‘Viraensano Orvgn NUTRENTS (Chapter 45, p. 64 ‘Subchapter 4 NUTRITIONALLY RELATED COMPONENTS: 45.4.01 AOAC Official Method 976.25 ‘Sodium In Foods for Special Dietary Use Jon Selective Electrode Method First Action 1976 Final Action 157 (Applicable to foods containing <100 mg Na/100 g.) A. Apparatus (@) Zectrode—Sodium combination ion selective electrode (Model 96-11, replacement Model 8611), Orion Research, Inc. OF equivalent). (b) Graph parer—Gran’s plot paper, 10% volume corrected (Wo, 9000-90, Orion Research, Ine. o equivalent (©) Magnetic stirer-—with 4. cm (1¥4 in) Teflon-coated stirring bar, Use mat to insulate sample from motor heat (@ pH meter—With expanded mV scale (Model TOLO1A, {replacement Model 290A, T10A, 720A, or 920A], Orion Research, Ine, or equivalent). B. Reagents (8) Bufer solution—pH 102. Total ionic suength adjustment batfer (TISAB), 0.5M triethanolamine. Dissolve 74.6 g wiethano- amine inca 900 ml, HO, adjust pH to 10.2 with HCI, and dilute to 1 Lwith H,0. (b) Sodium standard solutions —{1) Stock solution —10 mg/mL. Accurately weigh 2421 g NaCl, previously dried over- night at 100°, into 100 mL volumetric flask. Dissolve and dilute to volume with H,0, and mix. (2) Working soluions.— 0.1, 1.0, and 2.0 mghal. Pipet 1, 10, and 20 mL stock solution into separate 100 ‘mL volumetric asks, dilute each to volume with buffer solution, and ix . Preparation of Sample Blend entire contents of can, including H,O. Weigh 10.0 g sample and dilute to 100 mL with buffer solution. Mix and transfer to 150 mL beaker, D. Determination (@) For foods with 0-5 mg declared Na/100 g.—Immerse elec trode in sample beaker and sr magnetically 2-S min to equfibrate. Record alV potential on expanded mV scale. From 10 mi-buret add five 1.0 mL portions 0.1 mg Na/mL. working standard solution to beaker, stirring magnetically 30 s after each addition. Plot mV reading for0, 1,2,3,4,and S mL on graph paper, (b). Plot E directly for both blank and sample descending 5 mV for each major line crossing vertical axis. Plot most positive mV reading at top of vertical axis, Daw best straight line through poiats, extrapolate line to horizontal axis, and read mg Na in sample. Perform blank determination on 100 mL. buffer solution, adding five L.0 mL portions 0.1 mg Na/ml. working standard solution, as, above. Plot mV readings on graph paper, omitting 0 mL. reading, such that § mL reading falls near top of paper. Draw best straight line through points, extapolaze line to horizontal axis, and read mg Nain blank ‘AOAC Orricu Merioosor AuaLvsss (1995) _mg Na/100 g food = [(S~) x 100) where $= mg Nain sample, B= mg Na in blank. and W= g sample. (b) For foods with $-50 mg declared Na/100 g.—Proceed as in (a), except use 0 mg Na/ml working standard solution. (©) For foods with 50-100 mg declared Na/I00 g.— Proceed as in(a), except use 2.0 mg Na/mL. working sandard solution, Reference: JAOAC 59, 1131(1976) CAS-7440-23-5 (sodium) 45.4.02 AOAC Official Method 983.23, Fat in Foods CChioroformMethanol Extraction Method First Action 1983 Final Action 1984 (Method is applicable to composite foods and foods for which ‘methods of analysis for fat or lipids are not specified. Method is for lipids, not for fats (triglycerides and other ether-soluble materials.) (Caution: See Appendix B,safery notes on blenders, distillation, ‘and chloroform) A Apparatus and Reagents (a) Blending osembly—Se-mico, snes sel blending a sembly for Wag blender (Scientfie Produc Ca, Cat-No.SE395- Thorequivalen (@) Ene solution —1% Claas 40,00 in 05M sodium ace- tx solution Suspend 10 Clase 4,000 (Ges Laborato. ne. 11a? Myre St PO Box 70, Elkhart, IN 46514) in 200 ml. OSM sodium seetate Slaton in 1 Lvolumenie flask, due to volume with 0.5M sodium acetate solution, and mix thoroughly. (Stable at least 7 days when stored at 4°.) 8. Detormination ‘Accurately weigh ca 5 g well-minced sample (3 g if >10% fat) into 50 mL digestion tube, add enough enzyme solution so that total H,O content (enzyme solution added plus original moisture in sample) is 32 mL. Shake gently until sample is thoroughly mixed with enzyme solution and place tbe in 45-50" H,O bath 1 b. Mix thoroughly and quantitatively transfer digest to blending assembly ‘with 80.0 mL methanol and 40.0 mL CHC. Cover and blend? min at high speed. Remove cover, add 40.0 mL HCL, cover, nd blend 30s. Remove cover, add 40 mL. H,0, cover, and blend additional 30. ‘Transfer extract to 250 mL polypropylene centrifuge bottle and ccenrifge 10 min at ca 3000 rpm (when rotor diameter is 11.5 in.) toclarify bottom CHCI ayer. Pipe 20.0 mL CHCl, extract into tare 100 mL beaker. (Care should be taken to prevent ransferring aque ‘ous phase.) Evaporate to dryness on steam bath in fume hood, Dry fat residue in 101° oven to constant weight (ca 30 min), coo! in desiccator to constant weight (ca 30 min), and weigh. Calculate % fat = (g residuelg sample) x4 x 100. (Wore: Ratio of CHCL-methanol-H,0 is critical for quantitative extraction of fat. This may necessitate determination of moisture ‘content of sample to optimize amount of H.O [as enzyme solution) in initial extraction step. Reference source such as USDA Agricul- ture Handbook No. 8, Composition of Foods, or USDA Agriculture‘AAG OFRicis. METHODS OF ANALYSIS (1985) Handbook No. 456, Nuritive Value of American Foods, is adequate for this purpose. Also use as guide to pre-estimate fat conten so that proper sample weight canbe chosen fr analysis) Reference: IAOAC 66, 927(1983). 45,4.03 p AOAC Official Method 975.44 Lysine (Available) In Nutritional Supplements ‘Automated Method First Action 1975 Final Action 1976 A. Principle ) 1 Flooe-24-dinirobenzene (DNFB) reacts wih fre exnipe groups in proteins, forming DNFB-e-amino lysine which is stable to acid hydrolysis. Sample s acid hydrolyzed and unavailable lysine is determined with amino acid analyzer, total lysine is determined on untreated sample. Available lysine, which was bound by DNFB, is determined by difference. B. Reagents (Use deionized H,0 throughout) (@) Sodium ciate buffer-—pH 2.20 + 0.03. Dissolve 19.6 sodium citrate 2H,0 in H,0, add 16.5 mL HCI, 5.0 mL thiodiglyeol, (TG), 1.0 g Bri-35 dissolved in H,O by heating, and 0.1 mL. n-eaprylic acid and dilute to 1 L. Filter before using. (b) Sodium citrate buffer—pH 5.28 + 0.02. Dissolve 137.26 sodium citrate2H,0 in H,0, add 26.0 mL HCl, 4.0 g Bri-35 dissolved in HO by beating, and 0.4 mL n-caprylic acid, and dure to4 L. Fiker before using. (©) Sodium citrate buffer with 2% n-propanol—pHi $28 £0.02. Prepare as in (b), but add 80 mL n-propanol before dilution 4 L. (@) Sodium acetae buffer—AN, pH 5.51 + 0.03. Add 1088 NaCH;COO3H,0 to 800 mL H,O. Stir while heating on steam or HO bath until solution is complete and cool to room temperature. ‘Add 200 mL CH,COOH and stir. Add H,0 to final volume of 2L, adjust pH, if necessary (1g NaOH/2 L causes change of 0.02 pH). and filter before using. (©) Regeneration reagent.—Prepare 0.1N’ Brij-35/L. (0) Ninhydrin reagent —Prepare and introduce into reservoir in absence of O;. Bubble slow stream of N; through Sblution while mixing and string to displace any air. Use magnetic stirer with seamless Teflon bar, 053". Add3 L filtered peroxide-fre Methyl CCllosolve (colories solution must be produced when 3 mLis mixed ‘with 3 mL 4% KI solution) to 1 L filtered 4N NaCH,COO buffer solution, and stir magnetically 15 min while bubbling Na through solution atea I cufvb. Ad 80 g ninhydrin (Caution: Weigh inhood) ‘and sti magnetically with N, bubbling until ninhydrin is completely dissolved (asually 6-10 min). Weigh exactly 1.600 g SaCly2H,0 and add to solution, rinsing with MetiyTCellosolve to final volume. ‘Reageat turns dep ruby red as SnCl dissolves but color fades after ca 2ht0 yellow-green when fresh and yellow-brown later. Continue stirring with Ny bubbling until SaCl, dissolves completely (c2 3-8 nin). {30H containing 1 g \Vermuns ano Orr NUTRIENTS (Chapter 45, p. 65 (. Preparation of Protein Hydrolyeate Grind sample in Wiley mill with No, 20 seeen and mix well. ‘Weigh 0.1-1.0 g sample into No 510 crcible (1.3 ml). (Calculate sample weight to give final concentration of 0.72-0.88 mg pro- tein/nL after dilution with 100 mL sodium crate buifer, pH22. _mg Sample t use = [final concentration desired (mg/mL) 100 (aL) \% protein in semple/100) butdo nocuse>1 g) ‘Place sample or sample and crucible in S00 ml boing ask and add 4-5 glass beads. Add 10 ml freshly prepared 10% NaHCO, soliton, 10 mL aleobol, and (4 mL dinirofiuorobenzene (DNFB). Stopper flask and shake mechanically 53h. Carefully sity with NHC (ca2ml.).Evaporatetoolly dryness at 40" in vacmumrotary evaporator Release vacuum very slowly to avoid disturbing residue. ‘Add 50-75 ma anydrous ether, decent, end r-evaporas in rotary fevaporeor at 40° without vacuum. Repeat washing with eber and evaporation 3 ational times. (Caution: See Appendix B, safety ‘notes on distillation ad diethyl etx) "Aédca 125 ml GN HCL Heat careflly until all CO, i leased, and then boil under reflux 18 h maintaining constant seeam of 1H,0-pumped or prepurified N through Tygon capillary tbe which comes toca 25 om of surfce of solution. Cool 1 hand wah down residue in condenser. Evaporae to sticky paste in vacuum rotary evaporator a 40°. Repeat addition of 10 mL HO and evaporation 4 additional times, evaporating to drypess daring last evaporation. 1D. Preperation of Protein Hydrolysate Without DNFB ‘Weigh sample to give final conceatration of 0.18-0.22 mg pro- teivmL after dilution with 100 mL sodium citrate buffer, pH 2.2, {nto 5/0 crucible. Place sample or sample and eruible into 5C0 mi. boiling flask and add 4~$ glass beads. Add 200 mL. 6N HCl and distil off 100mL. Hydrolyze under reflux 24h maintaining constant stream of N, as in 97544C. Evaporate to sticky paste in vacuum, rotary evaporator at 40°. Repeat addition of 100 mL. H,0 and ‘evaporation 5 additional times, evaporating to dryness during last ‘evaporation Proceed asin 97S.44E, except use protein concentration of 0.18 0.22 mp/ml and 6 em prepared column. Determination Pipe in 100 sodium citrate buffer. pH2.2. Stopperflask shake 5 rnin, and filter through Whatman No. 43 paper into storage bore. Use 1.0 mb filtrate containing 0.72-0.88 mg protein/ml on 12 ém ‘column of Beckman Custom Research Resin Type PA-35, regener- sted after NH; peak of each sample with regeneration reagent and ‘equilibrated with sodium citrate butfer with 2% n-propy] slosbol. ‘Operate automatic amino acid analyzer (Beckman Model 120C {replacement Model 6300], or equivalent) in accordance with in- structions of manufacturer. Identify lysine peak by comparison with calibration aim with lysine or standard calibration mixture under same operating conditions as used in analysis. Determine area under curve for lysine or use iniéprato, and compare areas of samples wit those from calibration stndards containing known concentration of lysine (¢.g., 2500 + 0.004 m/nl O.LNHCD. ‘To calculate & available lysine, subwact % lysine of DNFB- treated sample from % lysine of non-DNFB-treated sample.