Native Lactoferrin Research Articles

Lactoferrin modulates oxidative stress and inflammatory cytokines in a murine model of dysbiosis induced by clindamycin.

Antibiotics, specifically clindamycin, cause intestinal dysbiosis, reducing the microbiota with anti-inflammatory properties. Furthermore, clindamycin can induce alterations in the immune responses and oxidative stress. Lactoferrin, among other activities, participates in the maintenance of intestinal homeostasis and reduces dysbiosis induced by antibiotic treatment. The aim of this study was to analyze the effect of native and iron-saturated bovine LF in a murine model of dysbiosis induced by clindamycin. Six groups of male C57BL/6 mice were treated with saline (control), clindamycin (Clin), native lactoferrin (nLF), iron-saturated lactoferrin (sLF), nLF/Clin or sLF/Clin. Oxidation caused in the intestinal cells of the ileum of animals subjected to different treatments was analyzed, focusing on lipid peroxidation and protein carbonyl content. The expression of inflammatory mediators was determined by qRT-PCR. Treatment with clindamycin did not modify lipid peroxidation, but significantly increased protein carbonyl levels up to almost 5-fold respect to the control, an effect that was reversed by orally administering sLF to mice. Furthermore, clindamycin increased the expression of interleukin-6 and TNF-α by 1- and 2-fold change, respectively. This effect was reversed by treatment with nLF and sLF, decreasing the expression to basal levels. In conclusion, this study indicates that lactoferrin can prevent some of the effects of clindamycin on intestinal cells and their associated immune system.

Synergistic action of lactoferrin and its derived functional fragments as a promising therapeutic agent in combating mucormycosis.

Aim: Currently, we have limited armamentarium of antifungal agents against Mucorales. There is an urgent need to discover novel antifungal agents that are effective, safe and affordable. Materials & methods: In this study, the anti-Mucorale action of native lactoferrin (LF) and its functional fragments CLF, RR6 and LFcin against three common Mucorale species are reported. The synergistic action of LF with antifungal agents like amphotericin B, isavuconazole and posaconazole was analyzed using checkerboard technique. Results: All the three mucor species showed inhibition when treated with fragments. The checkerboard assay confirmed that native LF showed the best synergistic action against Mucorales in combination with Amphotericin B. Conclusion: These results highlight the potential therapeutic value of native LF against Mucorales.

Quality attributes of whole milk powder processed using ultrasonication and UV-C radiation compared with equivalent thermal treatment

Nonthermal technologies have shown great potential in both reducing microbial loads and retaining bioactive components in milk. This study evaluated the quality of whole milk powder produced using UV-C and ultrasonication (US) compared to regular heat treatment (HT). The results showed that UV-C and US achieved an equivalent bactericidal effect (reduction of total bacteria count) of raw milk compared with HT. HT decreased the native whey protein concentration and led to significant protein and lipid oxidation. US decreased the particle size of milk powder particles but increased its solubility by 18% compared with HT. UV-C retained ∼80% of native lactoferrin and immunoglobulins in raw milk. UV-C and US increased short-chain fatty acid levels in reconstituted milk, probably due to the residual lipase.

A Gently Processed Skim Milk-Derived Whey Protein Concentrate for Infant Formula: Effects on Gut Development and Immunity in Preterm Pigs.

Processing of whey protein concentrate (WPC) for infant formulas may induce protein modifications with severe consequences for preterm newborn development. The study investigates how conventional WPC and a gently processed skim milk-derived WPC (SPC) affect gut and immune development after birth. Newborn, preterm pigs used as a model of preterm infants were fed formula containing WPC, SPC, extra heat-treated SPC (HT-SPC), or stored HT-SPC (HTS-SPC) for 5 days. SPC contained no protein aggregates and more native lactoferrin, and despite higher Maillard reaction product (MRP) formation, the clinical response and most gut and immune parameters are similar to WPC pigs. SPC feeding negatively impacts intestinal MRP accumulation, mucosa, and bacterial diversity. In contrast, circulating T-cells are decreased and oxidative stress- and inflammation-related genes are upregulated in WPC pigs. Protein aggregation and MRP formation increase in HTS-SPC, leading to reduced antibacterial activity, lactase/maltase ratio, circulating neutrophils, and cytotoxic T-cells besides increased gut MRP accumulation and expression of TNFAIP3. The gently processed SPC has more native protein, but higher MRP levels than WPC, resulting in similar tolerability but subclinical adverse gut effects in preterm pigs. Additional heat treatment and storage further induce MRP formation, gut inflammation, and intestinal mucosal damage.

Sterilization of yak milk by direct steam injection (DSI) and effects on milk quality

To facilitate development of new yak milk products with high added value, sterilization effects of direct steam injection (DSI) were evaluated. The Z value (temperature increase necessary to reduce inactivation time by 90%) of Geobacillus stearothermophilus spores (ATCC 7953; 106 CFU mL−1) in yak milk was 5.88 °C. Heating of the milk above 153.86 °C for 0.116 s achieved complete inactivation of ATCC 7953 spores; i.e., commercially-acceptable sterility. The native lactoferrin and β-lactoglobulin (β-Lg) contents of the milk decreased by 40 and 30% after DSI treatment, respectively, whereas the α-lactalbumin (α-La) content did not differ significantly; the overall extent of denaturation of heat sensitive proteins was lower than in UHT milk. The furosine and lactulose contents in DSI treated yak milk were ≤1.11 mg/10 g of protein and 0.368 mg/10 mL, respectively, similar to those in pasteurized milk. The pH, Hunter color values and aroma were significantly influenced by DSI treatment, but the overall sensory acceptability of DSI treated yak milk was assessed as good. These findings indicate that DSI has great potential for producing yak milk and processed yak milk products with considerable added value.

Encapsulation and stabilization of lactoferrin in polyelectrolyte ternary complexes

Effective delivery of the bioactive protein, lactoferrin (LF), remains a challenge as it is sensitive to environmental changes and easily denatured during heating, restricting its application in functional food products. To overcome these challenges, we formulated novel polyelectrolyte ternary complexes of LF with gelatin (G) and negatively charged polysaccharides, to improve the thermal stability of LF with retained antibacterial activity. Linear, highly charged polysaccharides were able to form interpolymeric complexes with LF and G, while coacervates were formed with branched polysaccharides. A unique multiphase coacervate was observed in the gum Arabic GA-LF-G complex, where a special coacervate-in-coacervate structure was found. The ternary complexes made with GA, soy soluble polysaccharide (SSP), or high methoxyl pectin (HMP) preserved the protein structures and demonstrated enhanced thermal stability of LF. The GA-LF-G complex was especially stable with >90% retention of the native LF after treatment at 90 °C for 2 min in a water bath or at 145 °C for 30 s, while the LF control had only ∼7% undenatured LF under both conditions. In comparison to untreated LF, LF in ternary complex retained significant antibacterial activity on both Gram-positive and Gram-negative bacteria, even after heat treatment. These ternary complexes of LF maintained the desired functionality of LF, thermal stability and antibacterial activity, in the final products. The ternary complex structure, particularly the multiphase coacervate, may serve as a template for the encapsulation and stabilization of other bioactives and peptides.

Новые подходы в создании функциональных продуктов питания на основе использования нетрадиционных региональных ресурсов и технологий

Purpose. Development of a scientifically based concept for the formation of an adaptive approach to the meat and dairy socially significant production based on deep processing of regional livestock raw materials, innovative ways to increase the shelf life of products, reduce the cost of technologies, increase consumer properties of products, incl. specialized and functional, are presented. Materials and Methods. The work is based on the convergence of experiments in vitro, in vivo, in silico. All studies were carried out on advanced equipment using modern methods of chromatography, mass spectrometry, voltammetry, physicochemical and biotechnological methods. Discussion. Minor components of animal raw materials can be effective in the treatment and prevention of lot diseases. Peptides with low molecular weight obtained from bovine lactoferrin have a more significant effect on gastric mucosa protection than native bovine lactoferrin in the same concentration. Using of angiogenin isolated from dairy raw materials, or serum concentrates enriched with angiogenin have a great social and practical importance. A complex of proteinoids obtained from collagen-containing raw materials, including animal skin processing waste, as a result of deep processing, can be widely used in the compounds of various food products: jellies, sausages, chopped meat semi-finished products, etc. Improving the safety of meat quality indicators when stored in a chilled state is possible using the case of its preliminary treatment of following antiseptic solutions: magnetized and electroactivated solution – catholyte; concentrate of mineral salts obtained after electrodialysis of whey. Meat treated with these solutions is more juicy compared with control. Its emulsifying ability is more high (on average 10%), which is important in sausage production. It make possible to exclude the using of third-party chemical reagents, and to increase the physical and chemical indices of meat quality and prevent its microbial contamination. In order to produce food products with the specified quality characteristics, it is proposed to use non-traditional vegetable raw materials with regional and price availability as part of the compositions. As a result of the performed research, enriched meat and dairy products of a new generation with established physiological effects were created. The developed formulations aimed to enrich food with natural bioactive complexes of macro- and micronutrients obtained from regional plant raw materials, secondary raw materials from food manufacturers, as well as to improve the organoleptics of the finished product, reduce its allergenicity, extend shelf life, reduce the cost of technologies, increasing the availability of socially significant products, including special-purpose products, for all segments of the population, consumer attractiveness due to functional properties (various types of yoghurts, cheese and cheese products, boiled and smoked sausages, jellies, chopped meat semi-finished products). Conclusion. As a result of the research, innovative technologies have been developed for the effective processing of livestock raw materials and the creation of new types of socially significant meat and dairy products through the use of non-traditional regional sources of biologically active components.

Myopia Is Suppressed by Digested Lactoferrin or Holo-Lactoferrin Administration.

Myopia is becoming a leading cause of vision impairment. An effective intervention is needed. Lactoferrin (LF) is a protein that has been reported to inhibit myopia progression when taken orally. This study looked at the effects of different forms of LF, such as native LF and digested LF, on myopia in mice. Mice were given different forms of LF from 3 weeks of age, and myopia was induced with minus lenses from 4 weeks of age. Results showed that mice given digested LF or holo-LF had a less elongated axial length and thinned choroid, compared to those given native-LF. Gene expression analysis also showed that the groups given native-LF and its derivatives had lower levels of certain cytokines and growth factors associated with myopia. These results suggest that myopia can be more effectively suppressed by digested LF or holo-LF than native-LF.

L-ergothioneine reduces nitration of lactoferrin and loss of antibacterial activity associated with nitrosative stress

Lactoferrin (LF) is a multifunctional antimicrobial, anti-inflammatory, and antioxidant protein that occurs naturally in mammals, most notably in exocrine gland tissues and fluids, such as in the eye. Nitrosative stress can promote changes to tyrosine and other amino acid residues of the protein, which also reduces the activity of LF. l-ergothioneine (ET) is a potent anti-inflammatory antioxidant present in the eye and other tissues through nutrition or supplementation and that may play a role in the prevention or treatment of a variety of diseases. Here we investigated the ability of ET to reduce 3-nitrotyrosine (NTyr) formation using two separate substrates, with the goal of determining whether ET can protect the antibacterial function of LF and other proteins when exposed separately to peroxynitrite and tetranitromethane as nitrating reagents. Native human LF was used as a simple protein substrate, and lamb corneal lysate was chosen as one example of mammalian tissue with a more complex mixture of proteins and other biomolecules. Nitration was monitored by absorbance and fluorescence spectroscopy as well as sandwich (nitrated LF) and direct NTyr (corneal lysate) enzyme-linked immunosorbent assays (ELISAs). We found that pretreatment with ET reduced chemical modification of both native LF and corneal lysate samples and loss of antibacterial LF function due to exposure to the nitrating reagents. These initial results suggest that ET, raised to sufficiently elevated levels, could be tailored as a therapeutic agent to reduce effects of nitrosative stress on LF and in turn sustain the protein activity.

Determination of native lactoferrin and other whey proteins at different pH conditions after UHT using reverse phase HPLC

Whey proteins such as lactoferrin are high value components in milk; however, under UHT conditions they can denature and lose their functionality and bioactivity. The heat stability of these proteins in water suspensions was investigated at different pH conditions using a pilot scale UHT plant. An RP-HPLC method was used to quantify β-lactoglobulin, α-lactalbumin and lactoferrin simultaneously. In a pH 3.0–5.0 lactoferrin-in-water suspension, at least 48% of undenatured lactoferrin remained after UHT. The highest level of undenatured lactoferrin was 81%, observed at pH 4.0; the iron binding capacity for this sample was 78%. In addition, mixtures of whey protein concentrate and lactoferrin in a lactose solution were investigated, which showed 84% and 70% nativity for whey at pH 3.0 and pH 3.5, respectively, and 78% nativity for lactoferrin at pH 3.5. These results provide useful information in product research and development to maintain bioactive protein nativity through UHT processing.

Antioxidant, lipase and ACE‐inhibitory properties of camel lactoferrin and its enzymatic hydrolysates

The antioxidant and the lipase and the angiotensin‐converting enzyme (ACE) inhibitory properties of camel lactoferrin and its hydrolysates elaborated with four proteolytic enzymes (trypsin, α‐chymotrypsin, pancreatin and papain) were assessed. Lactoferrin was purified from camel colostrum using cation exchange chromatography. Camel lactoferrin hydrolysates showed different degrees of hydrolysis, reverse phase‐HPLC profiles and molecular weight distributions, reflecting heterogeneity in terms of polarity and molecular weight of the generated peptides. Camel lactoferrin hydrolysates exhibited higher antioxidant, lipase and ACE inhibitory activities than native lactoferrin. Pancreatin‐generated hydrolysates showed the highest lipase inhibitory activity (48.1%), while papain‐generated hydrolysates presented the greatest ACE inhibitory activity (89.14%).

Optimal dose of lactoferrin reduces the resilience of in vitro Staphylococcus aureus colonies.

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of β–lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin’s method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ β–lactam antibiotic dose combinations and observed that at low doses (< 0.39 μM), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 μM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the β-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ β–lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from β-lactam-induced production of β-lactamase, which when sufficiently large reduces the population’s ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using β-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of β-lactams.

Synthesis, Physicochemical Characterization, and Antibacterial Performance of Silver—Lactoferrin Complexes

Antibiotic-resistant bacteria pose one of the major threats to human health worldwide. The issue is fundamental in the case of chronic wound treatment. One of the latest trends to overcome the problem is the search for new antibacterial agents based on silver. Thus, the aim of this research was to synthesize the silver-lactoferrin complex as a new generation of substances for the treatment of infected wounds. Moreover, one of the tasks was to investigate the formation mechanisms of the respective complexes and the influence of different synthesis conditions on the features of final product. The batch-sorption study was performed by applying the Langmuir and Freundlich isotherm models for the process description. Characterization of the complexes was carried out by spectroscopy, spectrometry, and separation techniques, as well as with electron microscopy. Additionally, the biological properties of the complex were evaluated, i.e., the antibacterial activity against selected bacteria and the impact on L929 cell-line viability. The results indicate the formation of a heterogeneous silver–lactoferrin complex that comprises silver nanoparticles. The complex has higher antibacterial strength than both native bovine lactoferrin and Ag+, while being comparable to silver toxicity.

Effect of chitosan oligosaccharide glycosylation on the emulsifying property of lactoferrin

There is fast increasing interest in the development of alimentary protein stabilized emulsions due to their potential applications in functional food fields. This work studied the effect of glycation degree with chitosan oligosaccharide (COS) on the emulsifying properties of lactoferrin (LF) through Maillard reaction. In the present study, SDS-PAGE and FT-IR were used to confirm LF and COS covalently binding together successfully. Intrinsic fluorescence showed that glycation with COS led more hydrophobic groups exposed to the surface of the structure and particle size increase of LF. Emulsions with 50% (v/v) oil phase and protein concentration of 2% (w/v) was fabricated through one-step shear method. Compared with native LF, emulsions stabilized by LF-COS conjugates showed smaller droplet size and lower creaming index (CI). Among these samples, LF-COS conjugates under 4 h had the best emulsifying efficiency and stability, the emulsion droplet size and the CI of which decreased 39.66% and 28.55% compared with LF, respectively. Furthermore, glycation with COS enhanced the interfacial activity of LF leading to more adsorbing amount and forming thicker layer on the droplets and gel network in the emulsions. This finding would make sense to further understand the modification of emulsifying properties of alimentary proteins through glycosylation with saccharides and develop novel protein-based emulsifiers.

Lactoferrin modulates gut microbiota and Toll-like receptors (TLRs) in mice with dysbiosis induced by antibiotics.

Background: Antibiotic administration can result in gut microbiota and immune system alterations that impact health. Bovine lactoferrin is a milk protein with anticancer, anti-inflammatory, antimicrobial and immune modulatory activities. The aim was to study the ability of native and iron-saturated lactoferrin to reverse the effects of clindamycin on gut microbiota and intestinal Toll-like receptor (TLR) expression in a murine model. Methods: Male C57BL/6 mice were treated with vehicle, clindamycin (Clin), native bovine lactoferrin (nLf), nLf + clindamycin (nLf_Clin), iron-saturated bovine lactoferrin (sLf) and sLf + clindamycin (sLf_Clin). Fecal samples of each group were collected, and bacterial DNA was extracted. Sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess the microbial composition. mRNA expression levels of TLRs (1-9) were determined in mouse colon by qPCR. Pearson's correlation test was carried out between bacteria showing differences in abundance among samples and TLR2, TLR8 and TLR9. Results: Beta-diversity analysis showed that the microbial community of the vehicle was different from the communities of Clin, nLf_Clin and sLf_Clin. At the family level, Bacteroidaceae, Prevotellaceae and Rikenellaceae decreased in the Clin group, and treatment with nLf or sLf reverted these effects. Clin reduced the expression of TLR2, TLR8 and TLR9 and sLf reverted the decrease in the expression of these receptors. Finally, TLR8 was positively correlated with Rikenellaceae abundance. Conclusion: In a situation of intestinal dysbiosis induced by clindamycin, lactoferrin restores the normal levels of some anti-inflammatory bacteria and TLRs and, therefore, could be a good ingredient to be added to functional foods.

High pressure processing inactivates human cytomegalovirus and hepatitis A virus while preserving macronutrients and native lactoferrin in human milk

The effect of high pressure processing (HPP) compared to Holder pasteurization (HoP) (62.5 °C, 30 min), on the inactivation of cytomegalovirus (CMV) and hepatitis A virus (HAV) inoculated human milk pools (n = 10) and culture media (n = 3) was studied. Samples were retained as untreated controls, treated by HoP (62.5 °C, 30 min) or with one of six different HPP protocols (350 MPa, 500 MPa, 600 MPa for 8- or 10-min at <10 °C). Macronutrient concentration and lactoferrin were measured to confirm milk quality. Both HPP and HoP reduced CMV by >4.8-log PFU/mL and >0.9-log PFU/mL in culture medium and human milk, respectively. HoP reduced HAV by 3.4-log PFU/mL and 3.1-log PFU/mL in culture medium and human milk, respectively. HPP treatments of 500 or 600 MPa reduced HAV by >5.7-log PFU/mL and >4-log PFU/mL in culture medium and in human milk, respectively. Macronutrients (fat, total protein, carbohydrate) and energy composition was not affected by any treatment. Lactoferrin concentration decreased by 35% ± 21% (SD) after HoP, but not HPP. Industrial relevanceThis study confirms that HPP is effective in inactivating representative enveloped and non-enveloped viruses in human milk and reducing bacterial load, with no adverse effect on macronutrient and energy composition. For these reasons, evidence it reduces bacteria, and increased efficiency in which milk can be processed, HPP shows great promise in replacing HoP in human milk banking.

Diverse Mechanisms of Antimicrobial Activities of Lactoferrins, Lactoferricins, and Other Lactoferrin-Derived Peptides.

Lactoferrins are an iron-binding glycoprotein that have important protective roles in the mammalian body through their numerous functions, which include antimicrobial, antitumor, anti-inflammatory, immunomodulatory, and antioxidant activities. Among these, their antimicrobial activity has been the most studied, although the mechanism behind antimicrobial activities remains to be elucidated. Thirty years ago, the first lactoferrin-derived peptide was isolated and showed higher antimicrobial activity than the native lactoferrin lactoferricin. Since then, numerous studies have investigated the antimicrobial potencies of lactoferrins, lactoferricins, and other lactoferrin-derived peptides to better understand their antimicrobial activities at the molecular level. This review defines the current antibacterial, antiviral, antifungal, and antiparasitic activities of lactoferrins, lactoferricins, and lactoferrin-derived peptides. The primary focus is on their different mechanisms of activity against bacteria, viruses, fungi, and parasites. The role of their structure, amino-acid composition, conformation, charge, hydrophobicity, and other factors that affect their mechanisms of antimicrobial activity are also reviewed.

An Efficient Heparin Affinity Column Purification Method Coupled with Ultraperformance Liquid Chromatography for the Quantification of Native Lactoferrin in Breast Milk

Lactoferrin (LF) is a bioactive multifunctional protein and found in the highest amounts in human milk. Several methods can be used to quantify LF. However, quantification of native LF has garnered relatively little interest to date. This study aimed to develop a novel efficient two-step method for quantifying native LF in breast milk. During the analysis, LF was first extracted with phosphate buffer (pH 5.0), purified using a heparin affinity column. Subsequently, LF was detected using ultraperformance liquid chromatography (UPLC) at a wavelength of 201 nm. A linear calibration curve was obtained in the range of 5–200 mg/L. The limit of detection and limit of quantitation were 1 mg/L and 5 mg/L, respectively, indicating that the validated method could be employed to quantify LF in breast milk. Compared with previous HPLC methods, this method demonstrated several remarkable advantages, including simple operation, low-cost detection, and high accuracy. Hence, the results demonstrate an efficient method that can be employed commercially to purify and analyze LF in human milk samples.

Oligosaccharide-lactoferrin shell-crosslinked particles for selective targeting of proteins to probiotic bacteria in the colon

Consumption of prebiotics seems to be a more effective approach for improving health compared to consumption of probiotic bacteria. However, these beneficial microorganisms require proteins to proliferate, while most consumed protein does not reach the gut. Still, no protein-containing prebiotics are commercially-available. Probiotic bacteria express sugar-binding-proteins (SBP) on their cell-membrane that mediate uptake of prebiotic oligosaccharides (OS). We have recently shown that Galactooligosaccharide-Lactoferrin hydrolysate (GOS-LFH) Maillard conjugates can serve as a vehicle for targeted delivery of protein to a model probiotic bacterium (Lactobacillus casei), boosting its growth rate. Herein, we aimed to advance this targeted protein delivery system by preparing three different compositions of prebiotic OS-LFH (2′-Fucosyllactose-LFH (2′-FL-LFH), Fructooligosaccharide-LFH (FOS-LFH), and Xylooligosaccharide-LFH (XOS-LFH)) self-assembled micellar particles, and characterizing their structure, physicochemical properties, and in-vitro digestibility. The OS shell of the particles is the molecular-recognition ligand for selective targeting to probiotic's SBP, and a shield against premature digestion of the delivered protein. We have demonstrated that these OS-LFH conjugates self-assembled into 10–25 nm particles, exhibiting good colloidal stability and lower susceptibility to proteolysis compared to the native lactoferrin (LF). Shell-crosslinking significantly reduced in-vitro gastrointestinal digestibility of the protein core. The proteolysis of the crosslinked 2′-FL-LFH, FOS-LFH, XOS-LFH progressed to only 55%, 57%, and 68% respectively, compared to native LF. Collectively, our findings suggest that these OS-LFH particles embody a promising strategy for high endurance and selective targeted delivery of protein to gut probiotics, which may enhance their competitive edge over non-beneficial/harmful colon bacteria, thereby promoting their potential to improve health.

Lactosmart: A Novel Therapeutic Molecule for Antimicrobial Defense.

The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as well as fungal pathogens such as Candida albicans, Candida tropicalis, and Candida glabrata. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique in vitro, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.