Patent Publication Number: US-2005129738-A1

Title: Infant formula supplemented with phospholipids

Description:
This application is a Continuation in part of U.S. application Ser. No. 10/275,748 filed on Nov. 8, 2002, which is currently pending and claimed priority from PCT application IL02/00674 filed on Aug. 15, 2002, which claimed priority from Israel application 150240 filed on Jun. 16, 2002. 
    
    
     FIELD AND BACKGROUND OF THE INVENTION  
      The present invention relates to an infant formula supplemented with glycerophospholipids (hereinafter phospholipids) and more particularly, to a formula supplemented with phosphatidylserine.  
      Breastfeeding, the natural feeding mode, has multiple beneficial effects on the infant and is known to be the most suitable diet for infant&#39;s nutritional requirements.  
      Jensen R G. (Textbook of Gastroenterology and Nutrition in Infancy, Second Edition, edited by E. Lebenthal. Raven Press, Ltd., pp. 157-208, New York 1989) reviewed measurements of the total lipid content of human milk and reports that phospholipids are found in the human milk lipids at levels of about 20 to 40 mg/dl.  
      G. Harzer et al. reports that the phospholipid composition of human milk lipids (weight % of total phospholipids) in day 36 postpartum is phosphatidylcholine (PC) 24.9%, phosphatidylethanolamine (PE) 27.7%, phosphatidylserine (PS) 9.3%, phosphatidylinositol (PI) 5.4%, sphingomyelin 32.4% (G. Harzer et al., Am J Clin Nutr., Vol. 37, pp. 612-621, 1983).  
      Phospholipids are essential components of growing tissue in particular nerve cells. In the latter, lack of phospholipids can cause serious impairment in development and problems in overt functions. The source of phospholipids in the buildup of neuronal tissue, originates in part from intracellular synthesis and in part from the diet. Therefore dietary supplementation of phospholipids in lactating infants could be critical for proper development (For comprehensive reviews, see: J. N. Hawthorne &amp; G. B. Ansell, Phospholipids 1982, Elsevier, Amsterdam, and M. Shinitzky, “Membrane Fluidity and Cellular Function”, in Physiology of Membrane Fluidity, Shinitzky M. Ed. Vol. I, pp. 1-53, 1984, CRC Press, Boca Raton, Fla.).  
      In the past, only pig or bovine phosphatidylserine was available in the market. However, due to the “mad cow disease” this source is generally unacceptable for usage with infant formulations today. In recent years a reliable and healthy source of phosphatidylserine, which is produced by reaction of vegetal lecithin, egg lecithin or milk phospholipids with phospholipase-D is available in the market. A specific process of enzymatic conversion is known and is described in, for example, the article by Eibl A. and Kovatchev S. (Eibl A. and Kovatchev S. “Methods in Enzymology” Vol. 72, pp.: 632-639, 1981).  
      U.S. Pat. No. 6,410,522 issued to Rutenberg has, as an inherent disadvantage, a strict requirement for at least 20% (w/w) phosphatidy-L-serine out of the total phospholipid content of the composition. These concentrations are ill suited for use in infant formula and would be prohibitively expensive to produce.  
      U.S. Pat. No. 5,591,479 issued to Ponroy teaches use of mammal brain derived phosphatidylserine in preparation of an infant formula. Such practice is both dangerous and prohibited. Thus the teacheings of Ponroy specifically preclude use of non-mammalian phospholipids. Further, Ponroy fails to teach the use of phospholipase-D to produce phosphatidylserine with a structural fatty acid chain derived from a raw material lecithin or crude phospholipid source. Further, Ponroy does not hint or fairly suggest that such an enzymatic reaction is necessary, advantageous or feasible as a means of preparing an infant formula, which has the potential to improve nerve cell development in an infant.  
      There is thus a widely recognized need for, and it would be highly advantageous to have an infant formula devoid of the above limitation(s).  
     REFERENCES  
     
         
          Jensen R G. “Textbook of Gastroenterology and Nutrition in Infancy”, Second Edition, edited by E. Lebenthal. Raven Press, Ltd., pp. 157-208, New York 1989.  
          G. Harzer et al., “Changing patterns of human milk lipids in the course of lactation and during the day”, Am J Clin Nutr., Vol. 37, pp. 612-621.  
          J. N. Hawthorne &amp; G. B. Ansell, Phospholipids 1982, Elsevier, Amsterdam.  
          Shinitzky M. Ed. “Membrane Fluidity and Cellular Function in Physiology of Membrane Fluidity”, Vol. 1, pp. 1-53, 1984, CRC Press, Boca Raton, Fla.  
          Eibl A. and Kovatchev S. “Preparation of phospholipids analogs by phospholipase-D” Methods in Enzymology” Vol. 72, pages: 632-639, 1981.  
          U.S. Pat. No. 6,410,522 “Anti-depressant, stress suppressor and mood improver” issued to Rutenberg.  
          U.S. Pat. No. 5,591,479 “Brain Phospholipid Composition and Method of making for an Infant Formula” issued to Ponroy  
       
    
     SUMMARY OF THE INVENTION  
      It is an object of the present invention to provide an infant formula which is more similar to human milk in a powder or solution form, having an effect of addresseing the shortcomings of the presently known infant formulas by the inclusion of a phospholipids supplement which contains phosphatidylserine as one of the effective ingredients. Phosphatidylserine is believed to be important in nerve cell development of neonates and dietary supplementation therof is believed to augment nerve cell devlopment.  
      The infant formula of the present invention contains phosphatidylserine or the salt thereof as the effective ingredient, wherein the phosphatidylserine has a structural fatty acid chain derived from at least one raw material lecithin or crude phospholipid source selected from the group consisting of soy bean lecithin, rapeseed lecithin, a milk phospholipids concentrate or egg yolk lecithin.  
      According to further features in preferred embodiments of the invention described below, the phospholipids include phosphatidylserine or the salt thereof as the effective ingredient, wherein the phosphatidylserine derived from at least one raw material lecithin selected from the group consisting of soy bean lecithin, rapeseed lecithin, or egg yolk lecithin, and which is produced by reaction with phospholipase-D.  
      The aforementioned phosphatidylserine as the effective ingredient in accordance with the present invention is produced by the enzymatic reaction with phospholipase-D using as the substrate soy bean lecithin, rapeseed lecithin or egg yolk lecithin. The process will now be illustrated. A raw material lecithin selected from soy bean lecithin, rapeseed lecithin or egg yolk lecithin is subjected to the process of transphosphatidylation with phospholipase-D in the presence of L-serine or D-serine or DL-serine and water, thereby substituting the choline group with the serine group through the hydroxyl group, to produce the rearranged phosphatidylserine.  
      Any commercially available soy bean lecithin, rapeseed lecithin or egg yolk lecithin may be used, with no limitation, as the raw material. As phospholipase-D for use in the process of enzymatic conversion, use may be made of for example those from cabbage and actinomyces, if they have an activity on lecithin or hydrogenated lecithin or lysolecithin in the presence of L-serine or D-serine or DL-serine and water to produce phosphatidylserine.  
      A specific process of enzymatic conversion is known and described in for example the article by Eibl A. and Kovatchev S. “Preparation of phospholipids analogs by phospholipase-D.” (“Methods in Enzymology” Vol. 72, pp.: 632-639, 1981), so no detailed explanation is described herein.  
      According to another aspect of the present invention there is provided a method of feeding an infant includes the steps of mixing an infant formula powder in water for obtaining a fluid including the nutritional components and the phospholipids supplement and feeding the infant with the fluid containing nutritional components and a phospholipid supplement wherein the phospholipids include phosphatidylserine or the salt thereof as the effective ingredient.  
      According to one aspect of the present invention there is provided a pharmaceutical or nutritional or food composition for feeding infants, the composition includes: (a) an infant formula base, the formula base formulated to provide at least a source of calories and protein; and (b) a phospholipid supplement includes a product of an enzymatic reaction between at least one raw material lecithin and phospholipase-D and including phosphatidylserine or salts thereof as an effective ingredient, the phosphatidylserine from the phospholipid supplement includes between 0.1% and 19.99% (w/w) of a total phospholipid content of the composition. Preferably, the phosphatidylserine has a structural fatty acid chain derived from the at least one raw material lecithin.  
      As a result, the composition is more similar to human milk with regard to its phospholipid composition. This similarity has the potential to improve nerve cell development in an infant fed the composition.  
      According to another aspect of the present invention there is provided a method of producing a composition for feeding infants, the method includes: (a) providing at least one raw material lecithin; (b) enzymatically processing the at least one raw material lecithin with phospholipase-D to produce a phosphatidylserine containing product and (c) combining the phosphatidylserine containing product with an infant formula base to produce the composition for feeding infants, the formula base formulated to provide at least a source of calories and protein; wherein phospatidylserine from the phosphatidylserine containing product includes between 0.1% and 19.99% (w/w) of a total phospholipid content of the composition.  
      According to further features in preferred embodiments of the invention described below, the composition is supplied in a form selected from the group consisting of a powder and a fluid which is ready for use.  
      According to still further features in the described preferred embodiments a structural fatty acid chain of the phosphatidylserine is a hydrogenated saturated fatty acid chain.  
      According to still further features in the described preferred embodiments the infant formula base includes at least one ingredient selected from the group consisting of milk derived ingredients and soy derived ingredients.  
      According to still further features in the described preferred embodiments the composition further includes at least one ingredient selected from the group consisting of lactose, vegetable oils, skimmed milk powder, whey protein concentrate, Sodium, Calcium, Phosphorus, Potassium, Chloride, Iron, Magnesium, Taurine, Vitamins, Glucose syrup, soy protein isolate, Sucrose, Maltodextrine, Methionine, Taurine, Carnitine, and trace elements.  
      According to still further features in the described preferred embodiments the composition further includes at least one ingredient selected from the group consisting of a pharmaceutical excipient and a food excipient.  
      According to still further features in the described preferred embodiments there is provided a fluid composition includes a powder of claim including the infant formula base and the phospholipid supplement and an amount of water sufficient for dilution of all ingredients to their desired cocentrations.  
      According to still further features in the described preferred embodiments the at least one raw material lecithin is selected from the group consisting of soy bean lecithin, rapeseed lecithin, and egg yolk lecithin.  
      According to still further features in the described preferred embodiments the composition is supplied as an article of manufacture and further includes packaging material and instructions for use.  
      The present invention successfully addresses the shortcomings of the presently known configurations by providing an infant formula which is more similar to human milk, adds protection from serious impairment in development and problems in overt functions and improve the development and maturation of tissue in nerve cells. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
      The present invention is of an infant formula supplemented with phospholipids, particularly which includes phosphatidylserine, which can be used to feed infants. Specifically, the present invention can be used to protect infants of syndromes associated with feed devoid of phospholipids fed to them in the first year of their lives. The present invention renders infant formulas more similar to human milk with regard to phospholipid composition. Specifically, it is believed that formulations in which phosphatidylserine from the phospholipid supplement represents between 0.1% and 19.99% (w/w), more preferably between 1% and 19% (w/w), more preferably between 1% and 15% (w/w), more preferably between 1% and 12.5% (w/w), more preferably between 2.5% and 12.5% (w/w), more preferably between 7.5% and 12.5% (w/w), most preferably about 10%(w/w) of a total phospholipid content of the composition are effective.  
      Thus, in accordance with one aspect of the present invention there is provided an infant formula in a powder or fluid form which formula includes nutritional components and a phospholipid supplement, particularly which includes phosphatidylserine.  
      In accordance with another aspect of the present invention there is provided a method of feeding an infant. The method is effected by executing the following steps: First an infant formula powder containing nutritional components and a phospholipid supplement is mixed in water for obtaining a mixture including the nutritional components and the phospholipid supplement. Second, the mixture is fed to the infant.  
      As exemplified in the Examples section below, the nutritional components may include milk or soy derived nutritional components. They may additionally include one or more of the following ingredients: lactose, vegetable oils, skimmed milk powder, whey protein concentrate, Sodium, Calcium, Phosphorus, Potassium, Chloride, Iron, Magnesium, Taurine, Vitamins, Glucose syrup, soy protein isolate, Sucrose, Maltodextrine, Methionine, Taurine, Carnitine, and trace elements.  
      Further exemplified in the Examples section below, is the preparation of phosphatidylserine and the phospholipid complex thereof.  
     EXAMPLE 1-1  
      Tables 1 and 2 below provide exemplary compositions of dry and fluid milk based infant formulas and dry and fluid soy based infant formulas according to the present invention.  
      Infant Formula I (Milk-Based)  
      The formula comprises the following ingredients: lactose, vegetable oils, skimmed milk powder, whey protein concentrate, Sodium, Calcium, Phosphorus, Potassium, Chloride, Iron, Magnesium, Taurine, Vitamins and phospholipids.  
                               TABLE 1                                       Powder   fluid           Unit   100 g   100 ml                                        General Comp.                                     Protein   gram   11.1   1.5           Fat   gram   25.9   3.5           Lactose   gram   55.5   7.5           Water   gram   2.5   —           Ash   gram   2.06   0.27                 Vitamins                                     Vitamin A   I.U.   1500   200           Vitamin D   I.U.   300   40           Vitamin E   mg   6   0.81           Vitamin K   .mu.g   15   2.01           Vitamin B1   .mu.g   350   47.03           Vitamin B2   .mu.g   450   60           Vitamin B6   .mu.g   222   30           Vitamin B12   .mu.g   0.66   0.09           Niacin   mg   2   0.27           Folic Acid   .mu.g   45   6           Calcium   mg   4.44   0.06                 Pantothenate                                     Biotin   .mu.g   11   1.5           Vitamin C   mg   45   6.08                 Minerals                                     Calcium   mg   326   44           Phosphorus   mg   219   29.6           Magnesium   mg   37   5           Iron   mg   7.4   1           Sodium   mg   120.7   16.3           Potassium   mg   373   50           Ca/p ratio   —   1.49   1.49                 Amino Acid Profile                                     Alanine   mg   522   69.6           Arginine   mg   368   49.1           Asparatic Acid   mg   11.10   1.5           Cystine   mg   191   25.5           Glutamic Acid   mg   1423   189.7           Glycine   mg   244   32.5           Histidine   mg   262   34.9           Isoleucine   mg   761   101.5           Leucine   mg   12.20   1.62           Lysine   mg   10.00   1.3           Methionine   mg   270   36           Phenylalanine   mg   461   62.3           Proline   mg   962   128.3           Serine   mg   681   90.8           Taurine   mg   37   4.9           Threonine   mg   686   91.5           Tryptophan   mg   180   24           Tyrosine   mg   463   61.7           Valine   mg   775   103.3                 Fatty Acid Profile                                     Caprylic (C8)   % from fat   2.6   2.6           Capric (C10)   %   2.1   2.1           Lauric (C12)   %   17.5   17.5           Meristic (C14)   %   6.7   6.7           Palmitic (C16)   %   11.2   11.2           Stearic (C18)   %   11.8   11.8           Oleic (C18:1)   %   37.0   37.0           Linoleic (C18:2)   %   10.0   10.0           Linolenic (C18:3)   %   1.2   1.2                 Phospholipid supplement which is added to the       aforementioned 100 g. of powder or fluid):                                     Phosphatidylcholine   mg   250.0   250.0           Phosphatidylethanolamine   mg   250.0   250.0           Phosphatidylserine   mg   100.0   100.0           Phosphatidylinositol   mg   50.0   50.0           Phosphatidic Acid   mg   50.0   50.0                      
 
 Infant Formula II (Soy-Based) 
 
      The formula comprises the following ingredients: Glucose syrup, vegetable oils, soy protein isolate, Sucrose, Maltodextrine, Sodium, Calcium, Phosphorus, Potassium, chloride, Iron, Magnesium, Vitamins, Methionine, Taurine, Carnitine, trace elements and phospholipids.  
                               TABLE 2                                       Powder   fluid           Unit   100 g   100 ml                                        General Comp.                                     Protein   gram   15   1.98           Fat   gram   27.54   3.64           Carbohydrate   gram   51.5   6.8           Linoleic Acid   gram   4.5   0.6                 Vitamins                                     Vitamin A   I.U.   1500   198           Vitamin D   I.U.   300   39.7           Vitamin E   I.U.   10   1.32           Vitamin C   mg   65   8.6           Vitamin K   .mu.g   77   10.2           Vitamin B1   .mu.g   345   45.6           Vitamin B2   .mu.g   445   58.9           Vitamin B6   .mu.g   327   43.3           Vitamin B12   .mu.g   1.5   0.2           Niacin   mg   7   0.93           Folic Acid   .mu.g   76           Pantothenic Acid   .mu.g   4.5   0.6           Biotin   .mu.g   25   3.3           Choline   mg   58   7.7                 Minerals                                     Calcium   mg   500   66.2           Phosphorus   mg   300   39.7           Magnesium   mg   45   6           Iron   mg   9.2   1.2           Zinc   mg   4   0.53           Manganese   .mu.g   150   19.8           Copper   .mu.g   400   53           Iodine   .mu.g   77   10.2           Sodium   mg   200   26.5           Potassium   mg   546   72.2           Chloride   mg   400   53           Inositol   mg   25   3.3           Carnitine   mg   10   1.3           Ca/P   ratio   1.67   1.67                 Amino Acid Profile                                     Alanine   mg   640   85.3           Arginine   mg   497   6.5           Aspartic Acid   mg   1385   184.7           Cystine   mg   242   32.3           Glutamic Acid   mg   3065   408.7           Glycine   mg   300   40           Histidine   mg   382   50.9           Isoleucine   mg   893   119.1           Leucine   mg   1600   213.3           Lysine   mg   1360   181.3           Methionine   mg   406   54.1           Phenylalanine   mg   650   86.7           Proline   mg   1113   148.4           Serine   mg   737   98.3           Taurine   mg   51   6.8           Threonine   mg   460   61.3           Tyrosine   mg   621   82.8           Valine   mg   947   126.3                 Fatty Acid Profile                                     Caprylic (C8)   % from fat   2.6   2.6           Capric (C10)   %   2.1   2.1           Lauric (C12)   %   17.5   17.5           Meristic (C14)   %   6.7   6.7           Palmitic (C16)   %   11.2   11.2           Stearic (C18)   %   11.8   11.8           Oleic (C18:1)   %   37.0   37.0           Linoleic (C18:2)   %   10.0   10.0           Linolenic (C18:3)   %   1.2   1.2                 Phospholipid supplement which is added to the       aforementioned 100 g. of powder or fluid):                                     Phosphatidylcholine   mg   250.0   250.0           Phosphatidylethanolamine   mg   250.0   250.0           Phosphatidylserine   mg   100.0   100.0           Phosphatidylinositol   mg   50.0   50.0           Phosphatidic Acid   mg   50.0   50.0                      
 
      Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.  
     EXAMPLE 2-1  
      Soybean lecithin (50 g; Epikuron 135 as the product name; Lucas Meyer GmbH, Germany) and soybean oil (10 g) were placed in a 300-ml vial, followed by addition of ethyl acetate (50 ml) for solubilization. Adding a solution (20 ml) of 0.30 g/ml L-serine dissolved in 0.1M sodium phosphate buffer, pH 7.0 to the resulting solution for thorough blending, a solution of 500 U/ml phospholipase-D from cabbage was added to the mixture solution for reaction at 25. degrees C. for 5 hours under stirring with a stirrer.  
      For inactivation of the enzyme in the reaction solution, the vial containing the reaction solution was immersed in hot water. Subsequently, the reaction solution was cooled in ice and treated with chloroform. The chloroform layer was collected and dried under reduced pressure.  
     EXAMPLE 2-2  
      Using egg yolk lecithin (DS-PL95E as the product name; manufactured by Doosan Corp. Venture BG Biotech BU. Korea) as the substrate, rearranged phosphatidylserine was produced by the same method as in Example 1-1.  
     EXAMPLE 3  
      Phosphatidylserine was prepared by Lipogen Products (9000) Ltd. via the process of enzymatic reaction from a substrate soybean lecithin according to Example 2-1. 50 grams of this preparation were added to 50 grams of soy lecithin containing high percentage of phosphatidylcholine (50 g; Epikuron 145V as the product name; Lucas Meyer GmbH, Germany). The mixture was vigorously stirred to produce a homogeneous phospholipids complex.