Search Results (28)

Background: Response speed refers to an individual’s ability to perceive and react to harmful stimuli, which can vary due to genetics, neural regulation, and environmental factors. Our previous study demonstrated that whey protein hydrolysate was a potential means to enhance cognitive function. Methods: This study used a variety of behavioral methods to evaluate the functional effects of whey protein hydrolysate on improving reaction speed, and revealed its potential mechanisms through proteomics analysis. Results: The results showed that whey protein hydrolysate improved response speed in mice when tested against thermal pain, mechanical strength stimuli, and prepulse inhibition. Proteomic analysis of the hippocampus revealed changes in proteins related to arginine and proline metabolism, as well as neuroactive ligand–receptor interactions. Conclusions: These findings provide new insights into the neuromodulatory effects of whey protein hydrolysate and support its potential role in enhancing response speed and cognitive performance. Full article

Milk is a nutritionally rich food and a frequent target of economically motivated adulteration, particularly through substitution with lower-cost milk types. Over the past decade, significant progress has been made in the authentication of milk using advanced proteomic and chemometric approaches, with a focus on the discovery and application of protein and peptide biomarkers for species differentiation and fraud detection. Recent innovations in both top-down and bottom-up proteomics have markedly improved the sensitivity and specificity of detecting key molecular targets, including caseins and whey proteins. Peptide-based methods are especially valuable in processed dairy products due to their thermal stability and resilience to harsh treatment, although their species specificity may be limited when sequences are conserved across related species. Robust chemometric approaches are increasingly integrated with proteomic pipelines to handle high-dimensional datasets and enhance classification performance. Multivariate techniques, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), are frequently employed to extract discriminatory features and model adulteration scenarios. Despite these advances, key challenges persist, including the lack of standardized protocols, variability in sample preparation, and the need for broader validation across breeds, geographies, and production systems. Future progress will depend on the convergence of high-resolution proteomics with multi-omics integration, structured data fusion, and machine learning frameworks, enabling scalable, specific, and robust solutions for milk authentication in increasingly complex food systems. Full article

The aim of this work is to highlight the beneficial effects of bioactive peptides present in fermented whey (FW) and carotenoids from pumpkin (P) against the pro-oxidant effects of aflatoxin B1 and ochratoxin A at the neuronal level. For this purpose, SH-SY5Y human neuroblastoma differentiated cells were exposed to (A) mycotoxins, (B) the digesta of mycotoxin-contaminated bread formulated with P, or (C) bread enriched with FW + P. A proteomic approach using HPLC-MS/MS-QTOF was then employed to characterize the metabolic pathways affected by the presence of these components, as well as their ability to modulate the toxic effects exacerbated by mycotoxins. Gene ontology functional analysis revealed proteins primarily associated with nucleosome structure, such as the H3-H4 tetramer, H2A-H2B dimer, and HIRA, which were overexpressed in the presence of mycotoxins and, interestingly, downregulated with the addition of the functional ingredients. Additionally, important metabolic pathways associated with the RHO GTPase family, estrogen-dependent gene expression, and androgen receptor transcription stimulated by PKN1 activation were discovered. Network interaction analysis highlighted the modulation of cytoskeletal dynamics, cell migration, and stress responses. These findings provide novel insights into the neuroprotective potential of functional food components, supporting their use in mitigating mycotoxin-induced neuronal damage and opening new avenues for dietary-based neuroprotection strategies. Full article

This study critically examines the limitations of the official Italian methodology used for detecting bovine adulteration milk in Protected Designation of Origin (PDO) Mozzarella di Bufala Campana (MdBC). This method focuses on the whey fraction of cheese samples, which comprises about 1% of total MdBC proteins, and is based on a high-performance liquid chromatography (HPLC) quantification of the bovine β-lactoglobulin A (β-Lg A) as a marker. Here, we have demonstrated that this official methodology suffers from measurement inconsistencies due to its reliance on raw bovine whey standards, which fail to account for β-Lg genetic polymorphisms in real MdBC samples and protein thermal modifications during cheesemaking. To overcome these limitations, we propose a dual proteomics-based approach using matrix-assisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS) and nano-HPLC-electrospray (ESI)−tandem mass spectrometry (MS/MS) analysis of MdBC extracted whey. MALDI-TOF-MS focused on identifying proteotypic peptides specific to bovine and buffalo β-Lg and α-lactalbumin (α-La), enabling high specificity for distinguishing the two animal species at adulteration levels as low as 1%. Complementing this, nano-HPLC-ESI-MS/MS provided a comprehensive profile by identifying over 100 bovine-specific peptide markers from β-Lg, α-La, albumin, lactoferrin, and osteopontin. Both methods ensured precise detection and quantification of bovine milk adulteration in complex matrices like pasta filata cheeses, achieving high sensitivity even at minimal adulteration levels. Accordingly, the proposed dual proteomics-based approach overcomes challenges associated with whey protein polymorphism, heat treatment, and processing variability, and complements casein-based methodologies already validated under European standards. This integrated framework of analyses focused on whey and casein fraction enhances the reliability of adulteration detection and safeguards the authenticity of PDO buffalo mozzarella, upholding its unique quality and integrity. Full article

The somatic cell count (SCC) is widely used to assess milk quality and diagnose intramammary infections. Several whey proteins have been shown to correlate significantly with SCC and are considered potential indicators of udder health. However, the relationship between milk whey proteins and SCC has not been fully elucidated. In this study, milk samples were grouped into five categories based on SCC levels. Subsequently, whey proteins were identified using a label-free proteomics approach, and the differential abundance of proteins was validated through a selected reaction monitoring (SRM) method. The levels of various proteins, including azurocidin 1 and kininogen-2, exhibited an increase, whereas topoisomerase I, tropomyosin-1, and desmin showed a significant decrease depending on the SCCs. Principal component analysis unveiled that these proteins contributed to the developmental alterations in milk proteins. A majority of these differentially abundant proteins were associated with response to stimulus, localization, and defense response. Our results provide fundamental information on the SCC that can be utilized for evaluating milk quality and serve as potential indicators for detecting intramammary infections. Full article

Aflatoxin B1 (AFB1) and Ochratoxin A (OTA) are considered the most important mycotoxins in terms of food safety. The aim of this study was to evaluate the hepatotoxicity of AFB1 and OTA exposure in Wistar rats and to assess the beneficial effect of fermented whey (FW) and pumpkin (P) as functional ingredients through a proteomic approach. For the experimental procedures, rats were fed AFB1 and OTA individually or in combination, with the addition of FW or a FW-P mixture during 28 days. For proteomics analysis, peptides were separated using a LC-MS/MS-QTOF system and differentially expressed proteins (DEPs) were statistically filtered (p < 0.05) distinguishing males from females. Gene ontology visualization allowed the identification of proteins involved in important biological processes such as the response to xenobiotic stimuli and liver development. Likewise, KEGG pathway analysis reported the metabolic routes as the most affected, followed by carbon metabolism and biosynthesis of amino acids. Overall, the results highlighted a strong downregulation of DEPs in the presence of AFB1 and OTA individually but not with the mixture of both, suggesting a synergistic effect. However, FW and P have helped in the mitigation of processes triggered by mycotoxins. Full article

Colostrum is essential for the survival and development of newborn mammals. This primary source of nourishment during the first days of infant life is rich in functional components conductive to the enhancement of neonate immunity and growth. Compared with mature milk, a higher protein and peptide content is observed in colostrum, whilst it is low in fat and carbohydrates. The functional properties of colostrum are closely linked to the release of bioactive peptides during the gastrointestinal digestion of colostrum proteins. Our study aimed to comprehensively analyze the whey proteome of colostrum from indigenous Greek goats and to examine the influence of bioactive peptides released during digestion on human metabolism. Colostrum and mature milk samples from healthy ewes were subjected to nanoLC-MS/MS analysis, revealing differentially expressed proteins. These proteins were functionally characterized and subjected to in silico digestion. Using machine learning models, we classified the peptide functional groups, while molecular docking assessed the binding affinity of the proposed angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPPIV)-inhibitory peptides to their target molecules. A total of 898 proteins were identified in colostrum, 40 of which were overexpressed compared with mature milk. The enzymatic cleavage of upregulated proteins by key gastrointestinal tract proteases and the downstream analysis of peptide sequences identified 117 peptides predicted (with >80% confidence) to impact metabolism, primarily through modulation of the renin–angiotensin system, insulin secretion, and redox pathways. This work advances our understanding of dietary bioactive peptides and their relevance to human metabolism, highlighting the potential health benefits of colostrum consumption. Full article

Human milk (HM) is a complex biofluid rich in nutrients and bioactive compounds essential for infant health. Recent advances in omics technologies—such as proteomics, metabolomics, and transcriptomics—have shed light on the influence of HM on bone development and health. This review discusses the impact of various HM components, including proteins, lipids, carbohydrates, and hormones, on bone metabolism and skeletal growth. Proteins like casein and whey promote calcium absorption and osteoblast differentiation, supporting bone mineralization. Long-chain polyunsaturated fatty acids like docosahexaenoic acid (DHA) contribute to bone health by modulating inflammatory pathways and regulating osteoclast activity. Additionally, human milk oligosaccharides (HMOs) act as prebiotics, improving gut health and calcium bioavailability while influencing bone mineralization. Hormones present in HM, such as insulin-like growth factor 1 (IGF-1), leptin, and adiponectin, have been linked to infant growth, body composition, and bone density. Research has shown that higher IGF-1 levels in breast milk are associated with increased weight gain, while leptin and adiponectin influence fat mass and bone metabolism. Emerging studies have also highlighted the role of microRNAs (miRNAs) in regulating key processes like adipogenesis and bone homeostasis. Furthermore, microbiome-focused techniques reveal HM’s role in establishing a balanced infant gut microbiota, indirectly influencing bone development by enhancing nutrient absorption. Although current findings are promising, comprehensive longitudinal studies integrating omics approaches are needed to fully understand the intricate relationships among maternal diet, HM composition, and infant bone health. Bridging these gaps could offer novel dietary strategies to optimize skeletal health during infancy, advancing early-life nutrition science. Full article

Research into mosquito-borne illnesses faces hurdles because feeding fresh animal blood to rear female mosquitoes presents logistical, economic, and safety challenges. In this study, a shelf-stable additive (spray-dried porcine blood; SDPB) hypothesized to supply accessible hemoglobin was evaluated within an alternative meal (AM) containing whey powder and PBS for rearing the yellow fever mosquito Aedes aegypti. LC–MS/MS proteomics, microbial assays, and particle reduction techniques confirmed and characterized the functionality of hemoglobin in SDPB, while engorgement, fecundity, egg viability, and meal stability bioassays assessed AM performance. Chemical assays supported hemoglobin as the phagostimulant in SDPB with aggregates partially solubilized in the AM that can be more accessible via particle reduction. Unpaired two-tailed t-tests indicate that the AM stimulates oogenesis (t₁₁ = 13.6, p = 0.003) and is stable under ambient (1+ y; t₁₂ = 0.576, p = 0.575) and aqueous (14 d; t₁₂ = 0.515, p = 0.639) conditions without decreasing fecundity. Egg hatch rates for the ninth generation of AM-reared Ae. aegypti were 50–70+%. With further development, this meal may serve as a platform for mass rearing or studying effects of nutritional additives on mosquito fitness due to its low cost and stability. Future work may examine tuning spray drying parameters and resulting impacts on hemoglobin agglomeration and feeding. Full article

Assessing the authenticity of PDO cheeses is an important task because it allows consumer expectations to be fulfilled and guarantees fair competition for manufacturers. A 3-year survey was carried out, analyzing 271 samples of grated Grana Padano (GP) PDO cheese collected on the European market. Previously developed analytical methods based on proteomics approaches were adopted to evaluate the compliance of market samples with selected legal requirements provided by the specification for this cheese. Proteolysis follows highly repeatable pathways in GP cheese due to the usage of raw milk, natural whey starter, and consistent manufacturing and ripening conditions. From selected casein breakdown products, it is possible to calculate the actual cheese age (should be >9 months) and detect the presence of excess rind (should be <18%). Furthermore, due to the characteristic pattern of free amino acids established for GP, distinguishing it from closely related cheese varieties is feasible. Cheese age ranged from 9 to 25 months and was correctly claimed on the label. Based on the amino acid pattern, three samples probably contained defective cheese and there was only one imitation cheese. Few samples (9%) were proven to contain some excess rind. Overall, this survey highlighted that the adopted control parameters can assure the quality of grated GP. Full article

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides (‘bottom-up’ proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and β-lactoglobulin (β-LG). Based on the findings, α-LA and β-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R² > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for β-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types. Full article

Whey is a by-product of the production of various types of cottage cheese and cheese, casein, and coprecipitates. Conditions of milk coagulation directly affect the physico-chemical properties of whey and the formation of its protein profile. This fact makes it difficult to standardize the protein profile of milk whey for its further processing. Whey proteins have a great potential to release a wide range of bioactive peptides (BAP), capable of reducing the risk of a number of chronic food-related diseases. Computer modeling of an enzymatic hydrolysis of proteins is one of the ways to increase the efficiency of BAP release studies and to reduce the number of labor consuming experiments. This research is aimed at generating a digital model of the peptide complex of different whey types with predicted bioactivity, safety, and sensory properties using bioinformatic modeling approaches. The study was performed with the use of the proteomic databases tools according to the algorithm of hybrid strategy of bioinformatic modeling developed earlier. As a result of the study, taking into account the ranking of the proteins ratio in the protein profile, the hydrolysis by the protease complex chymotrypsin C-subtilisin was characterized as the maximum efficacy method to release peptides with both antioxidant and ACE-inhibitory activity. It was also observed that the bioactive peptides obtained as a result of in silico hydrolysis after GI digestion simulation can be considered safe in terms of allergic reactions and toxicological effects. Full article

Whey protein and its hydrolysates are ubiquitously applied in the food system. However, their effect on cognitive impairment remains unclear. This study aimed to investigate the potential ability of whey protein hydrolysate (WPH) to ameliorate cognitive degeneration. WPH intervention in Crl:CD1 (ICR, Institute for cancer research) mice and aged C57BL/6J mice in a scopolamine-induced cognitive impairment model for 10 days were evaluated. Behavioral tests indicated that WPH intervention improved the cognitive abilities in ICR and aged C57BL/6J mice (p < 0.05). Scopolamine enhanced the Aβ_1-42 level in the brain tissue, and the WPH intervention exhibited a similar therapeutic effect to donepezil in ICR mice. A noticeable reduction occurred in serum Aβ_1-42 level of aged mice treated with WPH. The histopathological study of the hippocampus showed that WPH intervention alleviates neuronal damage. Hippocampus proteomic analysis suggested possible mechanisms of WPH action. The relative abundance of Christensenellaceae, a gut microbe related to Alzheimer’s disease, was altered by WPH intervention. This study demonstrated that short-term WPH intake protected against memory impairment induced by scopolamine and aging. Full article

This study investigated the effects of atmospheric spray drying (ASD), low-pressure spray drying (LPSD) and freeze drying (FD) on the properties of serum protein powder, including the basic characteristics of the powder, bioactive proteins and changes in protein profile, using a proteomics approach. The total solid and water activity of the powder obtained by FD was significantly higher than that obtained by ASD and LPSD. There was no significant difference in the content of fat, lactose or solubility between the three kinds of powders. The concentration and activity of the proteins/enzymes in the serum protein powder made from LPSD were not altered after drying, similar to FD, although both parameters decreased after ASD. The microstructure of the powder observed by scanning electron microscopy indicated that the powder manufactured by ASD and LPSD was spherical in structure, while that manufactured by FD was flake-like. In total, there were 245 proteins identified in the serum protein concentrate and powder from the three drying methods. These findings indicate that LPSD is an effective and cost-saving method for producing serum whey protein powder. Full article

Despite the well-known benefits of breastfeeding and the World Health Organization’s breastfeeding recommendations for COVID-19 infected mothers, whether these mothers should be encouraged to breastfeed is under debate due to concern about the risk of virus transmission and lack of evidence of breastmilk’s protective effects against the virus. Here, we provide a molecular basis for the breastfeeding recommendation through mass spectrometry (MS)-based proteomics and glycosylation analysis of immune-related proteins in both colostrum and mature breastmilk collected from COVID-19 patients and healthy donors. The total protein amounts in the COVID-19 colostrum group were significantly higher than in the control group. While casein proteins in COVID-19 colostrum exhibited significantly lower abundances, immune-related proteins, especially whey proteins with antiviral properties against SARS-CoV-2, were upregulated. These proteins were detected with unique site-specific glycan structures and improved glycosylation diversity that are beneficial for recognizing epitopes and blocking viral entry. Such adaptive differences in milk from COVID-19 mothers tended to fade in mature milk from the same mothers one month postpartum. These results suggest that feeding infants colostrum from COVID-19 mothers confers both nutritional and immune benefits, and provide molecular-level insights that aid breastmilk feeding decisions in cases of active infection. Full article