Search Results (93)

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in peptides and proteins, have widespread industrial applications, particularly in milk coagulation for cheese production. Microbial enzymes have been employed as alternatives to animal rennet, offering advantages such as cost-effectiveness, availability, and compliance with dietary, cultural, and religious requirements. Solid-state fermentation (SSF) is widely employed for microbial enzyme production because of its low operational costs, reduced water and energy requirements, high product concentrations, and the ability to utilize agro-industrial residues as low-cost substrates, thereby contributing to both process sustainability and waste valorization. We report the production and characterization of a novel milk-clotting protease produced by Trichoderma koningii FFT13. The protease was produced by SSF using wheat bran as the substrate, an agro-industrial residue. It was classified as a chymotrypsin-like serine protease and exhibited a specific caseinolytic activity of 9861 U/mg. The enzyme coagulated both reconstituted skim milk and pasteurized whole milk in the presence or absence of calcium. Coagulation was enhanced by increasing temperature, reaction time, enzyme concentration, and calcium levels. Scanning electron microscopy revealed destabilization of casein micelles, their progressive aggregation, and the formation of a well-defined gel network, confirming the effectiveness of the protease in milk coagulation. Therefore, these results demonstrate that the chymotrypsin-like protease from T. koningii is a promising enzyme for milk coagulation, with potential application in cheese production. The enzyme obtained constitutes an alternative to traditional coagulants, overcoming limitations related to animal rennet while potentially offering additional advantages in terms of process sustainability and industrial scalability. Full article

Nonfat dry milk (NFDM) powder was produced by spray drying a pulsed electric field (PEF)-treated solution of 48% (m/m) evaporated skim milk (ESM) that was treated with a field strength of 20 kV/cm and specific energy of 15 kJ/L at 150 L/h. PEF treatment induced reduction to particle size for whey proteins by 8.4% and casein micelles by 11.1% and increased conductivity by 10.6%. The PEF-treated ESM solution was less viscous than the non-PEF control (14.5% lower) and sedimentation was reduced by 40%. Increases to the tapped density (1.9%), solubility (4.7%), and emulsification stability (60%) of the NFDM were observed after PEF treatment. Evaluation of protein structure indicated no modification to the secondary structure, while minor changes to the tertiary structure were observed with increased fluorescence intensity and decreased transition temperatures. The reduction in casein micelle size for the PEF-treated ESM may be associated with the movement of minerals to the aqueous solvent. This study is the first to apply PEF technology to a highly concentrated ESM solution using a continuous flow commercial PEF system. The results of this study suggest that PEF technology may be beneficial to improving the dairy processing efficiency of ESM and product quality for NFDM. Full article

Drying significantly influences the quality of micellar casein (MC) powder. This study investigated the effects of three pre-freezing temperatures (−20 °C, −80 °C, and liquid nitrogen) prior to freeze drying on the structure and rennet-induced gelation properties of MC powder. The results showed that as the pretreatment temperature decreased, the degree of disruption to the secondary and tertiary protein structures was reduced, while the particle size gradually increased. In terms of rennet-induced gel properties, the untreated raw MC consistently outperformed all MC powder samples. Among the MC powders, the sample pre-frozen at −80 °C and then freeze-dried (FD-80) exhibited the highest gel strength and a relatively shorter rennet coagulation time. The observed microstructures of the rennet-induced gel were consistent with the rheological results, showing that samples with smaller particle sizes formed more regular and compact gel networks. In conclusion, the MC powder prepared via pre-freezing at −80 °C and then freeze-drying better preserved protein structure and demonstrated superior rennet-induced gel properties, which were closely related to particle size. This study provides theoretical insights for the application of MC powder in products such as cheese, processed cheese, and protein-fortified foods. Full article

Milk is a primary source of vital nutrients and bioactive components fundamental to the growth and development of both newborn animals and humans. Produced by economically significant livestock species (including cattle, buffaloes, goats, sheep and camels), milk is a complex matrix rich in caseins, vitamins, fats, and proteins. Beyond its classical nutritional profile, milk serves as a pivotal vehicle for milk-derived extracellular vesicles (mEVs). These specialized food-derived EVs (fEVs) exert pleiotropic effects that resonate with the One Health paradigm, linking animal well-being and human nutrition to broader ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stability, also rendering them potential therapeutic tools as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their molecular cargo. This review provides a comprehensive comparative analysis of mEVs across a diverse taxonomic range, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, highlighting their distinct functional signatures. Indeed, a critical issue in mEV research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing flow cytometry (FC) approaches are discussed. Key insights include the use of conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of bead-based assays to overcome resolution limits) and imaging flow cytometry (IFC). In both technical approaches, the application of different EV generic fluorescent markers and the strategic selection of tetraspanins (i.e., CD9, CD63, CD81), is mandatory: we emphasize that selecting the correct antibody clones and accounting for inter-species cross-reactivity are essential steps for ensuring the accuracy and reproducibility of mEV research across mammalian species. Full article

Handling dispersion of casein powders in water is widely encountered in the milk industry. However, in silico prediction of the apparent viscosity of these colloidal dispersions is not an easy task, especially when these micellar casein suspensions are highly concentrated, as in hyper-protein milk beverages, which are experiencing exponential market growth. In this work, Coarse-Grained (CG) models using Lennard-Jones potentials to model interactions were built for simulating rheological properties of colloidal micellar casein dispersions (native and demineralized). In a first approach, a polydisperse explicit CG model was developed. For this polydisperse CG model, the representation chain was composed of four large smooth spheres of different sizes mimicking the real distribution of casein colloids. The CG simulation results were validated by comparison with experimental rheological data for native colloidal casein dispersions. Both in-house experimental results and available data found in the literature were used for this purpose, covering a wide range of casein concentrations ([10 g/L–200 g/L], [8–20%] corresponding to casein concentration, colloid volume fraction and solid/liquid volume fraction, respectively). In a second approach, a simplified model using a monodisperse CG model was developed. This simplified model only included one type of soft sphere and was found to preserve the accuracy of the rheological prediction. Finally, a monodisperse CG model was set up to predict the behavior of demineralized micellar casein dispersions, for which a decrease in the average size of the micelle size distribution is observed when demineralization occurs. For all models, the comparison between the predicted and experimental rheological behavior is fully satisfactory, proving that the CG models proposed for casein-based micellar dispersions are physically well founded and that the proposed simplified representation chain, based on micelle size observation, makes sense. Full article

Curcumin is a polyphenolic compound isolated from Curcuma longa, which is widely recognized for its therapeutic properties: particularly its strong anti-inflammatory and antioxidant activities. However, its practical incorporation into functional foods, especially aqueous dairy beverages, is severely hindered by its extremely low water solubility, poor chemical stability (notably at the near-neutral pH of milk), and very limited oral bioavailability. This review provides a critical synthesis of the literature published in the last two decades, with a focus on the development and application of food-grade oil-in-water (O/W) nanoemulsions to advanced colloidal delivery systems. It covers the fundamental principles of nanoemulsion formulation, including the selection of the oil phase, surfactants, and stabilizers, as well as both high-energy and low-energy fabrication techniques. It further examines the integration of these nano-delivery systems into dairy matrices (milk, yogurt, cheese), highlighting key interactions between nanoemulsion droplets and native dairy constituents such as casein micelles and whey proteins. Critically, findings indicate that nanoencapsulation not only enhances curcumin’s solubility but also protects it from chemical degradation during industrial processes, including pasteurization and sterilization. Moreover, the dairy matrix structure plays a key role in modulating curcumin bioaccessibility, with fortified products frequently exhibiting enhanced stability, shelf life, and sensory attributes. Finally, key technological challenges addressed the heterogeneous global regulatory landscape surrounding biopolymers and future trends: most notably, the growing shift toward “clean-label” biopolymer-based delivery systems. Full article

Milk adulteration through centrifugation, which artificially reduces the somatic cell count (SCC), represents a significant challenge to food authenticity and public health. This fraudulent practice alters the native molecular architecture of milk, masking inflammatory conditions such as subclinical mastitis and distorting product quality. Conventional analytical and microscopic techniques remain insufficiently sensitive to detect the subtle physicochemical changes associated with centrifugation, highlighting the need for molecular-level, data-driven diagnostics. The dataset included 128 paired raw milk samples and approximately 25,000 bright-field micrographs acquired across multiple microscopes, of which 95% were confirmed to be of high quality. In this study, advanced machine learning (ML) and deep learning (DL) approaches were applied to identify centrifugation-induced alterations in raw milk microstructure. Bright-field micrographs (pixel size 0.27 µm) of paired unprocessed and centrifuged samples were obtained under standardized optical conditions and analyzed using convolutional neural networks (ResNet-18/50, Inception-v3, Xception, NasNet-Mobile) and hybrid attention architectures (MaxViT, CoAtNet). Model performance was evaluated using the harmonic average of recalls across five micrographs per sample (HAR₅). Human microscopy experts (n = 4) achieved only 18% classification accuracy—below the random baseline (25%)—confirming that centrifugation-induced modifications are not visually discernible. In contrast, DL architectures reached up to 97% accuracy (HAR₅, Xception), successfully identifying subtle molecular cues. Class activation and sensitivity analyses indicated that models focused not on milk fat globule (MFG) boundaries but on high-frequency nanoscale variations related to the reorganization of casein micelles and solid non-fat fractions. The findings strongly suggest that centrifugation adulteration constitutes a molecular reorganization event rather than a morphological alteration. The integration of optical microscopy with AI-driven molecular analytics establishes deep learning as a precise and objective tool for detecting fraudulent milk processing and improving food integrity diagnostics. Full article

The poor rehydration capacity of micellar casein (MC) powder due to the tightly cross-linked structure formed by colloidal calcium phosphate (CCP) limits its potential applications. This study aimed to improve the rehydration properties of MC powder by electrodialysis (ED) treatment. After being treated by ED for 0, 10, 30, 60, and 90 min, the casein micelle powder exhibited a reduction in calcium content (25.55 ± 0.08 g/kg to 17.47 ± 0.05 g/kg) with prolonged treatment, corresponding to the progressive dissociation of CCP bridges. Furthermore, the ED treatment significantly increased the solubility of casein micelle powder. Interestingly, after 60 min, the solubility of ED-treated micellar casein plateaued at approximately 80% within 30 min, likely due to modification on protein structure. Therefore, these results indicate that the structure of micellar casein can be modified through ED treatment, leading to improved rehydration properties. Full article

The enzymatic coagulation of milk, crucial in cheese production, entails the hydrolysis of κ-casein and subsequent micelle aggregation. Conventional assessment standards, such as the Berridge method, depend on visual inspection and are susceptible to operator bias. Recent methods for the identification of milk-clotting time rely on optical, ultrasonic, and image-based technologies. In the present work, the composition of milk was evaluated through standard methods from ISO and AOAC. Milk coagulation time (MCT) was measured through viscosimetry, Berridge’s operator-driven technique, and a machine learning approach employing computer vision. Coagulation was additionally observed using the Optigraph, which measures micellar aggregation through near-infrared light attenuation for immediate analysis. Sheep milk samples were analysed for their composition and coagulation characteristics. Coagulation times, assessed via Berridge (BOB), demonstrated high correlation (R² = 0.9888) with viscosimetry (Visc) and machine learning (ML). Increased levels of protein and casein were linked to extended MCT, whereas lower pH levels sped up coagulation. The calcium content did not have a notable impact. Optigraph assessments validated variations in firmness and aggregation rate. Principal Component Analysis (PCA) identified significant correlations between total solids, casein, and MCT techniques. Estimates from ML-based MCT closely align with those from operator-based methods, confirming its dependability. This research emphasises ML as a powerful, automated method for evaluating milk coagulation, presenting a compelling substitute for conventional approaches. Full article

Hybrid systems combining animal and plant proteins are promising for developing sustainable, high-protein foods. However, structural incompatibility between proteins like casein and pea protein hinders the formation of stable systems such as gels. This study explores pH-shifting (alkalization at pH 12 followed by neutralization) as an innovative strategy to improve pea protein functionality and compatibility in hybrid gels. Modified pea protein showed increased solubility, reduced particle size, higher zeta potential, and decreased intrinsic fluorescence intensity, indicating conformational changes and exposure of buried tryptophan residues. These structural changes influenced gel behavior depending on the protein ratio (casein/pea—80:20, 50:50, 20:80). Gels with higher pea content showed increased hardness and water-holding capacity, while in casein-rich gels, hardness decreased, likely due to altered protein–protein interactions. This is the first study to systematically apply pH-shifting to enhance the compatibility between pea protein and casein in high-protein gels, integrating structural and functional analyses. The results demonstrate the potential of pH-shifting as a sustainable and effective approach for improving plant protein performance in hybrid formulations. Full article

Casein, the main phosphoprotein in milk, has a multifaceted molecular structure and unique physicochemical properties that make it a viable candidate for biomedical use, particularly in wound healing. This review presents a concise analysis of casein’s structural composition that comprises its hydrophobic and hydrophilic nature, calcium phosphate nanocluster structure, and its response to different pH, temperature, and ionic conditions. These characteristics have direct implications for its colloidal stability, including features such as gelation, swelling capacity, and usability as a biomaterial in tissue engineering. This review also discusses industrial derivatives and recent advances in casein biomaterials based on different fabrication types such as hydrogels, electrospun fibres, films, and advanced systems. Furthermore, casein dressings’ functional and biological attributes have shown remarkable exudate absorption, retention of moisture, biocompatibility, and antimicrobial and anti-inflammatory activity in both in vivo and in vitro studies. The gathered evidence highlights casein’s versatile bioactivity and dynamic molecular properties, positioning it as a promising platform to address advanced wound dressing challenges. 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

Flavonoids, widely present in Artemisia argyi (AA), offer potential health benefits but are limited in food applications because of their bitter taste, inadequate absorption, and stability. Casein micelles encapsulation can enhance the flavonoid absorption, stability, and bioactivity. In this study, Artemisia argyi flavonoids (AAFs) were extracted using ultrasound-assisted extraction (UAE) to optimize the process. The glycosylation reaction between casein (CN) micelles and oat β-glucan (OBG) was employed to improve AAF’s emulsifying stability, sustained release during digestion, and cellular uptake. The maximum glycosylation degree of 32.33% was achieved at a CN-to-OBG ratio of 1:2, 120 min browning time, and 95 °C temperature. This glycosylated delivery system enhanced the emulsifying properties of the AAFs, digestive sustained release, and cellular uptake, showing potential as a cross-linking material for fat-soluble substances and medicines. Full article

In milk, casein proteins orientate themselves into spherical micellar structures with hydrophobic casein subtypes concentrated in the core, while hydrophilic casein subtypes populate the exterior. Previous research demonstrated that milk with the addition of emulsifying salts coupled with high-pressure homogenization induced an unprecedented amount of casein micelle dissociation. This research aims to quantify the extent of casein micelle dissociation in diluted skim milk and evaluate the functionality of these proteins following emulsifying salt treatment coupled with high-pressure homogenization. To evaluate the extent of micellar dissociation, dilute skim milk solutions (20% v/v) were prepared with a varying amount of treatment: no processing (control), just emulsifying salts (Treatment E, 100 mM sodium hexametaphosphate), just high-pressure homogenization (Treatment H, at 300 MPa), and EH (a combination of E and H treatments). Samples were then put through varying filter sizes (0.22 µm, 0.05 µm), and the permeates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the control group (20% skim milk), 9.35% ± 2.53% casein protein permeated through a 0.05 µm filter. Alternatively, 93.2% ± 7.71% casein protein was present in EH samples post-filtration through a 0.05 µm filter, demonstrating a significant processing-induced dissociation of casein micelles. A potential benefit to this casein micelle size reduction is the exposure of highly functional hydrophobic subunits from the core of the micelle. In agreement, compared to the control samples, the EH samples had higher foam expansion index values (138.3% ± 12.58% vs. 33.33% ± 14.43% at 0 h), foam stability (113.3% ± 5.774% vs. 21.67% ± 2.887% after 8 h), emulsifying activity (ca. two-fold higher), and interaction with caffeine. These data demonstrate that E, coupled with H, enhances skim milk system functionality, and these changes are likely due to micellar dissociation and protein conformational changes. This work has direct applications in dairy systems (e.g., dairy foams, dairy ingredients) as well as implications for potential processing strategies for other protein-rich systems. Full article

Fish oil, a rich source of omega-3 polyunsaturated fatty acids (PUFA), is a vital nutritional component, but considering its susceptibility to oxidation, it could benefit from an effective encapsulation system. This study aims to optimize high-pressure homogenization (HPH) parameters (pressure, number of passes) and wall material composition to maximize the encapsulation efficiency (EE) of fish oil, using different concentrations of maltodextrin with Arabic gum or sodium alginate. Key metrics such as emulsion droplet size, encapsulation efficiency, color, and oxidation in the final freeze-dried product were evaluated. Optimal values were achieved at 60 MPa, resulting in the lowest mean droplet diameter (369.4 ± 3.8 nm) and narrow distribution (0.197 ± 0.011) of the fish oil micelles prepared with a mixture of Tween80 and sodium caseinate as an emulsifier, without significant oxidation after four cycles of homogenization, while 80 MPa led to the highest EE (up to 95.6%), but increased oxidation. The combination of 10% w/w Arabic gum or 1% w/w sodium alginate with 20% w/w maltodextrin achieved the highest EE (79.1–82.9%) and whiteness index (82.5–83.0), indicating neutral-colored well-encapsulated fish oil without oxidation, which is desirable for product stability. Selecting optimal HPH conditions and wall material is crucial for the encapsulation efficiency and oxidation stability of omega-3 PUFA delivered in dehydrated forms. Full article