Quantitative Phosphoproteome of Infant Formula: New Insights into the Difference of Phosphorylation in Milk Proteins between Bovine and Goat Species.

Phosphorylation is a broad post-translational protein modification, and the level of phosphorylation of milk proteins is associated with lactation, coagulation properties, and digestibility. However, phosphoproteins in bovine milk-based and goat milk-based infant formula have not been systematically explored. Here, we have analyzed six bovine and six goat milk-based infant formula using a quantitative phosphoproteomics approach, from which we identified 200 phosphoproteins with 276 phosphorylation sites and 156 phosphorylation sites from 75 phosphoproteins, respectively. Of these, 99 phosphorylation sites from 26 shared phosphoproteins were differentially expressed between bovine and goat milk-based infant formula. Especially, CSN1S1 was the most phosphoprotein with 25 quantified phosphorylation sites. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the identified phosphoproteins not only provide nutrition to the infant but also have anti-inflammatory, antipathogenic, and other biological functions. Our results shed light on the composition, phosphorylation sites, and biological functions of phosphoproteins in bovine milk and goat milk-based infant formula, which provide new insights into the key role of protein modifications during infant development. It also helps us to better understand the differences in digestibility of infant formula from different animal milk sources and thus guides the choice of milk source for infant formula.

Comparative proteomics of whey proteins: New insights into quantitative differences between bovine, goat and camel species.

Whey proteins are the leading proteins class in milk and play an essential role in the immune defense of neonatal mammals. The aim of this study was to analyze whey proteins in bovine, goat and camel milk by label free proteomics techniques. Finally, 840 proteins were identified, which considerably increasing the number of whey proteins identified in these species. The results of the PCA revealed significant differences in whey proteome patterns between bovine, goat and camel milk. Proteins such as PAEP, CST3, SERPING1, CTSB and GLG1 play an important role as markers in the classification of bovine, goat and camel milk. Statistical analysis showed that the relative abundances of many whey proteins such as ALB, LALBA, LTF and LPO were significantly different among different species. GO and KEGG functional analysis have shown that while the distribution of biological functions involved in whey proteins was relatively similar across species, they differed in terms of protein quantity. These data shed light on the quantitative differences and potential physiological functions of whey proteins across species, and may point the way to the production of specific functional whey proteins.

Comparison of milk fat globule membrane protein profile among bovine, goat and camel milk based on label free proteomic techniques.

Milk fat globule membrane (MFGM) proteins, which are recognized to play a variety of biological roles; nevertheless, their composition and interspecies complexity remain unknown. This study aimed to identify and quantify proteins extracted from MFGM-enriched fractions of bovine, goat and camel milk by a label free proteomics approach. We identified 1579 proteins, substantially increasing the number of MFGM proteins identified for these species. The results of functional analysis showed that the identified MFGM proteins had similar functional annotations, with cellular processes, intracellular anatomical structures and binding being the predominant GO annotations. And most of the proteins were involved in KEGG pathways such as protein processing of ribosomes and endoplasmic reticulum. Subsequently, multiple statistical methods were used to compare the differences in MFGM protein quantification among different species. The results of principal component analysis showed that bovine, goat and camel milk MFGM proteins were significantly different, among which CSN1S1, XDH, LTF and PI4KA played an important role in taxonomic identification. Meanwhile the results of hierarchical clustering showed that the differences (p < 0.05) between bovine and goat milk were smaller than those of camel milk. These new data deepen the understanding of the protein composition of MFGM and its possible physiological roles, providing directions for the production of specific functional milk proteins.

Phosphoproteomics reveals that camel and goat milk improve glucose homeostasis in HDF/STZ-induced diabetic rats through activation of hepatic AMPK and GSK3-GYS axis.

Diabetes is a serious public health problem with global implications. Among many diabetes management therapies, non-pharmacological therapies such as those that focus on diet and exercise are gradually becoming more acceptable to patients. Within dietary management options, dairy products such as camel and goat milk are valued for their specific health benefits. The aim of this study was therefore to investigate the effect of camel and goat milk consumption on glucose homeostasis in high-fat diet and streptozotocin (HFD/STZ) induced diabetic rats. HFD/STZ-induced diabetic rats were fed with different milk for 35 days. Parameters related to glucose homeostasis, as well as hepatic proteome and phosphoproteome were investigated. The results of which showed that camel and goat milk consumption improved fasting glucose levels, glucose tolerance, and indicators related to lipid metabolism, while bovine and sheep milk consumption did not work. In addition, the hepatic phosphoproteome suggests that the ameliorative effect of both camel and goat milk was associated with the activation of AMPK. However, camel milk consumption further elevated the phosphorylation level of hepatic ACC, while goat milk consumption activated GSK3-GYS axis-related proteins. The present study investigated the possible mechanisms by which camel and goat milk consumption improves glucose homeostasis in HFD/STZ-induced diabetic rats and revealed their differences in the mechanism of antidiabetic effect.

Comparison of milk fat globule membrane and whey proteome between Dromedary and Bactrian camel.

Camel milk is rich in nutrients and its impact on human medicine and nutrition cannot be ignored. We conducted an in-depth analysis of milk proteins obtained from two camel breed (Camelus bactrianus, CB and Camelus dromedarius, CD). Label-free proteomic technology was performed to analysis the MFGM and whey proteomes of CB and CD milk. In total, 1133 MFGM proteins and 627 whey proteins were identified from camel milk. Results revealed that 216 MFGM proteins and 109 whey proteins were significantly different between them. In addition, the cellular process, cell and binding were the predominately GO annotations of milk proteins. KEGG analysis shown that most proteins were involved in metabolic pathways. Furthermore, many proteins were found to be involved in PI3K/AKT signaling pathway, which could be the possible reason for hypoglycemic effect of camel milk. These results could provide a further understanding for unique biological characteristics of camel milk proteins.

Comprehensive Identification and Absolute Quantification of Milk Oligosaccharides in Different Species.

Human and animal milk contains a rich variety of oligosaccharides (OSs), which are of great interest due to a similar biological efficacy. In this study, the OSs were identified and the concentrations of eight specific OSs in human and four animal milk (cow, goat, sheep, and camel) were analyzed. In general, 30, 42, 32, 34, and 35 OSs were identified in bovine, caprine, ovine, camel, and human milk, respectively. Camel milk was the most similar in type to human milk than other four animal milk. The concentration of eight OSs in human milk was approximately six times higher than that in camel milk, 20 times higher than that in bovine and caprine milk, and 75 times higher than that in ovine milk. Collectively, these findings revealed the characteristics and concentrations of OSs in the milk of different species, providing insights into the potential application of OSs in medical and functional foods.

Gestational Diabetes Mellitus-Induced Changes in Proteomes and Glycated/Glycosylated Proteomes of Human Colostrum.

Gestational diabetes mellitus (GDM) not only has a bad effect on the development of infants but also causes variations in breastmilk composition. This study aims to investigate the changes in the protein profile of colostrum between mothers with GDM and healthy mothers (H) by sequential windowed acquisition of all theoretical fragment ion proteomics techniques. A total of 1295 proteins were detected, with 192 proteins being significantly different between GDM and H. These significantly different proteins were enriched with the carbohydrate and lipid metabolism pathway as well as immunity. Some proteins had an AOC value of 1, such as apolipoprotein E and lipoprotein lipase. In addition, we identified 42 glycated and 93 glycosylated peptides in colostrum without any enrichment, with glycated peptides being upregulated and glycosylated peptides being downregulated in colostrum with GDM. These results help us to better understand the GDM-induced changes in proteomes and glycated and glycosylated level and provide guidance on infant formula adjustment for infants from mothers with GDM.

Peptidome comparison following gastrointestinal digesta of bovine versus caprine milk serum.

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), ß-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from αS1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.

Studying the effects of sea cucumber ovum powder on nonalcoholic fatty liver disease by proteomics techniques in a rat model.

Sea cucumber is a valuable marine food that has antioxidant, anti-diabetic, and anti-obesity functionalities. Sea cucumber ovum (SCO) may contain functional components, however, it is considered to be a waste product during industrial processing. In order to make good use of SCO, this work investigated the effects of freeze-dried SCO powder on NAFLD, using a rat model, through iBT labeling proteomics techniques, tracking changes in the hepatic protein profiles of rats whose diets were supplemented with SCO powder. Male rats were fed with standard food, a high fat diet (HFD), or a HFD supplemented with 150 mg per kg BW or 450 mg per kg BW SCO powder for 6 weeks. Compared with the HFD, low-dose SCO supplementation in the diet could significantly reduce body weight gain and liver weight. Furthermore, in total, 5922 proteins were identified, and 767 proteins were found to be significantly different proteins (p < 0.05) among all four groups. Most of the significantly different proteins were related to apoptosis and lipid metabolism. Fadd, Dci, and Aif1 have been identified as key proteins in the pathways related to apoptosis, lipid metabolism, and inflammation. The results in this study provide an overview of the SCO-induced changes in the liver proteome of NAFLD, which may help us to understand the molecular mechanism behind the effects of SCO on the alleviation of NAFLD.

Uncovering the antioxidant characteristics of black tea by coupling in vitro free radical scavenging assay with UHPLC-HRMS analysis.

Black tea (BT) is rich in dietary antioxidants, but its antioxidant composition has not been fully understood. To identify the true antioxidants occurring in BT, we established an approach based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay coupled with ultra-high performance liquid chromatography-high resolution mass spectrometry (DPPH-UHPLC-HRMS). The employment of HRMS enable us to detect trace antioxidants, resolve co-eluted antioxidants, and characterize chemical structures of unknown antioxidants. In total, 56 phenolic compounds were screened as potential antioxidants from 106 compounds identified in BT. Catechol and pyrogallol were revealed as the key substructures in enhancing the antioxidant abilities of phenolic compounds. During BT brewing, high temperature with extended time promote antioxidant leaching but may induce the degradation of esterified and glycosylated compounds such as theaflavin-3-gallate and rutin. In conclusion, this work identified the true antioxidant constituents of BT, their structural characteristics, and their dynamic changes under various brewing conditions.

Changes in bioactive milk serum proteins during milk powder processing.

This study mainly investigated changes in milk serum proteins by simulating the industrial processing of whole milk powder using a label-free proteomic approach. A total of 391 proteins were identified, 89 of which were quantified in all groups. Compared with raw milk, the milk subjected to the heating pasteurization process showed significantly decreased the serum protein profile, whereas that subjected to vacuum concentration and spray-drying showed minimal changes. The LC-MS/MS results were further confirmed by determining the activity of bioactive xanthine oxidase and retention of lactoferrin and immunoglobulins. The data showed that 70%-85% of lactoferrin and immunoglobulins were retained in vacuum-concentrated or spray-dried milk but were hardly identifiable in heat-pasteurized milk or whole milk powders. These findings indicate the need for improving the current milk powder-manufacturing techniques to allow the retention of active milk proteins.

Effects of Different Steeping Temperatures on the Leaching of Aroma Components in Black Tea by SPME-GC-MS Coupled with Chemometric Method.

Background: Black tea is famous for its unique aroma. The analysis of aroma components has attracted considerable attention worldwide because of its complex chemical composition and low concentration. Objective: Steeping temperature is one of the most important factors affecting the aroma of black tea. This study aims to evaluate the effects of four steeping temperatures [60, 70, 80, and 95°C (boiling water)]. Methods: Two major factors affecting extraction performance, including the type of extraction method [direct headspace injection (HS) and solid-phase microextraction (SPME)] and extraction time (50, 60, and 70 min), were optimized to enrich and analyze the aroma components of Congou black tea by GC-MS. In addition, heuristic evolving latent projection (HELP), an effective chemometric resolution method, was employed to resolve the overlapped peaks. Results: A total of 83 aroma components were tentatively identified by GC-MS, such as alcohol (42.06-50.52%), aldehyde (12.09-15.97%), and hydrocarbon (4.79-15.32%). Linalool and its oxides (25.49-36.24%) were the most abundant aroma components, followed by geraniol (2.55-8.54%), methyl salicylate (1.84-9.50%), and nerol (1.93-4.41%). Conclusions: The black tea steeped at 95°C smelled more pleasant with mild green, roast, and fruity aroma. Moreover, at 80°C, the tea had sweeter fragrance with floral aroma, while steeping at 60 and 70°C resulted in more reinforced woody and fatty aroma. Highlights: A total of 83 aroma components of black tea were tentatively identified by SPME-GC-MS. The overlapped peaks were resolved by the HELP method. Aroma characteristics of different steeping temperatures were revealed.

UPLC-Orbitrap-MS/MS combined with chemometrics establishes variations in chemical components in green tea from Yunnan and Hunan origins.

Multi-components of green tea from different origins were identified by UPLC-Orbitrap-MS/MS, including alkaloids, amino acids, catechins, flavones, flavone glycosides, phenolic acids and theaflavins. Quantitative chemical profiles of 72 samples of Yunnan green tea (YGT) and Hunan green tea (HGT) established the influence of origin on green tea. In addition, three variable selection methods, based on partial least squares-discriminant analysis (PLS-DA), were employed to screen characteristic components of YGT and HGT. The results of variable importance on projection (VIP) method showed better performance than coefficients ß and the least absolute shrinkage and selection operator (LASSO) for this dataset. Three characteristic components, named, gallocatechin gallate (GCG), epicatechin-(4ß → 8)-epigallocatechin-3-O-gallate, and vitexin were selected by VIP, with a correct rate of 98.61% and an AUC value of 0.9706. The results indicated that the combination of UPLC-Orbitrap-MS/MS and chemometrics could serve as a valid strategy to analyze complex analytical objects, such as green tea.

Comprehensive characterization and identification of antioxidants in Folium Artemisiae Argyi using high-resolution tandem mass spectrometry.

Antioxidants from natural sources, such as vegetables and fruits, are attracting more and more interest. In this work, we evaluated the antioxidant potential of Folium Artemisia Argyi, a traditional Chinese herb medicine and food supplement. The total phenolic content, total flavonoid content, and antioxidant ability of the crude extracts and fractions obtained from consecutively partition of n-hexane, ethyl acetate, and n-butanol were measured and compared. Ethyl acetate fraction shows the highest total phenolic and flavonoid contents and highest antioxidant capability with regard to DPPH, ABTS, superoxide anion free radical scavenging ability, and ferric-reducing antioxidant power. In addition, the potential antioxidant components were screened by DPPH-UHPLC-MS experiments and subsequently characterized by using high-resolution tandem mass spectrometry. This work finally identified 45 antioxidants, including organic acids, phenolic compounds, flavonoids, and methoxylated flavonoids. The results suggested that Folium Artemisiae Argyi is a potential inexpensive resource of natural antioxidants.

Combined application of chromatographic techniques for the separation of phenolic compounds from Stenoloma chusanum Ching.

High-speed countercurrent chromatography combined with preparative high-performance liquid chromatography was successfully used to separate seven phenolic compounds from Stenoloma chusanum Ching. A biphasic solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1:2, v/v) was used for the first step high-speed countercurrent chromatography separation in elution-extrusion mode. A mobile phase composed of acetonitrile (18%) and pure water (82%) was used for further preparative high-performance liquid chromatography purification. In total, the combined separation yielded seven compounds, including 3,4-dihydroxy benzoic acid, 3,4-dihydroxy benzaldehyde, esculetin, caffeic acid, syringic acid, luteolin, and apigenin, at a purity of over 90%. Esculetin was separated from Stenoloma chusanum Ching for the first time. The results suggest that the proposed combination method is a useful strategy for separating compounds from complex samples.