Degradation Of Lactoferrin Research Articles

Effects of heat treatment and simulated digestion on the properties and osteogenic activity of bovine lactoferrin

Lactoferrin (LF) is known to stimulate osteoblast proliferation and differentiation, potential to be applied in products improving skeletal health. However, LF is sensitive to thermal processing, and oral administration leads to hydrolysis in gastrointestinal tract, probably influencing the osteogenic activity. To determine the impacts of heat treatment, we explored the changes in secondary structure and surface hydrophobicity underlying aggregation along with surface charge alteration of bovine LF under four conditions commonly used in dairy production and found 72 °C/15 s, bringing the least structural alteration, was optimal to retain effect of LF promoting osteogenesis, while LF was completely deprived of the effect treated at 85 °C/10 min and 95 °C/10 min. In vitro adult and infant digestion models were established to identify the degradation of LF, and, despite impaired effect on osteoblast growth, LF hydrolysates from intestinal digestion stage presented stronger effect of boosting osteoblast differentiation independent of p38 and vitamin D receptor.

Lactoferrin directly scavenges hydroxyl radicals and undergoes oxidative self-degradation: a possible role in protection against oxidative DNA damage.

In this study, we examined the protective effect of lactoferrin against DNA damage induced by various hydroxyl radical generation systems. Lactoferrin (LF) was examined with regard to its potential role as a scavenger against radical oxygen species using bovine milk LF. Native LF, iron-saturated LF (holo-LF), and apolactoferrin (apo-LF) effectively suppressed strand breaks in plasmid DNA due to hydroxyl radicals produced by the Fenton reaction. In addition, both native LF and holo-LF clearly protected calf thymus DNA from fragmentation due to ultraviolet irradiation in the presence of H2O2. We also demonstrated a protective effect of all three LF molecules against 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymus DNA following ultraviolet (UV) irradiation with H2O2. Our results clearly indicate that native LF has reactive oxygen species-scavenging ability, independent of its nature as a masking component for transient metals. We also demonstrated that the protective effect of LF against oxidative DNA damage is due to degradation of LF itself, which is more susceptible to degradation than other bovine milk proteins.

Lactoferrin and Neonates: Role in Prevention of Neonatal Sepsis and Necrotizing Enterocolitis

Lactoferrin (LF) is a member of the transferring family with a molecular weight of 78-kDa and is present in numerous external secretions including human milk, saliva, tears, airway mucus, and the secondary granules of neutrophils [1]. It is an iron-binding glycoprotein found in abundance in premature milk with its concentration decreasing as the term age [2]. This protein has been establish to cause a number of biological roles, including antimicrobial (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus mutans, Pseudomonas aeruginosa, Haemophilus influenzae, Helicobacter pylori, Clostridium difficile, Shigella Flexnieria) anti-fungal (Candida albicans), anti-cancer (head and neck squamous cell carcinoma), anti-viral (HCV, HIV) antioxidant, and immunomodulatory effects [3]. Partial degradation of LF by pepsin enzyme give rise to peptides termed lactoferricin (LFcin) with most potent antimicrobial action [4].

Stability during in vitro digestion of lactoferrin-loaded liposomes prepared from milk fat globule membrane-derived phospholipids

Liposomes loaded with positively charged lactoferrin (LF) were prepared from milk fat globule membrane-derived phospholipids using a thin-layer dispersion method. The entrapment efficiency of LF in the liposomes and the stability during in vitro gastrointestinal digestion were characterized and examined using dynamic light scattering, transmission electron microscopy, and PAGE. The entrapment efficiency of LF encapsulated in the liposomes was about 46%. The entrapped LF remained unchanged as a function of time and pepsin concentration when the liposome samples were digested in a simulated gastric environment, suggesting that the liposomes prepared from milk fat globule membrane-derived phospholipids were stable and protected the entrapped LF from pepsin hydrolysis. In simulated intestinal fluid, the entrapped LF was more susceptible to hydrolysis by the protease in pancreatin, as shown by changes in the diameter and membrane structure of the liposomes. The release of free fatty acids from the liposomes during digestion in simulated intestinal fluid revealed that the phospholipids in the liposomes were partly hydrolyzed by pancreatic lipase. It was suggested that liposomes may prevent the gastric degradation of LF and reduce the rate of hydrolysis of LF in intestinal conditions.

Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17–41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.

Lactoferrin: an iron-binding antimicrobial protein against Escherichia coli infection

Escherichia coli (E. coli) are the most common aerobic gram-negative bacilli in a normal intestinal tract. They cause most of the intra-abdominal infections, wound infections associated with abdominal surgery, and septicemia. Most of these infections are of endogenous intestinal origin. Lactoferrin (LF) is an iron-binding glycoprotein found in milk and various external secretions. This protein has been found to have a number of biological functions, including antimicrobial, anti-cancer, antioxidant, and immunomodulatory effects. Partial degradation of LF by pepsin can give rise to peptides termed lactoferricin (LFcin) with more potent antimicrobial activity. LF and LFcin have been shown to inhibit the growth of a number of pathogenic bacteria (including E. coli and antibiotic-resistant strains), fungi, and even viruses in both in vitro and in vivo studies. We previously demonstrated that both recombinant porcine LF (pLF) produced from yeast and a synthetic 20-residue porcine LFcin peptide exhibit antimicrobial activity in vitro. In one of our recent studies, we performed pathogen challenges, including pathogenic E. coli, Staphylococcus aureus and Candida albicans, of the digestive tract of a transgenic milk-fed animal model. The results showed that LF has broad spectrum antimicrobial activity in the digestive tract and protects the mucosa of the small intestine from injury. Our following study also revealed that pLF as a feedstuff additive enhances avian immunity, including antibody formation and cell-mediated immunity. All of these results suggest that LF could be a novel natural protein in the treatment and prevention of infections with E. coli or antibiotic-resistant bacteria strains.

Recombinant Human Lactoferrin and Iron Transport Across Caco-2 Monolayers: Effect of Heat Treatment on the Binding to Cells

Recombinant human lactoferrin (rhLF) from Aspergillus awamori bound to Caco-2 cell membranes in a saturable manner. The dissociation constant for the apo form was (Kd)=2.2 x 10(-7) M; however, the specific binding of the iron-saturated rhLF and of lactoferrin from human milk (hLF) was too low to calculate the binding parameters. Recombinant human lactoferrin subjected to heat treatment did not lose the ability to bind to cell membranes except at high temperature and long time treatments (85 and 89 degrees C for 40 min) for which there was a slight decrease in the binding. No significant differences have been found in the transport of iron bound to rhLF or to hLF across Caco-2 cell monolayers. Nevertheless, the amount of iron-saturated hLF transported across Caco-2 monolayers was significantly higher than that of rhLF. For both lactoferrins, the amount of intact protein in the lower chamber was about 4.5% of the total radioactivity transported, indicating the degradation of lactoferrin in the passage across Caco-2 cells.

Degradation of lactoferrin by periodontitis-associated bacteria

The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens

The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via a high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species ( P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis, is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had orginated from healthy or diseased oral sites.