Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O-glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O-glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O-glycans from 1121 MFGM glycoproteins and 619 site-specific O-glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O-glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O-glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O-glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O-glycoproteins and their site-specific O-glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O-glycosylation.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.

Bioactive natural products in donkey and camel milk: a perspective review.

Mammalian milk has numerous components that exhibit chemical and functional activities. They support human homeostasis. Immunoglobulins, peptides with antibacterial and antimicrobial activities, carbohydrates, lipids, and minor molecules have positive effects on health. Beyond the nutritional values of milk, milk-borne biologically active compounds such as proteins and other minor constituents exhibit essential physiological and biochemical functions. Human milk guarantees a healthy development and improves immunity. It is hypoallergenic. Sometimes, it is necessary to substitute this food with other milk for different reasons. Cow, sheep, goat, camel and donkey milk are natural alternatives. We evaluated the different compounds within donkey and camel milk analysing their biomolecular characteristics and potential benefits for human health. Camel and donkey milk bioactive products could be good candidates for controlling several diseases and excellent substitutes in the case of milk protein allergies in infants. However, more research should be conducted to further evaluate their nutraceutical potential.

A new sight to explore site-specific N-glycosylation in donkey colostrum milk fat globule membrane proteins with glycoproteomics analysis.

Donkey colostrum milk fat globule membrane (DCMFGM) proteins are involved in multiple biological functions. However, the effect of N-glycosylation on their physiological properties are unknown. The aim of this study was to map the DCMFGM protein site-specific N-glycosylation landscape using a label-free glycoproteomic approach. A total of 1,443 unique intact N-glycopeptides mapping to 453 unique N-glycosites on 336 N-glycoproteins were identified. The macro- and microheterogeneity of DCMFGM glycoproteins were explored at the N-glycosite level and the site-specific N-glycan level, respectively, and it was found that the N-glycosylation profiles of the DCMFGM proteins varied based on subcellular localisation and protein domain types. Our findings reveal the heterogeneity and functional diversity of N-glycosylation of DCMFGM proteins and provide theoretical support for the promotion of DCMFGM proteins as a functional food ingredient.

A Systematic Review of the Bioactive Components, Nutritional Qualities and Potential Therapeutic Applications of Donkey Milk.

In addition to providing individualized, specific, and ample nutritional compounds, donkey milk (DM) offers immunological modulation during health and disease. Recently, DM has attracted major interest in preparing infant formulas due to its similarity to human milk in terms of high protein and lactose content and low-fat concentration. The antimicrobial, anti-inflammation, antioxidant, and hypo-allergenicity properties of DM in human infants are well-documented. The purpose of this review is to summarize the knowledge of studies done in characterizing the composition of DM, including bioactive macronutrient levels influenced by the lactation status. The manufacture of DM-based food products and promising therapeutic applications in humans will also be discussed. The beneficial health effects of DM have been extensively studied as a valuable alternative source to breast milk. DM has proven to be a suitable nutrient to relieve milk-related allergies in human infants as opposed to cow's milk. Factors that influence the levels of macronutrients in DM include lactation status, processing, and manufacturing techniques. A wide variety of dairy products have been prepared using DM, such as cheese, ice cream, milk powder, novel functional fermented beverages, and milk powder for infant formulas. The bioactive macromolecules of DM exhibit antibacterial, antiviral, and antifungal effects as well as hypo-allergenicity, anti-inflammation, and antioxidant properties.

Distribution of calcium, phosphorus, sulfur, magnesium, potassium, and sodium in major fractions of donkey milk.

The aim of this study was to evaluate the concentrations of Ca, P, S, Mg, K, and Na, and their distribution in major fractions of donkey milk (i.e., fat, casein, whey proteins, and aqueous phase). Individual milk samples were collected by mechanical milking from 16 clinically healthy lactating donkeys. Milk yield per milking was recorded and milk gross composition, casein content, and pH were determined. Whole milk samples were centrifuged to separate fat and to obtain skim milk. Skim milk samples were ultracentrifuged to separate a sedimentable casein pellet and to obtain a supernatant whey (soluble) fraction, which was then ultrafiltered to obtain the aqueous phase of donkey milk. Whole milk and the processed samples were analyzed for the aforementioned elements by inductively coupled plasma-mass spectrometry. The concentration of elements associated with fat, casein, and whey proteins was then calculated. All the Na was present in the aqueous phase. The fat fraction in donkey milk carried very little or none of the investigated elements. The majority of Ca (62.9%) and P (53.1%) was associated with casein, and the rest of these elements was mostly present in the aqueous phase. The majority of Mg was present in the aqueous phase, but a relevant part (32.6%) was associated with the casein fraction. No K was associated with casein. On a molar basis, the ratio of colloidal Ca and P to casein (mmol/g of casein) was more than double the values reported in literature for cow milk. The correlation coefficient was negative between milk pH and P in the ultracentrifuged (r = -0.81) and ultrafiltered (aqueous) fraction (r = -0.66). Milk pH correlated positively with colloidal Ca (r = 0.59) and with the ratio of colloidal Ca to casein (mmol/g of casein; r = 0.68). Colloidal Ca and P were positively correlated (r = 0.64). These data suggest that the high ratio of colloidal Ca and P to donkey casein micelles is due to a larger amount of colloidal calcium phosphate bound to casein micelles compared with literature data on cow milk. The percentage of elements associated with whey proteins was less than 5% for Ca, P, and K, but Mg reached approximately 9% of total Mg. The majority of S (63.6%) was associated with whey proteins, and only one-fourth of this element was associated with casein, indicating a higher content of sulfur-containing amino acids in donkey whey proteins than in casein.

Urinary Metabolomic Profile of Preterm Infants Receiving Human Milk with Either Bovine or Donkey Milk-Based Fortifiers.

Fortification of human milk (HM) for preterm and very low-birth weight (VLBW) infants is a standard practice in most neonatal intensive care units. The optimal fortification strategy and the most suitable protein source for achieving better tolerance and growth rates for fortified infants are still being investigated. In a previous clinical trial, preterm and VLBW infants receiving supplementation of HM with experimental donkey milk-based fortifiers (D-HMF) showed decreased signs of feeding intolerance, including feeding interruptions, bilious gastric residuals and vomiting, with respect to infants receiving bovine milk-based fortifiers (B-HMF). In the present ancillary study, the urinary metabolome of infants fed B-HMF (n = 27) and D-HMF (n = 27) for 21 days was analyzed by 1H NMR spectroscopy at the beginning (T0) and at the end (T1) of the observation period. Results showed that most temporal changes in the metabolic responses were common in the two groups, providing indications of postnatal adaptation. The significantly higher excretion of galactose in D-HMF and of carnitine, choline, lysine and leucine in B-HMF at T1 were likely due to different formulations. In conclusion, isocaloric and isoproteic HM fortification may result in different metabolic patterns, as a consequence of the different quality of the nutrients provided by the fortifiers.

Comparative exploration of free fatty acids in donkey colostrum and mature milk based on a metabolomics approach.

Donkey milk is an ideal substitute for human milk owing to its similar composition. Nevertheless, changes in the composition and related metabolic pathways of free fatty acids (FFA) in donkey milk between colostrum and mature milk have not been studied well. In this study, metabolomic methods based on gas chromatography tandem time-of-flight mass spectrometry (GC-TOF-MS) were used to explore and compare FFA in donkey colostrum (DC) and mature milk (DMM). A total of 24 FFA were characterized and quantified in DC and in DMM. Of these, 11 FFA differed significantly between DC and DMM, and there were 6 key differential metabolic pathways. These results demonstrated that the composition of FFA in donkey milk changed with lactation stage. The interactions and metabolic pathways were further analyzed to explore the mechanisms that altered the milk composition during lactation. Our results provide insights into the changes in milk of the nonruminant mammals during lactation. The results provide practical information for the development of donkey milk products and a foundation for future research on specific milk nutrients.

Dezhou donkey (Equus asinus) milk a potential treatment strategy for type 2 diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: Donkey (Equus asinus) milk has become a medical and nutrient product since ancient times. In addition, donkey milk was regarded as a medicinal food and substitute product for infant formula in some ancient western countries. Chinese ancient medical books documented the medicinal value of donkey milk, using donkey milk to treat diabetes, cough and jaundice. AIM OF THE STUDY: To investigate the donkey milk's components and anti-diabetic effect of donkey milk in vitro and in vivo and to study the molecular mechanism of donkey milk was an anti-diabetic medication. MATERIALS AND METHODS: In this study, the gastrointestinal digested donkey milk was simulated in vitro and its products of protein digestion were analyzed by SDS-PAGE. We then performed cell viability assay, insulin secretion assay, animal experiments and ELISA assays to study the anti-diabetic effect of donkey milk in vitro and in vivo. Donkey milk's anti-diabetic molecular mechanism and specific targets were detected by using quantitative real time PCR. RESULTS: Lysozyme (LZ) and α-lactalbumin (α-La) exhibited significantly lower digestibility and higher retention than the other components of donkey milk. In vitro, 500 µg/mL of donkey milk could improve damaged ß-cells viability significantly (P < 0.0001). In vivo, the blood glucose and HOMA-IR of diabetic rats treated with donkey milk were 14.23 ±â€¯5.18 mM and 74.94 ±â€¯23.62, respectively, whereas the diabetic group were 22.18 ±â€¯2.23 mM and 112.16 ±â€¯18.44, respectively (P < 0.01). The SOD value of donkey milk group was 265.87 ±â€¯21.29 U/L, while the SOD value of diabetic group was 193.20 ±â€¯52.07 U/L (P < 0.05). These results indicated that the blood glucose was reduced, the ability of the body to eliminate free radicals was enhanced, antioxidant levels in the body was increased, insulin resistance was improved in type 2 diabetic rats after donkey milk powder fed for 4 weeks. Furthermore, donkey milk could treat diabetes through down-regulating phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6PC). CONCLUSIONS: Donkey milk has played an important role in the treatment of type 2 diabetes, and contributed to the development of the donkey milk products.

Comparative metabolomics analysis of donkey colostrum and mature milk using ultra-high-performance liquid tandem chromatography quadrupole time-of-flight mass spectrometry.

Donkey milk has been widely shown to be an ideal substitute for human milk because of its similar composition. However, alterations to the composition of donkey milk during lactation have not been well studied. In this study, untargeted metabolomics with ultra-high-performance liquid tandem chromatography quadrupole time-of-flight mass spectrometry were used to analyze and compare the metabolites in donkey colostrum (DC) and mature milk (DMM). Two hundred seventy metabolites were characterized in both DC and DMM. Fifty-two of the metabolites in the DC were significantly different from those in the DMM; 8 were downregulated and 44 were upregulated. This demonstrated that the composition of the donkey milk changed with lactation. Additionally, the interactions and metabolic pathways were further analyzed to explore the mechanisms that altered the milk during lactation. Our results provide comprehensive insights into the alterations in donkey milk during lactation. The results will aid in future investigations into the nutrition of donkey milk and provide practical information for the dairy industry.

Comparative analysis of whey proteins in donkey colostrum and mature milk using quantitative proteomics.

Donkey milk is attracting increasing attention as a nutritional milk source similar to human milk. In this study, we carried out qualitative and quantitative analysis of the donkey whey proteome using a label-free proteomic approach, combined with parallel reaction monitoring (PRM) as a validation method. A total of 300 whey proteins were identified in donkey colostrum (DC) and donkey mature (DM) milk, of which 18 were differentially expressed (P < 0.05) between the two types of milk. Gene ontology (GO) analysis showed that differentially and uniquely expressed proteins were mainly involved in cellular processes, response to stimulus, metabolic processes, and biological regulation. Their molecular functions included binding, catalytic activity, and molecular functional regulation, and their main annotated areas of origin were the cell, cell-part, and the extracellular region. Most differentially and uniquely expressed proteins were linked with malaria, systemic lupus erythematosus, or antigen processing and presentation. Our results provide insight into the complexity of the donkey whey proteome and molecular evidence for nutritional differences between different lactation stages.

The nutritional ingredients and antioxidant activity of donkey milk and donkey milk powder.

The aim of this work was to shed light on the nutritional ingredients and antioxidant activity of donkey milk and powder. It indicated that basic components of donkey milk were closed to human milk, especially lactose. The fat content of donkey milk was lower than that of cow milk and human milk. The essential amino acids, fatty acids and taurine in donkey milk and powder were richer. The whey protein content of donkey milk was 49.08 g/100 g. The ratio of casein to whey protein in donkey milk and powder was 70.3:100. The cholesterol of donkey milk was 8.6 mg/100 g. Therefore, donkey milk and powder are typical low-fat and low-cholesterol foods. The precious values of donkey milk and powder are suitable calcium and phosphorus proportion, and strongly resistant to oxidation. In conclusion, donkey milk and powder are good supplementary of cow milk and powder, would be better for infant dietary.

Donkey milk as a supplement in infant formula: Benefits and technological challenges.

The aim of this review paper is to assess the applicability of donkey's milk to infants suffering from Cow Milk Protein Allergy (CMPA) compared to human and other available milk types. The bioactive and immune-supportive character which could be beneficial as a fortifier to the formula-fed infants is described while limitations of this type of milk are also discussed. Studies showed that human and donkey's milk have similar, overall, chemical composition as well as protein homogeneity and antigenic similarities. Several in vitro and in vivo studies showed that donkey's milk has nutraceutical and functional properties that can support immunity, alter metabolism and beneficially modify gut microbiota. Clinical studies illustrated that donkeys' milk is well tolerated (82.6%-88%) by infants. Finally, the effect that processing (i.e. thermal, non-thermal treatments, drying methods) has on donkey milk components is also discussed pointing out the need for minimally processing this type of milk.

Nutritional adequacy of a novel human milk fortifier from donkey milk in feeding preterm infants: study protocol of a randomized controlled clinical trial.

BACKGROUND: Fortification of human milk is a standard practice for feeding very low birth weight infants. However, preterm infants often still experience suboptimal growth and feeding intolerance. New fortification strategies and different commercially available fortifiers have been developed. Commercially available fortifiers are constituted by a blend of ingredients from different sources, including plant oils and bovine milk proteins, thus presenting remarkable differences in the quality of macronutrients with respect to human milk. Based on the consideration that donkey milk has been suggested as a valid alternative for children allergic to cow's milk proteins, due to its biochemical similarity to human milk, we hypothesized that donkey milk could be a suitable ingredient for developing an innovative human milk fortifier. The aim of the study is to evaluate feeding tolerance, growth and clinical short and long-term outcomes in a population of preterm infants fed with a novel multi-component fortifier and a protein concentrate derived from donkey milk, in comparison to an analogous population fed with traditional fortifier and protein supplement containing bovine milk proteins. METHODS: The study has been designed as a randomized, controlled, single-blind clinical trial. Infants born <1500 g and <32 weeks of gestational age were randomized to receive for 21 days either a combination of control bovine milk-based multicomponent fortifier and protein supplement, or a combination of a novel multicomponent fortifier and protein supplement derived from donkey milk. The fortification protocol followed is the same for the two groups, and the two diets were designed to be isoproteic and isocaloric. Weight, length and head circumference are measured; feeding tolerance is assessed by a standardized protocol. The occurrence of sepsis, necrotizing enterocolitis and adverse effects are monitored. DISCUSSION: This is the first clinical study investigating the use of a human milk fortifier derived from donkey milk for the nutrition of preterm infants. If donkey milk derived products will be shown to improve the feeding tolerance or either of the clinical, metabolic, neurological or auxological outcomes of preterm infants, it would be an absolute innovation in the field of feeding practices for preterm infants. TRIAL REGISTRATION: ISRCTN - ISRCTN70022881 .

Minor and potentially toxic trace elements in milk and blood serum of dairy donkeys.

The aim of this trial was to study the concentration of Ti, V, As, Rb, Sr, Mo, Cd, Cs, and Pb in donkey milk and blood serum. One hundred twelve individual milk and blood serum samples were collected from 16 lactating donkeys (Martina-Franca-derived population; 6 to 12 yr old; 3 to 7 parities; average live weight 205.4kg; 32 to 58 d after foaling at the beginning of the trial) during a 3-mo-long experiment. The samples were analyzed for the aforementioned elements by inductively coupled plasma-mass spectrometry. Feedstuff and drinking water were also analyzed for the investigated elements. Data were processed by ANOVA for repeated measures. Average milk concentrations (±SD) of Ti, Rb, Sr, Mo, Cs, and Pb were 77.3 (±7.7), 339.1 (±82.1), 881.7 (±270.4), 4.5 (±1.6), 0.49 (±0.09), and 3.2 (±2.7) µg/L, respectively. More than 80% of samples were below the limit of detection for V, As, and Cd in milk and for Cd, and Pb in blood serum. The lower bound calculated for milk V, As, and Cd was 0.03µg/L for the 3 elements, the upper bound was calculated at 0.23, 0.10, and 0.31µg/L and the maximum value was observed at 0.54, 0.15, and 0.51µg/L, respectively. The average milk concentrations of Ti, Rb, Sr, Mo, and Cs were 600, 458, 346, 16, and 294%, respectively, than those of blood serum. Yet, Cs concentrations were in the same order of magnitude in milk and serum. Moderate to strong positive and significant correlation coefficients were observed between milk and blood serum concentrations for Ti, Rb, Sr, and Cs. The effect of the stage of lactation was significant for all the investigated elements in milk and blood serum, but most of the elements showed only small changes or inconsistent trends, and only the concentrations of Rb and Sr showed decreasing trends both in milk and blood serum. The relationship between milk and blood serum element concentrations indicates that the mammary gland plays a role in determining the milk concentrations of Mo, Ti, Rb, Sr, Mo, and Cs. In the current experimental conditions, in agreement with the low levels in drinking water and feedstuff, donkey milk concentration of potentially toxic elements was very low and did not raise health concerns for human consumption.