determination of the degree of hydrolysis of food protein hydrolysates by

8/12/2019 Determination of the Degree Of Hydrolysis of Food Protein Hydrolysates By

1/8

Subscriber access provided by CHINA AGRI UNIV

Journal of Agricultural and Food Chemistry is published by the American Chemical

Society. 1155 Sixteenth Street N.W., Washington, DC 20036

Determination of the degree of hydrolysis of food

protein hydrolysates by trinitrobenzenesulfonic acidJens Adler-Nissen

J. Agric. Food Chem., 1979, 27 (6), 1256-1262 DOI: 10.1021/jf60226a042 Publication Date (Web): 01 May 2002

Downloaded from http://pubs.acs.org on February 19, 2009

More About This Article

The permalink http://dx.doi.org/10.1021/jf60226a042provides access to:

Links to articles and content related to this article

Copyright permission to reproduce figures and/or text from this article
http://dx.doi.org/10.1021/jf60226a042


2/8

1256 J. Agric. FoodChern., Vol. 27, No. 6 1979Tak en together, these da ta suggest tha t chemical treat-m en t of blood for reclamation of protein is feasible a t theindustrial level. Industrial a pplication of chemical coagu-lation techniques could eliminate or diminish the need forsecondary treatm ent of blood wastewaters since chemicalcoagulation is capab le of qua ntita tive rem oval of proteinin the primary step. We are currently studying otherchemical procedures of blood protein removal.

ACKNOWLEDGMENTWe are th ankfu l to Delbert Doty for his insight andsuggestions concerning thi s research. Th e authors alsowish to than k Jack Barensfeld (A. W. Stad ler, Inc.) andTom Dieter (E mge Packing Co.) for supplying industrialwhole blood samples. We are grateful to the F ats andPro tein s Research Fou ndation, Inc., Des Plaines, IL, forbringing the blood processing problem to our attention an dfor a grant which supported this research. This ma nuscriptwas taken from da ta contain ed in th e Masters thesis ofA. Ratermann, submitted to the Departm ent of Chemistry,Mu rray S tat e University, Aug. 1979.

LITERATURE CITEDAmerican Public Health A ssociation, Stan dard M ethods for theExamination of Water and Wastewater, 13th ed., Washington,DC , 1971, p 244.

Adler-NissenBlock, R. J., Bolling, D., to the Borden Co., U.S. Pat ent 2 710858,Bray , W. E., Clinical Lab orato ry Met hods , C. V. Mosby Co.,Cerbulis, J., J. Agric. Food Chem. 26, 806 (1978).Chandler, R. L., OShaugnessy, J. C., Blanc, F.C., J. Water Pollut.Control Fed. 48, 2971 (1976).Claggett, F. G., Wong, J.,Circular No. 42, Salmon W astewaterClarification Pa rt 11,Fish Res. Bd. of Canada, Feb. 1969.Hartman, G. H., Swanson, A. M., J. Dairy Sci. 49, 97 (1966).Hentad , O., Hvidsten, H., Acta Agn. Scandinauica,23,154 (1973).Hidalgo, J.,Kurseman, J., Bohmen, H . V., J.Dairy Sci. 56,988Hopwood, A. P., Rosen, G. D., Proc. Biochem. 7, 15 (1972).Jones, S. B., Kalan, E. B., Jones, T.C., Hazel, J. F., J. gnc. FoodChem. 20, 229 (1972).Kramer, S. L., Waibel, P. E., Behren ds, B. R., El Kandelgy, S.M., J. Agric. Food Chem. 26, 979 (1978).Riehert, S. M., Diss. Abstr. 33, 3128-B (1973).Sand ers, M. D., Znd. Eng. Chem. 6, 1151 (1948).Spinelli, J., Koury, B., J. Agric. Food Chem. 18, 284 (1970).Van Steenberg, W., De Laval Co., Inc., Poughk eepsie,NY ersonalWaibel, P. E., Cuperlovic, M., Hur rell, R, F., Carpen ter, K. J. ,

Jun e 14, 1955.St. Louis, MO , 1951, p 239.

(1973).

communication, 1979.J. Agric. Food Chem. 25, 1 71 (1977).

Received for review May 2, 1979. Accepted July 30, 1979.

Determination of the Degree of Hydrolysis of Food Protein Hydrolysates byTrinitrobenzenesulfonic Acid

Jen s Adler-Nissen

An accurate , reprodu cible an d generally applicable procedure for determ ining th e degree of hydrolysisof food protein hydrolysates has been developed. Th e protein hydrolysate is dissolved/dispersed inhot 1 odium dodecyl su lfate to a conce ntration of 0.25-2.5 X amino equivalents/L. A samplesolution (0.250 mL ) is mixed with 2.00 mL of 0.2125 M sodium phosp hate buffer ( pH 8.2) and 2.00 mLof 0.10% trinitrobenzenesulfonic acid, followed by incubation in th e dar k for 60 min at 50 C. Th ereaction is quenched by adding 4.00 mL of 0.100 N HCl, and the absorbance is read a t 340 nm. A 1.500mM L-leucine solution is used as the stan dard. Transformation of the measured leucine amino equivalentsto degree of hydrolysis is carried out by means of a stan dard curve for each particula r protein substrate.

Enzymatically hydrolyzed proteins possess functionalproperties, such as low viscosity, increased whippingability, and high solubility, which make them advantageousfor use in many food products. Recent experiments haveindicated that in order to obtain desirable organoleptic andfunctional properties of soy protein hydrolysates, the hy-drolysis must be c arried out unde r strictly controlled con-ditions to a specified (generally low) degree of hydrolysis(DH ) (Adler-Nissen, 1977; Adler-Nissen and Sejr O lsen,1979). DH is defined as the percentage of peptide bondscleaved (Adler-Nissen, 1976). Therefore, a need exists fora general method of determining DH of food protein hy-drolysates, in particular for quality control. An obviousmethod to consider for this purpose is the tr initro-benzenesulfonic acid (TNB S) method, by which the con-Novo Research Institute, E nzyme Applications Researchand Development, Novo Industri A/S, DK 2880 Bag-svaord, Denmark.

centration of primary amino groups in the hyd rolysate canbe determined.Basically, this m ethod is a spectroph otom etric assay ofthe chromophore formed by the reaction of TNBS withprimary amines (Figure 1). T he reaction takes place underslightly alkaline conditions and is terminate d by loweringthe pH . T N B S also reacts slowly with hydroxyl ions,whereby the b lank reading increases; this increase is stim-ulated by light (Fields, 1971).Since its introduction by S atake e t al. (1960), the TN BSmethod has enjoyed a widespread use for the determ ina-tion of free amino groups of proteins and protein hydro-lysates. However, th e presence of insoluble proteinaceousmaterial in, e.g., the commercially used whipping ag entsbased on hy drolyzed soy protein necessitates certain mod -ifications of th e various existing proce dures describe d inthe literature, as they seem to have been developed forsoluble ma teria ls only. Also, althoug h it is generally as-sumed th at a linear relationship between the color intensityand t he conce ntration of a-am ino groups exists, we have0021-856 1/79/ 1427-1256 01 OO/O 1979 American Chemical Society


3/8

Determination of Hydrolysis of Protein Hydrolysates J. Agric. Food Chem. Vol. 27, No. 6, 1979 1257trypsin (Novo Industri 1971).Th e st and ard sam ples of soy protein hydrolysate, whichwere used in most of th e experiments, were prepared ac-cording to the procedure described in th e last subsectionof the experimental section.Reagents Th e following reagents were used: 0.2125M p hosphate buffer [0.2125 M N aH 2P 04 s added to 0.2125M N a 2 H P 0 4until pH is 8.20 0.02 (th e propo rtion ofvolumes is approximately 43:1000)], 0.1 TN BS solution[TNBSis dissolved in deionized water in a volumetric flask100 or 150 mL) covered with aluminium foil; the solutionmus t be prepared immediately before use],0.100 N HCl,1 NaD odS 04, 1.500 mM leucine stand ard in 1 Na-

TNBS Reaction Unless otherwise stated , the T NB Sreaction was carried out as follows: 0.250 mL of a sample,containing between 0.25 X and 2.5 X aminoequiv/L, ismixed in a test tub e with 2.00 mL of phosp hatebuffer a t pH 8.2. Tw o milliliters of 0.10 TN BS solutionis added and the test tube is shaken and placed in a waterba th a t 50 1 C for 60 min. During incubation the testtubes and the water bath must be covered with aluminiumfoil because the blan k reaction is accelerated by exposureto light. After th e 60 min 4.00 mL of 0.100 N HC1 is addedto term inate the reaction, an d the test tu be is allowed tostan d a t room tem pera ture (cooling below room tem pera-ture may cause turbidity because of the NaD odS 04 pres-ent) for 30min before the absorbance is read against waterat 340 nm.Th e reactions on the blank an d the stan dard solutionsare carried ou t by replacing the sample with 1 NaDod-SO4 and 1.500 X M L-leucine in 1 NaD odS04, re-spectively. The absorbances of th e blank and the s tand ardare determ ined as the averag es of six individual d eterm i-nations.Th e conditions during the reaction are as follows: bufferconcentration = 0.10 M, TN BS concentration = 1.43 XM, leucine concentration = 0.088 X M, pH 8.2, tem-perature = 50 C. pH afte r the HC1 addition is 3.7-3.9;a pH below 3.5 will cause turb idity. Th e reaction can beconsidered pseudo-first-order with respect to th e am inogroups.Defiliitions and Symbols Only conce ph and symbolswhich are used more tha n once in the test are included:a intercept on the y axis of a regression line; AU, Ans onunits, a measure of proteolytic activity; b , slope of a re-gression line; DH, degree of hydrolysis, defined as thepercentage of peptide bonds cleaved, thu s DH = h/h,)X 100%; E /S , enzyme-substrate ratio, based on proteinsubstrate; A , absorbance; A,, maximum absorbance esti-mated in kinetic experiments; h, hydrolysis equivalent,defined as th e co ncentration in m illiequivalents/ g of pro-tein of a-am ino groups formed during hydrolysis; hbt,hy-drolysis equivalent a t com plete hydrolysis to am ino acids;hbt is calculated by summing up th e conten ts of th e indi-vidual amino acids in 1g of pro tein; k irst-orde r reactionconstant; A, spectrophotometric wavelength; so, standarddeviation within the groups in grou ped regression analysis;sl, stand ard deviation around regression line in groupedregression analysis; s h) , otal standard deviation on hdeterminations; S , substra te concentration defined as con-centration of K jeldahl nitrogen m ultiplied by th e appro-priate factor. Sensitivity AA/Amequiv of NH2 - X L-'(change in A for a given change in conc entratio n of am inoequivalents.Kinetic Experiments Th e kinetic experiments wereeither carried out by varying the reaction time in theTN BS reaction described above or by using th e following,

DodSO4.

0 - 0 4 2 1NO2

NO2 NO2Figure 1. Reaction of TN BS wi th amino groups.observed in this stud y tha t ther e is a considerable differ-ence between the different proteins with respect to theactual value of the slope and intercept of th is relationship.(Th e intercept, which is mainly due to the -a minogroups,is of considerable and varying magnitude.) Thu s, someway of standard izing th e assay is needed. Finally, aftersome initial expe rime nts using either a version of th e ori-ginal TN BS procedure (Satake et al., 1960) or a more rapidme thod desc ribed by Fields (1971),it was concluded thata more thorough study was needed in order to obtain amanual, accurate TN BS procedure for determining the D Hof food protein hy drolysates in general. In particular, whenusing the above-mentioned methods, a high spreading ofth e results from rep eated analysis on the same materialwas observed. Th is was soon ascribed to difficulties indispersing th e partially insoluble proteins and pro tein hy-drolysates. With a view to these considerations it wasdecided in advan ce tha t th e following features should beincorporated in th e modified T NB S procedure.(1)Th e sam ple should be dispersed in sodium dodecylsulfate (NaDodS 04)rather than buffer alone. Incorpora-tion of NaDodSOl in the TN BS m ethod has been reportedby Hab eeb (1966), who used this agent for dena turing theproteins in his samples. Th e use of mercaproethanol topreve nt protein aggregation is ruled out because this ag entreacts with TN BS (Kotaki et al ., 1964).(2) Th e buffer used in most versions of the TN BS me-thod is bicarbonate (p H 8.5). As we considered the form a-tio n of C 0 2 uring th e acidification step a nuisance, it wasdecided to use anothe r buffer system. Bora te, which wasapplied by, e.g., Fields (1971), does not seem to be recom-me ndab le if sugars are present (as hey may be in our case),whereas pho sphate is suitable (B urger, 1974). Also, phos-pha te allows th e choice of less alkaline pH values thanoften used, which is advantageous in practice, as th e b lankreaction increases considerably if p H is above 8.5 (Sata keet al., 1960).(3) A reaction tim e of 1h was considered optimal for th eman ual procedure, being neither so short as to make thecolor intensity sens itive to a few minute s of deviation onth e reaction time, nor so long as to make it difficult to runth e complete analysis in half a working day. Com pletereaction should be achieved within the reaction time toensure high reproducibility.E X P E R I M E N T A L S E C T I O N

Materials Trinitrobenzenesulfonic acid dihydrate(analytical grade) and NaDodSO, were obtained fromSigma. All the other analytical chemicals were fromMerck. Th e proteins (Kjeldahl nitrogen contents in par-entheses) were soy protein isolate (Purina 500 E fromRalston P urina (14.1% N), casein according to Ham mer-sten (Merck) (14.0% N ), and gelatin (a commercial, low-Bloom, alkaline extracted gelatin from E xtraco, Sweden;15.7% N). T he enzymes used for the hydrolyses werealcalase 6.0 FG and pancreatic trypsin Novo 6.0 S, bothfrom Novo Indus tri A/S. Th e declared proteolytic activityof both enzyme prepar ations was 6 Anson units (AU )/g;for comparison the proteolytic activities of the crystallineenzymes are approxim ately 25 AU/g for Alcalase (NovoIndu stri 1978a) and a pproximately 20 AU/g for porcine


4/8

1258slightly more accura te procedure: a 5-mL sam ple wasmixed with 40 m L of buffer an d 40 mL of TN B S solutionin an aluminium foil covered flask immersed in a waterbath. All solutions had been heated t o the temperatureof the water bath prior to the experiments. A t 5-minintervals a 4-mL sam ple was drawn and mixed with 4.00mL of 0.094 HC1 (this corresponds to mixing 4.25 mL with4.00mL of 0.1 N HC1 as in the stan dard procedure). Th eabsorbance was then im mediately measured a t 340 and 420nm (the two absorption maxima for TNBS-substitutedamino acids). A blank reaction was run simultaneouslyby the same procedure. Th e increase in the absorbanceof the blank during the reaction follows a zero-orderscheme, and the ra te constant was determined by a linearregression analysis. T he regression equation was then usedto correct the sample readings for the contribution fromthe blank. Th e corrected sample readings were plotted asa function of time, an d the first-order rate constant wasdetermined direct from the plot by the method of leastsquares as described below.Theoretically, the a bsorbance sho uld follow the first-or-der relationship

J. Agric. Food Chem., Vol. 27, No. 6, 1979

A = A,(1 - e-k t)T he sum of squared differences between th e n observedabsorbances, Ai i = 1, n), and their theoretical values forthe same t = ti i = l,-n) is called z. Assuming a fixed,estimated value of k , k becomes

Adler-Nissen

= C(Ai )2 + Am2?(1 - e - 2A,?Ai(l - e-n t i )1 1 1

By differentiation of z with respect to A, and settingdzldA, = 0 to cbtain a minimum value of z the bestestima te of A,, A,,,, is

and

A differe nt estim ate of k would give diffezent values of A,and zmin. By trial-and-e rror a value of k- is found, whichgives the low est value of z-. T he se t of k and A, therebyobtained is the n taken as the best e stimates of k and A,,respectively.Th e main advantages of the procedure above are tha tit assigns a constant variance to all Ai values, therebyreflecting the physical realities of the experim ental design,and that A, does not have to be independ ently estimated.The-disadvantage is primarily th at a confidence intervalfor k cannot be readily obtained.Th e kinetics of the TN BS reaction was studied undera varie ty of conditions: pH 8.2 vs. pH 8.5,40 OC vs. 50 OC,NaDodSOl vs. no NaDodS04. As samples were used 1.0X M L-leucine and a s tandard soy protein hydrolysatecontaining approximately 0.40 X equiv/L of a-aminogroups (t he experiments were designed in such a way th atan accurate knowledge of the concentration of aminogroups was of no importance in the deter mina tion of th erate constant). Because the kinetic experiments serve onlyto optimize the TN BS reaction, a very rough determinationof the average rate constant is sufficient, and it seemedjustified from the results obtained to treat the proteinhydrolysate as f all the amino groups (including the -am-

G - O Z L ~0 . 5 OD - ODblant0 . 4 1 A

0 . 3 A l eu , 340 nm, k 4 . 07 4 rn i n . - l6: h y d r . , 340 nm, k.0.105 rn in. -0 . 2

0 . 1

I m i n u t e sFigure 2. Kinetics of the pse udo-first-order reaction bttwe enNH2 and TNBS a t 40 OC and pH 8.2. The rate constants, k weredetermined by the method of least squares as described in theExperimental Section.ino groups) exhibit the same rate constant.Protein Hydrolysis. Th e proteins were hydrolyzed ina therm ostated, 1-L vessel equipped w ith stirrer an d pH -stat equipment, including a recorder for the base (4 NNaO H) consu mption (Adler-N issen, 1977). All hydrolyseswere carried o ut using the following hydrolysis parame ters:S = 5.0 w/w 7 (N X 6.25) in 800 g of aqueous solution,E/S = 12 mAU/g (in one experiment: 30 mAU /g), pH 9.5and 50 OC Protein concentrations were later recalculatedas Kjeldahl nitrogen multiplied by the appropriate con-version factors: soy protein, 6.25 (Smith and Circle, 1972);casein, 6.38 (Jones, 1931); gelatin, 5.55 (Jone s, 1931).During hydrolysis, 6-mL sam ples were drawn at specifiedtimes (typically after 5, 10, 20, 30,4 0, 60,80, 100, 120, andsometim es 160,200, and 240 min of hydrolysis time) andquickly transferred to a test tube. From th e test tube 2X 2.00 mL were rapidly pipetted in to two tes t tubes con-taining 10 mL of 1 NaD odS04 and kept at 75 C byimmersing in a water bath. Th e test tubes were shakenand m aintained in the water bath for at least 15 min. Itseems reasonable to assume that the enzymes werepromptly inactivated by the treatm ent with the hot Na-DodSOI solution, and the prolonged heat tre atme nt mainlyserved to disperse the protein hydrolysate. After the heattrea tm ent the contents of the te st tubes were transferredquantitatively to 50-m L volumetric flasks and the volumewas adjusted with 1% NaDodS04. The content of freeamino grou ps, expressed as leucine amino equivalents, wasassayed (single determination) by the TNBS reaction.From the base consumption recorded, the hydrolysisequivalent h)measured in milliequivalents/gram of pro-tein could be calculated. T he appro xima te pK value ofthe am ino groups in polypeptides is 7.5 a t 25 C and th eionization enth alpy is in the range of 40-50 kJ /eq ui v ofNH 2 (Steinha rdt and Beychock, 1964). Using the Gibbs-Helmholtz equation (Castellan, 1964) on these data , thepK value a t 50 C can be calculated to approxima tely 7.This means tha t the a-NH 2groups are fully titrated a ndthe equivalents of peptide bonds cleaved are equal to theequiv alents of base consumed. In these calculations, cor-rections were made for th e gradual, slight decrease in theamo unt of hydrolysis mixture caused by the drawing ofsamples (Novo Industri, 1978b).RESUL TS AND DISCUSSION

Kinetic Experiments. Figure 2 shows the course ofreaction between N H2 and T NB S a t 40 C during theexperiments carried out according to the modified proce-dure described in the Experimental Section. Th e solidcurves are the reaction curves calculated from the esti-ma ted values of k and A, (see the Experimen tal Section);it appear s th at t he course of reaction is reasonably close


5/8

Determination of Hydrolysis of Protein HydrolysatesTable I .

J. Agric. Food Chem. Vol. 27, No. 6, 1979 1250Determinat ion of Rate Co nstants for the Pseudo-Fi rst -Order React ion between NH, a nd TNBS in Excessa

NaDodSO, t emp , no . of blank,sample in sample C pH analyses A n m h , min- ' h, min- ' AA/min- 'L- leucine 50 8.2 6 340 0.115 0.133 see valuesbelow

- 6 420 0.130+ 6 340 0.115+ 6 420 0.120+ 340 0.145+ 420 0.1478.5 6 340 0.230 0.184- 6 420 0.145+ 6 340 0.175+ 6 420 0.185+ 40 8.2 12 340 0.074 0.072+ 12 420 0.069soy protein + 50 8.2 6 340 0.180 0.170 0.00195+ 6 4 20 0.160 0.00100+ 340 0.195 0.00172+ 420 0.145 0.000900.002178.5 6 340 0.160 0.150+ 6 420 0.140 0.00114+ 40 8.2 1 2 340 0.105 0.092 0.00132+ 12 420 0.079 0.00090

hydrolysates

a Concent rat ion of TNBS, 1.43 mM ; concen t r a t i on of NH,, Q 0.06 mM. Furth er detai l s in the Exper imental Sect ion,to an ideal first-or der scheme.Th e rate constants obtained in all the individual exper-imen ts are shown in Tab le I. A comparison between therate c onstan ts obtained a t 340 and 420 nm does not dis-close any systematic effect of t he choice of w avele ngth forthe estimation of th e rate constant, nor does the presenceof NaD odSOl seem to have any systematic effect on therate of reaction between TN B S and leucine. Th e valuesof the rate constants are therefore pooled to yield theaverage values shown in T able I. On the basis of thesevalues it can be concluded t ha t for both leucine and soyprotein hydrolysate th e reaction rate increases 1.85 timeswhen th e temp erature is increased from 40 to 50 C, aresult which seems very reasonable indeed. I t also appearsth at t he reaction rate is higher for the hydrolysate thanfor the leucine standard.With respect to the effect of pH, Table I shows thatincreasing the p H from p H 8.2 to 8.5 ha s a significantlypositive effect on the reaction rate of leucine, whereas tha tof soy protein hydrolysate has hardly changed. This canbe explained by the different pK values for the aminogroups in the two systems. For leucine, pK can be calcu-lated as approximately 9 at 50 C, whereas the pK of thesoy protein hydrolysate will be as low as 7, as explainedpreviously. Because TNB S reacts only with the aminogroups in their unpro tonated state (Freedman and R adda,1968), it is obvious th at t he pH increase from 8.2 to 8.5will increase the concentration of reactive amino groupsin the leucine system but not in the hydrolysate system.For practical purpo ses it is satisfactory tha t the reactionis 99-99.5% com plete, which is achieved when the pro ductof k and th e reaction tim e is above 5. For a reaction tim eof 60 min this requirement is fulfilled at 50 C and p H 8.2,but n ot at 40 OC and p H 8.2. Increasing the pH to 8.5 mayallow a slightly shorter reac tion time; however, this alsoincreases the zero-order rate constan t for the blank reac-tion. Furthe rmo re, the buffer capacity of the phosphatebuffer is also considerably lower at th at pH . T he condi-tions of pH 8.2,5 0 C, and 60-min reaction time thus seema reas onable comprom ise in the choice of reaction para me-ters.O p t i m i z a t i o n of P o s t r e a c t i o n T r e a t m e n t . A seriesof experim ents was carried o ut consisting of repea ted as-says of t he stan dard leucine solution and the s tandard

hydrolysate solution used in the kinetic expe riments. Allthe assays were carried out according to the standardTN B S reaction, except that the normality of HC1 (tovarythe end p H) an d the time interval between the HC1 addi-tion and the sp ectrophotom etric reading were varied. Th edata obtained during this optimization were treated bysim ple analyses of varian ce, an d it could be concluded tha tif th e end pH was below neutra l and the tim e interval wasbetween 30 min a nd 3 h, no significant effect of the se twoparam eters was observed. If the time interval was short-ened to 15 min, it appe ared, however, that the absorbance sof the standard leucine were slightly higher. It wastherefore co ncluded th at a period of at least 30 min sho uldelapse between the termination of the reaction and thespectrophotom etric reading to ensure reproducible results.Choice of W ave leng th. In the choice between 340 and420 nm, both sensitivity and reproducibility should beconsidered. Th e sensitivity is expressed as A Ablmk ora given concentration of amino groups, and th e reprod uc-ibility can be take n as the variation coefficient of A -Ab-obtained by repeated assays of th e same sample. Six re-peated an alyses on a number of leucine a nd soy proteinhydrolysate stan dar d samples covering a range of aminoconcentrations were therefore carried out. From thes estudies it could be concluded th at th e sensitivity for allsamples was 1.5-1.8 times higher a t 340 nm th an at 420nm. A comparison of th e va riation coefficients showed thatthese were fairly independ ent of the am ino concen trationand t ha t for leucine th e greatest reproducibility was ob-tained a t 340 nm (3% vs. 7% at 420 nm on a single de-termination). For the hydrolysate the reproducibility wasapproximately the s ame for both wavelengths (4% on asingle determination). T hree hundred and forty nanome-ters thus seemed superior to 420 nm, and 340 nm wastherefore chosen as the standard wavelength.D e m o n s t r a t i o n of Lineari ty. Dilut ion series (eightconcentrations) of a 4.0 mM leucine and of a soy proteinhydrolyzate containing approximately 2.9 mequiv ofNH,-/L were prepared and analyzed (triple determina-t ion). The 8 X 3 values of A - Ablmk (at 340 nm) weresubjec ted to a grouped regression analysis. In both casesvariance ratios close to unity were obtained an d th e as-sum ption of linearity between NH 2 concentration and A- Ablank ould thus be sustained.


6/8

1260 J. Agric. Food Chem. Vol. 27, No. 6, 1979 Adler-NissenTable 11. Resul t s f rom Groupe d Regression Analysis on the D ata f rom the H ydrolysis Exper imentsa

~soy protein hydrolysate casein hydrol . gelatinregression parameter I I1 I11 IV V hyd rol. , VIX (mean of X )Y (mean of Y )b (s lope)s, (SD regr. line)so ( residual SD )F (variance rat io)SS, (sum of ( X i - X ) )r (correl . coeff.)n ( n o . of doub l e de t . )signif. level fo r diff. betw een s lopesbsignif. level for diff. be twee n s I csignif. level for d iff. betwe en soc

0.53220.86791.01100.02630.01961.791.23730.998e

0.5488 0.8728 0.5920 0.36250.8595 1.1929 1.0067 0.75300.9968 0.9173 1.0148 1.04720.0401 0.0302 0.0396 0.02330.0289 0.0208 0.0409 0.02951.93 2.12 0.94 0.621.3761 2.1017 1.5329 0.38650.996 0.998 0.997 0.9949 9 9

0.950-0.975 - 0.7 0-0.80s , = 0.0333P A 0.40-0.50so= 0.0272P = 0.90-0.95

0.63250.96040.79570.03730.01744.62*2.43460.99611

a The fol lowing enzym es and enzyme-subst rate rat ios were appl ied: I and 11, alcalase 12 mA U/g; IV , alcalase 12 m A U / g :

* Tested according to HaldV, trypsin 12 mA U/g; VI, alcalase 12 mAU/ g .gression analyse s, X i s equal to t he value ( in mequiv/g) of h (hydrolysis equivalents) obtained f rom the pH-stat and Y ( t w ovalues of Y fo r each X ) s equal to th e measured amino c ontent expressed in leucine mequiv/g .(1951). Barlett 's test .

All hydrolyses were carr ied out a t pH 9.5 and 50 C . In the grouped re-

I .6

1 . 4

1 . 2

1 . 0

0 . 8

0.6

0.4

0 . 2

0 . 0

S o y P r o t e i n I s o l a t e - A l c a l a s el e u - N H 2 m e q v / g

I : E / S = 12 rnAU/g. 1: E / S = 12 rnAU/g

[ l e u - N H 2 1 = D.970x.h t 0 , 3 4 2r = 0 . 9 9 7

1 e h0 . 2 0.4 0.6 0.8 1.0 1 .2 1 . 4 m e q v / q

Figure 3. Correlation between hydrolysis equivalents h) ndleucine amino equivalents for the soy protein isolate-alcalasesystem.D emons t ra t i on of Qu an t i ta t iv e React ion . Dilut ionseries of leucine and soy protein hydrolysate were assayedby the usual procedure. Th e assays were repeated and th ereaction tim e was now prolonged from 60 to 90 min. Re-gression analyses of the A values vs. the concentration ofam ino groups yielded values of the sensitiv ity, expressedas the change in A for a given change in the concentrationof amino groups. No significant change in the sensitivityfor bo th leucine and soy protein hydrolysate was observed.Th is confirms the conclusion from the kinetic experiments,namely that a quanti tat ive reaction between T NB S andth e amino groups is achieved after a reaction time of 60min. It is impo rtant to realize, however, tha t this re sultdoes not mean th at th e reaction time should not be care-fully controlled. If the reaction times for the samples andthe blanks differ more th an a few minutes, a considerableerror will be introduced because of the blank reaction.Pr o t e in Hydro lysis Exp erim ents . Six hydrolyseswere carried ou t according to the procedure described in

1 . 6

I , 4

1 . 2

I o

0 . 8

C a s e i n , G e l a t i nl e u N H 2 m e q v / go I V C a s e i n - A l c a l a s e

V : C a s e i n - T r y p s i n

I 0 3 9 x h += 0 . 9 9 6

0.383

0 . 4

h0 . 0 0 . 0 0 . 2 0.4 0.6 0.8 1.0 1. 2 1.4 meqv/gFigure 4. Correlation between hydrolysis equivalents h ) ndleucine amino equivalents for casein and gelatin, respectively.the Experimental Section. In the hydrolyses I, 11,and 111,soy isolate was hydrolyzed with alca lase E/S = 12 mAU/g,12 mAU/g, an d 30 mA U/g, respectively). In IV and V,casein was hydrolyzed with alcalase and trypsin,respectively-both enzymes were applied in concentration sof 1 2 mA U/g. Finally, in VI, gelatin was hydrolyzed withAlcalase (12 mAU/g).During each of the hydrolyses, samples were drawn atregular intervals. For each sam ple a value of h (hydrolysisequivalents),expressed as milliequivalents/gram, was ob-tained from the p H- stat, as described in the Experim entalSection. In addition, the sample was assayed twice by theTN BS reaction. Thu s, for each hydrolysis a set of da tawas obtained which could be subjected to a grouped re-gression analysis. T he results of these regression analysesare summarized in Table 11,and al l the dat a are plot tedin Figures 3 and 4.It seems likely to expect th at there wll be no significantdifference between the three regression lines, I, 11,and 111.Th e tests for significance (H ald, 1951) obviously rest o n


7/8

Determination of Hydrolysis of Protein HydrolysatesTable 111. Correlat ion b etwe en H ydrolysis Equivalents (h) nd Leucine Am ino Equivalents for Three Protein Hydrolysa te

J. Agric. Food Chem. Vol. 27, No. 6, 1979 1261

sta nd ard cu rves: Leu- , = bh + aregression param eter soy protein hydrolys ate casein hydrolys ate gelatin hydrolys ate

b * l S Da SDLys mequiv/ghtot mequiv/ginverse curve libto tal S n h (4 e t erm. )

-albh = 0.1h = 0.4h = 0.7h = 1.0h = 1.3

0.970 * 0.0160.342 f 0.0120.42b7.75e1.031-0.3520.0190.0210.0240.0290.035

1.039 0.0220.383 i 0.0110.54'8 0e0.962-0.3680.0180.0200.0240.0310.038

0.796 f 0.0240.457 f 0.0170.32dll.le1.257-0.5740.0280.0270.0300.0360.044

a The stan dard curves were o btained by pool ing the data f r om th e individual hydrolyses (Table 11). The t o t a l s t andarddeviat ion on h is calculated under the as sum pt ion that the leucine amino equivalents are assayed by a quad ruple determ ina-t i on us ing t he s t andard p rocedure . The t o t a l s t andard dev ia t ion on h is composed of three inde pende nt sources of er ro r :the leucine s tandard, th e regression line for the s tan dard curve, and th e assay of the sam ple i tsel f .ses on th e raw mater ial , perform ed at the Inst i tu te of Protein Chemist ry , Horsholm, Denm ark, and a t Bioteknisk In st i tu t ,Kolding, Denmark,From ami no ac i d ana l y -

Value taken f rom Provansal et al . (1975), ssuming 14.0 N in casein according to Hammarsten.Val ue t aken f ro m Eas t oe and L each (1977). e Calculated f ro m th e am ino acid composi t ions g iven in the references above.the assump tion, which is inherent in all regression analyses,th at th e uncertainty associated with the independent var-iable (in this case h ) s zero. Th e standa rd deviation onh is in fact about 0.01me quivlg, which is one-th ird of thestand ard deviation on the dep endent variable (Table 11),an d thi s is sufficient to explain the w eak significance ob-serve d for hydrolysis VI. Th e picture is comp licated bythe fact th at when different hydrolyses are compared, asystem atic, relative error in h makes its contribution. Thissystematic error arises mainly from the uncertainty inestablishing the substrate concentration (1.5% relative)and from the uncertainty in determining the absorbanceof the standard leucine (1.8 elative). T his adds a rela-t ive uncertainty to the slope in the order of 2-2.5%,whereby th e weak significance for difference between th ethree slopes vanishes. Fur ther ca lculations according toHald (1951) shows th at the three regression lines can in-deed be regarded as expressing the same tru e relationship,and they a re therefore pooled to yield t he regression lineshown in Figure 3.Identical calculations as above show that there is nosignificant difference between th e regression lines IV andV, although V has been hydrolyzed with trypsin insteadof alcalase. The se two regression lines are therefore pooledto give the regression line for casein in Figure 4.Finally, it can be shown t ha t the pooled regression linesfor soy protein an d casein are significantly different fromeach othe r both with respect t o slope as well as intercept.Thes e two lines ar e of course also differe nt from the re-gression line for gelatin (VI), which has a m arkedly lowerslope than the two other lines. Thu s, it must be concludedth at th e slope and interce pt for the regression lines, whichdescribe the relationship between hydrolysis equivalentsand leucine amin o equivalents, assume particula r valuesfor each individual protein substrate. On the other ha nd,th e results for casein hydrolysate, where two enzymes withcom pletely different specificities (see, e.g., Henn rich, 1973)were applied, indicate tha t the enzyme has little or noinfluence on th e slope and interc ept. Table I11 gives theslope and interc ept for the thr ee differe nt regression linesassociated w ith hydro lysate s of soy protein isolate, casein,an d gelatin. As mentioned previously, the intercept isbelieved to be derived mainly from the presence of e N H 2groups. How ever, the c onte nt of lysine expressed in mil-liequiva lents/gra m does not agree too well with the in ter-cepts. For soy protein and casein hydrolysate the lysinecon tent is 1.23 and 1.41 times hig her, respectively. Now,it appears from the literature (Satake et al. 1960;Goldfarb,

1966) ha t there a re considerable differences in the molarabsorbance both between th e individual amino acids aswell as between am ino acids an d peptides, and the dis-crepancy may simply be caused by th e c groups inpeptides having a molar absor banc e of ab out three-fourthsof th at of L-leucine. Unf ortuna tely, we have been una bleto retrieve data on th e molar absorbances of leucine an dpeptide-bound lysine measured u nder identical conditions,and this problem deserves further study. Another expla-nation for the low values of t he inte rcepts could be tha tnot all the lysine has been available for reaction.Th e discrepancy between the intercept and the lysinecontent for gelatin hydrolysate may be due to the presenceof sm all peptides an d free amino acids in the raw material.In fact, it was ound that 9.2 of the n itrogen was solublein 0.8 M trichloroacetic acid, which suppo rts this ex plana-tion.From a practical point of view, however, the discussionabove may be settled by the fact that, when the intactproteins were assayed by the T NB S procedure , the follow-ing results were obtaine d: soy protein isolate, 0.35 me-quiv/g; casein, 0.43 mequivlg; gelatin, 0.40 mequivlg.These values lie reasonably close to th e values obtainedfor the intercepts.Accuracy of the Assay Th e close linear relationshipbetween the hydrolysis equivalent and the yield of th eTNBS assay, expressed in leucine amino equivalents,means tha t the T NB S reaction can be used to determineth e degree of hydrolysis (DH ) of food protein hy droly satesby determining the hydrolysis equivalent h) rom theinverse standar d curve.The uncertainty on the determination of h is derivedfrom three independe nt sources: th e uncertainty on de-termining the absorbance on the sample itself (the uncer-tainty on the blank is small in comparison and can beneglected), the unc ertainty on determ ining the absorbanceof th e leucine stan dar d, and finally the uncer tainty on thestandard curve for that particular hydrolysate.The first term (so in Table 11)appea rs to be fairly con-sta nt over a range of abso rbanc e values (cf. Choice ofWav elength ) and indep endent of the type of hydrolysate.Th e second term can be shown to add an uncertainty tothe absorbance which is proportional to the A -Ab&. Thethird term exhibits the usual curvilinear function of h. Allthree ter ms can be transformed to functions of h. I t ap -pears in this study tha t the three uncertainty terms areof th e sam e order of ma gnitude . Very little is theref oregained by increasing th e numbe r of sam ple determ ination s


8/8

1262to,e.g., six or eight, and a q uadruple determ ination shouldtherefore generally be applied.T he total stan dar d deviation on the thr ee types of hy-drolysate has been calculated as functions of h, and se-lected values are given in Table 111. I t appears tha t thedifferences in magnitude of s h) stan dard deviation onh) are relatively small. Th e standa rd deviations showncorrespond to a standard deviation on DH of 0.2-0.3%absolute, which should be satisfactory for all purposes (inthis calculation the systematic error arising from the un-certainty on h,, is neglected).CONCLUSION

Th e present work has shown tha t an accurate, reprodu-cible, and generally applicable determ ination of DH (th edegree of hydrolysis) can be based on a m odification of theTN BS reaction, as described in detail in the ExperimentalSection.It is our experience from the use of the T NB S assay thatnow and th en suddenly a large spreading of the resultsoccur. Th is spreading can generally be traced back toinhomogeneities in the N aD odS 04samp le solution. Cer-tain protein and p rotein hydrolysate products are difficultto disperse in N aDo dS0 4 without having been reduced bya disulfide reducing agent. Unfortunately, mercapto-ethanol a nd other reducing agents containing sulfhydrylgroups ruin the assay, as stated previously. Often it ispossible, however, to disperse th e protein without the useof mercaptoethanol by homogenizing the m aterial in hotNaD odS 04 and this procedure is therefore recommended.Another potential source of error is deterioration of th eleucine stand ard . Fresh stand ards should therefore beprepared regularly, and for accurate work it is recom-mended to use an additional standard as control, e.g.,glycylglycine.Except for th e TN BS , the reagents used are relativelyharmless. No particular safety precautions need to betaken except tha t gloves should be worn when making theTN BS solution.

J. Agric. Food Chem. Vol. 27, No. 6, 1979 Honig, RackisACKNOWLEDGMENTI thank Mrs. Gudrun Poulsen for her meticulous ana-lytical work and keen interes t in the developm ent of thi smethod. My thank s are also extended to my colleague,Hans Sejr Olsen, for valuable suggestions in this work.LITERATURE CITEDAdler-Nissen, J., J. Agric. Food Chem. 24, 1090 (1976).Adler-Nissen, J., Process Biochem. 12(6),18 (1977).Adler-Nissen, J., Sejr O lsen, H., ACS Symp. Ser . No. 92, 125Burger, W. C., Ana l. Biochem. 57, 306 (1974).Castellan, G. W., Physical Chem istry, Addison-Wesley, Reading,Eastoe, J. E., Leac h, A. A., in T he Science and Technology ofGelatin, Ward, A. G., Courts, A., Ed., Academic Press, NewYork, 1977, p 93.Fields, R., Biochem. J . 124, 581 (1971).Freedman, R. B., Radda, G. K., Biochem. J . 108, 383 (1968).Goldfarb, A. R., Biochemistry 5, 2570 (1966).Habeeb, A. F. S. A., Ana l. Biochem. 14,3 28 (1966).Hald, A., Statistical Theory with Engineering Applications,Hennrich, N., Kontakte (Merck) 1973(2), 3 (1973).Jones, D. B., US ep . Agric. Circ., 183 (1931).Kotaki, A,, Harada, M., Yagi, K., J . Biochem. 55, 553 (1964).Novo Industri, Crystalline Porcine Trypsin Novo, IB 051,Novo, Industri, Subtilisin A, IB 169, Bagsvaerd, Denmark,Novo Ind ustri, H ydrolysis of Food Proteins in the Laboratory,Provans al, M. M . P., Cuq, J.-L . A., Cheftel, J.-C., J . Agric. FoodSatake, K., Okuyama, T., Ohashi, M., Shinoda, T., J . Biochem.47, 654 (1960).Smith, A. K., Circle,S.J., Soybeans: C hemistry and Technology,Vol. 1, Avi P ublishing Co., We stport, CT , 1972, pp 62-63.Steinhardt, J., Beychock, S., The Proteins, Vol. 11, 2nd ed,Ne urath , H., E d., Academ ic Press, N ew Y ork, 1964, pp 160-171.

(1979).MA , 1964, pp 215-217.

Wiley, New York, 1951, pp 579-583.

Bagsvaerd, Denmark, 1971.1978a.IB 102, Bagsvaerd, Denm ark, 1978b.Chem. 23, 938 (1975).

Received for review Septem ber 7, 1978. A ccepted Jun e 27,1979.

Determination of the Total Pepsin-Pancreatin Indigestible Content Dietary Fiber)of Soybean Products, Wheat Bran, and Corn Bran

David H. Honig* and Joseph J. Rackis~ ~~~~~~

Successive pepsin a nd pa ncreatin digestions were used to determine indigestible content (IDC) of varioussoybean products and cereal brans. IDC included insoluble material as well as solubilized carboh ydrateand protein separated by ultrafiltration (molecular weight above 5000). To tal IDC as percent of drym atte r was: corn bra n, 97; soybea n hulls, 86; wheat b ran, 52; whole soybean, 23; soy protein co ncen trate,40; an d defatted soy flakes, 16. Th e IDC values include 3-25% soluble ma terial recovered by ultraf-iltration. Chem ical analyses of th e insoluble nondigestible fraction from soybean hulls indicated acom position of 71% c ellulose, 20% hem icellulose, 9% lignin plus ash. T he perce nt protein dige stibilitywas estim ated as: whole soybean, 68; def atte d soy flakes, 81; soybean hulls, 60; soy prote in con centr ate,61; corn bran, 43; and wheat b ran, 60. Th e large values for undigested protein in soy protein p roductswere unexpected.

Recent reports on the nutritional significance of indi-gestible compo nents of hum an food have stimula ted newinterest in the characterization of these components andtheir functionality, particularly th e pla nt polysaccharides.Trowel1 (1977) discusses the significance of fiber in th ehum an die t and defines dietary fiber as th e remnan ts ofplan t cells resistant to the alimentary enzymes of m an.Other discussions on th e definition and application of theterm dietary fiber include those of Van Soest and Rob-ertso n (19 77), Sou thgate (19771, and Meyer a nd C allowayNo rthe rn Regional Research C enter, Agricultural Re-search, Science and Educa tion Adm inistration, US. e-pa rt m en t of Agriculture, Peoria, Illinois 61604. (1977).

This article not subject to U S . Copyright. Published 1979 by the American Chemical Society

determination of the degree of hydrolysis of food protein hydrolysates by

Documents