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Foods (Basel, Switzerland) Sep 2021Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, α-lactalbumin (α-la)...
Selective thermal precipitation followed by a mechanical separation step is a well described method for fractionation of the main whey proteins, α-lactalbumin (α-la) and β-lactoglobulin (β-lg). By choosing appropriate environmental conditions the thermal precipitation of either α-la or β-lg can be induced. Whereas β-lg irreversibly aggregates, the precipitated α-la can be resolubilized by a subsequent adjustment of the solution's pH and the ionic composition. This study reports on the analytical characterization of resolubilized α-la compared to its native counterpart as a reference in order to assess whether the resolubilized α-la can be considered close to 'native'. Turbidity and quantification by RP-HPLC of the resolubilized α-la solutions were used as a measure of solubility in aqueous environment. RP-HPLC was also applied to determine the elution time as a measure for protein's hydrophobicity. DSC measurement was performed to determine the denaturation peak temperature of resolubilized α-la. FTIR spectroscopy provided insights in the secondary structure. The refolding of α-la achieved best results using pH 8.0 and a 3-fold stoichiometric amount of Ca per α-la molecule. The results showed that the mechanism of aggregation induced by gentle thermal treatment under acidic conditions with subsequent mechanical separation is reversible to a certain extent, however, the exact native conformation was not restored.
PubMed: 34574341
DOI: 10.3390/foods10092231 -
Applied Spectroscopy Jun 2021Characterization and quantification of individual whey proteins are of crucial importance to many industrial dairy processes. Labor intensive wet-chemical methods are...
Characterization and quantification of individual whey proteins are of crucial importance to many industrial dairy processes. Labor intensive wet-chemical methods are still being used for this purpose, but a rapid quantification method for individual whey proteins is highly desired. This work investigate the utility of Fourier transform mid-infrared spectroscopy and Fourier transform near-infrared spectroscopy for rapid quantification of the two main whey proteins (β-lactoglobulin and α-lactalbumin) in complex aqueous whey solutions simulating production process streams. MIR and NIR spectra obtained on whey samples with known and varying amounts of the proteins of interest and are used to develop partial least squares prediction models. Selection of informative wavelength regions allowed for prediction of β-lactoglobulin and α-lactalbumin concentrations with very high precision and accuracy (root mean square error of cross-validation, or RMSECV, of 0.6% and R of 0.99 for NIR), demonstrating the potential of being implemented for rapid in-line monitoring of protein composition in industrial whey streams. Two-dimensional MIR-NIR correlation spectroscopy is used to identify the most informative parts of the NIR spectra in relation to protein secondary structure. In addition multivariate curve resolution is applied to the MIR data to resolve mixture spectra and to elucidate the spectral ranges that were most useful in distinguishing between the two whey proteins. The study concludes that NIR spectroscopy has potential for accurate in-line protein quantification and overall secondary protein structure quantification which open new possibilities for in-line industrial applications.
Topics: Fourier Analysis; Lactalbumin; Lactoglobulins; Least-Squares Analysis; Spectroscopy, Fourier Transform Infrared; Spectroscopy, Near-Infrared; Whey; Whey Proteins
PubMed: 33231482
DOI: 10.1177/0003702820979747 -
Biomolecules Mar 2021Protein aggregation and misfolding are some of the most challenging obstacles, customarily studied for their association with amyloid pathologies. The mechanism of...
Protein aggregation and misfolding are some of the most challenging obstacles, customarily studied for their association with amyloid pathologies. The mechanism of amyloid fibrillation development is a dynamic phenomenon involving various factors such as the intrinsic properties of protein and the physical and chemical environmental conditions. The purpose of this study was to see the thermal aggregation profile of alpha-lactalbumin (α-LA) and to delineate the effect of trehalose on its aggregation profile. α-LA was subjected to thermal aggregation at high concentrations. UV-Vis spectroscopy, a turbidity assay, intrinsic fluorescence, Rayleigh scattering and a thioflavin T (ThT) assay explained the steady outcomes that 1 M trehalose repressed α-LA aggregation in the most effective way followed by 0.75 M and 0.5 M and to a significantly lesser degree by 0.25 M. Multi spectroscopic obser Sania ations were further entrenched by microscopy. Transmission electron microscopy confirmed that in the presence of its higher concentration, trehalose hinders fibril development in α-LA. In vitro studies were further validated by in silico studies. Molecular docking analysis indicated that trehalose occupied the binding pocket cavity of α-LA and offered several significant interactions, including H-bonds with important residues. This study provides a platform for trehalose in the therapeutic management of protein aggregation-related diseases.
Topics: Animals; Benzothiazoles; Cattle; Lactalbumin; Molecular Docking Simulation; Nephelometry and Turbidimetry; Protein Aggregates; Protein Binding; Scattering, Radiation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Temperature; Trehalose
PubMed: 33799517
DOI: 10.3390/biom11030414 -
Foods (Basel, Switzerland) Jul 2023Whey from goat and sheep have been gaining attention in the last few years for their nutritional properties. Unfortunately, β-Lg, not found in human milk, may trigger...
Whey from goat and sheep have been gaining attention in the last few years for their nutritional properties. Unfortunately, β-Lg, not found in human milk, may trigger infant allergies if used in infant food formulations, so there is a growing interest in developing ingredients derived from whey with higher α-La/β-Lg ratios. The objective of this work was to study the effect of high-pressure processing (HPP) on caprine and ovine native whey concentrates (NWC) in order to obtain α-Lactalbumin (α-La)-enriched fractions. NWCs were treated at 600 MPa (23 °C) for 2, 4, and 15 min and two pH conditions were studied (physiological pH and pH 4.60). The concentration of β-Lg in supernatant fraction after HPP significantly decreased after 2 min of treatment, while the concentration of α-La was unchanged in both goat and sheep samples. Longer HPP processing times (up to 15 min) progressively increased α-La purification degree but also decreased the α-La yield. Caprine and ovine NWCs treated at physiological pH provided better α-La yield, α-La purification degree, and higher β-Lg precipitation degrees than the corresponding acidified samples, while the corresponding NWC supernatant (NWC) showed lower values for both surface hydrophobicity and total free thiol indices, suggesting a higher extent of protein aggregation. Effects of sample acidification and the HPP treatment were opposite to those previously reported on bovine NWC, so further characterization of caprine and ovine β-Lg should be carried out to understand their different behavior.
PubMed: 37509780
DOI: 10.3390/foods12142688 -
International Immunopharmacology Dec 2022Cardiovascular aging is the most important factor leading to cardiovascular disease (CVD), and the incidence and severity of cardiovascular events increase with age....
Cardiovascular aging is the most important factor leading to cardiovascular disease (CVD), and the incidence and severity of cardiovascular events increase with age. Cardiovascular disease is one of the leading causes of death in the aging population. Therefore, it is extremely urgent to develop and explore effective drugs or bioactive molecules to prevent cardiovascular aging and related diseases. In the current work, the effect of bovine α-lactalbumin (α-lactalbumin is one of the major bioactive protein molecules in milk) on cardiovascular aging was investigated in vitro and in vivo. First, a cellular model of cardiovascular aging was established using HO-induced in vitro cellular models. It was found that α-lactalbumin could alleviate cardiovascular senescence by assessing Sa-β-gal and senescence-related markers (such as p16/p21/p53) in in vitro cellular models. Bovine α-lactalbumin attenuated aging-related inflammation and oxidative stress. Furthermore, aged mice were used as an in vivo cardiovascular aging model. We explored the effect of α-lactalbumin on cardiovascular aging and found that cardiovascular aging was significantly attenuated by evaluating Sa-β-gal staining and aging-related marker molecules. Mechanistically, we found that α-lactalbumin may alleviate cardiovascular aging by regulating the expression of Sirt1 (Sirtuin 1). In summary, in the current work, we systematically explored the potential biological activity of α-lactalbumin against cardiovascular aging and found that α-lactalbumin has good anti-aging potential in vitro and in vivo, suggesting that α-lactalbumin could be used as an antiaging functional food in the future.
Topics: Mice; Cattle; Animals; Lactalbumin; Cellular Senescence; Hydrogen Peroxide; Cardiovascular Diseases; Aging; Sirtuin 1
PubMed: 36252495
DOI: 10.1016/j.intimp.2022.109291 -
Animals : An Open Access Journal From... May 2021The increase of knowledge on the composition of donkey milk has revealed marked similarities to human milk, which led to a growing number of investigations focused on... (Review)
Review
The increase of knowledge on the composition of donkey milk has revealed marked similarities to human milk, which led to a growing number of investigations focused on testing the potential effects of donkey milk in vitro and in vivo. This paper examines the scientific evidence regarding the beneficial effects of donkey milk on human health. Most clinical studies report a tolerability of donkey milk in 82.6-98.5% of infants with cow milk protein allergies. The average protein content of donkey milk is about 18 g/L. Caseins, which are main allergenic components of milk, are less represented compared to cow milk (56% of the total protein in donkey vs. 80% in cow milk). Donkey milk is well accepted by children due to its high concentration of lactose (about 60 g/L). Immunomodulatory properties have been reported in one study in humans and in several animal models. Donkey milk also seems to modulate the intestinal microbiota, enhance antioxidant defense mechanisms and detoxifying enzymes activities, reduce hyperglycemia and normalize dyslipidemia. Donkey milk has lower calorie and fat content compared with other milks used in human nutrition (fat ranges from 0.20% to 1.7%) and a more favourable fatty acid profile, being low in saturated fatty acids (3.02 g/L) and high in alpha-linolenic acid (about 7.25 g/100 g of fat). Until now, the beneficial properties of donkey milk have been mostly related to whey proteins, among which β-lactoglobulin is the most represented (6.06 g/L), followed by α-lactalbumin (about 2 g/L) and lysozyme (1.07 g/L). So far, the health functionality of donkey milk has been tested almost exclusively on animal models. Furthermore, in vitro studies have described inhibitory action against bacteria, viruses, and fungi. From the literature review emerges the need for new randomized clinical trials on humans to provide stronger evidence of the potential beneficial health effects of donkey milk, which could lead to new applications as an adjuvant in the treatment of cardiometabolic diseases, malnutrition, and aging.
PubMed: 34067986
DOI: 10.3390/ani11051382 -
Frontiers in Genetics 2015Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC... (Review)
Review
Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants' udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).
PubMed: 26579195
DOI: 10.3389/fgene.2015.00323 -
Clinical and Molecular Allergy : CMA Apr 2023The accuracy of an atopy patch test (APT) for fresh cow's milk allergy is controversial. Few studies have focused on commercial extract solutions. We aimed to evaluate...
BACKGROUND
The accuracy of an atopy patch test (APT) for fresh cow's milk allergy is controversial. Few studies have focused on commercial extract solutions. We aimed to evaluate the diagnostic performance of the APT in cow's milk allergic children using fresh cow's milk and commercial extracts of cow's milk and its components including casein, α-lactalbumin, and β-lactoglobulin.
METHODS
A prospective study was carried out in children with a history of cow's milk allergy. Children underwent the skin prick test (SPT) and APT with fresh cow's milk, powdered cow's milk, and commercial extracts of cow's milk, casein, α-lactalbumin, and β-lactoglobulin. Oral food challenge (OFC) was confirmed in all children.
RESULTS
A total of 37 patients participated (mean age 13.14 ± 7.26 months). Only 5 (13.51%) patients had positive OFC to cow's milk. The sensitivity of the APT using fresh cow's milk was 40%, specificity was 65.6%, PPV was 15.4%, and NPV was 87.5%. The sensitivity of the APT using powdered cow's milk was 40%, 60.7% for specificity, 15.4% for PPV, and 58% for NPV. The sensitivity and PPV of the APT using commercial solutions of cow's milk, casein, α-lactalbumin, and β-lactoglobulin were zero. The specificities were 90.6%, 93.8%, 100%, and 100% for α-lactalbumin, cow's milk, casein, and β-lactoglobulin, respectively.
CONCLUSIONS
APT using commercial solutions showed higher specificity than fresh milk. The specificity increased using a protein component allergen.
PubMed: 37029394
DOI: 10.1186/s12948-023-00183-6 -
Journal of Dairy Science Nov 2023Cow milk is an important source of food protein for children; however, it could lead to allergy, especially for infants. α-Lactalbumin (α-LA) and β-lactoglobulin...
Cow milk is an important source of food protein for children; however, it could lead to allergy, especially for infants. α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) from whey protein make up a relatively high proportion of milk proteins and have received widespread attention as major allergens in milk. However, few studies have identified the epitopes of both proteins simultaneously. In this study, ImmunoCAP and indirect ELISA were first used for detection of sIgE to screen sera from allergic patients with high binding capacity for α-LA and β-LG. Subsequently, the mimotopes was biopanned by phage display technology and bioinformatics and 17 mimic peptide sequences were obtained. Aligned with the sequences of α-LA or β-LG, we identified one linear epitope on α-LA at AA 11-26 and 5 linear epitopes on β-LG at AA 9-29, AA 45-57, AA 77-80, AA 98-101, and AA 121-135, respectively. Meanwhile, the 8 conformational epitopes and their distributions of α-LA and β-LG were located using the Pepitope Server. Finally, glutamine and lysine were determined as common AA residues for the conformational epitopes both on α-LA and β-LG. Moreover, we found the addition of mouse anti-human IgE during the biopanning process did not significantly affect the identification of the epitopes.
PubMed: 37641259
DOI: 10.3168/jds.2022-23151 -
RSC Advances Nov 2019Some natural proteins can be complexed with oleic acid (OA) to form an active protein-lipid formulation that can induce tumor-selective apoptosis. The first explored... (Review)
Review
Some natural proteins can be complexed with oleic acid (OA) to form an active protein-lipid formulation that can induce tumor-selective apoptosis. The first explored protein was human milk α-lactalbumin (α-LA), called HAMLET when composed with OA in antitumor form. Several groups have prepared active protein-lipid complexes using a variety of approaches, all of which depend on target protein destabilization or direct OA-protein incubation to alter pH to acid or alkaline condition. In addition to performing vital roles in inflammatory processes and immune responses, fatty acids can disturb different metabolic pathways and cellular signals. Therefore, the tumoricidal action of these complexes is related to OA rather than the protein that keeps OA in solution and acts as a vehicle for transferring OA molecules to tumor cells. However, other studies have suggested that the antitumor efficacy of these complexes was exerted by both protein and OA together. The potential is not limited to the anti-tumor activity of protein-lipid complexes but extends to other functions such as bactericidal activity. The protein shell enhances the solubility and stability of the bound fatty acid. These protein-lipid complexes are promising candidates for fighting various cancer types and managing bacterial and viral infections.
PubMed: 35539089
DOI: 10.1039/c9ra07127j