Preparation, Composition and Functional Properties of Oat Protein Isolates1

Can. Inst. Food set. Technol. J. Vol. 16, No. 3, pp. 201-205, 1983

Preparation, Composition and Functional Properties 1 of Oat Protein Isolates Ching-Yung Ma Food Research Institute Agriculture Canada Ottawa, Ontario KIA OC6

al properties of oat proteins. In this work, protein isolates were prepared from a high and moderately high protein content oat cultivar, Hinoat and Sentinel, respectively, by two different procedures. One procedure involved isoelectric precipitation of an alkaline extract, and the other dialysis or dilution with water of a salt extract similar to that described by Murray et al. (1978, 1981). The chemical compositions and functional properties of the isolates were determined to assess the potential of oat proteins as a food ingredient.

Abstract Protein isolates were prepared from dehulled, defatted groats of Hinoat and Sentinel oats by two methods: isoelectric precipitation of an alkaline extract (alkaline isolate) and dialysis or dilution of a salt extract (salt isolate). Both types of isolates contained over 900/0 protein (N x 5.80), but the yield of N was much higher in alkaline isolate (over 60%) than salt isolate (25%). There was no sigificant difference in the proximate chemical compositions of the isolates from the two oat varieties. Both types of isolates had similar amino acid composition, although alkaline isolate had slightly higher lysine and total essential amino acid contents than salt isolate. Some functional properties of the isolates were assessed and compared to wheat gluten and soy protein isolate. The results indicate that the oat isolates had high fat binding capacity and good foaming properties.

Materials and Methods Two oat varieties, Sentinel and Hinoat, were grown at the Central Experimental Farm, Ottawa, in 1980 and 1981, respectively. The oats were dehulled, ground in a coffee grinder, and defatted by Soxhlet extraction with hexane. Salt soluble protein isolates were prepared by the procedure as described by Murray et al., (1978,1981). The ground groats were mixed with 0.5 M CaCl 2 at a groat solvent ratio of 1: 10 (w:v). The slurry was stirred for 60 min at room temperature and centrifuged at 6000 x g for 20 min. The supernatant was either dialyzed against cold running tap water or diluted with water. The saltsoluble proteins (salt isolate, SI) which precipitated, were collected by centrifugation and freeze-dried. Isoelectrically precipitated alkaline isolates were prepared by mixing the groats with dilute NaOH (0.015 N) at a groat solvent ratio of 1:8 (w:v); which gave an initial pH of 9.5. The slurry was stirred at room temperature for 60 min and centrifuged at 4000 x g for 10 min. The supernatant was neutralized with 2 N HCl to pH 5.5, recentrifuged, and the alkaline isolate (AI) residue and alkaline supernatant (AS) were freeze-dried. Nitrogen was determined by the microKjeldahl method (Concon and Soltess, 1973), converted to protein using a conversion factor of 5.80 (Tkachuk, 1969), and the protein content was expressed as a percentage of the sample dry weight. Carbohydrate contents were estimated by the phenol-sulphuric acid method (Dubois et al., 1956). Moisture, ash and fat were determined by AACC

Resume Des isolats de proteines ont ete prepares seIon deux procedes, a partir . de caryopses d'avoine (var. Hinoat et Sentinel) degraisses et decortiques: par precipitation isoelectrique d 'un extrait alcalin (isolat alcalin) et par dialyse ou dilution d'un extrait salin (isolat salin). Les isolats contenaient tous deux au-dela de 90% proteines (N x 5.80) mais le rendelnent en N etait de beaucoup superieur pour I' isolat alcalin (plus de 60%) en comparaison acelui de l'isolat salin (25%). On n'a pas observe de difference significative dans les analyses approximatives des isolats des deux varietes d' avoine. Les deux types d' isolats possedaient une composition semblable en acides amines; par contre, l'isolat alcalin avait un contenu de lysine et d' acides amines totaux superieur acelui de l'isolat salin. Quelques proprietes fonctionnelles des isolats ont ete etudiees et comparees acelles du gluten de ble et de I'isolat de proteines du soja. Les resultats demontrent une grande facilite a lier les gras et de bonnes proprietes mousseuses pour les isolats d' avoine.

Introduction Oats have been used essentially for animal feed, although oat proteins have been shown to have good nutritional value (Hischke et al., 1968) and can be used more widely for human consumption. Compared to other cereals, oats provide a relatively cheap source of proteins which do not contain antinutritional factors found in some plant protein sources. Protein concentrates and isolates have been prepared from oats by different procedures (Cluskey etal., 1973, 1978; Wu and Stringfellow, 1973; Youngs, 1974; Wu et al., 1977; Bell et al., 1978), but there is limited information on the chemical and functionIContribution No. 509. Food Research Institute, Agriculture Canada.

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1983 Canadian Institute of Food Science and Technology

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(1971) approved methods. Amino acid analyses were performed by hydrolyzing the samples according to the method of Liu and Chang (1971) and analyzing the hydrolysates on a Beckman Model 121 M amino acid analyser.

Functional Properties Solubility of the protein isolates was determined by dispersing the isolates in distilled water, stirring at room temperature for 20 min, and then adjusting the pH values between 1.5 and 10.0. The samples were then centrifuged at 10,000 x g for 30 min and the supematant analyzed for nitrogen by the microKjeldahl method. Emulsifying activity index (EAI) was determined by the Pearce and Kinsella (1978) turbidimetric method and water hydration capacity (WHC) was determined according to Quinn and Paton (1979). The procedure ofLin et al. (1974) was used to determine fat binding capacity (FBC) and the foaming properties were assessed by the procedure of Yatsumatsu et al. (1972). Surface (So) and exposed (Se) hydrophobicities were determined according to the methods of Kato and Nakai (1980) and Townsend (1982), respectively.

Results and Discussion Yields and Chemical Compositions Table 1 presents the yields and chemical compositions of the two types of protein isolates from Hinoat and Sentinal groats. SI constituted only 5-6% by weight of the groats while AI made up about 12-13%. Both isolates had a protein content of over 90% using a conversion factor of 5.80. The yield of Kjeldahl nitrogen was much higher in AI (over 60%) than SI (25%). The AS fraction from the alkaline extracts, constituting about 7.5 % of the groats by weight, had substantially less protein (ca 25%) than AI and represented about 10% of the total nitrogen. Although Hinoat groats had higher protein content than Sentinel, the yield of nitrogen in the respective isolates was about the same. All the oat isolates were low in fat, ash and carbohydrate. AS, on the other hand, had about 10% ash and 60% carbohydrate.

Amino Acid Composition Table 2 shows the amino acid composition of the protein isolates from the two cultivars along with the amino acid profiles of the defatted groats. Compared to the FAO/WHO (1973) recommendations, lysine is the limiting amino acid in both SI and AI, with AI having higher aromatic and sulphur containing amino acids than

SI. The total essential amino acid content of AI was about 40 g/16 g N; higher than that of the groats and the FAO recommended level (35 g/16 g N), and considerably higher than that of SI (33 g/16 g N).

Functional Properties The pH solubility curves of protein isolates from Sentinel groats are shown in Figure 1. The curves of the Hinoat isolates were not significantly different from the Sentinel samples, with both AI and SI showing bell shaped curves, with minimum solubilities around pH 6 and pH 5 for SI and AI, respectively. SI had higher solubility at acid pH than AI, while AI had significantly higher solubility than SI at alkaline pH, particularly at pH values between 7 and 9. Table 3 summarizes the results of some of the functional properties studied for the oat protein isolates. Vital gluten (Industrial Grain Products, Montreal, Quebec) and soy protein isolate (Supro 610, Ralston Purina Co., St. Louis, MO), two plant proteins widely used in foods, were also included for comparison. Results on EAI determination (Table 3) show that SI had a much lower EAI than AI. The Sentinel isolates had slightly higher EAI than the Hinoat isolates. The AS fraction had considerably higher EAI than AI. When compared to gluten and soy isolates, the oat isolates, particularly SI, had lower emulsifying activity. EAI of the oat isolates were also determined at various pH values (Figure 2). The pH EAI curves of both SI and AI resembled the pH solubility curves with minimum emulsifying activity between pH 4-6. However, the difference between the highest and lowest EAI was not as marked as the difference in solubility. Both SI and AI had considerably lower WHC than soy isolate, but was comparable to gluten. AS, in contrast, had a fairly high WHC. The FBC of the oat isolates was about the same as soy isolate and considerably higher than gluten; AS again having a much higher FBC than the isolates (Table 3). Except for SI from Hinoat, which had a low foamability, the oat isolates had a foamability equal to or higher than that of the other two plant proteins; AS also having fairly good foamability (Table 3). The foam stability of samples was determined at 30 and 60 min after foaming and all oat isolates had good foam stability when compared to gluten and soy isolates, with the exception of AS, which was poor in this regard. The foamability and foam stability of the oat isolates were also determined at various pH values. The pH

Table 1. Yields and chemical composition of oat protein isolates (% dry basis) I. Protein Hinoat Salt isolate (SI) Alkaline isolate (AI) Alkaline supematant (AS) Sentinel Salt isolate (SI) Alkaline isolate (AI) Alkaline supemantant (AS)

Yield of Protein (%)

Fat

Ash

Carbohydrate

(%)

(%)

(%)

(%)

6.1 13.4 7.5

98.9 95.7 22.8

26.2 65.9 8.9

0.8 1.3 1.0

0.2 1.0 11.4

0.1 0.3 59.6

5.1 11.9 7.5

95.9 90.8 24.7

24.8 67.2 10.0

1.0 2.3 0.9

0.3 1.2 10.6

0.1 0.2 58.2

I Average of duplicate determinations. 2Percentage by weight of defatted groats.

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J. Inst. Can. Sci. Technol. Aliment. Vol. 16, No. 3, 1983

Table 2. Amino acid composition of oat protein isolates (g/16 gN)'. Hinoat

Groat

Amino acid

3.4 2.2 6.4 7.8 3.0 4.4 19.8 4.7 4.2 4.2 5.0 1.8 1.2 3.7 7.3 3.3 6.1 34.8

Lysine Histidine Agrinine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine Methionine Isoleucine Luecine Tyrosine Phenylalanine Total essential

Sentinel

Salt isolate

Alkaline isolate

3.3 2.6 8.9 9.6 3.4 5.0 20.4 4.3 4.3 4.1 4.3 0.9 0.9 3.6 7.2 4.3 5.9 33.8

3.8 2.5 8.2 8.7 3.6 5.0 23.5 5.4 4.3 4.6 4.9 1.5 1.8 3.8 8.6 4.8 6.7 39.4

Groat

Salt isolate

Alkaline isolate

4.3 2.3 6.3 8.3 3.3 4.5 20.7 5.3 4.9 4.9 5.2 2.3 1.2 4.0 7.8 3.3 6.3 35.7

3.2 2.3 7.2 8.8 3.1 4.8 18.0 4.0 4.0 4.0 3.8 0.7 1.3 3.6 6.9 4.2 5.7 32.5

4.0 2.6 8.2 8.6 3.6 5.2 24.8 5.9 4.4 4.9 5.2 1.7 2.1 3.8 9.1 4.6 6.3 40.5

FAO Scoring Pattem 2 5.5

4.0

5.0 3.5 3.5 4.0 7.0 6.0 6.0 35.0

IAverage of duplicate determinations. 2Values taken from FAO/WHO (1973). Energy and protein requirements.

foamability curves for SI followed the shape of the solubility curves with minimum foamability occurring between pH 4.5 and 6.0 (Figures 3A, B), this parameter decreasing again at pH higher than 7.5, even though solubility was increased. The foam stability of SI was very low at acid pH and increased to a maximum at pH 7.5 (Figures 3A, B). For AI, the foamability was low at acid pH and increased gradually with increase in pH (Figures 100

90

80

3C, D). The curves showed a drop in foamability at pH 6 with the foam stability of AI increasing with a rise in pH (Figures 3C, D). The So and Se hydrophobicities of the oat isolates were also determined (Table 3), AI having a higher So than SI, with SI from Hinoat having an exceptionally low So value. When heated in the presence of 1.5% sodium dodecyl sulphate, the hydrophobicity (Se) of all isolates increased markedly to a common value. Gluten and soy isolates also had Se values comparable to the oat isolates. Preliminary experiments had shown that the yield of protein in AI was increased progressively with increase in alkali concentration, and no optimal pH was observed as reported previously (Wu et al., 1977). Although higher 60

70 50

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60

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0

50

40

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40

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30

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30

20

20 10

10

o

2

3

4

5

6

7

8

9

10

11

1.5

3.0

4.5

6.0

7.5

9.0

10.5

pH

pH

Fig. I. Nitrogen solubility curve of oat protein isolates from Sentinel groats (A-SI; e-AI). Can. Inst. Food Sci. Technol. J. Vol. 16. No. 3. 1983

Fig. 2. EAI of oat protein isolates at various pH 'alues ( O-AI from Hinoat; e-AI from Sentinel; D-SI from Hinoat; A-SI from Sentinel).

Ma /203

Table 3. Functional properties of oat protein isolates. gluten and soy isolate].

Hinoat Salt isolate Alkaline isolate Alkaline supemantant Sentinel Salt isolate Alkaline isolate Alkaline supemantant Gluten Soy isolate

EAI (m 2/g)

WHC (mUg)

FBC (mUg)

Foamability (mL)2

30 min

10.2 23.2 56.2

0.9 0.8 3.0

1.6 1.4 3.9

25 76 63

18 50 12

45 10

14.6 30.6 48.6 49.4 35.0

0.8 0.8 3.1 1.0 2.5

1.5 1.8 4.2 0.9 1.8

65 80

40 52 10 25 52

38 50 9 10 30

72

50 75

Foam stability (%) 60 min 17

So

Se

20 240

980 1010

nd 3

85 269

nd

915 1120

nd

nd

75 95

1080 900

]Average of duplicate determinations. 2Yolume of protein solution = 50 mL. 3Not determined.

yield can be obtained by increasing pH, at values greater than 10, the slurry became viscous and darkened. A pH of 9.5 was therefore chosen and the resulting isolates had a light brown colour. A yield of 53% was reported by Wu et al., (1977) for the preparation of protein isolates from hexane defatted Garland oat flour by alkali extraction. The yield obtained for Hinoat and Sentinel groats was considerably higher (66-67%), which could be due to a higher solid:solvent ratio (1 :6) used by the other workers. Using a conversion factor of 6.25, Wu et al., (1977) reported a protein content of 94-103% in the oat isolates, slightly higher than the present results (91-96%), based on a conversion factor of 5.8. For SI, 0.5 M CaCl z was a more effective solvent than 1.0 M NaCI. The yield of protein was affected slightly by the salt concentration and an ionic strength of 1.0 was found to be the optimum for protein extraction, yielding a colourless extract. Arntfield and Murray (1981) prepared fababean pro80 A

B

70

60 50 40

30 20 10

90

c 80 70

60 50 40

30 20 10

o

1.5

3.0

4.5

6.0

7.5

9.0

10.5

0

1.5

3.0

4.5

6.0

7.5

9.0

10.5

pH

Fig. 3. Foamability and foam stability of oat protein isolates at various pH values (e·foamability: a·foam stability: A: SI from Hinoat: B: SI from Sentinel: C: AI from Hinoat: 0: AI from Sentinel).

204/ Ma

tein isolates by both salt and alkaline extractions and found both the temperature of denuration and thermal transition decreased with increases in extraction pH, suggesting protein denaturation. For oat isolates prepared at pH 9.5, there may be some denaturation of the proteins. However, alkaline extraction gave a much higher yield than salt extraction which makes the salt extraction economically less attractive. The difference in the protein yield between the two processes could be due to the fact that salt only extracted globulins, while alkali extracted both globulins and other Osborne proteins from oats (Ma et al., 1981). Although globulins are the major soluble protein fraction in oats (Wu et al., 1972; Peterson and Smith, 1976), the other Osborne fractions made up a signfiicant portion of the proteins. The essential amino acid profiles of AI were similar to those reported in the isolates prepared from other oat varieties by alkaline extraction (Wu et al., 1977). A comparison of the amino acid compositions of the two isolates indicates that AI had a considerably better essential amino acid profile than SI. Apart from lysine, which was limiting in both isolates, SI was also limited in threonine and valine. The AI had a pH solubility pattern which closely resembled the isolate prepared from hexane defatted Dal oat groats (Wu et al., 1977) and was not much different from the SI. At pH 9.5 both isolates were over 90% soluble, indicating that the conditions employed for preparing AI can lead to substantial solubilization of the salt-soluble proteins. AI had a much higher EAI than SI and this could be due to the higher solubility of AI at ·pH 7.5, the pH at which EAI were determined. This fact may also explain the high EAI observed in AS which contained proteins soluble at neutral pH. The relationship between solubility and emulsifying activity is clearly illustrated in Figure 2 and these results suggest the dependence on soluble proteins for emulsification, as has been reported for cottonseed proteins (Cherry et al., 1979). In contrast to emulsifying activity, all oat isolates had similar WHC and FBe. AS had a much higher WHC which could be due to high fibre content in this fraction (Lapsley, 1980). The FBC of AS was also markedly higher than the isolates which may be attributed to a much lower bulk density in this fraction, leading to substantial entrapment of oil. J. Inst. Can. Sci. Technol. Aliment. Vo!. 16, No. 3. 1983

The foaming properties of the oat protein isolates compared favourably with the other two plant proteins, except for SI from Hinoat. The relationships between foaming properties and pH in the oat isolates did not exactly follow the pH solubility curves (e.g., the foamability and foam stability of AI were low at acid pH, although the protein solubility was fairly high). This was in contrast to oilseed proteins, in which the pH foaming properties relationship closely follows that of the protein solubility (Cherry et al., 1979). The results show that foaming properties of oat proteins do not depend entirely on the quantity of soluble proteins. No qualitative analyses (e. g., gel electrophoresis) were made on the oat proteins extracted at various pH levels, and it was not known whether the difference in foaming properties was attributed to specific type(s) of proteins solubilized at a particular pH. Hydrophobicity has been shown to have great influence on protein functionality (Voutsinas, 1982). In this study, So was found to differ considerably between the two types of isolates and between the two oat varieties, while Se remained fairly constant. The higher So values of AI could be due to some degree of denaturation or unfolding of the protein molecules. It has been demonstrated that proteins having similar solubilities have higher EAI if So is higher (Voutsinas, 1982). A good correlation has also been demonstrated between Se and foaming properties (Townsend, 1982). In the present study, however, So seems to correlate better than Se with foamability and foam stability of the SI. Protein concentrates had been prepared from Hinoat and Sentinel groats by alkaline extraction at pH 9.5 (Ma et al., 1981). The concentrates were found to have EAI, FBC and WHC intermediate between AS and AI. In the preparation of oat concentrates, the neutralized extracts were freeze-dried, without centrifugation to separate AS and AI. The AS, though a small fraction, would contribute to the overall functional performance of the protein concentrate. The present data indicate that protein isolates can be prepared from oats by salt and alkaline extraction. Although salt extraction was a milder procedure than alkaline extraction, thus less liable to denaturation, its yield was much lower. The alkaline isolates had a better amino acid profile and were functionally equal to or better than the salt-soluble isolates. The alkaline procedure also produces an AS fraction which has excellent functionality and hence, alkaline extraction at pH around 9.5 seems to be the more effective procedure to produce protein isolates from oats. Although the oat isolates had functional properties comparable to gluten and soy isolate, a knowledge of the full potential of oat protein isolates as food ingredient requires further assessment in a variety of food systems. Acknowled~ement

The technical assistance of A. Boisvert and G. Khanzada is acknowledged.

References AACC, 1971. Approved Methods. American Association of Cereal Chemists, St. Paul, MN. Amtfield, S.D. and Murray, E.D. 1981. The influence of processing

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