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Properties of protein concentrates and hydrolysates from Amaranthus and Buckwheat
Industrial Crops and Products 10 (1999) 175 – 183 www.elsevier.com/locate/indcrop
Properties of protein concentrates and hydrolysates from Amaranthus and Buckwheat Feliciano P. Bejosano, Harold Corke * Cereal Science Laboratory, Department of Botany, Uni6ersity of Hong Kong, Pokfulam Road, Hong Kong Accepted 20 April 1999
Abstract Protein concentrates and pepsin hydrolysates were made after isoelectric precipitation of the proteinaceous liquor from wet-milling of grain of five Amaranthus and one buckwheat genotype. The Amaranthus protein concentrates exhibited better solubility, foaming, and emulsification than two commercial soy protein controls. Many protein properties depend on solubility, and Amaranthus protein concentrates were more soluble than soy protein isolate. The buckwheat protein concentrate was highly soluble with excellent emulsification, but poor foaming ability. Partial pepsin hydrolysis further improved solubility of the protein concentrates and also altered their foaming property. Fractionation and subsequent characterization of protein concentrates revealed that glutelins, albumins, and globulins predominated, with prolamins present in minor quantity. SDS-PAGE showed that globulins and glutelins were comprised of several subunits with varying molecular weights from relatively high to low while albumins were mostly of low molecular weight. The prolamin fraction of the buckwheat concentrate was comprised of intermediate to low molecular weight subunits while those of Amaranthus concentrates were only of low molecular weight. This study demonstrated the feasibility of producing potentially useful functional protein concentrates as by-product of Amaranthus and buckwheat starch extraction. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amaranthus; Fractionation; Protein concentrate
1. Introduction New crops are those recognized to have potential for development into new markets or new products. Market demands may include special uses in industry, such as new functional starches or proteins. Grain Amaranthus has been recog* Corresponding author. Fax: +852-28583477. E-mail address: [email protected] (H. Corke)
nized as a crop of great potential for the 21st century (NRC 1984), but its adoption in US agriculture has been slow. However, driven by demand for animal feed uses, Amaranthus is increasing fast as a commercial crop in China with over 100 000 ha per year grown (Yue et al. 1993, S.X. Yue personal communication). Amaranthus and buckwheat (Fagopyrum esculentum) are pseudocereals producing seed high in starch content, similar to the major cereal crops like rice, maize,
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and wheat. In addition to starch, significant amounts of protein (Amaranthus, 13 – 18% w.b.; buckwheat, 12–15% w.b.) are also found in these seeds, and the proteins are of good quality. The nutritional quality of Amaranthus protein scored higher than that of other cereal grain and legume seeds by FAO/WHO protein scoring or by PER (Breene 1991; Bejosano and Corke 1998c). Pomeranz (1983) noted that buckwheat proteins are rich in lysine, and that buckwheat is one of the best plant sources of proteins of high biological value. Amaranthus and buckwheat are used as specialty starch sources, using methods such as alkali wet-milling, where the main concern is the maximum recovery of starch. The remaining material, including the protein fraction, may commonly be disposed of as waste. However, by a simple processing using isoelectric precipitation protein can be recovered, and thus another potentially important product can be obtained. We have shown the applicability of such by-products in cereal (noodle) and meat (sausage) products (Bejosano and Corke, 1998a,b). This study was made with the aim of investigating the basic properties of protein concentrates from Amaranthus and buckwheat as by-product of starch extraction, and the extent of variation among common genotypes. The specific aims were to: (a) determine the functional properties and hence potential use of the protein concentrates; and (b) test the effect of further protein modification (pepsin hydrolysis) on these properties.
2. Materials and methods
2.1. Materials Five Amaranthus samples were supplied by the Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing, China. The genotypes were: K112, K350, K459, and R104 (all A. cruentus with yellowish-brown seedcoat), and No. 3 (A. hybridus with a black seedcoat) which are all widely cultivated varieties in China. R104 is a widely-grown grain variety, and K112 is a widely-grown forage variety in
China (Wu et al., 1995). No. 3 was selected because of its unique grain color. The buckwheat sample was purchased in a retail outlet in Hong Kong, packed by Queenswood (United Kingdom) but its country of origin was China. Two brands of commercially available soy protein isolate were used as reference standards (Soy A, Protein Technologies International, St Louis, Missouri; and Soy B, Edward Keller, Hong Kong). These commercial samples were marketed for general usage without claims for particular functional characteristics.
2.2. Preparation of isoelectric protein concentrates Using an alkali wet milling procedure with subsequent isoelectric precipitation of the proteinaceous liquor, protein concentrates were made out of the five Amaranthus and one buckwheat samples. The procedure used was based on method C of Perez et al. (1993), essentially the same as used by Wu et al. (1995). The grain materials were steeped in NaOH solution at 5°C for 24 h, ground in a blender, and screened to separate the fibrous residue from the filtrate. The filtrate was then centrifuged to separate the proteinaceous liquor from the starch fraction. After acid precipitation of the proteinaceous liquor, a wet curd was obtained which was in an emulsified state containing a high oil content. In this condition, conventional drying was found to be very difficult, so the curd was first frozen and then thawed to break the emulsion and to facilitate separation of the water layer. After doing this, a large amount of water could be removed physically. This was followed by drying at 70°C in a forced convection dryer for about 12 h. The coarse pellets formed were crushed finely using mortar and pestle. The resulting powder was then defatted in petroleum ether (10 h with intermittent shaking; removing the petroleum ether layer; repeated twice). Commercially, defatting will be essential to exploit the high quality and squalene content of the oil co-processing by-product. This was followed by air drying and further grinding using an Udy Cyclone Mill (Udy, Boulder, CO, USA) with 0.5 mm mesh screen.
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2.3. Enzymatic hydrolysis Partial pepsin hydrolysis of protein concentrates was carried out based on the procedure of Hartnett and Satterlee (1990). The substrate was prepared by adjusting the protein concentration to 1% (w/v) in 0.01 N HCl (pH 2.0). 91 units of pepsin A (Sigma, St Louis, MO, USA) per mg solid was added to the suspension at the rate of 0.2 mg ml − 1. The mixture was incubated at 37°C with mild shaking for 16 h, after which pH was adjusted to 7.5 and kept at 5°C for 72 h. The pH was then adjusted to 6.5 followed by freeze drying. The degree of hydrolysis (DH) was measured according to Bombara et al. (1992). DH was based on the ratio of TCA (trichloroacetic acid; 12%) soluble proteins to the total proteins of the sample. This is a widely used method of DH measure (Adler-Nissen 1986) and we chose it because of its simplicity.
2.4. Functional properties The nitrogen solubility of proteins in water was determined as a function of pH. Tests were carried out from pH 3.0 to pH 9.0. This method was based on Mannheim and Cheryan (1993) with modifications: the volume of the protein suspension was reduced to 50 ml; a Kenwood mixer (Kenwood Ltd., Hampshire, United Kingdom) with blender cup (250 ml) was used for mixing; mixer was set at minimum speed for 10 min; and centrifugation was carried out at 1650× g for 15 min at room temperature (:25°C). The test on foaming property was also based on Mannheim and Cheryan (1993). After mixing as described in the N-solubility test above, contents were transferred to a 250 ml graduated cylinder, kept at room temperature (:25°C) for 30 min and the residual foam was measured. However, foaming properties were defined based on Vani and Zayas (1995) wherein foam expansion (FE) is the foam volume (ml) immediately after blending and foam stability (FS) is the volume after standing for 30 min. The test on emulsifying activity was based on the method of Yasumatsu et al. (1972). One hun-
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dred ml of 7% protein dispersion was added to an equal volume of oil and the mixture was homogenized. There were some modifications: corn oil was used instead of soybean oil; homogenization was carried out in Kenwood mixer with blender cup (250 ml) set at speed no. 4 for 1 min; centrifugation was carried out at 1650× g for 10 min at room temperature (: 25°C). Emulsifying activity was calculated as the height of emulsified layer divided by the height of the whole layer times 100%.
2.5. Protein characterization The proportion of the different protein fractions in the concentrates was determined according to their extractability in different solvents of the Osborne sequence. This was based on the procedures of Gorinstein et al. (1991) and Barba de la Rosa et al. (1992) except that we chose to start the sequential extraction with water (pH 7) in order to recover most of the water extractable albumins. This was followed by extraction with 0.1 M NaH2PO4 (pH 7) (globulins); 70% isopropanol (prolamins); and lastly with 0.1 M NaOH (glutelins). Thus, the globulin fraction referred to here is probably contaminated with some albumins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25% slab gels) of the proteins was carried out using a Mini-Protean II Electrophoresis System (Bio-Rad, Richmond, CA, USA) based on the instruction manual supplied. The gels were stained in Coomassie blue R-250. Molecular weight markers were used to estimate the molecular weight of the different fractions detected. Markers were purchased from Sigma (St Louis, MO, USA). Bovine albumin, pepsin, trypsinogen, b-lactoglobulin, and lysozyme with molecular weights of 66, 34.7, 24, 18.4, and 14.3 kDa, respectively were used. SDS-PAGE was carried out on the whole grains and the protein concentrates. Likewise, SDS-PAGE was carried out on the fractions collected from the Osborne fractionation and the hydrolysates of the buckwheat and the K350 Amaranthus protein concentrate.
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Fig. 1. Solubility (%) of Amaranthus and buckwheat protein concentrates at different pH levels (each data point represents the mean of two replicates)
2.6. Statistical analysis Analysis of variance (ANOVA) and correlation analysis were carried out using the SAS System for Windows release 6.10 (SAS Institute, Cary, NC, USA) software. Comparison of means was carried out using the least significant difference (LSD) test.
3. Results and discussion
3.1. Functional properties We compared the solubility of the six protein concentrates in water with that of the two soy protein isolates at four different pH levels (pH 3, 5, 7, 9). Most of the Amaranthus and buckwheat
protein concentrates were more soluble in water than the two brands of soy protein isolate except at pH 9.0 (Fig. 1). Soy protein isolates are commonly produced by alkali extraction similar to the method we used (Wolf and Cowan 1971). Therefore, like the protein concentrates we prepared, we expected them to be highly soluble at alkaline pH. A reduction in their solubility in such conditions would, therefore, indicate the effect of the drying procedure following extraction that might have induced denaturation. Another possible concern is the effect of residual lipids. However, as shown in Table 1, the level of lipids in the protein concentrates is minimal (0.9–1.8%) which is similar to that of commercially available soy protein isolates. We also found no correlation between the fat content and the functional properties of the protein concentrates. Since the protein concentrates we made were oven dried, they were expected to have undergone some degree of denaturation. As the results show, at pH 9, soy A and K350 were significantly the most soluble, while soy B was in the third most soluble group (grouping based on comparison of means by LSD). K112 was significantly the least soluble (LSD; PB 0.05) at such pH. This also indicates that the protein of the materials we used, except K112, may have useful thermal stability in withstanding the drying procedure used. Among the Amaranthus protein concentrates, No. 3 (A. hybridus) was generally the most soluble at all pH levels while K112 (A. cruentus) was the least. The buckwheat concentrate at pH 7 was the most soluble among all the proteins tested although its
Table 1 Crude protein (Kjeldahl) and fat contents of Amaranthus and buckwheat isoelectric protein concentrates and the degree of hydrolysis of their corresponding pepsin hydrolysates; distribution of protein fractions of Amaranthus and buckwheat concentrates Source of protein
K112 (A. cruentus) K350 (A. cruentus) K459 (A. cruentus) R104 (A. cruentus) No. 3 (A. hybridus) Buckwheat
Protein concentrate
Hydrolysate
Albumin+globulin*
Prolamin*
Glutelin+residue*
Protein (%)
Fat (%)
DH (%)
(%)
(%)
(%)
60.4 70.1 65.7 58.5 66.1 77.1
1.5 1.2 1.1 0.9 1.6 1.8
11.6 16.6 15.1 17.0 14.5 11.8
25.3 90.3 46.8 9 1.0 41.3 9 0.4 42.2 91.6 50.2 91.3 55.4 91.0
1.6 9 0.0 1.6 90.1 1.6 90.0 1.9 9 0.3 1.69 0.0 1.8 9 0.3
73.1 9 0.1 51.6 9 1.8 57.2 9 1.3 55.9 9 1.3 48.1 90.1 42.8 91.8
* Means of two replicates;9 denotes S.D.
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Table 2 Emulsifying activity (%) at pH 7.0 of Amaranthus and buckwheat protein concentrates, and effect of partial pepsin hydrolysis on the solubility (%), foam expansion (ml) and foam stability (ml) of the concentrates, compared to two soy protein isolatesa
K112 K350 K459 R104 No. 3 Buckwheat Soy A Soy B LSD*
Emulsifying activity
Solubility
Conc
Conc
55.6 73.3 61.9 56.0 62.2 63.8 50.6 45.7 3.1
19.9 56.5 42.1 47.2 60.5 64.2 33.3 21.3 4.5
Foam expansion
Foam stability
Hydrol
Conc
Hydrol
Conc
Hydro
64.5 76.1 76.0 72.8 76.8 86.4 N/A N/A 6.4
49 91 52 35 68 36 23 28 20
94 63 32 28 42 22 N/A N/A 14
34 56 26 18 26 14 20 22 9
30 25 16 16 17 14 N/A N/A 7
* Least significant (PB0.05) difference for comparison of means in the same column. Conc, concentrates; hydrol, hydrolyzates.
a
solubility was not significantly different from No. 3. It was however, significantly higher than K350. It was noted that the solubility of the two soy protein isolates was poor at pH 3, 5, and 7. This may appear to contradict literature reports (e.g. Mahmoud 1994). However, our result is not exceptional because similar results can also be found (e.g. Frokjaer 1994). Wolf and Cowan (1971) noted a wide variation in solubility of commercially prepared soy proteins. They attributed this to the heat treatment given during the manufacture. We therefore assume, that the reference soy protein isolates we used were perhaps significantly denatured during their manufacture, thus, their solubility is affected as shown in the results. Using pepsin in the protein concentrates for a period of 16 h, hydrolysates with 12 – 17% DH were obtained (Table 1). Protein solubility at pH 7.0 was increased by at least 27% to as much as 221% after partial pepsin hydrolysis (Table 2). The protein concentrates were also evaluated for their foaming stability at pH 3.0 – 9.0 and foam expansion at pH 7.0, again comparing them against soy proteins. Similar to the results on solubility, it was found that at all pH levels, most of the Amaranthus protein concentrates showed better foaming stability than soy proteins (Fig. 2) with the A. cruentus K350 protein giving the best result. However, the buckwheat protein concen-
trate gave the least amount of foam after 30 min standing, even less than those of the soy proteins. The measurement of foaming properties at pH 7.0 (Table 2) showed that the foams produced by the Amaranthus protein concentrates, except for No. 3, were able to maintain more than 50% of their initial volume after standing for 30 min. Although the volume of the foam produced by the soy proteins were relatively small, they were nevertheless able to keep about 80% of it after standing. However, buckwheat protein concentrate produced a small amount of foam which was also unstable.
Fig. 2. Foam stability (foam volume, ml) of Amaranthus and buckwheat protein concentrates (each data point represents the mean of two replicates)
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The effect of partial protein hydrolysis on foaming property was also noted. At pH 7, it was found that there was a reduction in foam expansion as a result of hydrolysis, although this was only significant in half of the cases. Furthermore, it was unexpectedly noted that hydrolysis of K112 actually caused an increase in foam expansion. It was also noted that after partial hydrolysis, it gave the highest increase in solubility (concentrate vs. hydrolysate) of 221% compared to the others with about 27–77% increase. This may therefore, indicate a large change in the surface active properties of the proteins which could have then caused a favorable effect on foam expansion. The ability of these protein concentrates to form emulsions at pH 7.0 was also determined. Here again, it was found that they all have significantly higher emulsifying activity than soy protein isolates (Table 2). Again, A. cruentus K350 protein concentrate had significantly the highest emulsifying activity followed by buckwheat, No. 3 and K459 protein concentrates. We investigated the reasons for the variation in the functional properties among the samples. The emulsifying activity of the proteins at pH 7.0 was significantly correlated with their solubility in water at the same pH level (r= 0.77; n =8; a = 0.05), showing that at least in our case, the ability of the protein concentrates to form emulsions is somehow dependent on their solubility in the medium. The trend in the results on foaming properties was found to be more complicated. Perhaps it is because this protein property is determined by many factors and their specific roles are not well defined. Schnepf (1992) found that attempts by several authors to correlate foam characteristics to molecular properties of proteins have not always been successful, although a study by Townsend and Nakai (1983) had indicated the main factors contributing to foaming capacity were viscosity, surface hydrophobicity, and solubility in descending order. Generally, it appeared that foaming was favored at acidic pH and that it decreased with increase in pH. The behavior of proteins at interfaces influences the formation and stabilization of foams which depend to a great extent on the properties of the interfacial film and that films produced near the protein’s isoelectric point are more condensed and
stronger (Kinsella and Phillips 1989). Hence we observe foaming properties to be favored near pH 4.0.
3.2. Protein characterization Proteins from different genotypes of the same species of Amaranthus were found to have different properties, and it is not surprising to find such differences in proteins from seeds of another species or genus. These differences are further complicated when modifications are carried out on the proteins. In the previous discussion, we attempted to correlate protein solubility with other functional properties. Solubility often depends, at least partially, on molecular size. Therefore, characterization techniques for estimation of molecular size, as well as more tests on solubility were carried out. SDS-PAGE comparison of the protein in the whole seeds and the protein concentrates was used to determine the effectiveness of the wet-milling procedure in recovering the seed proteins (Fig. 3A and B). For buckwheat, it appeared that all the protein fractions were recovered by the milling and subsequent protein precipitation procedure because the protein profiles of the whole seeds and the concentrate were very much alike. However, this was not so with all the Amaranthus samples because most of the higher molecular weight fractions were missing in the concentrates. The two soy proteins profiles were identical, although they showed some differences in functional properties. Furthermore, the soy proteins were generally comprised of higher molecular weight fractions compared to the Amaranthus and buckwheat proteins and were hence less soluble. The previous test on protein solubility was based only on solubility in water with pH adjustment, and most of the protein concentrates were not highly soluble except at strongly alkaline pH. Thus, we determined solubility in other common solvents by doing protein fractionation based on the Osborne procedure (Table 1). The fractionation procedure was carried out on the protein concentrates which were obtained as by-products of wet-milling for starch extraction, so it does not reflect the true picture of the total protein in the seeds, but only those which were recovered from the milling process. Gorinstein et al. (1991) found that the protein
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Fig. 3. (A) SDS-PAGE of buckwheat and Amaranthus whole grains and the protein concentrates (M-mol. wt. markers; 1-buckwheat grain; 2-buckwheat protein concentrate; 3-K112 grain; 4-K112 protein concentrate; 5-K350 grain; 6-K350 protein concentrate). (B) SDS-PAGE of Amaranthus whole grains and protein concentrates and soy protein isolates (M-mol. wt. markers; 1-Soy B; 2-Soy A; 3-No. 3 protein concentrate; 4-No. 3 grain; 5-K459 protein concentrate; 6-K459 grain).
of their sample of A. cruentus seeds had 58.8% albumin+globulin +non-protein-N; 1.0% prolamin; 23.2% glutelin; and 13.1% residue. Comparing this to our protein concentrates, we found a much higher proportion of the alkali soluble proteins. As a result of this, the proportion of the combined water and salt soluble proteins was lower. For buckwheat protein, Javornik et al. (1981) reported that it comprised 18% albumin; 43% globulin; 1% prolamin; and 38% glutelin + residue. In our buckwheat protein concentrate there was an increased proportion of the alkali solubles and thus a decrease in the albumin+ globulin fraction. As expected, the level of albumin +globulin fraction in the protein concentrates is highly correlated to their solubility (r= 0.99; P=0.0001). A large fraction of the total protein in our samples was found to be highly soluble in alkaline conditions (Table 1) simply because they were extracted using alkali wet-milling. However, this may not be the only reason for the large proportion of glutelin-type proteins as observed here. Another factor is perhaps the defatting procedure used. Barba de la Rosa et al. (1992) noted that defatting the wholemeal flour with hexane induced insolubilization of Amaranthus hypochondriacus proteins and caused an increase in the proportion of glutelin-type proteins (from
21–24% to 38–42%). Petroleum ether, which was used in this study to defat the protein concentrates, could have had the same effect. This apparent reduction in solubility might also have been caused by the heating involved in preparation of the concentrate. Drying at 70°C for 12 h would denature the protein to some extent, but we consider this treatment appropriate for the likely levels of technology to be applied in small scale industrial preparation of these products. However, defatting the protein concentrates was found to be necessary because their fat level is too high (31–41% for Amaranthus and 13% for buckwheat) hence, at such state their functional properties would be severely limited and that they could also easily deteriorate. Furthermore, through defatting, the oil by-product can be obtained. The SDS-PAGE profiles of the buckwheat and Amaranthus K350 protein concentrates (hydrolyzed, unhydrolyzed, and Osborne sequence fractions) (Fig. 4A and B) confirmed that in the protein concentrates, only a portion of the total protein was albumin, hence they were only moderately soluble in water. However, after limited pepsin hydrolysis, the protein profile was drastically changed such that it was only concentrated at the bottom end of the gel corresponding to low molecular weight. The cutting of the native
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proteins into low molecular weight peptide fragments and some amount of free amino groups, therefore, caused a great improvement in solubility. SDS-PAGE of the albumin fraction of the K350 Amaranthus protein concentrate showed that its molecular weight would be lower than 14 kDa. This could then be similar to the protein fraction that Marcone et al. (1994) purified and analyzed from A. hypochondriacus which they referred to as albumin-1 with a molecular weight of 12 kDa (SDS-PAGE). They noted that this was the major albumin fraction, that its monomer form has a molecular mass of 133.4 kDa, and that its protomer is a homo-oligomer with 12 subunits. SDS-PAGE of the salt-soluble fraction of the K350 Amaranthus protein concentrate showed that it comprised various subunits of relatively high to low molecular weight range. Marcone and Yada (1991) found that there were at least six major Amaranthus globulin subunits and that their molecular weights range from 67 to 14.5 kDa. However, no band was found in the Amaranthus protein concentrates with an approximate size of 67 kDa. SDS-PAGE of the prolamin fractions of the protein concentrates showed that in buckwheat it was spread over a wider molecular weight range (intermediate to low MW) than in Amaranthus. The glutelin fraction on the other hand comprise of various subunits with relatively high to low molecular weight for both buckwheat and Amaranthus protein concentrate.
4. Summary and conclusions Using isoelectric precipitation on the proteinaceous liquor from wet-milling of Amaranthus and buckwheat grains, protein concentrates were made. Compared with the reference soy proteins, Amaranthus protein concentrates exhibited good functional properties in three tests: solubility, emulsification, and foaming. The buckwheat protein concentrate was also good in the first two tests but not in the third. As expected, with the application of partial pepsin hydrolysis, the solubility of these protein concentrates was further improved. Foaming property was reduced for some proteins after hydrolysis while no significant effect was observed for the others. Some of the molecular attributes of the protein concentrates and hydrolysates were also determined. There were differences in the proportion of the protein fractions based on their extractability in solvents of the Osborne sequence in the protein concentrates. The particular fractions and the hydrolysates showed distinct SDS-PAGE profiles. Protein concentrates can, therefore, be easily obtained as by-product of the starch extraction process of Amaranthus and buckwheat grains. The procedure involved is simple, such that an ovendrying procedure would be sufficient although a defatting step is necessary to produce an acceptable product. The favorable functional properties
Fig. 4. SDS-PAGE of protein concentrate and hydrolysate and extracts from solvents of the Osborne sequence (M-molecular wt. markers; 1-buckwheat protein concentrate; 2-albumin; 3-globulin; 4-prolamin; 5-glutelin; 6-pepsin hydrolysate) (A) Buckwheat. (B) Amaranthus K350.
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of the protein concentrates and hydrolysates, therefore, indicate that they can be of potential usefulness as food ingredients. We believe that under current yield productivity of Amaranthus in China, commercially viable specialty wet-milling can be implemented to separate valuable starch, functional protein, and high-quality vegetable oil.
Acknowledgements Financial support was received from the University of Hong Kong Committee on Research and Conference Grants and the Hong Kong Research Grants Council.
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Properties of protein concentrates and hydrolysates from Amaranthus and Buckwheat Feliciano P. Bejosano, Harold Corke * Cereal Science Laboratory, Department of Botany, Uni6ersity of Hong Kong, Pokfulam Road, Hong Kong Accepted 20 April 1999
Abstract Protein concentrates and pepsin hydrolysates were made after isoelectric precipitation of the proteinaceous liquor from wet-milling of grain of five Amaranthus and one buckwheat genotype. The Amaranthus protein concentrates exhibited better solubility, foaming, and emulsification than two commercial soy protein controls. Many protein properties depend on solubility, and Amaranthus protein concentrates were more soluble than soy protein isolate. The buckwheat protein concentrate was highly soluble with excellent emulsification, but poor foaming ability. Partial pepsin hydrolysis further improved solubility of the protein concentrates and also altered their foaming property. Fractionation and subsequent characterization of protein concentrates revealed that glutelins, albumins, and globulins predominated, with prolamins present in minor quantity. SDS-PAGE showed that globulins and glutelins were comprised of several subunits with varying molecular weights from relatively high to low while albumins were mostly of low molecular weight. The prolamin fraction of the buckwheat concentrate was comprised of intermediate to low molecular weight subunits while those of Amaranthus concentrates were only of low molecular weight. This study demonstrated the feasibility of producing potentially useful functional protein concentrates as by-product of Amaranthus and buckwheat starch extraction. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Amaranthus; Fractionation; Protein concentrate
1. Introduction New crops are those recognized to have potential for development into new markets or new products. Market demands may include special uses in industry, such as new functional starches or proteins. Grain Amaranthus has been recog* Corresponding author. Fax: +852-28583477. E-mail address: [email protected] (H. Corke)
nized as a crop of great potential for the 21st century (NRC 1984), but its adoption in US agriculture has been slow. However, driven by demand for animal feed uses, Amaranthus is increasing fast as a commercial crop in China with over 100 000 ha per year grown (Yue et al. 1993, S.X. Yue personal communication). Amaranthus and buckwheat (Fagopyrum esculentum) are pseudocereals producing seed high in starch content, similar to the major cereal crops like rice, maize,
0926-6690/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 9 9 ) 0 0 0 2 1 - 7
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and wheat. In addition to starch, significant amounts of protein (Amaranthus, 13 – 18% w.b.; buckwheat, 12–15% w.b.) are also found in these seeds, and the proteins are of good quality. The nutritional quality of Amaranthus protein scored higher than that of other cereal grain and legume seeds by FAO/WHO protein scoring or by PER (Breene 1991; Bejosano and Corke 1998c). Pomeranz (1983) noted that buckwheat proteins are rich in lysine, and that buckwheat is one of the best plant sources of proteins of high biological value. Amaranthus and buckwheat are used as specialty starch sources, using methods such as alkali wet-milling, where the main concern is the maximum recovery of starch. The remaining material, including the protein fraction, may commonly be disposed of as waste. However, by a simple processing using isoelectric precipitation protein can be recovered, and thus another potentially important product can be obtained. We have shown the applicability of such by-products in cereal (noodle) and meat (sausage) products (Bejosano and Corke, 1998a,b). This study was made with the aim of investigating the basic properties of protein concentrates from Amaranthus and buckwheat as by-product of starch extraction, and the extent of variation among common genotypes. The specific aims were to: (a) determine the functional properties and hence potential use of the protein concentrates; and (b) test the effect of further protein modification (pepsin hydrolysis) on these properties.
2. Materials and methods
2.1. Materials Five Amaranthus samples were supplied by the Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing, China. The genotypes were: K112, K350, K459, and R104 (all A. cruentus with yellowish-brown seedcoat), and No. 3 (A. hybridus with a black seedcoat) which are all widely cultivated varieties in China. R104 is a widely-grown grain variety, and K112 is a widely-grown forage variety in
China (Wu et al., 1995). No. 3 was selected because of its unique grain color. The buckwheat sample was purchased in a retail outlet in Hong Kong, packed by Queenswood (United Kingdom) but its country of origin was China. Two brands of commercially available soy protein isolate were used as reference standards (Soy A, Protein Technologies International, St Louis, Missouri; and Soy B, Edward Keller, Hong Kong). These commercial samples were marketed for general usage without claims for particular functional characteristics.
2.2. Preparation of isoelectric protein concentrates Using an alkali wet milling procedure with subsequent isoelectric precipitation of the proteinaceous liquor, protein concentrates were made out of the five Amaranthus and one buckwheat samples. The procedure used was based on method C of Perez et al. (1993), essentially the same as used by Wu et al. (1995). The grain materials were steeped in NaOH solution at 5°C for 24 h, ground in a blender, and screened to separate the fibrous residue from the filtrate. The filtrate was then centrifuged to separate the proteinaceous liquor from the starch fraction. After acid precipitation of the proteinaceous liquor, a wet curd was obtained which was in an emulsified state containing a high oil content. In this condition, conventional drying was found to be very difficult, so the curd was first frozen and then thawed to break the emulsion and to facilitate separation of the water layer. After doing this, a large amount of water could be removed physically. This was followed by drying at 70°C in a forced convection dryer for about 12 h. The coarse pellets formed were crushed finely using mortar and pestle. The resulting powder was then defatted in petroleum ether (10 h with intermittent shaking; removing the petroleum ether layer; repeated twice). Commercially, defatting will be essential to exploit the high quality and squalene content of the oil co-processing by-product. This was followed by air drying and further grinding using an Udy Cyclone Mill (Udy, Boulder, CO, USA) with 0.5 mm mesh screen.
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2.3. Enzymatic hydrolysis Partial pepsin hydrolysis of protein concentrates was carried out based on the procedure of Hartnett and Satterlee (1990). The substrate was prepared by adjusting the protein concentration to 1% (w/v) in 0.01 N HCl (pH 2.0). 91 units of pepsin A (Sigma, St Louis, MO, USA) per mg solid was added to the suspension at the rate of 0.2 mg ml − 1. The mixture was incubated at 37°C with mild shaking for 16 h, after which pH was adjusted to 7.5 and kept at 5°C for 72 h. The pH was then adjusted to 6.5 followed by freeze drying. The degree of hydrolysis (DH) was measured according to Bombara et al. (1992). DH was based on the ratio of TCA (trichloroacetic acid; 12%) soluble proteins to the total proteins of the sample. This is a widely used method of DH measure (Adler-Nissen 1986) and we chose it because of its simplicity.
2.4. Functional properties The nitrogen solubility of proteins in water was determined as a function of pH. Tests were carried out from pH 3.0 to pH 9.0. This method was based on Mannheim and Cheryan (1993) with modifications: the volume of the protein suspension was reduced to 50 ml; a Kenwood mixer (Kenwood Ltd., Hampshire, United Kingdom) with blender cup (250 ml) was used for mixing; mixer was set at minimum speed for 10 min; and centrifugation was carried out at 1650× g for 15 min at room temperature (:25°C). The test on foaming property was also based on Mannheim and Cheryan (1993). After mixing as described in the N-solubility test above, contents were transferred to a 250 ml graduated cylinder, kept at room temperature (:25°C) for 30 min and the residual foam was measured. However, foaming properties were defined based on Vani and Zayas (1995) wherein foam expansion (FE) is the foam volume (ml) immediately after blending and foam stability (FS) is the volume after standing for 30 min. The test on emulsifying activity was based on the method of Yasumatsu et al. (1972). One hun-
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dred ml of 7% protein dispersion was added to an equal volume of oil and the mixture was homogenized. There were some modifications: corn oil was used instead of soybean oil; homogenization was carried out in Kenwood mixer with blender cup (250 ml) set at speed no. 4 for 1 min; centrifugation was carried out at 1650× g for 10 min at room temperature (: 25°C). Emulsifying activity was calculated as the height of emulsified layer divided by the height of the whole layer times 100%.
2.5. Protein characterization The proportion of the different protein fractions in the concentrates was determined according to their extractability in different solvents of the Osborne sequence. This was based on the procedures of Gorinstein et al. (1991) and Barba de la Rosa et al. (1992) except that we chose to start the sequential extraction with water (pH 7) in order to recover most of the water extractable albumins. This was followed by extraction with 0.1 M NaH2PO4 (pH 7) (globulins); 70% isopropanol (prolamins); and lastly with 0.1 M NaOH (glutelins). Thus, the globulin fraction referred to here is probably contaminated with some albumins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25% slab gels) of the proteins was carried out using a Mini-Protean II Electrophoresis System (Bio-Rad, Richmond, CA, USA) based on the instruction manual supplied. The gels were stained in Coomassie blue R-250. Molecular weight markers were used to estimate the molecular weight of the different fractions detected. Markers were purchased from Sigma (St Louis, MO, USA). Bovine albumin, pepsin, trypsinogen, b-lactoglobulin, and lysozyme with molecular weights of 66, 34.7, 24, 18.4, and 14.3 kDa, respectively were used. SDS-PAGE was carried out on the whole grains and the protein concentrates. Likewise, SDS-PAGE was carried out on the fractions collected from the Osborne fractionation and the hydrolysates of the buckwheat and the K350 Amaranthus protein concentrate.
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Fig. 1. Solubility (%) of Amaranthus and buckwheat protein concentrates at different pH levels (each data point represents the mean of two replicates)
2.6. Statistical analysis Analysis of variance (ANOVA) and correlation analysis were carried out using the SAS System for Windows release 6.10 (SAS Institute, Cary, NC, USA) software. Comparison of means was carried out using the least significant difference (LSD) test.
3. Results and discussion
3.1. Functional properties We compared the solubility of the six protein concentrates in water with that of the two soy protein isolates at four different pH levels (pH 3, 5, 7, 9). Most of the Amaranthus and buckwheat
protein concentrates were more soluble in water than the two brands of soy protein isolate except at pH 9.0 (Fig. 1). Soy protein isolates are commonly produced by alkali extraction similar to the method we used (Wolf and Cowan 1971). Therefore, like the protein concentrates we prepared, we expected them to be highly soluble at alkaline pH. A reduction in their solubility in such conditions would, therefore, indicate the effect of the drying procedure following extraction that might have induced denaturation. Another possible concern is the effect of residual lipids. However, as shown in Table 1, the level of lipids in the protein concentrates is minimal (0.9–1.8%) which is similar to that of commercially available soy protein isolates. We also found no correlation between the fat content and the functional properties of the protein concentrates. Since the protein concentrates we made were oven dried, they were expected to have undergone some degree of denaturation. As the results show, at pH 9, soy A and K350 were significantly the most soluble, while soy B was in the third most soluble group (grouping based on comparison of means by LSD). K112 was significantly the least soluble (LSD; PB 0.05) at such pH. This also indicates that the protein of the materials we used, except K112, may have useful thermal stability in withstanding the drying procedure used. Among the Amaranthus protein concentrates, No. 3 (A. hybridus) was generally the most soluble at all pH levels while K112 (A. cruentus) was the least. The buckwheat concentrate at pH 7 was the most soluble among all the proteins tested although its
Table 1 Crude protein (Kjeldahl) and fat contents of Amaranthus and buckwheat isoelectric protein concentrates and the degree of hydrolysis of their corresponding pepsin hydrolysates; distribution of protein fractions of Amaranthus and buckwheat concentrates Source of protein
K112 (A. cruentus) K350 (A. cruentus) K459 (A. cruentus) R104 (A. cruentus) No. 3 (A. hybridus) Buckwheat
Protein concentrate
Hydrolysate
Albumin+globulin*
Prolamin*
Glutelin+residue*
Protein (%)
Fat (%)
DH (%)
(%)
(%)
(%)
60.4 70.1 65.7 58.5 66.1 77.1
1.5 1.2 1.1 0.9 1.6 1.8
11.6 16.6 15.1 17.0 14.5 11.8
25.3 90.3 46.8 9 1.0 41.3 9 0.4 42.2 91.6 50.2 91.3 55.4 91.0
1.6 9 0.0 1.6 90.1 1.6 90.0 1.9 9 0.3 1.69 0.0 1.8 9 0.3
73.1 9 0.1 51.6 9 1.8 57.2 9 1.3 55.9 9 1.3 48.1 90.1 42.8 91.8
* Means of two replicates;9 denotes S.D.
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Table 2 Emulsifying activity (%) at pH 7.0 of Amaranthus and buckwheat protein concentrates, and effect of partial pepsin hydrolysis on the solubility (%), foam expansion (ml) and foam stability (ml) of the concentrates, compared to two soy protein isolatesa
K112 K350 K459 R104 No. 3 Buckwheat Soy A Soy B LSD*
Emulsifying activity
Solubility
Conc
Conc
55.6 73.3 61.9 56.0 62.2 63.8 50.6 45.7 3.1
19.9 56.5 42.1 47.2 60.5 64.2 33.3 21.3 4.5
Foam expansion
Foam stability
Hydrol
Conc
Hydrol
Conc
Hydro
64.5 76.1 76.0 72.8 76.8 86.4 N/A N/A 6.4
49 91 52 35 68 36 23 28 20
94 63 32 28 42 22 N/A N/A 14
34 56 26 18 26 14 20 22 9
30 25 16 16 17 14 N/A N/A 7
* Least significant (PB0.05) difference for comparison of means in the same column. Conc, concentrates; hydrol, hydrolyzates.
a
solubility was not significantly different from No. 3. It was however, significantly higher than K350. It was noted that the solubility of the two soy protein isolates was poor at pH 3, 5, and 7. This may appear to contradict literature reports (e.g. Mahmoud 1994). However, our result is not exceptional because similar results can also be found (e.g. Frokjaer 1994). Wolf and Cowan (1971) noted a wide variation in solubility of commercially prepared soy proteins. They attributed this to the heat treatment given during the manufacture. We therefore assume, that the reference soy protein isolates we used were perhaps significantly denatured during their manufacture, thus, their solubility is affected as shown in the results. Using pepsin in the protein concentrates for a period of 16 h, hydrolysates with 12 – 17% DH were obtained (Table 1). Protein solubility at pH 7.0 was increased by at least 27% to as much as 221% after partial pepsin hydrolysis (Table 2). The protein concentrates were also evaluated for their foaming stability at pH 3.0 – 9.0 and foam expansion at pH 7.0, again comparing them against soy proteins. Similar to the results on solubility, it was found that at all pH levels, most of the Amaranthus protein concentrates showed better foaming stability than soy proteins (Fig. 2) with the A. cruentus K350 protein giving the best result. However, the buckwheat protein concen-
trate gave the least amount of foam after 30 min standing, even less than those of the soy proteins. The measurement of foaming properties at pH 7.0 (Table 2) showed that the foams produced by the Amaranthus protein concentrates, except for No. 3, were able to maintain more than 50% of their initial volume after standing for 30 min. Although the volume of the foam produced by the soy proteins were relatively small, they were nevertheless able to keep about 80% of it after standing. However, buckwheat protein concentrate produced a small amount of foam which was also unstable.
Fig. 2. Foam stability (foam volume, ml) of Amaranthus and buckwheat protein concentrates (each data point represents the mean of two replicates)
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The effect of partial protein hydrolysis on foaming property was also noted. At pH 7, it was found that there was a reduction in foam expansion as a result of hydrolysis, although this was only significant in half of the cases. Furthermore, it was unexpectedly noted that hydrolysis of K112 actually caused an increase in foam expansion. It was also noted that after partial hydrolysis, it gave the highest increase in solubility (concentrate vs. hydrolysate) of 221% compared to the others with about 27–77% increase. This may therefore, indicate a large change in the surface active properties of the proteins which could have then caused a favorable effect on foam expansion. The ability of these protein concentrates to form emulsions at pH 7.0 was also determined. Here again, it was found that they all have significantly higher emulsifying activity than soy protein isolates (Table 2). Again, A. cruentus K350 protein concentrate had significantly the highest emulsifying activity followed by buckwheat, No. 3 and K459 protein concentrates. We investigated the reasons for the variation in the functional properties among the samples. The emulsifying activity of the proteins at pH 7.0 was significantly correlated with their solubility in water at the same pH level (r= 0.77; n =8; a = 0.05), showing that at least in our case, the ability of the protein concentrates to form emulsions is somehow dependent on their solubility in the medium. The trend in the results on foaming properties was found to be more complicated. Perhaps it is because this protein property is determined by many factors and their specific roles are not well defined. Schnepf (1992) found that attempts by several authors to correlate foam characteristics to molecular properties of proteins have not always been successful, although a study by Townsend and Nakai (1983) had indicated the main factors contributing to foaming capacity were viscosity, surface hydrophobicity, and solubility in descending order. Generally, it appeared that foaming was favored at acidic pH and that it decreased with increase in pH. The behavior of proteins at interfaces influences the formation and stabilization of foams which depend to a great extent on the properties of the interfacial film and that films produced near the protein’s isoelectric point are more condensed and
stronger (Kinsella and Phillips 1989). Hence we observe foaming properties to be favored near pH 4.0.
3.2. Protein characterization Proteins from different genotypes of the same species of Amaranthus were found to have different properties, and it is not surprising to find such differences in proteins from seeds of another species or genus. These differences are further complicated when modifications are carried out on the proteins. In the previous discussion, we attempted to correlate protein solubility with other functional properties. Solubility often depends, at least partially, on molecular size. Therefore, characterization techniques for estimation of molecular size, as well as more tests on solubility were carried out. SDS-PAGE comparison of the protein in the whole seeds and the protein concentrates was used to determine the effectiveness of the wet-milling procedure in recovering the seed proteins (Fig. 3A and B). For buckwheat, it appeared that all the protein fractions were recovered by the milling and subsequent protein precipitation procedure because the protein profiles of the whole seeds and the concentrate were very much alike. However, this was not so with all the Amaranthus samples because most of the higher molecular weight fractions were missing in the concentrates. The two soy proteins profiles were identical, although they showed some differences in functional properties. Furthermore, the soy proteins were generally comprised of higher molecular weight fractions compared to the Amaranthus and buckwheat proteins and were hence less soluble. The previous test on protein solubility was based only on solubility in water with pH adjustment, and most of the protein concentrates were not highly soluble except at strongly alkaline pH. Thus, we determined solubility in other common solvents by doing protein fractionation based on the Osborne procedure (Table 1). The fractionation procedure was carried out on the protein concentrates which were obtained as by-products of wet-milling for starch extraction, so it does not reflect the true picture of the total protein in the seeds, but only those which were recovered from the milling process. Gorinstein et al. (1991) found that the protein
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Fig. 3. (A) SDS-PAGE of buckwheat and Amaranthus whole grains and the protein concentrates (M-mol. wt. markers; 1-buckwheat grain; 2-buckwheat protein concentrate; 3-K112 grain; 4-K112 protein concentrate; 5-K350 grain; 6-K350 protein concentrate). (B) SDS-PAGE of Amaranthus whole grains and protein concentrates and soy protein isolates (M-mol. wt. markers; 1-Soy B; 2-Soy A; 3-No. 3 protein concentrate; 4-No. 3 grain; 5-K459 protein concentrate; 6-K459 grain).
of their sample of A. cruentus seeds had 58.8% albumin+globulin +non-protein-N; 1.0% prolamin; 23.2% glutelin; and 13.1% residue. Comparing this to our protein concentrates, we found a much higher proportion of the alkali soluble proteins. As a result of this, the proportion of the combined water and salt soluble proteins was lower. For buckwheat protein, Javornik et al. (1981) reported that it comprised 18% albumin; 43% globulin; 1% prolamin; and 38% glutelin + residue. In our buckwheat protein concentrate there was an increased proportion of the alkali solubles and thus a decrease in the albumin+ globulin fraction. As expected, the level of albumin +globulin fraction in the protein concentrates is highly correlated to their solubility (r= 0.99; P=0.0001). A large fraction of the total protein in our samples was found to be highly soluble in alkaline conditions (Table 1) simply because they were extracted using alkali wet-milling. However, this may not be the only reason for the large proportion of glutelin-type proteins as observed here. Another factor is perhaps the defatting procedure used. Barba de la Rosa et al. (1992) noted that defatting the wholemeal flour with hexane induced insolubilization of Amaranthus hypochondriacus proteins and caused an increase in the proportion of glutelin-type proteins (from
21–24% to 38–42%). Petroleum ether, which was used in this study to defat the protein concentrates, could have had the same effect. This apparent reduction in solubility might also have been caused by the heating involved in preparation of the concentrate. Drying at 70°C for 12 h would denature the protein to some extent, but we consider this treatment appropriate for the likely levels of technology to be applied in small scale industrial preparation of these products. However, defatting the protein concentrates was found to be necessary because their fat level is too high (31–41% for Amaranthus and 13% for buckwheat) hence, at such state their functional properties would be severely limited and that they could also easily deteriorate. Furthermore, through defatting, the oil by-product can be obtained. The SDS-PAGE profiles of the buckwheat and Amaranthus K350 protein concentrates (hydrolyzed, unhydrolyzed, and Osborne sequence fractions) (Fig. 4A and B) confirmed that in the protein concentrates, only a portion of the total protein was albumin, hence they were only moderately soluble in water. However, after limited pepsin hydrolysis, the protein profile was drastically changed such that it was only concentrated at the bottom end of the gel corresponding to low molecular weight. The cutting of the native
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proteins into low molecular weight peptide fragments and some amount of free amino groups, therefore, caused a great improvement in solubility. SDS-PAGE of the albumin fraction of the K350 Amaranthus protein concentrate showed that its molecular weight would be lower than 14 kDa. This could then be similar to the protein fraction that Marcone et al. (1994) purified and analyzed from A. hypochondriacus which they referred to as albumin-1 with a molecular weight of 12 kDa (SDS-PAGE). They noted that this was the major albumin fraction, that its monomer form has a molecular mass of 133.4 kDa, and that its protomer is a homo-oligomer with 12 subunits. SDS-PAGE of the salt-soluble fraction of the K350 Amaranthus protein concentrate showed that it comprised various subunits of relatively high to low molecular weight range. Marcone and Yada (1991) found that there were at least six major Amaranthus globulin subunits and that their molecular weights range from 67 to 14.5 kDa. However, no band was found in the Amaranthus protein concentrates with an approximate size of 67 kDa. SDS-PAGE of the prolamin fractions of the protein concentrates showed that in buckwheat it was spread over a wider molecular weight range (intermediate to low MW) than in Amaranthus. The glutelin fraction on the other hand comprise of various subunits with relatively high to low molecular weight for both buckwheat and Amaranthus protein concentrate.
4. Summary and conclusions Using isoelectric precipitation on the proteinaceous liquor from wet-milling of Amaranthus and buckwheat grains, protein concentrates were made. Compared with the reference soy proteins, Amaranthus protein concentrates exhibited good functional properties in three tests: solubility, emulsification, and foaming. The buckwheat protein concentrate was also good in the first two tests but not in the third. As expected, with the application of partial pepsin hydrolysis, the solubility of these protein concentrates was further improved. Foaming property was reduced for some proteins after hydrolysis while no significant effect was observed for the others. Some of the molecular attributes of the protein concentrates and hydrolysates were also determined. There were differences in the proportion of the protein fractions based on their extractability in solvents of the Osborne sequence in the protein concentrates. The particular fractions and the hydrolysates showed distinct SDS-PAGE profiles. Protein concentrates can, therefore, be easily obtained as by-product of the starch extraction process of Amaranthus and buckwheat grains. The procedure involved is simple, such that an ovendrying procedure would be sufficient although a defatting step is necessary to produce an acceptable product. The favorable functional properties
Fig. 4. SDS-PAGE of protein concentrate and hydrolysate and extracts from solvents of the Osborne sequence (M-molecular wt. markers; 1-buckwheat protein concentrate; 2-albumin; 3-globulin; 4-prolamin; 5-glutelin; 6-pepsin hydrolysate) (A) Buckwheat. (B) Amaranthus K350.
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of the protein concentrates and hydrolysates, therefore, indicate that they can be of potential usefulness as food ingredients. We believe that under current yield productivity of Amaranthus in China, commercially viable specialty wet-milling can be implemented to separate valuable starch, functional protein, and high-quality vegetable oil.
Acknowledgements Financial support was received from the University of Hong Kong Committee on Research and Conference Grants and the Hong Kong Research Grants Council.
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