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  Refining in vitro digestibility assays: Fractionation of digestibleand indigestible peptides 1  Y. Qiao 2 , X. Lin, J. Odle, A. Whittaker 3 , and T. A. T. G. van Kempen 4 Department of Animal Science, North Carolina State University, Raleigh 27695  ABSTRACT:  Typically, in vitro methods used for es-timating the amount of ileal digestible AA do not ex-haustively digest samples, and arbitrary methods forseparating digestible from indigestible protein areused. This may lead to over- or underestimation of di-gestibility coefficients. A method that exhaustively di-gests proteins using pepsin and pancreatin was devel-oped, and the first objective of this research was toconfirm that exhaustive digestion was indeed appro-priate and to determine the fractionation method forseparating digestible from indigestible proteins. Forthis, three homoarginine-labeled animal proteins wereprepared. Samples were subsequently digested in vivoand in vitro to determine which fraction should be con-sidered indigestible, and in vitro followed by in vivo todetermine whether the extent of digestion in vivo wasimproved by predigestion. In vivo, soluble but unab-sorbed peptides were smaller than 1 kDa, suggesting that the size of soluble peptides is not what preventstheir absorption. Thus, all in vitro-soluble proteinsshould be considered digestible. In vitro, 88  ±  3% of thesoluble peptides were smaller than 1 kDa, with theremainder between 1 and 5 kDa, suggesting that inKey Words: Amino Acid Digestibility, Animal Protein, In Vitro, In Vivo ©  2004 American Society of Animal Science. All rights reserved.  J. Anim. Sci. 2004. 82:1669–1677 Introduction Ileal AA digestibility is a well-accepted indicator of protein quality (Darragh and Hodgkinson, 2000). How-ever,measuringdigestibilityiscostlyandtimeconsum-ing. Due to this shortcoming, in vitro techniques have 1 Financial support for this research was provided by the Fatsand Protein Research Foundation, the Animal and Poultry WasteManagement Center, and the North Carolina Agric. Res. Service. 2 Current address: Ajinomoto (China) Co. Ltd., 2201 Tower A, FullLinkPlaza,No.18ChaoYangMenWaiDaJie,Beijing100020,China. 3 Current address: 50 E. 98th St., 14P, New York, NY 10029. 4 Correspondence: Campus Box 7621 (phone: 919 515 4016; fax:919 515 7780; e-mail: August 19, 2003. Accepted February 4, 2004. 1669  vitro digestion is less complete. Predigested sampleswere digested in vivo to the same size distribution asthe nonpredigested samples. The second objective wasto test whether in vitro digestibility assays based onthese principles equaled in vivo digestibility. For this,digestibilitydatafor25animalproteinswerecompared.Results showed a lack of correlation between lysinedigestibility coefficients; however, across samples, theextent of digestion did not differ for lysine (  P  = 0.71),threonine (  P  = 0.26), methionine (  P  = 0.18), or valine(  P  = 0.66), whereas in vitro digestibility coefficientswere lower for (the less water-soluble) histidine (  P  =0.05), isoleucine (  P  <  0.01), leucine (  P  <  0.01), and phe-nylalanine(  P =0.05).Inconclusion,invitrodigestibilityassays should exhaustively digest proteins to mimic in vivo digestibility. All in vitro-soluble peptides could beconsidered digestible, because in vivo, no large solublepeptideswereobservedwhosesizepreventedthemfrombeing absorbed. However, an in vitro assay based onthese principles lacked precision for highly water-solu-ble AA, and underestimated digestibility for other AA.Better solubilization of the digesta and more replicatesmay improve the in vitro assay further.beenexplored(BoisenandFerna´ndez,1995;BoisenandMoughan, 1996). These methods typically use pepsinand pancreatic proteases to mimic digestive functionsin vivo. Simulation of the in vivo absorption process isapplied based on the solubility or molecular sizes of digested peptides. The underlying assumption is thatpeptides that are insoluble or greater than a criticalsize (molecular weight cut-off) are not absorbed in vivo.The reported digestibility coefficients, however, aretypicallylowerbyasmuchas40%thanthosemeasuredin vivo (Brule and Savoie, 1988; Savoie et al., 1989).Incomplete digestion resulting from short incubationtime and/or insufficient enzyme use might be reasonsfortheunderestimation.Choiceofthemolecularweightcut-off for separating digestible from indigestible pep-tides(1kDainthesecases)mightalsoaffecttheestima-tion,astoosmallamolecularweightcut-offwouldallowonly very small peptides to be separated as digestible.  Qiao et al. 1670 Table 1.  Amino acid composition of three guanidinatedanimal proteins used in the in vivo and in vitro assays(%, as-fed basis)  Animal protein sample Amino acid A B C Arginine 3.65 3.13 3.94Cysteine 1.16 0.38 1.27Histidine 0.81 1.11 1.28Isoleucine 1.71 1.67 2.05Leucine 3.41 3.20 4.19Lysine 0.47 0.90 0.66Homoarginine a 1.86 1.69 2.50Methionine 0.63 0.77 0.84Phenylalanine 1.94 1.76 2.33Threonine 1.93 1.70 2.21 Valine 2.68 2.30 3.14 a Homoarginine, obtained through guanidination of the lysine resi-dues.Conversionsoflysinetohomoarginine,calculatedas[homoargi-nine/(homoarginine + lysine)  ×  100] were 80, 65, and 79% for samples A, B, and C, respectively. To quantify AA digestibility, a new method has beendeveloped that exhaustively digests proteins based onthehypothesisthat thedigestivecapacityof theanimalis not limiting the extent of digestion. Enzyme use inthis method is minimized, and potential contaminationfrom digested enzymes is corrected for (Qiao et al.,2002). Separation of indigestible and digestible pro-teins, however, remains contentious.The objectives of this research were 1) to study thesize distribution of soluble peptides in vitro and in vivosoastodefineafractionationmethodforinvitroproteindigestibilityassaysandtoconfirmwhetherproteinsareexhaustively digested in vivo, and 2) to validate theresulting in vitro method by comparing in vitro and in vivo digestibility. Materials and Methods  Materials Sodiumcitrate,citricacid,sodiumphosphatedibasic,sodium phosphate monobasic,methanol (HPLC grade),and trifluoroacetic acid (HPLC grade) were obtainedfrom Fisher Scientific (Atlanta, GA). Peptides (HPLCgrade, Table 1) used for size exclusion chromatographywere obtained from Sigma (St. Louis, MO).  O -Methyli-sourea was purchased from SKW Chemicals (Marietta,GA). Animal protein samples were obtained from theFats and Protein Research Foundation (Bloomington,IL). These included 25 samples that had been assayedinvivofordigestibleAAusingthececectomizedcockerelassay and a protein-free diet for determining basal en-dogenous losses (Parsons, 1986). Guanidination of Animal Protein To determine the extent of digestion of proteins inanimal meals, three meat and bone meal samples werelabeled with homoarginine (Table 1). Homoarginine isnot a naturally occurring AA, but because it acts likelysine, its digestion can be used as a marker for feed-stuff protein digestion (Nyachoti et al., 1997). Guanidi-nation of these samples was performed according topublished methods (Maga, 1981; Siriwan et al., 1994;Nyachoti et al., 1997). In brief, 15 L of 0.5 mol/L  O -methylisourea in 1 mol/L of NaOH solution was addedto 6 kg of animal protein (for an  O -methylisourea toprotein ratio of 0.43, wt/wt). The resulting broth wasstirred, and the pH was maintained at 10.5 while thereactionwasallowedtotakeplaceatroomtemperature. After 4 d, the animal protein mixture was neutralizedwith 6 mol/L of HCl, and then washed four times toremove remaining   O -methylisourea. The guanidinatedanimal protein was subsequently freeze-dried, and thehomoarginine content in each animal protein samplewas determined at the Experimental Station ChemicalLaboratories at the University of Missouri (Columbia)using AOAC methodology (AOAC, 1995) to verify theextent of guanidination. The AA compositions of theguanidinated animal protein samples are presented inTable 1. In Vitro Digestibility The in vitro assay that was used in this study was atwo-stage method that was developed to maximize thehydrolysis of the animal protein peptide bonds withminimal enzyme usage (Qiao, 2001). In brief, in Stage1, pepsin (120 U/mL, or 0.25% enzyme protein relativeto substrate protein) was used to digest the substrateproteins (12.5 mg/mL) in citrate buffer solution (pH 2)for 24 h. For Stage 2, phosphate buffer solution for afinal pH of 8 and trypsin-enriched pancreatin (activityequivalent to at least three U.S. Pharmacopeia/mL, or7.5% enzyme protein relative to substrate protein, finalsubstrate concentration 5 mg/mL) were added and thedigestion was continued for an additional 96 h. Thelength of these incubation times correspond to the timeneeded to loose over 95% of the activity of the enzymesin order to maximize their efficacy, thus allowing forminimal enzyme usage. Incubations were carried outat 38 ° C under continuous agitation.For the guanidinated animal proteins, the digestionwas carried out in large containers fitted with twoaquarium heaters to prepare samples for the feeding trial, and in triplicate using 50-mL centrifuge tubes formeasurement of digestibility. For the 25 animal mealsamples, the digestion was carried out in triplicate in50-mL centrifuge tubes. Ileal Digestibility ThisstudywasapprovedbytheNorthCarolinaStateUniversity Institutional Animal Care and Use Com-mittee.To determine whether the extent of in vivo digestionis improved by predigesting the sample, guanidinated  Refining in vitro digestibility assays  1671 Table 2.  Composition (%, as-fed basis) of experimentaldiets fed to ileum-cannulated pigs Ingredient ContentCornstarch 40.67Sucrose 20.70Corn oil 2.07Salt 0.35Dicalcium phosphate 3.11Limestone 0.52Mineral and vitamin mix a 0.35Chromic oxide 0.55 Animal protein b 31.68 a Provided per kilogram of diet: 2,000 IU of vitamin A; 300 IU of  vitamin D 3 ; 20 IU of vitamin E; 1.0 mg of vitamin K (menadione); 4mg of thiamine; 15 mg of niacin; 4 mg of riboflavin; 12 mg of panto-thenic acid; 15   g of vitamin B 12 ; 2 mg of pyridoxine; 0.1 mg of d-biotin; 0.5 mg of folic acid; 0.6 g of choline; 90 mg of Fe (ferroussulfate); 5 mg of Mn (manganese oxide); 8 mg of Cu (copper sulfate);0.20 mg of I (potassium iodate); 0.21 mg of Se (sodium selenite); and90 mg of Zn (zinc sulfate). b  Animal protein: Three animal proteins, A, B, and C, were used.Regularnonguanidinatedanimalproteinswerefedtopigsonadapta-tion days, whereas guanidinated animal proteins were fed to pigs onday of collection. meat and bone meal was tested for ileal digestibilityas is, or after predigestion in vitro. Failure to showimproved digestibility withpredigested material wouldsuggest that in vivo digestion is exhaustive and that in vitro assays should digest to exhaustion as well. Toestimatethesizedistributionofsolublebutunabsorbedpeptides, ileal and in vitro digestibility assays wereperformed with guanidinated animal protein samples.In vivo-soluble but undigested proteins and in vitrodigests were subsequently size separated for determi-nation of the faith of homoarginine, the assumptionbeingthatbesidesinsolubleproteins,largesolublepep-tides of a size to be quantified were indigestible.The design of this trial was a 6  ×  6 Latin square withsix diets (three guanidinated animal protein samples,as is or predigested in vitro), six periods (each of oneweek), and six castrated pigs (53  ±  1.3 kg BW) fittedwith ileal T-cannulae. Animal protein was the sole pro-tein source in the diets (Table 2). Each period consistedof a 6-d adaptation period and a 1-d collection period.During the adaptation period, pigs were fed diets com-posed of the corresponding nonguanidinated and undi-gested animal protein twice daily (0600 and 1600) atapproximately 45 g/kg  0.75 BW per meal (adjustedweekly).On collectiondays, pigswere fedguanidinatedanimal meal (fortified with chromic oxide as a markerfor the indigestible fraction) for the morning feeding,whereasnofeedwassuppliedintheevening.Collectionof ileal juices started at the morning feeding (0600) andcontinued for 19 h. This collection period was sufficientto collect ileal content corresponding to the test diet(as judged by chromic collection). The ileal juices werecentrifuged at 1,000  ×  g  for 30 min to separate solubleproteins from insoluble ones. These fractions were keptfrozen at  − 20 ° C until analysis for molecular weightsof peptides by size exclusion chromatography and AA contents by HPLC. All AA and chromium analyses were carried out atthe Experimental Station Chemical Laboratories using  AOAC-approved methodology (AOAC, 1995). Aminoacid recovery exceeded 96.4%, (T. Mawhinney, Univer-sity of Missouri, personal communication), and no cor-rections were made for incomplete recovery resulting from hydrolysis. The ileal AA digestibilities of the gua-nidinated animal protein samples in the solid feedswere calculated according to van Kempen et al. (2002).These digestibility figures are apparent for all AA ex-cept homoarginine, for which real digestibility is calcu-lated due to the absence of homoarginine in endoge-nous secretions.Digestibility, % = 100  −  [(M d  ×  AA  I )/(AA  d  ×  M I )]  ×  100where M d  = chromium concentration in the diet (mg/ kg),AA  I =AAconcentrationinilealdigesta(g/kg),AA  d = AA concentration in the diet (g/kg), and M I  = chromiumconcentration in the ileal digesta (mg/kg).The ileal AA digestibility coefficients were analyzedwith the PROC GLM procedure of SAS (Version 7, SASInst., Inc., Cary, NC). To compare treatment effects (in vitro vs. in vivo) on the digestibility coefficients of thethree guanidinated meat and bone meals samples, thein vitro and in vivo AA digestibility coefficients wereanalyzed with the PROC MIXED procedure of SAS us-ing the model: Y  ijk  =    +  S i  +  D  j  + (  DS ) ij  +  A (  D )  j(i)  +  P (  D ) k(i)  +  ε ijk where  Y  ijk  = response variable (AA digestibility coeffi-cient) for the  i th sample (  S i ,  i  = 1, 2, 3),  j th digestiontreatment (  D  j , digestion treatment = in vivo, in vitro),and  k th period ( k  = 6 levels [six animals, six periods,missing values were treated as such] for in vivo trial, k  = 1 level [three test tubes, one period] for in vitrotrial),  S i  = MBM sample effect,  D  j  = digestion effect,(  DS ) ij  = interaction between sample and digestion,  A (  D )  j(i)  = animal (test tube) effect within the digestiontreatment,  P (  D ) k(i)  = period effect within the digestiontreatment,    = overall mean,  ε ijk  = residual error withmean of 0 and variance of   σ 2 . The effects  A (  D )  j(i)  and  P (  D ) k(i)  were treated as random effects by using theRANDOM statement. Size Exclusion Chromatography Size separation of ileal and in vitro digesta was con-ducted with a Tosohaas 2000SWXL column (Montgom-eryville, PA) suitable for separation of 500- to 100,000-Da polypeptides (Swergold and Rubin, 1983). The col-umn (7.8 mm i.d.  ×  30 cm in length, silica-based pack-ing, particle size = 5   m, and pore size = 125 A ˚) wasfittedwithaguardcolumn(6mmi.d. × 4.0cminlength,silica-based packing, particle size of 7   m). The HPLCsystem consisted of a model 600 Controller, a model  Qiao et al. 1672 Table 3.  Size of peptides used to calibrate the size exclu-sion column a Item Molecular weight, DaLeucylglycine 176Homoarginine  HCl 225Gly-Gly-Phe-Phe 455Casein fragment 913Bombesin 1,620Diazepam binding inhibitor(DBI, fragment 51 to 70) 2,150Insulin 5,730Ubiquitin 8,565Cytochrome C 12,327Lysozyme 14,400 a Peptides were purchased from Sigma Chemical Co. (St. Louis,MO). 717 autosampler, and a model 996 photodiode arraydetector (Waters Inc., Milford, MA). The mobile phasewas an aqueous solution of 35% methanol and 0.1%trifluoroacetic acid, selected based on solubility of dif-ferent peptides (Swergold and Rubin, 1983; Irvine andShaw, 1986; Vijayalakshmi et al., 1986).Ten peptides of known molecular weight were usedfor calibration of the column (Table 3). These peptideswere dissolved in the mobile phase (0.1%, wt/vol) andfilteredthrougha0.1-  mMilliporefilter(Sigma)beforethey were individually run on the column (materialremaining on the filter was considered insoluble). Mo-bile phase was degassed with an in-line degasser (Wa-ters Inc.). The injection volume was20  L, and the flowrate was 0.51 mL/min. The detection wavelength wasset at 214 nm. Retention times of these peptides wereregressed against their molecular weight using linearregression (SAS), and the obtained regression equationwas used for predicting the molecular weight of size-fractionated digesta/digests.For size separation of the ileal indigestible peptides,ileal juices were first centrifuged at 1,000  ×  g  for 10min. The supernatants were then dried at 35 ° C using an evaporator (Centrivap Console, Labconco, KansasCity, MO). Dried supernatants were pooled on an equalweight basis according to dietary treatment and thenresuspended in mobile phase (with magnetic stirring)at 0.2 g/mL. The resuspensions were filtered with No.4 filter paper (Whatman Int., Ltd., Maidstone, U.K.)and washed with an equal volume of mobile phase. Thefinal concentrationof the re-suspension wasaround 0.1g/mL.Thefiltrateswerefurtherfilteredwitha0.45-  mMillipore filter (Sigma) before loading onto the column.For size separation of in vitro peptides, in vitro di-gests of the three guanidinated animal proteins alsounderwent centrifugation, drying, resuspension, andfiltration as described above for the ileal samples. Be-cause the in vitro digests contained a much higher con-centration of homoarginine than ileal juice, the driedsupernatant was resuspended in mobile phase at 0.05g/mL.In vitro and ileal samples were loaded on the columnandchromatographedunderconditionsasdescribedforthe calibration of the column. The eluent was collectedwithafractioncollector(modelFC-80K,GilsonMedicalElectronicsInc.;Middleton,WI)at1-minintervalsfromtime 0 to 36 min after loading. To ensure there wassufficienthomoarginineforAAanalysis,eachilealjuicesample was run at least 12 times, and each in vitrodigesta sample was run at least seven times. Theeluents were pooled according to sample and elutiontime and analyzed for homoarginine contents by theExperimental Station Chemical Laboratories using  AOAC methodology (AOAC, 1995). Comparison of In Vivoand In Vitro Digestibility Twenty-five animal meal samples with known stan-dardized digestibility were obtained. These sampleshad been evaluated for digestibility using cecectomizedcockerels (n = 5 per sample) and consisted of (as-fedbasis) 19 meat and bone meals (CP = 44.6  ±  4.1%) andsix feather meals (CP = 77.2  ±  3.0%).These samples were digested in vitro as describedabove. In vitro digestibility data were corrected for en-zymeaddedwhichwashydrolyzedintopeptidesconsid-ered digestible (enzyme contamination). This contami-nationwasdeterminedbyperformingtheinvitroassaywithout added substrate (Qiao et al, 2002).Soluble peptides, obtained after centrifugation at15,000  ×  g  for 20 min were considered as digestible. AminoacidsinthisfractionwereanalyzedattheExper-imental Station Chemical Laboratories using AOACmethodology (AOAC, 1995). The in vitro AA digestibil-ity was calculated with the following formula:  Digestibility  =  AA protein in supernatant  −  AA enzyme in supernatant  AA animal protein The in vitro AA digestibility coefficients of the 25reference samples were compared with the known in vivoAAdigestibilitycoefficient.Thenullhypothesis“in vitro  −  in vivo = 0” was tested by a two-tailed paired  t -test using the PROC TTEST procedure. Results Size Fractionation of Peptides In the size-fractionation study, a high correlation be-tweenlog  10 molecularweight(Da)andelutiontimewasfound (log  10  molecular weight =  − 0.223 ( ±  0.016)  ×  time(min) + 7.03 ( ±  0.28), R 2 = 0.96, Figure 1). For smallpeptides(belowthespecifiedlowersizeseparationlimitof the column), the elution time was affected by theirsize and their electrical charges (ion exchange), in linewith findings from Irvine and Shaw (1986). Therefore,elution times longer than 20 min were considered as20 min (383 Da).
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