Search Results (70)

Goat caseins are highly polymorphic proteins that affect milk functional properties. In this study, an in silico approach was employed to analyze the influence of goat casein allelic variants on the quantity and bioactivity potential of peptides released after enzymatic hydrolysis. The reported protein sequences from the most frequent allelic variants in Capra hircus caseins (α-S1, β, α-S2, and κ-casein) were analyzed in the BIOPEP-UWM database to determine the frequency of occurrence of bioactive fragments from each casein. After specific hydrolysis with pepsin, trypsin, and chymotrypsin A, important differences in the peptide profile and bioactivity potential were observed within and between the casein allelic variants. The β-casein A and C alleles, α-S1-casein allele E, and α-S2-casein allele F presented the highest bioactivity potential, and some allele-specific peptides were also released, highlighting the impact of genotype on the predicted bioactivity. The inhibition of angiotensin-converting enzyme (ACE-I) and dipeptidyl peptidase IV (DPP-IV) activities was the most frequent bioactivity of the released peptides, suggesting possible antihypertensive and antidiabetic effects. Once confirmed by experimental studies, the use of goat casein genotyping could direct efforts to enhance the functional quality of goat milk. 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

Background/Objectives: Research on postbiotics derived from probiotic fermented milk bases require further expansion, and the mechanisms through which they exert their effects have yet to be fully elucidated. This study utilized in vitro cell co-culture, digestion, and fermentation experiments, combined with targeted T500 technology, to elucidate the mechanism by which postbiotic Pa JY062 safeguards intestinal health. Compared to the LPS group, Pa JY062 boosted phagocytic ability in RAW264.7 macrophages, decreased NO levels, and alleviated LPS-induced excessive inflammation. Pa JY062 suppressed pro-inflammatory cytokines (IL-6, IL-17α, and TNF-α) while elevating anti-inflammatory IL-10. It prevented LPS-induced TEER reduction in Caco-2 monolayers, decreased FITC-dextran permeability, restored intestinal microvilli integrity, and upregulated tight junction genes (ZO-1, occludin, claudin-1, and E-cadherin). The hydrolysis rate of Pa JY062 progressively rose in gastrointestinal fluids in 0–120 min. At 5 mg/mL, it enriched gut microbiota diversity and elevated proportions of Limosilactobacillus, Lactobacillus, Pediococcus, and Lacticaseibacillus while augmenting the microbial production of acetic acid (120.2 ± 8.08 μg/mL), propionic acid (9.9 ± 0.35 μg/mL), and butyric acid (10.55 ± 0.13 μg/mL). Pa JY062 incorporated α_s-casein/β-lactoglobulin hydrolysate (L-glutamic acid, alanine, lysine, tyrosine, phenylalanine, histidine, and arginine) to mitigate protein allergenic potential while harboring bioactive components, including tryptophan metabolites, vitamin B6 (VB6), and γ-aminobutyric acid (GABA). Pa JY062 represented a novel postbiotic with demonstrated intestinal health-promoting properties. These findings advance the current knowledge on postbiotic-mediated gut homeostasis regulation and expedite the translational development of dairy-derived postbiotic formulations. Full article

Cow’s milk allergy (CMA) is the most common food allergy in infants. This study aimed to identify peptide biomarkers predictive of tolerance in a Spanish population of children with CMA. We investigated specific IgE and IgG4 binding to sequential epitopes of the five major CM allergens (α-s1-, α-s2-, β-, and κ-caseins as well as β-lactoglobulin) using a microarray-based immunoassay. Microarray analysis was performed in 118 patients at baseline and after 6, 18, 30, 42, and 54 months. Most patients tolerated CM at 6 months (40.7%) and 18 months (35.4%). We found significant differences in IgE and IgG4 binding intensity and diversity between allergic and tolerant patients. No differences were observed at baseline. Combining baseline IgE and IgG4 serology variables and peptide microarray analysis results, a predictive model was developed using the XGBoost algorithm to classify tolerance status at different time points. The generated models showed high predictive value at 6 and 30 months with AUCs of 0.883 and 0.833, respectively. Therefore, using IgE and IgG4 antibody-binding peptides at baseline, we generated two models predicting tolerance in children with cow’s milk allergy at 6 and 30 months. Full article

This study optimizes the process conditions for preparing angiotensin-converting enzyme (ACE) inhibitory peptides from skimmed goat milk (SGM) hydrolyzed by multi-enzymes using response surface methodology. When the enzymatic hydrolysis time was 90 min, the optimal hydrolysis conditions were a pH of 8.49, enzyme-to-substrate ratio (E/S ratio) of 8.04%, and temperature of 61.54 °C. The hydrolysis degree and ACE inhibitory activity were 65.39% ± 0.01% and 84.65% ± 0.03%, respectively. After purification by ultrafiltration, macroporous resin, and gel filtration, the ACE inhibitory activity of F2-2 in the two components of F2 was higher, with the ACE inhibitory rate of 93.97% ± 0.15% and IC₅₀ of 0.121 ± 0.004 mg/mL. The content of hydrophobic amino acids, fatty amino acids, and aromatic amino acids in component F2-2 accounts for 73.17%, 33.86%, and 33.72%, respectively. Eleven peptides were isolated and identified from the F2-2 components of the enzymatic hydrolysate of SGM, including two peptides without an established database. The peptides mainly came from β casein, α_S1 casein, and α_S2 casein. 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

Protein-based hydrogels have attracted considerable interest due to their biocompatibility, nontoxic properties, biodegradability, and renewable nature, as well as their being inexpensive and easy to obtain. Hydrogel properties depend on the temperature, polymer concentration, pH, crosslinking levels, salt concentrations, and aging. Casein is a natural protein present in bovine milk (about 80%), which exists in the form of various micelles. It is composed of α-s1, α-s2, β-, and κ-casein and tends toward self-assembly. Casein-based hydrogels are suitable for use in biomedical applications. Considering their potential applications in the field of medicine, in this work, our objective is to find the best conditions for the development of a casein hydrogel with tetracaine hydrochloride as the active compound. The tetracaine hydrochloride has anesthetic properties; therefore, it would allow for a painless and comfortable treatment to be offered to the patient. Accordingly, different hydrogel formulations were proposed. The selected components were casein, glycerol, tetracaine hydrochloride, potassium carbonate, and sodium alginate. Stability and swelling tests was carried out, and apparent density, pH, and moisture content were investigated. The formulation that allowed us to obtain hydrogel with the desired properties was composed of tetracaine hydrochloride 1%, casein 2%, glycerol 50%, sodium alginate 4%, and potassium carbonate solution 18% (the percentages use the casein as the basis). Full article

Objectives: The present study identified multiple proteins in early mature milk and explored the correlation between protein compositions in HM and maternal BMI during lactation. Methods: A total of 70 mothers giving birth to single-term infants from four representative cites were enrolled in this research. Milk samples were collected between 9 and 11 a.m. to avoid the influence of circadian rhythms. The concentration of total protein in the milk samples was determined using the Bradford method, and the concentrations of α-lactalbumin, lactoferrin, osteopontin, α_s−1 casein, β-casein, and κ-casein, butyrophilin, periodic acid Schiff 6/7, fatty acid-binding protein, and xanthine oxidoreductase in the milk samples were measured through a previously published method using ultra-performance liquid chromatography coupled with mass spectrometry. A semi-structured questionnaire investigation and body measurements were carried out by trained investigators to collect the information of subjects. Results: In the univariate models, the concentrations of TP (r = 0.306), α-La (r = 0.260), LF (r = 0.371), OPN (r = 0.286), and α_S1-CN (r = 0.324) were all positively and significantly correlated with maternal BMI. In the models’ adjusted covariates, the concentrations of TP (Lg β = 7.4 × 10⁻³), LF (Lg β = 19.2 × 10⁻³), α_S1-CN (Lg β = 8.2 × 10⁻³) and the proportion of LF (β = 0.20%) were positively correlated with continuous maternal BMI changes. TP concentrations in the HM of obese mothers were higher than in the other three groups (Lg β: 66.7 × 10⁻³~140.5 × 10⁻³), α-La concentrations were higher than in the underweight and normal groups (Lg β: 94.4 × 10⁻³~145.7 × 10⁻³), and OPN concentrations were higher than in the overweight groups (Lg β = 103.6 × 10⁻³). The concentrations of LF (Lg β: −298.2 × 10⁻³~−191.0 × 10⁻³), OPN (Lg β: −248.9 × 10⁻³~−145.3 × 10⁻³), and α_S1-CN (Lg β: −160.7 × 10⁻³~−108.3 × 10⁻³) in the HM of underweight mothers were lower than those in the other three groups. β-CN concentrations were lower than normal (Lg β = −125.1 × 10⁻³) and obese groups (Lg β = −165.7 × 10⁻³), κ-CN concentrations were lower than the overweight (Lg β = −132.5 × 10⁻³) and obese groups (Lg β = −147.9 × 10⁻³), and the proportion of LF was lower than that of the overweight (β = −2.80%) and obese groups (β = −2.52%). The proportion of LF in normal mothers was lower than that in the overweight group (β = −1.15%). No statistically significant associations between four MFGM proteins and maternal BMI were determined as the equation models could not be fitted (p for F-test < 0.05). Conclusions: Obese mothers had higher concentrations of multiple protein components than other groups, while underweight mothers had lower concentrations. The association between BMI and protein compositions may be more pronounced for certain protein types. Full article

The diminishing of food waste is gaining increasing importance, especially in context with a growing population and a need for the sustainable use of food resources. A more precise determination of the best-before date can contribute to this general aim. As proteoforms can be regarded as indicators for ecophysiological influences, their suitability for determining the spoilage and, consequently, the shelf-life of food is suggested. Proteoforms reflect the spoilage of food more accurately. The aim of the present study was to develop an efficient proteomics workflow to determine the shelf-life of milk as a prominent target. In this case, raw milk was chosen as model, as it degrades much faster. The integration of different multivariate analysis techniques was used to analyze the spoilage of raw milk with regard to aspects of its proteome. As the feasibility of such an approach has already been demonstrated in previous studies, it is further necessary to enable a robust and reproducible workflow, primarily gaining appropriate numbers and amounts of peptides when the research question differs and other dairy products are evaluated. In the present study, two approaches for gaining peptides were considered: In addition to a direct hydrolysis of a protein-rich sample solution, in-gel hydrolysis is another common approach in proteomics. By separating the proteins in a traditional gel electrophoresis before hydrolysis, the change in the individual proteins and, consequently, potential peptides can be monitored more specifically during storage. However, the traditional approach offers not only possibilities but also limitations that must be considered. The study showed that it is beneficial to apply a combination of different application strategies, as they complement each other and can thus increase the information content of a sample or confirm a theory. Mass spectrometric features, which represent a chemical–structural change of all kinds of compounds during storage, were selected, and three of them were identified as peptides, originating from α-s1-casein. Full article

The αs2-casein is a phosphoprotein secreted in the milk of most mammals, and it is the most hydrophilic of all caseins. Contrary to genes found in ruminants, in donkeys two different encoding genes for donkey αs2-casein (CSN1S2 I and CSN1S2 II) have been identified. However, unlike in ruminants, the variability at these loci has not been characterized in detail in donkeys until now. In this study, we analyze the transcript profile of the donkey CSN1S2 I and CSN1S2 II genes, and we identify and describe the variability of these loci in the Ragusana and Amiatina breeds reared in Italy. The analysis of the CSN1S2 I Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) products and subsequent sequencing showed, in addition to correctly spliced mRNA, seven other minor mRNAs resulting from differential splicing events involving, in various combinations, entire exons (4, 5, 6, and 11), parts of exons (5′ or 3′ end of exon 17), or the recognition of intronic sequences as an exon (exon 12′). Similarly, the transcription analysis of the CSN1S2 II gene revealed a remarkable variability in splicing events, mainly concerning the alternative insertion of an extra exon 7 (named 7′); the first 33 bp of exon 13; or the alternative skipping of exons 9, 10, 11, 12, and 15, and their combinations. At the mRNA level for CSN1S2 I, seven SNPs were observed, five of which led to amino acid changes: p.T73>A, p.I109>V, p.I130>V, p.I146>T, and p.D217>Y. Similarly, nine SNPs were observed at the CSN1S2 II locus, seven of which are non-synonymous: p.L63>F, p.H70>Q, p.D90>N, p.129A>T, p.H131>Y, p.E144>G, and p.F157>S. In addition, the DNA sequencing of exon 17 and flanking introns of the CSN1S2 I gene revealed a G>A transition at the splice acceptor site of CSN1S2 I exon 17 (FM946022.1:c.375-1G>A), resulting in an allele-specific skipping of the first 15 nucleotides of this exon, which encode the peptide 176NKINQ180, and the recognition of an in-frame cryptic splicing acceptor site: arAACAAAATCAACCAG. A genotyping method based on restriction fragment length polymorphism (XbaI PCR-RFLP) was set up for this SNP. In the total population studied (105 Ragusana and 14 Amiatina donkeys), the A allele had a frequency of 0.2437 with no evidence of deviation from the Hardy–Weinberg equilibrium. This study adds new knowledge regarding the genetic variability of αs2-caseins in donkeys and may contribute significantly to the genetic improvement of milk production for this species. Full article

In many parts of the world, goat milk has been part of the human diet for millennia. Allergy to goat’s milk, not associated with allergy to cow’s milk, is a rare disorder, although some cases have been described. Goat milk proteins have substantial homology with cow’s milk proteins and even show cross-reactivity; therefore, they are not advised as an alternative to cow’s milk for infants with IgE-mediated cow’s milk allergies. However, there are indications that, due to the composition of the goat milk proteins, goat milk proteins show lower allergenicity than cow’s milk due to a lower αS1-casein content. For this reason, goat milk might be a better choice over cow’s milk as a first source of protein when breastfeeding is not possible or after the breastfeeding period. Additionally, some studies show that goat milk could play a role in specific types of non-IgE-mediated cow milk allergy or even in the prevention of sensitization to cow’s milk proteins. This review discusses a possible role of goat milk in non-IgE mediated allergy and the prevention or oral tolerance induction of milk allergy. Full article

Human milk reduces risk for necrotizing enterocolitis in preterm infants. Necrotizing enterocolitis occurs in the ileocecal region where thousands of milk protein-derived peptides have been released from digestion. Digestion-released peptides may exert bioactivity, such as antimicrobial and immunomodulatory activities, in the gut. In this study, we applied mass spectrometry-based peptidomics to characterize peptides present in colostrum before and after in vitro digestion. Sequence-based computational modeling was applied to predict peptides with antimicrobial activity. We identified more peptides in undigested samples, yet the abundances were much higher in the digested samples. Heatmapping demonstrated highly different peptide profiles between undigested and digested samples. Four peptides (α_S1-casein [157–163], α_S1-casein [157–165], β-casein [153–159] and plasminogen [591–597]) were selected, synthesized and tested against common pathogenic bacteria associated with necrotizing enterocolitis. All four exhibited bacteriostatic, though not bactericidal, activities against Klebsiella aerogenes, Citrobacter freundii and Serratia marcescens, but not Escherichia coli. Full article

Adipose tissue is an active endocrine gland, synthesizing and secreting multiple signaling molecules termed adipokines. Following the detection of adipokines and their receptors in the mammary tissue of various species, it is indicated that adipokines play a role in the development of the mammary gland. The aim of the present study was to determine the concentration-dependent influence of three adipokines, leptin, adiponectin, and chemerin, on the viability, apoptosis, and secretory activity of BME-UV1 bovine mammary epithelial cells. The study confirmed that BME-UV1 cells contain the leptin receptor (Ob-R) protein, and express transcripts of adiponectin (ADIPOR1 and ADIPOR2) and chemerin (CMLKR1 and GPR1) receptors. Regardless of the administered dose, none of the three tested adipokines had an effect on the viability of BME-UV1 cells, and the number of apoptotic cells remained unchanged. However, chemerin (100 ng/mL) stimulated BME-UV1 cells to synthesize and secrete αS1-casein, the major protein component of milk. These results indicate that chemerin may be a potent regulator of the bovine mammary epithelial cells’ functional differentiation, contributing, along with the major systemic hormones and local growth factors, to the development of the bovine mammary gland. Full article

Introduction: Goat milk has poorer fermentation characteristics due to the absence or only traces of αs1-casein, due to which goat yoghurt contains a less dense gel structure. Moreover, the fermentation characteristics of the milk vary between the breeds of the same species. Therefore, it becomes imperative to explore a few metabolites which could regulate the techno-functional properties of goat yoghurt. Objectives: This study was aimed at relating the metabolite profile of yoghurt prepared from milk of Barbari, an indigenous goat breed of India, and its techno-functional properties (firmness, whey syneresis, and flow behaviour) using multivariate data analysis and regression models. Results: Goat yoghurt was prepared with two different total solids (TS) levels (12 and 16%) and cultures, namely, commercial culture comprising a thermophilic yoghurt culture (A) and NCDC-263 comprising a mixed yoghurt culture (B). Results demonstrated a significant difference (p < 0.05) in whey syneresis with the increase in the TS level. Flow behaviour of all yoghurt samples showed a decrease in viscosity with an increase in shear rate, which confirmed its non-Newtonian behaviour and shear thinning nature, whereas frequency sweep confirmed its viscoelastic nature. Firmness was the most affected under the influence of different TS and culture levels. It was higher (p < 0.05) for 16-A, followed by 16-3B, and minimum for 12-2B. GC-MS-based metabolomics of the yoghurt revealed a total of 102 metabolites, out of which 15 metabolites were differentially expressed (p < 0.05), including 2-hydroxyethyl palmitate, alpha-mannobiose, and myo-inositol. Multivariate data analysis revealed clear separation among groups using principal component analysis and several correlations using a correlation heat map. Further, regression analysis exhibited methylamine (0.669) and myo-inositol (0.947) with higher regression coefficients (R² values) exceeding 0.6, thus demonstrating their significant influence on the techno-functional properties, mainly firmness, of the yogurt. Conclusion: In conclusion, A gas chromatography-based metabolomics approach could successfully establish a relationship between the metabolome and the techno-functional properties of the yoghurt. Full article

Breast-milk α_S1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated α_S1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of α_S1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V⁷⁷-E⁹² of α_S1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that α_S1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of α_S1-casein, as described before, could be regulated by conformational changes induced by phosphorylation. Full article