Effect of thermal and non-thermal processing methods on the Structural and Functional Properties of Whey Protein from Donkey Milk.

This study evaluated the impact of thermal, ultrasonication, and UV treatment on the structural and functional properties of whey proteins from donkey milk (DWP). Whey proteins exhibited notable stability in non-heat-treated environments, while their structural and functional characteristics were notably impacted by excessive heat treatment. The application of high-temperature long-time thermal treatment (HTLT) resulted in a decrease in fluorescence intensity, foaming and emulsification stability, and considerable damage to the active components of the proteins. Specifically, the preservation of lysozyme activity was only 23%, and lactoferrin and immunoglobulin G exhibited a significant loss of 70% and 77%, respectively. Non-thermal treatment methods showed superior efficacy in preserving the active components in whey proteins compared with heat treatment. Ultrasonic treatment has demonstrated a notable capability in diminishing protein particle size and turbidity, and UV treatment has been observed to have the ability to oxidize internal disulfide bonds within proteins, consequently augmenting the presence of free sulfhydryl groups, which were beneficial to foaming and emulsification stability. This study not only offers a scientific basis for the processing and application of DWP but also serves as a guide to produce dairy products, aiding in the development of dairy products tailored to specific health functions.

Characterization and discrimination of donkey milk lipids and volatiles across lactation stages.

The lipid and flavor in milk are key factors that affect its quality, which profiles during donkey lactation are not yet clear. In this study, the lipids and volatile compounds (VOCs) in donkey milk from stages of lactation were analyzed by using LC-MS and GC-IMS. A total of 1774 lipids were identified in donkey milk, spanning over 6 major categories and attributed to 30 subclasses. The 233 differentially expressed lipids were identified between donkey colostrum and mature milk, which participate in 20 metabolic pathways, including glycerophospholipid, linoleic acid, and sphingolipid. Additionally, 35 VOCs in donkey milk were identified, including 28.57% aldehydes, 28.57% ketones, 25.71% esters, and 8.57% alcohols. Of these VOCs, 15 were determined to be characteristic flavors in donkey milk, mainly including methyl 2-methylbutanoate, 2-pentanone, and butyl acetate. 11 significantly different VOCs were found between the groups. Acetone, 2-heptanone, and ethyl acetate-m were considered potential discriminatory markers.

Donkey milk as a non-bovine alternative: a review of its nutri-functional properties, applications, and challenges.

Elevation in incidences of cow milk protein allergies warrants the need to investigate the suitability of non-bovine milk alternatives for human consumption. Donkey milk has emerged as a potential alternative attributed to its benefits to human health. Evidently, it is a great option for infants as it closely resembles human milk. Researchers have also investigated its suitability in producing numerous dairy products. This review discusses the various nutri-functional aspects of donkey milk, its applications and challenges in the manufacturing of infant formula, yogurt, cheese, ice cream, kefir, and fermented milk. Research updates on processing techniques (thermal and non-thermal) for donkey milk preservation are also delineated. Despite abundant nutrients and desirable functional properties, the growth of the donkey milk industry is not significant. This is due to the lower yield, scattered population, and lack of regulatory standards for both products and processing. Recommendation on research gaps and obstacles in its commercialization are also addressed.

Isolation of 60 strains from fermented milk of mares and donkeys in Algeria and identification by 16S rRNA sequencing of lactobacilli: Assessment of probiotic skills of important strains and aromatic productivity power.

Background and Aim: Donkey and mare milk have high nutritional and functional values, but their lactic acid bacteria (LAB) content remains poorly studied and undervalued in the Algerian dairy industry. This study aimed to isolate and select LAB strains that produce antimicrobial substances during fermentation and to characterize the probiotic profiles of each extracted strain to indicate their potential for antioxidant and proteolytic activity. Materials and Methods: This study focuses on isolating and identifying lactic acid bacterial strains from 10 Equid-fermented milk samples collected in two regions of El Bayed Wilaya (Algeria). Identification of LAB strains was obtained by 16S rRNA sequencing. The probiotic properties of important strains and their aromatic productivity power are assessed. To evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, Chryseobacterium joostei, Pseudomonas aeruginosa, and Escherichia coli, we selected 21 strains. Different induction methods have been used to amplify the antibacterial effects against these pathogenic strains. Results: Among a total of 60 identified strains, 31 had a probiotic profile, and most were catalase-negative. Aromatic productivity power was observed in eight strains: Lactiplantibacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Weissella confusa, Weissella cibaria, Leuconostoc mesenteroides, Leuconostoc lactis, and Lactobacillus sp1. Conclusion: Our results provide insight into the considerable diversity of LAB present in fermented donkey and mare milk. To meet the expectations of the Algerian dairy industry, it is important that the probiotic skills of the nine selected strains are met. In addition, a significant number of these strains may have important probiotic activity and biotechnological potential.

Characterisation and comparison of enzymatically prepared donkey milk whey protein hydrolysates.

This study evaluated the structural characteristics, processing properties, and antioxidant properties of hydrolysates prepared from donkey milk (DM) whey protein using different proteases (Alcalase, Neutrase, papain, and Flavourzyme). The results showed that enzymatic hydrolysis significantly increased hydrolysate solubility and reduced average particle size compared to those of DM whey protein. Neutrase and Flavourzyme hydrolysates exhibited higher degrees of hydrolysis (DH), along with elevated emulsification properties and surface hydrophobicity. The choice of protease influenced secondary and tertiary protein structures and amino acid composition. Enzymatic hydrolysis led to decreased molecular weight of DM whey proteins. Moreover, all hydrolysates exhibited higher fluorescence intensity at λmax compared to DM whey protein, implying distinct properties due to the varied impacts of the four proteases on DM whey protein structure. The preparation of hydrolysates from DM whey proteins using proteases contributes to the development of integrated-value DM products.

The Use of a Novel Donkey Milk-Derived Human Milk Fortified in the Neonatal Period Had No Effect on the Frequency of Allergic Manifestations During the First Years of Life: The "Fortilat Trial" Follow-Up.

Background: Since human milk contents does not meet the high need of very low birth weight infants, fortification of breast milk is a standard practice for this population. As donkey milk has been long considered for children allergic to cow's milk proteins due to its low allergic properties, a new donkey milk-derived fortifier (DF) has been recently evaluated as a valid alternative to bovine milk-derived fortifier (BF). It seems to improve feeding tolerance when compared with standard BF, with similar neurodevelopmental and auxological outcome at 18 months of age. The aim of this study is to evaluate the development of allergic manifestations occurring in the population of the "Fortilat Trial" at 6-8 years of age. Methods: Allergic manifestations were assessed by an ad hoc questionnaire administered to families. The occurrence of asthma, allergic rhinitis and oculorhinitis, rashes and atopic dermatitis, food allergies, accesses to an emergency department for allergic reactions, and the need of antihistamine have been investigated. Results: In total, 113 infants were enrolled in the study (BF arm: n = 60, DF arm: n = 53). No difference in risk was observed between the two groups for all the considered outcomes. In conclusion, our data suggest that DF does not impact the development of allergic manifestations in the first years of life. Clinical Trial Registration number: ISRCT N70022881.

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.

Analysis of the Differentially Expressed Proteins in Donkey Milk in Different Lactation Stages.

Proteins in donkey milk (DM) have special biological activities. However, the bioactive proteins and their expression regulation in donkey milk are still unclear. Thus, the differentially expressed proteins (DEPs) in DM in different lactation stages were first investigated by data-independent acquisition (DIA) proteomics. A total of 805 proteins were characterized in DM. The composition and content of milk proteins varied with the lactation stage. A total of 445 candidate DEPs related to biological processes and molecular functions were identified between mature milk and colostrum. The 219 down-regulated DEPs were mainly related to complement and coagulation cascades, staphylococcus aureus infection, systemic lupus erythematosus, prion diseases, AGE-RAGE signaling pathways in diabetic complications, and pertussis. The 226 up-regulated DEPs were mainly involved in metabolic pathways related to nutrient (fat, carbohydrate, nucleic acid, and vitamin) metabolism. Some other DEPs in milk from the lactation period of 30 to 180 days also had activities such as promoting cell proliferation, promoting antioxidant, immunoregulation, anti-inflammatory, and antibacterial effects, and enhancing skin moisture. DM can be used as a nutritional substitute for infants, as well as for cosmetic and medical purposes. Our results provide important insights for understanding the bioactive protein differences in DM in different lactation stages.

Effects of high hydrostatic pressure on antimicrobial protein stability and the rheological and shelf-life properties of donkey milk.

The effects of high hydrostatic pressure (HHP) and heat treatments on antimicrobial protein stability and on the physico-chemical, microbiological, rheological and shelf-life properties of donkey milk were investigated. Although heat treatment at 75°C for 2 min resulted in 1.50 log CFU ml-1 microbial inactivation, losses in activities of lysozyme (58%) and lactoferrin (82%) were observed due to whey protein denaturation. By contrast, HHP application at 400 MPa caused lower enzyme activity losses (22 and 37% respectively) whilst maintaining a significant reduction of microbial load (1.80 log CFU ml-1). Color analyses showed that the lightness values of all samples decreased during storage. Higher flow consistency (viscosity) and lower flow behavior indexes were observed in heat-treated samples compared to untreated and HHP-treated ones, which can be explained by advanced protein denaturation during heat-treatment. The results suggest that HHP is a more suitable process than heat treatment for preservation of donkey milk within the conditions studied.

Proteomics and Peptidomics As a Tool to Compare the Proteins and Endogenous Peptides in Human, Cow, and Donkey Milk.

Cow's milk is the most widely used ingredient in infant formulas. However, its specific protein composition can cause allergic reactions. Finding alternatives to replace cow's milk and fill the nutritional gap with human milk is essential for the health of infants. Proteomic and peptidomic techniques have supported the elucidation of milk's nutritional ingredients. Recently, omics approaches have attracted increasing interest in the investigation of milk because of their high throughput, precision, sensitivity, and reproducibility. This review offers a significant overview of recent developments in proteomics and peptidomics used to study the differences in human, cow, and donkey milk. All three types of milks were identified to have critical biological functions in human health, particularly in infants. Donkey milk proteins were closer in composition to human milk, were less likely to cause allergic reactions, and may be developed as novel raw materials for formula milk powders.

Identification and analysis of miRNAs expression profiles in human, bovine, and donkey milk exosomes.

The purpose of this study is to identify and characterize mirnas in mammalian exosomes. Using Illumina sequencing technology, we sequenced miRNAs in the exosomes of mammalian human milk, bovine milk, and donkey milk. 36 known mature miRNAs and 256 novel miRNAs were identified in human milk. 61 known mature miRNAs and 346 novel miRNAs were identified in milk. 16 known mature miRNAs and 196 novel miRNAs were identified in donkey milk, and miRNAs target genes were predicted. Gene Ontology analysis showed that the miRNAs of human, bovine and donkey milk exosomes all labeled the functions related to body metabolism. Kyoto Encyclopedia pathway analysis showed that human, bovine and donkey milk miRNAs enriched AGE-RAGE signaling pathway in Complications of diabetes. Diabetes is a Metabolic disorder. Based on this pathway, we screened out hsa-miR-8485, bta-miR-342, miR-29c and other genes related to diabetes. This study has a new understanding of the physiological function of mammalian milk miRNAs, and also provides a new way to explore diabetes related miRNAs.

Effects of Roughage on the Lipid and Volatile-Organic-Compound Profiles of Donkey Milk.

The lipid molecules and volatile organic compounds (VOCs) in milk are heavily influenced by diet. However, little is known about how roughage affects the lipid and VOC contents of donkey milk. Accordingly, in the present study, donkeys were fed corn straw (G1 group), wheat hulls (G2 group), or wheat straw (G3 group), and the lipid and VOC profiles of their milk were determined using LC-MS and GC-MS. Of the 1842 lipids identified in donkey milk, 153 were found to be differential, including glycerolipids, glycerophospholipids, and sphingolipids. The G1 group showed a greater variety and content of triacyclglycerol species than the G2 and G3 groups. Of 45 VOCs, 31 were identified as differential, including nitrogen compounds, esters, and alcohols. These VOCs were significantly increased in the G2 and G3 groups, with the greatest difference being between the G1 and G2 groups. Thus, our study demonstrates that dietary roughage changes the lipid and VOC profiles of donkey milk.

Donkey Colostrum and Milk: How Dietary Probiotics Can Affect Metabolomic Profile, Alkaline Sphingomyelinase and Alkaline Phosphatase Activity.

Positive results on animal health, feed efficiency, and milk's nutritional content have been obtained after oral administration of probiotics. The aim of the present study was therefore to evaluate the effect of dietary supplementation with high numbers of multispecies probiotic formulations on the milk metabolomic profiles of alkaline sphingomyelinase (alk-SMase) and alkaline phosphatase (ALP) in donkeys. Twenty animals were randomly allocated to receive either a normal diet (group B) or a supplemented diet (group A). Colostrum and milk samples were obtained within 48 h, at 15 days (supplementation start), and at 45 days after parturition. Different metabolomic profiles were observed between colostrum and milk, as were the concentrations of 12 metabolites that changed following 30 days of probiotic supplementation. Alk-SMase activity was found to be higher in donkey colostrum (vs. milk at 15 days); this enzyme, together with ALP, increased in milk after 30 days of probiotic supplementation. The results of the present study provide new insight into the complex changes in donkey colostrum and milk composition in the first 45 days of lactation and how the milk metabolome can be modulated by probiotic supplementation.

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.

Invited review: Human, cow, and donkey milk comparison: Focus on metabolic effects.

Milk is an important food of the daily diet. Many countries include it in their dietary recommendations due to its content in several important nutrients that exert beneficial effects on human health. Human milk is a newborn's first food and plays an important role in the growth, development, and future health of every individual. Cow milk is the type of milk most consumed in the world. However, its relatively high content of saturated fats raises concerns about potential adverse effects on human health, although epidemiological studies have disproved this association. Indeed, dairy consumption appear to be linked to a lower risk of mortality and major cardiovascular disease events. In the last few years many researchers have begun to focus their attention on both the production and quality of cow milk as well as the analysis of milk from other animal species to evaluate their effect on human health. The need to investigate the composition and metabolic effects of milk from other animal species arises from the adverse reactions of individuals in several groups to certain components of cow milk. It has emerged that donkey milk compared with that of other animal species, is the nearest to human milk and an excellent substitute for it. Milk from various animal species shows substantial differences in nutritional composition and distinct metabolic effects. In this review, we discussed the main compositional features and metabolic effects of 3 types of milk: human, cow, and donkey milk.

Effects of donkey milk on UVB-induced skin barrier damage and melanin pigmentation: A network pharmacology and experimental validation study.

Introduction: Dairy products have long been regarded as a controversial nutrient for the skin. However, a clear demonstration of donkey milk (DM) on skincare is required. Methods: In this study, spectrum and chemical component analyses were applied to DM. Then, the effects of DM on UVB-induced skin barrier damage and melanin pigmentation were first evaluated in vitro and in vivo. Cell survival, animal models, and expression of filaggrin (FLG) were determined to confirm the effect of DM on UVB-induced skin barrier damage. Melanogenesis and tyrosinase (TYR) activity were assessed after UVB irradiation to clarify the effect of DM on whitening activities. Further, a network pharmacology method was applied to study the interaction between DM ingredients and UVB-induced skin injury. Meanwhile, an analysis of the melanogenesis molecular target network was developed and validated to predict the melanogenesis regulators in DM. Results: DM was rich in cholesterols, fatty acids, vitamins and amino acids. The results of evaluation of whitening activities in vitro and in vivo indicated that DM had a potent inhibitory effect on melanin synthesis. The results of effects of DM on UVB­induced skin barrier damage indicated that DM inhibited UVB-induced injury and restored skin barrier function via up-regulation expression of FLG (filaggrin). The pharmacological network of DM showed that DM regulated steroid biosynthesis and fatty acid metabolism in keratinocytes and 64 melanin targets which the main contributing role of DM might target melanogenesis, cell adhesion molecules (CAMs), and Tumor necrosis factor (TNF) pathway. Discussion: These results highlight the potential use of DM as a promising agent for whitening and anti-photoaging applications.

Milk from Halari Donkey Breed: Nutritional Analysis, Vitamins, Minerals, and Amino Acids Profiling.

This current research set out to characterize Halari donkey milk by investigating its nutritional constituents, including its proximate analysis, water activity, titratable acidity, energy, and microbiological analysis. A comprehensive profiling of vitamins, minerals, and amino acids was also carried out. It was found that the composition of Halari donkey milk was consistent with previously published donkey milk literature and was comparable to that of human milk. Halari donkey milk has low 0.86 ± 0.04% fat content, 2.03 ± 0.03% protein content, 0.51 ± 0.05% ash content, and high 5.75 ± 0.15% lactose content making it sweet and palatable. The energy content of Halari donkey milk was 40.39 ± 0.31 kcal/100 g, and the water activity ranged from 0.973 to 0.975. Titratable acidity was 0.03 ± 0.01%. Halari donkey milk can be considered acceptable and microbiologically safe, having low total plate count and yeast and mould counts. Mineral testing revealed that Halari donkey milk included significant amounts of magnesium, sodium, calcium, potassium, phosphorus, and zinc. The concentration of different vitamins and amino acids such as isoleucine and valine also contribute to the nutritional value of Halari donkey milk.

Microbiological Quality of Raw Donkey Milk from Serbia and Its Antibacterial Properties at Pre-Cooling Temperature.

The aim of this study was to examine the microbiological quality of raw donkey milk of an indigenous Serbian breed as well as the changes in the microbial populations during storage at 4 °C. In addition, antibacterial activity of donkey milk against E. coli, L. monocytogenes and S. aureus at 15 °C as well as the content of the two main antibacterial proteins lysozyme and lactoferrin were investigated. Microbiological examination of 137 individual milk samples collected over a period of 21 months showed good microbiological quality since foodborne pathogens such as Salmonella spp. and L. monocytogenes were not detected in any of the analyzed samples, while the number of E. coli, Enterobacteriaceae, total coliform bacteria, sulfite-reducing Clostridia and aerobic sporogenic bacteria was below the limit of quantification (<1 cfu mL-1). During the six-days storage at 4 °C, total bacterial counts and the counts of lactic acid bacteria remained at the initial level while pathogenic bacteria were not detected. The strongest antibacterial activity of the tested milk was observed against E. coli, while S. aureus was the least sensitive to milk antibacterial compounds. Although further research is needed to fully elucidate the antibacterial mechanism and synergistic activity of different compounds in donkey milk, the high content lysozyme (2.63 ± 0.03 g L-1) and lactoferrin (15.48 mg L-1) observed in tested milk could contribute to its strong antibacterial activity and extension of the storage period during which it can be safely consumed.

Donkey Milk: An Overview of its Chemical Composition and Main Nutritional Properties or Human Health Benefit Properties.

The donkey milk has a remarkable similarity to human milk, in addition to its valuable nutritional composition and content of numerous immune factors. The donkey milk is the subject of research worldwide, and data from the literature suggest significant differences with respect to the contents of individual components. However, some basic characteristics of donkey milk have been established: low contents of fat and cholesterol, total proteins and casein and high contents of lactose, whey proteins, calcium, selenium, and Vitamin D3. The donkey milk is rich in various protective proteins (α-lactalbumin, lysozyme, lactoferrin, lactoperoxidase, and immunoglobulins), and shows strong antioxidant, antibacterial, antiviral, antifungal, hypoglycemic, antiparasitic, and antitumor activity. Donkey milk can be considered functional food, having in mind that its fatty-acid profile and content of essential fatty acids are beneficial for cardiac health. The total fat content in donkey milk is low compared to human milk, and for this reason it is necessary to enrich donkey milk with other fat in order to provide enough calories in a diet for children. Commercialization of donkey milk and dairy products is still limited due to low production levels, that is lack of products on the market, and lack of product information. Considering the research data from the literature, there is a need for human clinical trials in order to obtain a stronger evidence of the therapeutic properties of donkey milk.

Glycoproteomics analysis reveals differential site-specific N-glycosylation of donkey milk fat globule membrane protein during lactation.

N-glycosylation is a prevalent and complex post-translational modification of milk proteins with significant biological importance. However, the systematic characterisation of donkey milk fat globule membrane (MFGM) N-glycoproteins remains largely ill-defined. Here, 1443 intact N-glycopeptides from 336 MFGM glycoproteins in donkey colostrum (DC) and 489 intact N-glycopeptides from 86 MFGM glycoproteins in donkey mature milk (DM) were identified via label-free site-specific glycoproteomics. Mannosylation and fucosylation were predominant in DC MFGM N-glycoproteins compared to sialylation and mannosylation in DM. Among them, 22 site-specific N-glycans attached to 14 glycosites of eight glycoproteins were significantly increased, whereas 30 site-specific N-glycans attached to 19 glycosites of 16 glycoproteins were significantly decreased. Furthermore, the site-specific N-glycans with Neu5Gc moieties or simultaneous fucosylation and sialylation were not significantly increased, exhibiting significant site specificity. We provide new insights into the composition of donkey MFGM N-glycoproteins and their roles in donkey milk-related biological functions.