Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
• • A—HUMAN NECESSITIES
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  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
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  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/16—Otologicals
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
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  • A61P35/00—Antineoplastic agents
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2300/00—Processes
  • A23V2300/30—Ion-exchange
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2300/00—Processes
  • A23V2300/34—Membrane process
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to the quality of the lactoferrin (Lf) to reach an optimal of all its activities and avoid any secondary effects
• lactoferrin Since its first identification as a "red protein" in bovine milk more than 65 years ago, and its purification in 1960, lactoferrin has pointed and puzzled researchers. Subsequent determination of its amino acid sequence, three dimensional structure and detailed iron binding properties firmly established lactoferrin is a glycoprotein, as a member of the transferrin family, and reinforced the natural presumption that its biological function related to iron binding. [0003] Different research centers have played an important role stressing on some biological key functions of the protein. Lactoferrin was isolated as a major component in the specific granules of the polymorphonuclear leukocytes with an important role in the amplification of the inflammatory response.
• the Lf is extracted from milk or whey in presence of other Milk Basic Proteins (MBP) such as lactoperoxidase, some immunoglobulins and other contaminants of which the concentration is dependent of the specificity of the cationic ion exchange resin.
• MBP Milk Basic Proteins
• the elution of the different components bound on the resin will be performed using solutions containing different NaCI concentrations. Using such procedures, the industrial producers consider that a purity between 90 to 92% correspond to a Lf enough pure to be used for the different applications.
• This molecule is responsible to the creation of the blood vessel to feed the cancer cells, neo-vascularization indispensable to the growth of tumors and to the development of the metastasis. During the purification of the Lf, this molecule has been concentrated at least 4 times what is certainly not beneficial for the health of the consumers.
• Angiogenin contributes to an inflammatory process that allows the transmigration of endothelial and smooth muscle cells through basement membrane to enter a site of injury. Angiogenin promote the neovascular of the tumor cells and promote the proliferation of the metastasis of the cancer cells.
• Angiogenin is a protein of 15 kDa with an isoelectric pH of 9,5 that means very close to Lf. As decribed by Strydom et al., in 1997 (Eur. J. Biochem, 247, 535-544, angiogenin was applied to a CM-52 (cation-exchange chromatography resin) and was eluted with IM NaCI in 50 mM sodium phosphate, pH 6,6 solution. So it is not surprising that this molecule is co-purified with Lf and has been detected in the SDS-PAGE gel in presence of all the commercial Lf.
• lactoferrin Although originally identified as an abundant protein in milk secretion, lactoferrin is expressed predominantly by surface epithelia and secreted into the mucosal environment. As described, lactoferrin is produced at high levels not only in the milk but also in nasal and tracheal passages and in gastric, genital, and ophthalmic secretions. Lactoferrin is also produced at high levels in neutrophils where it is stored in secondary granules and released during inflammation and contribute to their antimicrobial activity.
• Lactoferrin contains 2 homologous iron binding domains that sequester available iron and can deprive iron-requiring domains bacteria of this essential growth element. In this manner, the protein exerts a bacteriostatic effect against a large range of microorganisms and certain yeast. Moreover, lactoferrin, by the presence of its cationic peptide located close to the amino-terminus of the protein, has shown to possess both bactericidal and anti-endotoxin activities that are independent of the iron binding function of the protein. This region acts by disrupting bacterial membranes and by binding and inactivating bacterial lipopolysaccharides containing the lipid-A called also endotoxins (see figure 1)
• Lactoferrin is also able to regulate cellular signaling pathways, which affect activities such as its alleviation of inflammation, promotion of bone growth and suppression of carcinogenesis.
• its anti-inflammatory activity is linked to an ability to inhibit the production of pro-inflammatory cytokines, but by several distinct mechanisms, and its regulation of bone growth that occurs through mitogen-activated protein kinase pathways.
• lactoferrin possesses anti-cancer properties, inhibiting the growth of cancer, that stem from its ability to modulate pathways that impinge on the cell cycle or result in upregulation of the expression of cytokines as interleukin-18.
• lactoferrin Moreover the antimicrobial functionality of lactoferrin is dependent on its protein conformational, metal binding and milieu conditions (Naidu AS and Arnold RR., 1995, Lactoferrin interactions and biological Functions pp 259-275 Totowa, NJ, Humana Press). Antimicrobial activity is enhanced when lactoferrin binds to the microbial surfaces.
• the specific lactoferrin binding microbial targets have been identified on different Gram-positive and Gram-negative bacterial pathogens (Naidu SS et al., 1991, APMIS, 99, 1142-1150).
• lactoferrin-mediated outer-membrane damage in Gram-negative bacteria and the lactoferrin-induced antibiotic potentiation by causing altered outer membrane permeation are typical examples of such antimicrobial outcomes (Naidu et al., Diagn Microbiol Infect, 1988, Infect Immun, 56, 2774-2781).
• Lactoferrin interaction with the microbial surface, the outer membranes in particular, has led to other antimicrobial mechanisms such as microbial adhesion-blockage to intestinal epithelia and specific detachment of pathogens from gut mucosa.
• Specific binding of lactoferrin could instantly collapse bacterial outer membrane barrier function and leads to the shutdown of pathogen colonization factors and enterotoxin production.
• Lf On its N-terminal (lactoferricin peptide) Lf binds to lipid-A, the toxic moiety of LPS with high affinity and works as a therapeutic agent to neutralize effects of LPS and endotoxins (Appelmelk BJ et al, 1994, Infect Immun, 62, 2628-2632). Lf could effectively reduce LPS and endotoxin influx into the bloodstream while toxins still are inside the intestinal lumen but to reach such result, it is important that Lf is manufactured free of LPS and endotoxin. Moreover, if the Lf feeds by the healthy person is covered by LPS, these LPS could be removed from the molecule and be transferred into the bloodstream.
• Lf is also depleted rapidly and may not be present in sufficient amounts to perform this function if LPS and endotoxins are continuously released in large quantities (Caccavo et al, 2002, J. Endotoxin Res, 8, 403-417).
• a protective effect for Lf against lethal shock induced by intravenously administrated endotoxin has been reported.
• Lf-mediated protection against endotoxin (if the molecule is itself free of endotoxin during its production) challenge correlates with both-resistance to induction to hypothermia and an overall increase in wellness.
• Lf also plays an important role in the intestinal absorption of iron and other trace essential elements such as zinc, copper (Lonnerdal B., 1989, J. Nutr. Suppl, 119, 1839-1844). Lf also protects the gut mucosa from excess uptake of heavy metal ions. Specific Lf binding receptors in the human duodenal brush border are involved in the iron absorption (Cox et al 1979, Biochem Biophy Acta, 588, 120-128). An intestinal Lf receptor was identified. Increased iron absorption via this Lf receptor from the intestinal brush-border membranes have been reported (Kawakami H et al., 1991, AmJ.
• Lf Lf is shown to enhance natural killer (NK) activity of monocytes in a dose-dependent manner. Lf strongly increases both NK and lymphokine-activated killer (LAK) cell cytotoxic functions.
• Lf is an effective modulator of cell-mediated immune response and serum cytotoxic factors at low dosages if the LPS are not bound on Lf structure and if Lf is not contaminated by the angiogenin.
• the Lf-mediated induction could lead to a positive or negative feedback according not necessary to the density and subsets of the immune cell population but also to the presence of the LPS on the Lf structure.
• the anti-inflammatory activity of the Lf is primarily associated with its ability to scavenge iron. It is known that accumulation of iron in inflammated tissues could lead to catalytic production of highly toxic free radicals. During an inflammatory response, neutrophils migrate to the challenge site to release their Lf containing acidic granules. This results in the creation of a strong acidic milieu at the inflamed tissue site to amplify iron-sequestering and detoxification capacities of Lf. Besides modulating iron homeostasis during inflammation, there is mounting evidence that Lf could directly regulate various inflammatory responses.
• Lf Lf binding to bacterial LPS, which is major pro-inflammatory mediator during bacterial infections and septic shock (Miyazawa et al., 1991, J. Immunol, 146, 723-729).
• Lf could play an important role in the modulation of gastric inflammation, since this protein is also expressed in the gastric mucosa of the stomach and interacts with receptors localized on gastric intestinal epithelial cells. This activity of the Lf is completely decreased or even eliminated when LPS cover the Lf structure.
• Several in vivo studies have shown that oral administration of Lf could reduce gastric induced by Helicobacter pillory and protect gut mucosal integrity during endotoxemia.
• the iron-independent activity of the Lf can be described as follows:
• One of the central proinflammatory functions of endothelial cells is the recruitment of circulating leukocytes at inflammatory tissue sites.
• Lipolysaccharides (LPS) or endotoxins is a predominant glycolipid in the outer membrane of Gram negative bacteria.
• the LPS are potent stimulators of inflammation that induce either directly or through the intermediary of cytokines, the expression of adhesions molecules such as endothelial-leukocyte adhesion molecule (E- selectin) and intracellular adhesion molecule 1 ICAM-I).
• Endotoxin stimulation of endothelial cells is mediated by soluble protein found CD14 (sCD14), a specific receptor.
• CD14 is a 55kDa glycoprotein that exists in the serum and as an anchored protein (mCD14) on the surface of monocytes-macrophages.
• LBP LPS-binding protein
• endothelial cells by the SCD14-LPS complex or by the LPS alone causes various pathophysiological reactions including fever and hypotension, promotes leukocytes infiltration and microvascular thrombosis and contributes, during septic shock, to the pathogenesis of disseminated intravascular inflammation.
• lactoferrin found in exocrine secretions of mammals and released from granules of neutrophils during inflammation is able to modulate the activation of the cells and avoid the severe damages causing by the presence of the LPS.
• lactoferrin concentrations higher than 20 ⁇ gr/ml, can be detected in blood.
• Lactoferrin is part of a primary defense system against the inflammation. Any presence of bacteria in the organism, is going to induce the inflammation, cancer and other pathologies. This induction is going to stimulate immune responses including cytokine production, increase of expression of cell adhesion molecules, and pro-inflammatory mediator secretion by monocytes, macrophages and neutrophils, which are into specific host tissues by systemic LPS exposure. The response of the host to LPS is mediated by immune modulator molecules such as tumor necrosis factor alpha (TNF-alpha), members of the interleukins (IL) family, reactive oxygen species, and lipids. Overproduction of those mediators induces tissue damage that precedes multiple organ failure.
• TNF-alpha tumor necrosis factor alpha
• IL interleukins
• Lactoferrin prevents the LBP-mediated binding of LPS to mCD14 and decreases the release of cytokines from LPS-stimulated monocytes. Lactoferrin might also modulate the inflammatory process. Indeed, studies reported the protective function of lactoferrin against sublethal doses of LPS in mice. In conclusion, the ability of lactoferrin to bind free LPS may account, in part, for the anti-inflammatory activities of the molecule.
• milk, whey or milk serum could carry through fermentative streptococci (Streptococcus thermophilus%) and a medium with an acidic environment could selectively enrich several yeast and molds.
• fermentative streptococci Streptococcus thermophilus
• these microbial populations are commonly known to proliferate and competitively limit the growth of several probiotics.
• Lf derived from milk with a contamination of the milk pool from mastitis source could introduce the presence of LPS from gram- positive cocci including Streptococcus uberis, Staphycoccus aureus and coagulate-negative staphylococci.
• the lipopolysaccharides (LPS) in the gram-negative bacterial outer membrane typically consist of a hydrophobic domain known as lipid-A (or endotoxin), a non-repeating core oligosaccharide, and a distal polysaccharide (or O-antigen) (Erridge et al., 2002, Microbes Infect, 4, 837-851).
• LPS and endotoxins could stimulate the induction of cytokines and other mediators of inflammation, which in turn could trigger a broad range of adverse physiological responses (Raetz et al., 2002, Annu Rev Biochem, 71, 635-700).
• Gram-negative bacterial bioburden of milk or its derivatives used in protein isolation, processing plant environment and conditions cumulative contribute to LPS and endotoxin levels in an Lf source material. It has been reviewed the potential reservoirs for endotoxin contamination during isolation of protein materials (Majde et al., 1993, Peptides 14, 629-632). Rylanders (Rylander 2002, J.
• Endotoxin Res, 8, 241-252 has also reviewed the occurrence of endotoxin level in different environmental conditions and further pointed out the risks associated with non-bacterial endotoxins, particularly 1-3- ⁇ -D-glucan from mold cell walls.
• the microbial keeping standards of chromatographic resins, sanitation practices of processing equipment even more significantly the water quality used in Lf purification could cumulatively contribute to the LPS and endotoxin levels of the purified Lf material and thereby could limit in vivo applications of commercial Lf.
• Pre-existence of Lf-LPS and endotoxin complexes reduce the potential of Lf interaction with gut epithelia and diminish its ability to control intestinal influx of LPS and endotoxins.
• the heat treatment denaturation follows a first order kinetic.
• the denaturation increases with the temperature.
• the iron-free lactoferrin (Apo-Lactoferrin) shows a more rapid denaturation than the iron- saturated lactoferrin (Holo-Lactoferrin). That reflects to a more stable conformation when it is bound to iron.
• the break of several binding provokes important changes in the Lf structure.
• the thermal stability is increasing in presence of other milk components due to the interaction between the lactoferrin and caseinates and other milk proteins.
• the lactoferrin that is extracted from milk has an iron saturation level between 9 to 20% of the iron-saturated lactoferrin.
• the protein which is extracted has not the same activity level and not the same values compared to the lactoferrin, which has been extracted before any heat treatment of the milk or the cheese whey.
• the heat treatment is able to destroy the glycan chains of the molecule which are important to protect the lactoferrin against proteolytic enzymes that are present in the stomach and is also able to produced Lf polymers. This effect has been also demonstrated by the fact that when lactoferrin is submitted to a heat treatment, the molecule has a higher absorbance power at 280 nm (Table 1).
• the Lf surface is also composed of two surfaces (surface A and surface B).
• the shoulder (surface C) is only observed in the commercial Lf.
• the shoulder or surface C has a higher absorbance power at 280 nm compared to the native Lf see Table lbelow.
• the peak A and peak B are parts of the pure Lf.
• the presence of the peak C cannot be detected with the use of the Reverse Phase chromatography.
• the absorbance of this peak C at 280 nm is almost double to the Lf one.
• Lactoferrin is usually purified from milk or whey (milk whey or cheese whey) by one or more different types of chromatography resins such as ion exchange, especially cation-exchange, affinity (immobilized heparin, single strand DNA, lysine or arginine) dye affinity and size exclusion.
• Ultrafiltration membrane can also be used to separate lactoferrin from milk or whey. Tornita and his collaborators (Tomita et al., 2002, biochem Cell Biol, 80, 109-112) have given an example of the industrial process which uses both cation-exchange chromatography and tangential-flow membrane filtration.
• He provides a method for purifying lactoferrin comprising the steps of contacting in a bind-and-elute mode and in an adsorptive fashion a solution of lactoferrin, with a hydrophilic absorbent and with a hydrophobic with the presence of surfactant, both in the presence of an excluded solute, and collecting a fraction containing lactoferrin substantially free of contaminant enzyme and/or lactoferrin inhibitor.
• He has demonstrated that compared to the purified lactoferrin using its methods, the commercial lactoferrin manufactured from the same supplier as well as other suppliers available on the market did not display the same activity.
• the purified lactoferrin did not lose its activity at higher concentration of lactoferrin in the medium (figure 4). None of the commercial lactoferrin preparations available on the market were able to display a minimum inhibition concentration and he has demonstrated that these commercial lactoferrin extracted from milk or whey have lost its growth inhibitory activity at high concentration. Nevertheless, Dr Petitclerc has concluded that such phenomenon was due to the presence of proteases or degraded peptides Lf but he has never mentioned the presence of angiogenin.
• the step b) is a step of submitting this raw material to a step of extraction on a cation exchange resin using an excluded solute concentration solution in order to obtain a solution of Lactenin (LN), (figure 5a)
• the steps of extraction or purification on a cation exchange resin are done in flow through or bind and elute mode.
• the excluded solute is sodium chloride.
• a step of concentration and diafiltration is done after the step b).
• the step c) comprises at least four steps of elution, a step to collect the contaminants, a step to collect the Lactoperoxidase, a step to collect LPS, endotoxins and angiogenin and a step to collect the Lactoferrin ( Figure 5 b).
• the steps to collect the contaminants, the Lactoperoxidase, the LPS, the endotoxins and the angiogenin are performed at a pH between 4 and 8, and preferably between 6 and 7.
• the steps to collect the Lactoferrin is performed at a pH comprised between 7 and 9.
• the solute is sodium chloride at a concentration comprised between 0,02 to 1,5 M.
• the Lactoferrin comprises less than 50 pg/mg of LPS, endotoxins and angiogenin.
• the Lactoferrin has an iron saturation level is comprised between 9% to 20 %.
• Figure 1 illustrates the localization of the LPS and lipid A
• Figure 3 illustrates the chromatogram on Reverse phase HPLC with a Lf which has not been submitted to a heat treatment and the same Lf which has been submitted to a heat treatment.
• Figure 4 Illustrates the determination of the Minimal inhibitory concentrations (MIC) using broth microdilution of bovine Lf purified on SP-Sepharose Fast Flow called Lf-NFQ compared to different commercial Lf preparations.
• MIC Minimal inhibitory concentration
• Figure 5 Illustrates a flow chart of the production of
• Figure 6. Illustrates the general diagram of the Lf purification.
• Figure 7 illustrates the measure of the iron-binding capacity by an optical density of the Lactoferrin gradually brought to complete saturation by successive addition of aliquots of a ferric iron solution.
• Figure 8 illustrates the iron binding activity of lactoferrin extracted from pasteurize milk, lactoferrin from UHT milk, lactoferrin from microfiltrated milk and lactoferrin of the invention (Lf-NFQ).
• Figure 9 illustrates the antibacterial activity. The presence of LPS on Lf structure (Lf) decreases its antibacterial activity on Escherichia coli compared to a free -LPS Lf (Lf-NFQ).
• Figure 10 illustrates the antibacterial activity.
• the presence of LPS on the Lf structure (Lf) decreased its antibacterial activity against Helicobacter pylori compared to a free-LPS Lf (Lf-NFQ)
• Figure 11 illustrates the antioxidant activity of Lf-NFQ as measured by kinetic assay and compared with Lf extracted from bovine milk (12.000 pg LPS/mg Lf) and with Lf extracted from bovine whey (30.000pg LPS/mg Lf) .
• Lf has a cationic nature according to its amino acid composition
• it can be purified by cation-exchange chromatography such carboxymethyl (CM)-Sephadex (Law et al., 1977; Yoshida et al., 2000) and this purification method is the most popular procedure for bLf purification in bLf-supplying companies.
• CM carboxymethyl
• bLf-supplying companies For example, skim milk (pH 6.7) or cheese-whey (pH 6.4) is filtered and applied to a cation-exchange chromatography column without pH adjustment.
• the material which will be eluded from a cation-exchange resin (Extraction resin) using a high NaCI concentration solution (8%), is called Lactenin (LN) or Milk Basic Protein (MBP).
• the LN is a solution containing Lactoferrin, Lactoperoxidase and some other molecules (+/- 5%).
• This LN will be microfiltrated, concentrated and diafiltrated before to be applied to another cation-exchange resin equilibrated with an acetate buffer at pH 5,5 and that we called the Purification Resin.
• the different molecules contained in the LN will be eluted by applications of different buffer solution containing different NaCI concentrations. This second chromatography is very important to obtain a lactoferrin as claimed. ( Figure 6)
• lactoferrin is concentrated by ultrafiltration and is separated from NaCl by diafiltration. Afterwards, the Lf solution will be dried at low temperature and under vacumn (freeze-dry technology) and stocked in food grade aluminium sachets.
• lactoferrin obtained by the claimed process is in the following description called Lf-NFQ
• the heat treatment denaturation follows a first order kinetic.
• the denaturation increases with the temperature.
• the iron-free lactoferrin (Apo-Lactoferrin) shows a more rapid denaturation than the iron- saturated lactoferrin (Holo-Lactoferrin). That reflects to a more stable conformation when it is bound to iron.
• the break of several binding provokes important changes in the Lf structure.
• the thermal stability is increasing in presence of other milk components due to the interaction between the lactoferrin and caseinates and other milk proteins.
• the lactoferrin that is extracted from milk has an iron saturation level between 9 to 20% of the iron-saturated lactoferrin.
• the protein which is extracted has not the same activity level and not the same values compared to the lactoferrin, which has been extracted before any heat treatment of the milk or the cheese whey.
• the heat treatment is able to destroy the glycan chains of the molecule which are important to protect the lactoferrin against proteolytic enzymes that are present in the stomach and to produce Lf polymers. This effect has been also demonstrated by the fact that when lactoferrin is submitted to a heat treatment, the molecule has a higher absorbance power at 280 nm.
• lactoferrin extracted from microfiltrated milk has been tested for its iron binding activity. Lactoferrin is gradually brought to complete saturation by successive addition of aliquots of a ferric iron solution. The rate of iron saturation is followed spectrophotometrically at 465 nm.
• a bactometer microbial Monitoring system is used to monitor the growth of probiotic either Lactobacillus or Bifidus strains by measuring impedance signals (a function of capacitance and conductance) in the cultivation media.
• a volume of 0,25 mlof Lf-NFQ followed by 0,25 ml of bacterial suspension (104 cells/ml) prepared in 0,9 % saline was added to the wells. Addition of 0,5 ml bacterial suspension serves as control.
• the inoculated modules were incubated at 32° C and impedance changes in the media was monitoring continuously by the Bactometer at 6 minute intervals for 48 hours., bacterial growth curves were graphifically displayed as percent changes of impedance signals versus incubation time. The amount of time required to cause a series of significant deviation from the baseline impedance value was defined as the detection time. If this detection time is lower than the control, the test samples was considered to elicit "prebiotic effect”. 2) Micro-scale optical density assay
• the free-LPS lactoferrin should shortened by 30 % the detection time compared to the control. Normally, the detection time for probiotic is estimated to 15h, the free-LPS lactoferrin should shortened by 4-5 h this detection time. Moreover, the multiplication of probiotic test strains is enhanced by > 100% with free-LPS lactoferrin, which is at least twice as effective as the lactoferrin covered by lipopolysaccharides
• the Lf solution should shortened by 25 to 30 % the detection time compared to the control and increase at a minium of 100 % compared to the control after 48 hours of incubation.
• Lf elicits microbial growth-inhibition by iron-deprivation stasis mechanism. Iron is critical for many life forms including intestinal pathogens to generate ATP by cytochrome-dependent electron transport system.
• This prebiotic effect by Lf in the intestinal environment is a phenomenon of natural selection to enrich beneficial probiotic flora and affect competitive exclusion of harmful pathogens by bacteriostasis.
• Prebiotic activity the commercial Lf extracted from whey or from milk has a lower prebiotic activity compared to the Lf-NFQ
• Ferric reducing/antioxidant power (FRAP BENZIE I.F.F. and STRAIN JJ. in Methods in Enzymology, VoI 299, 1990, p 15) assay as described hereafter has been used to measure the antioxidant activity of the Lf-NFQ.
• the FRAP reagent was prepared by mixing 40 ml of 0,3M acetate buffer (pH 3.6), 4 ml of 20 mM ferric chloride, and 4 ml of 10 mM TPTZ (2,4,6-tris(2-pyridyl-s-triazine).
• FRAP assay Serial solutions (0,1 to 1.0 mM) of 6-OH-2,5,7,8,-tetramethyl chroman-2-carboxylic acid were used as FRAP standards. All reagents were brought to 37° C prior to the assay. FRAP assay was performed in a 96-well microplate by mixing 20 ⁇ l of distillated water, 10 ⁇ l of Lf-NFQ sample, and 150 ⁇ l of FRAP reagent. In combination studies 10 ⁇ l distillated water and 20 ⁇ l of Lf-NFQ were mixed with 150 ⁇ l of FRAP reagent. After instant incubation at 37° C for 5 min (for ascorbic acid) and for a time lapse of 5 min to 24 hours for Lf-NFQ). The absorbance of reaction mixture was measured at 593 nm. The test compounds were given antioxidant (FRAP) scores compared to the FRAP value of ascorbic acid.
• FRAP antioxidant
• a free-LPS lactoferrin (Lf-NFQ) should reach a value of 0,660 mM in 6 hours and a peak to 0,994 mM in 24 hours ( Figure 11).
• a Lf can be considered as acceptable in reaching a value between 90 to 100 % of this value.
• LPS bacterial lipopolysaccharides
• LPS and endotoxin free Lf has a superior antioxidant activity compared to its original source, the Lf-whey or Lf-milk
• LPS-Lf is able to induce the production of Tumor Necrosis Factor (TNF- ⁇ ), Interleukin 6 (IL-6) and interleukin 8 (IL-8).
• TNF- ⁇ Tumor Necrosis Factor
• IL-6 Interleukin 6
• IL-8 interleukin 8
• Human colon carcinoma Caco-2 cells were growth as semiconfluent monolayer in Dulbecco's modified Eagle's medium supplemented with 1.2 gr of NaHCO3/litre, 2 mmol glutamine/litre, 100 U penicillin/ml, 0.1 mg of streptomycin/ml, and 20% heat inactivated fetal calf serum in a 5% CO2 incubator at 37°C. Twelve hours before infection, monolayer were washed with PBS without Ca2+ and Mg2+ and then cultured in fresh media without fetal calf serum to avoid the presence of transferrin during infection.
• coli HB101(pRI203) to kill extracellular bacteria, and cells were incubated for a further 2h at 37°C and washed extensively. Then the monolayers were treated with 0.3 ml trypsin-EDTA mixture (0.05% trypsin (1/250) and 0.02% EDTA) for 5 min at 37°C and lysed by the addition of 0.5 ml of 1% deoxycholic acid. Cell lysates were diluted in PBS without Ca2+ and Mg 2+ and plated on agar with ampicillin (100 ⁇ g/ml) to quantify the number of viable intracellular E. coli HB101(pRI203).
• IL-6, IL-8 and TNF- ⁇ were determined using standard ELISA Quantikine kits (R&D Systems, Wiesbaden, Germany) and HBT kits (Holland Biotechnology BV, Firma Bierman, Bad Nauheim, Germany).
• a significant portion of aerobic plate counts of whey- derived Lf were identified as Gram-positive microorganisms such as Penicillium spp and Aspergillus spp.
• the LPS and endotoxin levels of both commercial Lf preparations reflected their coliform and gram negative bacterial loads.
• the median LPS and endotoxin in whey- derived Lf were about 3 times higher than in the milk-derived Lf.
• the LPS and endotoxin contaminants in both Lf preparations were biologically active and induced TNF- ⁇ (Tumor Necrosis factor) production in stimulated monocytes and enterocyte cells (Caco-2 cells).
• the quantity of LPS and endotoxins which can cover the lactoferrin structure should be at a value between 50 to lOOpg/mgr of lactoferrin (figure 13) with a preference of a value ⁇ 50pg/mg Lf.
• the raw material is collected at maximum 1O 0 C in the farms having received a control safety number from the AFSCA (Federal Agency of the Security of the Food Chain).
• the raw material is skimmed at 50 0 C and does not need to be microfiltrated before to extract the LN containing Lf. Nevertheless, some milk cooperatives prefer to microfiltrate the raw material on l,4 ⁇ ceramic membranes which constitute for them a replacement of the pasteurization.
• the raw material is applied through this cation-exchange resin and the molecules under cationic ions from at pH 6,6 are bound on the resin, so this resin is allowed to extract the LN.
• the LN is a mixture mainly composed by basic proteins/enzymes. Taking into account of the color of the molecules, it is easy to observe their binding to the resine.
• Lf representing the highest isoelectric pH isoelectric
• the resin becomes red that means that the resin has almost only Lf bound on its support.
• the lactenin will be composed of 88% Lf, 10% LP and 2% contaminants.
• the LN is stocked at 4°C before to be applied to another cation-exchange resin.
• the LN solution will be applied on a cationic- exchange resin.
• the volume which will be applied will depend of the volume of the resin in relation with the concentration of lactoferrin in the LN.
• This purification resin is a Sepharose Fast Flow manufactured by Amersham.
• Lactoperoxidase eluted using a buffer sodium acetate, pH
• Lactoferrin eluted using a buffer ammonium acetate, pH 8, NaCI IM.
• lactoferrin is submitted to a final microfiltration using 0,22 ⁇ m membranes
• lactoferrin solution is freeze-dried at 45 0 C under vacuum
• the lactoferrin powder is crushed and micronisized to obtain a 80 mesh size powder.
• the lactoferrin powder is packed in food grade aluminium- polytethylene sachets.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to accelerate the maturation of the gastrointestinal tract in the newborn, or the tissue repair of the intestinal mucosa in conditions of the recovery of a gastroenterotitis.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to increase the hepatic synthesis in the new born. [000139] The invention also concerns the use of the Lactoferrin obtained by the method as above described, to enhance natural killer (NK) activity of monocytes and to increase both the NK and lymphokine-activated killer (LAK) cell cytoxicity functions. [000140] The invention also concerns the use of the Lactoferrin obtained by the method as above described, as a potential anti-tumor agent through its specific receptors on macrophages, T and B- lymphocytes and leukemia cells.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to reduce expression of some pro-inflammatory cytokines.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to inhibit or to kill bacteria or to treat diseases associated to biofilm bacteria, such diseases being cystic fibrosis or oral inflammation.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to prepare wound care solutions, wounds care solutions, ear care solutions, ointments for wound healing or eye care solutions.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, for the uptake of iron through the epithelial cells in case of Iron deficiency and Iron deficiency anemia patients and also for pregnant women.
• the invention also concerns the use of the Lactoferrin obtained by the method as above described, to treat respiratory infectious diseases (URTI and LRTI).

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