Effects of Severe Alkali Treatment of Proteins onAmino Acid Composition and Nutritive Value '
A. P. DE GROOT AND P. SLUMPCentral Institute for Nutrition and Food Research TNO (CIVO),Zeist, The Netherlands
ABSTRACT Amino acid analyses, protein quality assays, in vitro digestion and absorption tests and feeding studies with rats were conducted to study the effects ofalkali treatment on food proteins under varying conditions of pH, temperature andtime. Exposure of several high protein products to aqueous alkali at pH 12.2 resultedin the formation of the amino acid derivative lysinoalanine (LAL) which is poorly absorbed. The amount of LAL formed in isolated soy protein (ISP) upon exposure atpH 12.2 increased with rising temperature and a longer exposure period. The presenceof LAL in proteins was attended with decreased contents of cystine and lysine, anddecreased net protein utilization (NPU) values. More severe treatment of ISP withalkali of pH 12.2 at 60 or 80also caused a decrease in serine content and in digestibility of the protein. The LAL content of ISP treated under various conditions showeda highly significant negative correlation with NPU values (r = 0.96). The presenceof LAL in proteins was a sensitive criterion of alkali damage. The NPU assays of ISPsupplemented with amino acids showed methionine to be the first limiting amino acidand threonine the second. The decreased NPU of alkali-treated ISP could not be completely alleviated by amino acid supplementation, probably as a result of decreasedutilization of threonine. Most of the essential amino acids from alkali-treated ISP werereleased at a relatively slow rate by pepsin-pancreatin digestion. Threonine and methionine were the only essential amino acids in an enzymatic digest of ISP which showeddecreased absorption by everted intestinal sacs. Upon feeding rats diets with relativelyhigh levels of proteins treated at pH 12.2 and 40for 4 hours no clinical or histologicalabnormalities were observed other than an increased degree of nephrocalcinosis infemales which could be prevented by additional dietary calcium.
Exposure of proteins to alkali is increasingly applied in technological treatment offoods and feeds, e.g., for dissolving proteinsin the preparation of concentrates and isolates 2 ( 1) ; for obtaining proteins withspecific properties such as foaming, emulsifying or stabilizing (2); for destructionof aflatoxin in groundnuts (3); and for obtaining protein solutions suitable for spinning fibers (4). Several authors haveobserved that alkali treatment of wool, enzymes and serum albumin may inducechemical changes in these proteins whichlead to the formation of new amino acids :lysinoalanine (5, 6), lanthionine (7) andornithinoalanine (8). These modificationsinvolve the amino acids cystine, lysine,arginine and possibly serine.
During routine analyses of amino acidsin proteins Slump 3 observed an unknownpeak in the chromatograms of alkali-treatedfoods and feeds which upon identificationturned out to be lysinoalanine.4 Obviouslyalkali treatment of food proteins may result in chemical changes similar to those
mentioned above and very likely affectingthe same amino acids, e.g., cystine andlysine. Since these are the limiting aminoacids in the majority of food proteins, animpaired nutritive value may result fromexposure to alkali. A study was undertaken,therefore, to evaluate the effects on foodproteins, of alkali treatments varying inpH, temperature and duration, by aminoacid analysis, biological assays of proteinquality and in vitro tests of digestion andabsorption. In addition, attention was paidto possible harmful properties of alkali-treated proteins by feeding drasticallytreated proteins at relatively high levels
Received for publication December 6, 1968.1Presented in part at the 9th International Sym
posium of the Commission Internationale des Industries Agricoles et Alimentaires (C.I.I.A.) on "Newprotein sources in the human diet," Amsterdam,November, 1968.GaUiver, G. B., and A. W. Holmes 1959 Proteincontaining food product from fish. Assignors toUnilever N.V., Rotterdam. Dutch Patent 92.828(issued December 15).3P. Slump 1967 unpublished data.
4Thanks are due to Dr. Z. Bohak, Weizmann Institute of Science, Rehovoth, Israel, for a gift of purelysinoalanine.
J. NUTRITION,98: 45-56. 45
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46 A. P. DE GROOT AND P. SLUMP
to rats for periods up to 13 weeks, andexamination of the animals by clinical andpathological methods.
MATERIALS AND METHODSStandard alkali treatment. An amount
of material equivalent to 400 g crude protein was suspended in 3 liters 0.2 M sodiumhydroxide. The pH was adjusted at 12.2and the suspension kept at 40for 4 hours.Thereafter, the slurry was acidified to pH4.5 with 6 N HC1 and centrifuged at 2700rpm for 5 minutes; the residue was frozen(20)until used.
Amino acid analysis. Duplicate samplesof 200 to 300 mg were hydrolyzed underreflux with 200 ml 6 N HC1 for 22 hours.The acid was removed at reduced pressurein a rotary evaporator at 45.The residuewas dissolved in 0.2 N sodium citrate buffer,pH 2.2. In this hydrolysate all amino acidswere determined except cystine, methio-nine and tryptophan. The S-amino acidswere analyzed after oxidation with per-formic acid as described by Moore (9).Tryptophan was determined after autoclav-ing 1-g samples with 8 g Ba(OH)2-8H2Oand 16 ml water for 8 hours at 120,according to Slump and Schreuder (10).
The hydrolysates were chromatographedwith the CIVO-automatic analyzer usingthe ion exchange resins Aminex A4 5 foracid and neutral amino acids, Q15S ' forbasic amino acids and Sephadex G-257for tryptophan. The correction factors fordestruction and incomplete hydrolysis were1.05, 1.10, 1.07 and 1.08 for threonine,serine, isoleucine and valine, respectively.Upon chromatography with Q15S, lysino-alanine (LAL) appeared before lysine.Since this position is not specific for LAL,some chromatograms were developed witha PA35 ' column for separating basic aminoacids in physiological fluids. In these chromatograms LAL appears between ammoniaand lysine.
In vitro digestibility. Pepsin digestibilitywas examined by incubating 2-g sampleswith 425 ml 0.1 N HC1 and 100 mg pepsinpowder" at 38 to 40. Eight milliliters0.1 N HC1 were added after 16, 24 and 40hours. After 48 hours the digest was cooled,brought to 500ml and filtered. The totalnitrogen content of the filtrate was determined by the Kjeldahl method.
Pepsin-pancreatin I0digestibility of individual amino acids was determined as described by Akeson and Stahmann (11). Thedigests were deproteinized with 10% tri-chloroacetic acid, filtered and the nitratesanalyzed for amino acids as describedabove. The amino acids released from thetest protein were calculated after correctionfor the amino acids found in the blanks.
The rate of intestinal absorption ofamino acids from enzymatic digests wasexamined in vitro by the everted sac technique of Wilson and Wiseman (12) usingthe small intestine distal to the duodenumof four adult female rats (200 g) whichhad been fasted for 20 hours. Three evertedsacs (12cm) were made from each intestine, filled with 2 ml Krebs-Henseleit solution (13) and incubated in pepsin-pan-creatin digests of alkali treated protein,untreated protein and enzyme blanks withthe protein omitted, respectively. After incubation at 37for 45 minutes the fluidsinside the four sacs in each of the threemedia were pooled and analyzed for aminoacids.
Biological studies with rats. All feedingstudies were carried out with weanling albino rats from the CIVO-colony (Wistar-derived) which were caged in groups offour to five rats in stainless steel wire-screen cages with raised-screen bottoms.Food and tap water were available at alltimes. Individual body weights were recorded every week.
Protein quality. Evaluation was carriedout by the determination of net proteinutilization (NPU) and true digestibility(D) according to the carcass-water methodof Miller and Bender (14). The samplesto be tested were incorporated into experimental diets as the sole source of proteinto supply a protein level of 10% of the air-dried matter. The diets were made up tocontain (in percent): sucrose, 30; minerals (15), 4; cellulose, 4; B-vitamin mixture (table 1), 2.2; vitamin ADE-prepara-tion (table 1), 0.4; soybean oil, 5; andwheat starch to total 100.
s Bio-Rad Laboratories, Richmond, Calif., bulletinno. 115 A4, 1966.
6 See footnote 5.7 Pharmacia, Uppsala, Sweden.8 Beckman Instruments, Inc., Spinco Division, Palo
Alto, Calif., bulletin no. A-TB-009A.Orthana A. S., Kemisk Fabrik, Kastrup, Denmark.
1:10 000 BPC.10Hog pancreas powder, N.V. Organon, Oss, TheNetherlands, activity 5 x NF.
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