An Investigation of Pea Seed Albumins

An Investigation of Pea Seed Albumins D. R. Grant Department of Chemistry & Chemical Engineering

and A. K. Sumner and Janis Johnson! College of Home Economics, University of Saskatchewan, Saskatoon

, Present Address - School of Household Economics, University of Alberta, Edmonton

Abstract Albumins comprised 13-14% of the total N of both Century and Trapper varieties of peas. Water extraction at pH 5, dialysis, centrifugation and lyophilization yielded an albumin preparation (83% protein) with no detectable globulins. Ultracentrifugation showed two components with S,o w values of 0.53 and 3.55. Three components were resolved on 0-150 Sephadex, with molecular weights of 78,000, 47,700 and 26,000. Six major and several minor components were resolved by gel electrophoresis at pH 10. Sodium dodecylsulfate (SDS) promoted dissociation of the larger components and SDS-polyacrylamide gel electrophoresis showed only two major components with molecular weights of 25,000 and 15,500. Pea albumins were very soluble in water over a wide pH range. Less than 50% precipitated from solution by heat, depending on pH and concentration. An albumin concentrate prepared by acetone precipitation had foaming properties similar to egg white. Other functional properties are described. The amino acid composition is given.

Resume Les albumines comptent pour 13-14% de l'azote total des varietes de pois Century et Trapper. Une preparation d'albumine (83% proteine) exempte de globulines a ete obtenue par extraction aqueuse, dialyse, centrifugation et lyophilization. Deux constituants avec des indices de sedimentation S,o w de 0.53 et 3.55 ont ete reveles par ultracentrifugation. On a separe trois constituants sur Sephadex 0-150, avec des poids moleculaires de 78,000, 47,700 et 26,000. L'electrophorese sur gel il pH 10 a mis en evidence six constituants majeurs et plusieurs constituants mineurs. La dissociation des constituants plus gros a ete favorisee par Ie dodecylsulfate de sodium (DSS). L'electrophorese sur gel DSS-polyacrylamide n'a revele que deux constituants majeurs avec des poids moleculaires de 25,000 et 15,500. Les albumines de pois ont ete tres solubles dans I'eau sur une grande marge de pH. Dependant du pH et de la concentration, la precipitation thermique des albumines a ete inferieure il 50%. Un concentre d'albumine prepare par precipitation il l'acetone avail des proprietes de foisonnement semblables il celles du blanc d'reuf. On donne aussi d'autres proprietes fonctionnelles ainsi que la composition en acides amines.

Introduction Field peas are currently the object of great interest because of their potential as a source of protein for both human food and animal feed (e.g. Vaisey et al., 1975: Sarwar et ai, 1975 and Fan and Sosulski, 1974). The major protein components in pea seeds are globulins (Danielsson, 1950), but there is also a substantial fraction that displays the solubility characteristics of the albumins (Danielsson and Lis, 1950; Goa and Strid, 1959). On the basis of their solubility the pea albumins can readily be isolated. This protein fraction is known to be heterogeneous. Goa & Strid have resolved pea albumins into 3 major components by moving boundary electrophoresis. Two papers have appeared recently in the Russian literature (Shefyrste and Klimenka, 1973: Bezhan et al., 1973). Both Can. Ins!. Food Sci. Techno!. J. Vo!. 9. No.2, 1976

reports indicate that the albumin fraction is resolved into several components by ion exchange column chromatography. The results of our investigations of both physicochemical and functional properties of isolated pea albumin are described herein.

Material and Methods Seeds of Pisum sativum L. var. Century or Trapper were dehulled and ground to a floury texture. A pea protein concentrate and a high starch fraction were prepared from the Trapper variety by fine grinding and air classification (Anon, 1974). Table I gives some analytical data for these starting materials. The pea flour or air classified fraction was extracted by stirring with deionized water or with buffer at either room temperature or 5°C. Extraction times and the ratio of pea flour to extractant were varied. Insoluble material was removed by centrifugation at 15000 x g. The albumin fraction was separated from most of the soluble nonprotein material by dialysis for 48 h, against a large volume of deionized water at pH 5.0, using regenerated cellulose dialysis tubing. Adjustment of pH was with dilute solutions of HCI, acetic acid or NaOH. The dialysis also caused the precipitation of. the globulins (Danielsson, 1950). After further centrifugation, the dialyzed extract was lyophilized and designated as rea albumin (PA). Moisture content was determmed by the air oven method (AACC, 1967). Nitrogen and crude protein content were determined by the Kjeldahl method (AACC, 1961) and by the biuret method (Pinckney, 1961). The latter was standardized against the former. A factor of 5.7 for the conversion of nitrogen to protein was used as recommended by Holt and Sosulski (1974). The calculation of the amount of protein solubilized by the extraction procedure assumed that the extractant remaining with the residue, contained the same protein concentration as the recovered extract. Equilibrium dialysis was carried out as described by Hughs and Klotz (1956). An acetone precipitation method was developed to prepare a relatiyely large amount of an albumin-rich protein concentrate (ARPC) from the high protein fraction of air classified pea flour. One part of the starting material was stirred with 4 parts of water at room temperature and 84

the pH adjusted to 5.0. After 30 min the insoluble residue was removed by centrifugation at 1000 X g. The supernatent was cooled and cold acetone was added to 70% concentration vIv. The resulting precipitate was recovered and dried under vacuum at room temperature. The specific volume of PA at 10% wlv was measured using a 2.0 ml volumetric flask as a pycnometer. The calculated value was 0.715 ml/g. This value was used in calculating the total volume of solution, and the wIv concentration, when mixing a known weight of protein with a known volume of water. Solubility studies were conducted by stirring mixtures ofPA and water, centrifuging at 15000 X g and measuring the protein content of the solution, or the insoluble residue or both. The small amount of residue that was found to be insoluble in water, was soluble in the biuret reagent, and thus its protein content could be measured readily. PA solutions were heat treated by the immersion of small test tubes in a boiling water bath for 5 min. Sedimentation coefficients were determined using a Spinco Model E Analytical Ultracentrifuge. No corrections were made for concentration effects. Viscosities of protein solutions were measured with an Ostwald Viscosimeter having a flow time of 70 sec with water at 20.0°C. Reduced viscosity was calculated as described by Mysels (1959). Crystalline bovine serum albumin (Calbiochem) was used as a standard for comparison. Chromatography on Sephadex G-150 (Pharmacia Fine Chemicals) was carried out on a 2.5 X 90 cm column, which was prepared according to the procedure recommended by the manufacturer, and operated at room temperature by the reverse flow technique. Fractions of 5.0 ml were collected at the rate of 0.25 mllmin. The eluting buffer was O.oI M phosphate, pH 7.0, containing 0.2 M NaCl; or O.oI M glycine, pH 10.0, also containing 0.2 M NaCl. Polyacrylamide gel electrophoresis was performed in a water-cooled apparatus similar to that described by Lawrence et al (1970). The gels were prepared according to the procedure of Raymond and Wang (1960). Acrylamide and methylene bisacrylamide were mixed in the ratio of 22: 1. The best resolution of protein bands was achieved using a 0.05 M glycine buffer at pH 10.0. Typical running conditions were two h at a current density of 7 ma/cm 2 of gel cross section and an overall voltage of 400 V. Molecular weights of components separated on the G150 column were calculated by the method of Andrews (1965) using high purity grades of bovine serum albumin, egg albumin and myoglobin (Calbiochem) as standard proteins. Molecular weights were also determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), using the method of Weber and Osborne (1969). The proteins listed above were again used as standards. Fractions recovered from the Sephadex column were too dilute for direct polyacrylamide gel electrophoresis. They were dialyzed for 48 h against deionized water and lyophilized. Sufficient buffer was then added to give protein solutions of approximately 1% w/v. Amino acid analysis was performed on a Beckman 85

Model 120 C Auto-analyzer. The procedures for protein hydrolysis and for the analysis of tryptophan, methionine and cystine, were those described by Sosulski and Sarwar (1973). Several tests designed to evaluate the functional prop. erties of proteins, were performed on the ARPC. Compari. son tests were carried out with fresh egg white, the air clas. sified pea protein concentrate (Table I) and a soy concentrate (Promasoy-Central Soya Co.). Table I. Analysis of starting materials.

Sample Century Pea Flour . Trapper Pea Flour . Pea Protein Concentrate' . High Starch Fraction' .

% Moisture

% Protein' (N x 5.7)

8.7 6.7

23.3

5.1 4.4

18.9 53.2 3.9

I as is basis , prepared by air classification of Trapper flour.

Foaming characteristics were determined by mixing a known weight of test material, usually 1.0 g, in approximately 25 ml of water, adjusting to the desired pH, making to 35 ml in a 250 ml beaker and stirring with a laboratory stirrer at high speed for 1.0 min. Longer stirring did not increase the volume or stability of the foam produced. Foam volume was measured in a 250 ml graduate cylinder at 5 min after mixing and at timed intervals thereafter. The percentage volume increase was calculated by the method of Lawhon and Cater (1971). Foam viscosity was measured with a Brookfield Viscosimeter, Model RTV, at 5 rev I min using a TA spindle. Fat absorption was determined according to the method of Lin et al (1974). Oil emulsification values were determined by the method of Inklaar and Fortuin (1969). Mazola Corn Oil (Best Foods Div. - Canada Starch Co.) was used in both procedures. The nitrogen solubility index was determined by the AOCS Method (1970) except that protein in solution was measured by the biuret test (Pinckney, 1961). In order to evaluate binding capability the following test was used, based on a procedure recommended by Murray (1975). Texturized vegetable protein (TVP - a soya meat analogue from Archer Daniels Midland Co.) was screened to provide a fraction that passed through a 20, but not through a 40 W sieve. The TVP was mixed with the test protein in the ratio of 3:2 by weight and enough water was added so that after sufficient time had passed to allow the TVP to hydrate, the mixture had the consistency of a muffin batter. The batter was baked at 240°C for 15 min in greased aluminum pans, 6.5 cm in diameter, filled to a depth of 1.5 cm. After cooling the resulting cakes were tested for hardness and cohesiveness with a Food Technology Corp. Texture Test System, Model T-2I00, equipped with a multiple shear blade cell. Control experiments substituted washed fine sand (75% passed through a #40 W screen) for the test protein. A further set of experiments was carried out with mixtures of test protein, sand and TVP in a I: 1:3 ratio. J. Inst. Can. Sci. Technol. Aliment. Vol. 9. No.2, 1976

Results and Discussion Extraction of Pea Seed Proteins The use of dilute NaCI to extract pea seed protein followS the precedent of earlier investigators (Danielsson, 1950; Goa and Strid, 1959). Up to 80% of the nitrogen in Century peas was solubilized over a 48 h period at 5°C when the flour was extracted with 0.5 M NaCI solution, buffered to pH 7.0 with 0.1 M phosphate. The ratio of flour to extractant was I: 12 wIv. Shorter periods resulted in lower recoveries. The effect of stirring time on the extent of extraction is illustrated in Figure I. Exhaustive dialysis against the same buffer removed 15% of the nitrogen from the extract, that is 12% of the total N. This value was confirmed in an equilibrium dialysis experiment in which the amount of N in the dialysate was measured. The percentage of dialyzable N found in Trapper peas, when extracted with water, was slightly lower (Table 2).

90

•

Table 2. Percentage of total N present in water extracts at various stages of pea albumin preparation.

12

Trapper pea flour Pea protein concentrate' High starch fraction'

initially at pH 6.5

on adjustafter ment dialysis to pH 5.0 (albumin)

in the dialysate

% additional globulins'

85.2

25.0

13.7

9.7

1.6

81.8

20.9

12.3

7.0

1.6

78.6

50.0

17.9

30.1

2.0

, globulins not precipitated by pH adjustment but precipitated by dialysis. , prepared by air classification of Trapper flour.

Precipitation of the globulins by exhaustive dialysis against water left an albumin fraction that accounted for 13.5% of the total N in Century pea flour and 13.7% in Trapper pea flour. Carrying out the dialysis at pH 5.0 rather than pH 7.0 resulted in a more rapid and more efficient precipitation of the globulins. Procedures that include NaCI in the extractant are designed to maximize the solubilization of the globulins. Since the albumin fraction was of primary interest, deionized water was also investigated as an extractant. The percentage of total N solubilized by stirring one part of Trapper pea flour with 10 to 12 parts of water, for 30 min at room temperature ranged from 75 to 85%. The pH of the resulting extracts was 6.5 ± 0.1. Apparently the naturally occurring salts are r.resent in sufficient amount to promote the initial solubl1ization of the globulins along with the albumins. However, such a hypothesis does not agree with the data of Zarkadas et at (1965), who reported that very low concentrations of NaC 1 in the extractant actually depressed the amount of N that was solubilized. Using a 4: 1 ratio of water to pea flour, only 55-60% of the flour N was solubilized. Low temperature, or extended periods of stirring, did not increase the extent of N extraction. In fact, longer extraction periods resulted in a substantial decrease. After 48 h of stirring, less than one half as much N was found in solution as at 30 min. It was also observed that a considerable amount of precipitated protein would accumulate, when fresh extracts were allowed to stand for several h at either room temperature or 5°C. Can. Inst. Food Sci. Technol. J. Vol. 9, No.2, 1976

36

48

TIME,HRS.

% of total N present in the extract

Starting Material

24

Fig. 1.

Effect of time of stirring on the extent of protein extraction from Century pea flour at 5°C with 0.5 M NaCI, pH 7.0.

The isoelectric points of the two globulin components in pea seeds are 4.8 and 5.3 (Danielsson, 1950). Adjusting the pH of a water extract to the intermediate value of 5.0 resulted in the precipitation of most of these globulins. The extent of such precipitation was very similar over the pH range from 4.0 to 5.5. Exhaustive dialysis following pH adjustment resulted in very little additional precipitation of pea globulins. The percentage of the total N found in extracts of Trapper pea flour at various stages in the procedure for isolating the albumin fraction, is given in Table 2. A comparison is made with similar extracts of the high starch and high protein fractions, prepared from by air classification of the same flour. The percentage of total N that remained soluble at pH 5.0 agrees well with the value reported by Fan and Sosulski (1974). It is noted that the ratio of albumin to total N was slightly higher and the proportion of dialyzable N was considerably higher in the high starch fraction, than it was in either of the other starting materials. The albumin preparation designated PA was a white fluffy powder containing 7.8% moisture and 14.7% N, corresponding to a protein content of 83.6%. Thus it could almost be classified as a protein isolate. Indeed, if the customary protein conversion factor of N X 6.25 had been used, this material would have been classified as an isolate. Sedimentation velocity measurements of aqueous solutions in the analytical ultracentrifuge gave two peaks as shown in Figure 2, with sedimentation of coefficients (S20 w) of 0.53 Sand 3.55 S. There was no trace of globulins in the sedimentation velocity profile. The product obtliined by direct lyophilization of a pH 5.0 water extract contained only 33% of crude protein. This reflects the relatively large amounts of sugars and other water-soluble non-protein material known to be present in pea flour (Youngs, 1975). The material designated ARPC was a pale yellow powder with a crude protein content of 55%, of which only 78% was soluble in water. The water insoluble residue did 86

ing the pH had a minor effect on protein solubility. Increases to values above 5.0 did not cause precipitation to occur at any concentration of protein that was tested. Ad. justment of the pH below 5.0, created considerable turbid_ ity in the solutions, but on centrifugation it was found that most of the protein remained in solution. In a series of eight analyses, that covered the pfl range from 5.0 down to 2.6, the amount remaining in solution after adjustment was greater than 95% in all cases except one, where the result was 93%. In these experiments the starting concentration of PA was either 16.5 or 14.5% w/v, which was much higher than that which is customarily used in the nitrogen' solubility index test (AOCS, 1970). With further adjust. ment of the pH down to 1.9 only 75% of the protein remained in solution. Below pH 2.0, the precipitate changes from white and flocculent to a viscous, translucent, gel-like consistency. Adjustment to either a high or low pH followed by readjustment back to pH 4.7 resulted in the precipitation of a considerable portion of the protein originally in solution. Results of such experiments are in Table 3. Table 3. Effect of adjustment to extreme pH on the subsequent solubility of pea albumin'. pH history %of Original protein - - - - - - - - - - - - protein that concentration precipitates final pH % w/v original pH extreme pH

Fig. 2.

Ultracentrifuge diagram of pea albumin from Trapper peas. 60,000 rpm; 75 min after attaining speed; phase plate 40°; 20°C.

10.0 14.5 14.5 I

not dissolve in dilute NaCl so that it should not be concluded that this material was necessarily globulin contamination. The water soluble protein of the ARPC was more than 80% albumin as determined by ultracentrifugal analysis. In preparing this material, an acetone concentration of 70% was chosen because it caused virtually complete precipitation of dissolved protein, whereas lower acetone concentrations did not. Solubility Studies Pea seed albumin, (PA) was highly soluble in water, giving solutions that ranged from pale yellow to brown as the concentration increased. A direct linear relationship between the amount of protein in solution and the amount of dry protein added, was observed at concentrations up to at least 40% w/v. At this concentration, the small amount of wet, water insoluble residue accounted for only 1.2% of the total protein. Abover 40% wIv the solutions were so viscous that quantitative transfer of aliquots was difficult to carry out, and consequently, accurate protein concentration measurements could not be readily performed. At 55% w/v, a very viscous mixture, full of air bubbles, was obtained. Centrifugation at 15000 x g forced out the air to give a single, transparent and apparently homogeneous phase. Dissolution of PA in water occurred very rapidly and seemed to be a co-operative phenomena in that larger quantities dissolved even more readily than smaller quantities. Solutions of PA in water had a pH of 5.0 ± 1. Adjust-

87

5.0 5.0 5.0

2.6

4.6

20.6

1.9

4.7 4.7

41.2 31.1

10.5

Trapper peas

Heating pea albumin solution at 98°C caused very rapid precipitation of part of the protein, the amount depending on both the protein concentration and the pH. At pH 5.0 and 40% wi V, heating rapidly converts the entire solution to a firm gel, resembling cooked egg white. Although a 40% solution was fairly dark in colour, the gel produced by heating was white. Some of the protein in this gel remained soluble and could be extracted with water. At protein concentrations below 10% wi v, heating caused a gelatinous white precipitate to settle out of solution. The effect of protein concentration on the extent of protein precipitation, by heat, is shown in Figure 3. The modifying effect of pH on the extent of protein precipitation caused by heat is shown in Figure 4. Maximum precipitation occurs near pH 5.0. At pH 10.5 or higher and below approximately pH 3.0, none of the protein is precipitated by heating. The relationship between concentration effects and pH effects resulted in no precipitation on heating of a 7% w/v solution, at pH 3.5 but 19% precipitation ofa 16.5% w/v solution at pH 3.25. The time required for precipitation to occur as a result of heating, increased below pH 4.0 and the nature of the precipitate changed from opaque white to translucent. The effect of dry heat on the subsequent solubility of PA in water, was briefly investigated. Heating at 125°C for 1 h resulted in an increase in the amount of material that would not dissolve, particularly at lower protein concentrations. At 14% w/v, 12% of the protein would not go into soJ. Inst. Can. Sci. Technol. Aliment. Vol. 9, No.2. 1976

coagulated by calcium ions (de Man et al., 1975). It was found, however, that a 3% w/v solution of pea seed albumin did not coagulate when diluted 1: 1 with saturated CaSO., nor did the presence of calcium ions increase the proportion of pea seed albumins that could be precipitated by heating such solutions.

10

20

30

40

PROTEIN CONCENTRATION

V¥v

Fig. 3. The effect of protein concentration on the extent of heat precipitation of pea albumin solutions (Trapper peas) at pH 5.0. Heating was for 5 min. at 98°C.

50

Viscosity Studies The effect of pH and heat on the reduced viscosity (Mysels, 1959) of PA from Trapper peas is shown in figures 5 and 6. A comparison was made with bovine serum albumiri as a reference protein (Figure 5). These results indicate that in solution near neutral pH, pea albumin polypeptide chains appear to be somewhat less compactly folded, or more highly hydrated, than bovine serum albumin, but at extreme pH's the tendency for extensive unfolding to occur is less severe. The effect of heat on the viscosity of PA solutions was examined at both high and low pH levels, since no difficulty with protein precipitation is encountered under these conditions. Heating for 5 min in a boiling water bath had very little effect at high pH but at low pH and at 7% w/v, there was a very marked increase in the reduced viscosity (Figure 5). There was also a further increase as the heat treated solution was allowed to stand at 20°C. In contrast, at 1% w/v, heating caused a small decrease in reduced viscosity. For noninteracting protein molecules, reduced viscosity values show little dependency on solution concentrations (Mysels, 1959). The results cited above and the steepness of curve I in Figure 5 suggests that heating under mildly acid conditions promotes some type of concentration dependent interaction among pea albumin molecules.

80

2

4 pH

Fig. 4.

The effect of pH on the extent of heat precipitation of pea albumin solutions (Trapper peas) at 16.5% w/v. Heating was for 5 min. at 98°C.

lution. It was again observed that the ease of solution increased with increasing ratio of protein to water. At 36% W/v only 2% of the protein would not dissolve. Furthermore, a clear 36% solution became turbid on dilution with water. The solutions prepared from dry heated material appeared to be somewhat more viscous and darker in colour than those prepared from unheated material. One of the applications of vegetable protein concentrates is in the formulation of milk replacers and products such as soy milk. The use of pea seed albumins in such applications is conceivable. The conditions under which this type of product will curdle are of interest. Soy milk can be Can. Ins\. Food Sci. Techno!. J. Vo!. 9, No.2, 1976

...

'

L

! ...rC ,._ • .-'B

2

4

6

F

8

% PROTEIN W/V

Fig. 5. The effect of pH and heat on the reduced viscosity of Tra'pper pea albumin (PA) and bovine serum albumin (BSA) solutIons. A - BSA at pH 5.0; B - BSA at pH 11.2; C - BSA at pH 2.7; D - PA at pH 5.0; E - PA at pH 11.3; F - PA at pH 2.7; G - PA at pH Il.l after heating at 7% w/v; H - PA at pH 2.8 after heating at 7% w/v; 1- PA at pH 2.8 after heating and standing for 2 hrs. Heating was for 5 min. at 98°C.

88

14

12 I-

z

10

III

I-

Z

0

z

~

l;l :> Q

III

iii

l5

II:

Do

6

•

0

::)

•

Q

III

II:

C

0

> t: 8

4

120 2

Fig. 8. 2

4

6

8

10

12

pH

Fig. 6.

Effect of pH on the reduced visosity of pea albumin solutions at 1% w/v. (Trapper variety).

Gel Electrophoresis and Gel Chromatography Polyacrylamide gel electrophoresis revealed that pea seed albumins were a complex mixture in which at least 10 components could be resolved (Figure 7).

Fig. 7.

Polyacrylamide gel electrophoresis of the albumins from Trapper peas at pH 10.0 in glycine buffer. The anode is to the right.

On a 90 cm G-150 Sephadex column at pH 7.0, the mixture was resolved into only 4 peaks as shown in Figure 8. Peak A emerged at the void volume of the column. The elution volumes of peaks B, C and D correspond to molecular weights of 78,000,47,700 and 26,000 respectively (Andrews, 1965). The peak fractions were subjected to gel electrophoresis and the pattern of bands obtained, in comparison to that of the original mixture, is shown in Figure 9. Obviously the fractionation on Sephadex did not lead to the isolation of homogeneous components. The components in peak A were probably association complexes of the smaller components, or severely denatured material. Since gel electrophoresis at pH 10.0 gave better resolution than at lower pH's the effect of operating the G 150 Sephadex column at pH 10.0 was investigated. The number of peaks and the elution volumes were similar to that observed at pH 7.0 except that the peak emerging at the void volume was much larger. The higher pH probably caused some unfolding of the polypeptide chains, resulting in a higher proportion of the albumin being excluded from the gel particles. (Chao and Einstein, 1969). Further evaluation of pea albumin molecular weights, by gel electrophoresis in the presence of SDS, revealed 89

220

320

420

ELUTION VOLUME

Chromatography of albumin from Trapper peas on a 90 cm G150 Sephadex column at pH 7.0; 0.2 M NaCI.

only two major protein bands. Their migration rates correspond to molecular weight values of 25,000 and 15,500. The absence of major bands corresponding to the higher molecular weight components that were observed on the G-150 Sephadex column, suggest that these larger species may conSIst of molecular complexes that dissociate in the presence of SDS (Grant and Lawrence, 1964). Six additional, but very minor components were also detected in the SDS-acrylamide gels. Two of these migrated considerably more slowly than the slowest standard protein. Rough estimates of their molecular weights are 200,000 and 110,000. The other four correspond to molecular weights of 48,000; 32,000; 21,500 and 18,000. S PA

A B

II

i

i

c D

Fig. 9.

Sketches of the band patterns obtained by polyacrylamide gel electrophoresis at pH 10.0, of the peak fractions that were separated by G-150 Sephadex chromatography of pea albumins. PA is the unfractionated pea albumin mixture. A, B, C and D refer to peaks identified in Fig. 8. S is the position where the samples were inserted in the gel. The anode is to the right.

Amino Acid Composition The amino acid composition of PA from Century peas is given in Table 4 in comparison to that found in the protein of the original peas. The albumin fraction shows less of a deficiency in sulfur amino acids and is a superior source of lysine. Comparison of this data with that of Goa and Strid (1959) reveals several discrepancies, particularly with regard to glutamic acid and cystine, where they report values that are much lower and higher respectively. However, substantial differences in the amino acid composition of different varieties of peas, have been reported. (Zarkadas, 1965). J. lnst. Can. Sci. Technol. Aliment. Vol. 9, No.2, 1976

Table 6. Oil emulsification and fat absorption.

Table 4. Amino acid composition' of pea albumin'.

-

Amino Acid

ALA ARG ASP CYS

GW GLY HIS ISO LEU LYS

Pea Flour

Pea Albumin

Amino Acid

Pea Flour

Pea Albumin

4.9 7.9 12.6 1.5 18.6 4.9 2.3 4.1 7.2 7.7

6.4 5.5 12.1 1.8 15.0 5.5 2.6 3.8 5.2 9.3

MET PHE PRO SER THR TRY TYR VAL NH,

0.9 4.5 4.3 5.1 4.2

1.2 4.5 4.3 5.0 5.8 1.2 4.6 5.2

1.1 2.5 4.5 1.8

Sample

% Fat absorbed

%Oil emulsified

101 90 99

26.5 29.5 10.0

ARPC' Pea concentrate Soy concentrate

, Albumin rich protein concentrate - Trapper Peas.

Of the proteins tested for their ability to act as a binding agent for TVP, pea protein concentrate was the most effective? e?g white was next and the soy concentrate gave results similar to the controls. No data could be obtained for ~he test cakes containing ARPC because they were so fragile that they could not be removed from the pans without crumbling. This result infers that pea albumin had a negative effect on binding. Such a conclusion is questionable because it was also observed that the cakes containing ARPC rose up during baking, much more than any of the others. Their lack of cohesiveness may be a direct consequence of this leavening effect, rather than a lack of binding capacity. The nitrogen solubility index of ARPC in comparison with other concentrates is shown in Figure 10. Wnile the r~sults do indicate the small effect of pH on solubility, they give no clue to the larger amounts of pea albumin that will dissolve in water. Under the conditions of the test, the concentration of protein in solution was approximately 1% w/ v and more than 20% of the ARPC protein did not dissolve. Consequently, on this basis alone, one could easily be misled to believe that pea albumins are only moderate~y soluble in water. Actually, the .insoluble portion is an artifact of the method of preparation and has no direct bearing on the inherent solubility of the pea albumins. The experiments on PA solubility that were described earlier, were prompted in part, because the nitrogen solubility index test does not adequately characterize this property.

1.5

, g amino acid/ 16 g N from Century peas

1

Functional Properties

The available procedures for testing functional properties, require relatively large quantities of test material. The lack of suitable facilities for large scale dialysis made it impractical to prepare sufficient PA isolate. Therefore, an acetone precipitation procedure was used to prepare the required quantities of an albumin-rich concentrate (ARPC) for use in these tests. Large amounts of stiff foam were produced when mixtures of ARPC and water were stirred at high speed. The appearance and stability of the foam was comparable to that obtained with fresh egg white (Table 5). Visual observation could not distinguish one from the other. Under the microscope, the ARPC foam had smaller air cells. Egg white foam had a lower volume at its natural pH of9.1 but was similar in volume to ARPC at lower pH levels. There was a negligible effect of pH on viscosity for either protein. Egg white had a higher foam viscosity but in the data in Table 5, it must be pointed out that the protein concentration of fresh egg white is much higher than that which was used initially in testing ARPC. Further tests showed that increasing the ratio of ARPC to water so that the protein concentration was similar to egg white, had a rather small effect on either the volume or viscosity of the pea albumin foam. A 1:4 dilution of egg white at its natural pH reduced the volume and viscosity of the foam by approximately 25 and 30% respectively. The oil emulsification and fat absorption tests on ARPC gave results similar to that for pea protein concentrate (Table 6).

General Discussion Because of their high solubility, the incorporation of pea albumin offers a potential means of controlling the viscosity of liquid foods. Since solubility remains high over the normal pH range, they may be suitable for applications such as the protein fortification of carbonated beverages. Other properties, particularly the capacity to form

Table 5. Comparison of foaming characteristics of pea albumin concentrate and egg white.

Sample ARPC' ARPC ARPC fresh egg white fresh egg white fresh egg white

pH

protein cone. w/v

5.4' 4.0 8.0 6.1 4.0 9.1'

1.5

1.5 1.5 11.0 11.0 11.0

% volume increase' on whipping

10 min.

330 310 300 317 350 160

96 97 95 100 98 96

Foam stability Relative volume' after elapsed time of 30 min. 60 min. 92 92 88 97 88 87

86 86 85 93 69 79

120 min.

Foam Viscosity'

81 77 80 82 61 76

14,800 13,400 13,600 22,400 22,720 26,280

, volume after whipping - volume b.efore whipping X 100 (Lawhon and Cater 1970) volume before whlppmg , , foam volume at given elapsed time as percentage of foam volume 5 min. after whipping , Brookfield Viscosimeter units - see methods section • albumin rich protein concentrate from Trapper peas , pH as is. Can. lnst. Food Sci. Technol. J. Vol. 9, No.2. 1976

90

References

fa

~a: t
*20 2

4

6

8

10

12

pH Fig. 10.

Nitrogen solubility index: A - pea albumin concentrate (ARPC); B - pea protein concentrate; C - soy concentrate.

stable foams, and the nutritional balance of the amino acids, indicate that a pea albumin isolate may have many additional food uses. The fact that less than 15% of the protein in peas is albumin, and the anticipated high cost of the isolation procedure, make it questionable whether or not the commercial production of either an albumin isolate or albumin concentrate, would be economically feasible. It would almost certainly have to be the byproduct of some more general fractionation of pea flour. For example, the starch prepared by air classification of pea flour contains a residual protein content that is too high for some of the potential applications of this material. Extraction with water to upgrade this starch may be necessary. In turn, the extract may provide the starting material for the economic preparation of a pea albumin isolate.

A.A.CC. 1961. Method 46-10 A.A.C.C. Approved Methods 7th ed. American Association ofCe. real Chemists. St. Paul, Minnesola. A.A.CC. 1967. Method 44-15 ibid. Andrews, P. 1965. The gel filtration behavior of proteins related to their molecular weight Over a wide range. Biochem, J. 96:595. Anon. 1974. Pea flour and pea protein concentrate. P.F.P.S. Bulletin No. I, Prairie Regional Lab National Res. Council and College of Home Econontics, U. of Sask. Saskatoon, Canada.' AO.C.S. 1970. Method Ba 11-65. Official and Tentative Methods. 3rd Ed. American Oil Chemists Society, Champaign, Illinois. Bezhan, F. I., Gofman, Yu. Ya. and Klimenko, V. G. 1973. Chromatography on DEAE-cellulose of albumins of seeds of different pea species. Izv. Akad. Nauk Mold. SSR. Ser. BioI. Kim Nauk.4:22. Chao, L. P. and Einstein, E. 1969. Estimation of the molecular weight of flexible disordered proteins by exclusion chromatography. J. Chromatog. 42: 485. Danielsson, C. E. 1950. An electrophoretic investigation ofvicilin and legumin from seeds of peas. Acta. Chern. Scand. 4:762. Danielsson, C. E. and Lis H. 1952. Differences in the chemical composition of some pea proteins. Acta. Chern. Scand. 6: 139. de Man, J. M., Tanaka, M. and Stanley, D. W. 1975. Coagulation properties of soybean mille Can. Inst. Food Sci. Technol. 1. 8:9. Fan, T. Y. and Sosulski, F. W. 1974. Dispersibility and isolation of proteins from legume flou... Can. Inst. Food Sci. Technol. J. 7:256. Goa,.]. and Strid, L. 1959. Amino acid content ofleguminolls proteins as affected by genetic and nutritional factors 111. Arch. fur Mikrobiol, 33:253. Grant, D. R. and Lawrence, J. M. 1964. The effect of sodium dodecyl sulfate and other dissociating reagents on the globulins of peas. Arch. Biochem. Biophys. 108: 552. Holt, N. W. and Sosulski, F. W. 1974. Nitrogen to protein factors. A.A.CC. Conference, Montreal, paper 169, abstracted Cereal Sci. Today 19: (9) 409 (1974). Hughs, T. R. and Klotz, 1. M. 1956 in Methods of Biochem Anal. Vol 3. D. Glick Ed. Inlerscience N.Y. Inklaar, P. A. and Fortuin, J. 1%9. Determining the emulsifying and emulsion stabilizing capacity of protein meat additives. Food Techno!. 23: 103. Lawhon, J. T. and Cater, C. M. 1971. Effect of processing method and pH of precipitation on the yields and functional properties of protein isolates from glandless cottonseed. 1. Food Sci. 36: 372. Lawrence, J. M., Grant, D. R. and Herrick, H. E. 1970. Apparatus for vertical polyacrylamide gel electrophoresis. Cereal Chern. 47:91. Lin, M. J. Y., Humbert, E. S. and Sosulski, F. W. 1974. Certain functional properties of sunflower meal products. J. Food Sci. 39:368. Murray, E. D. 1975. Personal Communications. General Foods, Coburg, ant. Mysels, K. J. 1959. Introduction to Colloid Chemistry p. 276. Interscience, New York. Pinckney, A. J. 1961. The buiret test as applied to the estimation of wheat protein. Cereal Chern. 38:501. Raymond, S. and Wang, Yi, Ju. 1960. Preparation and properties of acrylamide gel for use in electrophoresis. Anal. Biochem. 1:391. Sarwar, G., Sosulski, F. W. and Bell, 1. M. 1975. Nutritive value of field peas and fababean proteins in rat diets. Can. Inst. Food Sci. Technol. 1. 8: 109. Shefyrtse, M. A. and Klimenko, V. G. 1973. Study of the albumins of the seeds of several pea varieties by column gradient extraction and chromatography on DEAE-cellutose and hydrox. yapatite. 1973. Belki Semyan Kul' T. Rast. P: 3-14. Sosulski, F. W. and Sarwar, G. 1973. Amino acid composition of oil seed meals and protein is0lates. Can. Inst. Food Sci. Technol. J. 6: I. Vaisey, M., Tassos, L., McDonald, B. E. and Youngs, C. G. 1975. Performance of fababean and field pea protein concentrates as ground beef extenders. Can. Inst. Food Sci. Technol. J. 8:74. Weber, J. and Osborn M. 1969. The reliability of molecular weight determinations by dodecylsulfate-polyacrylamide gel electrophoresis. J. BioI. Chern. 244:4406. Youngs, C. G. 1975. In preparation. Zarkadas, C. G., Henneberry, G. D. and Baker, B. E. 1965. The constitution of leguminous seeds V. Field peas (Pisurn sali,urn L.) J. Sci. Fd. Agric. 16:734. Received October 27, 1975

Acknowledgements The financial support of the National Research Council of Canada and the Saskatchewan Research Council, who provided a fellowship for Janis Johnson, is gratefully acknowledged. The technical assistance of Mr. R. G. Teed, Miss Lorna Ornawka, and Mrs. Marilyn Nielsen was greatly appreciated. Dr. E. Scheltgen kindly performed the ultracentrifugal analysis. Dr. F. W. Sosulski and Dr. C. Youngs generously provided pea flour and air classified fractions of pea flour.

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