Chemical Characterization and Nutritional Analysis of Protein Isolates

Mar 20, 2014 - Chemical Utilization of Albizia lebbeck Leaves for Developing Protein Concentrates as a Dietary Supplement. Lutful Haque Khan , V. K. ...
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Chemical Characterization and Nutritional Analysis of Protein Isolates from Caragana korshinskii Kom. Cheng Zhong,† Zhuo Sun,† Zhao Zhou,†,‡ Ming-Jie Jin,§ Zhi-Lei Tan,† and Shi-Ru Jia*,† †

Key Laboratory of Industrial Fermentation Microbiology (Ministry of Education), School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ‡ Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, P.O. Box 6888, Tianjin 300072, People’s Republic of China § Department of Chemical Engineering and Material Science, Biomass Conversion Research Laboratory, Michigan State University, Lansing, Michigan 48910, United States ABSTRACT: Plant-based proteins are valuable supplements to compensate the gap between supply and demand in the food or feed industry. However, they lack essential amino acids, such as lysine in cereal grains and sulfur-containing amino acids in legumes, which greatly limit their wider uses for human and animals. In this study, the contents of nutritional ingredients and antinutritional factors of Caragana korshinskii Kom. and its protein isolates were quantitatively investigated. It was shown that the crude protein contents of C. korshinskii Kom. and its protein isolates obtained by alkaline extraction method (Al-CPI) and acetone precipitation method (A-CPI) were 9.1, 50.1, and 42.6%, respectively. Amino acid contents in C. korshinskii Kom., AlCPI, and A-CPI basically exceeded the FAO/WHO (2007) reference pattern for adults except sulfur-containing amino acids. The lysine levels in C. korshinskii Kom., Al-CPI, and A-CPI were 4.1, 3.1, and 3.8 mg/100 mg crude protein respectively, which were higher than some other kinds of cereal grains. The methionine in A-CPI (1.39 mg/100 mg crude protein) was even higher than that in soybean. The antinutritional factors in C. korshinskii Kom. and Al-CPI were generally lower than those in some other kinds of legumes except total phenol and tannin. Total phenol and tannins in Al-CPI were 19.02 and 5.66 mg/g dry substance, respectively, but they were undetectable in A-CPI. This study provided a detailed analysis on nutritional and antinutritional factors in C. korshinskii Kom. and its protein isolates, indicating that they have a great potential on food and feed additives. KEYWORDS: Caragana korshinskii Kom., plant proteins, nutritional ingredients, antinutritional factors

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INTRODUCTION Caragana korshinskii Kom., a kind of deciduous shrub, which belongs to Galegeae (Br.) Torrey et Gray, is mainly distributed in arid and semiarid areas of northwestern China. It has excellent performances in cold hardiness, drought resistance, and vigorous growth, facilitating its wide application in controlling desertification of water-deficient areas.1 Previous studies on C. korshinskii Kom. have focused on biological taxonomy, genetic diversity, and ecological values.2 Meanwhile, it has been used as a green manure to improve soil fertility in agricultural farming, as well as firewood to offset energy shortages. Besides, C. korshinskii Kom. was also utilized as fodder to feed animals in rural areas. Plant tissues need to be first purified by some physical or chemical methods to form protein isolates, which have better purity and bioavailability than their raw materials. In recent years, plant proteins have been regarded as substitutes for human food or animal feeds to compensate the gap between supply and demand of proteins. Compared with animal-based proteins, plant-based proteins have advantages of lower cholesterol contents and abundant sources. However, they lack some essential amino acids, such as lysine in cereal grains or sulfur-containing amino acids in legumes, and show lower digestibility, which limited their wider uses for human and animals.3 To evaluate the nutrition and potential applications of protein isolates, their amino acid compositions, digestibility, and functional properties were common concerns. Our © 2014 American Chemical Society

previous studies investigated the functional properties and digestibility of protein isolates by alkaline extraction (Al-CPI) and acetone precipitation (A-CPI) from C. korshinskii Kom. It was shown that Al-CPI exhibited good performance on functional properties. The in vitro digestibility of A-CPI was as high as 83.7%, which was slightly higher than that of the commercial soybean protein isolate (82.5%).1 However, detailed information on the chemical characterization and nutritional analysis of Al-CPI or A-CPI has not been reported. On the other hand, C. korshinskii Kom., like other legumes, may have antinutritional factors (ANFs), such as trypsin inhibitor, tannins, and phytic acid, which may remain in its protein isolates and reduce in vivo digestibility and absorption of the proteins.4 In this study, the nutritional ingredients and ANFs (trypsin inhibitor, lectin, total phenol, tannins, phytic acid, and saponin) of C. korshinskii Kom., Al-CPI, and A-CPI were investigated. The nutritional values of protein isolates were compared to related products to find their potential applications on fodder sources or food additives. Received: Revised: Accepted: Published: 3217

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absorbance of the solution was observed at 660 nm with KH2PO4 as standard. Amino Acid Analysis. The derivatization method described by Hainida10 was followed by assay amino acids. The hydrolysate (1 mL) of amino acids was evaporated to dryness under vacuum and then redissolved with 1 mL of deionized water. Hydrolysate (50 uL), acetonitrile/triethylamine solution (25 μL, 43:7, v/v), and phenyl isothiocyanate (PITC)/triethylamine solution (25 μL, 1:80, v/v) were mixed and incubated for1 h at room temperature. The mixed mobile phase of acetonitrile, sodium acetate solution, and water was used. The essential amino acid (EAA) score was calculated as

MATERIALS AND METHODS

Chemicals. Benzyl- DL -arginine-p-nitroanilide hydrochloride (BAPNA), trypsin from porcine pancreas (1000−2000 benzyl arginine ethyl ester (BAEE) units/mg solid), and PHA-P (8754) were purchased from Sigma (St. Louis, MO, USA). All other reagents were purchased from Shanghai BOAO Biochemical Co. (China). Sample Preparation. C. Kkorshinskii Kom. was collected from Liang Cheng County of Inner Mongolia (China). Leaves and tissues were air-dried, milled, and passed through a 60 mesh screen. The milled C. korshinskii Kom. was stored at −20 °C until further use. Preparation of C. korshinskii Kom. Protein Isolates (CPI). Alkali extraction and acetone precipitation methods were used to prepare CPIs from the milled powder of C. korshinskii Kom.1 For an alkaline extraction method, the milled C. korshinskii Kom. was dispersed in 0.12 M NaOH with the solid to liquid ratio of 1:20 (w/v) and then incubated at 37 °C for 1 h. The slurry was centrifuged at 4000g for 20 min to obtain a supernatant. The pH of the supernatant was adjusted to 4.5 with 0.1 N HCl, and then the supernatant was heated in a water bath at 75 °C for 10 min. After centrifugation at 4000g for 20 min, the protein precipitate was freezedried to obtain Al-CPI. For an acetone precipitation method, the milled C. korshinskii Kom. was dispersed in an extraction buffer in the ratio of 1:20 (w/v). The slurry was incubated at 4 °C for 7 h and centrifuged at 4000g for 20 min to obtain a supernatant. Precooled acetone was added to the supernatant, which was then incubated at −20 °C overnight. The mixture was centrifuged at 4000g for 20 min, and the resulting precipitate was then rinsed with precooled acetone three times. The ACPI was obtained when the precipitate was dried under nitrogen gas. Analysis of Nutritional Ingredients. Approximate Composition Analysis. The contents of moisture, crude protein, crude lipid, and ash were determined according to the standard methods described by the Association of Official Analytical Chemists (AOAC).5 Crude fiber was determined by GB/T 5009.10-2003 (Recommended National Standard of China).6 Samples were successively dispersed in boiling solutions of 1.25% H2SO4 and 1.25% NaOH. The oven-dried residual was then weighed to calculate the content of crude fiber. The carbohydrate content was determined according to the data of moisture, crude protein, crude lipid, crude fiber, and ash contents. The gross energy was calculated according to the following formula:

EAA score = g of EAA in 100 g crude proteins g of EAA in 100 g FAO/WHO (2007) reference pattern

Analysis of Various Antinutritional Factors (ANFs). Trypsin Inhibitor. Trypsin inhibitor activity (TIA) was measured according to the method of Siddhuraju.11 Defatted ground samples (0.5 g) were incubated in 25 mL of 0.01 M NaOH at pH 9.4−9.5 for 3 h. The mixture was centrifuged at 4000g for 15 min, and the supernatant was collected to estimate the TIA content of material. Lectin. The lectin content was analyzed by using the hemagglutination assay.12 Samples (0.5 g) were incubated at room temperature in 10 mL of the phosphate buffer for 10 h with agitation using a magnetic bar. The slurry was centrifuged at 4000g for 20 min. The collected supernatant was then added in microtiter plates with 2% (v/v) rabbit blood erythrocyte suspension in phosphate buffer (pH 7.0). When >50% of the erythrocytes was aggregated, it was considered as agglutination. PHA-P was used as a positive control. The hemagglutination activity was defined as

C = (S0 × H × V )/(H0 × M )

(3)

where C is the content of lectin that produced agglutination (mg/g), S0 is the concentration of PHA-P solution (mg/mL), H is agglutinin titers (the minimum amount of samples producing hemeagglutination, 2n), V is the volume (mL), H0 is agglutinin titers (the minimum amount of standard producing hemeagglutination, 2n), and M is sample quantity (g). Phenolic Substances. The Folin−Ciocalteu method13 was adopted to determine the total phenol and tannin levels with some slight modifications. Samples (0.2 g) were incubated with 10 mL of 70% acetone in an ultrasonic bath (frequency, 35 kHz; acoustic power, 85 W) for 20 min at room temperature. The mixture was centrifuged at 4000 rpm for 10 min. Extraction processes were performed once again. The obtained supernatants were mixed together. A certain amount of the supernatant was used to determine the content of total phenols. The tannin content was reflected by the decrease of total phenol content in the supernatant after polyvinylpyrrolidone treatment. Gallic acid was used as equivalents for total phenols and tannins. Phytic Acid. The FeCl3·6H2O−sulfosalicylic acid colorimetric procedure11 was utilized to determine the content of phytic acid. Samples (0.5 g) were stirred with 10 mL of 3.5% HCl for 1 h at room temperature. The mixture was centrifuged at 4000 rpm for 10 min to obtain the supernatant. Phytic acid (98%, Guizhou Di-Da Biotech Co. Ltd.) was used as standard in the assay. Saponins. The extraction method of total saponins was reported by Chen14 with some slight modifications. Defatted samples (0.5 g) were incubated with 10 mL of 80% aqueous methanol in an ultrasonic bath (frequency, 35 kHz; acoustic power, 85 W) for 30 min at room temperature. The mixture was centrifuged at 3000 rpm for 10 min. Extraction processes were conducted once again. The obtained supernatants were mixed together. A certain amount of the supernatant was used to determine the content of total saponins. Statistical Analysis. All experiments are conducted in three replicates. One-way analysis of variance (ANOVA) was carried out to compare the mean values, followed by Duncan’s multiple-range tests. Significant differences in the mean values were determined at p < 0.05 (SPSS 19).

gross energy (kJ 100/g dry substance) = (protein × 16.7) + (lipid × 37.7) + (carbohydrates × 16.7)

(2)

(1)

Total Starch Analysis. Total starch was measured as described by Goni7 with some modifications. Samples (100 mg) were dispersed in 12 mL of 2 M KOH and vigorously shaken at room temperature for 30 min. The mixture was adjusted to pH 7.0 with 0.4 M sodium acetate buffer, followed by an addition of 0.5 mL of amyloglucosidase (Aladdin, 100000 U/mL), and then incubated at 60 °C for 45 min in a shaking water bath. Glucose oxidase/peroxidase reagent (Shang Hai Rong Sheng Biotech Co., Ltd.) was utilized to measure glucose content. The conversion factor from glucose to starch was 0.9. Soluble Sugar Analysis. Soluble sugar was extracted with 70% (v/ v) ethanol, and sugar content was determined by using a phenol− sulfuric acid method.8 Mineral Composition. C. korshinskii Kom. powder was digested with the mixed acids (concentrated nitric acid, sulfuric acid, and perchloric acid in the ratio 10:0.5:2, v/v). The contents of minerals (sodium, potassium, calcium, magnesium, iron, copper, zinc, and manganese) were determined by atomic absorption spectrophotometer (Shimadzu, AA-6800) accoring to the AOAC procedure.5 Total phosphorus was determined according to GB/T 5009.87-2003.9 Samples (0.5 g) were digested with mixed acids to obtain a digestive solution, and then digestive solution (1 mL), (NH4)6Mo7O24·4H2O reagent (2 mL), 0.5% hydroquinone solution (1 mL), 20% sodium sulfite reagent (1 mL), and water (15 mL) were added. After 0.5 h, the 3218

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Table 1. Proximate Analysis of C. korshinskii Kom., Al-CPI, and A-CPIa moisture (%) ash (%) crude protein (%) crude fiber (%) crude lipid (%) total available carbohydrate (%) gross energy (kJ/100 dry substance) a b

C. korshinskii Kom.

Al-CPI

A-CPI

6.25 ± 0.25c 3.63 ± 0.10c 9.09 ± 0.31c 56.86 ± 0.97a 1.77 ± 0.04a 22.40 ± 1.40c 592.65 ± 17.70b

8.6 ± 0.37b 8.95 ± 0.42a 50.06 ± 1.40a 0.79 ± 0.03b 0.91 ± 0.04b 30.70 ± 1.72b 1382.79 ± 12.65a

10.02 ± 0.49a 7.28 ± 0.27b 42.59 ± 1.17b 0.81 ± 0.02b NDb 39.30 ± 1.59a 1367.57 ± 8.75a

Values are expressed as mean ± SD of three measurements. Values with different letters are significantly different at p < 0.05 within the same row. ND, not detected.

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Table 3. Mineral Composition of C. korshinskii Kom., AlCPI, and A-CPI (Milligrams per 100 g Dry Substance)a

RESULTS AND DISCUSSION Analysis of Nutritional Ingredients. The proximate chemical compositions of C. korshinskii Kom., Al-CPI, and ACPI are summarized in Table 1. For C. korshinskii Kom., crude fiber constituted about 56.9%, followed by total carbohydrate (22.4%) and crude protein (9.1%). Protein content in the plants may be varied using different growth environments and harvest times. A recent study15 has shown that the protein level of phloem from C. korshinskii Kom. was about 11%. In our study, the feedstock of C. korshinskii Kom. was a mixture of leaves and tissues. The contents of crude protein in Al-CPI and A-CPI were 50.1 and 42.6%, respectively, which was lower than that of most protein isolate from beans, such as fava bean (81.9%), pea (88.8%), and soy (87.6%),16 but higher than that of barley flour (35.5%).17 As shown in Table 2, the contents of starch in C. korshinskii Kom., Al-CPI, and A-CPI were not significantly different

C. korshinskii Kom. powder sodium potassium calcium phosphorus magnesium iron copper zinc manganese

total starch soluble sugar

Al-CPI

A-CPI

24.12 ± 0.98a 5.50 ± 0.11b

24.8 ± 0.40a 2.60 ± 0.01c

25.44 ± 0.29a 6.47 ± 0.19a

± ± ± ± ± ± ± ± ±

0.11c 12a 44b 88a 0.10b 0.23c 0.02c 0.04c 0.06b

Al-CPI 1878 154 183 551 15.76 13.71 2.64 1.49 0.49

± ± ± ± ± ± ± ± ±

77a 11a 17c 17b 0.84b 1.01b 0.22b 0.11b 0.05b

A-CPI 145 58.82 1712 503 284 21.44 12.91 3.32 10.48

± ± ± ± ± ± ± ± ±

12b 5.21b 168a 16b 27a 0.40a 0.72a 0.32a 0.79a

Values are expressed as mean ± SD of three measurements. Values with different letters are significantly different at p < 0.05 within the same row.

a

those of soybean, wheat grains, rice, and FAO/WHO reference pattern. The important plant species used for humans or animals are mainly cereal grains and food legumes. Compared with animal-based proteins, many plant-based proteins do not provide essential amino acids in a balanced proportion, which restricts their potential application. The typical amino acids are lysine (Lys), threonine (Thr), and sulfur-containing acid amino acids (methionine and cysteine). The Lys content is normally deficient in cereal grains,3 such as wheat grains, rice, and maize, but it was relatively plentiful in C. korshinskii Kom. (4.1 mg/100 mg crude protein), Al-CPI (3.1 mg/100 mg crude protein), and A-CPI (3.8 mg/100 mg crude protein) relative to that in cereals, but lower than that in buckwheat seeds.29 In fish, poultry, or pig fodder, Lys was found to be the first limiting amino acid.21 Thr is the second limiting amino acid in grain sorghum, barley, and wheat, as well as the third limiting amino acid in maize. It also becomes the first limiting amino acid when animal fodder is supplemented with lysine.23 The content of Thr in C. korshinskii Kom. (4.7 mg/100 mg crude protein) or A-CPI (4.7 mg/100 mg crude protein) was even higher than that in soybean, rice, wheat grains, and FAO/WHO reference pattern for preschool children and adults.22 However, the Thr content in Al-CPI (2.8 mg/100 mg crude protein) exceeded only rice and FAO/WHO reference pattern for adults. According to EAA scores, sulfur-containing amino acids are the first limiting amino acids for C. korshinskii Kom. and CPIs. Some legumes had relatively lower levels of methionine (Met) and cysteine (Cys) than animal-based proteins.20 Met was the first limiting amino acid for soybean, the amino acid composition of which was mostly closed to animal-based protein. It has to be noted that the Met content in A-CPI (1.4 mg/100 mg crude protein) was slightly higher than that in soybean. The total amounts of aromatic amino acids (phenyl-

Table 2. Total Starch and Soluble Sugar in C. korshinskii Kom., Al-CPI, and A-CPI (Grams per 100 g Dry Substance)a C. korshinskii Kom. powder

1.17 140 1163 3562 24.82 8.11 0.22 0.40 0.61

Values are expressed as mean ± SD of three measurements. Values with different letters are significantly different at p < 0.05 within the same row.

a

(24.1−25.4 g/100 g dry substance). In addition, the soluble sugar in A-CPI (6.5%) was about 2.5-fold that in Al-CPI (2.6%). Table 3 shows that phosphorus and calcium existed in C. korshinskii Kom., which exceeds the demand for adults.18 Phosphorus and calcium are the essential ingredients in human and animal skeleton series and teeth. In addition, phosphorus is a necessary component for nucleic acid, protein, and lecithin. It also participates in the metabolism process of saccharides and lipids and forms high-energy phosphate bonds to store energy. Compared with C. korshinskii Kom., the content of sodium in Al-CPI shows a dramatic increase, which may have resulted from the extraction solution (NaOH). However, the contents of calcium and iron in Al-CPI decreased sharply. This result may be attributed to the possibility that calcium and iron were more likely to chelate with phytic acid when Al-CPI was precipitated at pH 4.5.19 Moreover, the contents of trace elements, such as Fe, Mn, Zn, and Cu, were higher in A-CPI than in C. korshinskii Kom. and Al-CPI. The compositions of essential amino acids in C. korshinskii Kom., Al-CPI, and A-CPI are shown in Table 4, together with 3219

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Table 4. Essential Amino Acid Composition of C. korshinskii Kom., Al-CPI, A-CPI, Soybean, Rice, Wheat Grain, and FAO/ WHO Recommended Patterns for Adults and Preschool Children essential amino acid content (mg/100 mg crude protein) material C. korshinskii Kom. Al-CPI A-CPI soybeanb ricec wheat grainsd FAO reference patterne FAO reference patternh EAA score C. korshinskii Kom. Al-CPI A-CPI

His

Thr

Val

Met

Ile

Leu

2.2 1.1 1.8 2.5 1.6 2.4 1.6 1.9

4.7 2.8 4.7 3.8 2.3 3 0.9 3.4

2.5 1.9 3.1 4.6 4.3 3.9 1.3 3.5

0.9 0.8 1.4 1.2 1.3 1.5 1.7f 2.5f

1.7 1.4 2.4 4.6 2.3 2.7 1.3 2.8

4.2 4.5 6.8 7.7 4.9 6.9 1.9 6.6

113.68 60.00 92.63

138.24 81.18 138.82

72.29 53.43 87.71

36.00 33.20 55.60

60.71 50.00 85.71

63.79 68.03 103.64

Phe + Tyr 7.5 5.6 8.6 6.1 5.7 7.4 1.9g 6.3g 119.05 88.73 135.71

Lys

Try

4.1 3.1 4.8 6.1 2.5 2.4 1.6 5.8

NDa ND ND 1.24 0.6 1.4 0.5 1.10

70.69 53.62 83.10

0 0 0

a ND, not detectable. bData from Bau et al. (1994). cData from Abdel-Aal and Hucl (2002). dData from Sosulski and Imafidon (1990). eFAO/WHO (2007) essential amino acids pattern for adults. fMethionine + cystine. gPhenylalanine + tyrosine. hFAO/WHO (2007) essential amino acids pattern for preschool children, individuals aged 2−5 years.

alanine and tyrosine) in C. korshinskii Kom. and A-CPI were 7.5 and 8.6 mg/100 mg crude protein, respectively. They are even higher than those in soybean, rice, wheat, and FAO/WHO pattern for children. Phenylalanine has been widely used as human food and animal feed additives, as well as resources to synthesize some active materials.24 For C. korshinskii Kom., AlCPI, and A-CPI, the contents of essential amino acids generally exceeded the FAO/WHO reference pattern for adults except tryptophan and sulfur-containing amino acids. The ample qualities of Lys, Thr, and aromatic amino acid make it a good alternative additive in the feed or food industry. The compositions of nonessential amino acids in C. korshinskii Kom., Al-CPI, and A-CPI are shown in Figure 1. In C. korshinskii Kom., compared with others, proline (10.1 mg/100 mg crude protein) and glycine (3.5 mg/100 mg crude protein) ranked first and last in nonessential amino acids. Proline was adequate in the plants with high abilities of cold hardiness and drought resistance. However, in Al-CPI or A-

CPI, the highest level of nonessential amino acid was aspartic acid, whereas the lowest was tyrosine. In general, the total amount of amino acids in A-CPI was higher than that in Al-CPI. Different pH values utilized in the protein extraction processes may account for this variance. A pH of 6.8 was utilized in the extraction process of A-CPI, and most amino acids were stable at this pH.40 The contents of essential and nonessential amino acids in C. korshinskii Kom. were higher than those in Al-CPI, but lower than those in A-CPI with the exception of histidine. Different extraction methods could lead to different amino acid contents and compositions in protein isolates. Therefore, a suitable extraction method for proteins should be chosen to satisfy the demand of food and feed additives. Analysis of Antinutritional Factors. Some ANFs exert their effects on the digestion and assimilation of some nutriments, as well as the immune system of animals. In our study, the ANF values of C. korshinskii Kom., Al-CPI, and ACPI are investigated in Table 5. Table 5. Antinutritional Factor Contents in C. korshinskii Kom., Al-CPI, and A-CPI (Milligrams per Gram Dry Substance)a C. korshinskii Kom. trypsin inhibitor lectin total phenol tannins phytic acid saponin

b

2.07 3.74 10.67 7.70 1.70 ND

± ± ± ± ±

0.10a 0.0b 0.24b 0.19a 0.09a

Al-CPI c

ND b NDc 19.02 ± 0.57a 5.66 ± 0.23b NDc ND

A-CPI 2.46 ± 0.10a 4.73 ± 0.0a NDc NDc 0.96 ± 0.04b ND

Values are expressed as mean ± SD of three measurements. Values with different letters are significantly different at p < 0.05 within the same row. bTrypsin inhibitor, mg pure trypsin inhibited per g of dry sample. cND, not detected.

a

The TIA contents of C. korshinskii Kom. and A-CPI were 2.07 and 2.46 mg pure trypsin inhibited/g dry substance, respectively. However, TIA was not detected in Al-CPI. Using the alkaline extraction, the TIA contents in protein isolates from Vicia faba were less than one-third of those in their raw materials.25 The TIA value of C. korshinskii Kom. in this study

Figure 1. Nonessential amino acid composition of C. korshinskii Kom., Al-CPI, and A-CPI (mg/100 mg crude protein). Values are expressed as mean ± SD of triplicate measurements. All samples were significantly different (p > 0.05) except for aspartic acid and proline. Asp, aspartic acid; Glu, glutamic acid; Ser, serine; Gly, glycine; Arg, arginine; Ala, alanine; Pro, proline; Tyr, tyrosine. 3220

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was lower than those of some kinds of mucuna beans (13.8− 19.2 mg/g).26,39 Combined with protease, trypsin inhibitor could lead to a decrease of protease activity and a low digestibility of proteins. The lectin contents in C. korshinskii Kom. and A-CPI were 3.74 and 4.73 mg/g dry substance, respectively. However, lectin was undetectable in Al-CPI. A previous study indicated that alkaline extraction led to decreased contents of lectin in pea protein isolates (14 mg/g) compared with their raw materials (35 mg/g).27 As a kind of glycoprotein, lectin was not digested by proteases, and it disturbed other protein digestion in humans and animals. Additionally, lectin can affect the human immune system and reduce the content of hormones secreted by the gut.28 The total phenols content of C. korshinskii Kom. (10.54 mg/ g dry substance) was higher than that in Vigna radiata (4.5 mg/ g) and lower than that in Sesbania (20.4−30.0 mg/g).11 The content of total phenols in Al-CPI (19.02 mg/g) was higher than that in C. korshinskii Kom. Our results compared well with a previous alkaline extraction process,29 in which the total phenols in protein isolates of buckwheat seeds (29.3 mg/g) were higher than those in their materials (6 mg/g). The content of tannins in C. korshinskii Kom. was 7.61 mg/g dry substance. It was lower than that in pea (27.80−30.93 mg/ g) and cowpea (22.63−26.14 mg/g).30 Tannin content in AlCPI was 5.66 mg/g, which was higher than that in protein isolate of mung bean (1.52 mg/g).31 Total phenols and tannins could be united with diet proteins and digestive enzymes to form indigestible compounds, which could result in a low digestibility of food. However, in recent years, some ANFs, such as tannins and phytic acids, were also found to have some beneficial biological functions, such as oxidation resistance, antitumor promotion, and antiviral activity. The contents of phytic acid were 1.70 and 0.96 mg/g dry substance in C. korshinskii Kom. and A-CPI, but it was undetectable in Al-CPI. Mwasaru32 had reported that phytic acid contents were not detected in pigeonpea and cowpea protein isolates extracted by alkaline extraction. The phytic acid content in C. korshinskii Kom. was less than that in many kinds of legume beans, such as mung bean seeds (5.8 mg/g), chickpea (12.1 mg/g), cowpea (14 mg/g), and horse gram (10.2 mg/g).33 Phytic acid could combine with minerals to form infusible precipitates, which could lead to interference with the absorption of mineral substances, especially calcium, zinc, and iron.19 Saponins was not detected in C. korshinskii Kom., A-CPI, and Al-CPI. Saponins could cause hemolysis and decrease the adsorption of nutrition in humans and animals.34 Hydroxy steroids or bile acids can form insoluble complexes with saponins. In an alkaline extraction process, protein extraction was followed by isoelectric precipitation. Hagerman35 reported that phenolic substances could effectively combine with proteins during the protein isoelectric precipitation. In our study, some ANFs, such as the total phenols and tannins, in Al-CPI were higher than those in C. korshinskii Kom. On the other hand, the phytic acid in Al-CPI was lower than that in C. korshinskii Kom. Proteins and phytic acid did not combine when the pH was >11, and most of the phytic acid was removed with insoluble materials.36 In addition, trypsin inhibitor and lectin in Al-CPI were not detected. The pH values used in the extraction process may lead to reduced bioactivity of proteins.41

In an acetone precipitation method, some ions derived from C. korshinskii Kom. may be entrapped in the extraction buffer, which might lead to an increase in the ionic strength of acetone and the precipitation efficiency of proteins.37 This may explain the higher contents of TIA and lectin in A-CPI than those in C. korshinskii Kom. Additionally, phenolic substances could be solubilized in acetone during the protein precipitation,38 which may lead to undetectable total phenols or tannins in A-CPI. In summary, the protein contents of C. korshinskii Kom., AlCPI, and A-CPI were 9.1, 50.1, and 42.6%, respectively. The contents of essential amino acids in the three materials were higher than those in FAO/WHO reference pattern for adult (2007) except sulfur-containing amino acids and tryptophan. As the limiting amino acids in cereal grains, the contents of Lys and Thr in C. korshinskii Kom., Al-CPI, and A-CPI exceeded those in the cereal grains, such as rice and wheat grains. The methionine in A-CPI (1.39 mg/100 mg crude protein) was even higher than that in soybean. The ANFs in C. korshinskii Kom. and Al-CPI were generally lower than those in other legumes except total phenols and tannins. Total phenols and tannins in Al-CPI were 19.02 and 5.66 mg/g dry substance, respectively, but they were undetectable in A-CPI. Our study provides a detailed analysis of the nutritional compositions and antinutritional factors in C. korshinskii Kom. and its protein isolates, indicating that they have great potential as food and feed additives.

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AUTHOR INFORMATION

Corresponding Author

*(S.-R.J.) E-mail: [email protected]. Fax: 86-22-60602298. Phone: +86-22-60601598. Funding

We are grateful for financial support from the International Cultivation of Excellent Postdoctor (2012) by Tianjin Municipal Human Resources and Social Security Bureau, the National Natural Science Foundation of China (No. 21106105), and Changjiang Scholars and Innovative Research Team in University (No. IRT1166). Notes

The authors declare no competing financial interest.

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REFERENCES

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