WO1999037167A2

WO1999037167A2 - Process of improving the stability of vitamin d in a nutritional product containing hydrolyzed protein and product produced thereby

Info

Publication number: WO1999037167A2
Application number: PCT/US1999/001381
Authority: WO
Inventors: Marlene Wynn Borschel; Benjamin D. Travis; Cindy Black; Jerry L. Hill; Steven T. Leubbers; Helen R. Churella
Original assignee: Abbott Laboratories
Priority date: 1998-01-22
Filing date: 1999-01-21
Publication date: 1999-07-29
Also published as: WO1999037167A3; NO20003225L; NO20003225D0; EP1049386A2; CA2319158A1; JP2002500869A

Abstract

A liquid enteral nutritional hypoallergenic formula is disclosed. The formula contains carbohydrates, lipids, protein hyrolysates, free amino acids, vitamins, minerals and L-methionine as an added component at therapeutically effective levels to provide a liquid dietary formula with improved Vitamin D stability and acceptable Protein Efficiency Ratio.

Description

PROCESS OF IMPROVING THE STABILITY OF VITAMIN D

IN A NUTRITIONAL PRODUCT

CONTAINING HYDROLYZED PROTEIN AND PRODUCT PRODUCED THEREBY

Technical Field

This invention relates to a method of improving the stability of Vitamin D in a liquid nutritional product that comprises hydrolyzed proteins and/or free amino acids. The process comprises the addition of methionine to the nutritional product to assure nutritional adequacy and the reduced or eliminated supplementation of cystine. The invention also relates to nutritional products made through the inventive process.

Background of the Invention

Some adults and infants have food allergies that include sensitivity to intact protein or protein maldigestion. These individuals require a special nutritional formula, such as a hypoallergenic formula, to meet their dietary needs. Hypoallergenic formulas, that are also sometimes referred to as elemental formulas, are characterized in that they typically contain hydrolysates such as soy protein hydrolysate, casein hydrolysate, whey protein hydrolysate or a combination of animal and vegetable protein hydrolysates as the major source of amino nitrogen. Hypoallergenic formulas may also use only free amino acids as the source of amino nitrogen. The protein hydrolysates comprise short peptide fragments and/or free amino acids instead of the intact protein found, for example, in cow's milk and soy protein isolate-based formulas. These short peptide fragments and free amino acids have been found to be less immunogenic and allergenic than intact proteins. Infants who experience adverse reactions to intact protein often are able to tolerate hydrolysate-based liquid nutritional products. Typical sources of hydrolyzed protein include casein, whey, rice, soy and beef collagen.

Alimentum^® is one such hypoallergenic nutritional formula that is manufactured by the Ross Products Division of Abbott Laboratories, Columbus, Ohio. Alimentum^® is a nutritionally complete formula that contains an extensively hydrolyzed casein which is approximately 60% by weight free amino acids, the remainder being small peptides. Such predigestion virtually eliminates allergenicity. During the manufacture of the protein hydrolysate, the concentration of a number of amino acids, such as tryptophan and tyrosine, is significantly reduced. Thus, fortification of the hydrolysate with these "lost" amino acids is needed to restore nutritional adequacy.

Although cystine is not an essential amino acid, products such as Alimentum^® have traditionally been fortified with cystine in order to mimic the amino acid profile of human breast milk. Thus, in addition to the casein that has been enzymatically hydrolyzed and charcoal treated, Alimentum has added L-cystine dihydrochloride, L-tyrosine, L-tryptophan, taurine and L-carnitine. Pregestimil^®and Nutramigen^®, which are hypoallergenic infant nutritionals manufactured by the Mead Johnson Division of Bristol Meyers Squibb of Evansville, Indiana, are supplemented with L-cystine, L-tryptophan, L-tyrosine, taurine and L-carnitine. All of these hypoallergenic pediatric formula are also fortified with Vitamin D at a level above 45 IU (label claim) per five fluid ounce (142 ml) or 100 calories.

In addition to the protein hydrolysate, most nutritionally balanced hypoallergenic formulas contain carbohydrates, lipids, vitamins, and minerals. These hypoallergenic formulas are utilized for feeding infants, children and adults who have allergies or sensitivities to intact protein, and are often medically used in the treatment of cystic fibrosis, chronic diarrhea, galactosemia, small bowel resection, steatorrhea and protein-calorie malnutrition.

Advera^®, a specialized, complete nutritional for the dietary management of people with HIV infection or AIDS; AlitraQ^®, a specialized, elemental nutritional with glutamine for metabolically stressed patients; and Perative^®, a specialized liquid nutritional for the dietary management of metabolically stressed patients all contain hydrolyzed protein and/or free amino acids as their source of amino nitrogen. All of these products are manufactured and distributed by the Ross Products Division of Abbott Laboratories, Columbus, Ohio. All of these products also contain Vitamin D and are susceptible to Vitamin D degradation when in liquid form due to the presence of protein hydrolysates and free amino acids.

The beneficial qualities of Vitamin D are well documented and accepted. For example, Vitamin D regulates the calcium level in the body and is responsible for depositing calcium and phosphorous into bone from the blood. Too little Vitamin D can cause soft bones, muscle weakness, poor growth, bone fractures and secondary hyperthyroidism. Although vitamin supplements in tablet form may be taken, Vitamin D is generally easily obtainable from the diet, especially a diet containing Vitamin D enriched foods.

The most common biologically active forms of Vitamin D are previtamins D2 and D3 and vitamins D2 and D3. Previtamin D2 and Vitamin D2 are produced from ergosterol and are biologically active in humans, cattle, swine and other mammals. Previtamin D3 and Vitamin D3 are biologically active and are produced in the skin of many animals following irradiation (i.e., exposure to the sun) of 7-dehydrocholesterol. The other isomeric forms of Vitamin D show no significant biological activity.

The isomerization of bio-available Vitamin D to inactive forms occurs in solution. The stability of Vitamin D in a nutritional matrix is dependent upon a number of factors. For example, light, high temperatures and iodine catalyze the conversion of the biologically active forms of Vitamin D to inactive forms.

One excellent source of Vitamin D is liquid nutritional products that are fortified with Vitamin D. With respect to fortification with Vitamin D and infant formula, Vitamin D levels in infant products are federally regulated in the U.S.A. under the Infant Formula Act (IFA). Nutritional products containing levels above or below those set forth in the IFA may not legally be offered for sale.

Hypoallergenic formulas encounter a major problem regarding Vitamin D stability. Vitamin D undergoes significant degradation in protein hydrolysate formulas and/or formulas containing free amino acids. In fact, research has shown that the more hydrolyzed the protein, the greater the Vitamin D degradation. Specifically, the inherent and fortified sources of free or combined cysteine and cystine are capable of forming the thiyl-free radical (in the presence of a free radical initiator such as oxygen or peroxides) that isomerizes Vitamin D into non-bioavailable forms. These free radical reactions also make Vitamin D more susceptible to oxidation that converts the Vitamin D to non-bioavailable forms. This ability to isomerize Vitamin D to non-bioavailable forms by a free radical reaction involving the thiyl radical is true for all sulfur compounds present as a thiol (such as cysteine) or as a disulfide (such as cystine). The reaction also occurs with cysteine-containing peptides or disulfide-linked peptides but is not generally observed with intact proteins due to steric hindrances.

The reactions do not occur in sulfur-containing molecules in which the sulfur atom is not bonded to hydrogen or sulfur, due to the inability of these compounds to readily form the thiyl free radical. Therefore, sulfur compounds such as the salts and esters of methionine, cystathionine, S-methylcystine and cysteine sulfonate do not induce Vitamin D degradation.

In view of the problems with Vitamin D degradation in liquid nutritional products containing hydrolyzed protein and/or free amino acids, some products are over-fortified with Vitamin D. Over fortification is used to meet the label claim for Vitamin D content over the shelf life of the product. However, as mentioned above, acceptable upper limits of Vitamin D concentration exist, since too much Vitamin D can cause hypercalcemia, hypercalciuria, urinary tract stones, extraskeletal calcifications and malfunction of the kidneys and other organs. In similar fashion, if the Vitamin D concentration drops below a certain limit due to degradation, the product must be removed from sale to the public. The costs associated with replacing, shipping and overall monitoring of the Vitamin D level in liquid nutritional products are significant. A need presently exists in the industry to enhance the stability of Vitamin D in a liquid, hypoallergenic formula.

U.S. Patent No. 4,836,957 to Nemoto, et al., discloses a preparation containing an active form of Vitamin D3 that is stabilized by incorporation of an amino acid that is neither containing a sulfur atom nor an acid amino group in its structure, nor an acidic amino acid, nor a salt of a basic amino acid. This patent is not concerned with Vitamin D stability in liquid nutritional formula that contain protein hydrolysates and/or free amino acids.

U.S. Patents 5,382,439 and 5,456,926 to Hill, et al., disclose a method for improving the stability of Vitamin D in liquid nutritional products that contain hydrolyzed protein or free amino acids. These patents teach that Vitamin C should be added at a concentration exceeding 300 mgs per liter and that cystine be added to the liquid nutritional product after completion of a preliminary heat treatment. While these patents disclose methods that address the problem of Vitamin D stability in a hypoallergenic product, they are less than totally satisfactory.

It is thus apparent that a need exists for a method to improve the stability of Vitamin D in the presence of protein hydrolysates and/or free amino acids without adversely affecting the nutritional quality of the formula.

Disclosure of the Invention

In general, this invention relates to the discovery that Vitamin D stability in liquid nutritional products can be dramatically improved by eliminating or limiting cystine fortification without sacrificing nutritional adequacy provided methionine is used to provide the requirements for sulfur-containing amino acids as assessed by animal growth are met. The present invention also provides an improved hypoallergenic formula that minimizes the need for over-fortification with Vitamin D and provides excellent protein quality for normal growth in humans.

Thus, there is disclosed a method for improving the stability of Vitamin D in a liquid nutritional product wherein said nutritional product comprises Vitamin D and a source of amino nitrogen selected from free amino acids, hydrolyzed protein and mixtures thereof, said method comprising the addition of L-cystine to said product at a level not to exceed 7.0 mgs (in addition to inherent levels) per gram of protein and the addition of L-methionine to said product at a level of at least 9.0 mgs (in addition to inherent levels) per gram of protein.

In a more preferred embodiment, the liquid nutritional product is an infant formula. In a yet more preferred embodiment less than two mgs of cystine is added to the nutritional product per gram of total protein and at least 12 mgs of L-methionine is added per gram of total protein. Most preferably, no more than 0.5 mgs per gram of L-cystine is added and at least 14 mgs of L-methionine is added per gram of total protein. In a yet most preferred embodiment, no L-cystine is added to the nutritional product and at least 15 grams of L-methionine is added. There is also disclosed a liquid nutritional product comprising a source of amino nitrogen selected from the group consisting of hydrolyzed proteins, free amino acids and mixtures thereof, Vitamin D and L-methionine as an added substance at a concentration of at least 9.0 mgs per gram of protein.

From another perspective the invention can be viewed and implemented in terms of total content of the sulfur-containing amino acids methionine, cystine and cysteine. The protein hydrolysate used in the hypoallergenic formula will contain inherent levels of methionine, cystine and cysteine. For example, intact casein inherently has 30 mgs of methionine per gram of protein and four mgs of cystine (cystine plus cysteine) per gram of protein. As used herein and in the claims the levels associated with cystine are to be understood as meaning the sum of cystine plus cysteine. This is a result of the analytical chemistry. In contrast, the casein hydrolysate used in Alimentum has an inherent level of methionine of about 26 mgs per gram of protein and about six mgs of cystine per gram of protein. To realize the benefits of improved Vitamin D stability and adequate growth, the inventors have determined that at least 45 mgs (320 micro-moles (μM)) of sulfur-containing amino acids (cystine, methionine and cysteine) per gram of protein be present in the inventive formula from the protein hydrolysate and amino acid supplementation. Further, at least 34 mgs of the 45 mgs of the sulfur-containing amino acids should be methionine. Thus, a hydrolysate containing 28 mgs of methionine per gram of protein and seven mgs of cystine per gram of protein (inherent levels) will be fortified with at least six mgs of methionine (34-28=6) per gram of protein and may, but not necessarily, be additionally fortified with four mgs of cystine (6+28+7+4=45).

Thus, there is also disclosed a method for improving the stability of Vitamin D in a liquid nutritional product wherein said nutritional product comprises Vitamin D and a source of amino nitrogen selected from hydrolyzed protein, free amino acids and mixtures thereof, said method comprising the steps of: a) providing a liquid nutritional product containing Vitamin D; b) adding L-methionine to said product such that the total level (inherent plus supplemental) of L-methionine is at least 34 mgs per gram of protein and the addition of at least one amino acid selected from the group consisting of lysine, tryptophan, tyrosine, taurine and L-carnitine.

There is further disclosed a method for improving the stability of Vitamin D in a liquid nutritional product wherein said nutritional product comprises Vitamin D and a source of amino nitrogen selected from hydrolyzed protein, free amino acids and mixtures thereof, said method comprising the addition of L-methionine to achieve a total level of L-methionine of at least 34 mgs per gram of protein and the addition of at least one amino acid selected from the group consisting of cystine, cysteine and mixtures thereof to achieve a concentration of at least 45 mgs of sulfur-containing amino acids per gram of protein.

There is also disclosed an improved hypoallergenic enteral nutritional comprising lipid, carbohydrate, Vitamin D and a source of amino nitrogen, the improvement characterized in that the source of amino nitrogen is selected from hydrolyzed protein, free amino acids and mixtures thereof wherein the sum of all sulfur-containing amino acids is at least 50 mgs per gram of protein and the level of methionine is at least 40 mgs per gram of protein. In a more preferred embodiment the level of methionine is at least 42 mgs per gram of protein and the level of cystine is less than 12 mgs per gram of protein. In yet a more preferred embodiment, the formula comprises at least 43 mgs of methionine (inherent plus supplemented) per gram of protein and less than ten mgs of cystine (inherent plus supplemented) per gram of protein. In a most preferred embodiment, the formula comprises less than eight mgs of cystine per gram of protein and at least 42 mgs of methionine per gram of protein.

This invention also relates to an enteral nutritional with an improved PER comprising a source of amino nitrogen, the improvement characterized in that the source of amino nitrogen is selected from hydrolyzed protein, free amino acids and mixtures thereof and wherein the concentration of L-methionine is at least 34 mgs per gram of protein.

In yet another view of the present invention, the inventors have determined that an improved hypoallergenic formula comprises Vitamin D and at least 34 mgs of total (inherent plus fortification) methionine per gram of protein and less than 12 mgs of total (inherent plus fortification) cystine per gram of protein. In yet a more preferred embodiment, the formula comprises at least 40 mgs of methionine per gram of protein and less than ten mgs of cystine. In a most preferred embodiment the formula comprises at least 42 mgs of methionine per gram of protein and less than eight mgs of cystine per gram of protein.

In similar fashion, a process for the production of an improved hypoallergenic enteral formula comprising protein hydrolysates is disclosed, said process comprising the steps of supplementing said formula with Vitamin D and free methionine to achieve a total (inherent plus fortification) concentration of methionine of at least 34 mgs per gram of protein, more preferably at least 40 mgs per gram of protein, and most preferably at least 42 mgs per gram of protein. The process may additionally comprise the addition of free cystine to achieve a total (inherent plus fortification) concentration of cystine not to exceed 12 mgs per gram of protein.

The hydrolyzed protein that is used in the present invention can be any edible source of protein such as animal (i.e., meat and fish), cereal (i.e., rice and corn) and vegetable proteins (i.e., soy). More specifically, the source of protein can be milk proteins such as casein and whey. The most preferred source of protein for hydrolysis is casein.

In addition to the hydrolyzed protein, the nutritional product of this invention can additionally contain free amino acids other than L-methionine. Any amino acid can be added to the formula, however, the addition of cystine and/or cysteine should be avoided or held to a minimum. Thus, in addition to L-methionine, the nutritional according to this invention is preferably fortified with tryptophan, tyrosine, taurine and L-carnitine.

There is further disclosed an improved hypoallergenic enteral nutritional comprising, based on total calories of the nutritional, about 30-65% carbohydrates, about 30-60% lipids, about 5-20% of a source of amino nitrogen selected from hydrolyzed protein, free amino acids and mixtures thereof; and about 400 to 1 ,000 IU per liter of Vitamin D, the improvement characterized in that said nutritional comprises L-methionine as an added component at a concentration of at least 9 mgs per gram of protein. In a more preferred embodiment, L-methionine is at a concentration of at least 12 mgs per gram of protein; and most preferably at a concentration of at least 14 mgs per gram of protein. The hypoallergenic nutritional according to this invention also may contain as an added component an amino acid selected from lysine, tryptophan, tyrosine, taurine, L-carnitine and mixtures thereof.

The present invention provides a method for improving the stability of Vitamin D in liquid nutritional products having hydrolyzed protein and/or free amino acids as the primary source of amino nitrogen. The method is extremely easy to practice, very reliable and does not compromise the nutritional quality of the formula. While the primary focus of the invention is directed to infant formula, the invention is also applicable to other liquid nutritional products that utilize hydrolyzed proteins and/or amino acids as a source of amino nitrogen. The formula may take the form of a ready-to-feed product, a concentrate or a powder.

In yet another embodiment, this invention relates to a method of improving the stability of Vitamin D in a liquid nutritional product that contains hydrolyzed protein and/or free amino acids as the source of amino nitrogen, the method comprising the step of adding L-methionine to said liquid nutritional product at a concentration of added L-methionine of at least 9 mgs per gram of protein.

There is also disclosed an improved liquid nutritional formula with enhanced Vitamin D stability and a source of amino nitrogen that provides for acceptable growth, made in accordance with the methods disclosed above. Other objects and advantages of this invention will be apparent from the following description and the appended claims.

As used herein and in the claims, the total protein upon which the level of amino acid fortification is based is determined through Kjeldahl's method for the determination of total nitrogen. The principle of Kjeldahl's method is the conversion of the nitrogen containing substances to ammonium sulfate by boiling with sulfuric acid in the presence of a catalyst, usually copper sulfate. Potassium sulfate is added to raise the boiling point. The mixture is then made alkaline and the ammonia distilled off into standard acid. Details of this technique can be found in numerous textbooks on chemical analysis or practical biochemistry. The nitrogen value is converted to grams of protein by multiplying grams of nitrogen by 6.25. As used herein and in the claims the levels of added methionine or any other amino acid are based on total protein content including the added amino acids. For example, if a formula contains ten grams of protein per liter, the ten grams is the sum of all proteins, protein fragments and free amino acids and a fortification level of 15 mgs per gram of protein includes the 15 mgs in each gram of protein.

Cystine or dicysteine (C₆Hι₂N₂0 S₂) has the chemical structure:

COOH COOH Cystine consists of two molecules of cysteine (cystine yields cysteine upon reduction). Thus, due to the two sulfur atoms, one mole of cystine provides two moles of sulfur-containing amino acid equivalents. Cystine is particularly abundant in the proteins of the skeletal, connective tissues, hair and wool of animals.

Methionine or 1-Amino-3-methylmercaptobutyric acid (C₅HnN0₂S) has the chemical structure:

CH₃ - S - CH₂ - CH2 - CH - COOH

I

Methionine is one of the natural sulfur-containing amino acids and is present in small quantities in the hydrolysis products of proteins compared to most of the other amino acids. It is an essential constituent of the food of mammals and is particularly important in that it and choline are the only compounds in the diet known to take part in methylating reactions. As used herein and in the claims, the term "methionine" includes the salts and esters of methionine, cystathionine, s-methyicystine and cysteine sulfonate. Also as used herein and in the claims, all of the recited amino acids, including cystine and methionine, are understood to be in the L- or laevorotatory form.

Detailed Description of the Invention

The hypoallergenic formula according to the present invention is made by blending carbohydrates, lipids and a protein hydrolysate, homogenizing the mixture into a stable emulsion, adding the supplemental free amino acids and sterilizing the product in the pH range from about 6 to about 7.

The protein hydrolysate of the invention may be any suitable protein hydrolysate utilized in a nutritional formula such as soy protein hydrolysate, cereal grain protein hydrolysate, casein hydrolysate, whey protein hydrolysate, animal and vegetable protein hydrolysates and mixtures thereof. The protein hydrolysate of the hypoallergenic formula of the invention is preferably a soy protein hydrolysate or a casein hydrolysate comprising short peptides and free amino acids. The immunogenicity of the formula of the present invention depends largely on the extent of hydrolysis of the selected protein. To insure hypoallergenicity of the formula, the protein hydrolysate should be extensively hydrolyzed to yield very short peptides and free amino acids. This is important since free amino acids and di-and tri-peptides are known to be absorbed through the small intestine without any digestive breakdown. High molecular weight peptides are preferably avoided because they are more allergenic and cause precipitation and emulsion destabilization of the liquid nutritional, In a preferred embodiment, the protein hydrolysate of the invention contains a high percentage of low molecular weight peptide fragments.

In general, any known technique can be used to produce the protein hydrolysate. Preferably, the protein is hydrolyzed to such an extent that the ratio of amino nitrogen (AN) from the free amino acids in the hydrolysate to total nitrogen (TN) from intact protein, protein fragments and free amino acids ranges from about 0.3 AN to 1.0 TN to about 0.8 AN to 1.0 TN. Such hydrolysis generally yields hydrolysates with the following representative molecular weight distribution.

Hypoallergenic formula may also use only free amino acids as the source of amino nitrogen or may be more hydrolyzed than that given above.

One aspect of this invention resides in the discovery that the hydrolyzed protein source be supplemented with significant levels of methionine and only sparingly, if at all, with cystine and still be nutritionally adequate for growth. The formula may be fortified with other free amino acids such as L-tryptophan, L-tyrosine, L-arginine, L-taurine and L-carnitine. The total caloric value of the protein hydrolysate and supplemented free amino acid mixtures in the hypoallergenic formula may range from about 8% to about 20% of the total calories of the formula and is preferably in the range of about 10% to about 14% of total calories.

While the presence of cystine in a hypoallergenic nutritional beverage is known to increase the degradation of the Vitamin D, a replacement is required to assure that proper growth and protein maintenance is achieved. One aspect of the invention resides in the discovery that methionine can be added to the nutritional formula without degrading Vitamin D while promoting acceptable growth of the animal.

"Protein Efficiency Ratio" (PER) is a measure of protein quality using laboratory rats. The inventors have discovered that the replacement of cystine with methionine in micro-moles (μM) of sulfur-containing amino acid equivalents (e.g., two moles of methionine for each mole of cystine), results in a formula that provides adequate nutrition for growth (amino nitrogen), has an excellent PER and dramatically improves the stability of Vitamin D in liquid nutritionals.

Analytical Method for Vitamin D

In order to determine whether the method of the invention is effective in reducing Vitamin D degradation, it was necessary to accurately determine the concentration of bio-active Vitamin D in the formula. The technique used by the inventors to quantitate Vitamin D was a modification of the method published by Sertl and Molitor, Journal of the Association of Official Analytical Chemists, Volume 68, Number 2, 177-182 (1985). The method consists of saponifying the sample, extracting the saponified sample, subjecting the extract to preliminary liquid chromatography, cleaning-up and subjecting the cleaned-up extract to quantitative liquid chromatography. Details of this analytical procedures can be found in U.S. Patent 5,456,926 to Hill, et al.

Under the Infant Formula Act (IFA) in the U.S.A. there are certain maximum and minimum levels assigned to Vitamin D in infant formula. For example, the upper limit for a 20 calorie per fl. oz. formula is 676 IU per liter, or 100 IU per 100 calories. The liquid nutritional product is typically over-fortified such that by zero-time (the time when the container is ready for sale) the Vitamin D concentration has dropped into the acceptable range. During the shelf life of the product, the Vitamin D concentration can fall below the acceptable minimum and in that event, the product would have to be recalled or exchanged. According to the Infant Formula Act in the U.S.A., the acceptable minimum level of Vitamin D is 40 IU per 100 calories or 270 IU per liter for an infant formula containing 20 calories per fl. oz.

EXAMPLE I Study of PER

This experiment was conducted to evaluate the PER of intact casein, casein hydrolysates and casein hydrolysates fortified with methionine at two levels, and with lysine and/or tryptophan. The investigation was conducted in accordance with AOAC Method 960.48, AOAC Official Methods of Analysis, 15th Edition, 1990. There were two Control groups used in this experiment; one was the casein (intact protein) based diet having the composition given in the Official AOAC method and the second was based on an unfortified casein hydrolysate that is used in Alimentum^® (neither shown in Table I). A total of six experimental diets were prepared that were based on the casein hydrolysate with varied levels and types of fortification. Table I sets forth the fortification of each diet based on milligrams of added amino acid per 1.0 g of total protein (nitrogen times 6.25).

TABLE I

Experimental Diets

Casein Hy drolysate base Hi JS Miinαram oτ Am no Acid Per i .υ u ram ot I otal Prote n

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

5.0 g MET* 5.0 MET 5.0 MET 5.0 MET 10 MET 10 10 LYS* 3.0 TRP* 10 LYS MET 3.0 TRP 10 LYS

3.0

TRP

* MET = methionine LYS = lysine hydrochloride TRP = tryptophan

Each diet was virtually identical in caloric content, source and distribution of calories from carbohydrate and fat, and in macro- and micro-nutrients. The casein hydrolysate Control diet contained an inherent total of about 27-28 mgs of methionine per gram of protein. Diets 1 through 4 contained 32-33 mgs of methionine as actually analyzed inherent plus supplemental) per gram of protein and Diets 5 and 6 contained 34 mgs of methionine per gram of protein as actually analyzed.

Weaning male Sprague-Dawley rats between 21 and 23 days of age with an initial weight between 40 and 60 grams were used in this experiment. The animals were placed in test cages for an acclimatization period of three to four days. During this time, the rats received a standard rat chow diet. After acclimatization, 10 rats per group were assigned a Control or Experimental diet. The rats were fed their assigned diet and water ad libitum. The animals were weighed at the beginning of the study and every week during the four-week study period. Consumption of the diets was measured and the PER was calculated by the method given in the AOAC PER procedure, 15th Ed., Method 960.48, 1990.

Table II sets forth the data generated in this experiment. For each diet, the average weight gain per animal, the average amount of protein consumed and the PER for each diet is reported. TABLE II

The data indicate that supplementation of the base casein hydrolysate with methionine at a total level 34 mgs per gram protein level significantly improves the PER of the protein system (Diets 5 and 6). Surprisingly, no response to lysine and/or tryptophan supplementation was evident. It thus appears that supplementation of a casein hydrolysate with methionine can greatly enhance the growth efficiency of the protein system. This interesting result forms an aspect of the present invention.

EXAMPLE II

Vitamin D Stability

Commercially available Alimentum® served as the Control in this experiment that contains a casein hydrolysate fortified with the amino acids L-cystine (11 mgs per gram of protein), L-tryptophan and L-tyrosine. The Experimental formula contained the same casein hydrolysate but was supplemented with 15 or 9.2 mgs of methionine (depending on the total protein concentration in the formula) in place of the 11 mgs of cystine per gram of total protein in the Control product. Table III sets forth the Control and Experimental products.

* median value of 20 lots

The Vitamin D levels were determined during shelf-life storage on product held at room temperature. The results are set forth in Table IV. TABLE IV

Vitamin D Concentrations (IU/L) After Storage at Room Temperature

Vitamin D Label Claim - 304 IU/L

NT - Not Tested

From Table IV, it is evident that the Experimental products in accordance with the invention experienced less degradation of Vitamin D than the Control product. Table V sets forth the Vitamin D loss from processing and over the 12-month study period. Total loss in IU per liter and % of original fortification is also set forth in Table V.

TABLE V

Vitamin D Loss (IU/L) 0 to 12 Months Loss

It is quite evident from Table V that hypoallergenic products using the present invention, (Samples l-lll) are significantly less likely to fall outside of Vitamin D acceptable ranges during storage than the Control product that used the techniques of the prior art. EXAMPLE III

PER Study

A formula in accordance with this invention was analyzed for PER substantially in the manner set forth in Example I. Groups of 10 rats each, were fed a diet for 28 days according to the invention or a casein based formula using AOAC Method-960.48, AOAC Official Methods of Analysis, 15th Edition, 1990. Every seven days, the rats were weighed and their food consumption was recorded. At the end of 28 days, the total weight gain and protein composition of the two groups were calculated. These values were used to calculate the PER. The composition of each diet is set forth in Table VI.

TABLE VI

Composition of Diets

+ added as cyst ne y roc or e

The intact casein contained inherent levels of methionine ranging from 26-30 mgs per gram of protein and cystine at about 4.0 mgs per gram of protein. The casein hydrolysate contained about 26 mgs methionine per gram of protein and about 4.0 mgs of cystine per gram of protein.

Diets 7 and 8 were identical except that the cystine was added prior to or subsequent to a heat treatment that was employed during the manufacture of the infant formula. The Control diet and Diets 7-10 contained the same blend of edible oils at about the same percent of total calories (34.2 to 39.8%). The carbohydrate was the same in all the diets and was from about 51.0 to 56.4% of total calories. As all components of the diets were similar, except for the source of amino nitrogen, differences in growth of the animals can be attributed to the efficacy of the protein system to support growth. Thus, the higher the PER, theoretically, the better the protein system. Table VII sets forth the PER for each diet and the percent increase over the Control.

TABLE VII PER V

The results of this experiment indicate that the protein system in accordance with the present invention (Diets 9 and 10) has a significantly greater (Tukey's Studentized Range Test for variable) protein efficiency ratio than the conventional protein system (Control and Diets 7 and 8) wherein cystine fortification is utilized.

Diets 9 and 10, that were fortified with methionine (39.6 and 44 mgs of total methionine per gram of total protein) and decreased or no cystine (total cystine of 4.0 and 10.4 mgs per gram of protein) dihydrochloride fortification tested the highest in the study. The other diets where no methionine fortification occurred, tested significantly lower than the methionine fortified diets. Diet 8 was very similar to the presently available commercial Alimentum^® product (30 mgs of total methionine and 15 mgs of total cystine per gram of protein) and had a PER of only 2.89. This experiment supports one aspect of the present invention; that being, increased fortification of sulfur-containing amino acids, other than cystine, has a beneficial effect on the growth of animals. This aspect, in combination with the reduction or elimination of cystine fortification, results in a hypoallergenic nutritional product with improved Vitamin D stability and a protein system for acceptable growth. As a result of this work, an improved hypoallergenic nutritional formula with acceptable protein quality and Vitamin D stability has been developed. EXAMPLE IV

Clinical Testing

As the protein quality and Vitamin D stability of a hypoallergenic nutritional formula according to the present invention had been demonstrated, the following clinical study was undertaken to demonstrate efficacy in human infants. The purpose of the study was to investigate if substituting methionine for cystine fortification or modifying the protein level and amino acid fortification would have any effect on the growth of healthy male, term infants.

The Control formula was commercially available Alimentum^®. Diet 11 was Alimentum^® with increased levels of protein and methionine while cystine fortification was reduced. Diet 12 was Alimentum^® with methionine fortification and no cystine fortification. Vitamin D stability was also measured in this study. Other biochemical responses of the infants such as plasma albumin, urea nitrogen and plasma amino acids were also evaluated.

This clinical trial was a controlled, blinded, randomized, 16-week growth study of male infants enrolled within the first five days of life. Shortly after parturition, parents of eligible infants were contacted by the investigators. The study was explained and parents of infants were requested to sign an Informed Consent Form. On Day One of enrollment (SD1), the infants were assigned to a feeding group by an investigator, typically a pediatrician, and seen again by the physician on weeks four, eight and 16 (+ three days) after enrollment. Anthropometric measurements were obtained on entry into the study and at subsequent visits.

The Control and Experimental formula were prepared in similar fashion. Table VIII sets forth the target addition levels of cystine, methionine, tyrosine and tryptophan for the Control and Diets 11 and 12.

TABLE VIII Target Addition Rates

Table IX sets forth the analyzed levels of protein, Vitamin D, Vitamin C, cystine, methionine, tyrosine and tryptophan for the Control and Experimental diets. Table IX also contains data on the selected amino acid content of representative human breast milk and a representative intact casein.

The equipment and procedures used to prepare the Control and Experimental formula are conventional and well known to those skilled in the art. All study feedings were supplied as ready-to-feed (RTF) in 32 fluid ounce (907 ml) cans and provided 20 kcal per fl. oz. All formulas met or exceeded levels of nutrients as recommended by the Committee on Nutrition of the American Academy of Pediatrics (1985) and the Infant Formula Act (1980) and subsequent amendments (1986).

Formula volume intakes, incidence of spit-up, vomiting and stool patterns were recorded on diaries completed by the parents for three days immediately prior to the study visits at four, eight and 16 weeks. Caloric and protein intakes were calculated based on formula volume intakes and nutrient composition of the formula fed.

Birth weight, length and head circumference were obtained from hospital records. Weight, length and head circumference were measured at SD1 and at each study visit (four, eight and 16 weeks) by the same examiner at each site, according to procedures in the Guide to Growth Assessment of Infants in Clinical Studies provided by Ross Products Division, Abbott Laboratories. Nude body weights were obtained using an electronic balance. Length boards were used to obtain lengths, measuring tapes were used to obtain the head and upper arm circumferences, while calipers were used to obtain triceps skin-fold thickness. Approximately 2 cc of blood was collected from the infants by venipuncture by a certified technologist, nurse or physician at four and 16 weeks of age for determination of plasma albumin, urea nitrogen and plasma amino acids. The sample was drawn 150 to 180 minutes after the beginning of the last feeding. Plasma was frozen at -70° C until analyzed.

The three feeding groups were evaluated for comparability at entry. Birth and entrance anthropometries were analyzed using ANOVA. Gestational age and age at SD1 were compared using ANOVA on the ranked data. Ethnicity and five-minute Apgar scores were analyzed using Fisher's Exact test.

Evaluation of feeding effects was carried out using Repeated Measures ANOVA. These were also analyzed separately at each visit using ANOVA. Ordinal variables were compared separately at each time point using ANOVA or Cochran Mantel-Haenszel tests. Categorical variables were analyzed at each time point using Fisher's Exact test.

Location of the study (site) was used as a blocking factor in the ANOVA models. Ranking transformations were used in the analyses of the percentage tolerance and stool variables, predominant stool consistency, volume intake and blood biochemistries. Mean rank stool consistency was also analyzed using the untransformed data. All tests were carried out using a 5% significance level. The primary analysis was done on the subset of completers only. An intent-to-treat analysis including available data on all subjects was done as a secondary analysis.

Subjects

A total of 73 infants successfully completed the study (23 on Control; 25 on Diet 11 ; 25 on Diet 12). A total of 27 infants were considered to be treatment failures due to intolerance to the study feeding (13 on Control; six on Diet 11 ; eight on Diet 12) and 22 infants were considered to be protocol failures.

No statistically significant differences were observed among groups for gestational appropriateness. One infant was considered small for gestation age, 88 infants were appropriate for gestational age and 33 infants were considered large for gestational age. No significant differences were observed among groups for age at SD1 , gestational age or five minute Apgar score. The mean age of infants at entry into the study ranged from 1.8 to 2.3 days (range 0 to five days of age). The mean gestational age at birth ranged from 39.3 to 39.4 weeks.

Anthropometric Measurements

No statistically significant differences were observed in birth weight, length or head circumference or in their respective NCHS Z-scores. No statistically significant differences in weight, length or head circumference or their respective NCHS Z-scores were observed at SD1 or at four, eight or 16 weeks. Most importantly, no statistically significant differences were observed among groups in weight gains, adjusted weight gains, length gains or head circumference gains at four, eight or 16 weeks. In addition, no statistically significant differences were observed among groups for total upper arm area, upper arm muscle area, upper arm fat area or arm fat index at SD1 , five, eight or 16 weeks or in gains of these at four, eight or 16 weeks.

The study also revealed that there were no significant differences among groups in the mean number of feedings per day, the mean volume of formula intake, mean adjusted volume of formula intake, adjusted caloric intake or caloric efficiency at four weeks or eight weeks. Adjusted mean protein intakes pooled across visits differed significantly (P<0.01) among the feeding groups. Infants fed Diet 12 had significantly greater mean adjusted protein intakes than infants fed Diet 11 at four weeks (P<0.01) and significantly greater mean adjusted protein intakes than infants fed Diet 11 and Control at eight weeks. At 16 weeks, infants fed Diet 12 had significantly greater volume of formula intakes, greater adjusted volume of formula intakes, and adjusted caloric intakes than those infants fed Diet 11 (P<0.05). Also, at 16 weeks, infants fed Diet 12 had significantly greater adjusted protein intakes than infants fed Diet 11 or Control (P<0.01). Infants fed Diet 11 had significantly greater caloric efficiency at 16 weeks than infants fed Diet 12 (P<0.05). No differences in the percent of feedings with spit-up and/or vomiting were observed among groups during the study.

No significant differences were observed among groups in the number of stools per day during the study. Also, no significant differences were observed among groups in the percent of stools that were watery, loose/mushy or formed at each visit during the study. However, infants fed Diet 12 had significantly higher plasma urea nitrogen concentrations than infants fed Diet 11 and Control at four weeks. For data pooled across visits, plasma methionine concentrations differed significantly between feeding groups (P<0.01). Infants fed Diet 12 had significantly higher plasma concentrations of asparagine, praline, valine, isoleucine, leucine and tyrosine than infants fed Control at four weeks. Infants fed Diet 11 and 12 had significantly higher serum methionine concentrations than infants fed Control at four weeks. When only the data of successful completers were analyzed, only plasma methionine differences were statistically significant. At 16 weeks, the only significant difference observed was that infants fed Diet 11 had a significantly higher plasma methionine level than infants fed Control.

Conclusion

The inventors herein had theorized that the amino acid methionine could be used to fortify a hypoallergenic formula in place of cystine to insure that the requirements for sulfur-containing amino acids were met. This study was conducted to determine the effects of modification of the protein system of a hypoallergenic formula on the growth and biochemical response of infants.

The results of this study confirmed that substituting methionine for cystine resulted in no alterations in growth of healthy term, male infants during the first four months compared to infants fed the Control formula, that was commercially available Alimentum^®. The study results also indicate that there was no advantage in feeding infants an increased protein-containing formula with methionine fortification and no cystine fortification. All groups grew in a remarkably similar fashion during the study and when weights of these infants were compared to the weights of infants fed Similac^® With Iron (an intact protein infant formula marketed by the Ross Products Division of Abbott Laboratories, Columbus, Ohio) in recent studies, the weights were also very similar. Most importantly, no adverse effects were observed in the group of infants fed a methionine fortified product.

Industrial Applicability

The results from these experiments demonstrate that the hypoallergenic enteral formula of this invention is effective in providing adequate growth without the need to over-fortify Vitamin D. The medical community is constantly searching for nutritional formulas that will benefit the infant/patient. The present invention can clearly fill that need. The manufacture of the formula utilizes conventional equipment and may be readily accomplished. Further, the discoveries of the invention have application to other liquid nutritional products that utilize hydrolyzed proteins and/or free amino acids and need an increased level of Vitamin D stability.

While the formula and method of making said formula disclosed herein constitute a preferred embodiment of this invention, it is to be understood that the invention is not limited to this precise formulation or method and that changes may be made therein without departing from the scope of the invention that is defined in the appended claims.

Claims

What is claimed is:

1. A method for improving the stability of Vitamin D in a liquid nutritional product wherein said nutritional product comprises Vitamin D and a source of amino nitrogen selected from free amino acids, hydrolyzed protein and mixtures thereof, said method comprising the addition of L-cystine to said product at a level not to exceed 7.0 mgs per gram of protein and the addition of L-methionine to said product at a level of at least 9.0 mgs per gram of protein.

2. The method according to claim 1 wherein said liquid nutritional product is an infant formula.

3. The method according to claim 1 wherein said hydrolyzed protein is selected from hydrolyzed casein, whey, soy, milk, rice, corn, vegetable protein, meat, fish and mixtures thereof.

4. The method according to claim 1 wherein said L-cystine is added to said product at a level not exceeding 2 mgs per gram of protein and said L-methionine is added to said product at a level of at least 12 mgs per gram of protein.

5. The method according to claim 1 wherein said L-cystine is added to said product at a level not exceeding 0.5 mgs per gram of protein and said L-methionine is added to said product at a level of at least 14 mgs per gram of protein.

6. A liquid hypoallergenic enteral nutritional comprising, based on total calories of the nutritional, about 30 to 65% carbohydrates, about 30 to 60% lipids, about 5 to 20% of a source of amino nitrogen selected from hydrolyzed protein, free amino acids and mixtures thereof; and about 400 to 1 ,000 IU per liter of Vitamin D, the improvement characterized in that said nutritional comprises L-methionine as an added component at a concentration of at least 9.0 mgs per gram of protein and the concentration of L-cystine, inherent and added, does not exceed 7.0 mgs per gram of protein.

7. The nutritional according to claim 6 wherein said L-methionine as an added component is at a concentration of at least 12 mgs per gram of protein.

8. The nutritional according to claim 6 wherein said L-methionine as an added component is at a concentration of at least 14 mgs per gram of protein.

9. The nutritional according to claim 6 wherein said nutritional additionally comprises as an added component an amino acid selected from lysine, tryptophan, tyrosine, taurine, L-carnitine and mixtures thereof.

10. A liquid nutritional product comprising:

a) a source of amino nitrogen selected from the group consisting of hydrolyzed proteins, free amino acids and mixtures thereof;

b) Vitamin D;

c) L-methionine as an added substance at a concentration of at least 9.0 mgs per gram of protein; and

d) L-cystine is present at a concentration not exceeding 7.0 mg per gram of protein based on both inherent and supplemental L-cystine.

11. The liquid nutritional according to claim 10 wherein said hydrolyzed protein is selected from hydrolyzed casein, whey, soy, milk, rice, corn, vegetable protein, meat, fish and mixtures thereof.

12. A method of improving the stability of Vitamin D in a liquid nutritional product that contains hydrolyzed protein as a source of amino nitrogen, the method comprising the steps of:

a) providing a liquid nutritional product containing Vitamin D;

b) adding L-methionine to said product such that the total concentration of

L-methionine is at least 34 mgs per gram of protein; and

c) adding at least one amino acid selected from the group consisting of lysine, tryptophan, tyrosine, taurine and L-carnitine.

13. The method according to claim 12 wherein said hydrolyzed protein is hydrolyzed casein.

14. The method according to claim 12 wherein said L-methionine is added such that the total concentration of L-methionine is at least 38 mgs per gram of protein.

15. The method according to claim 12 wherein said L-methionine is added such that the total concentration of L-methionine is at least 40 mgs per gram of protein.

16. A method for improving the stability of Vitamin D in a liquid nutritional product wherein said nutritional product comprises Vitamin D and a source of amino nitrogen selected from hydrolyzed protein, free amino acids and mixtures thereof, said method comprising the addition of L-methionine to achieve a total level of L-methionine of at least 34 mgs per gram of protein and the addition of at least one amino acid selected from the group consisting of cystine, cysteine and mixtures thereof to achieve a concentration of at least 45 mgs of sulfur-containing amino acids per gram of protein.

17. The method according to claim 16 wherein the sum of all said sulfur-containing amino acids is at least 50 mgs per gram of protein and the level of methionine is at least 40 mgs per gram of protein.

18. The method according to claim 16 wherein the total level of said methionine is at least 42 mgs per gram of protein.

19. An enteral nutritional with an improved protein efficiency ratio (PER) comprising a source of amino nitrogen, the improvement characterized in that the source of amino nitrogen is selected from hydrolyzed protein, free amino acids and mixtures thereof and wherein the concentration of L-methionine is at least 34 mgs per gram of protein.

20. A process for the production of an improved hypoallergenic enteral formula comprising protein hydrolysates, said process comprising the step of supplementing said formula with L-methionine to a total concentration of at least 34 mgs of methionine per gram of protein.

21. The enteral nutritional according to claim 19 wherein said L-methionine is at a concentration of at least 38 mgs per gram of protein.

22. The enteral nutritional according to claim 19 wherein said L-methionine is at a concentration of at least 40 mgs per gram of protein.

23. The process according to claim 20 wherein said L-methionine is at a concentration is at least 38 mgs per gram of protein.

24. The process according to claim 20 wherein said L-methionine is at a concentration of at least 40 mgs per gram of protein.

25. An enteral formula made in accordance with the process according to claim 20.