Analytical method for the determination of infant formula protein ...

Tag : corn syrup solids


Abstract
A method for in vitro determination of the digestibility ofproteins in a nutritional product. The method utilized gastric andintestinal enzymes that are standardized for in vitro digestionprocess that mimics the in vivo digestive process. Further,specificity in digestion is determined by an amino acid profile.
Description of the Invention

SUMMARY OF THE INVENTION

One embodiment of the invention is a method for determining thedigestibility of proteins, the method includes the steps ofdigesting a sample of a nutritional product and a reagent blankwith at least one enzyme; terminating the digestion process;determining the total concentration of each of a plurality of aminoacids for the sample and the blank; determining the totaltryptophan concentration for the sample and the blank; determiningthe soluble concentration of each of the plurality of amino acidsfor the sample and the blank; determining the soluble concentrationof tryptophan for the sample and the blank; and calculating thepercentage of soluble amino acids in the digested sample ofnutritional product.

In another embodiment, the method comprises the steps of separatingeach of the digested sample and the blank into a first portion anda second portion; determining the total concentration of each ofthe plurality of amino acids for the first portion of the sampleand the first portion of the blank; determining the totaltryptophan concentration for the first portion of the sample andthe first portion of the blank; separating each of the secondportion of the sample and the second portion of the blank into aliquid phase and a solid phase; determining a soluble concentrationof each of the plurality of amino acids in the liquid phase; anddetermining a soluble concentration of tryptophan in the liquidphase.

In another embodiment the separating step is selected from thegroup consisting of acidification, precipitation, centrifugation,filtration, and a combination of centrifugation and filtration.

In another embodiment, the step of calculating the percentage ofsoluble amino acids includes the steps of adding the totalconcentrations of the plurality of amino acids and tryptophanconcentrations for the sample and the blank; determining thedifference in total concentration of the plurality of amino acidsand tryptophan between the sample and the blank; adding the solubleconcentration of the plurality of amino acids and tryptophanconcentration in the sample and the blank; determining thedifference in soluble concentration of the plurality of amino acidsand tryptophan concentration between the sample and the blank;dividing the difference in soluble concentrations by the differencein total concentrations to determine a quotient; and multiplyingthe quotient by 100.

In one embodiment, the nutritional product is infant formula.

In one embodiment, the digesting step uses one or more enzymesintended to mimic the environment of a human gastrointestinaltract.

In a further embodiment, the enzymes are selected from the groupconsisting of pepsin, peptidases, pancreatin proteinase, papain,trypsin and chymotrypsin.

In yet a further embodiment, the step of digesting the sampleincludes the steps of obtaining the sample of nutritional product;adjusting the pH to about 4.5; adding pepsin; incubating thesample; increasing the pH to about 7.0; adding pancreatinproteinase; and incubating the sample.

In a further embodiment, the step of terminating the digestion isimmersing the sample in a boiling water bath.

DETAILED DESCRIPTION OF THE INVENTION

The proteins for which digestibility may be determined according tothe present invention may be in many forms, including but notlimited to, nutritional products, dietary supplements,pharmaceuticals or other products. They may be used at any age, forexample by infants, children or adults. There may be particularvalue in using them during periods of rapid growth, such asinfancy, childhood and adolescence. The proteins for whichdigestibility may be determined according to the invention may beincorporated into a nutritious "vehicle or carrier" whichincludes but is not limited to the FDA statutory food categories:conventional foods, foods for special dietary uses, dietarysupplements and medical foods. Suitable sources of protein fornutritional products include milk, soy, rice, meat (e.g., beef),animal and vegetable (e.g., pea, potato), egg (egg albumen),gelatin, and fish. Suitable intact proteins include, but are notlimited to, soy-based, milk-based, casein protein, whey protein,rice protein, beef collagen, pea protein, potato protein andmixtures thereof. Suitable protein hydrolysates also include, butare not limited to, soy protein hydrolysate, casein proteinhydrolysate, whey protein hydrolysate, rice protein hydrolysate,potato protein hydrolysate, fish protein hydrolysate, egg albumenhydrolysate, gelatin protein hydrolysate, a combination of animaland vegetable protein hydrolysates, and mixtures thereof.Hydrolyzed proteins (protein hydrolysates) are proteins that havebeen hydrolyzed or broken down into shorter peptide fragments andamino acids. Such hydrolyzed peptide fragments and free amino acidsare more easily digested. In the broadest sense, a protein has beenhydrolyzed when one or more amide bonds have been broken. Breakingof amide bonds may occur unintentionally or incidentally duringmanufacture, for example due to heating or shear. For purposes ofthis invention, the term hydrolyzed protein means a protein whichhas been processed or treated in a manner intended to break amidebonds. Intentional hydrolysis may be effected, for example, bytreating an intact protein with enzymes or acids. The hydrolyzedproteins that are preferably utilized in formulas according to thisinvention are hydrolyzed to such an extent that the ratio of aminonitrogen (AN) to total nitrogen (TN) ranges from about 0.1 AN to1.0 TN to about 0.4 AN to about 1.0 TN, preferably about 0.25 AN to1.0 TN to about 0.4 AN to about 1.0 TN. (AN:TN ratios given are forthe hydrolysate protein source alone, and do not represent theAN:TN ratios given in the final pediatric nutritional formulaproduct, since free amino acids may be added as a supplement andwould alter the reported value.) Protein may also be provided inthe form of free amino acids. A formula according to the inventionis preferably supplemented with various free amino acids in orderto provide a more nutritionally complete and balanced formula.

Amino acids are the individual building blocks of proteinbiosynthesis. Non-essential amino acids are those that aresynthesized in the body from ammonia and various carbon sources.Non-essential amino acids include: Alanine (ALA), Serine (SER),Aspartic Acid (ASP), Glutamic acid (GLU), Cysteine (CYS), Tyrosine(TYR), Asparagine (ASN), Proline (PRO), Glycine (GLY), andGlutamine (GLN). The abbreviation "GLX" refers to GLUplus GLN and the abbreviation "ASX" refers to ASP plusASN.

Essential amino acids are also required for protein synthesis invivo and must be obtained from dietary sources. They are Isoleucine(ILE), Leucine (LEU), Lysine (LYS), Methionine (MET), Phenylalanine(PHE), Threonine (THR), Tryptophan (TRP), Valine (VAL), Histidine(HIS) and Arginine (ARG) (essential in young growing animals, butnot in adults). Of the essential amino acids, tryptophan has thelowest daily intake requirement.

Direct inferences as to the digestibility of proteins may be drawnfrom analyses that determine the type and concentration of aminoacids in solution (the soluble or digestible portion) after invitro digestion and level of concentration of amino acids in thesolid phase (the non-digestible portion) after digestion. Examplesof suitable free amino acids for adding to formula include, but arenot limited to, L-tryptophan, L-tyrosine, L-cysteine, L-taurine,L-methionine, L-arginine, and L-carnitine.

Soy

One component of the nutritional formula of this invention is soyprotein. As described above, a number of soy protein sources may beconsidered. The soy protein is isolated from the soybean. Thesoybean is an excellent source of high quality protein where about38% to 40% of the soybean is protein. Briefly (as shown in SchemeI, see Original Patent), the processing of soybeans involves theextraction of the oil from the dehulled, and cracked soybeansleaving the defatted soybean flakes.

The defatted soybean flakes are typically milled into flours;alcohol-extracted or alkoline/H.sub.2O extracted to remove flavorcompounds and sugars to make protein concentrates; and processedwith water to remove sugars and flavor compounds, precipitated anddried to make protein isolates. Whey and protein fiber areby-products of the above processes.

Nutritional Products

Nutritional products contain macronutrients, ie. fats, proteins andcarbohydrates, in varying relative amounts depending on the age andcondition of the intended user, and often contain micronutrientssuch as vitamins, minerals and trace minerals. The term"food" includes solids and liquids. The term"nutritional product" includes but is not limited tothese FDA statutory food categories: conventional foods, foods forspecial dietary uses, medical foods and infant formulas."Foods for special dietary uses" are intended to supply aspecial dietary need that exists by reason of a physical,physiological, pathological condition by supplying nutrients tosupplement the diet or as the sole item of the diet. A"medical food" is a food which is formulated to beconsumed or administered enterally under the supervision of aphysician and which is intended for the specific dietary managementof a disease or condition for which distinctive nutritionalrequirements, based on recognized scientific principles, areestablished by medical evaluation.

In addition, a "dietary supplement" is a product intendedto supplement the diet by ingestion in tablet, capsule or liquidform and is not represented for use as a conventional food or as asole item of a meal or the diet.

Infant Formulas

Infant formula refers to nutritional formulations that meet thestandards and criteria of the Infant Formula Act, (21 USC.sctn.350(a) et. seq.) and are intended to replace or supplementhuman breast milk. Although such formulas are available in at leastthree distinct forms (powder, liquid concentrate and liquidready-to-feed ("RTF"), it is conventional to speak of thenutrient concentrations on an "as fed" basis andtherefore the RTF is often described, it being understood that theother forms reconstitute or dilute according to manufacturer'sdirections to essentially the same composition and that one skilledin the art can calculate the relevant composition for concentratedor powder forms.

"Standard" or "Term" infant formula refers toinfant formula intended for infants that are born full term as afirst feeding. The protein, fat and carbohydrate componentsprovide, respectively, from about 8 to 10, 46 to 50 and 41 to 44%of the calories; and the caloric density ranges narrowly from about660 to about 700 kcal/L (or 19-21 Cal/fl.oz.), usually about 675 to680 (20 Cal/fl.oz.). The distribution of calories among the fat,protein and carbohydrate components may vary somewhat amongdifferent manufacturers of term infant formula. SIMILAC.TM. (RossProducts Division, Abbott Laboratories), ENFAMIL.TM. (Mead JohnsonNutritionals), and GOOD START.TM. (Carnation) are examples of terminfant formula.

"Nutrient-enriched" formula refers to infant formula thatis fortified relative to "standard" or "term"formula. The primary defining characteristic that differentiatesnutrient-enriched formulas is the caloric density; a secondaryfactor is the concentration of protein. For example, a formula witha caloric density above about 700 Kcal/L or a protein concentrationabove about 18 g/L would be considered"nutrient-enriched". Nutrient-enriched formulas typicallyalso contain higher levels of calcium (e.g. above about 650 mg/L)and/or phosphorus (e.g. above about 450 mg/L). Examples includeSimilac NEOSURE.TM. and Similac Special Care.TM. formulas.

The liquid and powder nutritional products for which proteindigestibility of the present invention can be determined aremanufactured by generally conventional techniques known to thoseskilled in the art. Briefly, three slurries are prepared, blendedtogether, heat treated, standardized, spray dried (if applicable),packaged and sterilized (if applicable).

Liquid Products

A carbohydrate/mineral slurry is prepared by first heating water toan elevated temperature with agitation. Minerals are then added.Minerals may include, but are not limited to, sodium citrate,sodium chloride, potassium citrate, potassium chloride, magnesiumchloride, tricalcium phosphate, calcium carbonate, potassium iodideand trace mineral premix. A carbohydrate source, such as one ormore of lactose, corn syrup solids, sucrose and/or maltodextrin isdissolved in the water, thereby forming a carbohydrate solution. Asource of dietary fiber, such as soy polysaccharide, may also beadded. The completed carbohydrate/mineral slurry is held underagitation at elevated temperature until it is blended with theother slurries, preferably for no longer than about twelve hours.

An oil slurry is prepared by combining and heating the basic oilblend. The basic oil blend typically contains some combination ofsoy, coconut, palm olein, high oleic safflower or sunflower oil andmedium chain triglycerides. Emulsifiers, such as diacetyl tartaricacid esters of mono, diglycerides, soy mono, diglycerides, and soylecithin may be used. Any or all of the oil-soluble vitamins A, D,E (natural R,R,R form or synthetic) and K may be added individuallyor as part of a premix. Beta carotene, which can function as an invivo antioxidant, may also be added, as may a stabilizer such ascarrageenan. Oils containing specific LCPs important to thisinvention (e.g. DHA and AA) can be added to the oil slurry. Caremust be used with these LCPs since they easily degrade and becomerancid. The completed oil slurry is held under agitation until itis blended with the other slurries, preferably for a period of nolonger than about twelve hours.

A protein in water slurry is prepared by first heating water to anappropriate elevated temperature with agitation. The protein sourceis then added to the water with agitation. Typically this proteinsource is intact or hydrolyzed milk proteins (e.g. whey, casein),intact or hydrolyzed vegetable proteins (e.g. soy), free aminoacids and mixtures thereof. In general, any known source of aminonitrogen can be used in this invention. The completed proteinslurry is held under agitation at elevated temperature until it isblended with the other slurries, preferably for a period no longerthan about two hours. As an alternative, some protein may be mixedin a protein-in-fat emulsion rather than protein-in-water.

The protein in water and carbohydrate/mineral slurries are blendedtogether with agitation and the resultant blended slurry ismaintained at an elevated temperature. After a brief delay (e.g. afew minutes), the oil slurry is added to the blended slurry fromthe preceding step with agitation. As an alternative to addition tothe oil blend, the LCP oils can be added directly to the blendresulting from combining the protein, carbohydrate/mineral and oilslurries.

After sufficient agitation to thoroughly combine all constituents,the pH of the completed blend is adjusted to the desired range. Theblended slurry is then subjected to deaeration, ultra-hightemperature heat treatment, emulsification and homogenization, thenis cooled to refrigerated temperature. Preferably, after the abovesteps have been completed, appropriate analytical testing forquality control is conducted. Based on the analytical results ofthe quality control tests, and appropriate amount of water is addedto the batch with agitation for dilution.

A vitamin solution, containing water soluble vitamins and traceminerals (including sodium selenate), is prepared and added to theprocessed slurry blend with agitation. A separate solutioncontaining nucleotides is prepared and also added to the processedblended slurry with agitation.

The pH of the final product may be adjusted again to achieveoptimal product stability. The completed product is then filledinto the appropriate metal, glass or plastic containers andsubjected to terminal sterilization using conventional technology.Alternatively, the liquid product can be sterilized aseptically andfilled into plastic containers.

Powder Products

A carbohydrate/mineral slurry is prepared as was described abovefor liquid product manufacture.

An oil slurry is prepared as was described above for liquid productmanufacture with the following exceptions: 1) Emulsifiers (mono,diglycerides, lecithin) and stabilizers (carrageenan) typically arenot added to powder, 2) In addition to the beta carotene, otherantioxidants, such as mixed tocopherols and ascorbyl palmitate, canbe added to help maintain the oxidative quality of the productduring any subsequent spray drying process, and 3) The LCPs areadded after mixing the slurries, rather than to the oil slurry.

A protein in water slurry is prepared as was described above forliquid product manufacture.

The carbohydrate/mineral slurry, protein in water slurry and oilslurry are blended together in a similar manner as described forliquid product manufacture. After pH adjustment of the completedblend, LCPs are then added to the blended slurry with agitation.Desirably, the LCPs are slowly metered into the product as theblend passes through a conduit at a constant rate just prior tohomogenization (in-line blending).

After deaeration, ultra-high temperature heat treatment,emulsification and homogenization, the processed blend may beevaporated to increase the solids level of the blend to facilitatemore efficient spray drying. The blend then passes through apreheater and a high pressure pump and is spray dryed usingconventional spray drying technology. The spray dryed powder may beagglomerated, and then is packaged into metal or plastic cans orfoil/laminate pouches under vacuum, nitrogen, or other inertenvironment.

Variations on any of these manufacturing processes are known to orwill be readily apparent to those skilled in the art. It is notintended that the invention be limited to any particular process ofmanufacture. The full text of all US Patents mentioned herein isincorporated by reference.

Gastric and Intestinal Enzymes

The enzymes used for the in vitro digestion described herein wereproduced in accordance with United States Pharmacopeia standards.The activity of the enzymes is therefore consistent from lot tolot. The United States Pharmacopeia (USP) is a non-governmentorganization that promotes the public health by establishingstate-of-the-art standards to ensure the quality of medicines andother health care technologies. These standards are developed by aprocess of public involvement and are accepted worldwide. Thestandards developed by USP are published in the United StatesPharmacopeia and the National Formulary (USP-NF), which arerecognized in the Federal Food, Drug, and Cosmetic Act (21 U.S.C..sctn. 321 et seq.).

Enzymes suitable for use in the method described herein include,but are not limited to, pepsin, peptidases, pancreatin proteinase,papain, trypsin and chymotrypsin.

In Vitro Protein Digestion

The in vivo digestive process is difficult to reproduce exactly.However, several conditions that exist in vivo can be reproduced invitro. In vitro digestibility assays should be conducted underconditions that are as close as possible to in vivo conditions. Forexample, the pH and enzymes of the digestive system should beincorporated into the in vitro digestion process. Similarly, the invitro digestion should be of a duration corresponding to the timeproteins reside in the digestive tract. The in vitro digestionprocess described below mimics the pH and make up of gastric andintestinal enzymes of young infants. The time of in vitro digestionalso mimics the time required for food to pass through thedigestive tract of young infants. Protein digestions of nutritionalproducts were prepared by the following procedure: Prepare 80 mL ofa sample of nutritional product by obtaining a volume of ready tofeed nutritional product, reconstitution of powder or dilution ofliquid concentration. Quantitatively transfer the suspension into a100 mL volumetric flask and dilute to 100 mL with water. (Thesuspension should be prepared so that the 100 mL volumetric flaskcontains approximately 1.625 grams of protein, and so that thealiquot pipetted into the 20-mL screw cap vial containsapproximately 0.1625 grams of protein. The quality of the assayresults depend, to some extent, upon the use of a constant ratio ofenzyme to sample protein.) Pipette 10 mL of the diluted sample intoa 20 mL screw-cap vial. Adjust the pH of the 10 mL aliquot to 4.5with 1 M hydrochloric acid. Add 32 mg of USP Pepsin (U.S.Pharmacopeia, 12601 Twinbrook Parkway, Rockville, Md. 20852) andstir to thoroughly suspend the pepsin. Incubate in vial at37.degree. C. for thirty minutes. Raise the pH to 7.0 with 0.5 MNaHCO.sub.3. Add 3.0 mL of freshly prepared suspension of USPPancreatic Protease Amylase (U.S. Pharmacopeia, 12601 TwinbrookParkway, Rockville, Md. 20852) at 25 mg/mL in 0.1 M NaHCO.sub.3.Stir to thoroughly suspend the enzyme. Incubate the vial at37.degree. C. for sixty minutes. Immerse the vial in a boilingwater bath for 4 minutes. Cool to room temperature. Quantitativelytransfer the resulting digest into a tared 25 mL volumetric flaskusing water to assist the transfer. Dilute the digest in the flaskwith water and record the weight.

Reagent blanks are digested alongside the nutritional samples foruse in calculating the resulting total and soluble portions of theamino acids in the digested samples.

Separate aliquots of the digestion were taken to determine aminoacid profile and trytophan concentrations. However, if amino acidprofile and tryptophan concentrations are to be determined from thesame digestion aliquot, no separation steps are required.

Amino Acid Profile

Prior to testing for total amino acid profile, 100 .mu.L of thedigest was transferred by pipette into a tared 2 mL ampule, and theweight recorded. 2.0 mL of 6 M HCl were added and the ampule wasplaced under nitrogen blanket, sealed and heated at 110.degree. C.for 22 hours. The digest was then evaporated to dryness thenresuspended in 2 mL of Na--S buffer. The resuspension was filteredthrough a Gelman Acrodisc (0.45 um, Gelman P/N 4497).

Prior to testing for soluble amino acid profile, 10 mL of thedigest was transferred by pipette into a tared 50 mL centrifugetube. 10 mL of 24% trichloroacetic acid were added, the tube cappedand mixed well. The weight of the tube contents was recorded. Thetube and contents were centrifuged at 3000 times gravity for thirtyminutes, the liquid was then filtered through Whatman No. 41 paper.200 .mu.L of the filtrate was transferred by pipette into a tared 2mL ampule, and the weight of the sample was recorded. As with thetesting of total amino acid, 2.0 mL of 6 M HCl were added and theampule was placed under nitrogen blanket, sealed and heated at110.degree. C. for 22 hours. The digest was then evaporated todryness, then resuspended in 2 mL of Na--S buffer. The resuspensionwas filtered through a Gelman Acrodisc (0.45 um, Gelman P/N 4497).

If soluble and total amino acid profiles are to be determined fromthe same digestion aliquot, a separation step (such acentrifugation) is not required.

Proteins are hydrolyzed to their constituent amino acids by acidhydrolysis, and the acid hydrolyzate is tested by an automatedamino acid analyzer (Model 6300 Amino Acid Analyzer, BeckmanInstruments, Inc., Palo Alto, Calif.). The analyzer uses ionexchange chromatography to separate the individual amino acids, andpost-column derivatization with ninhydrin to generate amino acidderivatives which are then detected and quantified by acolorimeter.

Tryptophan Determination

Tryptophan determinations were made by the method described by S.E. Garcia and J. H. Baxter in Determination of Tryptophan Contentin Infant Formulas and Medical Nutritionals, J AOAC Int 1992November-December; 75(6):1112-9, incorporated herein by reference.

For tryptophan determination of total protein, 3.0 mL of digest wastransferred to a 50-mL volumetric flask. 1.0 mL of pronase solution(1.7 mg/mL in 0.05 M TRIS, pH 7.5) was added and the volume dilutedto 50 mL with pH 7.5 buffer. The 50 mL solution was then incubatedat 50.degree. C. for six hours. Tryptophan was then determined byHPLC procedure.

For tryptophan determination of soluble protein, 6.0 mL of thefiltrate as prepared for soluble amino acid determination wastransferred by pipette to a 50 mL beaker. 30 mL of 0.05 M TRIS, pHto 7.5, were added. The pH was adjusted to 7.5 using 45% potassiumhydroxide solution. As with the testing of tryptophan for totalprotein, 1.0 mL of pronase solution (1.7 mg/mL in 0.05 M TRIS, pH7.5) was added and the volume diluted to 50 mL with pH 7.5 buffer.The 50 mL solution was then incubated at 50.degree. C. for sixhours. Tryptophan was then determined by HPLC procedure.

HPLC System for Tryptophan Determination

Column: YMC ODS-AQ, 4.6.times.250 mm, 120A, 5 um, Waters#AQ12S052546WT.

Mobile Phase A: 900 mL 0.02 M KH.sub.2PO.sub.4, 100 mLacetonitrile; pH 3.1 with H.sub.3PO.sub.4.

Mobile Phase B: 200 mL laboratory water, 800 mL acetonitrile.

Flowrate: 0.5 mL/minute.

Temperature: 20.degree. C.

Detection: UV at 280 nm, 214 nm.

Injection: 10 .mu.L

Run time: 50 minutes

Elution Program: 0% B from 0-5 minutes, 0-25% B from 5-34 minutes,25-100% B from 34-35 minutes, 100% B from 35-37 minutes, 100-0% Bfrom 37-38 minutes.

Standard solutions: Abbott Laboratories PPD L-Tryptophan at about28 mg/L (High Standard), about 14 mg/L (Middle Standard), and about7 mg/L (Low Standard) in laboratory water.

Calculation of Protein Digestibility

Add the amino acid profile concentrations and tryptophanconcentration for the Reagent Blank Total Protein. Designate thisvalues as "RT".

Add the amino acid profile concentrations and tryptophanconcentration for the Reagent Blank Soluble Protein. Designate thisvalue as "RS".

Add the amino acid profile concentrations and tryptophanconcentration obtained for the Sample Digestion Total Protein.Designate this value as "IT".

Add the amino acid profile concentrations and tryptophanconcentration obtained for the Sample Digestion Soluble Protein.Designate this value as "IS".

Calculate Protein Digestibility as follows -- see Original Patent.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

The use of the terms "a" and "an" and"the" and similar referents in the context of describingthe invention (especially in the context of the following claims)are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into thespecification as if it were individually recited herein. Allmethods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradictedby context. The use of any and all examples, or exemplary language(e.g., "such as") provided herein, is intended merely tobetter illuminate the invention and does not pose a limitation onthe scope of the invention unless otherwise claimed. No language inthe specification should be construed as indicating any non-claimedelement as essential to the practice of the invention.

While some potential advantages and objects have been expresslyidentified herein, it should be understood that some embodiments ofthe invention may not provide all, or any, of the expresslyidentified advantages and objects.

Preferred embodiments of this invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations of those preferred embodimentswill become apparent to those of ordinary skill in the art uponreading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise thanas specifically described herein. Accordingly, this inventionincludes all modifications and equivalents of the subject matterrecited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements inall possible variations thereof is encompassed by the inventionunless otherwise indicated herein or otherwise clearly contradictedby context.

Claim 1 of 11 Claims
1. A method for determining the digestibility of proteins in anutritional product comprising the steps of: a) providing a sampleof a nutritional product that contains protein and a reagent blank;b) separately digesting the sample of a nutritional product and thereagent blank with at least one protein-degrading enzyme; c)terminating each said digestion process to obtain a digested sampleand a digested reagent blank; d) removing a first portion from eachof said digested sample and said digested reagent blank in step c);e) determining the concentrations of at least 18 different aminoacids, including tryptophan, for each of said first portionsobtained from said digested sample and said digested blank in stepd); f) removing a second portion from each of said digested sampleand said digested reagent blank in step c); g) separating each ofsaid second portions from step f) into a liquid phase and a solidphase; h) determining the concentrations of said at least 18different amino acids, including tryptophan, soluble in the liquidphases obtained from said second portions of said digested sampleand said digested reagent blank in step g); i) calculating thepercent digestibility of the protein in the nutritional sample by:1) adding the concentrations of said at least 18 different aminoacids, for each of said first portions obtained from said digestedsample and said digested reagent blank in step e) to provide a sumthat is the total amino acid concentration in each of said digestedsample and said digested blank; 2) determining the differencebetween the total amino acid concentrations of the first portionsof each of digested sample and the digested reagent blank in step1; 3) adding the concentrations of said at least 18 different aminoacids soluble in each of the liquid phases obtained from saidsecond portions of said digested sample and said digested reagentblank in step h); to provide a sum that is the soluble amino acidconcentration in each of said second portions of said digestedsample and said digested blank; 4) determining the differencebetween the soluble amino acid concentrations of the said secondportions from said digested sample and the digested reagent blank;5) dividing the difference between the soluble amino acidconcentrations of step 4) by the difference between the total aminoacid concentrations of step 2) to obtain a quotient; 6) multiplyingthe quotient by 100, thereby determining the protein digestibilityof a nutritional sample.

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