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Digestion of wheat gluten and potato protein by thepreruminant calf: digestibility, amino acid composition

and immunoreactive proteins in ileal digestaP Branco-Pardal, Jp Lallès, M Formal, P Guilloteau, R Toullec

To cite this version:P Branco-Pardal, Jp Lallès, M Formal, P Guilloteau, R Toullec. Digestion of wheat gluten and potatoprotein by the preruminant calf: digestibility, amino acid composition and immunoreactive proteinsin ileal digesta. Reproduction Nutrition Development, EDP Sciences, 1995, 35 (6), pp.639-654. �hal-00899809�

Original article

Digestion of wheat gluten and potato proteinby the preruminant calf: digestibility,

amino acid composition and immunoreactiveproteins in ileal digesta

P Branco-Pardal, JP Lallès, M Formal P Guilloteau R Toullec

INRA, laboratoire du jeune ruminant, 65, rue de Saint-Brieuc, 35042 Rennes cedex, France

(Received 6 March 1995; accepted 27 September 1995)

Summary &horbar; Three milk substitute diets, in which the protein was either provided exclusively by skimmilk powder or partially (52%) substituted by a native wheat gluten or a potato protein concentrate, weregiven to intact or ileo-caecal cannulated preruminant calves. The apparent faecal nitrogen digestibilitywas lower (P < 0.05) with the potato than with the gluten and control diets (0.90, 0.93 and 0.95, respec-tively). The same trend was observed at the ileal level (0.83, 0.87 and 0.91, respectively). Apparentdigestibilities of most amino acids were lower with the potato than with the control diet (P< 0.05 for glu-tamic acid, proline, cystine, methionine, isoleucine, leucine, tyrosine and lysine). The same trend wasobserved with the gluten diet. Apparent digestibilities of glutamic acid and cystine were also lower(P < 0.05) with the potato than with the gluten diet. Protein fractions of Mr 43 000 and below 14 000 weredetected immunochemically in ileal digesta corresponding to the potato diet, but no immunoreactivitywas found in digesta with the gluten diet. However, the considerable enrichment of digesta in glu-tamic acid and proline with gluten indicates that dietary protein fractions rich in these 2 amino acidsescaped digestion in the small intestine. With the potato diet, the undigested fractions contained highlevels of aspartic acid, glutamic acid and cystine.

digestion / preruminant calf / wheat gluten / potato protein / amino acid / immunoreactive pro-tein

Résumé &horbar; Digestion du gluten de blé et des protéines de pommes de terre chez le veau pré-ruminant : digestibilité, composition en acides aminés et en protéines immunoréactives desdigesta iléaux. Trois laits de remplacement dans lesquels les protéines étaient apportées en totalitépar de la poudre de lait écrémé ou partiellement (52%) par du gluten de blé vital ou un concentrat pro-téique de pomme de terre ont été distribués à des veaux préruminants intacts ou munis d’une canule!!!

*

Correspondence and reprints

réentrante iléo-coecale. La digestibilité apparente de l’azote a été moins élevée (p < 0,05) au niveau fécalavec le régime pomme de terre qu’avec les aliments gluten et témoin (respectivement 0,90, 0,93 et 0,95).Il en a été de même à la fin de l’iléon (respectivement 0,83, 0,87 et 0,91). La digestibilité apparente dela plupart des acides aminés a été moins élevée avec l’aliment pomme de terre qu’avec l’alimenttémoin (p < 0,05 pour l’acide glutamique, la proline, la cystine, la méthionine, l’isoleucine, la leucine,la tyrosine et la lysine). Il en a été de même pour l’aliment gluten, mais seule la différence concernantla lysine a été significative. La digestibilité apparente de l’acide glutamique et celle de la cystine ont éga-lement été moins élevées (p < 0,05) avec l’aliment pomme de terre qu’avec l’aliment gluten. Des frac-tions protéiques de Mr 43 000 et inférieur à 14 000 ont été détectées immunochimiquement dans lesdigesta correspondant à l’aliment pomme de terre, mais aucune immunoréactivité n a été observée dansceux obtenus avec l’aliment gluten. Toutefois, l’enrichissement considérable des digesta en acideglutamique et en proline avec le gluten indique que des fractions alimentaires riches en ces 2 acidesaminés ont échappé à la digestion dans l’intestin grêle. Dans le cas de l’aliment pomme de terre, lesfractions indigérées ont été caractérisées par des teneurs élevées en acide aspartique, acide glutamiqueet cystine.

digestion / veau préruminant / gluten de blé / pomme de terre / acide aminé / protéineimmunoréactive

INTRODUCTION

The replacement of milk protein with alter-native sources in milk replacers is of greatinterest because of the high cost of the for-mer. However, preruminant calves are verysensitive to protein quality. Inclusion of pro-tein sources such as soya (Paruelle et al,1972; Guilloteau etal, 1986; Khorasani etal,1989), methanol-grown bacteria (Guilloteauetal, 1980; Sedgman etal, 1985), fish (Guil-loteau et al, 1986) or whey (Toullec et al,1969) in the milk replacer caused adecrease in nutrient digestibility, growth andfeed efficiency. There has recently beenincreasing interest in the use of proteina-ceous crops grown in Europe and industrialproducts. However, the utilization of some ofthese crops such as peas (Nunes do Pradoet al, 1989; Bush et al, 1992a) or lupins(Tukur et al, 1995) may be limited by theirrelatively low protein content and perhapsreduced palatability or antigenic activity,which may decrease animal performance.

Wheat gluten is widely used for humanconsumption. It can be an alternative proteinto skim milk for the preruminant calf. Theflour may be extracted for the preparationof a product with a high protein content

(82%), which has unusual viscoelastic prop-erties. This characteristic may limit the glutenincorporation in diets to be given in liquidform. In addition, wheat gluten is devoid ofantinutritional factors, such as lectins and

oligosaccharides, but it does contain someprotease inhibitors (Liener and Kakade,1972). High digestibility values have beenfound at the faecal (Tolman and Demeers-man, 1991) and ileal levels (Bush et al,1992b) for diets rich in solubilised wheatprotein. However, little is known about thedigestion of native wheat gluten (Toullecand Grongnet, 1990).

Potato protein, a by-product of the potato-processing industry, is another ingredientused in milk substitutes. Recovering potatoproteins is of great interest not only toreduce their contribution to environmental

pollution, but also because it is consideredto be one of the more valuable plant pro-teins (Nuss and Hadziyev, 1980; Lindner etal, 1981 ). Industrial recovery of the proteinfrom the effluents of potato starch mills isgenerally carried out by heat coagulation(Oosten, 1976; de Boer and Hiddink, 1977;Knorr et al, 1977). Although potato proteinhas an adequate amino acid (AA) compo-sition and a low antitryptic activity, its use

in milk replacers for calves would probably

remain limited because of its poor sus-

pendability, low supply and digestibility (Kolarand Wagner, 1991). However, the ilealdigestibility of potato protein has not yet beenevaluated.

The aim of this study was to determinethe influence of native wheat gluten andpotato proteins on ileal and faecal

digestibilities of nutrients in preruminantcalves.

MATERIALS AND METHODS

Diets

Two commercial protein concentrates containing84 and 82% crude protein (CP; percentage ofdry matter (DM)) prepared from wheat flour(Triticum aestivum) and potato juice (Solanumtuberosum L), respectively, were used (table I). ).The wheat concentrate was native gluten. Thepotato concentrate was obtained by heat coagu-

lation of the juice. Both products were provided byRoquette Fr6res (Lestrem, France).

Three milk replacer diets (control, gluten andpotato) containing approximately 23% CP and21% fat (DM basis) were prepared (table I). Inthe control diet, all the protein was provided byskim milk powder (SMP) (table II). In the other 2diets, 51-52% of the protein was from either ofthe 2 concentrates; the remainder was provided bySMP and synthetic AA. L-Lysine-HCL andDL-methionine supplements were calculated inorder to obtain total levels of 1.8 and 0.9% for lysineand sulphur-containing AA, respectively. The sub-stitution rate was chosen in order to utilize the sameSMP-based fat premix in the 3 diets. Actually, thegluten and potato diets were obtained at each mealby mixing the protein concentrates and comple-mentary diets containing all the other ingredients.The AA composition of the protein concentratesand the whole diets is given in table III. l .

Animals, feeding and digesta collection

Six Holstein male calves (Experiment 1) wereobtained at about 8 d of age, and fed a SMP-

based milk replacer. The calves started to con-sume the experimental diets at about 8 weeks ofage. Each calf received each experimental diet for2 weeks in different orders. The animals werefed twice daily (at 0830 and 1630) using openbuckets. The amount of DM offered (58 g/kg° 75/d)was adjusted weekly to the body weight of theanimals. The switch between 2 test diets was

accomplished over 2 d as described by Bush et al(1992b). Total faeces were collected during thelast 5 d of each experimental period, and repre-sentative aliquots were frozen prior to subse-quent freeze-drying and analysis.

Experiment 2 involved the same experimentaldesign as Experiment 1 and used 5 Holstein heifercalves. At about 10 weeks of age, calves werefitted with an abomasal catheter and a reentrantileo-caecal cannula (Guilloteau et al, 1986). Twoweeks post-surgery, the calves started to receivethe experimental diets by means of abomasal

infusion twice daily. Abomasal infusion was usedin order to get a perfect control of intake. Theamount of DM offered was similar to thatdescribed in Experiment 1. Total digesta werecollected from the ileum over the last 4 d of each

experimental period (Guilloteau et al, 1986). Col-lection occurred under continuous stirring in flaskscontaining sodium benzoate (10 g/kg digesta) tolimit microbial activity. Digesta were weigheddaily, and representative aliquots were frozenand freeze-dried before analysis.

Chemical analyses

The protein sources, control and complementarydiets, faeces and ileal digesta were analysed forDM, nitrogen (N), fat and ash according to previ-ously described methods (Guilloteau et al, 1986).

Mineral elements were analyzed in the proteinsources, control and complementary diets byatomic absorption spectrometry, except phos-phorus and chloride, which were determined bythe phosphomolybdate method and potentio-metry, respectively.

The protein sources, control and comple-mentary diets, and ileal digesta were also anal-ysed for AA by ion exchange chromatographyafter acid hydrolysis of samples in 6 N HCI at110°C for 24 h, or 48 h in the case of valine andisoleucine. For the determination of sulphur-containing AA, oxidation with performic acid wascarried out prior to acid hydrolysis. Phenylala-nine was not accurately determined in ilealdigesta, probably because of their high muco-protein content; therefore, the results for that AAwere discarded. Tryptophan, which is destroyedby acid hydrolysis, was not analysed.

Proteins for immunoassays were extractedfrom the control diet and the corresponding

digesta in phosphate-buffered saline (PBS)(0.14 M NaCI, 0.02 M Na2P04, 0.0025 M KCI,0.0015 M KH2pO4, pH 7.4), from gluten and thecorresponding digesta using an ethanol/watersolution (70:30 vol/vol), and from the potato con-centrate and the corresponding digesta in boratebuffer (0.1 M NaB03, 0.15 M NaCl, pH 8.0). Theextracts were prepared by continually stirring afixed amount of sample (1 g/20 ml) for 60 min atroom temperature (25°C). They were then clearedby centrifugation at 12 000 g for 30 min. The sol-uble protein in the extracts was determined bythe procedure of Lowry et al (1951 ).

Antibodies were raised in rabbits against’native’ proteins, ie extracted as described abovefrom the gluten and potato concentrates. Eachantigen was injected subcutaneously in 30 dif-ferent sites in 3 rabbits. The amount of proteininjected was approximately 1 mg for each anti-gen emulsified (vol/vol) in complete (first injec-tion) or incomplete Freund’s adjuvant (Sigma, LaVerpillibre, France) (following injections). The

immunization was repeated 4 times at 21 d inter-vals.

Immunoreactive proteins from the gluten andpotato concentrates as well as from digesta weredetected by a dot-blotting technique (Stott, 1989).Nitrocellulose (NC) sheets (Hybond-C super,Amersham, Buckinghamshire, UK) were cut tofit a 96-hole dot-blotting apparatus (ConvertibleFiltration Manifold System, Gibco BRL, Gaithers-burg, MA, USA). After immersion in PBS, themembranes were placed in this system. Serialdilutions (1:1) of standardized protein solutions(0.15 mg/ml) were spotted (5 wl) under vacuum onthe NC disks in the wells. After 10 min, the mem-branes were removed and immersed in a block-

ing solution (SMP, 50 mg/ml) in Tris buffer (Tris10 mM, NaCl 150 mM, pH 8.0) and placed inshaking trays. After 30 min, the membranes wererinsed 3 times with a Tris-Tween 20 (0.5%,vol/vol) buffer. Specific sera (dilution 1:500) werethen incubated with the NC membranes. Theywere finally removed, washed in Tris-Tween 20buffer, and incubated with antirabbit lgs conju-gated with peroxidase (Sigma). After washing,the membranes were incubated with a solutionof diaminobenzidine (Sigma) as the enzyme sub-strate, until an optimal brown staining developed(usually after 5 min). The membranes were driedfor 2 h at 37°C prior to visual analysis. Skim milkpowder and digesta from the control calves wereused as negative controls. Antigen levels wererecorded as titres, eg, log2 dilutions, above whichthe brown colour disappeared. Protein concen-trates and digesta samples were also analysed byimmunoblotting after SDS-PAGE electrophore-sis and electro-transfer on NC membranes (Ger-shoni and Palade, 1982; Stott, 1989).

Statistical analyses

The results were subjected to analysis of vari-ance with repeated measures and the meanswere ranked according to Sheff6’s test. Fried-man’s test was also used when variances werenot homogeneous. Comparisons between theileal and faecal digestibilities were carried outby the U-test. Significance was declared atP < 0.05.

Amino acid profiles of digesta protein werecompared among themselves and with those ofdietary, endogenous and bacterial proteins bythe distance of x2 (Guilloteau et al, 1983). The

distance of X2 between 2 proteins i and j is cal-culated as follows:

where AAik and AAjk are the percentages of AAkin the sum of the assayed AA in protein i and j; krepresents the different AA and varies between 1and 16. As the x2 distance decreases, the simi-larity between the proteins increases. The pro-portions of dietary, endogenous and bacterialproteins which could be the main constituents ofdigesta protein were assessed by the methoddeveloped by Duvaux et al (1990). This methoduses multiple regression analysis to establish thetheoretical mixture of these proteins which mini-mizes the x2 distance with regard to the AA pro-file of digesta. The compositions of the proteinsources (table III), as well as literature data forgluten fractions and whole potato protein (listedbelow in table VI), were used for dietary protein.The mean composition of axenic lamb faeces(Combe, 1976) and calf meconium (Grongnet etal, 1981) was used as the model for undigestedendogenous protein. The mean composition ofpig (Mason et al, 1976) and sheep (Mason, 1979)faecal bacteria was used to represent the com-position of ileal bacteria. The common proteinescaping digestion in the small intestine of calvesgiven diets based on milk, fish or soyabean pro-tein (Guilloteau et al, 1986) was used as themodel for the mixture of undigested endogenousand bacterial proteins (MEBP).

RESULTS

Nutrient digestibility

The apparent faecal nutrient digestibilitywas significantly lower (P < 0.05) with thegluten than with the control diet, except forN and nitrogen-free extract (NFE) (table IV).A similar trend was observed between the

potato and the control diets, and the differ-ences were significant for all nutrients exceptNFE. The digestibility of N was lower (P <

0.05) with the potato than with the glutendiet.

Both the gluten and potato diets resultedin lower ileal digestibility values than the con-trol diet. However, the differences were onlysignificant with the potato diet for DM, organicmatter, N and minerals. Moreover, ilealdigestibility values tended to be lower withthe potato than the gluten diet, but the onlysignificant difference was for minerals.Digestibility was significantly lower at the ilealthan at the faecal level for the 3 diets, exceptfor fat with the control and gluten diets.

The apparent ileal digestibilities of AAwere lower with the gluten than the controldiet, except for cystine, but the differenceswere only significant for lysine (table V). Asimilar trend was observed between the

potato and the control diets (except for argi-nine) with significant differences for glutamicacid, proline, cystine, methionine, isoleucine,

leucine, tyrosine and lysine. The potato,compared with the gluten diet, also resultedin a lower digestibility (P < 0.05) of glutamicacid and cystine. The digestibility of aminoacid N (AAN) was also lower for the glutenand potato diets than for the control diet,but the differences were not significant (P >

0.05). Digestibilities for all 3 diets, as com-pared to AAN, were higher for valine,methionine, isoleucine, leucine, tyrosine andarginine, and lower for aspartic acid, threo-nine, glycine, alanine and cystine.

AA composition of protein sourcesand ileal digesta

The gluten concentrate contained consid-erably more glutamic acid, proline, glycine,

cystine and less aspartic acid, leucine andlysine than SMP (table 111). The potato con-centrate contained more aspartic acid andglycine, and less glutamic acid and prolinethan SMP. Consequently, large distancesof X2 (!! 88) were observed between diets.

The proportion of AAN (% total N) waslower in ileal digesta than in the corre-sponding diets (72, 71 and 65 vs 76, 78and 80, with the control, gluten and potatodiets, respectively). Digesta protein con-tained considerably more threonine, glycine,alanine, cystine (and glutamic acid with thepotato diet), and less methionine,isoleucine, leucine, tyrosine and lysine (andproline and arginine with the control and

potato diets, respectively) than the corre-sponding diets (fig 1; table III). This resultedin very large distance of x2 between digestaand the corresponding diets (> 161)(table VI).

Compared to control digesta, glutendigesta contained more proline and less ala-nine, valine and lysine (P < 0.05) (fig 1 ). Inpotato digesta, the proportions of asparticacid, glycine and cystine were higher (P <

0.05) than in control digesta. Finally, theproportion of proline was higher and that ofaspartic acid lower (P < 0.05) in gluten thanin potato digesta. The global differenceswere larger between gluten digesta on theone hand and control or potato digesta on

the other (x2 = 83 or 81) than between con-trol and potato digesta (x2 = 45) (table VI).However, these values were small whencompared to those recorded betweendigesta and the corresponding diets. Digestawere also more different from the proteinsources (x2 > 264). Gluten digesta differedlargely from the major fractions or subunitsof gluten (x2 143). Purified glutenin wasthe protein the least different from glutendigesta, but the considerable x2 distance

(143) showed that this protein, as a whole,was poorly represented in digesta. The AAcomposition of digesta was also very dif-ferent from that of endogenous and bacterialproteins (x2 176).

The amounts of AA recovered at the endof the ileum relative to DM intake are pre-sented in table VII. These amounts were

always higher with the gluten and the potatodiets than with the control diet. The differ-ences between the potato and the controldiets were significant for glutamic acid, pro-line, histidine and arginine. Differencesbetween the 2 plant proteins were also sig-nificant for aspartic acid and lysine. Theadditional undigested protein fractions, com-

pared with the control diet, were rich in glu-tamic acid and proline with the gluten diet,and in arginine and glutamic acid with thepotato diet. These braces of AA repre-sented 54.6 and 37.5% of the total, respec-tively. These fractions were very differentfrom the endogenous, bacterial, gluten andpotato proteins (x2 223). These werecloser to the corresponding whole digesta inthe case of potato than for gluten (x2 = 55

and 377, respectively).

Immunoreactive proteins in ileal digesta

Immunoreactive proteins, determined byimmuno-blotting analysis, were detected inthe extract of the gluten concentrate but notin the corresponding digesta. In contrast,immunoreactive proteins were detected inboth the potato concentrate and digesta.These represented approximately 6% of theamount present in the potato protein extract(fig 2). However, the detected immunore-active proteins belonged to the soluble pro-tein fraction which represented only 2% oftotal potato protein. Finally, immuno-blot-

ting analysis showed that the immunoreac-tive proteins detected in the soluble frac-tion of the potato concentrate had relativemolecular weights (Mr) of 43 000, 22 000,20 000 and 14 000, respectively after SDSdenaturation. The immunoreactive proteinsfound in ileal digesta when the potato dietwas used had Mr values of 43 000 andbelow 14 000.

DISCUSSION

The digestibility of nutrients was lower withthe gluten and potato diets compared to thecontrol diet, both at the faecal and ileal lev-els (table IV). Nitrogen digestibility was alsolower with the potato than with the glutendiet. The calculated value of the apparentfaecal N digestibility for the gluten concen-

trate was 0.91, which is slightly below thevalue obtained by Toullec and Grongnet(1990) (0.94). This difference could beattributed to different inclusion levels (14.5vs 5.5%). In contrast, the calculated value ofN digestibility for potato concentrate (0.86)was slightly higher than previously recorded(0.83) with a similar product from this labo-ratory (R Toullec and JY Coroller, unpub-lished data).

The increased apparent digestibility ofmost nutrients between the ileal and the

faecal levels is in line with earlier reports onmilk replacers diets containing milk and non-milk protein sources (Guilloteau etal, 1986;Nunes do Prado et al, 1989), except for fat,which was expected to show no apparentabsorption in the hind gut. This situationcould indicate that the fat digestion in fistu-lated calves was disturbed as compared to

that in intact calves. However, as the

changes were similar for the 3 diets thisshould not affect the other comparisons.The apparent ileal digestibilities of N for thegluten and potato concentrate were 0.84and 0.76, which represented 92 and 83%,respectively, of that observed for SMP. Thevalue for gluten is similar to previous dataobtained with solubilised wheat protein (RToullec and P Guilloteau, unpublishedresults), whey, alcohol-treated soyabeanand hydrolysed fish protein concentrates(Guilloteau etal, 1986; Caugant etal, 1993a,b). In contrast, the value for the potato con-centrate was lower than values quoted pre-viously and values obtained for a processedsoyabean flour and pregelatinized peas(Nunes do Prado et al, 1989; Caugant etal, 1993a). Therefore, native gluten appears

to be easily digested by preruminant calves,whereas additional processing couldimprove the digestibility of the potato pro-tein concentrate.

The amounts of undigested AA recov-ered at the distal ileum were always higherfor the gluten and potato than for the controldiet (table VII). Consequently, the truedigestibility of AA for the gluten concen-trate and especially for the potato concen-trate was always lower than 1 (0.92 and0.87 for NAA; table V). Essential and semi-essential AA, except histidine and cystine,were more digestible than NAA for bothconcentrates. The relatively poor digestibil-ity of cystine seems to be usual since thiswas previously observed with soya, peasand fish (Guilloteau et al, 1986; Nunes doPrado et al, 1989; Bush et al, 1992a). How-

ever, solubilised wheat protein does notpresent this characteristic (R Toullec andP Guilloteau, unpublished data). The resultsconcerning the true digestibility of proteincan be compared with those obtained insimilar experiments in this laboratory withwhey and alcohol-treated soya protein con-centrates (Caugant et al, 1993a, b). Over-all, the values obtained with the gluten con-centrate were lower than those of whey for rthe essential AA, except for threonine,methionine, isoleucine and lysine. The high-est differences were found for histidine and

cystine (-0.11 and -0.06). The gluten con-centrate was digested to the same extentas the alcohol-treated soya concentrate,except for histidine (-0.05); it even showed

slightly higher values for threonine, cystine,valine and isoleucine (+0.03 to +0.05). Incontrast, the true digestibility of potato pro-tein was clearly lower than that of wheyconcentrates, except for arginine. The dif-ference was particularly noticeable for cys-tine (-0.34). The potato concentrate wasalso less digestible than the soya concen-trate, but differences were small except forcystine (-0.24).

In milk-fed calves, the protein whichescapes digestion in the small intestineappears to be almost entirely from endoge-nous and bacterial origin (Guilloteau et al,1986). Replacing 52% of SMP by gluten orpotato protein induced some changes in theAA composition of ileal digesta (fig 1 Dif-ferences between gluten and potato digestawere also noteworthy. The theoretical mix-ture between control digesta and gluten pro-tein which minimized the x2 distance (x2 =

31) with gluten digesta contained 33% ofgluten concentrate (P < 0.05) (table VI). Thiswould suggest that the lower apparent Ndigestibility of the gluten diet might be due toincomplete digestion of gluten protein. Thex2 distance between the major proteins ofgluten and their respective digesta sug-gested that glutenin could be the major undi-gested fraction. However, the AA composi-

tion of the additional undigested proteinobtained with the gluten diet, compared withthe control diet, was very different fromwhole gluten, endogenous or bacterial pro-tein (X2 > 495). This fraction was particu-larly rich in glutamic acid and proline asfound in a previous experiment with hydro-lysed gluten (Bush et al, 1992b). It could bemainly constituted by the repetitive patternof Pro-Gin-Gin-Pro-Phe-Pro-Gln which isabundant in the N-terminal domain of the

major proteins of gluten (Tatham andSchrewry, 1985). When calculations werecarried out, after excluding glutamic acidand proline, the x2 distance between controland gluten digesta decreased (x2 = 41,instead of 83). Moreover, the x2 distancebetween gluten digesta and MEBP wasdrastically reduced (x2 = 39, instead of 228).This further supports the hypothesis thatthe background AA composition would beclose between these digesta.

The theoretical mixtures between con-trol digesta and dietary potato protein whichminimized the x2 distance with potatodigesta contained only 1 % potato protein.Therefore, the lower apparent N digestibilityobserved for the potato diet might be moredue to increased losses of undigestedendogenous and bacterial proteins than tothe incomplete digestion of dietary protein.The AA composition of the additional undi-gested protein due to potato protein wasvery different from whole dietary protein(x2 > 223) but was closest to whole digesta(x2 = 55). In addition, no theoretical mixtureof that protein with endogenous and bacte-rial proteins could be found to give a satis-factory fit with additional undigested frac-tion. Therefore, only small amounts ofprotein fractions having AA profiles similar tothat of intact potato protein probablyescaped digestion. These fractions wererich in aspartic acid, glutamic acid and cys-tine which appeared to be of relatively lowapparent availability.

Immunoreactive proteins

No immunoreactivity could be found ingluten digesta with the antibodies preparedagainst the native form of gluten protein.This probably means that the partiallydigested gluten fractions present in digestahad lost their reactive epitopes. Moreover, inileal digesta from calves given a diet con-taining soyabean flour, evidence for theacidic polypeptides from soyabean glycinincould only be demonstrated indirectlythrough the use of anti-basic polypeptideantibodies, following SDS-PAGE elec-trophoresis under non-reducing conditions(HM Tukur and JP Lall6s, unpublished data).The immunoreactive proteins detected inthe soluble fraction from the potato con-centrate had Mr values in agreement withthe results of Ahid6n and TrAgArdh (1992)who described 3 major potato proteins (44000, 20 000 and 14 000), and with theobservations of Suh et al (1990) about themolecular polymorphism of medium-Mr pro-teins (22 000, 23 000 and 24 000). Specificproteins of Mr 43 000 and below 14 000were detected in ileal potato digesta. Onecould therefore hypothesize that some pro-teins with the highest Mr are found virtuallyintact at the end of the small intestine of the

calf, as it has been shown for pea legumin(Bush et al, 1992a) and soyabean glycinin(HM Tukur and JP Lalles, unpublished data).The potato proteins with a low Mr in digestamight be digestion products, but a more pre-cise characterisation is necessary. Althoughthe immunoreactive protein detected hererepresented only a small fraction of thedietary protein, it could cause immunologi-cal reactions and impair calf performance, asshown with soya (Sissons, 1982) and peas(Bush et al, 1992a).

CONCLUSION

Replacing 52% of SMP protein by nativegluten or potato protein resulted in a

decrease in the apparent ileal and faecal

digestibilities of nutrients. However, valuesobtained in the present experiment weresimilar to those of previous experiments withpartially hydrolysed wheat or potato proteinconcentrates. The lower apparent digestibil-ities of gluten and potato protein might bedue, at least partially, to a lower truedigestibility. Epitopes of immunoreactiveproteins from potato protein, but not fromgluten, were probably sufficiently intact atthe end of the small intestine to be recog-nized by specific antibodies raised againstthe native structures. The true digestibility ofpotato protein was lower than that of glutenprotein, especially for cystine. Finally, theutilization of more specific methods, suchas the 15N technique (Souffrant, 1991), arerequired to get a more quantitative estima-tion of endogenous protein losses and resid-ual dietary protein in ileal digesta.

ACKNOWLEDGMENTS

The authors wish to thank all the staff of the

experimental facilities for their care of the ani-mals, J Quillet for gathering the literature, andRoquette Freres, 62136 Lestrem, France for finan-cial support.

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