Bovine Protein Research Articles

Neonatal Gut and Immune Responses to β-Casein Enriched Formula in Piglets

Backgroundβ-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less β-casein than HM, but whether different concentrations of β-casein affect tolerability and gut and immune maturation in newborns is unknown. ObjectivesUsing near-term piglets as a model for newborn infants, we investigated whether increasing the β-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation. MethodsThree groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) standard skim milk casein (BCN-standard, 35% β-casein of total casein, n = 18); 2) β-casein enrichment to HM concentrations (BCN-medium, 65%, n = 19); and 3) high β-casein enrichment (BCN-high, 91%, n = 19). A reference group was fed 100% whey protein concentrate (WPC) as protein (WPC, n = 18). Intestinal and immune parameters were assessed before and after euthanasia on day 5. ResultsClinical variables (mortality, activity, body growth, and diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of β-casein (BCN-high) had lower small intestine weight and tended to have more intestinal complications (highest gut pathology score, permeability, and interleukin-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte, and reticulocyte counts were increased with higher β-casein, whereas eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups. Conclusionsβ-casein enrichment of bovine-based formula to HM concentrations is clinically safe, as judged from newborn, near-term pigs, whereas no additional benefits to gut maturation were observed. However, excessive β-casein supplementation, beyond concentrations in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural β-casein concentrations or pure whey-based formula.

Comparative analysis of goat and bovine milk proteins in the improvement of sarcopenia based on host-microbial interactions

Comparative analysis of goat and bovine milk proteins in the improvement of sarcopenia based on host-microbial interactions

Screening of specific binding peptide for β-lactoglobulin using phage display technology

β-lactoglobulin (β-Lg) is a major food allergen, there is an urgent need to develop a rapid method for detecting β-Lg in order to avoid contact or ingestion by allergic patients. Peptide aptamers have high affinity, specificity, and stability, and have broad prospects in the field of rapid detection. Using β-Lg as the target, this study screened 11 peptides (P1–11) from a phage display library. Using molecular docking technology to predict binding energy and binding mode of proteins and peptides. Select the peptides with the best binding ability to β-Lg (P5, P7, P8) through ELISA. Combining them with whey protein, casein, and bovine serum protein, it was found that P7 has the best specificity for β-Lg, with an inhibition rate of 87.99%. Verified by molecular dynamics that P7 binds well with β-Lg. Therefore, this peptide can be used for the recognition of β-Lg, becoming a new recognition element for detecting β-Lg.

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Protein and oligosaccharide composition of colostrum and transition milk from pasture-based dairy cows supplemented prepartum with inorganic selenium, organic selenium or rumen-protected choline

Prepartum diet and mineral supplementation can influence bovine colostrum proteins and oligosaccharides (OS). The objective of this study was to evaluate the effects of prepartum supplementation with 1) inorganic selenium (INORG), 2) organic selenium (ORG) or 3) rumen-protected choline (RPC) on these components. Fifty-seven (12 primiparous and 45 multiparous) cows were supplemented daily from 49 ± 12.9 d prior to calving until d of calving. Colostrum was collected within 1 h postpartum and transition milk (TM) samples 1–5 were collected over the following 2–3 d postpartum. Prepartum supplementation with INORG, ORG or RPC had no significant effect on the gross, OS or IgG composition of colostrum. Animals supplemented with ORG or RPC produced significantly higher concentrations of colostrum αs2-Casein and β-Casein. Each milking postpartum had a significant effect on the protein and OS composition of milk. This research demonstrates that supplementing these micro-nutrients prepartum does not have a deleterious effect on subsequent colostrum or TM composition.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased β-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Binding of Per- and Polyfluoroalkyl Substances to β-Lactoglobulin from Bovine Milk.

Per- and polyfluoroalkyl substances (PFAS) are known for their high environmental persistence and potential toxicity. The presence of PFAS has been reported in many dairy products. However, the mechanisms underlying the accumulation of PFAS in these products remain unclear. Here, we used native mass spectrometry and molecular dynamics simulations to probe the interactions between 19 PFAS of environmental concern and two isoforms of the major bovine whey protein β-lactoglobulin (β-LG). We observed that six of these PFAS bound to both protein isoforms with low- to mid-micromolar dissociation constants. Based on quantitative, competitive binding experiments with endogenous ligands, PFAS can bind orthosterically and preferentially to β-LG's hydrophobic ligand-binding calyx. β-Cyclodextrin can also suppress binding of PFAS to β-LG owing to the ability of β-cyclodextrin to directly sequester PFAS from solution. This research sheds light on PFAS-β-LG binding, suggesting that such interactions could impact lipid-fatty acid transport in bovine mammary glands at high PFAS concentrations. Furthermore, our results highlight the potential use of β-cyclodextrin in mitigating PFAS binding, providing insights toward the development of strategies to reduce PFAS accumulation in dairy products and other biological systems.

A combination of polypeptide and magnetic molecular imprinted electrochemical sensor based on BSA-AuNPs/Fe3O4@ZIF-8 for the detection of norfloxacin

Norfloxacin (NOR) is now extensively used for the treatment of human and animal diseases. Unfortunately, large accumulations of NOR will pose a significant threat to the human body. Thus, it is urgent to develop a rapid and sensitive detection technique for the trace determination of NOR. In this study, an electrochemical molecular imprinting sensor with great properties was fabricated for the determination of NOR in milk and environmental water. The incorporation of bovine serum proteins produced a hybrid recognition interface consisting of molecularly imprinted polymers and peptides that provided good recognition properties for norfloxacin. The Fe3O4@ZIF-8 was distributed orderly on the electrode after the embedding of the magnets inside the electrode, then the BSA was coated on the electrode with the electrodeposited AuNPs through Au-S bonding. The sensor constructed in this study achieved ultra-sensitive detection of norfloxacin with an effective area of 0.08433 cm2, a limit of detection (LOD) of 0.108 pmol·L−1, and a wide linear range (1 ∼ 100, 100 ∼ 1000 and 1000 ∼ 8000 pmol·L−1). The prepared sensors exhibited satisfactory performance in the detection of milk and environmental water, with acceptable recoveries ranging from 96.24 % to 104.88 %, and the relative standard deviation (RSD) was no more than 4.84 %.

Antidiabetic Activities and GC-MS Analysis of 4-Methoxychalcone

Diabetes mellitus is a chronic metabolic disease that is mainly characterized by hyperglycemia. Chalcones and their derivatives have demonstrated promising pharmacological potential for the treatment of diabetes. The aim of the study was to evaluate antidiabetic activities and analyze 4-methoxychalcone (MPP) using GC-MS. The compound was characterized using mass spectroscopy, nuclear magnetic resonance and headspace with gas chromatography coupled to mass spectrometry (HS-GC-MS). MPP was evaluated via the inhibition of the alpha-glucosidase enzyme, cell viability and antiglycation and hemolytic activities in vitro. The study of the interaction between the bovine serum albumin protein and MPP was investigated via molecular docking. Oral sucrose tolerance and oral glucose tolerance tests were performed in streptozotocin (STZ)-induced diabetic mice. The HS-GC-MS method was able to accurately detect and characterize the compound, and the interaction between MPP and BSA revealed the remarkable affinity for the two main binding sites of BSA. This was confirmed by the in vitro antiglycation test, since MPP showed activity through both oxidative and non-oxidative stress. MPP significantly attenuated the increase in glycemia after glucose loading in STZ-induced diabetic mice. These results confirm that MPP has antihyperglycemic activity and may be an alternative for the treatment of diabetes mellitus.

Diazo-functionalised immunoelectrochemical sensor for the detection of ochratoxin a in foods

Ochratoxin A (OTA) is a toxic fungal metabolite that is commonly found in cereals and animal feed. It is economically damaging and potentially hazardous to human health. Herein, we propose an electrochemical immunosensor for the rapid detection of OTA using anti-OTA antibodies and diazonium-functionalized, screen-printed electrodes. We attached 4-aminobenzoic acid to an electrode surface, activated the carboxyl groups on the surface with carbodiimide, and attached an antibody to the diazo layer. Subsequently, we used bovine serum protein as a blocker to prevent non-specific antigens from binding to the antibody. We evaluated the performance of the sensor by cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry. The sensor is highly specific and sensitive, has good linear responses in the range 20–200 ng/mL, a limit of detection of 0.5 ng/mL, and good recoveries of 90.5%–100.9% in spiked samples. It can be stored at 4 °C for approximately 2 weeks, and is highly stable, with a current response variation of no more than 4.6%.

Hydrolysis of Beef Sarcoplasmic Protein by Dry-Aged Beef-Isolated Penicillium oxalicum and Its Associated Metabolic Pathways.

Yanbian cattle have a unique meat flavor, and high-grade meat is in short supply. Therefore, in this study, we aimed to improve the added value of Yanbian cattle low-fat meat and provide a theoretical reference for the subsequent development of an excellent starter. Rump meat from Yanbian cattle was dry-aged and then screened for protease-producing fungi. Three protease-producing fungi (Yarrowia hollandica (D4 and D11), Penicillium oxalicum (D5), and Meesziomyces ophidis (D20)) were isolated from 40 d dry-aged beef samples, and their ability to hydrolyze proteins was determined using bovine sarcoplasmic protein extract. SDS-PAGE showed that the ability of Penicillium oxalicum (D5) to degrade proteins was stronger than the other two fungi. In addition, the volatile component content of sarcoplasmic proteins in the D5 group was the highest (45.47%) and comprised the most species (26 types). Metabolic pathway analysis of the fermentation broth showed that phenylalanine, tyrosine, and tryptophan biosynthesis was the most closely related metabolic pathway in sarcoplasmic protein fermentation by Penicillium oxalicum (D5). Dry-aged beef-isolated Penicillium oxalicum serves as a potential starter culture for the fermentation of meat products.

2′-FL glycosylation of bovine milk protein matrices alters protein structure and reduces antibody affinity

Three glycosylated proteins were prepared from 2′-fucosyllactose (2′-FL) with α-lactalbumins, β-lactoglobulin and whey protein isolate, respectively, and their protein structures, glycosylation sites, digestion and transport capacities, and antibody binding capacities were determined. The findings indicate that following heating at 68 °C for 90 min, 2′-FL can form stable bonds with amino acid residues in milk proteins, leading to enhanced antioxidant properties, reduced hydrophobicity, and modified secondary structure content of the proteins. The glycosylation sites were mainly located in the amino acid sequence of β-lactoglobulin (K47, K91 and K135) and significantly reduce the affinity of its specific antibodies. 2′-FL glycosylation did not have a significant effect on the digestibility of cow's milk proteins, but it could significantly reduce the affinity of their specific antibody, indicating that it could be used in producing hypoallergenic dairy product.

Artificial Simulation of Saliva's Astringency Removal Effect on Squid

Astringency is a common issue in squid processing and consumption. Various techniques such as soaking in salt water, adding acids, and using enzymes have been used to eliminate astringency. However, these methods have their limitations, and the search for a better solution is ongoing. In recent years, ultrasound technology has been proposed as an effective method for removing astringency in squid. This study used four proteins, including lysozyme, bovine serum albumin, collagen, and whey protein, to simulate saliva in the human oral cavity. This study aimed to determine the removal effect of squid astringency after saliva soaking and ultrasound treatment. Physicochemical indicators such as polyphenols and flavonoids, antioxidant activity, relative polymerization degree, and solution zeta potential were used as physicochemical indicators. Sensory evaluation and volatile salt nitrogen content were used as quality indicators of squid. The results indicated that artificial simulated saliva treatment significantly reduces polyphenols and antioxidant activity in squid muscle, reduces the content of volatile base nitrogen, eliminates the astringency of squid, and improves the taste and overall quality of squid. The technique of using ultrasound technology and artificial simulated saliva is a scientific and effective method for removing astringency in squid. This method has several advantages over traditional methods, including being a non-invasive method that does not require adding any chemicals, making it an environmentally friendly solution. The use of ultrasound technology allows for removing astringency in a shorter time than traditional methods. Finally, the technique is cost-effective and easily scaled up for industrial applications. In conclusion, using ultrasound technology and artificial simulated saliva treatment is a promising method for removing astringency in squid. The technique effectively removes astringency in squid muscle, improves the taste and overall quality of the squid, and is a non-invasive, environmentally friendly, cost-effective solution that can be easily scaled up for industrial application.

Covalently grafting multihydroxy hyperbranched polymer on the surface of Ti3C2Tx MXene with remarkably enhanced colloidal stability and functionality for photothermal therapy

While MXenes have garnered acknowledgment for their remarkable photothermal properties, their practical usage has been hindered by their limited stability in aqueous mediums and vulnerability to external environmental factors. To address this issue, herein, we propose a simple and feasible method to enhance Ti3C2Tx MXene by utilizing its Ti-OH groups to directly initiate the ring-opening polymerization of glycidol. This process leads to covalently grafting multihydroxy hyperbranched polyglycerol (HPG) on the surface of MXene. The resulting Ti3C2-HPG nanohybrid demonstrates excellent aqueous dispersibility, favorable colloidal stability, and a customizable surface, thanks to the introduction of HPG. Moreover, it can withstand challenging conditions, such as extreme pH levels, high concentrations of electrolyte and bovine serum protein, and long-time storage. The abundant hydroxyl groups allow for easy modification, as demonstrated by the conjugation of rhodamine B. Preliminary in vitro and in vivo studies have unveiled that Ti3C2-HPG exhibits low cytotoxicity and exceptional photothermal performance for effectively destroying cancer cells, thus establishing it as a highly promising photothermal agent for photothermal therapy. This study thus open a new avenue for improving the stability and surface functionality of MXenes, enabling them to meet the specific requirements of practical applications.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Novel salty peptides derived from bovine bone: Identification, taste characteristic, and salt-enhancing mechanism

Excessive sodium intake is a major contributor to the incidence of cardiovascular diseases. The objective of this study was to prepare, isolate, and characterize peptides from bovine bone protein and investigate the salty/salt-enhancing mechanism of peptides. 1032 peptides were identified in the enzymatic hydrolysates of bovine bone protein and were further screened by the composition of amino acid residues and molecular docking analysis. 5 peptides were finally selected for solid-phase synthesis, and KER showed a better salty taste by sensory verification. Moreover, the synergistic effect of KER in NaCl and MSG solution could enhance the salty intensity by 65.26 %. The binding of KER to the salty receptor (TMC4) was driven by hydrogen bonding and electrostatic interactions with a binding energy of −88.0734 kcal/mol. This work may provide a new approach to efficiently screen salty peptides from natural food materials, which were expected as a taste enhancer used in salt-reducing foods.

VPBrowse: Genome-based representation of MS/MS spectra to quantify 10,000 bovine proteins.

SWATH is a data acquisition strategy acclaimed for generating quantitatively accurate and consistent measurements of proteins across multiple samples. Its utility for proteomics studies in nonlaboratory animals, however, is currently compromised by the lack of sufficiently comprehensive and reliable public libraries, either experimental or predicted, and relevant platforms that support their sharing and utilization in an intuitive manner. Here we describe the development of the Veterinary Proteome Browser, VPBrowse (http://browser.proteo.cloud/), an on-line platform for genome-based representation of the Bos taurus proteome, which is equipped with an interactive database and tools for searching, visualization, and building quantitative mass spectrometry assays. In its current version (VPBrowse 1.0), it contains high-quality fragmentation spectra acquired on QToF instrument for over 36,000 proteotypic peptides, the experimental evidence for over 10,000 proteins. Data can be downloaded in different formats to enable analysis using popular software packages for SWATH data processing whilst normalization to iRT scale ensures compatibility with diverse chromatography systems. When applied to published blood plasma dataset from the biomarker discovery study, the resource supported label-free quantification of additional proteins not reported by the authors previously including PSMA4, a tissue leakage protein and a promising candidate biomarker of animal's response to dehorning-related injury.

Phosphoproteomics Revealed Differentially Expressed Sites and Function of the Bovine Milk Fat Globule Membrane in Colostrum and Mature Milk.

One type of large and intricate post-translational modification of milk proteins that has significant biological implications is phosphorylation. The characterization of phosphoproteins found in the bovine milk fat globule membrane (MFGM) is still mostly unknown. Here, label-free phosphoproteomics was used to identify 94 phosphorylation sites from 54 MFGM phosphoproteins in bovine colostrum (BC) and 136 phosphorylation sites from 91 MFGM phosphoproteins in bovine mature milk (BM). αs1-Casein and β-casein were the most phosphorylated proteins in bovine colostrum. In bovine mature milk, perilipin-2 was the protein with the greatest number of phosphorylation sites. The results show that bovine colostrum MFGM phosphoproteins were mainly involved in immune function, whereas bovine mature MFGM phosphoproteins were mainly involved in metabolic function. Plasminogen and osteopontin were the most strongly interacting proteins in colostrum, whereas perilipin-2 was the most strongly interacting protein in bovine mature milk. This work demonstrates the unique alterations in the phosphorylation manner of the bovine MFGM protein during lactation and further expands our knowledge of the site characteristics of bovine MFGM phosphoproteins. This result confirms the value of MFGM as a reference ingredient for infant formula during different stages.

Identification of pasteurized mare milk and powder adulteration with bovine milk using quantitative proteomics and metabolomics approaches

Adulteration in dairy products presents food safety challenges, driven by economic factors. Processing may change specific biomarkers, thus affecting their effectiveness in detection. In this study, proteomics and metabolomics approaches were to investigate the detection of bovine milk (BM) constituents adulteration in pasteurized mare milk (PMM) and mare milk powder (MMP). Several bovine proteins and metabolites were identified, with their abundances in PMM and MMP increasing upon addition of BM. Proteins like osteopontin (OPN) and serotransferrin (TF) detected adulteration down to 1 % in PMM, whereas these proteins in MMP were utilized to identify 10 % adulteration. Biotin and N6-Me-adenosine were effective in detecting adulteration in PMM as low as 10 % and 1 % respectively, while in MMP, their detection limits extend down to 0.1 %. These findings offer insights for authenticating mare milk products and underscore the influence of processing methods on biomarker levels, stressing the need to consider these effects in milk product authentication.