Isolation of Rapeseed Protein Using Sodium Hexametaphosphate

Isolation of Rapeseed Protein Using Sodium Hexametaphosphate L. U. Thompson, P. Allum-Poon and C. Procope Department of Nutrition and Food Science University of Toronto Toronto. Ontario

Abstract The suitable conditions for the extraction and precipitation of proteins from rapeseed flour (RF) using sodium hexametaphosphate (SHMP) were determined. Rapeseed protein isolate (RI) was then prepared and analyzed for chemical composition, biological value, color and yield. The highest nitrogen yield was obtained when RF was double extracted with 2% SHMP at pH 7.0, first with a RF to solvent ratio of I: 10 and second with a ratio of 1:6 at 25°C for 30 min. The maximum precipitation of the extracted nitrogen was observed in the extract diluted with an equal volume of distilled water and adjusted to pH 2.5. The RI contained on dry basis. 72.6% protein (N x 6.25). 12.2% ash, 0.7% crude fiber, 7.6% nitrogen free extract. 3.21% phosphorus, trace amounts of glucosinolate and no myrosinase activity. Its PER was equal to that of cheese whey protein concentrate and greater than that of casein. RI was yellow at pH 2.5 and tan at pH 7.0. Ether extraction lightened the color of the neutralized RI. Thirty-four percent total solids yield and 53% protein yield were observed in the preparation of RI.

Resume Les conditions convenables ont ete ebalies pour I'extraction et la precipitation des proteines de farine de colza (Fe) it I'aide d'hexametaphosphate de sodium (HMPS). Un isolat proteique de colza (lC) a ensuite ete prepare et analyse pour sa composition chimique, sa valeur biologique, sa couleur et son rendement. Le plus fort rendement azote a ete obtenu lorsque FC a ete extraite it deux reprises avec 2% HMPS it pH 7.0 d'abord avec un rapport FC it solvant de I: 10 et en reprise avec un rapport de 1:6 it 25°C pendant 30 min. La precipitation de I'azote extrait a ete maximum lorsque l'extrait a ete dilue avec un egal volume d'eau distillee et son pH ajuste it 2.5. L'IC contenait sur base seche. 72.6% de proteine (N x 6.25), 12.2% d'e!ements mineraux, 0.7% de fibre brute. 7.6% d'extrait non azote. 3.21% de phosphore. des traces de glucosinolate et aucune activite de myrosinase. Son coefficient d'efficacite proteique a ete egal it celui d'un concentre proteique de petit lait de fromage et plus eleve que celui de la caseine. L'IC etait jaune au pH 2.5 et beige au pH 7.0. La couleur de l'IC neutralise a ete palie par extraction it I'ether. Des rendements de 34% d'extrait sec et de 53% de proteine ont ete obtenus dans la preparation de l'Ie.

Introduction Rapeseed, a major oilseed in Canada, is grown primarily for its edible oil. The meal which remains after oil extraction contains approximately 40% protein and is currently used for animal feeds and fertilizers (Burrows et aI., 1972). The potential value of rapeseed protein for human consumption is well documented (Burrows et al., 1972; Lo ~nd Hill, 1971 a, b; Girault, 1973). The amino acid pattern mdlcates that among oilseeds, rapeseed is the best for the supplementation of human diets (Sosulski and Sarwar, 1973)'. Nevertheless, the presence of glucosinolates reduces the bIOlogIcal value of the protein from the untreated rapeseed meal and flour (Girault, 1973; Josefson and Munck, 1973). . The isolation of the proteins can improve the versatilIty of rapeseed meal for use as nutritional and functional additives in human foods. Girault (1973) and Kodagoda et 15

al. (1973) reviewed some of the methods employed in t preparation of rapeseed protein concentrates and isolat differing mainly in the media used for protein extracti and precipitation. Sodium chloride (Lo and Hill, 1971 Owen and Chichester, 1971) and salino-alcoholic solutio (Eklund et al., 1971) had been used; so with alkali extra tion and acid precipitation (Pokorny, 1963; Sosulski an Bakal, 1969; Girault, 1973; Kodagoda et al., 1973). Li ited work has been reported on the use of polyphosphat although it has previously been applied to other oilsee with good results (Shemer et al., 1973). Bhatty et al. (196 extracted 45% of the rapeseed nitrogen with the use of s dium pyrophosphate but the use of high molecular weigH polyphosphates as sodium hexametaphosphate (SHMP) y improve the nitrogen extraction yield has not been trie~ In this study the effect of various processing param~ ters on the extraction and precipitation of rapeseed proteu using SHMP were determined. Rapeseed protein isolad (RI) was then prepared and evaluated for chemical com! position, biological value, color and yield.

Experimental Methods Materials Dehulled, hexane extracted rapeseed (Brassica nap~ L. var Oro) flour (RF) was provided by the Food Researc Institute, Canada Department of Agriculture, Ottawa, 0 tario. Analytical grade SHMP (m.w. 611.77, Fisher. Scie tific Co.) was used for protein extraction. Extraction of Proteins from Rapeseed Flour The effect of several parameters were systematicalll studied in order to determine the suitable conditions f~ protein extraction using SHMP. The best pH for protei~ extraction was first determined using as criteria the per~ centage yield in the extract and the color lightness of the subsequent rapeseed protein isolate. The effect of SHMf concentration, RF to solvent ratio, temperature, time and double extraction on extraction yield at this pH were then established. The effect on yield of varying one parametel was determined while holding others at specified values. All protein extractions, unless otherwise specified were carried out according to the following procedures Twenty-five ml of 2% aqueous SHMP were added to 2.5 I of RF and stirred for 10 min at 25°C. The slurry was adjusted to the desired pH using 3N NaOH or 3N HCl shaken in a Dubnoff metabolic shaking incubator for 3( min at 25°C and centrifuged at 1000 x g for 20 min. A I( m1 aliquot of the supernatant was analyzed for nitrogen bJ the Kjeldahl method (A.O.A.c., 1970). The percentage eX' traction yield was determined as that portion of the tola J. Inst. Can. Sci. Techno\. Aliment. Vol. 9. No. l. 1971

RAPESEED FLOUR (RF) Double extract with 2% SHMP First with I: 10 RF: 2% SHMP Second with 1:6 RF: 2% SH MP Stir for 10 min, 25°C Adjust to pH 7.0 with 3N NaOH Stir for 30 min, 25°C Centrifuge 1000 x g, 20 min RESIDUE

SUPERNATANT Dilute I: 1 with dis!. H,O Adjust to pH 2.5 with 3N HCI Centrifuge 1000 x g, 20 min SUPERNATANT

PRECIPITATE Wash 2 times with dis!. H,O . Centrifuge 1000 x g, 20 min

I

rl-----------~-----

PRECIPITATE

I

SUPERNATANT

Disperse in dis!. H,O Adjust to pH 7.0 with IN NaOH Freeze dry Grind to uniform size (20 E1esh) RAPESEED PROTEIN ISOLATE (RI) Fig. I. Preparation of rapeseed protein isolate

nitrogen in the RF which was extracted. All the data shown represent duplicate analyses using two samples of

RF. Precipitation of Extracted Rapeseed Nitrogen Precipitation experiments were carried out as follows: Twenty-five grams of RF were extracted first with 250 ml and second with 150 ml of2% SHMP at pH 7 for 30 min at 25°C as previously described. Twenty ml aliquots of the combined protein extracts were then acidified to various pH levels using 3N HCI and centrifuged at 1000 x g for 20 min. One series of protein extract was diluted with an equal volume of distilled water prior to pH adjustment, while another was undiluted. Ten ml of the supernatant was analyzed for nitrogen by the Kjeldahl method (AOAC., 1970). The precipitation yield was that portion of the total nitrogen of the extract which was recovered in the precipitate. The means of duplicate analyses of extracts from two samples of RF are reported in this paper. Preparation of Rapeseed Protein Isolate . The method adopted for the preparation of RI is outhned in Figure I. Chemical Analysis . Moisture, fat, ash, protein and crude fiber were determmed by A.O.A.c. (1970) methods and phosphorus by the method of Fiske and Subbarow (1925). For amino acids, samples were hydrolyzed with 6N HCI for 24 hours at llOoC under vacuum and analyzed in a Beckman model 121 amino acid analyzer. Oxazolidinethione was estimated aCcording to the spectrophotometric method of Wetter (1957), except that the wavelengths 235, 245 and 255 nm :Were selected, and the glucosinolates and myrosinase activl~y by unpublished methods of Jones (1974). The glucoStnolates were determined by enzymatic hydrolysis of these Can.lnsl. Food Sci. Technol. J. Vol. 9. No. I. 1976

compounds to the aglycones and the reaction of the liberated isothiocyanates with ammonia to form thioureas. The thioureas were extracted from the aqueous solution into dichloromethane, and their absorption at 235, 245 and 255 nm was determined. Values were expressed as n-butyl isothiocyanate. Myrosinase activity was determined by incubating the test material in the presence of sinigrin at pH 7, and extracting the released allyl isothiocyanate into dichloromethane with subsequent determination of the thiourea compounds. All determinations were done at least in duplicate.

Biological Evaluation of Protein Quality The criteria used to assess the nutritive value were the protein efficiency ratio (PER) as g weight gain of mice per g protein consumed and the feed efficiency ratio (FER) as g feed consumed per g weight gain. Casein and cheese whey protein concentrate (WPC) prepared by previously described methods (Hartman and Swanson, 1966; Wingerd, 1971; Hidalgo et al., 1973) were used as controls. The latter was included since it is also a protein-hexametaphosphate complex like the prepared RI. Eighteen weanling, male Swiss mice of 15-18 g weight were randomly divided into three experimental groups of six mice each and fed with the protein sources casein, WPC and RI respectively. The basal non-protein diet was similar to that given by Sarwar et al. (1973), except for the omission of chromic oxide and the addition of mineral premix (Salt mixture USP XIV, Nutritional Biochemicals Corp., Cleveland, Ohio) at the 5% level. The protein sources were incorporated in the diet to provide a 10% protein content at the expense of corn starch. Diet and distilled water were given ad libitum. Weight gain and feed consumption were recorded every two days during a ten day feeding period. The six mice per experimental group were treated as separate evaluations. Statistical analysis 16

was by analysis of variance and s~mple means were compared using Duncan's (1955) multIple range test at the 5% level of significance. . ,. Lightness of Color The Commission InternatlOnale de I Eclauage (CIE) tristimulus value Y, representing the ligh~ness of a substance, was determined in dupli.cate accordmg to the spectrophotometric method descnbed by Clydesdale and Francis (1969).

Results and Discussion The effect of pH on the e~traction of rapese~d nitrogen using SHMP is shown m Figure 2. AI~ extract~on conditions were arbitrarily chosen. The maximum yield was 90% at pH 12. However, initial studies showed that t~e very dark alkaline extract subsequently produced undesirable dark brown protein isolates. Similar observations were reported when sodium hydroxide solution was used as extractant (Sosulski and Bakal, 1969). The pH chosen for future studies was 7.0 because the extract was greenish yellow and the succeeding protein isolates were lighter in color. At pH 7.0 the extraction yield of 82% was still much higher than that obtained using 0.01 M sodium pyrophosphate, 10% sodium chloride (Bhatty et al., 1968), or water (Sosulski and Bakal, 1969).

Table I shows the effect of various processing para eters on the extraction of nitrogen from rapeseed at p 7.0. Under the conditions of the experiment, nitrogen e~ traction yield increased to a maximum of 82% with 2 SHMP. This was considered as the suitable concentratio for future nitrogen extractions. The same concentratio was observed by Shemer et al. (1973) in the extraction o' cottonseed protein. Results with various RF to solvent ratio (Table I) in dicate a ratio of 1: 10 to be best for nitrogen extraction. In creased proportion of the solvent has no further effect 0 the extraction yield. The selected time for extraction w 30 min because increase in yield after 120 min was negli gible. Approximately 3% increase in extraction yield was 0 served as the temperature was raised from 12 to 40° (Table I). The observed decrease in yield at temperatur above 40°C can be attributed to denaturation of the mor heat labile rapeseed proteins. Shemer et al. (1973) reporte a 2% increase in the yield of extracted cottonseed nitroge with elevation in temperature from 30 to 60°C but no de. creases at the higher temperatures. A temperature of 40° appeared best for nitrogen extraction under the conditio of the experiment. Table I. Effect of various processing parameters on the extraction of n' trogen from rapeseed flour at pH 7.0. Variable

Nitrogen extraction yield %

SHMP concentration', %

o

~o

30

Conditions: ' I: 10 rapeseed flour to solvent ratio, 30 min. 25°C. • 2% SHMP. 30 min, 25°C. c 2% SHMP, I: 10 rapeseed flour to solvent ratio, 25°C. d 2% SH MP, I: 10 rapeseed flour to solvent ratio. 30 min.

80

-...

""0

tI

'70

U

0

><

tI

60

c:: tI

50

0)

-

0 ... .c::-

~

0

25 0 2

3

~

5

6

7

8

9

10

11

12

pH Fig. 2. Effect of pH on extraction of nitrogen from rapeseed flour. Conditions: 2% SHMP, I: to rapeseed flour: solvent ratio, 25°C, 30 min.

17

61.25 64.75 71.69 73.95 78.16 81.65 81.16

0.1 0.5 1.0 1.5 2.0 3.0 Rapeseed flour to solvent ratio· 1:5 I: to I: 15 1:20 Time', min 30 60 90 120 d Temperature • °C 12 25 40 50 60

90

80.10 81.65 81.57 81.62 81.65 81.64 81.30 82.23 80.59 81.65 83.89 82.37 82.37

However, double extraction of RF at pH 7.0, first wit~ a RF to 2% SHMP ratio of I: 10, and second with a ratio 011 1:6 at 25°C for 30 min, gave an extraction yield of 97%.j This value is higher than the 91 % reported for double ex': traction of RF with sodium hydroxide solution (Girault, 1973), the 67% with sodium chloride, and the 41% with, 0.0 I M sodium pyrophosphate (Bhatty et al., 1968) and also the 83% observed for single extraction at 40°C (Table I). Based on the foregoing results, the conditions selected J. Inst. Can. Sci. Technol. Aliment. Vol. 9. No. I. 1976'

for extraction of rapeseed nitrogen were double extraction at pH 7.0 with 2% SHMP at 25°C for 30 min.

Table 2. Composition of rapeseed flour and protein isolate (dry basis).

Protein (Nx6.25), % Fat, % Ash, % Crude fiber, % NFE,% Phosphorus, % Total glucosinolates', mg! g Oxazolidinethione, mg! g Myrosinase activity

Flour

Prolein isolate

47.04 (51.04)" 7.82 6.72 4.83 33.59 1.41 12.11 9.97 very active

72.64 (77.97)" 6.84 12.20 0.68 7.64 3.21 0.14 none

'As equivalent n-butyl isothiocyanate. "Oil-free dry basis. '-Not detectable.

The effect of pH and dilution with water on the precipitation of rapeseed nitrogen extracted under the selected conditions were determined and the results are given in Figure 3. The maximum precipitation of protein in the diluted and undiluted extracts occured at pH 2.5. The shift in the reported isoelectric point of rapeseed proteins (pH 4-6) (Shaikh et al., 1968; Sosulski and Bakal, 1~69) to a more acidic region (pH 2.5) may be associated with changes in acid-base equilibrium due to the presence of.hexametaphosphate anion (Shemer et al., 1973). Similar shifts to more acidic pH were noted in cottonseed (Shemer et al., 1973), gluten (Finney et al., 1973), cheese whey (Hidalgo et al., 1973) and fish sarcoplasmic proteins (Spinelli and Koury, 1970). Dilution of the protein extract with water had a !llarked effect on the precipitation yield particularly at the Isoelectric pH 2.5 (Figure 3). The precipitation yield of extr~cted nitrogen increased from 42 to 73% when the protem extract was diluted with an equal volume of water before precipitation. The strong dependence of protein ~exam:taphosphate interaction on pH and ionic strength IS consistent with reports by other investigators (Nitschman et al., 1959; Spinelli and Koury, 1970; Finney et al., 1973). These results indicate that dilution and adjustment of t~e protein extract to pH 2.5 are suitable conditions for maximum precipitation of the rapeseed proteins. .RI "-,,as prepared by the method described in Figure I consldenng the results obtained in the protein extraction ~nd precipitation experiments. The composition of RI and F are given in Table 2. Can.lnst. Food Sci. Techno!. J. Vol. 9. No. I, 1976

The RI contained approximately 27% more protein than its starting material. The protein content of 72.6% (N x 6.25, dry basis) was lower than the reported values of 8290% by Sosulski and Bakal (1969), Owen and Chichester (1971), Girault (1973) and Kodagoda et at. (1973), but higher than the 38-68% reported by Lo and Hill (1971 a) and Eklund et al. (1971). The ash content of RI (12.2%) was almost double that of RF (6.72%) probably due to the formation of a hexametaphosphate-protein complex, the entrainment of hexametaphosphate from the extraction medium, and the introduction of alkali during pH adjustment. RI was 4% lower in crude fiber content than RF. The inclusion pf phosphorus in the protein hexametaphosphate complex and to a lesser extent, residual polyphosphate impurity, account for the high phosphorus in RI (3.21 %). This value is similar to that of acceptable commercial food ingredient lactalbumin phosphate (4% P) (Ellinger, 1972) and should not pose a problem when used in foods at the same level. Table 3. Amino acid composition of rapeseed flour, protein isolate and FAO human requirements'. (g amino acid! 16 g nitrogen).

Essential lysine threonine cys + met valine isoleucine leucine tyr + phen Non-essential histidine arginine aspartic acid serine glutamic acid proline glycine alanine

4.2 2.8 3.3 4.2 4.2 4.8 5.6

5.1 3.8 2.2 4.9 4.1 7.0 5.8

5.7 4.4 3.1 6.0 5.1 8.6 7.6

3.0 5.9 6.3 3.7 17.0 7.9 4.8 4.2

3.6 7.6 8.7 4.4 18.7 9.9 5.5 4.9

'FAO, 1965.

The glucosinolate content was substantially lower in RI than RF (Table I). More than 98% of the glucosinolate was left in the residual flour and/or was removed by the precipitation and washing steps. Myrosinase activity was detected in the RF but was non-existent in RI. Only glucosinolate levels greater than 1-3 mg (Lo and Hill, 1971 b; Jo18

Table 4. Protein and feed efficiency ratios of rapeseed isolate, cheese whey concentrate and casein. Protein source Rapeseed protein isolate Cheese whey protein concentrate Casein ;:-M~;~~ ± standard error.

Weight gain'

Feed intake'

g/mouse

g/mouse

FER'

PER'

6.83 ±0.13

45.52 ± 7.08

6.66 ± 1.77

1.50 ±0.26

8.06 ±0.44

54.50 ±0.20

6.76 ±0.04

1.48 ±0.13

5.20 ±0.09

40.00 ± 0.06

7.69 ±0.02

1.30 ±0.01

ser~son and M unck, 1973) potential isothiocyanate and oxazolidinethione per gram of diet were ~eported to reduce wcight gain and feed consumptIOn of m~ce. Hence, t~e total glucosinolate level (0.14.mg/g) orRI In thIs stu~y tS ~n­ likely to cause any negative ~utntlOnal effects m mIce when incorporated mto theIr dIets at the 10% level. Individual amino acid content of RI was either comparahle to or slightly higher than the original flour (Ta~le 3). Sosulski and Sarwar (1973) obtamed hIgher essentIal amino acid contents in flax and safflower meals than their isolates hut found the opposite in soybean, rapeseed and sunllower meals and isolates. The most limiting amino acids in RF and RI were methionine and cystine, in agreemcnt with Appelqvst and Ohlson (1972) but not with Sosulski and Sarwar (1973) who reported isoleucine. The FER and PER values (Table 4) showed the nutritive value of RI to be equal to that of WPC and significantly grcatcr than that of casein. Accordingly, RI prepared hy our method has a high biological value since the nutritional superiority of WPC is now well established (Wingerd, 1971). RI was prcparcd both in the isoelectric (pH 2.5) and Ileutrali/ul (pH 7.0) form in order to determine the effect of pH on the color of the product. lsoelectric RI was yellow and with Y.. II.: value equal to 60.~2 while the neutralizcd form was tan and with Y"I' value equal to 45.88. This l!ldlGltes that the compounds responsible for color is a functloll of pH. When the neutralized RI was ether extracted, the color was improved (Y"II': = 57.55) but obViously not to the same lightness as the isoelectric Rl. It is Inkn:stlng to note that the RI in any of the prepared forms werc conSiderably lighll'r in color than the brown RI (Y"II': = 23AO) prepared in our laboratory using 0.2% NaOH as protem extractant accordina to the methods of Sosulski and Bakal (1969). b The yield of total solids in neutralized RI was 34%. In comparison, Owen and Chichester (\971), Girault (\973), Sosulskl and Bakal (1969) and Lo and Hill (\97Ia) recovered 6, 15, 26 and 50% total solids respectively. R I protem yield of 71 % was expected based on the 9~(Y(J nllrogen extraction yield from the double extraction ot ~F, an9 the 73% precipitation yield from diluted protein extract (FIgure 3). The observed 53% yield may be due to II1completc r;covery of.the extract after centrifugation and losses ~unng. the washll1g process. Nevertheless, the protell1,1 le,ld IS either comparable to or greater than the values obt~ln~d bY?ther lI1vestigators (Sosulski and Bakal, 1969; Owen and Chtchester 1971' Eklund et al 1971' Girault 1973). " .",

Acknowledgement The authors thank Dr. J. D, Jones of Food Researc' Institute, Ottawa for providing the rapeseed flour used ij this study, and for the glucosinolate analysis,

References Association of Official Analytical Chemists. 1970. Official Methods or AO.A.C.. The Associati Washington. D.C. Appelqvst. L. A. and Ohlson. R. 1972. Rapeseed. Cultivation. Composition, Processing and Uti zation. Elsevier Pub!. Co.. New York. Bhatty. R. S.. McKenzie. S. L. and Finlayson. A. J. 1968. The proteins of rapeseed (Brassica na L.) soluble in salt solutions. Can. J. Biochem. 46: 1191. Burrows. V. D.. Green. A. H .. Korol. M. A .. Melnychyn. P.. Pearson. G. G. and Sibbald. I. 1972. Food Protein from Grains and Oilseeds - A Development Study Projected 10 19 Dept. of Trade. Industry and Commerce. Ottawa. Clydesdale. F. M. and Francis. F. J. 1969. Transmission·rdkl·tion spectrophotometry and tris mulus colorimetry. Food Prod. Dev. 3: 67. Duncan. D. B. 1955. Mulliple range and multiple F tests. Biomt:trics II: I. Eklund. A.. Agren. G. and Langler. T. 1971. Rapeseed protein fral"lions. I. Preparation of a delO. ified lipid-protein concentrate from rapeseed (Brassil'
J

Received January 9. 1975

T~e comparable yield of light colored, good quality pro~u~t 1I1 thiS lI1vestigation indicates the feasibility of the

met 19

0

used for the isolation of rapeseed protein.

J. Inst. Can. Sci. Teehnol. Aliment. Vol. 9, No. l, 19761