerance to cow's milk (CM) reduces the burden of cow's milk allergy (CMA). In this randomised controlled intervention study, we aimed to investigate the tolerance induction of a novel heated cow milk protein, the iAGE product, in 18 children with CMA (diagnosed by a paedriatric allergist). Children who tolerated the iAGE product were included. The treatment group (TG: n = 11; mean age 12.8 months, SD = 4.7) consumed the iAGE product daily with their own diet, and the control group (CG: n = 7; mean age 17.6 months, SD = 3.2) used an eHF without any milk consumption. In each group, 2 children had multiple food allergies. The follow-up procedures consisted of a double-blind placebo-controlled food challenge (DBPCFC) with CM t = 0, t = 1 (8 months), t = 2 (16 months), and t = 3 (24 months). At t = 1, eight (73%) of 11 children in the TG had a negative DBPCFC, versus four out of seven (57%) in the CG (BayesFactor = 0.61). At t = 3, nine of the 11 (82%) children in the TG and five of seven (71%) in the CG were tolerant (BayesFactor = 0.51). SIgE for CM reduced from a mean of 3.41 kU/L (SD = 5.63) in the TG to 1.24 kU/L (SD = 2.08) at the end of intervention, respectively a mean of 2.58 (SD = 3.32) in the CG to 0.63 kU/L (SD = 1.06). Product-related AEs were not reported. CM was successfully introduced in all children with negative DBPCFC. We found a standardised, well-defined heated CM protein powder that is safe for daily OIT treatment in a selected group of children with CMA. However, the benefits of inducing tolerance were not observed.

Heat-induced unfolding facilitates plant protein digestibility during in vitro static infant digestion.

Soy protein is the main protein source for plant-based infant formula, whereas pea protein is considered as a potential alternative plant protein source. This study assessed the structural changes of soy and pea proteins after heating between 65 °C and 100 °C, and its effects on the in vitro digestibility in the context of infant digestion. We found that with increased heating intensity, both soy and pea proteins unfolded, manifested as the increased surface hydrophobicity, thereby potentially improving the accessibility to digestive enzymes. Their final in vitro digestibility increased from âˆ¼ 30% of non-treated samples to âˆ¼ 60% of 100 °C-heated samples for soy protein, and from âˆ¼ 52% to âˆ¼ 65% for pea protein. Surface hydrophobicity was strongly positively correlated to the overall digestibility. Therefore, the heating temperatures that enabled protein unfolding promoted the digestibility of soy and pea proteins under infant digestion conditions.

Assessment of milk protein digestion kinetics: effects of denaturation by heat and protein type used.

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a ß-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including ß-casein, increases the rate of intact protein loss, and that changing the protein composition (by including ß-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM: <0.5 kDa peptides and free AA, TIM-1: bioaccessible AA) were monitored. Additionally, in SIM, intermediate product amounts and their characteristics were determined. DWPI showed considerably faster intact protein loss, but similar DH and absorbable product release kinetics compared with NWPI in both models. Furthermore, more, relatively large, intermediate products were released from DWPI than from NWPI. PBI showed increased intact protein loss, similar DH, and absorbable product release kinetics, but more, relatively small, intermediate products than NWPI. In conclusion, both whey protein denaturation and ß-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during in vitro digestion. Our results suggest that intermediate digestion product characteristics are important in relation to postprandial AA responses.

Gastrointestinal Protein Hydrolysis Kinetics: Opportunities for Further Infant Formula Improvement.

The postprandial plasma essential amino acid (AA) peak concentrations of infant formula (IF) are higher than those of human milk (HM) in infants. In addition, several HM proteins have been recovered intact in infant stool and appeared digestion resistant in vitro. We, therefore, hypothesized that gastrointestinal protein hydrolysis of IF is faster than HM and leads to accelerated absorbable digestion product release. HM and IF protein hydrolysis kinetics were compared in a two-step semi-dynamic in vitro infant digestion model, and the time course of degree of protein hydrolysis (DH), loss of intact protein, and release of free AA and peptides was evaluated. Gastric DH increase was similar for IF and HM, but the rate of intestinal DH increase was 1.6 times higher for IF than HM. Intact protein loss in IF was higher than HM from 120 min gastric phase until 60 min intestinal phase. Intestinal phase total digestion product (free AA + peptides <5 kDa) concentrations increased ~2.5 times faster in IF than HM. IF gastrointestinal protein hydrolysis and absorbable product release are faster than HM, possibly due to the presence of digestion-resistant proteins in HM. This might present an opportunity to further improve IF bringing it closer to HM.

The human milk proteome and allergy of mother and child: Exploring associations with protein abundances and protein network connectivity.

Background: The human milk proteome comprises a vast number of proteins with immunomodulatory functions, but it is not clear how this relates to allergy of the mother or allergy development in the breastfed infant. This study aimed to explore the relation between the human milk proteome and allergy of both mother and child. Methods: Proteins were analyzed in milk samples from a subset of 300 mother-child dyads from the Canadian CHILD Cohort Study, selected based on maternal and child allergy phenotypes. For this selection, the definition of "allergy" included food allergy, eczema, allergic rhinitis, and asthma. Proteins were analyzed with non-targeted shotgun proteomics using filter-aided sample preparation (FASP) and nanoLC-Orbitrap-MS/MS. Protein abundances, based on label-free quantification, were compared using multiple statistical approaches, including univariate, multivariate, and network analyses. Results: Using univariate analysis, we observed a trend that milk for infants who develop an allergy by 3 years of age contains higher abundances of immunoglobulin chains, irrespective of the allergy status of the mother. This observation suggests a difference in the milk's immunological potential, which might be related to the development of the infant's immune system. Furthermore, network analysis showed overall increased connectivity of proteins in the milk of allergic mothers and milk for infants who ultimately develop an allergy. This difference in connectivity was especially noted for proteins involved in the protein translation machinery and may be due to the physiological status of the mother, which is reflected in the interconnectedness of proteins in her milk. In addition, it was shown that network analysis complements the other methods for data analysis by revealing complex associations between the milk proteome and mother-child allergy status. Conclusion: Together, these findings give new insights into how the human milk proteome, through differences in the abundance of individual proteins and protein-protein associations, relates to the allergy status of mother and child. In addition, these results inspire new research directions into the complex interplay of the mother-milk-infant triad and allergy.

Introduction of Heated Cow's Milk Protein in Challenge-Proven Cow's Milk Allergic Children: The iAGE Study.

The introduction of baked milk products in cow's milk (CM) allergic children has previously been shown to accelerate induction tolerance in a selected group of children. However, there is no standardized baked milk product on the market. Recently, a new standardized, heated and glycated cow's milk protein (HP) product was developed. The aim of this study was to measure safety and tolerability of a new, well characterized heated CM protein (HP) product in cow's milk allergic (CMA) children between the age of 3 and 36 months. The children were recruited from seven clinics throughout The Netherlands. The HP product was introduced in six incremental doses under clinical supervision. Symptoms were registered after introduction of the HP product. Several questionnaires were filled out by parents of the children. Skin prick tests were performed with CM and HP product, sIgE to CM and α-lactalbumin (Bos d4), ß-lactoglobulin (Bos d5), serum albumin (Bos d 6), lactoferrin (Bos d7) and casein (Bos d8). Whereas 72% percent (18 out of 25) of the children tolerated the HP product, seven children experienced adverse events. Risk factors for intolerance to the HP product were higher skin prick test (SPT) histamine equivalent index (HEP) results with CM and the HP product, higher specific IgE levels against Bos d4 and Bos d8 levels and Bos d5 levels. In conclusion, the HP product was tolerated by 72% of the CM allergic children. Outcomes of SPT with CM and the HP product, as well as values of sIgE against caseins, α-lactalbumin, and ß-lactoglobulin may predict the tolerability of the HP product. Larger studies are needed to confirm these conclusions.