Search Results (83)

Young child formula (YCF) products are important sources of nutrients for children 1–3 years of age. Salt equilibria and protein glycation are two of the crucial aspects affecting nutritional properties and digestive behaviors of YCF, but detailed insights into these two aspects of YCF products remains limited. This study analyzed the distribution of salts and the level of protein glycation in 25 commercial YCF products from the retail market in China. The YCF products were reconstituted (12 g of powder per 100 g of water) and the distribution of calcium and phosphorus between the sedimentable (at 200× g), protein-associated and soluble (10 kDa-permeable) fractions were determined. Blocked lysine and 5-hydroxymethylfurfural were analyzed using reversed-phase high-performance liquid chromatography. Varying proportions of calcium (3.0–39.3%) and phosphorus (1.2–29.8%) were sedimentable for the products. Notable proportions of calcium (28.9–62.7%) and phosphorus (27.4–57.9%) were associated with the proteins. The remainder of the calcium (24.9–41.4%) and phosphorus (34.2–62.1%) were soluble. When expressing the protein-associated calcium as a function of casein, i.e., casein mineralization, large differences (~1.7 fold) were found among products. Variation in blocked lysine (7.4–19.2% of total lysine) and 5-hydroxymethylfurfural contents (3.0–7.0 mg/100 g protein) among products was also observed, suggesting notable differences in heat-load during processing. This study revealed notable variation in salt distribution and protein glycation among the YCF products. These findings underscore the critical need for manufacturers to optimize formulation and processing approaches, e.g., using milk with a low level of casein mineralization and using milk protein sources as concentrated liquid rather than powder to reduce protein glycation, to improve nutritional properties of the products. Full article

Background: Skin aging is mainly associated with oxidative stress and enzymatic degradation of collagen and elastin by protease activity. Peptides have antioxidant capacity and inhibitory effects on protease enzymes. Objective: The purpose of this study was to obtain peptides with in vitro anti-aging activity from the enzymatic hydrolysis of bovine casein with actinidin, a protease extracted from the green kiwi fruit (Actinidia deliciosa) Methodology: The enzyme actinidin was extracted from the pulp of the kiwi fruit, purified by ion exchange chromatography and characterized by polyacrylamide electrophoresis (SDS-PAGE). Subsequently, the extracted enzyme was used to hydrolyze commercial bovine casein at 37 °C for 30 min, precipitating the peptide fraction with trichloroacetic acid (TCA), and centrifuged. To determine the anti-aging potential of the peptides in vitro, antioxidant activity was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical. Additionally, the inhibitory capacity of the peptides against collagenase and elastase enzymes was also studied. To complement the in vitro results, the enzymatic hydrolysis of casein with actinidin was simulated. The binding energy (ΔG) of each of the hydrolysates with the collagenase and elastase enzymes was calculated using molecular docking to predict the peptide sequences with the highest probability of interaction. Results: Actinidin was extracted and purified exhibiting a molecular weight close to 27 kDa. The enzyme hydrolyzed the substrate by 91.6%, and the resulting hydrolysates showed moderate in vitro anti-aging activity: antioxidant (17.5%), anticollagenase (18.55%), and antielastase (28.6%). In silico results revealed 66 peptide sequences of which 30.3% consisted of 4–8 amino acids, a suitable size to facilitate interaction with structural targets. The sequences with the highest affinity were FALPQYLK and VIPYVRYL for collagenase and elastase, respectively. Conclusions: Despite the modest inhibition values, the use of a fruit-derived enzyme and a food-grade substrate is in line with current trends in sustainable and natural cosmetics. These findings highlight the great potential for laying the groundwork for future research into actinidin-derived peptides as multifunctional and eco-conscious ingredients for the development of next-generation anti-aging formulations. Full article

The covalent cross-linking of caseins by the enzyme transglutaminase (Tgase) stabilizes the structure of casein micelles. In our study, the effects of a pretreatment of skim milk (SM) by Tgase on milk protein fractionation by microfiltration were tested. Tgase was found to induce amount-dependent modifications of all milk proteins in SM and a reduction in deposit resistance for laboratory dead-end filtrations of up to 20%. This improvement in process performance could partially be confirmed in pilot-scale cross-flow filtrations of Tgase-pretreated SM and micellar casein solutions (MCC). These comparative trials with untreated retentates under a variation of ΔpTM (0.5–2 bar) at 10 and 50° revealed distinct differences in deposit behavior and achieved the reduction in deposit resistance in a range of 0–20%. The possibility of pre-fouling with enzymatically pretreated MCC prior to SM filtration was also investigated. Under different pre-fouling conditions, practical modes of retentate change, and pre-foulant compositions, a switch to untreated SM consistently resulted in an immediate and major increase in deposit resistance by 50–150%. This was partially related to the change in the ionic environment and the protein fraction. Nevertheless, our results underline the potential of Tgase pretreatment and pre-fouling approaches to alter filtration performance for different applications. Full article

Mastitis significantly impacts both the yield and quality of milk. The somatic cell count (SCC) and differential somatic cell count (DSCC), which are related to immune cells, are primary indicators for assessing mammary gland health. In this study, eight previously established mid-infrared spectroscopy models were utilized to predict the content of milk protein fractions (αs1-CN, β-CN, κ-CN, total CN, α-LA, β-LG, IgG, and LF) in milk samples from 21,388 lactating cows across 33 herds. Four linear mixed models were applied to analyze the secretion patterns of milk protein fractions by days in milk (DIM) and parity, their variations under different mastitis conditions, and their associations with the somatic cell score (SCS), DSCC, and immune cell counts (PMN + LYM score (PMN + LYMS) and MAC score (MACS)). The primary findings of the investigation comprised the following: (1) IgG was higher in early lactation, decreased with advancing lactation days, and slightly increased in late lactation, while seven other protein factions decreased from early to peak lactation and increased during mid-to-late lactation. Parity influenced all milk protein fractions except αs1-CN, with total CN, β-CN, and α-LA decreasing and κ-CN, β-LG, IgG, and LF increasing as parity increased (p < 0.05). (2) Mastitis significantly reduced the milk yield, fat percentage, protein percentage, and the contents of total CN, β-CN, κ-CN, and α-LA while increasing β-LG, IgG, and LF. (3) The SCS was negatively correlated with milk yield and α-LA but positively correlated with the fat percentage, protein percentage, κ-CN, β-LG, IgG, and LF. (4) When the DSCC increased to 50%, the milk yield decreased, while the milk protein percentage and κ-CN content significantly increased (p < 0.05). When the DSCC exceeded 50%, the fat percentage, protein percentage, total casein, αs1-CN, β-CN, κ-CN, β-LG, IgG, and LF decreased, while the α-LA content increased (p < 0.05). (5) When the PMN + LYMS increased, the milk yield and α-LA content rose, while the milk fat percentage, the milk protein percentage, and the contents of αs1-CN, β-CN, κ-CN, total CN, β-LG, IgG, and LF decreased (p < 0.05). Conversely, when the MACS increased, the milk yield and α-LA content declined, whereas the milk fat percentage, the milk protein percentage, and the contents of αs1-CN, β-CN, κ-CN, total CN, β-LG, IgG, and LF increased (p < 0.05). This study offers valuable insights into enhancing milk product quality, advancing the early diagnosis and mechanistic research of bovine mastitis, and the sustainable development of the dairy farming industry. Full article

This study examined the impact of extensive and intensive farming systems on the rheological behavior of rennet-induced goat milk coagulation in the indigenous Greek Skopelos goat breed. Milk samples were obtained from Skopelos dairy goats reared under extensive and intensive farming systems at two farms in Greece. Rennet-induced coagulation kinetics (at 35 °C) and curd rheological properties were assessed using dynamic rheometry. Milk from the extensive system exhibited longer rennet coagulation time (RCT) but resulted in curds with higher storage modulus (G′) and complex viscosity (η*), indicating formation of stronger coagulated structures compared to curds from the intensive system. The differences can be attributed to variations in milk composition and the structural characteristics of protein components, e.g., casein fractions, between the two systems. Principal component analysis (PCA) revealed that the farming systems could be differentiated based on the rheological properties of the curds, specifically on structure-related parameters (loss tangent, tanδ, apparent yield stress, τ_y). Strong correlations (p < 0.01) were noted between G′max and caseins/total proteins (positive), as well as with pH (negative), in both farming systems. These findings offer valuable insights into animal farming practices and cheese production, providing evidence of the relationships between goat-rearing systems and rheological properties of rennet-coagulated milk products. Full article

Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. 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

Background: Cow’s milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine α_S1-casein protein. Methods: A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine α_S1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant α_S1-casein production. After increasing the molecular abundance of the recombinant α_S1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine α_S1-casein. Results: Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant α_S1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mg_casein/g_CDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native α_S1-casein showed pH-mediated differences in aggregation behavior. Conclusions: The study demonstrates the potential of B. subtilis for the recombinant production of bovine α_S1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins. 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

The salt fractions of milk consist of cations (e.g., Ca, Mg, and Na) and anions (e.g., phosphate, citrate, and chloride). These salts are present as free ions or in complexes with other ions or proteins, primarily the caseins. Furthermore, significant levels of Ca and phosphate are also found in insoluble form, inside the casein micelles. The distribution of salts between this micellar phase and the soluble phase is important for the stability and properties of milk and dairy products. Various processes, such as (ultra-)centrifugation, (ultra-)filtration, dialysis, and selective precipitation have been used to separate the micellar and soluble phases in milk and dairy products to allow for studying the salts’ distribution between these phases. These different methods can lead to different levels of soluble salts because the salts in the supernatant from centrifugation, the permeate from ultrafiltration, and the diffusate from dialysis can differ notably. Hence, understanding which components are fractionated with these techniques and how this affects the levels of the soluble salts determined is critical for milk and dairy products. Applying the aforementioned methods to cheese products is further challenging because these methods are primarily developed for fractionating the soluble and micellar phases of milk. Instead, methods that analyze salts in water-soluble extracts, or soluble phases expressed from cheese by pressing or centrifugation are typically used. This review focuses on the significance of salt distribution and variations in salt fractions obtained using different methodologies for both milk and cheese. Full article

The aim of the study was to develop casein–fucoidan composite nanostructures through the method of polyelectrolyte complexation and subsequent spray drying. To determine the optimal parameters for the preparation of the composite structures and to investigate the influence of the production and technological parameters on the main structural and morphological characteristics of the obtained structures, 3(k-p) fractional factorial design was applied. The independent variables (casein to fucoidan ratio, glutaraldehyde concentration, and spray intensity) were varied at three levels (low, medium, and high) and their effect on the yield, the average particle size, and the zeta potential were evaluated statistically. Based on the obtained results, models C1F1G1Sp.30, C1F1G2Sp.40, and C1F1G3Sp.50, which have an average particle size ranging from (0.265 ± 0.03) µm to (0.357 ± 0.02) µm, a production yield in the range (48.9 ± 2.9) % to (66.4 ± 2.2) %, and a zeta potential varying from (−20.12 ± 0.9) mV to (−25.71 ± 1.0) mV, were selected as optimal for further use as drug delivery systems. Full article

Severe gastrointestinal symptoms (GIS) and food hypersensitivity are tightly associated in young individuals with autism spectrum disorders (ASD). Here, we explored the relationship of GIS (gastrointestinal severity index, ROMA IV criteria, Bristol scale), ASD-like behaviors (Childhood Autism Rating Scale), and certain sociodemographic/clinical traits (epidemiological survey) with serum immunoreactivity (IgG, IgA, IgE titers) towards bovine milk caseins (BMC; by ELISA) and subfractions (by immunoblotting) in thirty-one pediatric patients (~3–15 y, 77% male) with mild-to-severe GIS and ASD-like behaviors. In total, 42%, 25%, and 23% of all participants exhibited no (IgG−/IgA−), mono (IgG+/IgA−), or dual (IgG+/IgA+) immunoreactivity to BMC, respectively; the trend was significantly associated with the severity of the GIS and ASD-like behaviors, regurgitations, and self-reported allergies (OR: 1 → (1.9–3.1) → 13.5–16.0)]. No IgE+ response to BMC was found. Dual responders were α > κ > β-casein, though nonspecific reactivity to other protein fractions was also observed. The IgA+ > IgG+ but not IgE+ response to BMC (mainly α-casein) seems to be related to the severity of GIS and ASD-like behaviors, although a larger number of ASD patients are needed to draw a causal association. Full article

The objective of this work was to study the influence of three dietary fibre fractions (pectin, gum arabic and cellulose) and three protein fractions (casein, lactalbumin and soy) on the trace element bioaccessibility (Fe, Mn, Ni, Se and Zn) of turnip tops (B. rapa subsp. Rapa) growing under Mediterranean conditions. Then, it aimed to promote the use of this vegetable not only for direct fresh consumption but also as a main ingredient in the development of food mixtures. The results showed that soluble fibre fractions, such as pectin and gum arabic, can enhance the bioaccessibility of trace elements, such as Fe, Mn, Se and Zn. This effect was not proved for cellulose (an insoluble fibre fraction), in which, at best, no bioaccessibility effect was observed. Regarding the protein fractions, with the exception of Se, caseins and lactalbumin had a neutral effect on improving the trace element bioaccessibility. This did not hold true for soy protein, in which a considerable improvement in the bioaccessibility of Fe, Mn, Se and Zn was determined. Full article

This study aimed to evaluate the influence of low-concentration rennet on the chemical, rheological characteristics, and protein fractions of skim milk (SM) at 4 ± 1 °C. Skimmed milk (SM) was divided into four lots of 500 mL, and diluted rennet (1:10,000) was added at different levels at 4 ± 1 °C. The treatments included control (no rennet), T1 (0.001 mL/rennet), T2 (0.01 mL rennet), and T3 (0.1 mL rennet) treatments, which were incubated for 24 h. The sampling was performed at 0, 1, 2, 6, 12, and 24 h, and the SM after incubation time was heated to 73 °C/16 s to denature the rennet enzyme. Skim milk samples (SMS) (control and rennet-added samples) were evaluated for proximate composition, capillary gel electrophoresis (CGE), hydrodynamic diameter, zeta potential, and rheology at 0, 1, 2, 6, 12, and 24 h. Foaming ability, foaming stability, water-holding capacity (WHC), oil emulsifying activity (OEA), and emulsion stability (ES) were performed at 0, 12, and 24 h of incubation time. There was a significant (p < 0.05) increase in non-proteins by 0.50% and in non-casein nitrogen by 0.81% as incubation progressed. The results showed that aggregation or curd was not formed during storage time. The CGE data indicated that increasing the rennet concentration had a significant (p < 0.05) effect on decreasing κ-CN, and breakdown increased at higher levels of rennet usage. There was a significant (p < 0.05) increase in the hydrodynamic diameter and a decrease in the zeta potential values in rennet-added samples at the end of the incubation time (24 h). The rheological results showed no changes in the storage modulus (G′), loss modulus (G″), or viscosity values. Increasing the rennet amount and storage time led to a significant (p < 0.05) decrease in the foaming ability and foaming stability and a significant (p < 0.05) increase in the oil emulsifying activity and emulsion stability of rennet-added SMS. This study concluded that milk protein functionality can be changed without aggregating or curd formation, and rennet milk can be processed. Full article