Background of the Invention All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Human milk fat Human milk fat (HMF) is composed of about 30-40g/L lipids. Of those, approximately 98% are triglycerides, 0. 3-1% phospholipids, and 0.4% cholesterol.

The phospholipids are composed of four major moieties: sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI).

Although glycerophospholipids, sphingomyelin, cholesterol and their derivatives are found in relatively small amounts in mother's milk, they play an important role in the nutrition of developing infants, and play essential roles in all physiological systems and cycles of the human body.

Total fat content increases gradually from colostrum (2.0%) through transitional (2.5% to 3.0%) to mature milk (3.5% to 4. 5%) (Bitman et al., Am. J. Cli71. Nutr.

1983, 38 : 300-312).

Triglycerides Fat, or triglycerides, is the main energy source of the newborn infant (Hamosh et al. , Pediatrics 1985,75 (suppl) : 146-150). In addition to providing 40% to 50% of the total calories in human milk or formula, triglycerides are essential to normal development since they provide fatty acids necessary for brain development, are an integral part of all cell membranes, and are the sole vehicle for fat soluble vitamin and hormones in milk. Furthermore, these energy rich lipids can be stored in the body in nearly unlimited amounts in contrast to the limited storage capacity for carbohydrates and proteins.

Mature human milk has a fat content of 3.5% to 4.5%. Triglycerides levels remain almost unchanged throughout lactation and at all times account for 98% to 99% of milk lipids.

More than 98% of the lipids in human milk is present in 11 major fatty acids from C10 : 0 to C20: 4. Medium chain fatty acids amount to 10% of the total fat in mature milk. Saturated fatty acids constitute 42% and unsaturated fatty acids account for 57% of total lipid. Essential fatty acids levels are higher in colostrum and transitional milk than in mature milk. Long-chain polyunsaturated fatty acids derived from linoleic acid (20: 2w6,20 : 2,20 : 4,22 : 5w6) and from linolenic acid (20: 5,22 : 5w3,22 : 6) show a similar trend throughout lactation. These fatty acids are important in brain development, cell proliferation, myelination, and retinal function.

The triglycerides composition of human milk fat is unique in its fatty acid composition and distribution. This fat is characterized in a total palmitic acid (C16 : 0) content of about 25%, of which about 70% are positioned at the spi-2 position of the triglycerides. Additionally, sn positions 1 and 3 are rich in unsaturated fatty acids, especially monounsaturated fatty acids, such as oleic acid (C18 : 1). This structure is of extreme importance to the infant's nutrition and development. The triglycerides are digested in the infant by lipases which release the sn-1, 3 fatty acids. When these fatty acids are released they tend to create salts of dietary calcium. This calcium is crucial for normal skeletal and bone development of the infant, as well as other aspects of normal health and development. Calcium salts of saturated fatty acids are insoluble and tend to precipitate and to be secreted from the body. This results in the loss of crucial calcium, as well as of fatty acids, which are a major source of energy. Calcium salts of unsaturated fatty acids are soluble, and can be further metabolized with no loss of calcium neither of energy, in the form of fatty acids.

The six-1, 3 positions of vegetable fats are rich in saturated fatty acids and are not appropriate to be used in infant nutrition. Hence, advanced infant formulas include structured fats produced to mimic the unique structure and characteristics of human milk fat. Such structured fats include Betapole (of Loders-Croklaan) which provides 22% total palmitic acid of which 43% are at the sn-2 position. Another advanced fat is provided in the form of InFat (of Enzymotec Ltd. ), which can provide an even superior solution by a fat with a total of 25% palmitic acid, up to 68% of which are at the sn-2 position.

The fat in milk is contained within membrane enclosed milk fat globules. The core of the globules consists of triglycerides (98% to 99% of total milk fat), whereas the globule membrane is composed mainly of phospholipids, cholesterol, and proteins.

Glycerophospholipids The role of phospholipids, and especially the role of the phospholipid backbone, in human breast milk is poorly understood. Most scientific research on human breast milk phospholipids in fact uses them as an assay to the intake and incorporation of different fatty acids on the phospholipid skeleton.

It is known that phospholipids are involved in the micro-structure of human breast milk. Phospholipids are involved in the structure of human milk fat globule membrane (HMFGM), representing 23% of the membrane mass.

Interestingly, in contrast to the significant changes in fatty acid composition from woman to woman, related mainly to race and diet, the phospholipid composition remains constant, and is not influenced by diet. The level of phospholipids in human breast milk changes with the age of the infant. This further suggests that phospholipids are an essential nutritional component of human breast milk.

Phospholipids show a decrease from high levels in colostrum (1.10%) to lower levels in mature milk (0.60%). The decline in phospholipids is consistent with an increase in the fat globule size (Ruegg et al., Biochim. Biophys. Acta 1981,666, 7-14). The phospholipids composition of breast milk from mothers of term and preterm infants during lactation was thoroughly studied by Bitman et al.

(Bitman et al., The Am. J. of Clin. Nutr. 1984,40, 1103-1119).

Milk phospholipids do not exhibit any marked differences attributable to length of gestation after day 21. This remarkable constancy in class distribution of phospholipids indicates that the composition of the membrane of the milk fat globules is identical at all stages of lactation.

The amount of phospholipids (sphingomyelin and glycerophospholipids) in human milk fat is about 15-20 mg/dL. Phosphatidylcholine (PC) is found at 28% of total polar lipids (including sphingomyelin). Phosphatidylethanolamine (PE) is found to be about 19%, phosphatidylserine (PS) at 9% and phosphatidylinositol (PI) at 6%.

No attempt has been made so far to mimic the phospholipid composition of human breast milk. Needless to say, no commercial product of such sort exists on the market.

Some phospholipids, and especially those extracted from soybean, are used as dietary supplements and a variety of health benefits are associated with their intake. These benefits include the improvement of cognitive functions, improvement of memory and concentration, maintenance of cellular membrane composition, and contribution to general well-being. Phospholipids and lecithins are a source of choline and they enhance the bio-availability of other nutrients and therapeutics.

Phospholipids are used, additionally, as food emulsifiers, anti-oxidants, stabilizers, as well as in other food application such as mold-release and anti- caking agents. Phospholipids confer unique physical properties on food products as well as personal care products. Phospholipids are used in pharmaceutical formulation as carriers and delivery systems.

Thus, it is an object of the present invention to provide a dietary supplement which guarantees the sufficient and recommended intake of phospholipids, in the form of a mimetic substitute of the phospholipids from human breast milk lipid, aimed especially for infants and young children consumption, as well as pregnant women. Other uses and objects of the invention will become clear as the description proceeds.

Sphi7igomyelit-b Sphingomyelin is the major polar lipid in milk fat. As with the glycerophospholipids, most of the sphingomyelin is a building block of the milk fat globule membrane. Its levels are about 37% of total polar lipids (15-20 mg/dL). Sphingomyelin is also an important building block required by the infant for brain and other tissues development and plays an important role in many biochemical pathways.

Sphingomyelin is currently not produced on a commercial level and is produced only from animal sources, such as bovine milk, eggs, or animal brains. Animal sources, especially those related to brain tissues, are of course not used in infant nutrition.

Cholesterol and cholesteryl esters Human milk has a high cholesterol content. Cholesterol is the major milk sterol, contributing 90% of the total sterol content. The cholesterol is also an important lipid, and most of it is located at the milk fat globules. Cholesterol is usually found at levels of 10-15 mg/dL in human milk. Maternal diet has no appreciable effects on milk cholesterol. Breastfed infants have a relatively large cholesterol intake of about 25 mg/Kg body weight relative to adults (about 4 mg/Kg body weight).

Cholesterol content is highest in colostrum (approximately 1.30% of total lipids) and decreases to lower levels in transitional and mature milks (approximately 0.5-0. 4%). It is distributed as 87% free cholesterol and 13% cholesteryl esters. As with the phospholipids, this decline is consistent with an increase in the fat globule size (see below).

During most of the lactation period, the level of cholesterol in premature milk is about 12mg/100 ml, and greater than that present in term milk. There is a close association between cholesterol and phospholipids in the membrane of the milk fat globule. About 60% of the phospholipids and 85% of the cholesterol are associated with milk fat globules.

The cholesterol esters are located mainly in the hydrophobic core of the fat globules. Unsaturated fatty acids contribute 70% w/w of fatty acids in cholesteryl esters, considerably higher than in milk triglycerides. The major fatty acids in this group are linoleic and linolenic.

Cholesterol, and its derivatives, is mainly obtained from animal sources which are not encouraged in the use in infant nutrition for a variety of health concerns.

Lipid soluble nutrients Human breast milk contains a variety of nutrients and active ingredients crucial to the normal health and development of infants. Some of these ingredients and nutrients are lipid soluble and are found along with the major lipid ingredient of human milk-the triglycerides. Some of these important lipid soluble nutrients include hormones, cholesteryl esters, retinyl esters, vitamins, carotenoids, etc.

The human milk fat globule The milk fat globule has been studied most extensively in bovine milk (Patton et <BR> <BR> <BR> al. , Biochim. Biophys. Acta 1975,415, 273-309; McPherson et al. , J. Dairy Res.

1983, 50,107-133) and in the milk of experimental animals.

Lipids occur in milk primarily as triglycerides contained within small emulsified globules with a mean diameter of 2 to 4, um (Ruegg al., Periodical Food Microstruct 1982, 1,25-47). These globules are surrounded by a structural membrane composed of phospholipids (0.2 to 1%), cholesterol, enzymes, proteins, and glycoproteins.

The core of the globules is composed mainly of triglycerides. The latter contain mostly long-chain fatty acids (90% of fatty acids in mature human milk). The core also contains cholesterol esters, retinyl esters and other lipid soluble nutrients, such as vitamins.

The membrane of the globules, composed of phospholipids and cholesterol, provide these important ingredients of milk, as well as the proteins, enzymes, and glycoproteins associated with the milk fat globules. This amphipathic surface is required for the dispersion of milk fats in the watery environment of milk and for stability of the oil-in-water emulsion.

The milk fat globule membrane is a layer, consisting of many different compounds (mainly polar and neutral lipids, proteins and enzymes), that surrounds a more or less spherical core that predominantly consists of neutral lipids. The milk fat globule membrane originates from three sublayers, namely, an innermost layer that existed on the intracellular fat droplet, and outer bilayer membrane that originated from the apical plasma membrane and a proteinaceous coat located at the inner leaflet of the bilayer membrane that presumably originated in part from the cytoplasm of the secretory cell (Evers, J. M.; Int. Dairy J. 2004,14, 747-760).

The size and distribution of fat globules in human colostrum and milk indicate the presence of three subpopulations (Ruegg et al., Biochim. Biophys. Acta 1981, 666,7-14). The diameter of the globules increases from an average of 1.5 Om in colostrum to 4. 0 um in mature milk. The latter contain almost all the milk fat but amount to only 10% to 30% of total globules. In mature milk, globules less than 1 Elm contain only a few percent of total fat but amount to 70% to 90% of the total number of globules. The milk fat globule membrane comprises about 2% of the total weight of milk fat. Its thickness, which varies in different areas, is in the range of 5 to 50 nm.

The relative constancy attained in levels of fat, phospholipids, and cholesterol after 21 days suggests that the fat droplets in mature milk have a constant core lipid/membrane lipid ratio.

The exact structure of human milk fat globules membranes is not fully understood in light of the high complexity involved in their separation and analysis. Patton and McPherson have thoroughly investigated these globules and their membranes but with no conclusive results regarding their exact structure. McPherson suggested an asymmetric structure as the inner membrane is adjacent to the core fat and the more hydrophobic components are likely to be in this region. The outer membrane surface is situated in an aqueous environment with the more hydrophilic components expected near this surface.

Function and importance The milk fat globules and its structure are responsible to the delivery of lipids and lipid-soluble nutrients, regulation of the intake of its nutrients as well as other more complex functions.

The large surface area of the milk fat globules (4.5 m2/dL) can bind various lipases, and, thereby, contributes to effective triglyceride digestion.

Fat digestion requires adequate lipase activity and bile salts levels, the former for the breakdown of triglycerides, the latter for emulsification of fat prior to and during lipolysis. In the newborn, and especially the premature infant, pancreatic lipase and bile acid levels (the major components of intestinal fat digestion) are low. The efficient fat absorption in the newborn depends on alternate mechanisms for the digestion of dietary fat. Of special importance is intragastric lipolysis, in which lingual and gastric lipases compensate for low levels of pancreatic lipase, whereas the product of lipolysis, fatty acids and monoglycerides, compensate for low bile salt levels by emulsifying the lipid mixture. Intragastric lipolysis is catalyzed by lingual lipase, an enzyme secreted from lingual serous glands, and by gastric lipase secreted from glands within the gastric mucosa. These lipases have a special function in the hydrolysis of milk ft.

Milk fat globules are resistant to the action of pancreatic lipase, but are readily hydrolyzed by lingual lipase, which penetrates into the core of the fat particles and hydrolyzes the triglycerides without disrupting the globule membrane (Patton et al., Biochim. Biophys. Acta 1982,712, 400-407). As much as 15% of core triglycerides is hydrolyzed without producing any change in the microscopic appearance of the milk fat globules.

Hydrolysis of milk fat by lingual lipase also produces relatively large amounts of monolauryl, a substance with antibacterial, antiviral, and antifungal activity, suggesting that antibacterial agents are formed in the infant's stomach during fat hydrolysis.

Initial hydrolysis of the fat within the core of the milk fat globule by lingual lipase, probably facilitates the subsequent action of pancreatic lipase and of the bile-salt-stimulated lipase of human milk. This is probably associated with alteration of the surface topography of the milk fat globule membrane during hydrolysis of its triglyceride core by lingual lipase. Free fatty acids and monoglycerides, the products of intragastric lipolysis, are relatively polar, and they can locate in the surface layer, dislocating phospholipids and proteins and thereby making the core triglyceride more accessible to pancreatic lipase and human milk bile-salt-stimulated lipase. The latter hydrolyzes milk fat at pH 7.0 to 8.0, in the presence of bile salts, thus acting in the intestine to complete the digestive process initiated in the stomach by lingual and gastric lipases.

Higher developmental scores were associated to prolonged breast feeding and milk fat content (Agostoni et al. Bioactive Components of Huma71 Milk, ed.

Newburg, Plenum Publishers, NY, 2001). This work, as well as others, states that prolonged breast feeding during the weaning process may result in a better developmental performance at 12 months, possibly due to the supply of fats contributing energy and/or affecting brain composition. The improved development is mostly attributed to polyunsaturated fatty acids, such as DHA.

However, the addition of phospholipids, sphingomyelin and cholesterol, and especially phosphatidylserine, can also contribute to enhanced development.

Furthermore, the feeding through the natural delivery system of the milk fat globule and its membrane can also be responsible, at least in part, to these developmental benefits.

Human milk provides protection against infections. Both humoral and cellular immune elements are present in milk. Inhibition of adhesion of various microorganisms to their target cells has been shown for different milk components, some known to reside in the milk fat globule membrane. Schroten et al. have shown that human milk fat globules have anti-infectious as well as nutritional properties (Schroten et al., Nutrition 1998, 14, 52-53). Secretory immunoglobulin A (sIgA), the predominant antibody fraction of human milk, represents a major protective factor against neonatal infection. It was demonstrated by Schroten et al. (Bioactive Components of Human Milk, ed.

Newburg, Plenum Publishers, NY, 2001) that sIgA is truly an integral part of human milk fat globule membrane. Furthermore, the large surface area of these membranes can assist in inhibition of bacterial adhesion. Even undigested milk fat globules, found in stools of breast fed neonates, still has the capability to inhibit bacterial adhesion. The sphingolipids and glycolipids of the fat globule membrane appear to contribute to the host defense by binding bacterial toxins.

Other studies (Ogundele, M. O. , 1998, Immunology and immunological disorders poster) have shown evidence for the central role of milk fat globule membrane in its interaction both with Complement system and the pathogenic organism, thereby contributing significantly to the antibacterial effects of human breast milk.

Summary of the Invention The invention relates to a substantially homogenous lipid preparation comprising a combination of glycerophospholipids being phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), wherein the quantitative ratio between said glycerophospholipids essentially mimics their corresponding ratio in naturally occurring human milk fat (HMF).

In a preferred embodiment, the lipid combination of the invention optionally further comprising sphingomyelin (SM) and/or cholesterol, wherein the quantitative ratio between the glycerophospholipids in said combination and the sphingomyelin and/or cholesterol essentially mimics their corresponding ratio in said naturally occurring HMF.

In a further embodiment, the lipid combination of the invention is characterized in that upon dispersion or emulsification (with other ingredients of infant formula, especially protein and fat) in an essentially aqueous medium under suitable conditions, forms a substantially homogenous dispersion or emulsion having a fat globule-containing microstructure which is essentially mimetic of the corresponding globular microstructure of naturally occurring HMF, said globules having a membrane which is essentially mimetic of naturally occurring human milk fat globule membrane (HMFGM). The said suitable conditions are at least one of temperature, pressure and physical manipulation.

In a specific embodiment, the lipid combination of the invention comprises PC at 41-50 %, PE at 20-35 %, PS at 11-19 %, and PI at 6-17 %, of the total glycerophospholipid weight thereof.

In another preferred embodiment, the lipid combination of the invention comprises PC at 44-47 %, PE at 23-26 %, PS at 13-17 %, and PI at 11-16 %, of the total glycerophospholipid weight thereof.

In a yet further specific embodiment, the combination of the invention comprises about 38% sphingomyelin of the total glycerophospholipids and sphingomyelin.

In a further specific embodiment, the lipid combination of the invention comprises about 24% sphingomyelin and about 37% cholesterol of the total polar lipids, about 15% of said cholesterol content being in the form of cholesteryl ester.

In another specific embodiment, the lipid combination of the invention comprises about 37% cholesterol, about 15% of said cholesterol content being in the form of cholesteryl ester.

The said glycerophospholipids are derived from any one of vegetal, animal or synthetic source, preferably a vegetal source, preferably said glycerol- phospholipids are derived from soybean.

In a specific embodiment, in the lipid combination of the invention the dimensions of said mimetic HMFG are similar to those of naturally occurring HMFG at any desired stage of lactation.

In the lipid combination of the invention, the content of said glycerophospholipids is preferably similar to that of naturally occurring HMFG at any desired stage of lactation. In specific embodiments, the said mimetic HMFG have an average diameter of about 1. 51lm and are essentially similar to those of naturally occurring HMFG in colostrum milk, or the said mimetic HMFG have an average diameter of about 4llm and are essentially similar to those of naturally occurring HMFG in mature milk.

In a further aspect, the lipid combination of the invention further comprises triglycerides at an amount essentially mimicking the amount of triglycerides in said naturally occurring HMF, wherein the major part of said triglycerides have a palmitoyl group at the sua-2 position of the glycerol backbone, wherein upon dispersion or emulsification of said combination, with other ingredients of infant formula, especially protein and fat, in an essentially aqueous medium, it forms a dispersion or emulsion having a microstructure which is essentially mimetic of the corresponding microstructure of naturally occurring HMF under similar conditions, said dispersion or emulsion comprising globules having a microstructure which is essentially mimetic of the microstructure of naturally occurring human milk fat globules (HMFG), said globules having a polar lipid membrane which is essentially mimetic of naturally occurring human milk fat globule membrane (HMFGM) and a core comprising said triglycerides and any other non-polar lipids.

The said polar lipid may be any one of glycerophospholipids, a mixture of glycerophospholipids with sphingomyelin, a mixture of glycerophospholipids with cholesterol and a mixture of glycerophospholipids, sphingomyelin and cholesterol.

The lipid combination of the invention may optionally further comprise cholesterol derivatives, preferably cholesteryl ester, most preferably cholesteryl ester comprising fatty acid moieties of linoleic and/or linolenic acids, at a quantitative ratio essentially mimicking the quantitative ratio of cholesterol or cholesterol derivative/s in said naturally occurring HMF, said cholesterol being contained mainly in said mimetic HMFGM and said cholesteryl ester being contained mainly in said core.

The lipid combination of the invention may optionally further comprise long- chain polyunsaturated fatty acids (LC-PUFA), preferably selected from omega-3 DHA and omega-6 ARA, said omega-3 DHA or omega-6 ARA being contained essentially in said core of said mimetic HMFG.

The said omega-3 DHA or omega-6 ARA may be esterified to the said glycerophospholipids and/or sphingomyelin comprised in the combination of the invention.

In another embodiment, the lipid combination of the invention may optionally further comprise long-chain polyunsaturated fatty acids (LC-PUFA), preferably selected from omega-3 DHA and omega-6 ARA, said omega-3 DHA or omega-6 ARA being esterified to the said glycerophospholipids and/or sphingomyelin wherein said omega-3 and/or omega-6 moieties are contained essentially in said core of said mimetic HMFGM.

Still further, in the lipid combination of the invention the bioavailability and intake of said omega-3 DHA or omega-6 ARA are essentially similar to those of omega-3 DHA or omega-6 ARA in naturally occurring HMF, and/or improved compared to their bioavailability when contained in non-globular preparations.

In another embodiment, the lipid combination of the invention is characterized in that at least part of said PS is contained in the outer leaflet of said mimetic HMFGM.

In yet a further aspect, the invention relates to a process for preparing an essentially mimetic HMF comprising HMFG and HMFGM thereof, said process comprising the step of : mixing at specific ratios, glycerophospholipids sources, preferably commercial soy lecithins, fractionated soy lecithins and soy-derived phosphatidylserine, and optionally sphingomyelin and/or cholesterol, wherein the mixture obtained forms a lipid combination which is essentially mimetic to the composition of glycerophospholipid in naturally occurring HMF.

In the process of the invention, the said glycerophospholipids sources and optionally sphingomyelin and/or cholesterol may be provided in emulsion or dispersion form, said mixing being conducted under conventional emulsification or dispersion mixing techniques. Alternatively, the said glycerophospholipids sources are provided in powder form, said mixing being conducted under conventional powder mixing techniques.

In a further embodiment, the invention relates to a process for preparing an essentially mimetic HMF comprising HMFG and HMFGM thereof, said process comprising the step of : dissolving in an organic solvent, at specific ratios, glycerophospholipids sources, preferably commercial soy lecithins, fractionated soy lecithins and soy- derived phosphatidylserine, and optionally sphingomyelin and/or cholesterol, to give a mixture which forms a lipid combination which is essentially mimetic to the composition of glycerophospholipid in naturally occurring HMF, and optionally evaporating the solvent, to give said mimetic lipid combination in solid, preferably powder, form.

In a further embodiment, the invention relates to a dietary supplement comprising a lipid combination of the invention. The said combination is comprised in the dietary supplement of the invention in emulsified or dispersed form, preferably in the form of an essentially aqueous emulsion or dispersion, or said combination is comprised in dried form, preferably spray dried form.

The invention further relates to a method for preparing a dietary supplement according to the invention, said method comprising the steps of admixing a combination of the invention with at least one of additives, emulsifiers or carriers. This method may further comprise admixing said lipid combination with an aqueous liquid medium, said dietary supplement essentially being in an aqueous liquid form. Further, this method may additionally comprise dispersing, preferably dissolving said lipid combination in an organic medium, preferably an oil conventionally used in infant formulas, particularly an oil which mimics HMF. Alternatively, the method may further comprise spray-drying said liquid dietary supplement, to provide said dietary supplement in powder form.

The invention also provides a method for preparing a dietary supplement in accordance with the invention in powder form, said method comprising the steps of dissolving all the lipid components of the combination of the invention in an organic solvent and removing said solvent.

The dietary supplements of the invention are intended for use as an ingredient of a lipid constituent of infant formulas.

The dietary supplement of the invention are particularly intended for use in the enhancement of infants and/or children development, in the enhancement of infants and/or children cognitive development, in the enhancement of fetal development and in the enhancement of infants and/or children vision development.

In a further aspect, the invention relates to a food article comprising the dietary supplement of the invention, for example infant formula.

The food article comprising the dietary supplement of the invention is further characterized in that said dietary supplement enhances the intake of nutrients contained in said food article.

The food article comprising the dietary supplement or the lipid combination of the invention is particularly characterized in having a mimetic microstructure of naturally occurring HMF.

The invention particularly relates to a dry infant formula comprising the lipid combination of the invention, wherein said formula maintains said globule- containing microstructure upon re-dispersion in an aqueous medium.

The invention also relates to a dry infant formula comprising the lipid combination of the invention, wherein said formula maintains said globule- containing microstructure upon re-dispersion in an aqueous medium, and wherein the PS contained in said formula is substantially contained in the membrane of said globules, with preferably at least part of it on the outer leaflet.

In addition, the invention relates to a ready-to-feed liquid infant formula comprising the lipid combination the invention, wherein said formula maintains said or globule-containing microstructure.

The invention also relates to a ready-to-feed liquid infant formula comprising the lipid combination of the invention, wherein said formula maintains said globule- containing microstructure upon re-dispersion in an aqueous medium, and wherein the PS contained in said formula is substantially contained in the membrane of said globules, with preferably at least part of it on the outer leaflet.

The lipid combination, dietary supplements and infant formulas of the present invention, are further useful in maintaining infant health by supporting immunological and anti-bacterial body defense and other aspects and physiological process related to the immune system.

Description of the Drawings Figure 1: Confocal laser scanning micrographs of infant formula emulsions prepared by re-dispersion of infant formula powders produced on a pilot scale with triglycerides mimicking human breast milk fat (InFat) and the glycerophospholipids composition (Test 1, upper panel) and glycerophospholipids and cholesterol and cholesterol esters composition (Test 2, lower panel) of the invention. Confocal laser microscopy was performed using dual staining as followed. Globules fat was stained with Nile Red (A, C), membranes were stained with FM 1-43 (B, D) and proteins were stained with Fast Green FCF (A-D).

Micorgraph colors: red=fat; green=membrane; blue=protein. Micrographs were taken using a Radiance 2000 BioRad Confocal Laser Scanning Microscope at X63 oil immersion objective, at excitation wavelengths of 488 nm (FM 1-43), 548 nm (Nile red) and 568 nm (Fast Green FCF). Bars = 5 p. m.

Figure 2: Confocal laser scanning transmission micrographs of reconstituted milk mimicking human breast milk. Bars represent 5 p. m. Micrographs were taken using a Radiance 2000 BioRad Confocal Laser Scanning Microscope at X63 oil immersion objective.

Figure 3: Confocal laser scanning micrographs of fats staining of reconstituted milk mimicking human breast milk. Infant formula was emulsified and fats globules were stained with Nile Red, as described in text. The staining was examined by Confocal Laser Scanning Microscope using transmitted light (panel A), at exciting wavelength of 548 nm (panel C) and as a merge micrograph (panel B). Bars represent 5 p. m.

Figure 4: Confocal laser scanning micrographs of fats and protein staining of reconstituted milk mimicking human breast milk. Infant formula was emulsified and then both the proteins and the fats fractions were stained using Fast Green FCF and Nile Red, respectively, as indicated in text. The results were observed in Confocal Laser Scanning Microscope using transmitted light (panel A) and a merge of micrograph of the dual staining at exciting wavelengths of 548 nm and 568 nm, respectively (panel B). Micrograph colors: red=fat; blue= protein. Bars represent 5) im.

Figure 5: Confocal laser scanning micrographs of membranes staining of reconstituted milk mimicking human breast milk. Infant formula was emulsified and fat globules membranes were stained with FM 1-43, as described in text.

Micrographs of Confocal Laser Scanning Microscope were taken at exciting wavelength of 488 nm for membranes staining (panel A) or with transmitted light (panel B). Bars represent 5 um.

Figure 6: Confocal laser scanning micrographs of membranes and protein staining of reconstituted milk mimicking human breast milk. Infant formula was emulsified and both fat globules membranes and formula protein fractions were stained with FM 1-43 and Fast Green FCF, respectively. Confocal Laser Scanning Microscope images of fat globules membranes (green) and protein (blue) were obtained at exciting wavelength of 488 nm (panel A) or using dual wavelengths of 488 nm and 568 nm (panel B). Bars represent 5 pLM.

Figure 7: Confocal laser scanning micrographs of membranes and/or protein staining of human mother milk or reconstituted milk mimicking mother milk.

The globules membranes and/or the protein fraction of fresh mother milk (panel A) and an emulsion containing a mixture of glycerophospholipids (panel B) or glycerophospholipids with cholesterol and cholesterol ester (panel C) were stained using FM 1-43 or Fast Green FCF, as described in text. Micrographs colors: green=membranes ; blue=protein. Note that the emulsion was prepared by re-dispersion of spray-dried powder of a semi-industrially emulsion containing the indicated lipids.

Figure 8: Confocal laser scanning micrographs of reconstituted milk mimicking human breast milk was emulsified with 1 mg of NBD-phosphatidylcholine and 1 mg of BODIPY-cholesterol ester (panel A), 1 mg of NBD-sphingomyelin and 1 mg of BODIPY-cholesterol ester (panel B) or 1 mg of NBD-cholesterol and 1 mg of BODIPY-cholesterol ester (panel C). In addition, the proteins were stained with Fast Green FCF, as described in text. Micrographs colors: green=NBD tagged lipids; red=BODIPY tagged cholesterol ester; blue=Protein. Bars represent 5 >m.

Figure 9: Confocal laser scanning micrographs of Alexa Fluor Annexin V conjugates were incubated for half an hour with fresh human mother milk (panel A), emulsion of infant formula containing the mimicking composition including glycerophospholipids, cholesterol and cholesterol esters produced on a pilot scale (panel B) or emulsion of a commercially available infant formula (panel C) at 37°C. Confocal laser scanning micrographs were obtained at exciting wavelength of 495 nm. Bars represent 5 um.

Detailed Description of Preferred Embodiments The present invention describes infant food products which include a breast milk lipid mimetic, especially a glycerophospholipid mimetic, that provides most or all of the lipids found in human breast milk and is used in the preparation and formulation of these food products.

In a first aspect, the present invention relates to a combination lipids, wherein said combination is a mimetic substitute of the lipids from human breast milk (human milk fat-HMF). These mimetic lipids include glycerophospholipids, essentially PC, PE, PS, and PI. The mimetic combination of the invention may preferably further include a triglyceride portion comprising structured or natural triglycerides that mimic the unique structure and fatty acid composition of human milk triglycerides. The mimetic combination of the invention may further include sphingomyelin, and/or cholesterol and/or cholesteryl esters, as well as other dietary ingredients.

The preparation of a mimetic lipid combination in accordance with the invention may take various routes. As mentioned, the lipids of the invention comprise some or all of various lipids, or lipids belonging to the same classes, as well as other ingredients forming mimetic human milk fat combinations. The preparation of such combinations can utilize in the process solely or partly man- made ingredients and techniques, as well as naturally occurring components, all of which are within the scope of this application Thus, man-made combinations would be a product of a method of preparing such HMF mimetic combination according to the present invention.

Preferably, said mimetic lipid combination comprises lipids which are derived from a vegetal source, most preferably from soybean. Nonetheless, lipids from animal sources are not excluded from the scope of the present invention, particularly for furnishing sphingomyelin or cholesterol, or cholesterol derivatives ingredients for the mimetic lipid combination of the invention. For example, sphingomyelin may be provided from bovine milk, and cholesterol from various animal sources.

Further, the lipids of the present invention, e. g. glycerophospholipids, sphingomyelin, cholesterol, or cholesterol esters can be accompanied with fatty acid compositions, essentially evolving from the same sources. Such compositions may further be altered to comprise only some of or different fatty acids or fatty acid moieties, for example so as to contain nutrients such as omega-3 or omega-6 fatty acids at appropriate levels (content). It is to be noted that the terms"level" and content"are used herein interchangingly.

Thus, a typical lipid combination of the invention comprises glycerophospholipids in the following proportions: PC at 41-50%, preferably 44- 47%, PE at 20-35%, preferably 23-26%, PS at 11-19%, preferably 13-17% and PI at 6-17%, preferably 11-16% (all percentages are from total glycerophospholipids). A most preferred composition of glycerophospholipids of the dietary supplement of the invention is as detailed in the following Table 1.

The lipid combination of the invention may also include sphingomyelin, at levels (proportional amounts) similar to those found in human milk. Other phospholipids may also be included, such as phospholipids found in soybean, providing additional phospholipid backbones.

The lipid combination of the invention may sometimes include phosphatidic acid (PA), particularly PA originating from the starting material In such cases, the PA can preferably or optionally be removed. The removal may be carried out at any stage of preparation, either from the final lipid combination or from the starting material lipids sources.

Table 1 exhibits a typical combination of glycerophospholipids in accordance with the invention, compared to human milk fat (HMF). The product is a pre- designed mixture of glycerophospholipids, preferably soy-derived, which creates a"close to mother's milk"glycerophospholipids combination for use in nutritional formulations. Such combination is shown in Table 1. This combination, as well as other compositions mimicking the glycerophospholipids of HMF, can be obtained by mixing commercially available glycerophospholipids as well as structuring/synthesizing glycerophospholipids by chemical or enzymatic methods.

Table 1 Human Milk Fat Invention Glycerophospholipids (% of total Combination glycerophospholipids) (% of total glycerophospholipids) PC 45.4 46 PE 30. 8 24 PS 14. 1 16 Pi 9. 7 14 The combination of Table 1 was obtained by mixing 13.8% SharpPS70P (Enzymotec Ltd.), 83% Epikuron 130P (Degussa BioActives), 3.2% Phospholipon 80 (Phospholipids GmbH). Presented weight percent of each of the four main phospholipids is a calculation of the ratio of each from the sum.

In a second aspect, the present invention provides a method for the preparation of a dietary supplement comprising a mixture of phospholipids, wherein said mixture, and the phospholipids therein, are a mimetic substitute of human breast milk phospholipids. This method comprises the step of admixing phospholipids, preferably soybean-derived, and at least one of additives, emulsifiers or carriers, wherein said phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI), in any combination thereof which mimics that of HMF.

Most of the phospholipids found in mother's milk can be obtained from vegetal sources, particularly soybean. However, phosphatidylserine (PS) is not found in soybean and is present in small amounts in the brains of different mammals, such as bovine. Evidently, bovine PS cannot be used in infant nutrition.

Enzymotec (Migdal HaEmeq, Israel) is a producer of high quality PS derived from soybean phospholipids, using natural enzymatic processes. These high quality PS products can be preferably used to achieve the final formulation of the mixture of phospholipids of the lipid combination of the invention.

An additional advantage provided by the dietary supplement of the invention is that the mimicking of the phospholipid composition of human breast milk in infant formula may have beneficial contribution to the absorption of important nutrients, such as calcium. This may be linked to the formation of the milk lipid globules in infant nutrition formulas.

Furthermore, the incorporation of phospholipids will lead to the intake of phospholipid backbones, important to the buildup of membranes in the brain and other tissues.

The phospholipids supplement will be acylated by the infant's metabolic cycles with additional necessary fatty acids from the triglyceridic portion of the formula. The phosphatidylcholine ingredient is a natural and bioavailable source of choline. Soy phospholipids, which are preferably the source for the phospholipids in the lipid combination of the invention, have high levels of long chain unsaturated fatty acids.

A proposed product mimicking human breast milk lipids includes the above proposed glycerophospholipids, optionally mixed or solubilized in a triglyceride composition mimicking the triglycerides of human breast milk. Such triglycerides can be obtained in the form of BetapolR (Loders Croklaan) or InFatTM (Enzymotec, Israel). InFat is the subject of co-owned, co-pending PCT Application, claiming priority from IL158555, fully incorporated herein by reference. The triglyceride composition can be enriched with omega-3 and omega-6 fatty acids, especially DHA and ARA.

The product of the invention can include also sphingomyelin from animal sources, plant sources, microbial sources, or synthetic sphingomyelin produced by enzymatic or chemical methods. Table 2 exhibits a typical composition of human milk polar lipids and the proposed combination of this invention.

Table 2 Polar lipids Human milk Mimetic Combination (% of total polar (% of total polar lipids) lipids) Sphingomyelin 37. 5 38. 7 PC 28 29. 9 PE 19. 5 13. 8 PS 9. 6 PI 6 8 The formulation of Table 2 is obtained by mixing 10% SharpPS70P (Enzymotec Ltd. ), 55% Epikuron 130P (Degussa BioActives), 6% Phospholipon 80 (Phospholipids GmbH), 29% Bovine Milk Sphingomyelin (Northern Lipids). Presented weight percent of each of the five main phospholipids is a calculation of the ratio of each from the sum.

The product of the invention can also include cholesterol, obtained from animal, plant, and microbial sources or a synthetic cholesterol, obtained by chemical or enzymatic manipulations. Table 3 exhibits a typical composition of human milk polar lipids including cholesterol and the proposed composition of this invention.

Table 3 Human milk lipids Human milk lipids Mimic Sphingomyelin 23. 5 24. 2 PC 17. 5 18. 7 PE 12. 1 8. 6 PS 5. 7 6 PI 3. 7 5 Cholesterol* 37. 5 37. 5 * 15% of cholesterol in form of cholesteryl esters as described in Table 3.

The formulation of Table 3 is obtained by mixing 6.25. % SharpPS70P (Enzymotec Ltd. ), 34.4% Epikuron 130P (Degussa BioActives), 3.75% Phospholipon 80 (Phospholipids GmbH), 18. 1% Bovine Milk Sphingomyelin (Northern Lipids), 31.9% Cholesterol (Sigma), 5.6% cholesteryl esters (as described in Table 4). Presented weight percent of each of the five main polar lipids is a calculation of the ratio of each from the sum.

The cholesterol of this invention can also include cholesteryl esters, preferably with a fatty acid composition mimicking the unique fatty acids composition of cholesteryl esters found in human breast milk, characterized in their high levels of unsaturated fatty acids, especially linolenic acid. The cholesteryl esters are preferably added at the same ratio to cholesterol as found in human breast milk, approximately 15% of the cholesterol.

Cholesteryl esters were prepared in the following way: 1. A mixture of vegetable oils and fats was prepared in order to yield a total fatty acid composition that in most cases is within the range of the human milk cholesteryl esters maiu fatty acids. See Table 4.

Fatty acid composition was determined by gas chromatography (GC) after converting sample to methyl esters.

2. Oil blend was transferred to methyl esters by basic hydrolysis followed by acidic methylation.

3. Methyl esters where reacted with cholesterol with sodium methoxide, at 100°C and under vacuum. Reaction end was determined by following the disappearance of free cholesterol in GC. Product was purified and cleaned from catalyst residues.

Table 4 Fatty acid % w/w in human milk % w/w in mimetic cholesteryl esters* combination C12 0. 9-3. 4 4 C14 2. 4-6. 3 2 C16 8. 7-27. 4 20 C18 7. 6-11 10 C18 : 1 28. 9-37 30 C18: 2 21-33 25 C18: 3 0.5-2 5 * ranges covering the main fatty acids weight percent found in colostrum and mature milk. (Hamosh, M. et al. id ibid.).

The lipid mimetics of this invention can be provided as one ingredient.

Alternatively, the mimetic combination may contain only part of these lipids. For example, the product of the invention may include mimetic mixtures of glycerophospholipids, mimetic mixtures of glycerophospholipids, and sphingomyelin, mimetic mixtures of glycerophospholipids and cholesterol, mimetic mixtures of glycerophospholipids, sphingomyelin and cholesterol, mimetic mixtures of sphingomyelin and cholesterol, sphingomyelin, cholesterol, etc. In all cases these lipids may be combined with HMF mimetic triglycerides.

The ingredients comprised in the mimetic combination of this invention are aimed at providing important nutrients found in breast milk, and that nutrients which are important to the nutrition, health, and development of infants.

Furthermore, these ingredients are important in facilitating the intake or improving the intake/bioavailability of other nutrients, preferably of lipid-soluble nutrients. These nutrients may include omega-3 and omega-6 fatty acids, preferably DHA and ARA, lipid soluble vitamins, preferably vitamin E and carotenoids, retinyl esters, etc. Such nutrients are currently already added to infant nutrition. Thus, the mimetic combination of this invention may also include these nutrients, and their intake may be assisted by the lipids of the invention.

The lipid ingredients of the invention can be added to formulas or baby foods at levels mimicking the levels of same ingredients found in human breast milk.

Preferably the ingredients can be used at different levels, mimicking the different levels found in human milk at different stages of lactation.

Furthermore, the lipid ingredients can be added to or used to create formulas aimed especially at preterm infants. In the latter case, the ingredients of the combination of this invention may be added at levels similar to those found in human milk of mothers of preterm infants.

The lipid combination of the invention can be produced by mixing the different sources of the lipidic constituents, either plant, such as soy, animal, such as bovine, microbial, or synthetic, dispersing them in an appropriate aqueous medium, optionally homogenizing and/or at elevated temperatures (including pressure homogenizing), and drying to obtain a homogenous mixture. Optionally the drying can be done by spray-drying or lyophilization.

The lipid ingredients of the combination of the invention can be used in producing infant formulas by using the homogenous dry mixture as described above and re-dispersing it in an aqueous mixture together with other infant formula components. The complete ingredients are then processed according to common production techniques of infant formulas, preferably pressure homogenizing at elevated temperatures, followed by spray drying. In another aspect of the invention, the ingredients of the combination of the invention can be used for infant formula production by the addition of the different sources of the lipids at the appropriate levels to infant formula mixture, dispersion in aqueous media, followed by common formula production techniques and processes. The lipids of this invention can be used to produce a variety of infant food products, preferably powder or liquid infant formulas, and follow-on formulas.

As mentioned in the Summary of the Invention, the mimetic lipid combination of the invention has a globular microstructure when dispersed or dissolved in an essentially aqueous medium, with other ingredients of infant formula, especially protein and fat. Interestingly, the fat globules created during the formula production process were maintained in the powder form of formula and also after dispersion of said powder in water at conditions mimicking the preparation of formula by end-customers prior feeding.

Final infant formulas were produced using the lipid mimic of the invention on a pilot scale representing the standard methods and equipment used in industrial infant formula production. Different compositions of the milk lipid fractions where introduced into a final infant nutrition product.

For example, 243 gr of lipid fraction and 757gr of powder consisting mainly of low minerals whey powder, low fat milk powder, lactose, minerals and vitamins were dispersed with high shear agitator at 60-70°C in water. The slurry was homogenized in a 2 stage APV Rannie type 10.50 homogenizer with pressures ranging from 100-250bar in stage 1 and 30-70 bar in stage 2.

Particle size distribution was measured for the slurry in order to characterize the fat globules size. The slurry was dried with a Niro PM spray dryer.

The dried product was analyzed for moisture, free fat content, stability after dispersion in water, particle size distribution after dispersion in water. The lipid fractions were composed of triglycerides with high content of palmitic acid at the s/t-2 position (see Table 5), phospholipids in composition as described in Table 1, optionally with cholesterol and cholesteryl esters with fatty acid composition as described in Table 6.

Table 5 Triglycerides fatty acid composition used in infant formula pilot % w/w in Fatty acid formulation C12 8 C14 3 C16 22 C16 esterified in 61% of total sn-2 sn-2 position position C18 4 C18 : 1 42 C18: 2 13 C18 : 3 1. 2 ARA 0. 9 DHA 0. 9 Table 6: Summary of different pilot trials Formula Test Test spec 1 2 lipids from total % 24. 3 24.3 product Composition of lipid fraction % Triglycerides 98. 8 98.61 % Phospholipids 0. 8 0.8 % Cholesterol 0 0. 5 % Cholesterol 0 0.09 esters Others 0. 4 0.4 Moisture after % 1. 5-4 2.7 2.6 drying Active Water after 0.2-0. 6 0.255 0.263 drying Free fat 1. 5-2.5 2.72 2.57 Stability after Stable after Pass Pass Pass dispersion 24hrs (Aroma, taste, fat float) fat globules size mean (micron) None 1.7 3.2 after dispersion of powder in water median None 1.3 2. 5 (micron) The above results show that the introduction of the unique triglyceride and glycerophospholipids composition enabled the production of a final infant formula powder within specifications and with globule size mimicking those of human milk.

It can be seen in Figure 1 that powders of infant formula produced on a pilot scale by spray drying of infant formula emulsions containing commercial infant formula ingredients as well as the lipid mimetics of the invention, re-dispersed in water, successfully mimic the human milk fat globule-containing microstructure, including the presence of a polar lipid membrane and a neutral lipid core (as compared to human milk studied by CLSM, Figures 6A and 8A).

The re-dispersed formulations of powders produced in Test 1 and 2 (Table 6) have been stained by different selective fluorescent dyes (Figure 1, and details below) and scanned by Confocal Laser Scanning Microscopy (CLSM) to exhibit their content and micro-structure. The latter was compared to the globular microstructure of fresh human milk which was similarly stained and scanned.

Comparison reveals the ability of the resulting powders to mimic the micro- structure of human milk, as well as lipid composition, with great success even upon simple re-dispersion in water. Throughout the specification and claims, the terms"globular"and"globule-containing"are used interchangingly.

The ingredients of the combination of the invention can be further used to create and mimic the milk fat globule by providing the building blocks of its membrane.

It is a further object of this invention to provide means for producing such milk fat globules, mimicking the structure and optionally the size of human milk fat globules.

The structure and size of the globules can be created, mimicking the fat globules of human milk, simply by dispersing the ingredient of this invention with other ingredient/s of infant formulas under conditions (temperature, agitation, pressure, concentration, time) that would facilitate the formation of said globules. Preferably these conditions coincide with the conditions used to produce infant formula.

One procedure for creation of milk fat globules includes a first stage of mixing the lipid fraction in water. Optionally, in order to create a stable emulsion and to enable a final composition typical to infant formulas, the powder containing (among others): whey powder, milk powder, sugars, minerals and vitamins is added as well. The mixture is then homogenized by a high shear homogenizer that can be a typical milk industry equipment (e. g. APV 2 stage) or other types such as the Microfluidizer HC-8000 with 1 pressure stage. Pressures can vary between 20 bars to 300 bars and would create milk fat globules in the 0.1-8 microns range. Order of addition can be altered in order to improve the formation of the milk fat globules membrane (MFGM). Lipid fraction can be either added with glycerophospholipids and cholesterol dissolved in triglycerides or, optionally, the membrane constituents (glycerophospholipids, cholesterol) can be added after the triglycerides so the feasibility of an MFGM formation is higher. Other MFGM ingredients in the form of powders that are to be incorporated should be added in the proper ratios and timing in order to enable a stable formulation but as to enable the polar lipids to cover the core of the fat globules. The formed globules were analyzed by different techniques in order to exhibit their structure mimicking the structure of human milk fat globules.

The HMFG plays an important role in the delivery of fat soluble nutrients, such as LC-PUFA and vitamins. It is the object of this invention to mimic this delivery which ensures optimal intake and absorption of such nutrients by infants. It has now been shown that LC-PUFAs are indeed carried in the mimicked HMFG, prepared as described above, separated and analyzed for their fatty acid composition.

The lipid ingredients of the combination of the invention were enriched with standard levels of omega-3 and omega-6 fatty acids in the form of triglycerides.

The omega-3 fatty acid is preferably DHA and the omega-6 fatty acid is preferably ARA. Globules produced with the DHA/ARA enriched additives of the invention in the presence of all infant formula ingredients were separated from the liquid formula plasma and isolated. The globules were treated with a Folch lipid extraction mixture and the lipids were analyzed for their fatty acid composition. This analysis shows that most of the DHA/ARA added to the formula has been located in the fat globules. This in turn assists in the intake and bioavailability of said Omega-3/6 nutrients.

Thus, a 100 ml serving of a fluid ready-to-feed preparation of infant food was prepared by emulsifying all ingredients of an infant formula, as described above, with 3 g of the required fats and the lipid ingredient of the invention, containing glycerophospholipids, sphingomyelin, cholesterol and cholesterol esters, constituting 0.8% of the total lipids. In addition, 135 mg solution containing 21% of DHA and 21% of ARA were supplemented to the fats portion, before the emulsification, to give a final level of 0.9% out of total fatty acids of DHA and ARA. The final fluid ready-to-feed preparation of infant food was skimmed from the fat component by three centrifugations at 6000xg for 10 minutes, in which the cream was removed after each centrifugation (Attaie et al. J Dairy Sci 2000; 83,940-944). The lipids from the collected fractions were extracted according to the method of Folch et al. 1957 (JBC 1957 226,497-509). Methyl ester of pentadecanoic acid was added as an internal standard, and the lipids were converted to methyl ester by mild alkaline hydrolysis overnight, followed by gas- liquid chromatography.

As expected, the relative area of the DHA and ARA in the GC trace was found to be 0.9% of the total fatty acids composition. This shows that these important nutrients, crucial to infant cognitive and vision development, among other benefits, are carried and delivered by the MFG, as is the case with the same nutrients, and others, in human milk. The delivery of oil soluble nutrients, such as LC-PUFA and vitamins, is also mimicked by the globules formed through the utilization of the lipid ingredient of the invention. This delivery assists in the intake and absorption of such nutrients and benefits the development of infants.

Fluorescent markers of the different components were used in order to exactly locate the position of the different additives of the invention. Fluorescent markers of cholesterol, glycerophospholipids (as phosphatidylcholine-PC), sphingomyelin, and cholesteryl esters were obtained from Molecular Probes as NBD or BODIPY tagged derivatives of the abovementioned lipids. The tagged lipids were used to enrich the different lipid additives of the invention at milligram levels, assuming that their sorting will follow that of the enriched lipids that were used to generate the milk fat globules. The markers'localization was analyzed by confocal laser scanning microscopy.

Membrane and core selective fluorescent markers were also used, such as Nile red and Fast Green dyes, as well as FM 1-43 membrane selective dye by Molecular Probes. These were added to an aqueous mixture of pre-formed fat globules and the mixture was analyzed by laser confocal scanning microscopy.

The presence oi PS, a unique glycerophospholipids added to the additives of this invention and not provided by commercial infant formulas, in the milk fat globule membrane was analyzed by using the Annexin V cellular apoptosis kit designed to locate extra-membrane PS usually presented by apoptotic cells.

The mimicking of the fat globules and their structure was elucidated and analyzed by the analysis of the globules for their lipid composition (see above) as well as by.. confocal laser scanning microscopy (CLSM), accompanied by selective staining with fluorescent dyes as well as by using fluorescent tagged analogs of the different fat globule constituents.

CLSM has already been used in the past to investigate milk fat globules, mainly of bovine milk (Evers et al., Int. Dairy Journal 2004,14, 747-760; Michalski et al. J. Dairy Sci. 2002,85 : 2451-2461).

The inventors have mimicked human breast milk fat by mixing 6 gr of lipid fraction (the different compositions as detailed in tables 1-3). The lipid fraction was dispersed in 200 ml of warm (50-60°C) deionized water using a standard crude homogenizer to obtain an emulsion. To this emulsion was added 18 grs mixture of proteins, carbohydrates and minerals commonly used in infant formulas, to fully mimic human breast milk. The mixture was further homogenized. The resulting emulsion was transferred to a medium pressure micro-fluidizer (by MicroFluidics, USA) equipped with one flow chamber, passed 1 to 5 cycles, under 250bar, to obtain a stable emulsion.

The emulsion obtained was further analyzed by a CLSM. Microscopic analysis reveals a similar structure to that of human breast milk as also presented in Michalski et al. (J. Dairy Sci. 2002, 85 : 2451-2461).

The confocal micrographs exhibit a network of protein mass and fat globules at a variety of sizes, ranging from below 1 micron to 10 microns. As with human milk the majority of the globules are very small, around 1 micron or below. These how ever hold only a small portion of the milk fat. The majority of the milk fat is contained within the larger globules.

In order to establish the composition and structure obtained in their attempts to mimic human milk in both composition and microstructure, the inventors have used advanced staining techniques for the elucidation of the different components and their structure within the formulation.

Initially it was necessary to indeed show that the globules that appear in the mimicking formulation are indeed of fat. This was carried out by a common lipid dye, Nile Red (obtained from Molecular Probes, Oregon, USA). The phenoxazine dye Nile Red is used to localize and quantitate lipids, particularly neutral lipid droplets within cells. It is selective for neutral lipids such as cholesteryl esters (and also, therefore, for lipoproteins) and is suitable for staining lysosomal phospholipid inclusions. Nile red is almost non-fluorescent in water and other polar solvents but undergoes fluorescence enhancement and large absorption and emission blue shifts in non-polar environments. Its fluorescence enhancement upon binding to proteins is weaker than that produced by its association with lipids.

The fat fraction of the globules was stained by Nile red (Aldrich, St-Louise) as followed. 1 mg of Nile red were supplemented to the fats used to formulate the globules, prior to the homogenation. Alternatively, for formulated globules staining we employed 5 il of 0.1% (w/u) Nile red solution in ethanol that was incubated with 1 ml of the final formula for 10 minutes. The stained formula was then further stained by Fast Green FCF (see below) and a 50 ul sample was transferred to microscope slides with concave cavities covered with a cover slip.

Observations of the formula were performed with a x63 oil immersion objective at wavelength of 548 and 568 nm, which are close to the excitation maximum of Nile red and Fast Green FCF, respectively.

As can be seen in Figure 3, the globules are fully stained by Nile Red, exhibiting that these globules indeed contain the fat fraction of the formulation thus mimicking the milk fat globules of human breast milk.

In order to locate and assign the protein fraction of the formulation the inventors have used the Fast Green FCF stain. This stain is used to selectively dye protein aggregates. Figure 4 exhibits a formulation of this invention stained with Fast Green FCF and Nile Red. Thus, the resulting merged micrograph exhibits the milk fat globules (red) and the protein mass between these fat globules (blue).

This is again in perfect match with the microstructure of native milk.

The proteins in the infant formulation were stained with Fast Green FCF (Sigma, St-Lousie), by incubation of 5 ml formula with 0.5% (w/v) solution in de- ionized water for 5 minutes in room temperature. The stained formula was analyzed under confocal microscope as described above.

The inventors have established that the formulation of the invention is composed of globules containing lipids and a proteinaceous mass between the globules. As discussed above, the fat globules of milk fat are surrounded by the milk fat globule membrane, the latter is composed of glycerophospholipids, sphingomyelin, cholesterol and proteins. In order to verify that the globules created by this invention are indeed surrounded by a membrane-like layer, the inventors employed a selective dye, FM 1 (Molecular Probes, Oregon, USA).

Cell membranes provide a convenient conduit for loading live and fixed cells with lipophilic dyes. Not only can cells tolerate a high concentration of the lipophilic dye, but also lateral diffusion of the dye within the membrane can serve to stain the entire cell, even if the dye is applied locally. Lipophilic tracers are used to label cells, organelles, liposomes, viruses and lipoproteins in a wide variety of long-term tracing applications, including cell transplantation, migration, adhesion and fusion studies. FM 1-43 has been used to outline membranes in sea urchin eggs. This styryl dye has also proven extremely valuable for following synaptic recycling. FM dyes are highly useful as general- purpose probes for investigating endocytosis and for simply identifying cell membrane boundaries. These water-soluble dyes, which are nontoxic to cells and virtually nonfluorescent in aqueous medium, are believed to insert into the outer leaflet of the surface membrane where they become intensely fluorescent.

Globules membranes were labeled with FM 1-43 (Molecular Probes), by incubating infant formulas with 1, uM FM 1-43 for 15 to 30 minutes in 37°C. The stained formula was then observed under confocal microscope as described above at excitation wavelength of 488 nm.

As can be seen in Figures 4 and 5, staining a formulation with a FM 1 dye results in the clear fluorescent marking of a membranal structure surrounding each of the fat globules in the emulsion. Only a true membrane surrounding the neutral lipid core would result in the fluorescent emission of this selective dye.

Human milk was obtained and analyzed using the FM 1-43 and Fast Green FCF dyes. The resulting micrograph can be seen in Figure 7A. It can be seen that the mimetic lipid composition of the invention when used to create an emulsion, mimics the microstructure of human milk to a high degree (Figure 6B). Human milk fat was also compared to an emulsion prepared by the re-dispersion in water of a spray-dried emulsion produced by a mimetic lipid mixture of the invention, containing triglycerides and glycerophospholipids, dyed with FM 1-43 (Figure 7B). The emulsion and the resulting dry powder were produced using a semi-industrial pilot plant characteristic of the equipment, methods and process conditions of infant formula manufacturing. Again, high degree of similarity exists between human milk and the reconstituted product produced by the lipid composition of the invention. It should be emphasized that upon using the mimicking composition of the glycerophospholipids, other lipids can be left out while still being able to mimic to a high degree the micro-structure of human milk, if not the full lipid composition. Thus in this-example, the mimicking composition was based only on glycerophospholipids, and was successfully used to mimic the micro-structure of human milk fat (Figure 7B). Similar results were obtained by using a mimicking composition containing glycerophospholipids, cholesterol and cholesterol esters but not sphingomyelin (Figure 7C). Human milk micro-structure mimicking is successfully obtained by glycerophospholipids, with or without other lipids, but such compositions do not mimic the composition of human milk and its fat globules.

In order to further demonstrate the similarity between the composition and microstructure obtained by the inventors and the composition and microstructure of human breast milk, the inventors used fluorescent-tagged analogs of the different building blocks of the milk fat globule. NBD tagged phosphatidylcholine, sphingomyelin, and cholesterol were used in order to establish the location of these milk fat globule membrane building blocks.

BODIPY tagged cholesterol ester was used to elucidate the location of this important nutrient which is mostly found in human breast milk in the core of the milk fat globule.

In each scan 1 mg of each of the different NBD tagged lipid analogs and 1 mg of the BODIPY tagged cholesterol ester were added to 3 grams of total lipid mixture of the invention containing a human breast milk fat mimic (InFat, by Enzymotec), a mimetic mixture of breast milk glycerophospholipids (PC+PE+PS+PI), sphingomyelin, cholesterol and cholesteryl esters. The resulting mixture was added to a warm (50-60°C) homogenized emulsion of infant formula protein, carbohydrate and mineral powder. The mixture was further homogenized and passed through a micro-fluidizer pressure homogenizer (MicroFluidics) at 200bar. The resulting emulsion was scanned using a Radiance 2000 Confocal Laser Scanning Microscope by BioRad. Additional Fast Green FCF staining was utilized in order to dye protein regions in the emulsion.

Figure 7A exhibit the clear presence of phosphatidylcholine, as a representative of the 4 glycerophospholipids mimicking the glycerophospholipids of human milk, in the membrane of the resulting milk fat globules of the invention. Figure 7B exhibit the clear presence of sphingomyelin, one of the major MFGM building blocks, in the membrane of the resulting milk fat globules of the invention.

Figure 7C exhibit the clear presence of cholesterol, one of the major MFGM building blocks, in the membrane of the resulting milk fat globules of the invention. In all scans of Figure 7 cholesterol ester is clearly shown to be present in the core of the resulting milk fat globules. It should be noted that since the NBD tagging of cholesterol results in increased lipophylicity the fluorescent tagged cholesterol is present both in the MFGM and its core. This also represents the natural composition of human milk where some of the cholesterol is also present in the globules'core.

Human milk is further characterized by the presence of phosphatidylserine (PS) in its milk fat globule membranes. This PS, unlike to cellular PS, is found to some extent on the outer leaflet of the milk fat globule membrane. This aspect of human milk fat has not been thoroughly explored nor understood but the inventors believe that this structural feature is of great importance and may play an important role in the metabolic cascade in the infant as well as in other more complex roles of the MFGM (see above).

Human milk was analyzed by using the Annexin V selective stain. The human vascular anticoagulant, Annexin V, is a 35-36 kD Ca+2-dependent phospholipid- binding protein that has a high affinity for PS (J. Biol. Chef. 1990,265, 4923).

Annexin V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet (Blood 1994,84, 1415).

As can be seen in Figure 9A, staining with AlexaFluor 488 conjugated Annexin V (Molecular Probes, Oregon, USA) clearly reveals the presence of PS on the outer leaflet of the MFGM by the green fluorescence around the fat globules. Annexin V analysis of the emulsion of this invention produced by the mimicking composition including glycerophospholipids, sphingomyelin, cholesterol and cholesterol esters reveals the clear presence of PS on the outer leaflet of the milk fat membranes (Figure 9B) and high resemblance to human milk in this aspect as well. The same analysis performed on an emulsion of commercially available infant formula reveals practically no evidence to any MFGM PS (Figure 9C).

A 100 gel aliquot of fresh human mother milk or infant emulsions were incubated with 10 fLl of Annexin V conjugate solution, according to the manual of Annexin V conjugates for apoptosis detection kit (Molecular Probes), for half an hour at 37°C. The stained membranes images were obtained in confocal microscope as described above at excitation wavelength of 495 nm.

In a further aspect of the invention the globules are formed using other techniques, such as sonication of additives in an aqueous medium at appropriate conditions (temperature, concentration, time, frequency). Other methods can be used to create milk fat globules that mimic the milk fat globules of human breast milk.

The size and structure of the globules can be controlled by different techniques and process conditions. Preferably, the size is controlled by the ratio between lipid additives (glycerophospholipids, sphingomyelin, cholesterol) and the triglycerides as is the case in human milk where at later stages of lactation the level of membranal lipids is reduced while triglycerides levels are maintained or even raised, creating larger globules with thinner membranes. Thus, the size and structure of globules at different stages of lactation, as well as in cases of preterm infants, can also be mimicked by this invention.

Table 7 Fat globules size change with preparation conditions Homogenizer Pressure % lipids Particle size Fat globules size (bar) from distribution total Measured.. solids Stage Mean Median (micro 1/stage2 (micron) n) in water after Gaulin 15M 150/50 100 4.3 3.7 homogenization in water after Gaulin 15M 350/50 100 4 3.9 homogenization Gaulin 15M 200/50 25 in water after 1.7 1.1 homogenization after drying APV Rannie 240/70 25 powder and re-1. 7 1. 3 type 10.50 dispersion in water after drying APV Rannie 100/40 25 powder and re- 3.2 2.5 type 10.50 dispersion in water after drying APV Rannie 100/40 44 powder and re-3. 4 2. 6 type 10.50 dispersion in water Microfluidizer in water after HC-8000 homogenization Microfluidizer 550 25 in water after 2 1. 1 HC-8000 homogenization Microfluidizer in water after 200 25 5.1 4.6 HC-8000 homogenization The table shows that size can be controlled by both the formulation and the homogenization equipment and conditions. It is thus proved that the required size of about 5 micron average size can be readily obtained.

Particle size distribution was measured using a Coulter LS230 (small volume module) laser diffraction instrument equipped with PIDS system. Sample preparation: dilution 1 : 1 with EDTA solution 35mM pH 7. Measuring cell filled with 0.05% SDS solution in DI water." The additives of this invention as ingredients of a formula or as globule mimicking agents may assist in the absorption of a variety of other nutrients, preferably lipid soluble nutrients. The membrane of human milk fat globules is also composed of other ingredients or attaches other ingredients from the milk, such as glycolipids, glycoproteins, proteins, enzymes, immunoglobulins, etc.

These additives are not customarily provided in infant formula. However, it should be noted that the mimicking of milk fat globules and its membrane paves the way to include these ingredients which their position in the milk are as important as their inclusion itself.

Other biofunctional ingredients, such as hormones, are also found in human milk globules, either at the core, the membrane or its outer surface. It should be noted that the additives of this invention and the ability to mimic milk fat globules and their membranes paves the way to the inclusion of such ingredients. The intake and absorption of such ingredients as well as obtaining their proper functionality is dependant on their association to the milk fat globules.

The combination of the present invention, or dietary supplements comprising it, are aimed for use in the enhancement of children development, especially of infant development, as well as fetal development. Thus, the combination of the invention is aimed for consumption of young children, infants, pregnant women (expecting mothers), as well as breast-feeding women.

More specifically, the combination of the present invention, or dietary supplements comprising it, are intended for use in the enhancement of brain development. Since the combination of the present invention, or dietary supplements comprising it, contain phosphatidylserine (PS), an important phospholipid building block of brain tissues, the addition of PS to infants and young children will benefit their cognitive development. In addition, PS has also been shown to have beneficial effects for the elderly population and other populations. The supply of PS to infants and young children in the exact ratio to other mother's milk phospholipids will enhance its intake and efficacy. The additional phospholipids of the combination of the present invention, or dietary supplements comprising it, may also provide important nutrients such as choline. Besides, the human body can bio-transform certain phospholipids into other phospholipids, according to its needs and thus, the supplementation of a variety of phospholipids, especially in a ratio that mimics their levels in mother's milk, is of great importance.

The combination of the present invention, or dietary supplements comprising it may also be used, in a preferred embodiment, to mimic the milk fat globules that are important in the delivery of fat and other nutrients to infants and young children. This globule structure may be achieved by adding the ingredient to all other ingredients of infant formulas, dispersing the mixture in an aqueous or organic environment in such a way that globules are formed. Further drying, using spray-drying for example, will result in a dry product that upon its dispersion in aqueous media prior to its consumption will retain its fat globule structure. Furthermore, the fat globules may be formed in the final preparation of the formula in aqueous media while the phospholipid ingredient of this invention is added as powder or any other form to the infant nutrition product with no special pre-treatment.

The combination of the present invention, or dietary supplements comprising it are used in the preparation of any food product, or food article, which may or not contain phospholipids as one of its ingredients or components prior to the addition of the combination or dietary supplement comprising it described herein.

This food product may be aimed and consumed by infants and young children, such as formula, biscuits, candy, bars, cereals, instant drink products, prepared cooked mashed vegetables and/or fruits, etc.

Alternatively, this food product is any food product, such as dairy products, ice cream, biscuits, soy products, pastry and bread, sauces, condiments, oils and fats, margarines, spreads, cereals, drinks and shakes, infant formulas and foods, bars, snacks, candies or chocolate products.

In another aspect, the combination of the present invention, or dietary supplements comprising it are intended for use in the prevention and/or treatment of disorders associated with depletion of phospholipids.

The consumption of the combination of the present invention, or dietary supplements comprising it shall enhance the intake of calcium and other nutrients. Thus, combination of the present invention, or dietary supplements comprising it are also intended for the enhancement of physical development.

Additionally, the production of food products containing phospholipids based in the human milk fat phospholipid content comprised in the dietary supplement of the invention shall motivate the population to incorporate such products in their nutrition, in order to assist in the absorption and bio-availability of a variety of supplemented nutrients, especially calcium. This shall enhance the absorption and bio-availability of both supplemented and naturally occurring nutrients, preferably calcium, in the normal human nutrition.

As mentioned before, phospholipids are an important nutrient during child development, especially for proper brain development. Therefore, dietary supplements containing phospholipids should be an integral part of children's nutrition, preferably infants and children until the age of 3, as well pregnant mothers, since a significant part of human brain development happens in term.

The present invention is defined by the claims, the contents of which are to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms"a","an"and"the"include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word"comprise", and variations such as"comprises"and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The examples herein are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.