Bovine Whey Protein Research Articles

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine β-Lactoglobulin Identified by NMR Spectroscopy.

Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. β-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS–milk protein interactions, structural information on BLG–EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave KD values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG–EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.

Anxiety-like behavior and intestinal microbiota changes as strain-and sex-dependent sequelae of mild food allergy in mouse models of cow’s milk allergy

A number of studies have reported comorbidity of food allergies with various neuropsychiatric disorders, such as anxiety, depression, attention-deficit hyperactivity disorder, and autism. However, inconsistent results across clinical studies have left the association between food allergy and behavioral disorders inconclusive. We postulated that the heterogeneities in genetic background among allergic cohorts affect symptom presentation and severity of food allergy, introducing bias in patient selection criteria toward individuals with overt physical reactions. To understand the influence of genetic background on food allergy symptoms and behavioral changes beyond anaphylaxis, we generated mouse models with mild cow’s milk allergy by sensitizing male and female C57BL/6J and BALB/cJ mice to a bovine whey protein, β-lactoglobulin (BLG; Bos d 5). We compared strain- and sex-dependent differences in their immediate physical reactions to BLG challenge as well as anxiety-like behavior one day after the challenge. While reactions to the allergen challenge were either absent or mild for all groups, a greater number of BLG-sensitized BALB/cJ mice presented visible symptoms and hypothermia compared to C57BL/6J mice. Interestingly, male mice of both strains displayed anxiety-like behavior on an elevated zero maze without exhibiting cognitive impairment with the cross maze test. Further characterization of plasma cytokines/chemokines and fecal microbiota also differentiated strain- and sex-dependent effects of BLG sensitization on immune-mediator levels and bacterial populations, respectively. These results demonstrated that the genetic variables in mouse models of milk allergy influenced immediate physical reactions to the allergen, manifestation of anxiety-like behavior, levels of immune responses, and population shift in gut microbiota. Thus, stratification of allergic cohorts by their symptom presentations and severity may strengthen the link between food allergy and behavioral disorders and identify a population(s) with specific genetic background that have increased susceptibility to allergy-associated behavioral disorders.

Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk

Background:The world production of whey was estimated to be more than 200 million tons per year. Although whey is an important source of proteins with high nutritional value and biotechnological importance, it is still considered as a by-product of the dairy industry with low economic value due to low industrial exploitation. There are several challenges in the separation of whey proteins: low concentration, the complexity of the material and similar properties (pI, molecular mass) of some proteins.Methods:A narrative review of all the relevant papers on the present methodologies based on ion-exchange and adsorption principles for isolation of whey proteins, known to the authors, was conducted.Results:Traditional ion-exchange techniques are widely used for the separation and purification of the bovine whey proteins. These methodologies, based on the anion or cation chromatographic procedures, as well as combination of aforementioned techniques are still preferential methods for the isolation of the whey proteins on the laboratory scale. However, more recent research on ion exchange membranes for this purpose has been introduced, with promising potential to be applied on the pilot industrial scale. Newly developed methodologies based either on the ion-exchange separation (for example: simulated moving bed chromatography, expanded bed adsorption, magnetic ion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activated carbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’ purification processes.Conclusion:Many procedures based on ion exchange are successfully implemented for separation and purification of whey proteins, providing protein preparations of moderate-to-high yield and satisfactory purity. However, the authors anticipate further development of adsorption-based methodologies for separation of whey proteins by targeting the differences in proteins’ structures rather than targeting the differences in molecular masses and pI. The complex composite multilayered matrices, including also inorganic components, are promising materials for simultaneous exploiting of the differences in the masses, pI and structures of whey proteins for the separation.

Antioxidant and antihypertensive activity of bovine whey protein concentrate enzymatic hydrolysates

Whey is a highly polluting by-product of cheese processing. However, it has valuable nutritional properties since it is a rich and balanced source of proteins and amino acids. Therefore, it has a broad range of functional properties that can be exploited for diverse applications. Research has shown how the enzymatic hydrolysis of whey proteins releases bioactive peptides. In the present study, the hydrolysis of whey protein concentrate (WCP) was performed using purified Sporisorium reilianum aspartyl protease (Eap1), commercial enzymes chymotrypsin (C) and trypsin (T), as well as different enzymatic combinations in order to determine which enzyme or combination allowed for the release of peptides presenting the highest antioxidant and antihypertensive activity levels; our results indicated that hydrolysis with Eap1 releases the best-performing peptides in comparison with individual enzymes and their combinations.

Variations in N-linked glycosylation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) whey protein: Intercow differences and dietary effects

In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.

Effect of surfactant-induced competitive displacement of whey protein conjugated to acid- or alkali-extracted potato pectin on emulsion stability

Covalently linked protein–polysaccharide conjugate pre-adsorbed to the interface often shows more enhanced resistance to displacement induced by competitively adsorbing small molecule surfactant than the pure protein component. The objective of this study was to investigate the effect of surfactant-induced competitive displacement of whey protein–potato pectin conjugate pre-adsorbed to oil droplet surfaces on the stability of the oil-in-water emulsion. Potato pectin extracted in either an acidic or alkaline condition was conjugated to bovine milk whey protein using the Maillard reaction. The formation of covalent linkage between the protein and pectin moieties was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Atomic force microscopy studies of the interfacial films of the Maillard conjugates formed on flat water surfaces revealed that the alkali-extracted potato pectin provided more enhanced resistance to surfactant-induced competitive displacement from the air–water interface than the acid-extracted potato pectin. These results indicate that galactan side chains having an ability to self-assemble and form network structures had been preserved to a larger degree during the alkali-extraction. Furthermore, the alkali-extracted potato pectin prevented coalescence-induced increases in the oil droplet diameter in the oil-in-water emulsions prepared using the Maillard conjugate as the emulsifier upon the addition of up to 38.7 mM of the Tween 20 surfactant while maintaining relatively high levels of the surface load of protein (>0.8 mg/m2). The present results demonstrate that the molecular structure of the polysaccharide moiety of protein–polysaccharide conjugate has significant impacts on its resistance to competitive displacement and emulsion stability.

Optimisation of bovine β-lactoglobulin hydrolysis using cardosins from dried flowers of Cynara cardunculus

Bovine whey protein was hydrolysed using cardosins A and B purified from dried flowers of Cynara cardunculus by combining diafiltration, anion-exchange chromatography and ultrafiltration. The proteolysis experiments were performed using different whey protein concentrations and enzyme/substrate (E/S) ratios. Complete hydrolysis of the main whey proteins, β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La), was achieved after 4 h, at E/S ratios of 1/150 U/mg, regardless the initial protein concentration. In previous reports, the authors suggested that cardosins could not hydrolyse β-lactoblogulin. However, our promising results open up new possibilities to further explore the action of cardosins on whey proteins for the production of bioactive peptides.

Assessment of the Bioactive Potential of Cheese Whey Protein Hydrolysates Using Immobilized Alcalase

The hydrolysis of bovine cheese whey (BCW) proteins was performed by the Alcalase immobilized on the support glyoxyl-corn-cob-powder (AGCCP). The whey protein hydrolysates (WPH) obtained with AGCPP were fractionated by RP-HPLC according to the hydrophilicity of the peptides (F1, hydrophilic; F2, intermediate hydrophilicity; and F3, hydrophobic). The fractions were analyzed by MALDI-TOF, and molecular weight peptides (< 1500 m/z) were identified. Total WPH obtained by AGCCP presented high capacity reduction of the ABTS radical (57.82%) and high chelating activity of iron II (76.2%). The obtained peptides showed high antimicrobial activity (87.75–100%) against the species Escherichia coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC = 10 mg/mL). The results of this study show that the hydrolysis of BCW proteins using the AGCCP derivative represents a very interesting and viable way for obtention of bioactive peptides from cheese whey.

Region-specific regulation of central histaminergic H3 receptor expression in a mouse model of cow’s milk allergy

Central histaminergic H3 receptor (H3R) has been extensively investigated as a potential therapeutic target for various neurological and neurodegenerative disorders. Despite promising results in preclinical rodent models, clinical trials have not provided conclusive evidence for the benefit of H3R antagonists to alleviate cognitive and behavioral symptoms of these disorders. Inconsistent pharmacological efficacies may arise from aberrant changes in H3R over time during disease development. Because H3R is involved in feedback inhibition of histamine synthesis and secretion, the expression of the autoreceptor may also be reciprocally regulated by altered histamine levels in a pathological condition. Thus, we investigated H3R expression in a mouse model of cow’s milk allergy, a condition associated with increased histamine levels. Mice were sensitized to bovine whey proteins (WP) over 5 weeks and H3R protein and transcript levels were examined in the brain. Substantially increased H3R immunoreactivity was observed in various brain regions of WP-sensitized mice compared to sham mice. Elevated H3R expression was also found in the thalamic/hypothalamic region. The expression of histaminergic H1, but not H2, receptor subtype was also increased in this and the midbrain regions. Unlike the brain, all three histaminergic receptors were increased in the small intestine. These results indicated that the central histaminergic receptors were altered in WP-sensitized mice in a subtype- and region-specific manner, which likely contributed to behavioral changes we observed in these mice. Our study also suggests that altered levels of H3R could be considered during a pharmacological intervention of a neurological disease.

The effect of atmospheric cold plasma treatment on the antigenic properties of bovine milk casein and whey proteins

Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, β-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the β-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of β-sheet and β-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of β-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.

Effects of an herbal extract composed of white tea, roasted yerba mate and fermented rooibos on the antioxidant activity and sensory properties of popsicles manufactured with different protein sources

Popsicle-type edible ice cream is consumed worldwide for its sensory properties. However, its nutritional composition is limited to carbohydrates, sweeteners and synthetic flavors. In this work, the objective was to develop popsicles manufactured with different protein sources (rice protein, concentrated bovine milk whey protein and a mixture of both proteins) and added with an herbal lyophilized extract (LME) composed of white tea, fermented rooibos, and roasted yerba mate. Six formulations were produced and their proximate composition, physicochemical properties, sensory acceptability, total phenolic content, condensed tannins, and in vitro antioxidant activity determined. Popsicles added with LME showed a higher total phenolic content compared to the controls (without LME). The popsicles formulated with animal protein and LME showed the highest antioxidant activity as measured by the DPPH and FRAP assays. In relation to sensory analysis, the highest acceptance rates, 91 and 88%, were observed in formulations added with animal protein without and with LME, respectively. On the other hand, the vegan formulation added with LME had the lowest acceptance rate (69%). Overall, the addition of LME and concentrated bovine whey protein provides a viable option for the development of phenolic-rich protein-based popsicles.

In vitro cytomodulatory and immunomodulatory effects of bovine colostrum whey protein hydrolysates

SummaryBovine colostrum possesses biological compounds involved in the development of the newborn. Among them, the proteins have drawn attention as a source of bioactive peptides. This study shows the in vitro cytotoxic and immunostimulatory potential of two colostrum whey protein (CWP) hydrolysates obtained by in vitro digestion with pepsin and pancreatin. MTT cell viability, apoptosis induction, polymorphonuclear proliferation and phagocytic activity assays were performed. Treatment with the hydrolysates induced a significant decrease in the viability of MDA‐MB‐231 cell lines due to apoptosis and also a significant increase in the proliferation of blood mononuclear cells. It could also be observed that for the RM‐1 and PC‐3 prostate cancer cell lines and for the two times of exposure (24 and 48 h), the hydrolysate H1 is significantly more cytotoxic than CWP. These results showed the potential of bovine CWP and its hydrolysates for the treatment of chronic diseases such as cancer.

The effect of non-covalent interactions of xylitol with whey protein and casein on structure and functionality of protein

The mechanisms underlying the interactions of xylitol (XY), as a functional food ingredient, with bovine whey protein isolate (WPI) and casein (Cs) were investigated via multiple spectroscopic methods, and the structural and functional changes of the proteins were explored. The interactions were evidenced by fluorescence quenching, accompanied by a blue-shift. WPI had a slightly stronger binding affinity than Cs toward XY, with respective KA values of 16.95 × 104 L mol−1 and 6.69 × 104 L mol−1. H bonds and van der Waals forces may have significantly contributed to these interactions. Additionally, changes the environment around the corresponding amino acid residues of WPI and Cs, and decline in the α-helix structures were demonstrated by simultaneous fluorescence, three-dimensional fluorescence spectroscopy and circular dichroism, which resulted increased solubility, emulsification, and foaming, and decreased surface hydrophobicity of both the proteins. These results provide a basis for the development of a functional dairy-based product.

Evaluation of concentration process of bovine, goat and buffalo whey proteins by ultrafiltration.

In this research, the protein concentration, the permeate flux, and the predominant fouling mechanisms were investigated during ultrafiltration of different whey samples. The research was carried out at different values of transmembrane pressure and temperature using an experimental design, and a protein concentration of approximately 37gL-1 was obtained for the bovine whey powder solution, at 60kPa and 40°C. The maximum flux observed was 8.9 and 7.9kgm-2h-1, respectively, for the bovine whey powder solution and bovine whey, at 50kPa and 30°C. Although goat and buffalo whey presented lower permeate flux, probably due to high solutes and calcium contents, protein concentrates of around 40gL-1 were obtained using the ultrafiltration process. This demonstrates the potential of ultrafiltration to obtain non-bovine protein concentrates. The best fit,verified by Ho and Zydney model, suggests that the fouling for all analyzed whey occurs due to pore blocking and subsequent deposit on the membrane surface.

Age- and duration-dependent effects of whey protein on high-fat diet-induced changes in body weight, lipid metabolism, and gut microbiota in mice.

Bovine whey protein has been demonstrated to exert a positive effect on energy balance, lipid metabolism, and nutrient absorption. Additionally, it affects gut microbiota configuration. Thus, whey protein is considered as good dietary candidate to prevent or ameliorate metabolic diseases, such as obesity. However, the relationship that links energy balance, metabolism, and intestinal microbial population mediated by whey protein intake remains poorly understood. In this study, we investigated the beneficial effects attributed to whey protein in the context of high‐fat diet (HFD) in mice at two different ages, with short or longer durations of whey protein supplementation. Here, a 5‐week dietary intervention with HFD in combination with either whey protein isolate (WPI) or the control nonwhey milk protein casein (CAS) was performed using 5‐week or 10‐week‐old C57BL/6J mice. Notably, the younger mice had no prior history of ingestion of WPI, while older mice did. 5‐week‐old HFD‐WPI‐fed mice showed a decrease in weight gain and changes in the expression of genes within the epidydimal white adipose tissue including those encoding leptin, inflammatory marker CD68, fasting‐induced adipose factor FIAF and enzymes involved in fatty acids catabolism, relative to HFD‐CAS‐fed mice. Differences in β‐diversity and higher proportions of Lactobacillus murinus, and related functions, were evident within the gut microbiota of HFD‐WPI mice. However, none of these changes were observed in mice that started the HFD dietary intervention at 10‐weeks‐old, with an extended period of WPI supplementation. These results suggest that the effect of whey protein on mouse body weight, adipose tissue, and intestinal parameters depends on diet duration and stage of life during which the diet is provided. In some instances, WPI influences gut microbiota composition and functional potential, which might orchestrate observed metabolic and physiological modifications.

Effect of pH on the physicochemical characteristics and the surface chemical composition of camel and bovine whey protein’s powders

This study investigated the effect of pH on the denaturation extent, the surface chemical composition, the water sorption isotherm and the glass transition temperature of camel and bovine whey protein’s powders. The LC-MS analysis indicated that the β-Lactoglobulin was the most denatured protein in bovine whey powders regardless the pH value, while this protein was totally absent in camel whey. The α-Lactalbumin was relatively heat stable after drying and predominated the powder surface (X-ray photoelectron spectroscopy results) in both camel and bovine whey powders regardless the pH (neutral (6.7) or acidic (4.3 and 4.6)). Analysis of the water sorption isotherms indicated that decreasing the pH induced the increase of the water activity of lactose crystallization for camel and bovine whey powders. Finally, decreasing the pH led to the decrease of the glass transition temperature of camel and bovine whey powder (at 0.13, 0.23, and 0.33 of water activity).

The Physico-Chemical Properties of Bovine and Buffalo Whey Proteins Milk by Using Ultrafiltration Membrane Technology

The current study aims to prepare whey from bovine and buffalo fresh milk to make three types of cheese, namely: thermal, acidic and enzymatic. Afterward, whey proteins have been separated, then the concentration process of whey proteins has been conducted by using ultrafiltration membrane technology. Through the previous step, two products have been obtained; first, concentrated whey proteins which is called (Retentate), while the other is called (Permeate). Applying rotary evaporator, whey proteins are concentrated and then drying in two methods: spray-drying and freeze-drying in a form of white and soft powder. The chemical composition has been studied at each phase. The results show the separation, purification, and concentration of bovine and buffalo whey proteins by using ultrafiltration membrane technology. The results show that buffalo whey proteins produced by the method of enzymatic and dried with spray-drying are better than bovine whey protein. Finally, the results show a low ratio of lactose, salts and moisture content at the stages of filtration and concentration. The results present a high proportion of protein to 80 .and low ratio lactose and salt.

Hydrolysis of whey protein as a useful approach to obtain bioactive peptides and a β-Lg fraction with different biotechnological applications

Most of the studies related to the susceptibility of the main bovine whey proteins to the action of cardosins of Cynara cardunculus have underestimated the potential of these proteases to hydrolyse β-Lactoglobulin (β-Lg). These works used the one-factor-at-a-time methodology, which ignores the interactions and can lead to misleading conclusions. In this work, the hydrolysis of bovine whey protein, exploiting the joint action of cardosins A and B of C. cardunculus, was optimised. For that purpose, a second-order rotatable design (with two variables at five levels) was used to study the combined effect of pH and temperature on the degree of hydrolysis (DH), the number of peptides generated and the angiotensin I-converting enzyme (ACE)-inhibitory activity.The complete hydrolysis of α-Lactalbumin (α-La) was achieved at pH 6.99/35.86 °C and pH 5.01/64.14 °C, and contrary to the previous studies, cardosins were able to hydrolyse β-Lg (up to ~40%) at pH 6.29/50 °C. In addition, the statistical analysis revealed that the same pH and temperature values that led to a minimum DH for α-La also provide a maximum DH for β-Lg.At pH 5.3 and 60 °C, we have obtained abundant peptides with high angiotensin I-converting enzyme (ACE)-inhibitory activity, but also a non-hydrolysed β-Lg. So, the purification and identification of the bioactive peptides, produced under these hydrolysis conditions, was performed. Moreover, the biotechnological potential of the β-Lg obtained was successfully proved for 3 promising applications: heat-induced gels, nanofibrils (self-assembly) and microparticles (spray-dryer).

Tandem Mass Tag (TMT)-based quantitative proteomics reveals potential targets associated with onset of Sub-clinical Mastitis in cows

Bovine milk is vital for infant nutrition and is a major component of the human diet. Bovine mastitis is a common inflammatory disease of mammary gland in cattle. It alters the immune profile of the animal and lowers the quality and yield of milk causing huge economic losses to dairy industry. The incidence of sub-clinical mastitis (SCM) is higher (25–65% worldwide) than clinical mastitis (CM) (>5%), and frequently progresses to clinical stage due to lack of sensitive and specific detection method. We used quantitative proteomics to identify changes in milk during sub-clinical mastitis, which may be potential biomarkers for developing rapid, non-invasive, sensitive detection methods. We performed comparative proteome analysis of the bovine milk, collected from the Indian hybrid cow Karan Fries. The differential proteome in the milk of Indian crossbred cows during sub-acute and clinical intramammary gland infection has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins, we identified a total of 1459 and 1358 proteins in biological replicates, out of which 220 and 157 proteins were differentially expressed between normal and infected samples. A total of 82 proteins were up-regulated and 27 proteins were down-regulated, having fold changes of ≥2 and ≤0.8 respectively. Among these proteins, overexpression of CHI3L1, LBP, GSN, GCLC, C4 and PIGR proteins was positively correlated with the events that elicit host defence system, triggering production of cytokines and inflammatory molecules. The appearance of these potential biomarkers in milk may be used to segregate affected cattle from the normal herd and may support mitigation measures for prevention of SCM and CM.

Influence of high-pressure homogenization on structural properties and enzymatic hydrolysis of milk proteins

This study investigated the effects of HPH processing parameters on the conformational structure of bovine serum albumin (BSA) and whey protein isolate (WPI).BSA and WPI dispersions (1% w/v in phosphate buffer, pH = 7.5) were treated in a bench-scale HPH unit at different pressures (100, 150, 200 MPa) and number of passes (1, 2, 3, 5). The modifications of proteins' primary structure (carbonyl groups), secondary structure (α-helix, β-sheet, turn), tertiary and quaternary structure (free -SH groups) were analyzed together with their particle size distribution (PSD). HPH-assisted hydrolysis with trypsin and α-chymotrypsin was performed (200 MPa, 1 pass). Hydrolysis degree and molecular weight distribution of peptides were determined and compared with those of untreated protein dispersions.HPH treatments did not affect proteins' primary structure while slight modifications of secondary structure were detected. Interestingly, free - SH groups in BSA increased with increasing the pressure due to partial unfolding, while decreased in WPI, possibly due to disaggregation and compaction of native WPI aggregates. HPH pre-treatment allowed enhancing BSA hydrolysis reaction rate, while the compaction of WPI aggregates caused a reduced hydrolysis extent. In conclusion, HPH caused an appreciable modification of protein conformation and affected the degree of enzymatic hydrolysis.