Search Results (109)

Background/Objectives: This randomized controlled trial aimed to evaluate the effects of casein-enriched milk (CEM) consumption and its timing (presleep vs. during the day) in the early postoperative period on body composition, muscle strength, physical function, and biochemical parameters in individuals undergoing laparoscopic sleeve gastrectomy (SG). Methods: Forty-five adults (60% female, 40% male; mean age 35.1 ± 9.7 years; mean BMI 41.4 ± 4.9 kg/m²) undergoing SG were randomly assigned to three groups: (1) 15 g protein CEM (12 g casein) presleep, (2) the same CEM during the day, or (3) standard-protein diet without supplementation. The primary endpoint was change in fat-free mass (FFM) at 12 weeks; secondary endpoints included handgrip strength, 30 s sit-to-stand test, and serum total protein, albumin, and prealbumin. Assessments were performed preoperatively and at weeks 4, 8, and 12. Results: No significant differences were found between the groups in terms of body composition, muscle strength, or physical performance measurements (p > 0.05). However, a significant increase in handgrip strength was observed over time in Groups 1 and 2 (p < 0.05), which was not observed in Group 3. Prealbumin levels at week 12 were 0.3 ± 0.0 mg/dL in Group 1 and 0.2 ± 0.0 mg/dL in Group 2, both higher than 0.2 ± 0.0 mg/dL in Group 3 (p < 0.05). No significant differences were found in albumin and total protein levels (p > 0.05). Conclusions: Early postoperative CEM consumption following SG did not significantly affect body composition or physical performance; however, the higher prealbumin levels indicate that this marker may be more sensitive in detecting early protein response, highlighting its potential clinical relevance in monitoring nutritional status after bariatric surgery. Full article

This work reports a label-free analytical strategy based on protein-induced modulation of salt crystallization patterns upon drying. This method relies on the consistent observation that protein-containing saline samples produce distinct salt deposition morphologies compared to protein-free controls. The work first demonstrates the concept of this phenomenon and characterizes the structural features of the resulting salt patterns. Then, systematic experiments with different solution compositions, substrates, surface coatings, and protein types confirm the generality of this differential deposition behavior and its dependence on total protein concentration. Two complementary measurement approaches are evaluated: a custom laser-scattering setup for optical attenuation measurements and a digital image analysis method based on pixel intensity distributions. Both strategies enable quantitative protein detection in simple (casein) and complex (human serum) samples, offering good correlations between signal and concentration and detection limits in the range of 2–18 µg·mL⁻¹ for digital image analysis and 162–205 µg·mL⁻¹ for optical attenuation measurements. These findings introduce an appealing paradigm for protein quantification exploiting drying-mediated crystallization phenomena, with potential for simple and label-free bioanalytical assays. Full article

Alcalase was immobilized–stabilized on carrageenan beads following a previously described protocol. Then, the activities of free and immobilized enzymes were compared using different protein substrates (casein, (CS), bovine serum albumin (BSA), or hemoglobin (HG)) at different pH values and temperatures. The observed activity depended on the substrate protein and enzyme formulation used. The highest enzyme activity could be observed at pHs 5, 7, or 10, depending on the substrate protein and the Alcalase formulation. The effect of the temperature at these pHs on the activity versus the different substrate proteins showed a common pattern. At low temperatures, the immobilized enzyme presented higher (mainly at acidic-neutral pH values and using BSA) or similar specific activity than the free enzyme. At temperatures near the optimal for the free enzyme, it became the most active, while at higher temperatures, the immobilized enzyme recovered the lead, although differences in the optimal temperature were not very significant. This may be explained by the lower mobility of the immobilized–stabilized enzyme. The immobilized enzyme could be much more active than the free enzyme or slightly less active, even using mild conditions, depending on the substrate protein, pH, and temperature used to determine the enzyme activity. Full article

Alzheimer’s Disease (AD) is a multifactorial process. Amyloid plaque formation constitutes the main characteristic of the disease. Despite the identification of numerous factors associated with AD, the mechanism remains unclear in several aspects. Disturbances in intestinal and blood–brain barrier (BBB) penetration, observed in AD, may facilitate immunologic response to food-derived antigens. In the present study, antibodies against egg albumin, bovine-casein, and N-Glycolyl-Neuraminic acid (Neu5Gc) were measured in the cerebrospinal fluid (CSF) and serum of the patients using an enzyme-linked immunosorbent assay (ELISA). Zero anti-Neu5Gc and low concentrations of anti-casein antibodies were detected. Increased anti-native egg albumin antibodies were present in the serum of patients of all stages with 65% positivity (p < 0.001) in mild disease and a higher percentage in females (81.9%, p < 0.001). Lower serum positivity to anti-denatured egg albumin antibodies was observed, showing a gradual increase with severity and higher prevalence also in females. In the CSF, anti-native and anti-denatured egg albumin antibodies were mainly observed in severely ill patients with accumulative positivity to either antigen, reaching 61.8% in severe vs. 15% in mild disease (p < 0.001). Increased values were mainly observed in males. Anti-egg albumin antibodies may be implicated in the disease mechanism through sequence/structural similarity with human proteins, mainly serpins, and it would be worth consideration in further investigations and therapeutic strategies. Full article

Protein phosphorylation is one of the most common and important post-translational modifications (PTMs) and is highly involved in various biological processes. Ideal adsorbents with high sensitivity and specificity toward phosphopeptides with large coverage are therefore essential for enrichment and mass spectroscopy-based phosphoproteomics analysis. In this study, a newly designed IMAC adsorbent composite was constructed on the graphene matrix coated with mesoporous silica. The outer functional 3D-network layer was prepared by free radical polymerization of the phosphonate-functionalized vinyl imidazolium salt monomer and subsequent metal immobilization. Due to its unique structural feature and high content of Ti⁴⁺ ions, the resulting phosphonate-immobilized adsorbent composite G@mSiO₂@PPFIL-Ti⁴⁺ exhibits excellent performance in phosphopeptide enrichment with a low detection limit (0.1 fmol, tryptic β-casein digest) and superior selectivity (molar ratio of 1:15,000, digest mixture of β-casein and bovine serum albumin). G@mSiO₂@PPFIL-Ti⁴⁺ displays high tolerance to loading and elution conditions and thus can be reused without a marked decrease in enrichment efficacy. The captured phosphopeptides can be released globally, and mono-/multi-phosphopeptides can be isolated stepwise by gradient elution. When applying this material to enrich phosphopeptides from human lung cell lysates, a total of 3268 unique phosphopeptides were identified, corresponding to 1293 phosphoproteins. Furthermore, 2698 phosphorylated peptides were found to be differentially expressed (p < 0.05) between human lung adenocarcinoma cells (SPC-A1) and human normal epithelial cells (Beas-2B), of which 1592 were upregulated and 1106 were downregulated in the cancer group. These results demonstrate the material’s superior enrichment efficiency in complex biological samples. Full article

This study investigated the IgA antibodies targeting bovine serum albumin (BSA) in 27 adult celiac disease (CD) patients adhering to a gluten-free diet (GFD), compared to 123 controls (including individuals with autoimmune disorders, those with gastrointestinal cancers, and healthy donors). Serum samples were evaluated using a multiplex assay based on a microarray comprising 66 immobilized antigens, including autoantigens associated with autoimmune diseases, different albumins, cytokines, and inflammatory markers. Elevated IgA-BSA levels were detected in 22% of CD patients versus 3.25% of controls. IgA-BSA did not cross-react with milk proteins like casein, β-lactoglobulin, and γ-globulin, nor with autoantigens and human albumin, ruling out autoimmunity against self-proteins. The observed cross-reactivity with porcine albumin suggests that antibodies target epitopes shared by bovine and porcine albumin. Increased IgA-BSA levels may interfere with immunoassays performed using BSA as a stabilizer, necessitating protein-free buffers to avoid false results when testing CD patients. Elevated IgA-BSA levels may reflect ongoing gut barrier dysfunction in CD patients on a GFD, allowing dietary proteins like BSA to trigger immune responses. This study identifies a novel immune response in CD patients on a GFD, emphasizing the need for tailored diagnostic approaches (BSA-free assays) and further research into the clinical and dietary implications of IgA-BSA elevation. Full article

Bovine milk protein preparations (MPPs), namely micellar casein concentrate (MCC), serum protein concentrate (SPC), and MCC with ultrafiltrated buttermilk permeate (MBP), were analyzed as sources of inhibitors of angiotensin-converting enzyme (i.e., ACE) and dipeptidylpeptidase IV (i.e., DPP-IV) as well as antioxidative peptides. The studies involved in silico predictions of the release of biopeptides from bovine milk proteins. Then, all MPPs were subjected to the simulated gastrointestinal digestion using the INFOGEST protocol. Results using a BIOPEP-UWM database tool indicated that 59 biopeptides exhibiting the above-mentioned activities could be produced upon the action of pepsin, trypsin, and chymotrypsin. Thirty-six biopeptides were identified in at least one of the three MPPs subjected to the INFOGEST protocol. MCC before simulated digestion exhibited the strongest ACE-inhibiting activity among all MPPs (IC₅₀ = 1.856 mg/mL). The weakest ACE inhibitory effect was demonstrated for MBP after duodenal digestion (i.e., MBP D; IC₅₀ = 7.627 mg/mL). The above MPP showed the strongest DPP-IV-inhibiting activity (IC₅₀ = 0.0067 mg/mL). All MPPs exhibited antioxidative activity, with the strongest ABTS^•+ (i.e., 2,2′-azino-bis(3-ethylbenzotialozline-6-sulfonic acid) radical scavenging effect shown for MBP D (IC₅₀ = 2.754 mg/mL), and the strongest DPPH^• (i.e., 2,2-diphenyl-β-picrylhydrazyl) radical scavenging activity (IC₅₀ = 1.238 mg/mL) demonstrated for SPC D. Among all MPPs, SPC D also exhibited the highest FRAP (i.e., Ferric Reducing Antioxidant Power) bioactivity (IC₅₀ = 13.720 mg/mL), whereas MBP D was the MPP with the lowest FRAP potential (IC₅₀ = 20.140 mg/mL). The present study results show the potential of all MPPs as functional additives to support health-beneficial functions of dairy products. Full article

Mozzarella di Bufala Campana is an Italian protected designation of origin cheese characterized by a stretched structure, high moisture (<65%), and short shelf life (<30 days). This cheese is generally stored refrigerated in a covering liquid that is an aqueous solution containing NaCl and organic acids. Although microbial growth has been reported as the main cause of quality deterioration, physico-chemical phenomena (water/solute migration, enzymatic reactions, etc.) also play a role in determining the cheese quality and its storability. This study investigates the effect of covering liquids formulated with different percentages of NaCl (1, 2%) and types of organic acids (lactic acid, citric acid, and a 1:1 mix of both) on the evolution of some physico-chemical characteristics of the cheese (moisture, pH, electrical conductivity, color, expressible serum, texture, rheology) during a 30-day storage period. Eight cheese batches collected from different dairies were considered as replicates of the study. The % of NaCl in the covering liquid showed a strong, significant effect on the evolution of different structural, physico-chemical characteristics of the cheeses; in particular, a NaCl concentration of 2% caused the greatest extent of moisture content increase because of casein swelling during storage, accompanied by softening of the structure. Full article

This study utilized MAC-T cells cultured in vitro as a model to investigate the effects of varying concentrations of valine on α-casein synthesis and its underlying regulatory mechanisms. In this experiment, MAC-T cells were subjected to a 12 h starvation period, followed by the addition of valine in a range of concentrations (a total of seven concentrations: 0.000, 1.596, 3.192, 6.384, 12.768, 25.536, and 51.072 mM, as well as in 10% Fetal Bovine Serum). The suitable range of valine concentrations was determined using enzyme-linked immunosorbent assays (ELISAs). Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot analyses were employed to evaluate the expression levels and phosphorylation states of the casein alpha s1 gene (CSN1S1), casein alpha s2 gene (CSN1S2) and mTOR signaling pathway-related genes. The functionality of the mTOR signaling pathway was further validated through rapamycin (100.000 nM) inhibition experiments. Results indicated that 1× Val (6.384 mM), 2× Val (12.768 mM), 4× Val (25.536 mM), and 8× Val (51.072 mM) significantly enhanced α-casein synthesis (p < 0.01). Within this concentration range, valine significantly upregulated the expression of CSN1S1, CSN1S2, and mTOR signaling pathway-related genes including the RagA gene (RRAGA), RagB gene (RRAGB), RagC gene (RRAGC), RagD gene (RRAGD), mTOR, raptor gene (RPTOR), and 4EBP1 gene (EIF4EBP1), eukaryotic initiation factor 4E (EIF4E), and S6 Kinase 1 (S6K1) (p < 0.01). Notably, the expression of the eukaryotic elongation factor 2 (EEF2) gene peaked at 1× Val (6.384 mM), while the expression of other genes reached their maximum at 4× Val (25.536 mM). Additionally, valine significantly increased the phosphorylation levels of mTOR, S6K1, 4E-binding protein-1 (4EBP1), ribosomal protein S6 (RPS6), and eEF2 (p < 0.01), with the highest phosphorylation levels of mTOR, S6K1, and RPS6 observed at 4× Val (25.536 mM). Rapamycin treatment significantly inhibited mTOR phosphorylation and α-casein synthesis (p < 0.01); however, the addition of 4× Val (25.536 mM) partially mitigated this inhibitory effect. In conclusion, valine promotes α-casein synthesis by activating the mTOR signaling pathway, with an optimal concentration of 4× Val (25.536 mM). Full article

Mycotoxins are frequent food contaminants posing health risk to humans and animals. Since these interactions have been barely studied yet, we examined the potential complex formation of mycotoxins with human α₁-acid glycoprotein (AGP) and with bovine milk proteins (including casein (CSN), β-lactoglobulin (LG), and α-lactalbumin (LA)) based on fluorescence spectroscopic and ultracentrifugation techniques. Only weak interactions (logK = 2.7 to 3.5) of certain mycotoxins were observed with CSN, LG, and/or LA. Ultracentrifugation experiments demonstrated that aflatoxin M1, zearalenone, and α-zearalenol form more stable complexes with CSN than with LG or LA. These mycotoxins bound to bovine serum albumin with more than a tenfold higher affinity compared to CSN; nevertheless, it has likely limited importance due to the relatively low levels of BSA in bovine milk. Zearalenone, zearalenols, and sterigmatocystin showed strong interactions with AGP (logK = 5.5 to 6.4), suggesting that AGP may play an important role in the plasma protein binding of these mycotoxins. Full article

Protein-enriched yogurts have become increasingly popular among consumers seeking to boost their daily protein intake. The incorporation of milk proteins and protein preparations in yogurt production not only enhances nutritional value but also improves texture, viscosity, and overall sensory properties—key factors that influence consumer acceptance. The main objective of this study was to evaluate the influence of casein and whey protein preparations on the physicochemical properties, viability of lactic acid bacteria, and sensory attributes of yogurts. Yogurts were enriched with 2% (w/w) protein preparations, including micellar casein preparation (CN85), whey protein isolate (WPI), whey protein concentrate (WPC60), and protein preparations obtained from skim milk by membrane filtration: micellar casein concentrate (CN75) and serum protein concentrate (SPC). The yogurts were produced using the thermostatic method, and their chemical composition, rheological properties, syneresis, firmness, lactic acid bacteria population, and sensory attributes were evaluated. The effects of high-protein preparations derived from skim milk through laboratory-scale membrane filtration processes (SPC, CN75) were compared with those of commercially available protein preparations (SMP, CN85, WPI, and WPC). Obtained results demonstrated that the membrane filtration-derived preparations (SPC and CN75) exhibited advantageous physicochemical properties and supported robust viability of yogurt and probiotic bacteria. However, their sensory quality was marginally inferior compared to the commercial preparations (SMP, CN85, WPI, and WPC). These findings indicate the potential applicability of membrane filtration-derived protein preparations in yogurt production while underscoring the necessity for further investigation to enhance and optimize their sensory characteristics. Full article

Phenylalanine (Phe) is a potentially limiting amino acid for lactating cows. The mechanism by which Phe regulates milk protein synthesis remains unclear. The present study elucidates the mechanisms by which phenylalanine affects milk protein synthesis, amino acid utilization, and related signaling pathways in bovine mammary epithelial cells (BMECs). The BMECs were treated with five concentrations (0, 0.22, 0.44, 0.88, 1.76 mM, and serum free). Rapamycin inhibitors and RNA interference (RNAi) were used to inhibit the phosphorylation of the mammalian target of rapamycin (mTOR) signaling pathway and the expression of relevant amino acid transporters, respectively. The results showed that 4×Phe (0.88 mM) significantly increased (p < 0.05) both the mRNA and protein expression of α-casein (CSN1S1), β-casein (CSN2), and κ-casein (CSN3), as well as L-type amino acid transporter-1 (LAT1) mRNA expression. Protein expression and modification assays of mTOR-related proteins showed that 4×Phe could increase (p < 0.05) the expression of α-casein and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and tended to increase the expression of ribosomal protein S6 protein kinase (S6K1, p = 0.054). The general control nonderepressible 2 (GCN2) signaling pathway factor, eukaryotic initiation factor 2 (eIF2α), was downregulated by 4×Phe treatment (p < 0.05). The rapamycin inhibition test showed that Phe regulated casein synthesis via the mTOR signaling pathway. RNAi experiments showed that LAT1 mediated the entry of Phe into cells. Moreover, 4×Phe treatment tended to decrease (0.05 < p < 0.10) the consumption of valine, leucine, histidine, tyrosine, cysteine, alanine, asparagine, and serine in the medium. Collectively, phenylalanine enhanced α-casein synthesis by regulating the phosphorylation of 4EBP1 and eIF2α and promoting the formation of the mTOR-centered casein translation initiation complex. Full article

A plant-based diet is considered a promising approach for managing hyperuricemia (HUA). This study examined the effects of soy protein and plant-based oils on HUA-induced inflammation and immune dysfunction. Male Wistar rats, induced with HUA using oxonic acid and uric acid (UA), were fed casein or soy protein with palm or safflower oil (2 × 2 factorial design) for 8 weeks. HUA rats had lower serum albumin and T cell percentages in peripheral blood leukocytes (PBLs) and splenocytes, along with increased leukocyte counts and spleen weights, compared to healthy rats (p < 0.05). Soy protein improved HUA-induced reductions in albumin, while safflower-seed oil ameliorated reductions in albumin, plasma interleukin (IL)-4, and T-suppressor splenocytes, and mitigated elevated serum UA, plasma IL-6, and B leukocytes (two-way ANOVA, p < 0.05). In PBL, soy protein alleviated HUA-induced decreases in TNF-α, casein and palm oil increased IL-6, and casein further reduced IFN-γ production. Under Con A stimulation, casein and safflower-seed oil alleviated decreases in IL-6 and IL-10, respectively, while under LPS stimulation, casein further increased TNF-α production. In splenocytes, soy protein and safflower-seed oil reduced HUA-induced increases in TNF-α and increased IL-10, and safflower-seed oil increased IL-6 production. Under Con A stimulation, soy protein and safflower-seed oil reduced TNF-α and increased IL-10 production in splenocytes. The findings suggest that soy protein and safflower-seed oil may counteract HUA-related inflammation, alleviate monocyte activation, and enhance Th2 immune response in HUA. A plant-based diet rich in soy protein and safflower-seed oil may help manage HUA and associated inflammation and immune dysfunction. Full article

The influence of heating as a pretreatment on the structural and functional attributes of milk protein concentrate (MPC) powders derived from ultrafiltered/diafiltered (UF/DF) skim milk is under-reported. This research delves into the impact of pH and heat treatment on skim milk’s properties before UF/DF and how these changes affect the resulting MPC powders. By adjusting the pH of skim milk to 6.5, 6.8, or 7.1 and applying thermal treatment at 90 °C for 15 min to one of two divided lots (with the other serving as a control), we studied the protein interactions in MPC. Post-heat treatment, the skim milk’s pH was adjusted back to 6.8, followed by ultrafiltration and spray drying to produce MPC powders with protein content of 82.38 ± 2.72% on a dry matter basis. MPC dispersions from these powders at 5% protein (w/w) were also evaluated for particle size, viscosity, and heat coagulation time (HCT) to further understand how the protein interactions in skim milk influence the properties of MPC dispersions. Capillary electrophoresis was used to assess the casein and whey protein distribution in both the soluble and colloidal phases. Findings revealed that preheating skim milk at pH 7.1 increased serum phase interactions, while heating skim milk preadjusted to a pH of 6.5 promoted whey protein–casein interactions at the micellar interface. Notably, the D (4,3) of casein micelles was larger for dispersions from milk with a preheated pH of 6.5 compared to other pH levels, correlating positively with enhanced dispersion viscosity due to increased volume fraction. These results support the potential for tailoring MPC powder functionality in various food applications through the precise control of the milk’s pre-treatment conditions. Full article

The shear flow and solid–liquid transition of mixed milk protein dispersions with varying concentrations of casein micelles (CMs) and serum proteins (SPs) are integral to key dairy processing operations, including microfiltration, ultrafiltration, diafiltration, and concentration–evaporation. However, the rheological behavior of these dispersions has not been sufficiently studied. In the present work, dispersions of CMs and SPs with total protein weight fractions (ω_PR) of 0.021–0.28 and SP to total protein weight ratios (R_SP) of 0.066–0.214 and 1 were prepared by dispersing the respective protein isolates in the permeate from skim milk ultrafiltration and then further concentrated via osmotic compression. The partition of SPs between the CMs and the dispersion medium was assessed by measuring the dry matter content and viscosity of the dispersion medium after separating it from the CMs via ultracentrifugation. The rheological properties were studied at 20 °C via shear rheometry, and the sol–gel transition was characterized via oscillatory measurements. No absorption of SPs by CMs was observed in dispersions with ω_PR = 0.083–0.126, regardless of the R_SP. For dispersions of SPs with ω_PR ≤ 0.21, as well as the dispersion medium of mixed dispersions with ω_PR = 0.083–0.126, the high shear- rate-limiting viscosity was described using Lee’s equation with an SP voluminosity (v_SP) of 2.09 mL·g⁻¹. For the mixed dispersions with a CM volume fraction of φ_CM ≤ 0.37, the relative high shear-rate-limiting viscosity was described using Lee’s equation with a CM voluminosity (v_CM) of 4.15 mL·g⁻¹ and a v_SP of 2.09 mL·g⁻¹, regardless of the R_SP. For the mixed dispersions with φ_CM > 0.55, the relative viscosity increased significantly with an increasing R_SP (this was explained by an increase in repulsion between CMs). However, the sol–gel transition was independent of the R_SP and was observed at φ_CM ≈ 0.65. Full article