Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Modulation of the electrostatic potential around α-lactalbumin using oligoelectrolyte chains, pH and salt concentration.

In this study, we conducted a comprehensive computational investigation of the interaction between α-lactalbumin, a small globular protein, and strong anionic oligoelectrolyte chains with a polymerization degree from 2 to 9. Both the protein and oligoelectrolyte chains are represented using coarse-grained models, and their properties were calculated by the Monte Carlo method under constant pH conditions. We were able to estimate the effects of this interaction on the electrostatic potential around the protein. At acidic pH, the protein had a net positive charge; therefore, the electrostatic potential around it was also positive. To neutralize or reverse this electrostatic potential, oligoelectrolyte chains with a minimum size of six monomers were necessary. Simultaneously, low salt concentrations were required as elevated salt levels led to a significant attenuation of the electrostatic interactions and the corresponding electrostatic potential.

Genetic variability among and within domestic Old and New World camels at the α-lactalbumin gene (LALBA) reveals new alleles and polymorphisms responsible for differential expression.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

Evidence of additive genetic variation for major milk proteins in dairy cows: A meta-analysis.

In the past, there have been reports of genetic parameters for milk proteins in various dairy cattle populations. The high variability among genetic parameter estimates has been caused by this. This study aimed to use a random-effects meta-analysis model to compile published estimates of genetic parameter for major milk proteins of α-lactalbumin, ß-lactoglobulin, sum of whey proteins, casein, αs1-casein, αs2-casein, ß-casein, and κ-casein in dairy cows. The study used a total of 140 heritability and 256 genetic correlation estimates from 23 papers published between 2004 and 2022. The estimated range of milk protein heritability is from 0.284 (for α-lactalbumin in milk) to 0.596 (for sum of whey proteins). The genetic correlation estimates between casein and milk yield, milk fat and protein percentages were -0.461, 0.693, and 0.976, respectively (p < 0.05). The genetic correlation estimates between milk proteins expressed as a percentage of milk were significant and varied from 0.177 (between ß-lactoglobulin and κ-casein) to 0.892 (between αs1-casein and αs2-casein). Moderate-to-high heritability estimates for milk proteins and their low genetic associations with milk yield and composition indicated the possibility for improving milk proteins in a genetic selection plan with negligible correlated effects on production traits in dairy cows.

Whey proteins as multifunctional food materials: Recent advancements in hydrolysis, separation, and peptidomimetic approaches.

Whey protein derived bioactives, including α-lactalbumin, ß-lactoglobulin, bovine serum albumin, lactoferrin, transferrin, and proteose-peptones, have exhibited wide ranges of functional, biological and therapeutic properties varying from anticancer, antihypertensive, and antimicrobial effects. In addition, their functional properties involve gelling, emulsifying, and foaming abilities. For these reasons, this review article is framed to understand the relationship existed in between those compound levels and structures with their main functional, biological, and therapeutic properties exhibited either in vitro or in vivo. The impacts of hydrolysis mechanism and separation techniques in enhancing those properties are likewise discussed. Furthermore, special emphasize is given to multifunctional effects of whey derived bioactives and their future trends in ameliorating further food, pharmaceutical, and nutraceutical products. The underlying mechanism effects of those properties are still remained unclear in terms of activity levels, efficacy, and targeted effectiveness. For these reasons, some important models linking to functional properties, thermal properties and cell circumstances are established. Moreover, the coexistence of radical trapping groups, chelating groups, sulfhydryl groups, inhibitory groups, and peptide bonds seemed to be the key elements in triggering those functions and properties. Practical Application: Whey proteins are the byproducts of cheese processing and usually the exploitation of these food waste products has increasingly getting acceptance in many countries, especially European countries. Whey proteins share comparable nutritive values to milk products, particularly on their richness on important proteins that can serve immune protection, structural, and energetic roles. The nutritive profile of whey proteins shows diverse type of bioactive molecules like α-lactalbumin, ß-lactoglobulin, lactoferrin, transferrin, immunoglobulin, and proteose peptones with wide biological importance to the living system, such as in maintaining immunological, neuronal, and signaling roles. The diversification of proteins of whey products prompted scientists to exploit the real mechanisms behind of their biological and therapeutic effects, especially in declining the risk of cancer, tumor, and further complications like diabetes type 2 and hypertension risk effects. For these reasons, profiling these types of proteins using different proteomic and peptidomic approaches helps in determining their biological and therapeutic targets along with their release into gastrointestinal tract conditions and their bioavailabilities into portal circulation, tissue, and organs. The wide applicability of those protein fractions and their derivative bioactive products showed significant impacts in the field of emulsion and double emulsion stabilization by playing roles as emulsifying, surfactant, stabilizing, and foaming agents. Their amphoteric properties helped them to act as excellent encapsulating agents, particularly as vehicle for delivering important vitamins and bioactive compounds. The presence of ferric elements increased their transportation to several metal-ions in the same time increased their scavenging effects to metal-transition and peroxidation of lipids. Their richness with almost essential and nonessential amino acids makes them as selective microbial starters, in addition their richness in sulfhydryl amino acids allowed them to act a cross-linker in conjugating further biomolecules. For instance, conjugating gold-nanoparticles and fluorescent materials in targeting diseases like cancer and tumors in vivo is considered the cutting-edges strategies for these versatile molecules due to their active diffusion across-cell membrane and the presence of specific transporters to these therapeutic molecules.

Development of UV-Vis Imaging Compatible Chromatographic Matrix with Application for Injectable Formulation Characterization.

Transport within human tissue matrices, e.g., the subcutaneous tissue, exhibits some resemblance to chromatographic processes. Here, a porous matrix comprising agarose beads compatible with UV-vis imaging was developed for a parallel piped rectangular flow cell (4 mm light path). Introduction of high-molecular weight dextrans (Mr ∼ 200000 and ∼500000) at 10% (w/v) rendered imaging possible by providing optical clearing of the turbid porous matrix, resulting in improved transmittance as well as resolution (from 400 to 180 µm) at 280 nm, as well as 520 nm. The interplay between diffusive and convective transport at 0 < Pe ≤ 28 was visualized at 280 nm upon injection of dexamethasone suspensions. Real-time UV-vis imaging showed in-flow cell the effect of incorporating ion-exchange resins on the retention of infliximab, lysozyme, and α-lactalbumin. The ion-exchange matrix may serve as a surrogate for polyelectrolytes in the subcutaneous tissue, assessing the potential role of electrostatic interactions of biotherapeutics upon injection. UV-vis imaging of size-exclusion chromatographic matrixes may be of interest in its own right and potentially develop into a characterization tool for injectables.

Effects of α-lactalbumin on strength, fatigue and psychological parameters: a randomised double-blind cross-over study.

PURPOSE: The neurotransmitter serotonin has a strong effect on behaviour and motor control. Regarding motor control, serotonin contributes to the development of fatigue and is also involved in the ability of motor neurones to operate across a large range of forces (gain control). The consumption of tryptophan-rich supplements (such as α-lactalbumin) is of interest because this amino acid is the only precursor for brain serotonin synthesis. Therefore, the purpose of this study was to determine the effects of α-lactalbumin supplementation on neuromuscular performance. METHODS: Using a randomised double-blind cross-over design, 16 healthy participants performed plantar flexor and handgrip maximal voluntary contractions, a 30-s submaximal handgrip contraction, and a plantar flexor fatigue protocol before and 90 min after consuming either 40 g of α-lactalbumin, an isonitrogenous beverage (Zein) or an isocaloric beverage (corn-starch). Sleepiness, mood, and cognition were assessed to evaluate any psychological effects. RESULTS: α-Lactalbumin decreased force steadiness by 25% during the sustained submaximal handgrip contraction (p < 0.01) and induced greater fatigue (15% reduction in total torque-time integral, p = 0.01) during the fatigue protocol. These effects were not observed for the other control beverages. No effects were found for maximal or explosive strength, or psychological measurements. CONCLUSIONS: 40 g of α-lactalbumin increased handgrip force variability and reduced performance during fatiguing muscle contractions but did not influence brief maximal contractions or psychological parameters in healthy individuals. These findings support the hypothesis that the consumption of α-lactalbumin can increase motor neurone input-output gain and exacerbate central fatigue during sustained maximal exercise.

The effects of α-lactalbumin supplementation and handgrip contraction on soleus motoneuron excitability.

INTRODUCTION: We tested two strategies that hypothetically increase serotonin availability (α-lactalbumin consumption and a remote submaximal handgrip contraction) on estimates of persistent inward currents (PICs) amplitude of soleus muscle in healthy participants. METHODS: With a randomised, double-blind, and cross-over design, 13 healthy participants performed triangular-shaped ramp contractions with their plantar flexors (20% of maximal torque), followed by a 30-s handgrip sustained contraction (40% of maximal force) and consecutive repeated triangular-shaped contractions. This was performed before and after the consumption of either 40 g of α-lactalbumin, an isonitrogenous beverage (Zein) or an isocaloric beverage (Corn-starch). Soleus motor units discharge rates were analysed from high-density surface electromyography signals. PICs were estimated by calculating the delta frequency (ΔF) of motor unit train spikes using the paired motor unit technique. RESULTS: ΔF (0.19 pps; p = 0.001; d = 0.30) and peak discharge rate (0.20 pps; p < 0.001; d = 0.37) increased after the handgrip contraction, irrespective of the consumed supplement. No effects of α-lactalbumin were observed. CONCLUSIONS: Our results indicate that 40 g of α-lactalbumin was unable to modify intrinsic motoneuron excitability. However, performing a submaximal handgrip contraction before the plantar flexion triangular contraction was capable of increasing ΔF and discharge rates on soleus motor units. These findings highlight the diffused effects of serotonergic input, its effects on motoneuron discharge behaviour, and suggest a cross-effector effect within human motoneurons.

Effects of the maize-derived protein zein, and the milk proteins casein, whey, and α-lactalbumin, on subjective measures of satiety and food intake in normal-weight young men.

Protein is considered to be the most satiating food macronutrient and the satiating effect may be dependent on the source of the protein. The maize-derived protein zein and milk protein casein have been shown previously to lower stomach emptying rate more than dairy whey protein, but the effect of zein on satiety has not been evaluated. The objective was to compare the satiating effects of zein and casein, with whey protein and its protein component α-lactalbumin. The study was a randomised crossover design with thirteen normal-weight men (mean age 27.8 years and mean BMI 24.4 kg/m2) consuming isoenergetic (∼4000 kJ, ∼990 kcal) preload mixed meals enriched with Zein, Casein, whey protein isolate (Whey), α-lactalbumin (ALac), or maltodextrin carbohydrate (Carb). Consumption of an ad libitum standardised test meal of chicken fried rice and water provided 360 min following ingestion of the preload meal was measured, and subjective feelings of appetite (hunger, fullness, desire to eat, and prospective food consumption) were assessed using 100-mm visual analogue scales (VAS). There were no differences among the five preload mixed meals in the amount of chicken fried rice consumed at the ad libitum test meal (mean ± sem: 531.6 ± 35.0 g, p = 0.47) or total (preload + test meal) energy intakes (mean ± sem: 5780.5 ± 146.0 kJ, p = 0.29). The subjective VAS appetite ratings and total area under the curve responses for hunger, fullness, desire to eat, and prospective food consumption, were not different following consumption of all five preload mixed meals (p > 0.05). The findings indicate that the effects of zein and casein on satiety were not different from the satiating effects of whey protein and α-lactalbumin.

Molecular allergy diagnostics in predicting oral cow's milk challenge outcome in Finnish children.

Background: Oral food challenges (OFC) are required to diagnose food allergies but are resource-intensive. Objective: To reduce the need for OFCs, we sought to determine serum specific immunoglobulin E (sIgE) cutoff levels for cow's milk and its major allergens predicting oral milk challenge outcomes in children with suspected cow's milk allergy. Methods: A total of 135 Finnish children (median age, 1.8 years [range, 1.0-14.1 years]) with suspected cow's milk allergy underwent open OFC with unheated cow's milk. The sIgE levels to milk (f2), casein (Bos d 8), alpha-lactalbumin (Bos d 4), beta-lactoglobulin (Bos d 5), and bovine serum albumin (BSA) (Bos d 6) were measured and compared with the challenge outcomes. Results: Of the 135 OFCs, 5 were excluded from the study due to purely subjective symptoms. Of the 130 remaining OFCs, 98 results (75%) were positive. In a receiver operating characteristic analysis with 1-2-year-old children, no individual allergen sIgE had a better area under the curve than milk sIgE (0.824). A milk sIgE level > 6.30 kU/L gave 94% specificity and 33% sensitivity for positive OFCs. In 3-14-year-old children, a cutoff value >13.9 kU/L predicted a positive OFC result with 93% specificity and 25% sensitivity. Children with moderate-to-severe reactions had higher sIgE levels to milk, alpha-lactalbumin, and BSA than did children with mild reactions. Conclusion: Molecular allergy diagnostics did not improve the predictive performance compared with milk sIgE. The milk sIgE value that exceeds the cutoff for 95% specificity in combination with the clinical history may help to reduce the need for OFCs. The severity of an allergic reaction cannot reliably be predicted from sIgE measurements.

Tandem mass tag-based quantitative proteomics analysis reveals the effects of the α-lactalbumin peptides GINY and DQW on lipid deposition and oxidative stress in HepG2 cells.

The objective of this study was to investigate the mechanism by which the α-lactalbumin peptides Gly-Ile-Asn-Tyr (GINY) and Asp-Gln-Trp (DQW) ameliorate free fatty acid-induced lipid deposition in HepG2 cells. The results show that GINY and DQW reduced triglyceride, total cholesterol, and free fatty acid levels significantly in free fatty acid-treated HepG2 cells. Based on proteomic analysis, GINY and DQW alleviated lipid deposition and oxidative stress mainly through the peroxisome proliferator-activated receptor (PPAR) pathway, fatty acid metabolism, oxidative phosphorylation, and response to oxidative stress. In vitro experiments confirmed that GINY and DQW upregulated the mRNA and protein expression of fatty acid ß-oxidation-related and oxidative stress-related genes, and downregulated the mRNA and protein expression of lipogenesis-related genes by activating peroxisome proliferator-activated receptor α (PPARα). Meanwhile, GINY and DQW reduced free fatty acid-induced lipid droplet accumulation and reactive oxygen species generation, and enhanced the mitochondrial membrane potential and ATP levels. Furthermore, GINY and DQW enhanced carnitine palmitoyl-transferase 1a (CPT-1a) and superoxide dismutase activities, and diminished acetyl-coenzyme A carboxylase 1 (ACC1) and fatty acid synthase (FASN) activities in a PPARα-dependent manner. Interestingly, GW6471 (a PPARα inhibitor) weakened the effects of GINY and DQW on the PPARα pathway. Hence, our findings suggest that GINY and DQW have the potential to alleviate nonalcoholic fatty liver disease by activating the PPARα pathway.

The effect of heat treatment on the lactosylation of milk proteins.

Protein lactosylation is a significant modification that occurs during the heat treatment of dairy products, causing changes in proteins' physical-chemical and nutritional properties. Knowledge of the detailed lactosylation information on milk proteins under various heat treatments is important for selecting appropriate thermo-processing and identifying markers to monitor heat load in dairy products. In the present study, we used proteomics techniques to investigate lactosylated proteins under different heating temperatures. We observed a total of 123 lactosylated lysines in 65 proteins, with lactosylation even occurring in raw milk. The number of lactosylated lysines and proteins increased moderately at 75°C to 130°C, but dramatically at 140°C. We found that 6 out of 10, 9 out of 16, 6 out of 12, and 5 out of 15 lysine residues in κ-casein, ß-lactoglobulin, α-lactalbumin, and αS1-casein, respectively, were lactosylated under the applied heating treatment. Moreover, different lactosylation states of individual lysines and proteins can indicate the intensity of heating processes. Lactosylation of K14 in ß-lactoglobulin could distinguish pasteurized and UHT milk, while lactosylation of lactotransferrin can reflect moderate heat treatment of products.

Comparison of interaction, structure, and cell proliferation of α-lactalbumin-safflower yellow complex induced by microwave heating or conventional heating.

BACKGROUND: The protein-polyphenol interaction mechanism has always been a research hotspot, but their interaction is affected by heat treatment, which is widely applied in food processing. Moreover, the effects of microwave or water-bath heating on the protein-polyphenol interaction mechanism have been not clarified. The pasteurization condition (65 °C, 30 min) was selected to compare the effects of microwave or water bath on binding behavior, structure, and cell proliferation between α-lactalbumin (α-LA) and safflower yellow (SY), thus providing a guide for the selection of functional dairy processing conditions. RESULTS: Microwave heat treatment of α-LA-SY resulted in stronger fluorescence quenching than that of conventional heat treatment. Moreover, the binding constant Ka of all α-LA-SY samples was augmented significantly after microwave or water bath treatment, and microwave-heated α-LA-SY showed the maximum Ka . Fourier transform infrared spectroscopy showed that microwave heating resulted in more ordered structures of α-LA into its disordered structures than water bath heating. However, the ferric reducing antioxidant power and chroma value of α-LA-SY were more reduced by microwave heating than by water bath heating. Moreover, microwave heating facilitated the cell proliferation of α-LA-SY compared with water bath treatment. CONCLUSION: It was demonstrated that microwave heating promoted interaction between α-LA and SY more than water bath heating did. Microwave heat treatment was a safe and effective way to enhance the binding affinity of α-LA to SY, being a potential application in food industry. © 2022 Society of Chemical Industry.

Ultrasound-assisted glycation and the allergenicity of α-lactalbumin.

BACKGROUND: Ultrasound-assisted glycation is a promising method for decreasing the allergenicity of α-lactalbumin (ALA). However, there is a lack of in vivo studies on the allergenicity of ultrasound-assisted glycated ALA. In this study, the effects of the ultrasound-assisted glycation of ALA on the allergenicity and intestinal microflora were characterized using a BALB/c mouse model. RESULTS: Increased immunoglobulin -G/ immunoglobulin-E (IgG/IgE) and interleukin-4/6 (IL-4/6) secretions, and reduced interferon-γ (IFN-γ) secretions were found in the serum of ALA sensitized and challenged, mice in comparison with a control group. However, there was no significant difference between the mice fed with ultrasound-assisted glycated ALA and the control group. Mice that were sensitized and challenged with ALA showed disrupted intestinal microflora, manifesting in significantly decreased Firmicutes and significantly increased Proteobacteria. It was found that 100ALA-gal could maintain the intestinal microflora of mice in a normal state. Pearson's rank correlation showed that Proteobacteria and Spirochaetota were correlated positively with the IL-4/IL-6 level and were correlated negatively with the expression of IFN-γ. Proteobacteria were also significantly positively correlated with IL-6 and negatively correlated with IFN-γ (P < 0.05). CONCLUSION: These results suggested that ultrasound-assisted glycation on ALA can maintain the intestinal microflora in a normal state thus balancing the proportion of Th1/Th2 to decrease allergic reaction. © 2022 Society of Chemical Industry.

Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and ß-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells.

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.

The influence of cations on α-lactalbumin amyloid aggregation.

There is limited knowledge regarding α-lactalbumin amyloid aggregation and its mechanism. We examined the formation of α-lactalbumin amyloid fibrils (α-LAF) in the presence of cations (Mg²⁺, Ca²⁺, Na⁺, K⁺, NH₄⁺, and Cs⁺) in the form of chloride salts at two concentrations. We have shown that studied cations affect the conformation of α-lactalbumin, the kinetics of its amyloid formation, morphology, and secondary structure of α-LAF in a different manner. The higher salts concentration significantly accelerated the aggregation process. Both salt concentrations stabilized α-lactalbumin's secondary structure. However, the presence of divalent cations resulted in shorter fibrils with less ß-sheet content. Moreover, strongly hydrated Mg²⁺ significantly altered α-lactalbumin's tertiary structure, followed by Na⁺, NH₄⁺, K⁺, and weakly hydrated Cs⁺. On the other hand, Ca²⁺, despite being also strongly hydrated, stabilized the tertiary structure, supposedly due to its high affinity towards α-lactalbumin. Yet, Ca²⁺ was not able to inhibit α-lactalbumin amyloid aggregation.

Infant formula containing bovine milk-derived oligosaccharides supports age-appropriate growth and improves stooling pattern.

BACKGROUND: Adding bovine milk-derived oligosaccharides (MOS) enhances the oligosaccharide profile of infant formula. This study aimed to evaluate the safety and efficacy of a MOS-supplemented infant formula. METHODS: In this double-blind randomized controlled trial, healthy infants 21-26 days old were either assigned to bovine milk-based, alpha-lactalbumin, and sn-2 palmitate enriched infant formula (control, n = 115) or the same formula with 7.2 g MOS/L (test, n = 115) until aged 6 months. Co-primary endpoints were weight gain through 4 months and stool consistency (validated scale: 1 = watery to 5 = hard). Secondary endpoints included parent-reported GI tolerance, health-related quality of life (HRQoL), and adverse events (AEs). RESULTS: Weight gain was similar (p = 0.695); the difference between test and control (mean; 95% CI: 0.29; -1.15, 1.73 g/day) was above the non-inferiority margin (-3 g/day). Test had softer stools than control (mean difference in stool consistency score: -0.31; 95% CI: -0.42, -0.21; P < 0.0001); fewer parental reports of harder stools (OR = 0.32, 95% CI: 0.20, 0.49; P < 0.0001) and less difficulties in passing stool (OR = 0.25, 95% CI: 0.09, 0.65; P = 0.005). Parent-reported GI tolerance and HRQoL were similar between groups as were the overall low AEs. CONCLUSIONS: MOS-supplemented infant formula is safe and well-tolerated while supporting normal infant growth and promotes softer stooling pattern without increasing parent-reported and physician-confirmed adverse health concerns. IMPACT: This is the first study investigating the addition of bovine milk-derived oligosaccharides to an infant formula enriched with alpha-lactalbumin and elevated levels of sn-2 palmitate, providing safety and efficacy data for such a formula. Term infant formula supplemented with 7.2 g bovine milk-derived oligosaccharides per liter supported normal infant growth, was well-tolerated and safe. Addition of bovine milk-derived oligosaccharides to term infant formula promoted softer stooling pattern and reduced difficulties in passing stool. The study shows that bovine milk-derived oligosaccharide supplemented infant formula is a safe and effective option for healthy term infants who are formula-fed.

Recent perspective on cow's milk allergy and dairy nutrition.

Cow's milk is a highly nutritious biological fluid that provides nourishment and immunity to infants when breastfeeding declines. However, some infants, children, and adults are allergic to cow's milk because milk contains potential allergens in the form of proteins. Casein and whey proteins and their coagulated sub-fractions in the milk such as αS1-casein, αS2-casein, ß-casein, κ-casein and α-lactalbumin, ß-lactoglobulin, bovine serum albumin, immunoglobulins, lactoferrin, respectively are the major etiological determinant of cow's milk allergy (CMA). Moreover, milk processing techniques such as homogenization and pasteurization alter the milk fat and whey protein's molecular structure and serve them as allergens to the immune system of allergic individuals. Strict exclusion of nutrient-rich milk and other dairy products from diet puts children with CMA at higher nutritional risk. Thus, regular nutritional monitoring, the inclusion of protein and mineral-rich supplements as a substitute for cow's milk, management of animal genetics (sheep, goats, buffaloes, camel, mare, donkey, yak), and milk processing to produce non-allergenic milk by inactivating allergic proteins for designer nutrition is essentially required. This review paper details the prevalence, molecular profiling of milk allergens (proteins), body immune response against CMA, consequences of milk processing, treatment, and novel role of galectins as potentially allergy suppressors.

Gastric aspiration in sudden unexpected infant death of Prader-Willi syndrome: immunohistochemical detection of feeding components.

Prader-Willi syndrome (PWS) in infants is characterized by hypotonia and poor sucking with feeding difficulties. Two autopsy cases of sudden unexpected death during sleep after tube feeding are described herein. For one, gastric aspiration caused by the possible milk regurgitation was suspected. Immunohistochemical examination of lung sections was performed using three antibodies to human α-lactalbumin, human gross cystic disease fluid protein 15, and cow whey ß-lactoglobulin. Five cases of sudden unexpected infant death occurring earlier than at 6 months old were selected as controls. Marked immune-staining for infant formula in one PWS subject was evident within terminal bronchioles and alveoli with granular and amorphous features. However, no positive staining was apparent in the other subject, who exhibited contrasting features in milk distribution. Among control cases, one showed mild staining in the bronchiole, but the others did not. The antibody to ß-lactoglobulin reacted specifically with formula, with no nonspecific background. Gastric contents in the airway can be a difficult issue because of the consequent terminal gasping. However, because of an episode of antemortem symptoms of potential regurgitation, and from findings at autopsy such as petechiae, we inferred that fatal regurgitation occurred in this PWS infant after tube feeding. Several clinical reports have described milk aspiration, but this pathological report is the first related to aspiration in PWS during tube feeding.