Comparative proteomic analysis of donor human milk treated by high-pressure processing or Holder pasteurization on undigested proteins across dynamic simulated preterm infant digestion.

High-pressure processing (HPP) of donor human milk (DM) minimally impacts the concentration and bioactivity of some important bioactive proteins including lactoferrin, and bile salt-stimulated lipase (BSSL) compared to Holder pasteurization (HoP), yet the impact of HPP and subsequent digestion on the full array of proteins detectable by proteomics remains unclear. We investigated how HPP impacts undigested proteins in DM post-processing and across digestion by proteomic analysis. Each pool of milk (n = 3) remained raw, or was treated by HPP (500 MPa, 10 min) or HoP (62.5 °C, 30 min), and underwent dynamic in vitro digestion simulating the preterm infant. In the meal, major proteins were minimally changed post-processing. HPP-treated milk proteins better resisted proximal digestion (except for immunoglobulins, jejunum 180 min) and the extent of undigested proteins after gastric digestion of major proteins in HPP-treated milk was more similar to raw (e.g., BSSL, lactoferrin, macrophage-receptor-1, CD14, complement-c3/c4, xanthine dehydrogenase) than HoP.

Pneumococcal surface protein A (PspA) prevents killing of Streptococcus pneumoniae by indolicidin.

Pneumococcal surface protein A (PspA) is an important virulence factor in Streptococcus pneumoniae that binds to lactoferrin and protects the bacterium from the bactericidal action of lactoferricins-cationic peptides released upon lactoferrin proteolysis. The present study investigated if PspA can prevent killing by another cationic peptide, indolicidin. PspA-negative pneumococci were more sensitive to indolicidin-induced killing than bacteria expressing PspA, suggesting that PspA prevents the bactericidal action of indolicidin. Similarly, chemical removal of choline-binding proteins increased sensitivity to indolicidin. The absence of capsule and PspA had an additive effect on pneumococcal killing by the AMP. Furthermore, anti-PspA antibodies enhanced the bactericidal effect of indolicidin on pneumococci, while addition of soluble PspA fragments competitively inhibited indolicidin action. Previous in silico analysis suggests a possible interaction between PspA and indolicidin. Thus, we hypothesize that PspA acts by sequestering indolicidin and preventing it from reaching the bacterial membrane. A specific interaction between PspA and indolicidin was demonstrated by mass spectrometry, confirming that PspA can actively bind to the AMP. These results reinforce the vaccine potential of PspA and suggest a possible mechanism of innate immune evasion employed by pneumococci, which involves binding to cationic peptides and hindering their ability to damage the bacterial membranes.

Investigating the modulatory effects of lactoferrin on depressed rats through 16S rDNA gene sequencing and LC-MS metabolomics analysis.

Lactoferrin is a natural multifunctional glycoprotein with potential antidepressant-like effects. However, the mechanism of its antidepressant effect has not been explored from the perspective of gut flora metabolism. Therefore, we employed both 16S rDNA gene sequencing and LC-MS metabolomics analysis to investigate the regulatory effects and mechanisms of lactoferrin in a rat model of depression. After one week of acclimatization, twenty-four 7-week-old male Sprague-Dawley rats were randomly and equally assigned into three groups: the control group, the model group, and the lactoferrin intervention group. The control group rats were housed under standard conditions, while the rats in the model and lactoferrin intervention groups were individually housed and exposed to chronic unpredictable mild stress for 44 days simultaneously. The lactoferrin intervention group was provided with water containing 2% lactoferrin (2 g/100 ml). Behavioural tests were conducted at week 7. Upon completion of the behavioral tests, the rats were anesthetized with isoflurane, humanely euthanized using a rat guillotine, and tissue samples were collected for further experiments. The results indicated that lactoferrin intervention led to an increase in sucrose solution consumption, horizontal movement distance, number of cross platforms, and residence time in the target quadrant. Additionally, it resulted in an increase in jejunal tight junction protein ZO-1 expression and a suppression of serum expression of inflammatory factors, Lipopolysaccharide and Diamine oxidase. In summary, lactoferrin can regulate the metabolic disorder of intestinal flora, reduce intestinal permeability, and further regulate the metabolic balance of hippocampal tissues through the microbiota-gut-brain axis. This process ultimately alleviates the depression-like behavior in rats.

Lactoferrin-Based Heteroprotein Systems, From Their Formation Mechanism, Properties, To Applications.

Lactoferrin (LF) is an important iron-binding glycoprotein found in milk and mucosal secretions. The alkaline lactoferrin can interact with some acidic proteins to form heteroprotein systems with multifunctional properties and a wide range of applications. Lactoferrin can interact with animal and plant proteins mainly through the electrostatic forces, dipolar attraction, and hydrophobic interactions. In this review, the types of heteroprotein complexes formed by the complex coacervation of lactoferrin with other proteins are introduced, including the preparation, structure, and applications. The factors affecting the formation of heteroprotein complexes are described, such as pH, ionic strength, mixing ratio, total protein concentration, and temperature. The issues and challenges in the formation of heteroprotein complexes are also discussed.

Bovine lactoferrin inhibits Plasmodium berghei growth by binding to heme.

Bovine lactoferrin (bLF) is a 77 kDa glycoprotein that is abundant in bovine breast milk and exerts various bioactive functions, including antibacterial and antiviral functions. Few studies have explored bLF activity against parasites. We found that bLF affects hemozoin synthesis by binding to heme, inhibiting heme iron polymerization necessary for Plasmodium berghei ANKA survival in infected erythrocytes, and also binds to hemozoin, causing it to disassemble. In a challenge test, bLF administration inhibited the growth of murine malaria parasites compared to untreated group growth. To determine whether the iron content of bLF affects the inhibition of malaria growth, we tested bLFs containing different amounts of iron (apo-bLF, native-bLF, and holo-bLF), but found no significant difference in their effects. This indicated that the active sites were located within the bLFs themselves. Further studies showed that the C-lobe domain of bLF can inhibit hemozoin formation and the growth of P. berghei ANKA. Evaluation of pepsin degradation products of the C-lobe identified a 47-amino-acid section, C-1, as the smallest effective region that could inhibit hemozoin formation. This study highlights bLF's potential as a novel therapeutic agent against malaria, underscoring the importance of its non-iron-dependent bioactive sites in combating parasite growth.

A microfluidic model for infantile in vitro digestions: Characterization of lactoferrin digestion.

We present a miniaturized, flow-through model for infantile in vitro digestions, following up on our previously published in vitro digestive system for adults. Microfluidic 'chaotic' mixers were employed as microreactors to help emulate the biochemical processing going on in the infantile stomach and intestine. Simulated digestive fluids were introduced into these micromixers, and the mixtures were incubated for 60 min after both the gastric phase and the intestinal phase. The pH of the infantile stomach was set at 5.3, which is higher than that of adults. This leads to entirely different patterns of digestion for the milk protein, lactoferrin, used in our study as a model compound. It was found that lactoferrin remained undigested as it passed through the gastric phase and reached the intestinal phase intact, unlike in adult digestions. In the intestinal phase, lactoferrin was rapidly digested. Our miniaturized, infantile, in vitro digestive system requires much less labor and chemicals than standard approaches, and shows great potential for future automation.

Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Non-covalent interaction between lactoferrin and theaflavin: Focused on the structural changes, binding mechanism, and functional properties.

In this study, non-covalent binding mechanism of lactoferrin (LaF)-theaflavin (TF) complex and its functional properties were investigated. Multi-spectroscopic analyses showed that the secondary structure of LaF was altered with increasing TF concentration. The non-covalent binding of TF to LaF resulted in a reduction in the content of the α-helix and ß-sheet, as well as a decrease in the fluorescence intensity of LaF. DSC result showed that non-covalent binding of TF improved thermal stability of LaF. Molecular dynamics simulations confirmed that the stable binding of LaF-TF was driven by hydrogen bonding and hydrophobic interactions. Additionally, non-covalent binding of TF increased the antioxidant capacity and emulsifying properties of LaF. Dynamic interfacial tension indicated that the strong interaction between LaF and TF reduced the interfacial tension, but improved the rheological properties of LaF. The functional characteristics of the non-covalent complex was effectively enhanced, paving the way for its potential use in the food industry.

The Iron Binding Ability Maps the Fate of Food-Derived Transferrins: A Review.

As the demand for lactoferrin increases, the search for cost-effective alternative proteins becomes increasingly important. Attention naturally turns to other members of the transferrin family such as ovotransferrin. The iron-binding abilities of these proteins influence their characteristics, although the underlying mechanisms remain unclear. This overview systematically summarizes the effects of the iron-binding ability on the fate of food-derived transferrins (lactoferrin and ovotransferrin) and their potential applications. The findings indicate that iron-binding ability significantly influences the structure of food-derived transferrins, particularly their tertiary structure. Changes in structure influence their physicochemical properties, which, in turn, lead to different behaviors in response to environmental variations. Thus, these proteins exhibit distinct digestive characteristics by the time they reach the small intestine, ultimately performing varied physiological functions in vivo. Consequently, food-derived transferrins with different iron-binding states may find diverse applications. Understanding this capability is essential for developing food-derived transferrins and driving innovation in lactoferrin-related industries.

Deciphering the role of HLF in idiopathic orbital inflammation: integrative analysis via bioinformatics and machine learning techniques.

Idiopathic orbital inflammation, formerly known as NSOI (nonspecific orbital inflammation), is characterized as a spectrum disorder distinguished by the polymorphic infiltration of lymphoid tissue, presenting a complex and poorly understood etiology. Recent advancements have shed light on the HLF (Human lactoferrin), proposing its critical involvement in the regulation of hematopoiesis and the maintenance of innate mucosal immunity. This revelation has generated significant interest in exploring HLF's utility as a biomarker for NSOI, despite the existing gaps in our understanding of its biosynthetic pathways and operational mechanisms. Intersecting multi-omic datasets-specifically, common differentially expressed genes between GSE58331 and GSE105149 from the Gene Expression Omnibus and immune-related gene compendiums from the ImmPort database-we employed sophisticated analytical methodologies, including Lasso regression and support vector machine-recursive feature elimination, to identify HLF. Gene set enrichment analysis and gene set variation analysis disclosed significant immune pathway enrichment within gene sets linked to HLF. The intricate relationship between HLF expression and immunological processes was further dissected through the utilization of CIBERSORT and ESTIMATE algorithms, which assess characteristics of the immune microenvironment, highlighting a noteworthy association between increased HLF expression and enhanced immune cell infiltration. The expression levels of HLF were corroborated using data from the GSE58331 dataset, reinforcing the validity of our findings. Analysis of 218 HLF-related differentially expressed genes revealed statistically significant discrepancies. Fifteen hub genes were distilled using LASSO and SVM-RFE algorithms. Biological functions connected with HLF, such as leukocyte migration, ossification, and the negative regulation of immune processes, were illuminated. Immune cell analysis depicted a positive correlation between HLF and various cells, including resting mast cells, activated NK cells, plasma cells, and CD8 T cells. Conversely, a negative association was observed with gamma delta T cells, naive B cells, M0 and M1 macrophages, and activated mast cells. Diagnostic assessments of HLF in distinguishing NSOI showed promising accuracy. Our investigation delineates HLF as intricately associated with NSOI, casting light on novel biomarkers for diagnosis and progression monitoring of this perplexing condition.

Meconium Proteins Involved in Iron Metabolism.

The lack of specific biological materials and biomarkers limits our knowledge of the mechanisms underlying intrauterine regulation of iron supply to the fetus. Determining the meconium content of proteins commonly used in the laboratory to assess the transport, storage, and distribution of iron in the body may elucidate their roles in fetal development. Ferritin, transferrin, haptoglobin, ceruloplasmin, lactoferrin, myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), and calprotectin were determined by ELISA in meconium samples obtained from 122 neonates. There were strong correlations between the meconium concentrations of haptoglobin, transferrin, and NGAL (p < 0.05). Meconium concentrations of ferritin were several-fold higher than the concentrations of the other proteins, with the exception of calprotectin whose concentration was approximately three-fold higher than that of ferritin. Meconium ceruloplasmin concentration significantly correlated with the concentrations of MPO, NGAL, lactoferrin, and calprotectin. Correlations between the meconium concentrations of haptoglobin, transferrin, and NGAL may reflect their collaborative involvement in the storage and transport of iron in the intrauterine environment in line with their recognized biological properties. High meconium concentrations of ferritin may provide information about the demand for iron and its utilization by the fetus. The associations between ceruloplasmin and neutrophil proteins may indicate the involvement of ceruloplasmin in the regulation of neutrophil activity in the intrauterine environment.

Effects of novel lactoferrin peptides on LPS-induced alveolar bone destruction in a rat model.

To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Digestive Profiles of Human Milk, Recombinant Human and Bovine Lactoferrin: Comparing the Retained Intact Protein and Peptide Release.

Lactoferrin (LF) is a major component of human milk. LF supplementation (currently bovine) supports the immune system and helps maintain iron homeostasis in adults. No recombinant human lactoferrin (rhLF) is available for commercial food use. To determine the extent to which rhLF (Effera™) produced by Komagataella phaffii digests similarly to hmLF, a validated in vitro digestion protocol was carried out. Bovine LF (bLF) was used as an additional control, as it is approved for use in various food categories. This study compared the extent of intact protein retention and the profile of peptides released in hmLF, bLF and rhLF (each with low and high iron saturation) across simulated adult gastric and intestinal digestion using gel electrophoresis, ELISA and LC-MS. Intact LF retention across digestion was similar across LF types, but the highest iron-saturated hmLF had greater retention in the simulated gastric fluid than all other sample types. Peptides identified in digested hmLF samples strongly correlated with digested rhLF samples (0.86 < r < 0.92 in the gastric phase and 0.63 < r < 0.70 in the intestinal phase), whereas digested bLF samples were significantly different. These findings support the potential for rhLF as a food ingredient for human consumption.

Real-Time Study of the Specific Interactions of Lactoferrin with Mimicked Heparan Sulfate Meshes Using Ordered Porous Layer Interferometry.

Heparan sulfate (HS) meshes within the glycocalyx on cell surfaces have protein recognition ability and have been crucial for gaining insights into vital bioprocesses, such as viral infection, cancer development, and inflammation. The protein recognition ability is determined by the mesh property and compositions of HS, although little attention has been paid to the effect of the mesh property on the recognition. An in-depth specificity study of protein-HS-mesh recognition is essential to illustrate related biological functions. Here, ordered porous layer interferometry is applied to study the interaction behavior between mimicked HS meshes and lactoferrin (LF). Our work aimed at mimicking HS meshes with heparin, a widely used substitute of HS, and analyzing the specific LF-heparin-mesh interaction mechanism by inhibiting the nonspecific interaction in a blended sample. We found that the counterion release-based electrostatic interaction is dominant in the specific LF-heparin-mesh recognition. Furthermore, we detail the contributions of nonspecific and specific interactions to the recognition. We illustrate that the concentrated charge distribution of the proteins appears to be primarily related to this robust, specific recognition.

[The expression and secretion of human lactoferrin in Bacillus subtilis].

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 µg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 µg/L, with 498.68 µg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 µg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

Fluorescence-based reagent and spectrum-based optical reader for lactoferrin detection in tears: differentiating Sjögren's syndrome from non-Sjögren's dry eye syndrome.

Identification of an early biomarker and effective testing device to differentiate dry eye disease secondary to autoimmune disease (Sjögren's syndrome dry eye disease) from non-Sjögren's dry eye disease are prerequisites for appropriate treatment. We aimed to demonstrate the capacity of a new photo-detection device to evaluate tear lactoferrin levels as a tool for differentiating systemic conditions associated with dry eye disease. Patients with non-Sjögren's and Sjögren's syndrome dry eye disease (n = 54 and n = 52, respectively) and controls (n = 11) were enrolled. All participants completed the Ocular Surface Disease Index questionnaire. Tear collection was performed with Schirmer test, and tear break-up time was examined using a slit lamp. Tear lactoferrin was evaluated using our newly developed photo-detection device. The average lactoferrin concentration was significantly lower in samples from patients with non-Sjögren's dry eye disease (0.337 ± 0.227 mg/mL, n = 54) and Sjögren's syndrome dry eye disease (0.087 ± 0.010 mg/mL, n = 52) than in control samples (1.272 ± 0.54 mg/mL, n = 11) (p < 0.0001). Further, lactoferrin levels were lower in patients with Sjögren's syndrome dry eye disease than in those with non-Sjögren's dry eye disease (p < 0.001). Our cost-effective, antibody-free, highly sensitive photo-detection device for evaluating tear lactoferrin levels can assist ophthalmologists in differentiating different types of dry eye diseases.

Lactoferrin-chia seed mucilage complex coacervates for intestinal delivery of quercetin and fortification of set yogurt.

This study aimed to develop complex coacervates utilizing lactoferrin (LF) and chia seed mucilage (CSM) for promoting intestinal delivery of quercetin (Q) and fortification of set yogurt. Three cross-linkers, including calcium chloride (CC), transglutaminase (TG), and polyphenolic complex (HP), were used to further reinforce the coacervate network. Cross-linked coacervates had higher values of coacervate yield, encapsulation efficiency, and loading capacity. They efficiently preserved Q under gastric condition (⁓87%-99%), with CSM-TG-Q-LF being most effective for intestinal delivery of Q. Moreover, digested pellets of the cross-linked coacervates displayed better antioxidant activity than the uncross-linked coacervates with CSM-TG-Q-LF pellets showing maximum bioactivity. The Q-loaded coacervates demonstrated superior assembly in the yogurt matrix compared to the unencapsulated Q. Moreover, the coacervate systems, especially CSM-TG-Q-LF significantly improved the textural properties of yogurt and the stability of Q in it. Therefore, CSM-TG-LF is a promising carrier to promote intestinal delivery and food application of hydrophobic molecules.

Longitudinal Correlations between Molecular Compositions of Stratum Corneum and Breast Milk Factors during Infancy: A Prospective Birth Cohort Study.

Breast milk contains numerous factors that are involved in the maturation of the immune system and development of the gut microbiota in infants. These factors include transforming growth factor-ß1 and 2, immunoglobin A, and lactoferrin. Breast milk factors may also affect epidermal differentiation and the stratum corneum (SC) barrier in infants, but no studies examining these associations over time during infancy have been reported. In this single-center exploratory study, we measured the molecular components of the SC using confocal Raman spectroscopy at 0, 1, 2, 6, and 12 months of age in 39 infants born at our hospital. Breast milk factor concentrations from their mothers' breast milk were determined. Correlation coefficients for the two datasets were estimated for each molecular component of the SC and breast milk factor at each age and SC depth. The results showed that breast milk factors and molecular components of the SC during infancy were partly correlated with infant age in months and SC depth, suggesting that breast milk factors influence the maturation of the SC components. These findings may improve understanding of the pathogenesis of skin diseases associated with skin barrier abnormalities.