Antibacterial, anti-invasive, and anti-inflammatory activity of bovine lactoferrin extracted from milk or colostrum versus whole colostrum
Abstract
Lactoferrin (Lf), a multifunctional cationic glycoprotein extracted from milk or colostrum, is able to chelate two ferric ions per molecule, inhibit the formation of reactive oxygen species, interact with the anionic components of bacteria or host cells, and enter inside host cell nucleus, thereby exerting antibacterial, anti-invasive, and anti-inflammatory activities. By virtue of Lf presence, bovine colostrum is expected to perform analogous functions to pure Lf, along with additional activities attributable to other bioactive constituents. The present research aims to compare the antibacterial, anti-invasive, and anti-inflammatory activities of bovine Lf purified from milk (mbLf) and colostrum (cbLf) in comparison to those exhibited by whole bovine colostrum (wbc). The results demonstrated a major efficacy of mbLf in inhibiting pathogenic bacteria and in exerting anti-invasive and anti-survival activities with respect to cbLf and wbc. Furthermore, mbLf lowered IL-6 levels to those of uninfected cells, while a less evident decrease was observed upon cbLf treatment. Conversely, wbc managed to slightly lower IL-6 levels compared to those synthesized by infected cells. These data demonstrate that, to obtain maximum effectiveness in such activities, Lf should be formulated/used without addition of other substances and should be sourced from bovine milk rather than colostrum.
Introduction
In 1939, lactoferrin (Lf), identified from bovine milk as “an unknown red fraction” (Sorensen and Sorensen 1939) and isolated in 1960 from both human (Johanson 1960) and bovine milk (Groves 1960), is considered as the foremost glycoprotein of milk due to its multifunctional activities. While colostrum possesses the highest Lf concentration, its levels decrease in mature milk (Czosnykowska-Łukacka et al. 2019). Apart from colostrum and milk, Lf is constitutively synthesized by exocrine glands and excreted in various biological fluids including tears, saliva, bronchial secretions, bile, gastrointestinal fluids, urine, plasma, and amniotic fluid (Alexander et al. 2012). Lf is also synthesized and stored in secondary granules of neutrophils, being released at the sites of infection and inflammation upon induction (Masson et al. 1969; Berliner et al. 1995). Several structural studies have elucidated that Lf is a cationic glycoprotein folded into two lobes (Lobe N and Lobe C) connected by an α-helix (Moore et al. 1997). Each lobe possesses a deep task with high affinity for iron (Kd = 10–20 mol/L). Upon iron binding, Lf undergoes a conformational change from an open to a closed form (Baker and Baker 2004). The presence or absence of ferric ions in the chelation sites significantly affects Lf activities, including its antibacterial and antiviral properties. The iron-binding capability of both human (hLf) and bovine Lf (bLf) is also influenced by glycosylation chains: de-N-glycosylation notably reduces iron-binding capacity (Legrand et al. 1990). Thus, glycans play a crucial role not only in cellular processes (Varki 2017), but also in iron binding (Zlatina and Galuska 2021). It is noteworthy that, unlike hLf, which possesses only three glycosylation sites (Asn138, Asn479, and Asn624), bLf possesses five glycosylation sites (Asn233, Asn281, Asn368, Asn476, and Asn545).
Additionally, two variants of Lf have been identified in bovine colostrum and mature milk: Lf-a (MW 84 kDa), always glycosylated at Asn281, and Lf-b (MW 80 kDa), which is never glycosylated at Asn281 (Zlatina and Galuska 2021). Remarkably, in bovine colostrum, approximatively 30% of Lf is represented by Lf-a (105.2 mg/L) and 70% by Lf-b (230.8 mg/L), whereas in mature milk, 15% is Lf-a (52.6 mg/L) and 85% is Lf-b (115.4 mg/L) (Yoshida et al. 2000).
Among the plethora of glycoproteins present in colostrum and milk, Lf stands out as one of the most important, being multifunctional like all natural proteins (Valenti and Antonini 2005).
The activities of Lf are diverse and linked to different physico-chemical characteristics. For instance, its antimicrobial and antibiofilm activity stems from its ability to sequester iron, vital for microbial replication. Similarly, viruses rely on iron for their replicative cycle, as crucial enzymes require iron (Rosa et al. 2023). Furthermore, the synthesis of the extracellular exopolysaccharide, constituting biofilm along with microorganisms, requires available iron; hence, in its absence, biofilm is diminished or absent. Additionally, owing to its positive charge, Lf can interact with the anionic structures on bacterial surface, leading to microbial damage. An example is the interaction between Lf and the lipopolysaccharide of Gram-negative bacteria, resulting in bacterial lysis and subsequent death. Moreover, Lf can bind to cell glycosaminoglycans, impeding the entry of intracellular bacteria or viruses. Unlike transferrin, which remains in the cytoplasm, Lf also enters inside cells and penetrates the nucleus where it binds to specific DNA sequences encoding pro-inflammatory cytokines, thereby exerting an anti-inflammatory action (Rosa et al. 2017). Regarding its immunomodulatory and antitumor action, the activity of Lf is influenced by many factors and the data, sometimes contradictory, are still under investigation (Legrand 2016; Cutone et al. 2020c).
Most international publications are performed using Lf extracted from bovine milk, which is available in large quantities and at a price suitable for marketing as a food supplement (Cutone et al. 2020b). bLf shares 69% similarity with hLf and performs identical biological functions (Pierce et al. 1991). Different preparations derived from bovine milk and colostrum are commercially available, claiming similar or even enhanced efficacy with respect to pure constituents, including Lf. However, to date, no study has adequately addressed this issue.
The present study aims to compare the antibacterial, anti-invasive, and anti-inflammatory activity of bLf purified from milk (mbLf) and from colostrum (cbLf) with whole bovine colostrum (wbc).
This comparison is of great importance as it would clarify the effectiveness of wbc in various pathologies, including those affecting the oral cavity, where convincing studies in the literature on the functions of wcb is lacking. The mere presence of Lf in wbc is insufficient to ascertain whether wbc exerts the same functions as pure Lf, including iron chelation, inhibition of microbial multiplication and invasion, neutralization of lipopolysaccharide, and anti-inflammatory action (Arslan et al. 2021; Mehra et al. 2022). Conversely, other functions of colostrum are carried out by its bioactive constituents such as caseins, α-lactalbumin, β-lactoglobulin, lysozyme, lactoperoxidase, colostrinin (proline-rich polypeptide), and immunoglobulins.
Thus, this study aims to delineate the similarities or discrepancies in the antibacterial, anti-invasive, and anti-inflammatory activity between mbLf and cbLf compared to wcb containing an equivalent concentration of Lf.
Materials and methods
Reagents
The experiments have been carried out with pure bLf extracted by milk (mbLf) (Saputo Dairy, Australia, batch No. 1002096136), colostrum (cbLf) (Sigma–Aldrich, Italy, batch No. L4765-50MG) in comparison with wbc (Colostrum Bulk, Colchester, UK, batch No. 000185877).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
The purity and the concentration of the mbLf, cbLf compared with bLf present in wbc were checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
In particular, 10 mg of mbLf and cbLf were dissolved in 10 mL of distilled water to obtain a theorical concentration of 1 mg/mL. 20 µL of these two solutions, corresponding to theorical 20 µg bLf, were collected and loaded on SDS-PAGE.
Concerning wbc, 60 mg of powder were dissolved in 12 mL of distilled water to obtain a 5 mg/mL solution. From this untreated solution, an aliquot was centrifuged at 5000 g × 5 min, while another aliquot was filtered by 0,45 µm filters. 10 µL of each solution, untreated, centrifuged, or filtered, were collected and loaded on SDS-PAGE.
The concentration of protein solutions was also confirmed by Bradford assay (Biorad, Milan, Italy). The densitometry analysis of the three products was performed with ImageJ software.
Western blotting
Protein samples were separated by SDS-PAGE and electroblotted onto nitrocellulose (GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, UK). Following transfer, the membrane was incubated in TBS-T (Tris Buffer Saline: 20 mmol/L Tris-HCl pH 7.4; 137 mmol/L NaCl; 0.1% Tween 20) containing 5% nonfat dried milk powder (Blotting-Grade Blocker, PanReac AppliChem, ITW reagents, Monza, Italy) for 1 h at room temperature. The membrane was incubated overnight at 4 °C with primary antibody, monoclonal anti-bLf (sc-53498, Santa Cruz, CA, USA, 200 µg/mL) (1:1000), dissolved in TBS-T containing 5% cow milk. Milk, despite containing Lf as a natural constituent, did not appear to influence the detection of the immune-reactive band, as also evident from the absence of a relevant background (see Results section). This could be explained by: (i) the much higher amount of bLf blotted onto the nitrocellulose with respect to the concentration of bLf contained into the blocking medium; (ii) the structural integrity of the full glycoprotein contained into the medium, and in particular of its antibody-binding epitope, may have been compromised by the industrial procedures applied for the preparation of the product.
The membrane was then incubated with the HRP-conjugated secondary antibody (Biorad, Milan, Italy) (1:1000) in TBS-T containing 2.5% milk for 1 h at room temperature. The reagent used for detection was Clarity Western ECL substrate (170-5061, Biorad, Milan, Italy).
Iron titration assay
The percentage of bLf iron saturation for the three commercial samples was detected by spectrophotometric analysis at 468 nm of 1% solutions for both mbLf and cbLf, and of a 5% solution for wbc. At 468 nm, the extinction coefficient of a 1% solution of Lf, completely saturated in iron, corresponds to an optical density (OD) of 0.540. After dissolution, the mbLf and cbLf samples were soluble and the resulting solutions were clear. Instead, the wbc sample had a high turbidity, which affected the OD at 468 nm. To remove turbidity, an aliquot of this solution was centrifuged at 5000 g for 5 min, while a further aliquot was filtered with 0.45 µm filters. Centrifugation at 5000 g × 5 min did not change the turbidity, while the filtration at 0.45 µm allowed to obtain a clear solution, suitable for the assay.
The concentration of the active protein was assessed by the repeated additions of 2.5 µL of 0.2 mol/L of ferric ions to 1 mL of each solution (until the equilibrium), followed by the OD lectures at 468 nm.
Antibacterial activity
Strains
Streptococcus salivarius, commensal Gram-positive bacterium isolated from oral cavity of healthy subject and Streptococcus pyogenes, facultative intracellular pathogenic Gram-positive bacterium isolated from a patient suffering from pharyngitis were used.
Antibacterial assay
The antibacterial assays have been performed utilizing different concentrations (5, 2, 1, 0.5, 0.2, and 0.1 mg/mL) of bLf extracted from milk, colostrum and present in wbc.
The relative quantities of bLf in these products were normalized according to the data obtained from iron chelating assay and densitometry deriving from SDS-PAGE.
Streptococcus salivarius and S. pyogenes were overnight grown in brain heart infusion (BHI) (Oxoid, UK) broth a 37 °C. After this growth, 5 × 105 bacteria/mL were inoculated in 1 mL di BHI broth in the absence and the presence of decreasing concentrations (5, 2, 1, 0.5, 0.2, and 0.1 mg/mL) of mbLf, cbLf, and of wbc and incubated at 37 °C for 6 and 24 h. The number of bacteria has been detected, after appropriate dilutions, by counting the colony forming units (CFU)/mL on BHI agar.
Anti-invasive and anti-survival activity
For the experiments, S. pyogenes, facultative intracellular pathogenic bacterium, was grown in BHI broth for 18 h at 37 °C. After 18 h, a new inoculum of S. pyogenes broth culture was carried out in a new medium for 2 h at 37 °C to have the bacterial strain in the exponential growth phase to be used to infect the cell monolayers.
Cell monolayers
Human primary gingival fibroblast (hPGF) (ATCC, USA) cell line was propagated in 75 cm2 flask (Corning, Italy) containing 10 mL of Fibroblast Basal Medium (ATCC, USA) culture medium supplemented with the Fibroblast Growth Kit–Low Serum kit (ATCC, USA) and incubated at 37 °C in an incubator with a 5% CO2 atmosphere. The Fibroblast Growth Kit–Low Serum contained 7.5 mmol/L L-glutamine, 5 ng/mL recombinant human fibroblast growth factors, 5 µg/mL recombinant human insulin, 1 µg/mL hydrocortisone, 50 µg/mL ascorbic acid, 2% fetal bovine serum (FBS), and 1% penicillin/streptomycin.
Invasiveness and intracellular survival assay
The anti-invasive assay of mbLf, cbLf, and wbc against S. pyogenes was detected on hPGF cell line at a concentration of 100 µg/mL of bLf. Before in vitro experiments, all solutions were sterilized using 0.2 µm Millex HV filters (Millipore Corp., Bedford, USA).
For the invasion assay, 24-well plates containing 1 × 104 cells/well were used and incubated for 48 h at 37 °C in a humidified incubator with a 5% CO2. After 48 h of incubation, the medium was removed from the wells, the cell monolayers were washed with phosphate buffered saline (PBS) (Oxoid LTD, England) and new medium without FBS was added to avoid the possible interaction between Lf and serum transferrin. Penicillin/streptomycin was omitted to prevent those possible traces of antibiotic affecting the entry efficacy of bacteria. The multiwells were incubated at 37 °C for 2 h in an incubator with a 5% CO2. After 2 h of incubation, the invasion assay was carried out. In particular, after eliminating the medium and washing with PBS, a well containing the cell monolayer was incubated at 37 °C for 5 min with trypsin (Merck, Italy) to detach the cells and carry out cell counting. The cell count, equal to 3.1 ± 0.6 × 104 cells/well, is useful for establishing exactly the number of bacteria to inoculate. For this assay, the multiplicity of infection (MOI) of S. pyogenes was chosen as 100. The MOI indicates how many bacteria are put in contact with a known number of cells per well (3.1 ± 0.6 × 104 cells/well). In our experiment, to have a MOI 100, 3.1 ± 0.6 × 106 bacteria were in contact with 3.1 ± 0.6 × 104 cells. The anti-invasive activity of mbLf, cbLf, and wbc against S. pyogenes, at MOI 100, was tested by adding each substance to the cell monolayer together with S. pyogenes. After inoculating the bacteria on the cell monolayers in the absence and presence of the different substances, the multiwells were incubated for 2 h at 37 °C in a 5% CO2. After 2 h of incubation, the culture medium was removed and cell monolayers were washed three times with PBS. 200 µg/mL of gentamicin (Sigma–Aldrich, Italy) were added to cell medium without FBS and penicillin/streptomycin to kill extracellular bacteria. The multiwells were incubated for 1 h at 37 °C in a 5% CO2. After 1 h of incubation, for the invasion assay, the cell medium containing 200 µg/mL of gentamicin was removed and stored at −80 °C for the anti-inflammatory activity assay and five washes were carried out with PBS. The cell monolayer was then lysed with 0.1% (v/v) Triton X-100 and plated, after appropriate dilutions, on BHI plates to count the intracellular CFU/mL. The invasion efficiency was calculated as intracellular CFU/mL of S. pyogenes and as the ratio between the number of intracellular bacteria at 3 h and that of the inoculum. Furthermore, the anti-invasive activity of the different substances against S. pyogenes was also calculated.
For the survival assay, cell medium containing 200 µg/mL gentamicin was removed. Cell monolayers were incubated for 24 h at 37 °C in a 5% CO2 atmosphere with 100 µg/mL gentamicin in new medium without FBS and penicillin/streptomycin. After 24 h of incubation, the medium containing 100 µg/mL of gentamicin was removed from each well and stored at −80 °C for the anti-inflammatory activity assay and five washes were carried out with PBS. The cell monolayer was then lysed with 0.1% (v/v) Triton X-100 and plated, after appropriate dilutions, on BHI plates to count the intracellular CFU/mL. Survival efficiency was calculated as CFU/mL of S. pyogenes and as the ratio between the number of intracellular bacteria at 24 h and those at 3 h. In this case the control was set to 100% and the treated samples were normalized to 100% of the control. Furthermore, the anti-survival activity of the different substances against S. pyogenes was also calculated.
Anti-inflammatory activity
The anti-inflammatory activity assay of the three products against the synthesis of the pro-inflammatory cytokine IL-6 by hPGF cells infected with S. pyogenes was carried out using the commercial kit Human ELISA Max Deluxe Sets (BioLegend, San Diego, CA, USA). For the analysis of IL-6 levels, the culture media of uninfected cells and cells infected with S. pyogenes, in the absence and presence of mbLf, cbLf, and wbc after 3 h (2 h of infection plus 1 h of gentamicin at 200 µg/mL) and 24 h post-infection were analyzed with ELISA assay.
Statistical analysis
For the antibacterial, anti-invasive, anti-survival, and anti-inflammatory activity, the statistical analysis of the experiments was performed via one-way Analysis of variance and Tukey’s post-hoc test. Statistical analysis was performed using Prism v7 software (GraphPad software, San Diego, CA, USA). Results are expressed as the mean ± standard deviation of three independent experiments. A p-value ≤ 0.05 is considered statistically significant.
Results
Quality control of milk and colostrum bovine lactoferrins and whole bovine colostrum
As a protein nutraceutical, Lf activities strictly rely on its physico-chemical properties, including purity, integrity, and iron saturation rate (Rosa et al. 2018). Quality control of the three commercial samples have been carried out by SDS-PAGE, Western blot, and iron titration assay.
The results obtained by SDS-PAGE and Western blot to analyze the concentration and integrity of bLf in the three commercial products are reported in Fig. 1. In Fig. 1A, the bands corresponding to mbLf and cbLf are reported. The electrophoretic analysis of mbLf showed a single band of about 80 kDa, whereas cbLf presented two different bands, around 80 and 45 kDa, respectively.
Fig. 1.
From the electrophoretic gel (Fig. 1), it was possible to determine through densitometry that the quantitation of mbLf was equal to its theoretical value (20 µg with respect to 20 µg), while the amount of cbLf is a little lower than the theoretical value (19.3 µg with respect to 20 µg).
Concerning wbc, in 50 µg of colostrum the amount of bLf corresponded, according to the performed treatments, to 4.9 µg when wbc was untreated, to 4.7 µg when wbc was centrifuged, and 3.8 µg when wbc was filtered, with respect to 10 µg of pure bLf used as control (Fig. 1B).
Table 1 shows the percentage of intact bLf present in the mbLf, cbLf, and wbc untreated, centrifuged, and filtered.
Table 1.
As reported from Table 1, the mbLf appears 100% intact, as the theoretical value of 1 mg/mL of mbLf corresponds to a real value of 1 mg/mL. As regards cbLf, the concentration of intact bLf is equal to 96.5%, as the theoretical value of 1 mg/mL of cbLf corresponds to a real value of 0.965 mg/mL. The lower concentration of cbLf is conceivable due to the presence of a degradation band at approximately 45 kDa (line 3 of Fig. 1A).
As regards wbc, the untreated sample had a percentage of intact bLf equal to 9.8%, the centrifuged sample is equal to 9.4% and the filtered one is equal to 7.6%. In addition, as evident from the electrophoresis gel of wbc (Fig. 1B), several bands are highlightable, and the band at about 80 kDa could contain also other proteins, such as lactoperoxidase, and, consequently, bLf concentration could be even lower with respect to that reported. Finally, the lowest concentration of bLf detected after filtration could be due to nonspecific absorption of bLf on the filter.
The presence of bLf in the filtered wbc, which has been employed in further functional experiments, was confirmed by Western blot (Fig. 1C), and relative densitometry provided a concentration around 80 ng/µg of powder, corresponding to a percentage of intact Lf equal to 8%, similar to that obtained by SDS-PAGE.
In order to verify the activity of the bLf present in the three products, the ability to chelate iron has been assessed through the iron titration assay (Table 2).
Table 2.
The initial value of the OD at 468 nm of the mbLf sample was found to be 0.051, which corresponds to an iron saturation of the mbLf equal to 9.44%. The initial value of OD at 468 nm of cbLf sample was found to be 0.340, which corresponds to an iron saturation of the cbLf equal to 62.97%.
The initial reading of the OD at 468 nm of the filtered wbc was found to be 0.104, which corresponds to an iron saturation of the bLf equal to 19.26%.
From the titration performed after the subsequent additions of ferric ions, it can be stated that:
•
out of 10 mg of mbLf, 10 mg are active, since the maximum absorbance of the sample at 468 nm is equal to OD 0.540;
•
out of 10 mg of cbLf, 9.55 mg are active, since the maximum absorbance of the sample at 468 nm is equal to OD 0.510;
•
out of 50 mg of wbc, 5.74 mg of bLf, contained in filtered wbc, are active, since the maximum absorbance of the sample at 468 nm is equal to OD 0.310.
The iron binding activity showed that in 50 mg of wbc the amount of active bLf (5.74 mg) was major than that detected by densitometry analysis deriving from SDS-PAGE and Western blot on the same filtered wbc (3.80 and 4 mg, respectively).
In order to functionally compare the three products, the subsequent experiments have been carried out by normalizing the bLf concentration according to the values obtained from the titration assay and densitometries derived from SDS-PAGE and Western blot assays (Table 3): the mean value for bLf concentration in filtered wbc sample was equal to 4.50 mg per 50 mg of wbc (9.0%).
Table 3.
Antibacterial activity of mbLf, cbLf, and wbc
The antibacterial activity of the three commercial products was tested on two different bacterial strains, S. salivarius and S. pyogenes. These two strains were chosen as commensal and pathogenic bacteria belonging to the oral microbiota. As matter of fact, S. salivarius is commensal bacterium and S. pyogenes is a facultative intracellular pathogenic bacterium.
The results of antibacterial activity of cbLf, mbLf, and wbc against S. salivarius are reported in Fig. 2.
Fig. 2.
As shown in Fig. 2A, a statistically significant decrease in the load of S. salivarius was found in the presence of mbLf, cbLf, and wbc at all concentrations tested after 6 h of incubation. These decreases were particularly evident when the S. salivarius strain was put in contact with high concentrations (5, 2, 1 mg/mL) of bLf contained in wbc, while for mbLf and cbLf these decreases were less evident (Fig. 2A).
A statistically significant decrease in S. salivarius growth was also observed after 24 h of contact with cbLf, mbLf, and wbc at bLf concentrations of 5, 2, 1, and 0.5 mg/mL (Fig. 2B). Also in this case, these decreases were particularly evident when the S. salivarius strain was put in contact with wbc (Fig. 2B).
The results of the antibacterial activity of mbLf, cbLf, and wbc against S. pyogenes are shown in Fig. 3.
Fig. 3.
Figure 3A showed that mbLf, cbLf, and wbc at all concentrations tested, excluding the concentration of 0.1 mg/mL at 24 h, performed a statistically significant antibacterial activity against S. pyogenes at both 6 and 24 h. In particular, at 6 h of incubation, a significant decrease in S. pyogenes was observed in the presence of mbLf at concentrations of 5, 2, 1, and 0.5 mg/mL of bLf, while for cbLf and wbc the inhibition was less evident (Fig. 3A). After 24 h of contact with the S. pyogenes strain, cbLf, mbLf, and wbc presented statistically significant antibacterial activity at concentrations of 5, 2, 1, 0.5, and 0.2 mg/mL of bLf (Fig. 3B). This activity appears to be dose-dependent and similar for the three substances tested (Fig. 3B).
Anti-invasive and anti-survival activity of mbLf, cbLf, and wbc
The results of the anti-invasive activity of cbLf, mbLf, and wbc against S. pyogenes on the hPGF cell line are shown in Fig. 4.
Fig. 4.
MbLf and cbLf significantly decreased the entry of S. pyogenes into the hPGF cell line, while wbc did not influence the entry of the bacterial strain (Figs. 4A and 4B). In particular, mbLf and cbLf inhibited the entry of S. pyogenes by approximately 75% and 60%, respectively, while wbc did not present a statistically significant anti-invasive activity, showing an inhibition effect of approximately 15% (Fig. 4C).
The results of the anti-survival activity of mbLf, cbLf, and wbc against S. pyogenes on the hPGF cell line are shown in Fig. 5.
Fig. 5.
Also in the survival assay, mbLf and cbLf presented a statistically significant intracellular killing activity against S. pyogenes strain compared to wbc (Figs. 5A and 5B). Specifically, mbLf and cbLf presented an anti-survival activity of 60% and 40%, respectively (Fig. 5C). Wbc showed an anti-survival activity of approximately 15%, which was not statistically significant (Fig. 5C).
Anti-inflammatory activity of mbLf, cbLf, and wbc
As regards the anti-inflammatory activity, the analysis of the supernatant at 3 h post-infection did not highlight IL-6 levels determinable by ELISA assay (<0 pg/mL). The results at 24 h post-infection are shown in Fig. 6.
Fig. 6.
Preliminarily, IL-6 levels were measured on uninfected hPGF cells treated with mbLf, cbLf, and wbc, showing similar values to uninfected and untreated cells. Therefore, only the uninfected and untreated control cells have been reported in Fig. 6, where it is shown that S. pyogenes induced a significant production of IL-6 (approximately 100 pg/mL) compared to uninfected cells, and both mbLf and cbLf were able to decrease the levels of this pro-inflammatory cytokine. Specifically, mbLf was able to lower IL-6 levels to the concentration found in uninfected cells, while a less evident but statistically significant decrease was also observed upon cbLf treatment. Although less efficient than mbLf and cbLf, wbc managed to slightly lower IL-6 levels compared to infected cells. Of note, the anti-inflammatory activity of mbLf was statistically significant higher compared to the other two products tested (Fig. 6).
Discussion
Among the proteins involved in host innate defense, Lf has gained attention for its multifunctionality, multitargeting activities, and, importantly, its safety profile and high tolerability. To date, Lf is widely employed as a food supplement in different dietary products targeting the enhancement of both intestinal and immune health. However, the claimed beneficial activities of these products are often inadequately substantiated, relying on the presumption that if a bioactive substance is present, its functional properties remain intact regardless of the formulation.
Wbc is a rich source of micro- and macronutrients known to exert beneficial functions for human health. Marketed in powder form, either as such or in enriched formulations, wbc is also integrated into dietary supplements. Compared to mature milk, wbc contains higher content of fat, protein, peptides, nonprotein nitrogen, ash, vitamins and minerals, hormones, growth factors, cytokines, and nucleotides (Playford and Weiser 2021). The bioactive components within wbc confer protection against the development of various diseases and disorders, such as infections, cancers, inflammatory diseases, caries and periodontal disease, iron deficiency anemia, metabolic syndrome and complications in healing wounds, bone fractures, and microbial injuries. Notably, the composition of wbc and its derivatives undergoes significant alterations based on processing conditions and techniques such as heating, freezing, homogenization, and other chromatographic methods (Mehra et al. 2022). Consequently, each component, depending on the production process, may strictly influence each other’s functionality, potentially agonizing or antagonizing specific bioactivities, including those of Lf.
Here, for the first time, a functional comparison between pure bLf, both extracted from milk and colostrum, and wbc has been conducted.
First, quality control of the three commercial preparations has been performed by SDS-PAGE, Western blot, and iron titration assay. Both milk- and colostrum-derived Lfs exhibit an intact band at 80 kDa through SDS-PAGE, whereas cbLf sample showed an additional band at 45 kDa, possibly attributed to a degradation fragment of bLf itself or other protein contaminants. The SDS-PAGE analysis of wbc reveals an intact band at 80 kDa, with a relative Lf concentration of approximately 10% (w/w) in the raw powder. This quantitation was further confirmed by Western blot, which evidenced a single intact band of bLf in the filtered wbc. Interestingly, bLf from wbc seemed to possess a slightly higher molecular mass than mbLf, used as standard for the densitometry. This could be ascribed to differences in the glycosylation patterns, which have been described for Lfs deriving from milk and colostrum (Yoshida et al. 2000).
In the titration assay, mbLf presented an initial iron saturation of 9.44%, falling within the saturation range of native hLf in vivo, with 100% iron chelation ability. Conversely, cbLf exhibits a notably high initial iron saturation rate of 62.97%, potentially impacting its iron-dependent antibacterial activity. Its ability to chelate iron is 95.5% active compared to mbLf (100%), possibly attributed to the presence of the 45 kDa degradation band observed in the SDS-PAGE assay, which may lack iron-chelating activity and thus remain inactive. As regards the wbc, the titration assay showed an initial iron saturation rate equal to 19.26%, attributable to the Lf content present in the sample. Its iron chelation ability, wbc reached a final value equal to 57.4% of the activity, resulting in a relative concentration of Lf of 11.5% in the row powder, slightly higher than that obtained by SDS-PAGE and Western blot.
The lower contents of bLf obtained by SDS-PAGE and Western blot, compared to those assessed by iron titration assay, could be related to the high interaction capacity of the glycoprotein with other colostrum components, which can induce the formation of higher molecular weight complexes, thus globally interfering with the densitometry analysis. As a matter of fact, bLf was found to form noncovalent complexes with beta-lactoglobulin or albumin (Lampreave et al. 1990), and bind to osteopontin through a complex mechanism involving multiple cationic interactions (Yamniuk et al. 2009). All these interactions could significantly affect the biological functions of bLf contained in wbc. Therefore, we conducted functional studies by normalizing the concentration of the three products based on their relative content of bLf.
The antibacterial assay was assessed on two different strains from Streptococcus genus, S. salivarius and S. pyogenes, representing commensal and facultative pathogenic species, respectively, commonly found in the microflora of the oral cavity. Anti-invasive and anti-inflammatory activities were carried out only with the facultative intracellular pathogenic S. pyogenes.
First, data obtained from the antibacterial assay on the two strains at different concentrations of bLf (5, 2, 1, 0.5, 0.2, and 0.1 mg/mL) highlighted the selective bactericidal activity of bLf against the pathogenic S. pyogenes, with mbLf and cbLf treatments only partially affecting the growth of the commensal strain.
In particular, the antibacterial assay against S. salivarius showed a mild inhibition of approximately 1.5 log at 6 h and 1 log at 24 h of contact by mbLf and cbLf (at the highest concentrations tested), while wbc, at the highest concentrations tested, inhibited its growth by approximately 3 logs at 6 h and by approximately 2 logs at 24 h of contact. Conversely, when S. pyogenes was taken into account, a potent antibacterial activity of mbLf, especially at the highest concentrations tested, by approximately 2.5 log both at 6 and 24 h of contact was observed. CbLf and wbc treatments exerted lower, but still statistically significant, antibacterial activity with respect to mbLf. These data indicate that mbLf induces mild inhibition of the growth of S. salivarius, an essential component of the oral mucosa’s defense against pathogenic bacteria, while it strongly inhibits the growth of a pathogenic bacterium such as S. pyogenes. The antimicrobial activity of Lf can be both dependent and independent on its ability to bind iron (Rosa et al. 2021). Thanks to its ability to chelate two ferric ions per molecule up to pH values 3.0 (Ianiro et al. 2023b), bLf exerts a bacteriostatic action producing an iron-deficient environment and thus reducing bacterial growth. The bactericidal activity of bLf against Gram-positive bacteria, such as S. pyogenes, is due to electrostatic interactions between the negatively charged bacterial lipid layer and the positive surface of bLf, thus causing changes in permeability of the bacterial membrane and subsequent cell lysis (Epand and Vogel 1999). In contrast, wbc activity extends beyond bLf, displaying nonselective inhibition of both strains, significantly suppressing the growth of S. salivarius and potentially compromising the oral microbial barrier.
Regarding the entry and survival of S. pyogenes in the hPGF cell line, the most significant anti-invasive and anti-survival activities were carried out by mbLf, which managed to inhibit both the entry of S. pyogenes in this cell line, probably by binding to a component of the bacterial structure and/or human cells, and the strain survival, through an intracellular killing action. CbLf also demonstrates statistical significant inhibition, albeit to a lesser degree, while wbc shows no significant activity against S. pyogenes. Finally, the same trend was also observed in the anti-inflammatory assay, where hPGF cells infected by S. pyogenes showed an increase in IL-6 levels compared to the uninfected control, and mbLf treatment was able to restore IL-6 levels to physiological levels. These data indicate that the pure and intact mbLf is able to lower IL-6 levels in human cells, as demonstrated in a large number of studies (Cutone et al. 2017, 2019, 2022; Valenti et al. 2017). Such an ability has been widely recognized to be linked to its ability to enter the cell nucleus and regulate the expression of pro-inflammatory cytokines such as IL-6 (Cutone et al. 2020a; Ianiro et al. 2023a). Regarding cbLf, it decreased IL-6 levels compared to the infected control although to a lesser extent than to mbLf, and a similar trend is observed in the experimental condition in which wbc was employed.
Although wbc contains many proteins and peptides exerting both antibacterial and anti-inflammatory activities, this product was not effective as mbLf and cbLf in inhibiting invasion and survival of S. pyogenes in hPGFs cells as well as in abolishing the related pro-inflammatory response.
Overall, mbLf showed more potent antibacterial, anti-invasive, anti-survival, and anti-inflammatory activities when compared to cbLf and wbc, although the molecular mechanisms underlying these major activities need to be further explored. It is important to underline that, in addition to protein integrity and purity, iron saturation rate, iron binding ability and LPS contamination, other physico-chemical properties of Lf, depending on the natural source (e.g., cow breed, seasonality, day of colostrum collection) and the industrial procedures for its purification, could greatly impact the biological activities of the glycoprotein. In this respect, more commercial products should be analyzed and tested to obtain a more complete picture of the problem addressed by the present study.
In conclusion, all data obtained experimentally demonstrate that, to obtain the maximum effectiveness of Lf in its multiple activities against bacterial replication, invasion and survival, as well as inflammation, this protein must be formulated and used without the addition of other substances, as for the wbc, and must be preferentially extracted from bovine milk and not from colostrum.
References
Alexander D.B., Iigo M., Yamauchi K., Suzui M., Tsuda H. 2012. Lactoferrin: an alternative view of its role in human biological fluids. Biochem. Cell Biol. 90: 279–306.
Arslan A., Kaplan M., Duman H., Bayraktar A., Ertürk M., Henrick B.M., et al. 2021. Bovine colostrum and its potential for human health and nutrition. Front. Nutr. 8(8): 651721.
Baker H.M., Baker E.N. 2004. Lactoferrin and iron: structural and dynamic aspects of binding and release. Biometals, 17(3): 209–216.
Berliner N., Hsing A., Graubert T., Sigurdsson F., Zain M., Bruno H.R. 1995. Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression. Blood, 85: 799–803.
Cutone A., Rosa L., Lepanto M.S., Scotti M.J., Berlutti F., Bonaccorsi di Patti M.C., et al. 2017. Lactoferrin efficiently counteracts the inflammation-induced changes of the iron homeostasis system in macrophages. Front. Immunol. 8(8): 705.
Cutone A., Lepanto M.S., Rosa L., Scotti M.J., Rossi A., Ranucci S., et al. 2019. Aerosolized bovine lactoferrin counteracts infection, inflammation and iron dysbalance in a cystic fibrosis mouse model of Pseudomonas aeruginosa chronic lung infection. Int. J. Mol. Sci. 20(9): 2128.
Cutone A., Colella B., Pagliaro A., Rosa L., Lepanto M.S., Bonaccorsi di Patti M.C., et al. 2020a. Native and iron-saturated bovine lactoferrin differently hinder migration in a model of human glioblastoma by reverting epithelial-to-mesenchymal transition-like process and inhibiting interleukin-6/STAT3 axis. Cell. Signal. 65: 109461.
Cutone A., Ianiro G., Lepanto M.S., Rosa L., Valenti P., Bonaccorsi di Patti M.C., et al. 2020b. Lactoferrin in the prevention and treatment of intestinal inflammatory pathologies associated with colorectal cancer development. Cancers, 12: 3806.
Cutone A., Rosa L., Ianiro G., Lepanto M.S., Bonaccorsi di Patti M.C., Valenti P., et al. 2020c. Lactoferrin’s anti-cancer properties: safety, selectivity, and wide range of action. Biomolecules, 10: 456.
Cutone A., Rosa L., Bonaccorsi di Patti M.C., Iacovelli F., Conte M.P., et al. 2022. Lactoferrin binding to SARS-CoV-2 spike glycoprotein blocks pseudoviral entry and relieves iron protein dysregulation in several in vitro models. Pharmaceutics, 14(10): 2111.
Czosnykowska-Łukacka M., Orczyk-Pawiłowicz M., Broers B., Królak-Olejnik B. 2019. Lactoferrin in human milk of prolonged lactation. Nutrients, 11: 2350.
Epand R.M., Vogel H.J. 1999. Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta Biomembr. 1462: 11–28.
Groves M. L. 1960. The isolation of a red protein from milk. J. Am. Chem. Soc. 82: 3345–3350.
Ianiro G., D'Ezio V., Carpinelli L., Casella C., Bonaccorsi di Patti M.C., Rosa L., et al. 2023a. Iron saturation drives lactoferrin effects on oxidative stress and neurotoxicity induced by HIV-1 tat. Int. J. Mol. Sci. 24: 7947.
Ianiro G., Rosa L., Bonaccorsi di Patti M.C., Valenti P., Musci G., et al. 2023b. Lactoferrin: from the structure to the functional orchestration of iron homeostasis. Biometals, 36(3): 391–416.
Johanson B. 1960. Isolation of an iron-containing red protein from human milk. Acta Chem. Scand. 14: 510–512.
Lampreave F., Piñeiro A., Brock J.H., Castillo H., Sánchez L., Calvo M. 1990. Interaction of bovine lactoferrin with other proteins of milk whey. Int. J. Biol. Macromol. 12(1): 2–5.
Legrand D. 2016. Overview of lactoferrin as a natural immune modulator. J. Pediatr. 173: S10–S15.
Legrand D., Mazurier J., Colavizza D., Montreuil J., Spik G. 1990. Properties of the iron-binding site of the N-terminal lobe of human and bovine lactotransferrins. Importance of the glycan moiety and of the non-covalent interactions between the N- and C-terminal lobes in the stability of the iron-binding site. Biochem. J. 266(2): 575–581.
Masson P.L., Heremans J.F., Schonne E. 1969. Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J. Exp. Med. 130: 643–658.
Mehra R., Garhwal R., Sangwan K., Guiné R.P.F., Lemos E.T., Buttar H.S., et al. 2022. Insights into the research trends on bovine colostrum: beneficial health perspectives with special reference to manufacturing of functional foods and feed supplements. Nutrients, 14(3): 659.
Moore S.A., Anderson B.F., Groom C.R., Haridas M., Baker E.N. 1997. Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution. J. Mol. Biol. 274: 222–236.
Pierce A., Colavizza D., Benaissa M., Maes P., Tartar A., Montreuil J., Spik G. 1991. Molecular cloning and sequence analysis of bovine lactotrans-ferrin. Eur. J. Biochem. 196(1): 177–184.
Playford R.J., Weiser M.J. 2021. Bovine colostrum: its constituents and uses. Nutrients, 13: 265.
Rosa L., Cutone A., Lepanto M.S., Paesano R., Valenti P. 2017. Lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis. Int. J. Mol. Sci. 18(9): 1985.
Rosa L., Cutone A., Lepanto M.S., Scotti M.J., Conte M.P., Paesano R., et al. 2018. Physico-chemical properties influence the functions and efficacy of commercial bovine lactoferrins. Biometals, 31: 301–312.
Rosa L., Lepanto M.S., Cutone A., Ianiro G., Pernarella S., Sangermano R., et al. 2021. Lactoferrin and oral pathologies: a therapeutic treatment. Biochem. Cell Biol. 99(1): 81–90.
Rosa L., Cutone A., Conte M.P., Campione E., Bianchi L., Valenti P. 2023. An overview on in vitro and in vivo antiviral activity of lactoferrin: its efficacy against SARS-CoV-2 infection. Biometals, 36(3): 417–436.
Sorensen M., Sorensen S. 1939. The proteins in whey. C. R. Trav. Lab. Carlsberg. 23: 55–99.
Valenti P., Antonini G. 2005. Lactoferrin: an important host defence against microbial and viral attack. Cell. Mol. Life Sci. 62(22): 2576–2587.
Valenti P., Frioni A., Rossi A., Ranucci S., De Fino I., Cutone A., et al. 2017. Aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of Pseudomonas aeruginosa lung infections. Biochem. Cell Biol. 95(1): 41–47.
Varki A. 2017. Biological roles of glycans. Glycobiology, 27(1): 3–49.
Yamniuk A.P., Burling H., Vogel H.J. 2009. Thermodynamic characterization of the interactions between the immunoregulatory proteins osteopontin and lactoferrin. Mol. Immunol. 46: 2395–2402.
Yoshida S., Wei Z., Shinmura Y., Fukunaga N. 2000. Separation of lactoferrin-a and -b from bovine colostrum. J. Dairy Sci. 83(10): 2211–2215.
Zlatina K., Galuska S.P. 2021. The N-glycans of lactoferrin: more than just a sweet decoration. Biochem. Cell Biol. 99: 117–127.
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Published In
Biochemistry and Cell Biology
Volume 102 • Number 4 • August 2024
Pages: 331 - 341
History
Received: 25 January 2024
Accepted: 21 April 2024
Accepted manuscript online: 29 May 2024
Version of record online: 19 June 2024
Notes
This paper is part of a Collection based on presentations at the 2023 XVI International Lactoferrin Conference held on November 6 to 10 2023 in Rome, Italy. https://cdnsciencepub.com/topic/bcb-lactoferrinconference2023.
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© 2024 The Author(s). Permission for reuse (free in most cases) can be obtained from copyright.com.
Data Availability Statement
Data generated or analyzed during this study are provided in full within the published article.
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Conceptualization: LR, PV, AC
Data curation: LR, MPC, LO, PV, AC
Formal analysis: LR, GI, ALC
Methodology: LR, GI, ALC
Supervision: PV
Validation: AC
Writing – original draft: LR, PV, AC
Writing – review & editing: GI, MPC, LO, PV, AC
Competing Interests
The authors declare no conflict of interest.
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The authors declare no specific funding for this work.
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Luigi Rosa, Giusi Ianiro, Antonietta Lucia Conte, Maria Pia Conte, Livia Ottolenghi, Piera Valenti, and Antimo Cutone. 2024. Antibacterial, anti-invasive, and anti-inflammatory activity of bovine lactoferrin extracted from milk or colostrum versus whole colostrum. Biochemistry and Cell Biology.
102(4): 331-341. https://doi.org/10.1139/bcb-2024-0021
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