Human Milk Proteins Research Articles

Cow’s Milk Antigens Content in Human Milk: A Scoping Review

The functionality of breast milk in terms of immunity is well-known. Despite this, a significant proportion of breastfed infants exhibit sensitization to different potentially allergenic proteins and clinical reactivity (including anaphylaxis) early in life and before the introduction of complementary feeding for the first time. The potential induction of early oral tolerance to overcome early allergic sensitization through exposure to allergens in breast milk also remains controversial and not yet well-established. The objective of this scoping review is to provide a critical appraisal of knowledge about the content of cow’s milk antigens in human milk. The amount of dietary derived milk antigens found in human milk and the analytical methodologies used to detect and quantify these antigens, the allergic status of the mother, the stage of lactation, the time of sampling (before or after ingestion of food), and the impact of human milk allergen on the infant were the outcomes that were assessed. Allergy risk was explored in all reviewed studies and could help to better elucidate its role in the context of allergic disease development. According to the included literature, we can conclude that there are mainly fragments derived from bovine proteins in human milk, and the presence of potentially allergenic molecules is greater in the milk of mothers with an allergic tendency. A clear relationship between maternal diet and allergen content in breast milk could not be firmly concluded though. Also, infants receiving milk from human milk banks, where donor milk is pasteurized for preservation, may be subject to greater risk of allergy development, especially for β-lactoglobulin.

Microbial antigen in human milk: a natural vaccine?

Microbial antigen in human milk: a natural vaccine?

Quantitative glycoproteomics of human milk and association with atopic disease.

The prevalence of allergic diseases and asthma is increasing rapidly worldwide, with environmental and lifestyle behaviors implicated as a reason. Epidemiological studies have shown that children who grow up on farms are at lower risk of developing childhood atopic disease, indicating the presence of a protective "farm effect". The Old Order Mennonite (OOM) community in Upstate New York have traditional, agrarian lifestyles, a low rate of atopic disease, and long periods of exclusive breastfeeding. Human milk proteins are heavily glycosylated, although there is a paucity of studies investigating the milk glycoproteome. In this study, we have used quantitative glycoproteomics to compare the N-glycoprotein profiles of 54 milk samples from Rochester urban/suburban and OOM mothers, two populations with different lifestyles, exposures, and risk of atopic disease. We also compared N-glycoprotein profiles according to the presence or absence of atopic disease in the mothers and, separately, the children. We identified 79 N-glycopeptides from 15 different proteins and found that proteins including immunoglobulin A1, polymeric immunoglobulin receptor, and lactotransferrin displayed significant glycan heterogeneity. We found that the abundances of 38 glycopeptides differed significantly between Rochester and OOM mothers and also identified four glycopeptides with significantly different abundances between all comparisons. These four glycopeptides may be associated with the development of atopic disease. The findings of this study suggest that the differential glycosylation of milk proteins could be linked to atopic disease.

Prosit-TMT: Deep Learning Boosts Identification of TMT-Labeled Peptides.

The prediction of fragment ion intensities and retention time of peptides has gained significant attention over the past few years. However, the progress shown in the accurate prediction of such properties focused primarily on unlabeled peptides. Tandem mass tags (TMT) are chemical peptide labels that are coupled to free amine groups usually after protein digestion to enable the multiplexed analysis of multiple samples in bottom-up mass spectrometry. It is a standard workflow in proteomics ranging from single-cell to high-throughput proteomics. Particularly for TMT, increasing the number of confidently identified spectra is highly desirable as it provides identification and quantification information with every spectrum. Here, we report on the generation of an extensive resource of synthetic TMT-labeled peptides as part of the ProteomeTools project and present the extension of the deep learning model Prosit to accurately predict the retention time and fragment ion intensities of TMT-labeled peptides with high accuracy. Prosit-TMT supports CID and HCD fragmentation and ion trap and Orbitrap mass analyzers in a single model. Reanalysis of published TMT data sets show that this single model extracts substantial additional information. Applying Prosit-TMT, we discovered that the expression of many proteins in human breast milk follows a distinct daily cycle which may prime the newborn for nutritional or environmental cues.

Partial Hydrolyzed Protein as a Protein Source for Infant Feeding: Do or Don’t?

Exclusive breastfeeding until the age of six months is the recommended feeding method for all infants. However, this is not possible for every infant. Therefore, a second choice of feeding, as close as possible to the gold standard, is needed. For historical reasons, this has been cow’s-milk-based feeding. This paper discusses if this second-choice feeding method should contain intact protein or partially hydrolyzed proteins. The limited data available indicates that mother’s milk is relatively rich in bioactive peptides. Whether partially hydrolyzed protein might be a protein source closer to human milk protein content than intact cow’s milk needs further research. However, more research on protein and bioactive peptides in mother’s milk should be a priority for future scientific development in this field. Results of such research will also provide an answer to the question of which option would be the best second choice for infant feeding if sufficient breast milk is not available.

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.

Efficient Bioproduction of Human Milk Alpha-Lactalbumin in Komagataella phaffii.

Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, Komagataella phaffii (K. phaffii) was selected as the production host. We found that the K. phaffii host X33 was suitable for expressing the target protein, yielding 5.2 mg·L-1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters AOX1, GAP, and GAP(m) were compared and applied. The strain with the promoter AOX1 produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (hPDIA3) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L-1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in K. phaffii and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.

Food Proteins in Human Breast Milk and Probability of IgE-Mediated Allergic Reaction in Children During Breastfeeding: A Systematic Review

Previous reports suggested that food proteins present in human milk (HM) may trigger symptoms in allergic children during breastfeeding, but existing evidence has never been reviewed systematically. To assess the probability of food proteins in HM to trigger allergic reactions in infants with IgE-mediated food allergy. Electronic bibliographic databases (MEDLINE, EMBASE) were systematically searched from inception to November 3, 2021. The data regarding the levels of food proteins detected in HM were extracted and compared with data from the Voluntary Incidental Trace Allergen Labelling (VITAL 3.0) guide to assess the probability of food-allergic individuals to experience immediate type allergic reactions on ingesting HM. A total of 32 studies were identified. Fourteen studies assessed excretion of cow's milk proteins into HM, 9 egg, 4 peanut, and 2 wheat; 3 measured levels of cow's milk and egg proteins simultaneously. We found that levels of all food proteins across the studies were much lower than the eliciting dose for 1% of allergic individuals (ED01) in most of the samples. The probability of an IgE-mediated allergic reaction in a food-allergic infant breastfed by a woman consuming the relevant food can be estimated as ≤1:1000 for cow's milk, egg, peanut, and wheat. To our knowledge, this is the first systematic review that assesses and summarizes evidence on food proteins in HM and potential for IgE-mediated allergic reactions. Our data suggest that the probability of IgE-mediated allergic reactions to food proteins in HM is low.

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.

Human Milk Fortifiers for Preterm Infants: Do We Offer the Best Amino Acid Mix?

For preterm and small-for-gestational age infants on enteral nutrition, the best solution is to add human milk fortifier (HMF) to human milk (HM) which is provided by the mother or a milk bank. HMF provides a means to add additional protein, energy, and micronutrients, while maintaining HM as the main source of nutrition. Because of their rapid increase of lean body mass, preterm infants have much higher protein requirements than term infants. Recommendations on protein requirements of preterm infants are available, but protein quality - i.e. the amino acid (AA) profile in HMFs has not been systematically assessed. Present guidelines for enteral nutrition recommend protein intakes around 4 g/kg body weight (BW) for preterm infants <1,500 g, an intake that is not achievable with unfortified HM intakes <200 mL/kg BW/day. It is generally assumed that the AA profile of HM is the best reference for the AA profile of HMF. We calculated advisable intakes of AAs for preterm infants between 400-2,500 g which are based on AA increments of the fetus. Corrections for absorption, inevitable losses, oxidation, and variation of AAs in HM were introduced. Our calculations indicate that extremely low birth weight (ELBW <1,000 g) and very low birth weight (VLBW <1,500 g) infants have substantially higher AA requirements than low birth weight (LBW) infants growing from 1,900 to 2,400 g. In ELBW infants, daily intakes of the different indispensable AAs (IAA) with 4 g of (term) HM protein/kg BW range between 59 and 125% of the respective advisable intakes. Intakes of 7 IAAs and 3 conditionally indispensable AAs (CIAA) are below advisable intakes. On the other hand, with 4 g HM protein per kg BW/day, the IAAs isoleucine and leucine and some dispensable AAs are already supplied in abundance. In VLBW infants, daily intakes of the IAA methionine and 3 CIAAs are still below the advisable intakes. In LBW infants (<2,000 g) receiving 3.5 g HM protein per kg BW daily intakes of 1 IAA and 3 CIAAs would be too low. Preterm infants should receive HMFs which provide adequate amounts of AAs which are needed for their rapid growth and development while avoiding excessive intakes. In particular, very high AA requirements of ELBW infants are a challenge. AA composition of present HMFs for preterm infants should be reconsidered: spiking HMF protein with the AAs which are presently undersupplied or providing targeted AA-based HMF are options to further improve the AA profile in fortifiers.

Интенсивность процессов окислительной модификации белков женского и коровьего молока

Breast milk is a source of all the essential nutritional components necessary for the full growth and development of the child, therefore, it is necessary to study its composition and physical and chemical properties in order to adapt human milk substitutes. Adapted infant milk formulas are produced mainly from cow's milk, bringing formula nutrient composition closer to the composition of women's milk, adapting it in accordance with the requirements of the infant body. However, technological processes for the production of dairy products contribute to the activation of oxidative reactions, the violation of protein conformation. The purpose of the study was to compare the intensity of formation of carbonyl derivatives of human and cow's milk proteins during spontaneous and metal-catalyzed oxidation. Material and methods. The object of the study were samples of mature milk of healthy nursing mothers (n=12), and samples of drinking ultra-pasteurized milk for baby nutrition (n=8) which were used as a comparison material. The intensity of oxidative modification of milk proteins was determined spectrophotometrically by the interaction of carbonyl derivatives of amino acid residues with 2.4-dinitrophenylhydrazine to form 2.4-dinitrophenylhydrazone derivatives in a native sample of biological material and under induction of protein oxidation in vitro by the Fenton reaction by adding FeSO4 and hydrogen peroxide solutions. The content of nonprotein sulfhydryl groups was determined after protein precipitation spectrophotometrically with 5.5'-dithio-bis-2-nitrobenzoic acid. Results. The intensity of spontaneous (basic) oxidation doesn't have significant differences between the indicators of breast and cow's milk. Significant differences were established in the content of carbonyl derivatives of amino acid residues of human and cow's milk proteins during metal-catalyzed oxidation. Incubation with iron ions caused 1.5-2.5 fold more formation of both aldehyde and ketone derivatives of cow's milk proteins, recorded in the visible and ultraviolet spectrum. In cow's milk during spontaneous oxidation and induction of oxidation by a metal, the percentage of aldehyde-dinitrophenylhydrazones was lower than in breast milk and, conversely, the proportion of ketone-dinitrophenylhydrazones, late markers of oxidative degradation of proteins, was significantly higher. The content of non-protein sulfhydryl groups in cow's milk was 2 times less than in fresh human milk. A significant excessive content of aldehyde-dinitrophenylhydrazones (2 times) and ketone-dinitrophenylhydrazones (2.6 times) undet metal-catalyzed protein oxidation of cow's milk in comparison with breast milk indicates a lower level of antioxidant reserves of cow's milk. This is confirmed by the reduced level of non-protein sulfhydryl groups. The results obtained indicate the need to improve the antioxidant status of dairy products for infant nutrition.

Investigation of the Effect of Human Milk Expression Techniques on Milk Content: A Systematic Review.

If the mother and infant cannot meet after birth, it is recommended to express milk and give it to the infant. There was evidence indicating that there might be decrease in essential nutrient values in human milk content depending on the expression technique in literature. The goal of this systematic review was to investigate the effect of human milk expression techniques on the macronutrient milk content and establish an evidence base for future studies. Studies investigating the effect of human milk expression techniques on milk content were reviewed without year limitations. A literature review was conducted in six electronic databases (MEDLINE, Web of Science, PubMed, ScienceDirect, CINAHL and Cochrane) until 30 May 2021, using the keywords of breast milk expression techniques, milk content and breast milk pumping. From 258 articles initially screened, we included 6 articles in the systematic review. The fat, protein and lactose content of human milk was analyzed in the studies reviewed. It was concluded that there was no significant effect on the protein (9.7-9.8 g/dl and 2.1-2.1 g/dl, respectively) and lactose (6.50-6.53% and 6.1-6.1 g/dl, respectively) content of milk. However, the fat (58.30, 48.81g/l; 2.6-2.2 g/dl) content was affected. This study investigated the effect of milk expression techniques on the macronutrient content of human milk, and it was concluded that there was no significant effect on the protein and carbohydrate content of milk. However, the fat content was affected. Limitation of this study is that some factors that might affect the content of human milk were not standardized sufficiently in the included studies.

Impact of holder, high temperature short time and high hydrostatic pressure pasteurization methods on protein structure and aggregation in a human milk protein concentrate

This work evaluated the impact of high temperature short time (HTST, 72 °C, 15 s), high hydrostatic pressure (HHP, 400–600 MPa at 5 and 10 min) and Holder pasteurization (HoP, 62.5 °C, 30 min) on protein profile and aggregation in a human milk protein concentrate (HMPC). The structural changes induced in milk proteins were investigated in HMPC as well as in sedimentable and non-sedimentable fractions recovered after ultracentrifugation. The results showed that heat treatments induced more protein denaturation and aggregation than did HHP treatments. Indeed, heat-induced protein aggregates observed in HMPC and the sedimentable fraction were mainly composed of lactoferrin and α-lactalbumin. More specifically, the concentration of lactoferrin in HMPC decreased by 86% after HTST and HoP whereas no effect was observed after HHP treatment. These results show the potential of HHP processing as a pasteurization method for HMPC since it minimizes the impact on protein structure, which generally correlates to protein quality and bioactivity.

Effect of convection and microwave heating on the retention of bioactive components in human milk

Bioactive substances are very important components of human milk (HM), especially for premature newborns. The effects of convection (CH) and microwave heating (MWH) at 62.5 and 66 °C, on the level of selected bioactive components of HM: lysozyme (LZ), lactoferrin (LF), secretory immunoglobulin A (sIgA), basal lipase (BL), cytokine TGF-2, vitamin C and total antioxidant capacity (TAC) was compared. Regardless of the used heating methods the TAC of HM, determined by TEAC and ORAC-FL assay, proved to be insensitive to temperature pasteurization, in contrary to BL. MWH in the conditions of 62.5 for 5 min and 66 °C for 3 min are ensuring microbiological safety with a higher retention of most of the tested active HM proteins compared to CH. Only in the case of LZ the MWH had a more degradative effect on its concentration. Controlled conditions of MWH preserve the bioactive components of the HM better than CH.

Quantification of FAM20A in human milk and identification of calcium metabolism proteins.

BackgroundFAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel‐renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low‐abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism.MethodThis study used mass spectrometry‐driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions.ResultsShotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC‐MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism.ConclusionUsing mass spectrometry‐driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk.

Digestibility of proteins in camel milk in comparison to bovine and human milk using an in vitro infant gastrointestinal digestion system

The absence of β-lactoglobulin, high β-/αs-casein ratio and protective proteins make camel milk a promising alternative protein base for making human infant formulae. In this study, protein digestibility of camel milk was compared with that of bovine and human milk using an in vitro infant gastrointestinal digestion system. A low degree of gastric proteolysis was observed in all three kinds of milk, and a single clot was formed in camel milk. The soluble milk proteins remaining in the gastric digesta were digested rapidly and extensively in the intestinal phase, while the proteins in the camel milk clot were hydrolysed gradually. Despite several similarities, bioactive peptides unique to individual milk were identified in the three intestinal milk digesta. The results suggest that camel milk proteins are equally digestible as bovine and human milk proteins under infant gastrointestinal digestion conditions, and it may be a prospective substitute for infant formula base.

Processing Human Milk to Increase Nutrient Density for Preterm Infants.

Human milk is the optimal food for newborns. Choices to feed preterm infants in neonatal intensive care units are mother's milk, donor milk, or formula. Preterm infants have better tolerance for human milk, but the lower caloric density of donor milk might not meet preterm infant growth needs. Preterm infants have higher protein and energy requirements with a limited stomach capacity. Therefore, there is a need for human milk with increased nutrient density. To concentrate donor milk to have a higher caloric and protein density while avoiding side effects of high osmolality by precipitating lactose at low temperatures. We investigated the results of volume reduction and lactose removal processes on the lactose, protein, osmolality, and viscosity of human milk. Donor milk was obtained from WakeMed Mothers' Milk Bank. Homogenization and evaporative condensation were applied to samples (N = 36) before they were stored frozen overnight, followed by refrigerated centrifugation for lactose removal at 0 °C. Supernatants were separated and compared to the composition of controls. A significant reduction of lactose (SW = -262, p < .0001) and osmolality (SW = -211.5 p < .01) was achieved in the concentrated milk without a significant protein loss from centrifugation (SW = -44.5, p = .49). A 30%-40% volume reduction is within the American Academy of Pediatrics recommended osmolality for infant feeding. Concentrating human milk in a milk bank setting for feeding preterm infants might be a simple and low-cost process to achieve a product with higher nutrient density and no non-human components.

Proteomics analysis reveals digestion-resistant proteins from colostrum are associated with inflammatory and cytotoxic responses in intestinal epithelial cells.

Although human-milk feeding reduces the risk of necrotizing enterocolitis (NEC) in preterm infants compared with formula feeding, the exact risk-reduction mechanism remains unknown. As NEC occurs at the distal small intestine in which digestion has occurred, we applied proteomics to examine the extent to which colostrum proteins survive simulated infant in vitro-digestion and, thus, have potential to exert biological function. Ten preterm colostrum samples were left undigested or in vitro-digested, and lipopolysaccharide (LPS)-binding protein, soluble cluster of differentiation 14, and tumor necrosis factor (TNF) receptors I and II were measured using enzyme-linked immunosorbent assay in all undigested and in vitro-digested samples. Fully differentiated Caco-2 cells were exposed to digested colostrum samples before stimulation with LPS or TNF or no stimulation. Inflammation (interleukin-8) and cytotoxicity (lactate dehydrogenase) were measured. Proteomic analyses of undigested and in vitro-digested samples were done using mass spectrometry. We found that most proteins in colostrum are significantly, if not completely, degraded after in vitro-digestion. We found select individual and combination digestion-resistant proteins that were positively correlated with LPS- and TNF-induced inflammation. These results indicate the importance of considering the extent to which specific dietary compounds survive digestion to reach their site of claimed action (distal intestine) and that some digestion-resistant proteins may be contributing toward "low-grade" inflammation that is necessary to promote intestinal growth and maturation during early infancy. This work provides the most detailed understanding of human-milk protein degradation with simulated infant in vitro-digestion to date.

Breast Milk Protein Percentiles According to Gestational and Postnatal Age: Could It Be Used as a New Alternative in Guided Fortification?

Human milk is the first choice for infant nutrition but it must be multinutrient fortified for optimum growth and neurodevelopment in preterm infants. However, there is no consensus on ideal fortification method. The authors aimed to generate the human-milk protein content percentiles during the first five postnatal weeks in four preterm groups (n = 108) with median gestational age of 32 (23-36) wk, who were fed adjustably fortified breast milk in the NICU between October 2011 and June 2013. Total 540 breast milk samples of mothers of 108 infants were weekly analyzed for protein intake. It was observed that the median human-milk protein levels decreased throughout the five postnatal weeks in all groups. None of the preterm infants was able to take the recommended daily protein intake with the fortification protocol of the authors' unit. Preterm human-milk protein charts can be used as a new practical individualized fortification guiding method instead of laborious targeted or adjustable approaches currently in use.

Transcriptome Atlas of 16 Donkey Tissues.

Donkeys (Equus asinus) are important livestock with great economic value in meat, skin, and milk production. However, a lack of knowledge of the transcriptome landscape across a wide range of donkey tissues limits genetic selective breeding and conservation. Here we used transcriptomics to describe the transcriptome landscape, classify the tissue-specific gene expression across all primary donkey tissues, and present supplementary analyses on the protein level of additional donkey milk samples. Overall, 16,013 protein-coding genes and 21,983 transcripts were mapped to the reference genome, including 6,778 ubiquitously expressed genes and 2,601 tissue-enriched genes. Functional analysis revealed that the function of the tissue-enriched genes was highly tissue specific. Tissue-elevated genes that could be associated with unique phenotypes in donkey were analyzed. The results showed that, compared with those in human and other livestock, the lysozyme gene in donkey breast was specifically and highly expressed. The calcium-binding lysozyme, encoded by the lysozyme gene, was also detected in high amounts in donkey milk. Given those intact lysozyme genes that predict potentially functional calcium-binding lysozyme found in only a few species (e.g., donkey and horse), the high expression of the lysozyme gene in donkey breast may contribute to the high lysozyme content in donkey milk. Furthermore, 71% of the proteins in donkey milk overlapped with human milk protein, higher than the overlapping rates of bovine, sheep, and swine with humans. The donkey transcriptomic resource contributes to the available genomic resources to interpret the molecular mechanisms underlying phenotype traits.