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Agricultural and Biological Sciences » "Milk Proteins - From Structure to Biological Properties and Health Aspects", book edited by Isabel Gigli, ISBN 978-953-51-2537-2, Print ISBN 978-953-51-2536-5, Published: September 7, 2016 under CC BY 3.0 license. © The Author(s).

Chapter 8

Bioactive Lactoferrin-Derived Peptides

By Adham M. Abdou and Hend A. Elbarbary
DOI: 10.5772/62569

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Bioactive Lactoferrin-Derived Peptides

Adham M. Abdou and Hend A. Elbarbary
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Lactoferrin (LF) is a member of the transferrin family that is a cationic iron-binding protein. It is an 80-kDa glycoprotein that is found in many secretions in the body and is highly present in milk and colostrums. It exerts antibacterial effects and has a wide range of biological activities. Moreover, it is considered as a precursor of different peptides that have multifunctional bioactivities. During the last decade, several applications of LF and its peptides have been discovered, which has led to its commercial production. Therefore, LF and its peptides can offer a variety of specialized ingredients that can be tailored to meet the needs of natural food preservatives and functional food ingredients.

Keywords: lactoferrin, structure, purification, food preservation, therapeutic activities

1. Introduction

Milk is the first complete functional food devised by nature for the protection and development of newborn mammals. The antimicrobial and bioactive components in milk have received strong attention in recent years. These components have been isolated from a variety of sources including cheese whey and colostrums from hyperimmunized cows [1]. Lactoferrin (LF), lactoperoxidase system, lactoglobulin, and lactolipids are the most common antimicrobial preservatives in dairy industry [2].

The biodefensive properties of mammalian milk proteins have been well described and widely acknowledged for many years. As early as 1930, it was reported that milk possessed active inhibitors [3]. LF was originally discovered in cow’s milk in 1939, which was identified as a red iron-binding protein, so it was called LF (lacto=milk; ferrin= iron) [4]. Later, Schade and Caroline [5] isolated an iron-binding protein from human serum “serotransferrin,” which was later named transferrin.

Milk is the major source of LF. It is abundantly excreted in colostrums of human milk in a concentration up to 7 g/L and the LF concentration in mature milk declines approximately 7-fold in time during lactation to 1 g/L [6]. LF concentration in milk varies among different mammals. In bovine milk, LF occurs in relatively low amounts from 0.02 to 0.03 g/L, although, in colostrums, this amount is much higher from 2 to 5 g/L [7], whereas, in rats and dogs, no LF has been detected so far [8].

LF, a member of the transferrin family, is a cationic iron-binding protein that is found in many exocrine secretions, including milk, tears, saliva, and serum [9]. It exerts antibacterial effects by limiting iron availability for microorganisms [10]. In addition, it shows some biological activities as immunomodulatory effect and chemoprevention of carcinogenesis [11].

It has been demonstrated that peptides derived from LF by pepsin digestion showed higher antimicrobial activity against both Gram-positive and Gram-negative bacteria than intact LF. However, other proteases such as trypsin, chymotrypsin, and plant and microbial proteases do not generate significant antimicrobial peptides from LF [12]. Antimicrobial peptides purified by high-performance liquid chromatography (HPLC) have been extensively used for the fractionation and identification of peptides on a laboratory scale [13]. This system allows the high resolution of peptides in a short time. Therefore, these peptides are potential food preservatives that can suppress microbial growth. However, liquid chromatography is a relatively expensive system for the large-scale preparation of peptides; moreover, some solvents used in chromatography are harmful to humans and not suitable for food processing [14]. Therefore, there is a large-scale, low-cost, and biocompatible approach to peptide fractionation based on the amphoteric nature of the sample peptides dissolved in water [14]. This approach is referred to as autofocusing that is considered as a potential industrial peptide fractionation process [15]. In the sight of these facts, the concerns of this chapter were

  • Structure of LF;

  • Methods of production, isolation and purification of peptides derived from LF;

  • Stability of LF and its derived peptides; and

  • Bioactive properties and applications of LF and its derived peptides.

2. Structure

LF is an iron-binding monomeric glycoprotein with a molecular weight of approximately 80 kDa [7]. Moore et al. [16] demonstrated some physicochemical characteristics of bovine LF. Its carbohydrate content is 11.2%, maximal iron content is 1.4 mg/g, isoelectric point is 8, and absorbance at 465 nm of 1% solution (iron saturation) is 0.58.

LF consists of 692 amino acids. The protein contains a single polypeptide chain folded into two distinct lobes called N- and C-lobe, referring to the N- and C-terminal parts of the molecule, respectively [17]. Both lobes have the same fold, consistent with their high level of sequence identity; in each case, the lobe is subdivided into two sublobes or domains called N1, N2, C1, and C2, respectively. It is separated by a deep interdomain cleft that surfaces an iron-binding site (Fe3+), which is tightly bound in synergistic cooperation with a bicarbonate anion [18]. The use of this anion can affect the metal binding capacity, giving pH control of iron release [19]. Furthermore, LF retains iron to as low as pH 3.5. This gives LF a more potent iron withholding capacity than other transferrins [7]. There is just a single covalent link between the two lobes, which is called a hinge region that forms a short helix conformation with three turns and represent the sequences 334 to 344. In the N-terminal lobe of LF, the sequences start in one domain, move into the second domain, and then back into the first domain before running through a short helix to the C-lobe where the folding pattern is repeated [17].

The two domains fold with a ligand-binding site that is exploited by many binding proteins, including the large family of bacterial periplasmic binding proteins [20]. Moreover, LF is also highly basic due to a unique basic region in the N-terminal of the molecule. One important consequence of this property is that LF can bind with many acid molecules, including heparin and various cell surface molecules [21]. Also, there is a positive charge concentrated in the interlobe region associated with the short helix. This appeals as a likely DNA and many cell types binding region [22].

3. Methods of production, isolation, and purification of peptides derived from LF

3.1. Enzymatic hydrolysis

The antimicrobial sequence of LF was found mainly near its N-terminus, which has an important role in iron chelation. The high proportion of basic residues in the identified domain is allowing its interaction with surface components of microbial cells [23]. Cationic peptide generated upon gastric pepsin cleavage of bovine LF with porcine pepsin, cod pepsin, or acid protease from Penicillium duporzti showed strong activity against Escherichia coli O111, whereas hydrolysates produced by trypsin, papain, or other neutral proteases were much less active [12]. Bellamy et al. [24] reported that a 25-residue peptide could be released from the N-terminal region of bovine LF with potent bactericidal activity. Interestingly, this reaction is catalyzed at acidic pH by pepsin. After cleavage by this enzyme, the region from amino acids 17 to 41 (FKCRRWQWRMKKLGAPSITCVRRAF) is released as lactoferricin B (Lf-cin B) from bovine LF. Lf-cin B showed broad-spectrum antibacterial activity against both Gram-negative and Gram-positive species, even strains that were resistant to native LF. This indicated that the antibacterial activity of hydrolysate was greater than that of undigested LF with all strains tested approximately 8-fold [24].

3.2. Rennet enzyme and autofocusing technique

LF-cin (RRWQWRMKKLG) as well as other three peptides (KLLSKAQEKFGKNKSRSFQL, APRKNVRWCTISQPEWFKCR, and TRVVWCAVG) was also isolated in a single step from calf rennet hydrolyzed LF by autofocusing technique and were purified by reverse-phase HPLC. They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination [25]. The autofocusing is conducted based on the amphoteric nature of the sample using 5 L autofocusing apparatus without using any chemical substances [26]. The basic autofocusing fractions exhibited potent antibacterial activity against E. coli and Bacillus subtilis in comparison to acidic ones and much higher than the crude LF. In addition, they had greater antibacterial activities than those resulted from autofocusing fractions of LF hydrolyzed by both porcine pepsin and fungal rennet. The most active autofocusing fraction was purified by standard chromatography techniques and assessed for antibacterial activity. The synthetic peptides were found to inhibit the growth of E. coli and B. subtilis with minimum inhibitory concentration (MIC) ranging from 25 to 100 μg/ml and from 6.25 to 50 μg/ml, respectively. Moreover, the autofocusing can produce stronger antimicrobials than those in crude ones [25].

3.3. Chromatographic techniques

LF has a cationic nature according to its amino acid composition. This gives it suitability to be purified by cation-exchange chromatography such as carboxymethyl-Sephadex [27]. It is considered the most popular procedure for LF peptide purification in most companies. For example, skim milk or cheese-whey is applied to a cation-exchange chromatography column without pH adjustment after its filtration. First, the column is washed with a low concentration (1.6%) NaCl solution in which lactoperoxidase is eluted. Then, LF is eluted with a high concentration (5%) NaCl solution. Ultrafiltration is applied to concentrate LF and is separated from NaCl by dia-filtration. The LF is then freeze- or spray-dried to make a powder [27]. In the same manner, LF can bind transition metals and anionic compounds such as heparin and DNA. These materials have been used to purify LF. Metal (copper)-chelate affinity chromatography followed by gel filtration was reported for the purification of LF from human whey [28]. Heparin-agarose affinity chromatography was used to purify bovine LF and its peptides from the secretions of the involuting mammary gland [29].

4. Stability and solubility of LF and its derived peptides

4.1. Temperature

It is well known that heat treatment of milk and milk protein solutions affect the functional properties of the native proteins. A consideration of heat stability is very important if LF is to be used as a bioactive component of foods [30]. Several studies were recorded and varied about the heat stability of LF. Heating of milk at 65°C for 30 min had no significant effect on LF. However, the entire activity of LF was lost at 85°C/30 min in all kinds of milk. However, camel’s milk antimicrobial factors were significantly more heat resistant than cow’s and buffalo’s milk proteins because it contains a higher concentration of LF [31]. Similarly, Paulsson et al. [32] found that milk LF was unaffected by pasteurization but completely denatured by ultra-high temperature (UHT) treatment and decrease the interaction capacity of LF with bacteria. Luf and Rosner [33] found that heat treatment at 63°C/30 min reduced the native LF content by 40%, whereas high temperature short time treatment of milk had no significant effect on LF denaturation.

LF is denatured more rapidly in its apo-form than in the iron-saturated form. Both apo- and iron-saturated LF are more heat sensitive when treated in milk than in phosphate buffer [34]. Abe et al. [35] reported that LF resisted heating at 80°C/5 min without any significant loss of the iron-binding capability. After heating at 100°C/5 min, LF was still able to bind approximately 85% of the amount of iron bound by the unheated LF. All iron-binding capability was lost when LF was heated to 120°C in phosphate buffer.

The thermostability of LF was dependent on both pH and ionic strength. LF was thermostable at acidic pH than neutral pH [35]. However, under more extreme conditions (120°C, pH 2), LF is acid hydrolyzed; however, it retained antimicrobial activity independent of iron binding [36]. Kawakami et al. [37] reported that, at pH 3.5, LF was resistant to heating at ionic strength 0.37 or below, but turbidity and precipitation occurred at ionic strength above 0.47. LF and its derived peptides’ antibacterial activity are varied with the growth temperature. Decreased temperature resulted in a corresponding decrease in MIC and minimum bactericidal concentration (MBC). This was most pronounced for E. coli, which increased its sensitivity for LF with decreased growth temperature [38].

4.2. pH

pH is an important factor affecting the denaturation of LF. The rate of LF denaturation was highest at pH above 5.2 in which it mainly affects the rate of the unfolding and aggregation stages of denaturation [39]. In the same aspect, Saito et al. [40] reported that LF and its hydrolysates were denatured to an insoluble state by heat treatment under neutral or alkaline conditions above pH 6. In contrast, they remain soluble after heat treatment under acidic conditions at pH 2 to 5.

LF retained their functional activities at pH ranging from 2 to 7.4 [41]. LF is very stable at pH 4 and high temperature. It resisted heating at 90°C for 5 min at pH 4 without any loss of iron-binding capacity, antigenic activity, or antibacterial activity. LF treated at pH 2 or 3 and 100°C or 120°C for 5 min was apparently degraded, but antibacterial activity was equal to or stronger than that of unheated one [35].

LF was demonstrated to bind to the surface of some pathogenic microorganisms associated with intramammary infections. This binding is optimal at acidic pH with time-dependent binding surface varied according to bacterial species [42]. Griffith and Humphreys [43] concluded that LF was active near neutral pH only in the presence of bicarbonate ions. Because bicarbonate is secreted in the lumen of the intestine, the conditions are favorable for the antimicrobial activity of LF [44].

In the combination between pH and temperature, Murdock and Mathews [45] reported that, under low pH and refrigeration conditions, LF and its pepsin-digested peptides could limit the growth or reduce the population of pathogenic bacteria in dairy products. Moreover, Troost et al. [46] concluded that LF resisted stomach pH and survive gastric transit after oral administration.

4.3. Solubility

The iron-binding capability of bovine LF affects mainly its solubility. Ferrous iron was not stable in solution and was easily changed to insoluble ferric state, but the solubility of the ferrous iron was stabilized in solution in the presence of LF [47].

At acidic pH 2 to 5, LF hydrolysate in the heated samples at 80°C to 120°C remained soluble and the solutions were clear. On the other aspect, at neutral and alkaline pH 6 to 10, turbidity and gel formation occurred, which is markedly increased with temperature rise. At pH 11, the LF hydrolysate in the heated samples remained soluble, but the color of the samples became dark [35].

4.4. Salts

Reiter et al. [48] suggested that citrate reduces the antibacterial activity of LF by competing with the iron-binding proteins. In contrast, bicarbonate enhances the iron-binding properties of LF.

Salanlah and al-Obaidi [49] studied the effect of pH, temperature, magnesium, and calcium on the bactericidal activity of LF against Yersinia pseudotuberculosis. The bactericidal activity of LF was higher at acid pH. However, it was not efficient at 4, 15, and 25°C, but it was effective at 37°C. The activity of LF-derived peptides was time and concentration dependent. Calcium did not affect their activity up to 60 mM, whereas magnesium reduced the activity of LF only. Furthermore, Branen and Davidson [50] reported that the addition of EDTA enhanced the activity of hydrolyzed LF in tryptic soy broth against Enterohemorrahgic E. coli, Listeria monocytogenes, and Salmonella enteritidis. The EDTA chelate the excess of cations in broth that reduces the activity of hydrolyzed LF.

4.5. Proteases

Holo-LF is more resistant to proteolysis than apo-LF. The five different fragments with molecular weight that ranged from 25 to 53 kDa were generated from trypsin digest of LF. However, β-lactoglobulin has the ability to reduce the susceptibility of LF to tryptic digestion, suggesting that complex formation is not a mechanism for protecting LF against intestinal degradation [51].

4.6. Interaction with other antimicrobial agents

The anti-Candida activity of LF and its peptides or Lf-cin in combination with clotrimazole was shown synergistic by checkerboard analysis. These results indicate that LF-related substances function cooperatively with azole antifungal agents against Candida albicans. The concentration of Lf-cin required for inhibiting the growth of C. albicans decreased in the presence of relatively low concentrations of clotrimazole [52].

The concomitant use of LF with antibiotic cefodoxime proxetil resulted in a synergistic activity against Staphylococcus aureus, E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and an additive activity against E. coli strain. The antibiotic MIC in the presence of LF was reduced to <1/64 with an efficacy rate of 53/57 (92.9%) in a patient group with infections [53].

Soukka et al. [54] reported that both LF or its peptides and lactoperoxidase system showed bactericidal activity against Streptococcus mutans at low pH. LF hydrolysate enhanced lactoperoxidase enzyme activity against S. mutans but decreased the yield of antimicrobial components. Denisova et al. [55] reported that the combined administration of LF, lactoperoxidase, and lactoglobulin obtained from cow milk’s as well as LF obtained from human milk contributed to the elimination of Shigella sonnei from the lungs of infected mice. They prevented the death of the animals. On the contrary, each one has no protective action. López-Expósito and Recio [56] reported that there is a synergistic effect when Lf-cin B was combined with bovine LF against both E. coli and Staphylococcus epidermidis. Bovine LF increased its antimicrobial activity when it was assayed with bovine αs2-casein f (183-207).

5. Bioactive properties and applications of LF and its derived peptides

5.1. Antimicrobial activity

The role of LF in innate defense enhances the broad spectrum of antimicrobial activity. Accordingly, various modes of antimicrobial effects have been reported for LF [2].

5.1.1. Stasis effect

The bacteriostatic mechanism is related to the high iron-binding affinity of the protein that deprives iron-necessary for microbial growth. Because the bacteriostatic properties of LF are due to its iron-binding ability, the protein is capable of retarding the growth of a broad range of Gram-negative and Gram-positive bacteria (E. coli, Salmonella typhimurium, L. monocytogenes, B. subtilis, Bacillus stearothermophilus, and Shigella dysenteriae) and certain yeasts [57]. However, bacteriostasis is often temporary because some Gram-negative bacteria adapt to iron-restrictive conditions by synthesizing low molecular weight iron chelators (siderophores) that can remove iron from LF [58]. In the same way, Bishop et al. [59] stated that 0.02 mg/ml apo-LF showed marked inhibition of growth of coliforms. In addition, it can inhibit the growth of Klebsiella spp. and Aerobucter aerogenes at concentrations of 0.2 and 2 mg/ml, respectively.

5.1.2. Cidal effect

The bactericidal effect of Lf-cin against some Gram-negative and Gram-positive bacteria has not any relation simple iron deprivation. It causes a rapid loss of bacterial viability [60]. It was observed that Lf-cin could bind to the outer membrane of Gram-negative bacteria causing alterations in microbial permeation that lead to bacterial death [61]. Moreover, it has the ability to interfere with the carbohydrate metabolism of invading bacteria or LF’s ribonuclease activity, which may interfere with microbial protein synthesis [10].

5.1.3. Cationic effect

Current opinion on the mechanism of action of basic antibacterial peptides is focused on their interaction with the negatively charged elements in the membranes of susceptible bacteria causing an increase in cell permeability [62, 63]. These elements are lipopolysaccharide in Gram-negative bacteria and lipotechoic acid in Gram-positive bacteria. This is in agreement with the study that demonstrates that only basic autofocusing fractions have antibacterial activity [25]. Notably, 6 of the 20 amino acid residues in peptide (KLLSKAQEKFGKNKSRSFQL) and 5 of 11 amino acid residues of peptide (RRWQWRMKKLG) (Lf-cin B fragment) revealed from rennet digestion of LF are basic residues and showed antibacterial activity and this feature probably has an important role in determining their potent bactericidal properties [25]. In addition, Hoek et al. [64] reported that the first 10-amino acid residues of Lf-cin B had more potent antibacterial activity than the rest of 25-amino acid residues) FKCRRWQWRMKKLGAPSITCVRRAF )against E. coli L361. In the same way, antimicrobial domain was identified in the N1-domain of LF, designated as lactoferrampin (WKLLSKAQEKFGKNKSR), corresponding to the amino acids 268 to 284 of LF. This peptide exhibited candidacidal activity, which was substantially higher than the activity of LF. Furthermore, lactoferrampin was active against B. subtilis, E. coli, and P. aeruginosa [65].

Morten et al. [66] reported that the activity increases with an increased amount of tryptophan (Trp) residues, especially against B. subtilis. Trp is thought to function as an anchor in membrane proteins. The aromaticity of Trp has been suggested to interact with both hydrophobic and hydrophilic constituents of the membrane due to its amphipathic nature [67]. Trp is also suggested to act as a needle that pulls α-helices across phospholipid membranes. In cationic peptides, the insertion of Trp residues into bacterial cell membranes disturbs the packing of the phospholipids and causes a weakening of the membrane [68]. The presence of several Trp residues in the 11-residue Lf-cin B peptide (RRWQWRMKKLG) clearly leads to a more effective disruption of the membrane [69]. Moreover, the presence of Trp in Lf-cin B fragment revealed from rennet digest may be responsible for the antibacterial activity by this mechanism [25].

The hydrophobicity, amphipathicity, and net positive charge have been described as important characteristics of antimicrobial peptides [70]. The penetrating ability of antimicrobial peptides to the surface membrane is known as the carpet model [71, 72]. In this model, the peptides first bind to the phospholipid head groups at the surface of the membrane. The hydrophobic core portion of the peptide is then able to interact with the lipid bilayer when hydrophobicity threshold concentration is above 3, causing a disruption of membrane permeability [72]. However, some basic and hydrophobic peptides generated from rennet digested LF have no antibacterial activity [25, 73]. These facts suggest that not only the basic and hydrophobic nature but also other factors may contribute to the antibacterial activity.

5.1.4. Adhesion-blockade effect

Bacterial adherence to target epithelia is an important step in the pathogenesis of the disease. There are many forms of adhesion sites according to the nature of microorganisms. Adhesion-blockade of enteric pathogens

Several carbohydrates, such as 0.1% fructose or 0.5% glucose, strongly inhibit the adherence of shigellae to guinea pig colonic cells. Fructose-containing peptides from LF also inhibit the adhesion of Shigella flexneri to colonic epithelial cells [74]. LF-derived peptides have the ability to inhibit the hemagglutination activity of type 1 fimbriated E. coli [75]. The agglutination reaction was specifically inhibited by glycopeptides derived from LF or α-methyl-D-mannoside. These observations indicate that the glycans of LF could serve as receptors for type 1 fimbrial lectin of E. coli. Furthermore, more than 3 log CFU/g E. coli reduction in feces resulted from the oral administration of LF (20 mg/ml in 20% sucrose solution) [75]. Bovine LF inhibited the binding of fibronectin, fibrinogen, collagen type I, collagen type IV, and laminin to bacteria. Electron microscopy revealed the loss of type 1, CFA-I and CFA-I1 fimbria of E. coli grown in broth containing 10 μM LF. These data suggest a strong influence of LF on adhesion-colonization properties of E. coli [2]. Adhesion-blockade of oral pathogens

LF-derived peptides have the ability to adsorb hydroxyapatite, which resulted in interfering with the attachment of S. mutans to hydroxyapatite [76]. Also, LF-derived peptides inhibit the growth of Prevotella intermedia by interfering with the binding of this bacteria to fibronectin, collagen type I and type IV, and laminin [77]. LF-dependent adhesion-inhibition of Actinomyces comitans and P. intermedia to fibroblasts and Matrigel could involve the binding of LF to both the bacteria and substrata. The decreased adhesion may be due to the blocking of both specific adhesin-ligand and nonspecific charge-dependent interactions [78].

5.2. Food preservation

LF possesses an intrinsic bactericidal activity that is unrelated to its capacity to bind iron [62]. From this point, there has been an increased interest in the food industry for using LF and its peptides as a preservative in a wide variety of food products. Furthermore, LF has been used as a functional food ingredient in some products such as in infant formula, supplemental tablets, yoghurt, skim milk, drinks, and pet foods. In addition, these products have many therapeutic effects, including anti-infection, improvement of gastrointestinal microflora, immunomodulation, anti-inflammation, and antioxidation [52].

A diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to the inhibition and inactivation by Lf-cin B. It had the ability to reduce the bacterial population by approximately 3 log10 CFU/ml for both E. coli and B. subtilis at concentration 31 μg/ml [64]. In a study reported by Shin et al. [79] about the activity of bovine LF and its peptides, they demonstrated that MIC against E. coli O157:H7 was 3000 μg/ml for LF, 100 to 200 μg/ml for LF hydrolysate, and 8 to 10 μg/ml for Lf-cin B in 1% bactopeptone broth. In addition, Lf-cin B killed these bacteria at a concentration above 10 μg/ml.

In the meat industry, LF-cin B was primarily tested in ground beef at concentrations of 50 or 100 μg/ml, where it was found to cause a maximum of 2 log CFU/g reductions at 4°C or 10°C [80]. The LF-derived peptides can also be sprayed onto carcasses at a concentration of 3.26 ml spray/kg beef or 65.2 mg/kg beef for preventing bacterial contamination during processing or can be applied to finished beef surface before final packaging to extend the shelf life [81]. In addition, it has been used in the preservation of poultry, pork, fish, and other seafood [82].

The addition of EDTA has shown a positive effect on the antimicrobial activity of Lf-cin. Lf-cin B was unable to inhibit the growth of E. coli at 37°C at a concentration of 1600 μg/ml, whereas the addition of 100 or 400 μg/ml EDTA, depending on the strain of E. coli, totally prevented the growth [50]. However, Murdock and Mathews [45] reported that there was no effect for EDTA on the antimicrobial action of Lf-cin B in UHT milk even at 4000 μg/ml Lf-cin and 10 mg/ml EDTA.

On the contrary, LF and its peptides have been used in the preservation of raw, pasteurized, and UHT milk, butter, and cheese due to its antimicrobial activity against pathogenic and spoilage bacteria [83]. Moreover, Hugunin [84] reported that LF should be added after pasteurization and fermentation of yoghurt to extend the shelf life of yoghurt and stimulate the growth of its starter cultures. Chen and Allen [85] reported that LF preserves milk with a concentration of 2 mg/ml. Suzuki et al. [41] recorded that LF at a concentration of 1 mg/ml inhibited the formation of hydroperioxides by 48%, whereas the application of 11 mg/ml completely inhibited both the formation of hydroperioxides and several microorganisms. In 2009, Elbarbary reported that the 11-residue Lf-cin B fragment (RRWQWRMKKLG) generated from calf rennet digest of LF has a significant activity in reduction of approximately 3 log10 CFU/ml of E. coli and B. subtilis populations in milk.

Nowadays, the surface of vegetables, fruit, and carrot juice was coated with Lf-cin B to extend their shelf life. It is more significant when it was added to juice filtrate after its dialysis. As the concentration of cations in filtrate decreases, the antimicrobial efficacy of LF-cin B increases [9].

Moreover, LF can be incorporated into edible films. This is applied for dual purposes that enhance food safety and preservation, as this film acts as a physical barrier and prevents lipid oxidation as well as an antimicrobial substance [86].

Emerging preservation technologies, such as the combination of high hydrostatic pressure with LF, have proven promising in the effort to overcome the limitations in LF and its peptide application [120]. The use of LF and LF-cin B in combination with high pressure (155–400 mPa) enhanced the bactericidal activity against E. coli, S. enteritidis, S. typhimurium, S. sonnei, S. flexneri, Pseudomonas fluorescens, and S. aureus in potassium phosphate buffer at 20°C and showed more marked bacterial reduction than the application of LF or Lf-cin B alone [87].

On the contrary, a novel LF derivative [activated LF (ALF)] can be applied as alternative to LF. Naidu [82] reported that ALF has more potent bacteriostatic efficacy against E. coli in contaminated beef steak, and the MIC of ALF and LF was 62 and >1000 μg/ml, respectively. In the same manner, Ransom et al. [88] recorded that ALF inhibited the growth of L. monocytogenes, S. typhimurium, and E. coli O157:H7 in beef carcass adipose tissue after its dipping into ALF followed by dipping into 2% lactic acid in comparison to LF, which had no effect on the growth of bacterial population.

5.3. Antimicrobial effect in human therapy

5.3.1. Antibacterial effect

The antibacterial function of LF has been substantiated by both in vitro [89] and in vivo [90]. It appears that two different mechanisms involving two separate domains of the protein contribute to the antibacterial function of LF, which are its bacteriostatic and bactericidal mechanisms [89].

The ingestion of milk containing LF at 40 mg/ml will result in the formation of LF-cin B at a molar concentration corresponding to 4.5% of ingested LF. The highest concentration of Lf-cin B was found mainly in the stomach then in the upper small intestine then the lower one. Not all ingested LF was digested and some LF were detected in lower gastrointestinal tract [91].

Feeding the infants with LF or its peptides-enriched formula at a concentration of 1 mg/ml for 2 weeks leads to the increase and establishment of bifidobacterium-predominant flora in infant [92], whereas the ratios of Enterobacteriaceae, Streptococcus, and Clostridium showed a tendency to decrease [93].

The oral administration of LF hydrolysate prepared by porcine pepsin in mice showed an inhibitory effect on the proliferation of Clostridium ramosum [94]. Furthermore, supplementation of the milk diet with LF or a pepsin-generated hydrolysate of LF resulted in a significant suppression of bacterial translocation from the intestines to the mesenteric lymph nodes, and the bacteria involved were mainly members of the family Enterobacteriaceae. This ability of LF to inhibit bacterial translocation may be due to its suppression of bacterial overgrowth in the guts of milk-fed mice [95].

Lf-cin B was identified as the most active antimicrobial peptide generated from pepsin digest. It has significant bacteriostatic and bactericidal activities against multiple strains of Helicobacter pylori [96], which is mainly associated with chronic gastritis, peptic ulcer, and gastric cancer. There are numerous possible mechanisms by which Lf-cin B alone may suppress H. pylori infection. First, it possesses a direct antimicrobial activity against Helicobacter species [97]. Second, Lf-cin B may interfere with H. pylori adhesion to the gastric surface [98]. Third and last, Lf-cin B may suppress the production of proinflammatory cytokines, tumor necrotic factor, as a part of a regulatory role in the host immune response [99]. There is also evidence that Lf-cin B can exert a direct bactericidal effect on certain S. mutans and Vibrio cholerae [100].

5.3.2. Antiviral activity

In addition to the antibacterial activity of LF and its peptides, they display antiviral activity against a wide range of human and animal viruses, both DNA and RNA viruses, including rotavirus, respiratory syncytial virus, herpes virus, HIV, hepatitis C virus (HCV), and poliovirus. The antiviral effect of LF lies in the early phase of infection [101]. The antiviral mechanism of LF against viral infection depends on preventing the entry of virus in the host cell either by interfering with adsorption of the virus to the target cell, as LF is capable of binding to viral particles or its envelope protein [102], or by interfering with the viral-coreceptor interaction [103]. The N-terminal region of LF proved to be essential for its antiviral activity that is responsible for binding to the receptor specific to viral infection, which in turn act as a binding site for the initial interaction of virus with host cells [104] .

Another mechanism for the antiviral effect of LF is the ability to block viral replication in target cells [105]. Further studies indicated that the protective effect of LF was due to the up-regulation of natural killer (NK) cells, monocytes, and granulocytes, which eliminated the infection [104]. Tanaka et al. [106] reported that patients with chronic viral infection, such as HCV, when given LF orally at a dose 1.8 or 3.6 mg/day for 8 weeks showed a decrease in their serum alanine transaminase and viral concentration. In another study, 25 patients with chronic hepatitis C genotype 1b received a 6-month course of LF. The serum level of HCV RNA significantly decreased in patients given LF during the 6 months of treatment [107].

Fujihara and Hayashi [108] reported that LF and its peptides could inhibit the infection in mouse with herpes simplex virus type-1 (HSV-1) by preventing HSV-1 plaque formation after a topical application of 1% LF. However, it did not inhibit the propagation of the virus.

The administration of LF (1 mg/g body weight) protected mice from death due to infection with murine cytomegalovirus (MCMV) through the augmentation of NK cell activity. LF-treated mice showed a significant increase in the NK cell activity but not of the cytolytic T lymphocytes that recognize an MCMV-derived peptide [109].

Bovine LF and its peptides could also inhibit the integrin-mediated internalization of adenovirus into host cells through its binding to viral polypeptides III and IIIa [110].

5.3.3. Antifungal activity

The antifungal activity of LF has been studied. It is mainly through the ability of LF to change the structure of the cell wall of fungi, inactivate the enzyme, and deprive fungi from iron [111].

C. albicans and other Candida species are common pathogens that frequently cause oral infections in immunocompromised individuals due to the suppression of local as well as systemic defense mechanisms [112]. LF with concentrations ranging from 0.5 to 100 mg/ml is able to inhibit the growth of several Candida species [113]. Moreover, Takakura et al. [114] showed the protective activities of LF administered orally against oral candidiasis. LF at a dose of more than 0.5 g/kg/day exerts therapeutic activity and facilitated the recovery from oral candidiasis within 5 to 7 days. Furthermore, Masci [115] reported that a mouthwash containing LF and lysozyme was effective against oral candidiasis in immunocompromised patients rather than oral administration.

LF has also been shown to improve inflammatory diseases such as dermatophytosis caused by the fungus Trichophyton mentagrophytes and intractable stomatitis [116].

5.4. Antiparasitic activity

The role of LF in parasitic diseases is not well defined and may involve multiple mechanisms. Preincubation of Toxoplasma gondii and Eimeria stiedae sporozoites with LF peptides reduced their infectivity in animal models [117]. Mice challenged with T. gondii survived for 35 days as a result of oral administration of 5.0 mg Lf-cin. Furthermore, mice challenged with T. gondii cysts were still alive after an intraperitoneal injection of 0.1 mg Lf-cin. In contrast, 80% of untreated mice died due to acute toxoplasmosis within 14 days [118]. This suggests an effect of basic peptides on parasite membrane integrity and/or interaction with host tissues. Moreover, LF inhibits some parasites via the stimulation of the process of phagocytosis where immune cells engulf and digest foreign organisms [118]. Also, LF has the ability to reduce the binding of Plasmodium berghei to the surface of the target cells [119]. Other reported antiparasitic activities appear to involve interference with parasite iron acquisition, such as Pneumocystis carinii [120]. In other parasites, such as Trichomonas foetus, LF appears to act as a specific iron donor and could thus be expected to enhance infection [121].

5.5. Enhancer of iron absorption

The role of LF as an enhancer of iron absorption was proposed because LF binds a majority of iron in breast milk and therefore could facilitate the uptake of iron in the small intestine of infants [122]. The notion that LF is involved in iron absorption was supported by studies that showed the presence of specific LF receptor on brush membranes from human intestine [123]. Lönnerdal and Bryant [124] showed that iron is equally well absorbed from LF (whether heat-treated or untreated) and ferrous sulfate. Thus, iron provided by dietary LF is likely to be well utilized in human adults. The ability of LF to bind iron naturally support its function in intestinal iron absorption [123].

5.6. Anticancer activity

LF has shown anticancer activity in experimental lung, bladder, tongue, colon, and liver carcinogenesis on rats [125]. Also, in another study, LF suppressed the incidence of oral cancer by 50% [126]. In vivo studies have indicated that LF may induce apoptosis in cancer cells; in addition, the immunomodulatory activity of LF is critical in preventing cancer growth by enhancing the activity of NK cells [127]. Therefore, LF offers promise as a potential chemopreventive agent for oral cancer. Currently, LF is used as an ingredient in yogurt, chewing gums, infant formulas, and cosmetics [126].

5.7. Immunomodulatory activity

The immunomodulatory activity of LF is attributed to its ability to up-regulate T-cell proliferation, boost NK cell number, promote lymphocyte maturation, and also play a role in the myelopoietic process [128]. Moreover, LF released from neutrophils forms an integral part of the innate immunity protecting against infection through its antimicrobial activity [129].

6. Conclusion

Several studies proved that LF and its peptides have great potential for use as natural antimicrobials for food preservation and other health benefits in addition to the well-known iron-binding activity. Because LF is commercially available from bovine milk (~90 MT/year worldwide) and considered as a highly safe food additive, it may be an excellent agent for administration to humans. The enzymatic hydrolysis of LF results in the elaboration of functional peptides that exert, in some cases, greater functionality than the native LF. These peptides could serve as an attractive model for the development of new natural food preservatives, functional food ingredients, food supplements, and drug discovery.


1 - Farrell HM, Jamenez-Flores R, Bleck GT, Brown EM, Butler JE, Creamer LK, Hicks CL, Hollar CM, Ngkwai-Hang KF, Swaisgood HE: Nomenclature of the proteins of cow’s milk—sixth revision. J Dairy Sci 2004; 87: 1641–1647.
2 - Naidu AS. Natural Food Antimicrobial Systems. CRC Press, Boca Raton; 2000.
3 - Lyer S, Lonnerdal B: Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr 1993; 47: 232–241.
4 - Sorensen M, Sorensen MPL: The proteins in whey. C R Trav Lab Carlsberg 1939; 23: 55–59.
5 - Schade AL, Caroline L: An iron-binding component in human blood plasma. Science 1946; 104: 340–341.
6 - Hennart PF, Brasseur DJ, Delogne-Desnoeck JB, Dramax MM, Robyn CE: Lysozyme, lactoferrin and secretory immunoglobulin A content in breast milk: Influence of duration of lactation, nutrition status, prolactin status and parity of mother. Am J Clin Nutr 1991; 53: 32–39.
7 - Baker EN, Baker HM, Kid RD: Lactoferrin and transferrin: Functional variations on a common structural framework. Biochem Cell Biol 2002; 80: 27–34.
8 - Masson L, Heremans JF: Lactoferrin in milk from different species. Comp Biochem Physiol B 1971; 39: 119–129.
9 - Chantaysakorn P, Richter RL: Antimicrobial properties of pepsin-digested lactoferrin added to carrot juice and filtrates of carrot juice. J Food Protect 2000; 63: 376–380.
10 - Ye XY, Wang HX, Liu F, Ng TB: Ribonuclease cell free translation-inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk. Int J Biochem 2000; 32: 235–241.
11 - Tomita M, Wakabayashi H, Yamauchi K, Teraguchi S, Hayasawa H: Bovine lactoferrin and lactoferricin derived from milk: Production and applications. Biochem Cell Biol 2002; 80: 109–112.
12 - Tomita M, Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K: Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci 1991; 74: 4137–4142.
13 - Mant CT, Zhou NE, Hodges RS: Amino acids and peptides. In: Chromatography Part B: Applications. Heftman E, editor. Elsevier Science Publishers: Amsterdam, The Netherlands, 1992; pp. 75–150.
14 - Yata M, Sato K, Ohtsuki K, Kawabata M: Fractionation of peptides in protease digests of proteins by preparative isoelectric focusing in the absence of added ampholyte: A biocompatible and low-cost approach referred to as autofocusing. J Agric Food Chem 1996; 44: 76–79.
15 - Hashimoto K, Sato K, Nakamura Y, Ohtsuki K: Development of continuous type apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in protein hydrolysates. J Agric Food Chem 2006; 54: 650–655.
16 - Moore SA, Anderson BF, Groom GR, Haridas M, Baker EN: Three dimensional structure of bovine lactoferrin at 2.8 Å resolution. J Mol Biol 1997; 274: 222–236.
17 - Powell MJ, Ogden JE: Nucleotide sequence of human lactoferrin DNA. Nucleic Acids Res 1990; 18: 4013–4013.
18 - Vorland LH: Lactoferrin: a multifunctional glycoprotein. Acta Pathol Microbiol Immunol Scand 1999; 107: 971–981.
19 - Baker HM, Masson AB, He QY, Baker EN: Ligand variation in the transferring family: The crystal structure of the H249Q mutant of the human transferring N-lobe as a model for iron binding in insect transferrins. Biochemistry 2001; 40: 11670–11675.
20 - Quiocho FA, Ledvina PS: Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: Variation of common themes. Mol Microbiol 1996; 20: 17–25.
21 - Zakharova ET, Shavlovski MM, Bass MG, Gridasova AA, Pulina MO, De Filippis V, Beltramini M, Di Muro P, Salvato B, Fontana A, Vasilyev VB, Gaistskhoki VS: Interaction of lactoferrin with ceruloplasmin. Arch Biochem Biophys 2000; 374: 222–228.
22 - Wu HF, Monroe DM, Church FC: Characterization of the glycosaminoglycan binding region of lactoferrin. Arch Biochem Biophys 1995; 317: 85–92.
23 - Tomita M. Active peptides of lactoferrin. In: Proceedings of the International Dairy Federation Seminar. Indigenous Antimicrobial Agents of Milk: Recent Developments; 1994. pp. 7–13.
24 - Bellamy W, Takase M, Yamaauchi K, Wakabayashi H, Kawase K, Tomita M: Identification of bactericidal domain of lactoferrin. Biochim Biophys Acta 1992; 1121: 130–136.
25 - Elbarbary HA, Abdou AM, Park EY, Nakamura Y, Mohamed HA, Sato K: Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique. Int Diary J 2010; 20: 646–651.
26 - [26]Hashimoto K, Sato K, Nakamura Y, Ohtsuki K: Development of a large-scale (50 L) apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in enzymatic hydrolysates of food proteins. J Agric Food Chem 2005; 53: 3801–3806.
27 - Yoshida S, Ye-Xiuyun Y: Isolation of lactoperoxidase and lactoferrins from bovine milk acid whey by carboxynlethyl cation exchange chromatography. J Dairy Sci 1991; 74: 1439–1444.
28 - Lonnerdal B, Carlsson J, Porath J: Isolation of lactoferrin from human milk by metal-chelate affinity chromatography. FEBS Lett 1977; 75: 89–92.
29 - Rejman JJ, Hegarty HM, Hurley WL: Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: Identification of multiple molecular forms. Comp Biochem Physiol 1989; 93B: 929–934.
30 - Mata L, Sanchez L, Headon DR, Calvo M: Thermal denaturation of human lactoferrin and its effect on the ability to bind iron. J Agric Food Chem 1999; 46: 3964–3970.
31 - Elagamy EI: Effect of heat treatment on camel proteins with respect to antimicrobial factors: A comparison with cow’s and buffalo’s milk proteins. Food Chem 2000; 68: 227–232.
32 - Paulsson MA, Svensson U, Kishore AR, Naidu AS: Thermal behaviour of bovine lactoferrin in relation to bacterial interaction and antibacterial activity.Bull Int Dairy Fed 1994; 215.
33 - Luf W, Rosner E: Thermal stability of lactoferrin in bovine milk. Wien Tierarztl Monatsschr 1997; 84: 70–73.
34 - Sanchez L, Peiro JM, Castillo H, Perez MD, Ena JM, Calvo M: Kinetic parameters for denaturation of bovine milk lactoferrin. J Food Sci 1992; 57: 873–879.
35 - Abe H, Saito H, Miyakawa H, Tamura Y, Shimamura S, Nagao E, Tomita M: Heat stability of bovine lactoferrin at acidic p H. J Dairy Sci 1991; 74: 65–71.
36 - Saito H, Miyakawa H, Tamura Y, Shimamura S, Tomita M: Potent bactericidal activity of bovine lactoferrin hydrolysate produced by heat treatment at acidic p H. J Dairy Sci 1991; 74: 3724–3730.
37 - Kawakami H, Tanaka M, Tatsumi K, Dosako S: Effect of ionic strength and p H on the thermostability of lactoferrin. Int Diary J 1992; 2: 287–298.
38 - Vorland LH, Ulvaten H, Andersen J, Haukland HH, Rekdal Q, Svendsen JS, Gutteberg TJ: Antibacterial effects of lactoferricin B. Scand J Infect Dis 1999; 31: 179–184.
39 - Law A J, Leaver J: Effect of pH on the thermal denaturation of whey proteins in milk. J Agric Food Chem 2000; 48: 672–679.
40 - Saito H, Takase M, Tamura Y, Shimamura S, Tomita. M: Physicochemical and antibacterial properties of lactoferrin and its hydrolysate produced by heat treatment at acidic p H. Adv Exp Med Biol 1994; 357: 219–226.
41 - Suzuki YA, Kelleher SL, Yalda D, Wu L, Huang J, Huang N, Lönnerdal BO: Expression, characterization, and biologic activity of recombinant human lactoferrin in rice. J Pediatr Gastroenterol Nutr 2003; 36: 190–199.
42 - Naidu AS, Miedzobrodzki J, Andersson M, Nilsson LE, Forsgren A, Watts JL: Bovine lactoferrin binding to sex coagulase- negative Staphylococci isolated from bovine intramammary infections. J Clin Microbiol 1990; 28: 2313–2319.
43 - Griffith E, Humphreys J: Bacteriostatic effect of human milk and bovine colostrums on Escherichia coli: Importance of bicarbonate. Infect Immun 1977; 15: 396–401.
44 - Reiter B: Review of the progress of dairy science: Antimicrobial systems in milk. J Dairy Res 1978; 45: 131–147.
45 - Murdock CA, Mathews KR: Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA. J Appl Microbiol 2002; 93: 850–856.
46 - Troost FJ, Steijns J, Saris WHM, Brummer RJM: Gastric digestion of bovine lactoferrin in vivo in adults. J Nutr 2001; 131: 2101–2104.
47 - Nagasako Y, Shimamuro S, Tomita M, Saito H, Tamura Y: Iron-binding properties of bovine lactoferrin in iron rich solution. J Dairy Sci 1993; 76: 1876–1881.
48 - Reiter B, Brock JH, Steel ED: Inhibition of Escherichia coli by bovine colostrum and postcolostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Immunology 1975; 28: 83–95.
49 - Salanlah AA, al-Obaidi AS: Effect of some physical and chemical factors on the bactericidal activity of human lactoferrin and transferrin against Yersinia pseudotuberculosis. N Miaobiol 1995; 18: 275–281.
50 - Branen J, Davidson PM: Activity of hydrolysed lactoferrin against foodborne pathogenic bacteria in growth media: The effect of EDTA. Lett Appl Microbiol 2000; 30: 233–237.
51 - Brock JH, Pineiro A, Lampreave F: The effect of trypsin and chymotrypsin on the antibacterial activity of complement, antibodies and lactoferrin and transferrin m bovine colostrum. Ann Vet Res 1978; 9: 287–294.
52 - Wakabayashi H, Abe S, Okutomi T, Tansho S, Kawase K, Yamaguchi H: Cooperative anti-Candida effects of lactoferrin or its peptides in combination with azole antifungal agents. Microbiol Immunol 1996; 40: 821–825.
53 - Chimura T, Hirayama T, Nakahara M: In vitro antimicrobial activities of lactoferrin, its concomitant use with cefpodoxime proxetil and clinical effect of cefpodoxime proxetil. Jpn J Antibiot 1993; 46: 482–485.
54 - Soukka T, Lumikari M, Tenovuo J: Combined inhibitory effect of lactoferrin and lactoperoxidase system on the viability of Streptococcus mutans, serotype c. Scand J Dental Res 1991; 99: 390–396.
55 - Denisova II, Gennadeva TI, Zhdanova, TV, Khristova M, Khazenson LB: The action of lactoperoxidase, lactoferrin and lactoglobulin on Shigella sonnei in an in vivo experiment. Z Mikrobiol Epidemiol Immunobiol 1996; 1: 66–69.
56 - López-Expósito I, Recio I: Protective effect of milk peptides: antibacterial and antitumor properties. Adv Exp Med Biol 2008; 606: 271–293.
57 - Pakkanen R, Aalto J: Growth factors and antimicrobial factors of bovine colostrum. Int Diary J 1997; 7: 285–297.
58 - Crosa JH: Genetics and molecular biology of siderophore-mediated iron transport in bacteria. Microbiol Mol Biol Rev 1989; 53: 517–530.
59 - Bishop JG, Schanbacher FL, Fergnson LC, Smith KL: In vitro growth inhibition of mastitis- causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo lactoferrin. Infect Immun 1976; 14: 911–918.
60 - Arnold RR, Russell JE, Champion WJ, Gauthier JJ: Bactericidal activity of human lactoferrin: influence of physical conditions and metabolic state of the target microorganism. Infect Immun 1981; 32: 655–660.
61 - Chapple DS, Mason DJ, Joannou CL, Odell EW, Gant V, Evans RW: Structure-function relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli serotype O111. Infect Immun 1998; 66: 2434–2440.
62 - Epand RM, Vogel HJ: Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta 1999; 1462: 11–28.
63 - Ulvatne H, Haukland HH, Olsvik O, Vorland LH: Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposome. FEBS Lett 2001; 492: 62–65.
64 - Hoek KS, Milne JM, Grieve PA, Dionysius DA, Smith R: Antibacterial activity of bovine lactoferricin-derived peptides. Antimicrob Agents Chemother 1997; 41: 54–59.
65 - Van der Kraan MIA, Groenink J, Nazmi K, Veerman ECI, Bolscher JGM, Amerongen AVN. Lactoferrampin: A novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides. 2004; 25: 177–183.
66 - Morten BS, Haug BE, Rekdal Q, Skar ML, Stensen W, Svendsen JS: Important structural features of 15-residue lactoferricin derivatives and methods for improvement of antimicrobial activity. Biochem Cell Biol 2002; 80: 65–74.
67 - SchifferM, Chang CH, Stevens FJ: The functions of tryptophan residues in membrane proteins. Protein Eng 1992; 5: 213–214.
68 - Wimley WC, White SH: Determining the membrane topology of peptides by fluorescence quenching. Biochemistry 2000; 39: 161–170.
69 - Schibli DJ, Hwang PM, Vogel HJ: The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles. FEBS Lett 1999; 446: 213–217.
70 - Hong J, Oren Z, Shai Y: Structure and organization of hemolytic and nonhemolytic diastereomers of antimicrobial peptides in membranes. Biochemistry 1999; 38: 16963–16973.
71 - Shai Y: Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell nonselective membrane-lytic peptides. Biochim Biophys Acta 1999; 1462: 55–70.
72 - Stark M, Liu L, Deber CM: cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents Chemother 2002; 46: 3585–3590.
73 - Elbarbary HA. Emerging opportunities of natural antimicrobials in the dairy industry (Ph.D. thesis). Egypt: Faculty of Veterinary Medicine, Benha University; 2009.
74 - Izhar M, Nuchanlowitz Y, Mlrelman D: Adherence of Shigella flexneri to guinea pig intestinal cells is mediated by a mucosal adhesin. Infect Immun 1982; 35: 1110–1118.
75 - Teraguchi S, Shin K, Fukuwatari Y, Shimanlura S: Glycans of bovine lactoferrin function as receptors for the type 1 fimbrial lectin of Escherichia coli. Infect Immun 1996; 64: 1075–1077.
76 - Visca P, Berlutti F, Vittorioso P, Dalmastri C, Thaller MC, Valenti P: Growth and adsorption of Streptococcus mutans 6715-13 to hydroxyapatite in the presence of lactoferrin. Med Microbiol Immunol (Berl) 1989; 178: 69–79.
77 - Alugupalli KR, Kalfas S, Edwardsson S, Forsgren A, Arnold RR, Naidu AS: Effect of lactoferrin on interaction of Prevotella intermedia with plasma and subepithelial matrix proteins. Oral Microbiol Immunol 1994; 9: 174–179.
78 - Alugupalli KR, Kalfas S: Characterization of the lactoferrin-dependent inhibition of the adhesion of actinomycetemcomitan, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane. APMIS 1997; 105: 680–688.
79 - Shin K, Yamauchi K, Teraguchi S, Hayasawa H, Tomita M, Otsuka Y, Yamazaki S: Antibacterial activity of bovine lactoferrin and its peptides against enterohemorrhagic Escherichia coli O157:H7. Lett Appl Microbiol 1998; 26: 407–411.
80 - Venkitanarayanan KS, Zhao T, Doyle MP: Antibacterial effect of lactoferrin B on Escherichia coli O157:H7 in ground beef. J Food Protect 1999; 62: 747–750.
81 - Food and Drug Administration (FDA). Center for Food Safety and Applied Nutrition. Agency Response Letter. GRAS Notice no. GRN 000067. 2001.
82 - Naidu AS: Activated lactoferrin—A new approach to meat safety. Food Technol 2002; 56: 40–45.
83 - Champagne CP, Laing RR, Roy D, Mafu AA: Psychrotrophs in dairy products: Their effects and their control. Crit Rev Food Sci Nutr 1994; 34: 1–30.
84 - Hugunin A. Whey Products in Yoghurt and Fermented Dairy Products. U.S. Dairy Export Council: California, USA; 1999. pp. 1–8.
85 - Chen HY, Allen JC: Human milk antibacterial factors: the effect of temperature on defense systems. Adv Exp Med Biol 2001; 501: 341–348.
86 - Medina I, Tombo I, Satué-Gracia MT, German JB, Frankel EN: Effects of natural phenolic compounds on the antioxidant activity of lactoferrin in liposomes and oil-in-water emulsions. J Agric Food Chem 2002; 50: 2392–2399.
87 - Masschalck B, van Houdt R, Michiels CW: High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin. Int J Food Microbiol 2001; 64: 325–332.
88 - Ransom JR, Sofos JN, Belk KE, Scanga JA, Hossner KL, Childs KD, Yemm RS, Smith GC. Susceptibility of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes, inoculated onto beef tissues, steaks and RTE products, to lactic acid, lactoferrin, and activated lactoferrin. Final Report to National Cattlemen’s Beef Association, Centennial, CO. Center for Red Meat Safety, Colorado State University: Fort Collins, CO; 2003. pp. 1–41.
89 - Brock JH: The physiology of lactoferrin. Biochem Cell Biol 2002; 80: 1–6.
90 - Van Hooijdonk ACM, Kussendrager KD, Steijnsa JM: In vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence. Br J Nutr 2000; 84: 127–134.
91 - Kuwata H, Yip TT, Yamauchi K, Teraguchi S, Hayasawa H, Tomita M, et al.: The survival of ingested lactoferrin in the gastrointestinal tract of adult mice. Biochem J 1998; 334: 321–323.
92 - Roberts AK, Chierici R, Hill MJ, Volpata S, Vigi V: Supplementation of an adapted formula with bovine lactoferrin: I. Effect on the infant fecal flora. Acta Paediatr 1992; 81: 119–124.
93 - Kawaguchi S, Hayashi T, Masano J, Okuyama K, Suzuki T, Kawase K: A study concerning the effect of lactoferrin enriched infant formula on low birth weight infants with special reference to changes in intestinal flora. Perinat Med 1989; 19: 557–562 (in Japanese).
94 - Teraguchi S, Slun K, Ogata T, Kingaku M, Kaino A, Miyauchi H, Fukuwatari Y, Shimamura S: Orally administered bovine lactoferrin inhibits bacterial translocation in mice fed bovine milk. Appl Environ Microbiol 1995; 61:4131–4134.
95 - Teraguchi S, Shin K, Ozawa K, Nakamura S, Fukuwatari Y, Tsuyuki S, Namihira H, Shimamura S: Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk. Appl Environ Microbiol 1995; 61: 501–506.
96 - Dial EJ, Lichtenberger LM: Effect of lactoferrin on Helicobacter felis induced gastritis. Biochem Cell Biol 2002; 80: 113–117.
97 - Appelmelk BJ, An YQ, Geerts M, Thijs BG, De Boer HA, Maclaren DM, De Graaf J, Nuijens JH: Lactoferrin is a lipid A-binding protein. Infect Immun 1994; 62: 2628–2632.
98 - Wada T, Aiba Y, Shimizu K, Takagi A, Miwa T, Koga Y: The therapeutic effect of bovine lactoferrin in the host infected with Helicobacter pylori. Scand J Gastroenterol 1999; 34: 238–243.
99 - Shimtzu N, Ikehara Y, Nozaki K, Kaminishi M, Tsuda H, Sugiyama A, Katsuyama T, Tatematsu, M. Effects of lactoferrin administration in Helicobacter pylori infection animal model. In: Lactoferrin: structure, function and applications. Shimazaki K, Tsuda H, Tomita M, Kuwata T, Perraudin JP, editors. Elsevier Science Publishers B.V.: Amsterdam, The Netherlands; 2000. pp. 209–215.
100 - Losnedahl KJ, Wang H, Aslam M, Sixiang Z, Hurely WL. Antimicrobial proteins in milk. Illinois Dairy Report, Department of Animal Sciences, University of Illinois; 1996.
101 - Van der Strate BWA, Beljaars L, Molema G, Harmsen MC, Meijer DKF: Antiviral activities of lactoferrin. Antiviral Res 2001; 52: 225–239.
102 - Superti F, Ammendolia MG, Valenti P, Seganti L: Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29. Med Microbiol Immunol (Berl) 1997; 186: 83–91.
103 - Berkhout B, van Wamel JLB, Beljaars L, Meijer DKF, Visser S, Floris R: Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides. Antiviral Res 2002; 55: 341–355.
104 - Swart PJ, Kuipers ME, Smit C, Pauwels R, DE Bethune MP, De Clercq E, Huisman H, Meijer DKF: Antiviral effects of milk proteins: acylation results in polyanionic compounds with potent activity against human immunodeficiency virus type1 and 2 in vitro. AIDS Res Hum Retroviruses 1996; 12: 769–775.
105 - Groot F, Geijtenbeek TBH, Sanders RW, Baldwin CE, Sanchez-Hernandez M, Floris R, Van Kooyk Y, de Jong E, Berkhout B: Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN-gp 120 interaction. J Virol 2005; 79: 3009–3015.
106 - Tanaka K, Ikeda M, Nozaki A, Kato N, Tsuda H, Saito S, Sekihara H. Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn J Cancer Res 1999; 90: 367–371.
107 - Iwasa M, Kaito M, Ikoma J, Takeo M, Imoto, I, Adachi Y, et al: Lactoferrin inhibits hepatitis C virus viremia in chronic hepatitis C patients with high viral loads and HGV genotype 1b. Am J Gastroenterol 2002; 97: 766–767.
108 - Fujihara T, Hayashi K: Lactoferrin inhibits herpes simplex virus type-1 (HSV-1) infection to mouse cornea. Arch Virol 1995; 140: 1469–1472.
109 - Shimizu K, Matsuzawa H, Okada K, Tazume S, Dosako S, Kawasaki Y, Hashimoto K, Koga Y: Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell dependent augmentation of natural killer cell activity. Arch Virol 1996; 141: 1875–1889.
110 - Pietrantoni A, Di Biase AM, Tinari A, Marchetti M, Valenti P, Seganti L, Superti F: Bovine lactoferrin inhibits adenovirus infection by interacting with viral structural polypeptides. Antimicrob Agents Chemother 2002; 47: 2688–2691.
111 - Nikawa H, Samaranayake LP, Tenovuo J, Pang KM, Hamada T: The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei. Arch Oral Biol 1993; 38: 1057–1063.
112 - Samaranayake LP. In: Host factors and oral candidosis. Mac Farlane TW, editor. Wright: London, United Kingdom; 1990. pp. 66–103.
113 - Kuipers ME, de Vries HG, Eikelboom MC, Meijer DKF, Swar PJ: Synergistic fungistatic effects of lactoferrin in combination with antifungal drugs against clinical Candida isolates. Antimicrob Agents Chemother 1999; 43: 2635–3641.
114 - Takakura N, Wakabayashi H, Ishibashi H, Teraguchi S, Tamura Y, Yamaguchi H, Abe S: Oral lactoferrin treatment of experimental oral candidasis in mice. Antimicrob Agents Chemother 2003; 47: 2619–2623.
115 - Masci JR: Complete response of sever, refractory oral candidiasis to mouthwash containing lactoferrin and lysozyme. AIDS 2000; 14: 2403–2404.
116 - Sato R, Inanami O, Tanaka Y, Takase M, Naito Y: Oral administration of bovine lactoferrin for treatment of intractable stomatitis in feline immunodeficiency virus (FIV)-positive and FIV-negative cats. Am J Vet Res 1996; 57: 1443–1446.
117 - Omata Y, Satake M, Maeda R, Saito A, Shimazaki K, Yamauchi K, Uzuka Y, Tanabe S, Sarashina T, Mikami T: Reduction of the infectivity of Toxoplasma gondii and Eimeria stiedai sporozoites by treatment with bovine lactoferricin. J Vet Med Sci 2002; 63: 187–190.
118 - Isamida T, Tanaka T, Omata Y, Yamauchi K, Shimazaki K, Saito A: Protective effect of lactoferricin against Toxoplasma gondii infection in mice. J Vet Med Sci 1998; 60: 241–244.
119 - Shakibaei M, Frevert U: Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparin sulfate proteoglycans. J Exp Med 1996; 184: 1699–1711.
120 - Cirioni O, Giacometti A, Barchiesi F, Scalise G: Inhibition of growth of pneumocystis carinii by lactiferrins alone and in combination with pyrimethamine, clarithromycin and minocyclin. J Antimicrob Chemother 2000; 46: 577–582.
121 - Tachezy J, Kulda J, Bahnikova I, Suchan P, Razga J, Schrevet J: Trichomonas foetus: iron acquisition from lactoferrin and transferring. Exp Parasitol 1996; 83: 216–228.
122 - Iyer S, Lönnerdal B: Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr 1993; 47: 232–241.
123 - Davidson LA, Lönnerdal B: Fe-saturation and proteolysis of human lactoferrin: Effect on brush-border receptor-mediated uptake of Fe and Mn. Am J Physiol 1989; 257: G930–G934.
124 - Lönnerdal B, Bryant A: Absorption of iron from recombinant human lactoferrin in young US women. Am J Clin Nutr 2006; 83: 2305–2309.
125 - Tsuda H, Sekine K, Fujita K, Ligo M: Cancer prevention by bovine lactoferrin and underlying mechanisms—A review of experimental and clinical studies. Biochem Cell Biol 2002; 80: 131–136.
126 - Chandra Mohan KV, Kumaraguruparan R, Prathiba D, Nagini S: Modulation of xenobiotic-metabolizing enzymes and redox status during chemoprevention of hamster buccal carcinogenesis by bovine lactoferrin. Nutrition 2006; 22: 940–946.
127 - Lönnerdal B, Iyer S: Lactoferrin: molecular structure and biological function. Annu Rev Nutr 1995; 15: 93–110.
128 - Actor JK, Hwang SA, Kruzel ML: Lactoferrin as a natural immune modulator. Curr Pharm Des 2009; 15: 1956–1973.
129 - Zimecki M, Artym J, Chodaczek G, Kocieba M, Kruzel ML: Protective effect of lactoferrin in Escherichia coli-induced bacteremia in mice: Relationship to reduced serum TNF alpha level and increased turnover of neutrophils. Inflamm Res 2004; 53: 292–296.