Mineral Elements, Lipoxygenase Activity, and Antioxidant Capacity of

Article pubs.acs.org/JAFC

Mineral Elements, Lipoxygenase Activity, and Antioxidant Capacity of Okara as a Byproduct in Hydrothermal Processing of Soy Milk Sladjana P. Stanojevic,*,† Miroljub B. Barac,† Mirjana B. Pesic,† Sladjana M. Zilic,§ Mirjana M. Kresovic,# and Biljana V. Vucelic-Radovic† †

Department of Chemistry and Biochemistry, Faculty of Agriculture, Institute for Food Technology and Biochemistry, University of Belgrade, Belgrade, Serbia § Department of Technology, Maize Research Institute, “Zemun Polje”, Belgrade, Serbia # Department of Agrochemistry and Plant Physiology, Faculty of Agriculture, University of Belgrade, Belgrade, Serbia ABSTRACT: Minerals and antioxidative capacity of raw okara that was obtained as a byproduct from six soybean varieties during hydrothermal cooking (HTC) of soy milk were assessed. Lipoxygenase (Lox), an enzyme deteriorating the sensory characteristics of okara, was also investigated. All genotypes had very similar concentrations of Lox (4.32−5.62%). Compared to raw soybeans, the applied HTC significantly reduced Lox content in okara (0.54−0.19%) and lowered its activity to 0.004−0.007 μmol g−1 min −1. Correlation between the content of Lox in soybeans and that in okara (r = 0.21;p < 0.05) was not registered. This indicates that the content of this enzyme in okara depended much more on the technological process than on soybean genotype. Very strong correlation (r = 0.99; p < 0.05) between okara Lox content and its activity was found. The most abundant minerals in raw okara were potassium (1.04−1.21 g/100g), phosphorus (0.45−0.50 g/100 g), calcium (0.26−0.39 g/100 g), and iron (5.45−10.95 mg/100 g). A very high antioxidant capacity (19.06−29.36 mmol Trolox kg−1) contributes to the nutritional value of raw okara. KEYWORDS: okara, HTC processing, soybean genotype, lipoxygenase, antioxidant capacity, mineral content

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INTRODUCTION The search for alternative food and feed ingredients for man and livestock continues to attract the attention of researchers all over the world. Okara (soy pulp) is the main byproduct from soy milk and tofu preparation. Raw okara is a white-yellowish material consisting of insoluble parts of the soybean seed remaining in the filter sack when pureed soybean seeds are filtered in the production of soy milk. Okara is rich in proteins (24.5−40.36 g/100 g of dry matter (dm), lipids (9.3−23.2 g/ 100 g of dm), and dietary fiber (14.5−54.5 g/100 g of dm).1−4 It also contains isoflavones (0.14 g/100 g of dm), minerals, and vitamins as well as essential fatty and amino acids.2,4−9 In addition, okara is characterized by a low energy value (about 3 kcal/g of fresh matter).7 Due to this specific composition, okara might have a potential use in the food industry in the fortification of tortillas,6 cakes,8 and biscuits.9 The quality of soybean proteins is limited by the high content of bioactive compounds that may have antinutritional activity (trypsin inhibitors (TIs), lectins)10 or undesirable effects on the sensory characteristics of soy foods (lipoxygenases).11,12 Heat treatment significantly reduces TI activity in okara (4.61−14.93 TUI/mg) compared to soybeans (95.61−197.68 TUI/mg), although the TI contents are higher in okara (5.19−13.65% of extractable okara proteins) than in soybeans (3.10−12.17% of extractable soybeans proteins)7 thus greatly improving the nutritional value of okara because the TIs are cysteine-rich proteins.13 In addition, an appropriate heat treatment can significantly reduce the content of lectins in okara (0.07−1.73% of extractable okara proteins) compared to grain (5.48−7.74% extractable soybeans proteins).7 © 2014 American Chemical Society

The beneficial effects of the consumption of certain foods are attributed to the ability of bioactive compounds in food of vegetable origin to avoid or ameliorate the oxidative stress caused by the imbalance between reactive oxidative species and the endogenous antioxidant system in a living organism. Oxidative stress and inflammation are linked to the risk of developing cardiovascular diseases.14 Agricultural byproducts are attractive sources of antioxidant components.15 The primary waste fractions, which are seed coat and residues, contain high amounts of bioactive components that can be potentially exploited as antioxidant agents and nutraceuticals. Several studies indicated that okara is a source of antioxidant components.16−20 Yokomizo et al.17 showed that protease hydrolysates from okara yield antioxidant activity, MateosAparicio et al.18 found antioxidant activity of polysaccharide fractions of okara, and Jiménez-Escrig et al.19 have shown the effect of a fiber-rich okara diet on mineral balance and antioxidant status in healthy rats. Several methods to measure the antioxidant activity have been published in the past 20 years. Many attempts using solvent mixtures or physical treatments have been made to increase the solubility of food components to assess their antioxidant activity.21,22 The antioxidant activity of fermented okara or some of its fractions (proteins, polysaccharides, phenolics, isoflavones) has been reported.1,17−19,23,24 Also, literature data show that the total Received: Revised: Accepted: Published: 9017

April 15, 2014 August 20, 2014 August 20, 2014 August 20, 2014 dx.doi.org/10.1021/jf501800s | J. Agric. Food Chem. 2014, 62, 9017−9023

Journal of Agricultural and Food Chemistry

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antioxidant capacity of methanolic-water okara extracts was examined.16 It is clear that many food items have insoluble components that cannot be solubilized without altering their molecular nature by chemical or enzymatic treatments.25,26 However, the insoluble components are not necessarily chemically inert. The contribution of insoluble matter to total antioxidant capacity is especially high for cereal-based and dietary-fiber-rich foods.27 So far, the total antioxidant capacity of both soluble and insoluble components of okara has not been reported. The mineral content in okara is relatively well researched. Okara contains macro- and microelements that increase its nutritional value.1,3,28,29 Mateos-Aparicio et al.28 emphasize that within the group of macroelements potassium is the main mineral, whereas iron is the predominant microelement. In the literature there are no data on the contents of Cd, Co, Cr, and Pb in okara, although the amounts of these microelements in foods are strictly limited.30 Lipoxygenase (Lox) is an important bioactive protein in soybean. This enzyme catalyzes the addition of molecular oxygen to polyunsaturated molecules containing a cis,cis-1,4pentadiene structure to pentadienyl and further results in a pentadienyl radical intermediate, which could interact with other lipids to form many other derivatives such as aldehydes and ketones.11 Because of this activity of Lox, sensory characteristics of soy foods are significantly affected. Lox produces off-flavors in soybeans by hydroperoxidation of fatty acids and by interaction with protein in flours, concentrates, and isolates.31 The undesirable flavors, characterized as beany, green, grassy, painty, astringent, and bitter, reduce acceptance of soy foods by many consumers who may prefer a bland flavor in soy products. Nowadays, efforts are made to produce soy foods with more efficient Lox inactivation because in this way the off-flavor in soy foods could be eliminated.32 Traditionally prepared soy milk has beany and painty flavors owing to lipid oxidation catalyzed by Lox during soaking and grinding. In Western societies, this flavor is unacceptable for most consumers. A steam infusion cooking process, known as hydrothermal cooking (HTC), was developed to produce soy milk continuously from ground full-fat soybeans. During HTC processing the temperature is higher and the processing time shorter than for conventional methods. HTC-processed soy milk had less beany flavor because of the much shorter time for Lox activity and because steam flashing stripped volatiles. The process also increased recoveries of dry matter and protein in the soy milk from 60 and 70% for traditional soy milk to 87 and 90%, respectively.33 Characteristics with advantages over conventionally processed soy milk have been demonstrated by physical and chemical analyses of hydrothermally cooked soy milk.33 Hydrothermal cooking results in an acceptable decrease in activities of trypsin inhibitors7 as well as in levels of lysinoalanine and browning products. However, previous papers have not documented the nutritional performance in mineral element content of hydrothermally processed okara. Also, total antioxidant capacity arising from both soluble and insoluble matters of okara has not been studied. Therefore, the aim of this study was to assess the influence of a pilot plant method that uses high-pressure hydrothermal conditions on mineral composition and antioxidative capacity as well as lipoxygenase activity in okara produced from different soybean genotypes. The results obtained in this study would complement our previous work on bioactive proteins and nutritional properties of okara produced by this proccess.2,7

Article

MATERIALS AND METHODS

Materials. For okara preparation six commercial soybean genotypes grown in field conditions were used: ZPS-015 (0 maturity group), Krajina (00 maturity group), Novosađanka and Balkan (I maturity group), and Nena and Lana (II maturity group). Three genotypes (Lana, Nena, and ZPS-015) were selected by the Maize Research Institute Zemun Polje (Belgrade, Serbia) and the others (Novosadjanka, Krajina, and Balkan) by the Institute of Field and Vegetable Crops (Novi Sad, Serbia). All of the genotypes were of food grade and were characterized by high protein content in grain. Okara Processing. Okara was made on the pilot plant scale using the production method that includes HTC34 for soy milk preparation, as modified by Stanojevic et al.35 Briefly, soybeans were soaked for 14 h in water at 5−7 °C. Soaked beans were ground and cooked by steam injection system at 1.8 bar/110 °C/8 min (SoyaCow VS 30/40, model SM-30, Russia). Thereafter, the slurry was filtered to separate soy milk from okara. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE). The soybean powder and defatted raw okara were prepared for SDS-PAGE analyses according to the method of Stanojevic et al.7 Briefly, the soybean powder and raw okara were defatted using hexane. Thereafter, proteins were extracted for 120 min at room temperature with 0.03 M Tris-HCl buffer, pH 8.00, which contained 0.01 M β-mercaptoethanol. For defatted soy flour, the sample to buffer ratio was 1:20; for okara, the sample to buffer ratio was 1:10. The mixture was centrifuged at 7558g for 15 min at room temperature. The protein extracts were diluted with sample buffer (pH 6.80) to a concentration of 2 mg/mL, then heated at 90 °C for 5 min, and cooled to room temperature. Dissociating electrophoresis for all samples was performed according to the Fling and Gregerson36 procedure, as modified by Stanojevic et al.,2,7 using 5% (w/v) (pH 6.80) stacking and 12.5% (w/ v) separating gels (pH 8.85). Briefly, 25 μL of sample containing 0.05 mg of protein was loaded onto each well, and then the gels were run at 80 mA per gel for 6 h in a buffer solution (pH 8.30). The gels were fixed for 50 min, stained with 0.23% (w/v) Coomassie brilliant blue R250, and destained with 8% (v/v) acetic acid and 18% (v/v) ethanol. Molecular weight markers that were used were phosphorylase B (94 kDa), bovine albumin (67 kDa), ovoalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa) (Pharmacia, Uppsala, Sweden). Densitometric Analysis. All gels were destained, then scanned, and subsequently analyzed by SigmaGel software version 1.1 (Jandel Scientific, San Rafael, CA, USA). Quantitative analysis of each identified polypeptide was calculated as the percentage of the respective area with respect to total area of the densitogram. Lipoxygenase Activity. The Lox (EC 1.13.11.12) was extracted from fresh okara with 5 volumes (w/v) of 0.2 M sodium phosphate buffer (pH 6.5) at 4 °C for 120 min. Supernatant obtained by centrifugation at 10000g for 10 min was used to measure the activity of Lox I. The reaction was carried out at 25 °C in a quartz cuvette with a 1.0 cm light path. The assay mixture contained 0.060 mL of 10 mM linoleate substrate and 0.1 mL of the extract in 2.440 mL of 0.2 M borate buffer, pH 9.0. The initial rate of the absorbance changes at 234 nm (ε = 2.5 × 104 M−1 cm−1) was recorded, and the activity of Lox I was expressed in micromoles of conjugated diene formed per minute and per gram of dry matter.37 Total Antioxidant Capacity. The antioxidant capacity was measured by QUENCHER method described by Serpen et al.27 using 7 mM aqueous solution of 2,2-azinobis(3-ethyl-benothiazoline6-sulfonic acid) (ABTS). Radical cation was generated by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration), and the mixture was kept in the dark at room temperature for 12−16 h before use. The working solution of ABTS•+ was obtained by diluting the stock solution with water/ethanol (50:50, v/v). Finely ground sample (10 mg) was mixed with 20 mL of ABTS•+ working solution, and the mixture was vigorously shaken for 25 min. After centrifugation at 10000g for 5 min, the absorbance was recorded at 734 nm. The total antioxidant capacity was expressed as Trolox 9018

dx.doi.org/10.1021/jf501800s | J. Agric. Food Chem. 2014, 62, 9017−9023


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equivalent antioxidant capacity (TEAC) in millimoles of Trolox per kilogram of dry matter. Mineral Content. The mineral content in fresh okara was analyzed by atomic absorption spectroscopy (AAS) (Varian Spectra AA 220). The microelement Fe, Mn, Cu, Zn, Cr, Pb, Co, and Cd contents in okara were analyzed after the destruction in concentrated HNO3 and HClO4 with the addition of 33% H2O2 by wet ashing.38 Contents of Ca and Mg in okara were detected after the destruction with concentrated acids HNO3 and HCl by dry ashing.39 Contents of P, K, and Na in okara were determined after the destruction with concentrated acids H2SO4 and HClO4 by dry ashing. Phosphorus was determined by the colorimetric method, whereas K and Na were determined by the flame-photometry method.39 Other Analyses. Total nitrogen content in okara was determined by using the micro-Kjeldahl method,40 and protein content was calculated (N × 6.25). Ash content was calculated from the weight of okara after burning at 550 °C for 2 h in a muffle furnace.41 Okara moisture and volatiles were determined according to standard AACC procedures.42 The pH of a 1:10 okara water suspension (w/v) was measured using a Consort-C931 pH meter (Belgium) with automatic temperature compensation. Statistical Analysis. Data were assessed by analysis of variance (ANOVA) using the Statistical software version 7.0 (StatSoft Co., Tulsa, OK, USA). The Tukey multiple-range test was used to separate means, and significance was accepted at p < 0.05. Regression analyses were also carried out. The different parameters were correlated with each other by Pearson two-tailed significance correlation at the p < 0.05 level. The experiments were performed in triplicate.

Figure 2. SDS-PAGE analysis of protein composition in okara from investigated genotypes. Lanes: mws, molecular weight standards; 1, Nena; 2, Krajina; 3, Novosadjanka; 4, Balkan; 5, Lana; 6, ZPS-015.

Table 1. Lipoxygenase Content of Soybeans and Okara from Investigated Genotypesa % extractable protein

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genotype

RESULTS AND DISCUSSION Lipoxygenase Content and Activity. The sensory quality of soy foods is limited by the soybean’s content of Lox. By SDSPAGE analysis of investigated soybean genotypes and okara extractable protein composition, we registered the presence of Lox (Figures 1 and 2); the first significant single broad band

soybean

Nena Krajina Novosadjanka Balkan ZPS-015 Lana

4.95 5.42 5.11 4.32 5.51 5.62

± ± ± ± ± ±

0.04 0.03 0.02 0.01 0.03 0.04

okara c b bc d ab a

0.21 0.31 0.54 0.03 0.05 0.19

± ± ± ± ± ±

0.001 0.003 0.002 0.000 0.000 0.002

c b a d d c

a

Means in the same column with different letters are significantly different (p < 0.05).

the development of lipoxygenase-free soy genotypes, there is permanent striving to reduce the content or activity of this enzyme by technological process.32 Our results showed that thermal/pressure treatment applied to soybeans was sufficient to get such a result in okara. When compared to soybeans, the applied treatment significantly reduced Lox content in okara (0.03−0.54%; Table 1). This result was expected because the denaturation temperature of Lox is at 69−74 °C,45 and the applied temperature in the production of okara was 110 °C. We did not register a significant correlation between the content of Lox in soybean and in okara (r = 0.21; Table 2). This indicates that the content of this enzyme in okara significantly depended on the technological process but not on soybean genotype. On the basis of this very low Lox content in okara, a very low activity of this enzyme was expected. Our results confirmed that applied thermal/pressure treatment was sufficient to inactivate Lox activity in okara. Because the most stable isomer is Lox-I, we registered only its activity. Because of its stability, most kinetic, structural, and spectroscopic studies have involved this isoform.43 Okara Lox-I activity was registered in traces (0.004− 0.007 μmol g−1 min −1; Table 3). The okara’s pH value might contribute to such low Lox-I activity. Okara pH was from 6.88 to 7.04 (Table 4), whereas the value for the optimum activity of Lox-I is about 9.0.44 These results suggested that the applied thermal/pressure treatment produced okara with very low Lox content and activity. Therefore, when used for the preparation of food, this okara would not give undesirable sensory effects.

Figure 1. SDS-PAGE analysis of protein composition in soybeans from investigated genotypes. Lanes: mws, molecular weight standards; 1, Lana; 2, Krajina; 3, Novosadjanka; 4, Balkan; 5, Nena; 6, ZPS-015.

corresponds to lipoxygenase. Soybeans have three Lox isoforms (Lox-I, Lox-II, and Lox-III), which have homologous sequences and MW of about 96 kDa.43,44 According to the results of densitometric analysis (Table 1) all genotypes had very similar contents of Lox, which ranged from 4.32 to 5.62% of total extractable proteins. Nowadays, lipoxygenase-free soy genotypes have been developed. Soy milk made from lipoxygenasefree soybeans has a less beany aroma, a less beany flavor, and less astringency and was rated darker and more yellow than that made from soybeans synthesizing lipoxygenase.12 In addition to 9019



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Content of Mineral Elements. The okara ash content of only 3.50−4.25% suggested that during production minerals of the soybeans mainly found their way into the soymilk, Table 5. Osthoff et al.47 have come to the same conclusion. This result is in agreement with literature data.1,28,29,47 Total ash content in okara was not significantly correlated with the content of certain mineral elements Table 2. Smith and Circle48 pointed out direct dependence of ash and phosphorus contents in soybeans, but this was not the case with the investigated okara (r = −0.25; p = 0.05; Table 2). We have registered a slightly higher correlation coefficient between the okara ash content and calcium, iron, and chrome contents, but they were not in significant dependence (Table 2). This result of correlation analysis showed that the content of total minerals could not indicate a trend change in the content of certain mineral elements. Smith and Circle 48 noted that the mineral compositions of soybeans vary depending on the origin of the crop or the specific cultivar. Our results have shown that there were differences in the content of certain mineral components of the okara from different genotypes. However, these differences were not for all mineral elements statistically significant (e.g., P, Na). These results of statistical analysis suggest that the technological process of okara production has a much greater influence on mineral composition of okara than soybean genotype. With the exception of nitrogen, within the group of macroelements, potassium was the main mineral in the okara (1.04−1.21 g/100 g dm), approximately 2 times higher than the amount of phosphorus (0.45−0.50 g/100 g dm; Table 5). Calcium also appeared in significant amounts (0.26−0.39 g/ 100 g dm). Okara contains significant amount of dietary fiber (54.3%).28 Van der Rietet et al.29 indicated a possible association of calcium, potassium, phosphorus, and sodium with the fiber. Because of this, it is possible that these elements are bound for fiber and remain in okara, thus comprising its nutritional value. In addition, nondigestible polysaccharides can positively affect the absorption of calcium.50 Jiménez-Escrig et al.20 evaluated the metabolism of calcium, magnesium, and zinc in rats fed a diet supplemented with okara, and a significant enhancement on the absorption of calcium was found. In addition, literature data indicate that okara contains very small amounts of phytic acid (0.5−1.2%).29 Van der Riet et al.29 note that phytic acid largely remains in tofu. Phytic acid is nutritionally significant because of its potential to chelate essential dietary minerals such as calcium and zinc.49 Due to the low phytic acid content, and hence the lower chelating capacity, calcium remained in available form. Iron is the predominant microelement ranging from 5.45 to 10.95 mg/100 g dm, whereas the manganese content is very similar in all samples (1.43−1.85 mg/100 g dm; Table 5).

Table 2. Correlation Coefficients

a

relationship

r

soybean Loxa content/okara Lox content okara Lox content/okara Lox activity okara ash content/okara K content okara ash content/okara P content okara ash content/okara Ca content okara ash content/okara Mg content okara ash content/okara Na content okara ash content/okara N content okara ash content/okara Fe content okara ash content/okara Cu content okara ash content/okara Mn content okara ash content/okara Zn content okara ash content/okara Cr content okara ash content/okara Pb content

0.21 0.99b 0.11 −0.25 −0.50 −0.30 −0.40 0.06 −0.54 −0.37 −0.30 0.15 0.73 0.14

Lox, lipoxygenase; bSignificant at p < 0.05.

Literature data show that the correlation between Lox content in soybeans and respective enzymatic activity was positive but not significant (r = 0.57, p < 0.05).11 On the contrary, our results showed very strong correlation (r = 0.99; Table 2) between okara Lox content and activity. This indicates that the most stable isoform of this enzyme survived the heat/ pressure treatment that resulted in the proportional relationship between Lox content and activity. Total Antioxidant Capacity. To examine the influence of the applied heat/pressure treatment on okara total antioxidant capacity arising from both soluble and solid okara particles, we implemented the direct procedure developed by Serpen et al.27 to include the activity arising from insoluble matter. According to our results (Table 3), very high values of okara antioxidant capacity were registered. The lowest antioxidant capacity was found for genotype Lana (19.06 mmol Trolox kg−1 dm) and the highest for genotype Novosadjanka (29.36 mmol Trolox kg−1 dm). Other genotypes had very similar antioxidant capacities ranging from 23.31 to 29.31 mmol Trolox kg−1 dm. When compared to raw soybeans. the antioxidant capacity of okara was decreased by around 34−58%. When compared with the published results for different foods, our values are higher than for oat flour or breakfast cereals.27 Our results are similar to the antioxidant capacity of pumpkin or beet and much higher than those of broccoli, tomato, onion, or carrot.46 Registered okara antioxidant capacity is more than twice higher than the antioxidant capacity of green beans, a plant belonging to the same family of legumes as soybeans.46 Registered antioxidant activity certainly contributed to the functional value of fresh okara.

Table 3. Lipoxygenase (Lox-I) Activities and Antioxidant Capacitya lipoxygenase activity (μmol g−1 min genotype Nena Krajina Novosadjanka Balkan ZPS-015 Lana a

−1

)

okara 0.005 0.006 0.007 0.004 0.004 0.005

± ± ± ± ± ±

0.00 0.00 0.00 0.00 0.00 0.00

antioxidant capacity (mmol Trolox equiv kg−1 dm) beans

a a a a a a

63.47 38.99 58.42 42.19 36.92 49.05

± ± ± ± ± ±

2.17 1.24 2.11 0.56 0.79 2.09

okara a e b d d c

26.57 23.31 29.36 27.84 19.06 29.31

± ± ± ± ± ±

0.26 1.07 1.22 1.32 0.24 0.71

b c a ab d a

Values are given on a moisture-free, full-fat basis. Means in the same column with different letters are significantly different (p < 0.05). 9020



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Table 4. Some Characteristics of Raw Okara (Percent)a total proteinsbb

genotype Nena Krajina Novosadjanka Balkan ZPS-015 Lana a

30.89 32.06 32.06 27.81 28.12 27.44

± ± ± ± ± ±

1.37 0.03 0.42 0.11 0.61 1.57

ashb

a a a b b b

3.50 3.50 4.25 4.00 3.50 3.75

± ± ± ± ± ±

0.00 0.08 0.29 0.00 0.00 0.09

moisture b b a ab a ab

79.19 83.63 75.00 79.76 73.68 76.86

± ± ± ± ± ±

pH

1.82 1.15 1.10 1.85 1.18 1.85

7.04 6.93 6.93 6.96 7.01 6.88

± ± ± ± ± ±

0.04 0.02 0.03 0.05 0.06 0.07

Values are given on a moisture-free, full-fat basis. bMeans in the same column with different letters are significantly different (p < 0.05).

Table 5. Content of Mineral Elements in Raw Okaraa Nena

Krajina

Novosadjanka

K P Ca Mg Na N

1.04 0.46 0.28 0.11 0.01 4.94

± ± ± ± ± ±

0.01 0.00 0.00 0.00 0.00 0.02

c a c b a a

1.08 0.50 0.32 0.14 0.02 5.13

± ± ± ± ± ±

0.03 bc 0.02 a 0.01 b 0.00 a 0.00 a 0.05a

Fe Cu Mn Zn

7.05 1.70 1.58 5.53

± ± ± ±

0.08 0.08 0.05 0.08

c a b b

10.95 0.78 1.50 2.15

± ± ± ±

0.10 0.05 0.08 0.08

Cd Co Cr Pb

ndc nd 0.43 ± 0.02 e 3.04 ± 0.10 b

a d b c

nd nd 1.13 ± 0.10 b 2.78 ± 0.27 b

Balkan

ZPS-015

Macroelements (g/100 g dry matter)b 1.04 ± 0.03 c 1.21 ± 0.07 a 0.50 ± 0.01 a 0.46 ± 0.01 a 0.26 ± 0.01 c 0.30 ± 0.01 cb 0.13 ± 0.01 ab 0.11 ± 0.00 b 0.01 ± 0.00 a 0.01 ± 0.00 a 5.13 ± 0.06 a 4.45 ± 0.02 b Microelements (mg/100g dry matter)b 5.45 ± 0.06 d 7.80 ± 0.05 bc 0.70 ± 0.00 d 1.10 ± 0.00 b 1.43 ± 0.05 b 1.45 ± 0.01 b 1.93 ± 0.05 c 7.97 ± 0.01 a Microelements (mg/kg dry matter)b nd nd nd nd 2.04 ± 0.07 a 0.91 ± 0.04 c 3.19 ± 0.41 ab 3.47 ± 0.30 a

Lana

1.01 0.48 0.39 0.15 0.02 4.50

± ± ± ± ± ±

0.04 0.01 0.01 0.00 0.00 0.09

c a a a a b

1.15 0.45 0.33 0.13 0.02 4.39

± ± ± ± ± ±

0.04 0.02 0.01 0.00 0.00 0.05

ab a b ab a b

7.20 0.90 1.52 1.67

± ± ± ±

0.02 0.08 0.05 0.05

c c b c

9.03 0.78 1.85 1.50

± ± ± ±

0.04 0.05 0.01 0.08

b d a c

nd nd 0.62 ± 0.05 d 3.07 ± 0.26 b

nd nd 0.46 ± 0.07 e 3.04 ± 0.11 b

a

Values are given on a moisture-free, full-fat basis. bMeans in the same row with different letters are significantly different (p < 0.05). cnd, not detected.

best scores for sensory characteristics were given to cookies prepared with the addition of 60%9 and 70%8 okara. If we calculate the daily intake of both of these harmful elements for the consumption of 100 g of cakes, our results suggest that the lead content can be problematic in biscuits containing 70% okara, because the result was on the border or slightly increased than the allowable value for Pb (Table 6). These results suggest that the content of potentially harmful microelements should be controlled when okara is incorporated in foods and feeds.

Legumes are generally rich in iron; nevertheless, the bioavailability of this microelement is low, and therefore its value as a source of iron is diminished. The content of zinc significantly depended on the soybean genotype (1.50−7.97 mg/100 g dm). Zinc bioavailability is relatively good in legumes, approximately 25% from the zinc intake;50 however, there is no evidence of an increase of zinc absorption in rats fed diets supplemented with okara during 4 weeks.20 Copper is in low amount in all of the samples (0.70−1.70 mg/100 g dm; Table 5). Okara micro- and macroelements present similar values in this study as those given by Van der Riet et al.29 Because the plant material may contain harmful substances, we examined the contents of Cd, Co, Cr, and Pb in okara produced from the investigated genotypes using hydrothermal processes. The content of these microelements in raw okara is presented in Table 5. Our results have shown that chromium (0.43−2.04 mg/kg dm) and lead (2.78−3.47 mg/kg dm) were both registered in okara (Table 5). In the literature there are no data on the content of these microelements in okara, but our results indicated their presence in very low amounts, and therefore their content should be controlled in food and feed products. According to the FAO/WHO Expert Committee on food additives,30 acceptable daily intakes (ADI) of these elements are for Cr, 200 μg/daily, and for Pb, 214 μg/daily. Okara is considered to be a functional food, a rich natural source of nutritional ingredients. Okara is mainly incorporated into baked goods to enhance their dietary fiber and protein contents. New bakery products such as cakes and cookies fortified with high amounts of okara have been developed. The

Table 6. Daily Intake (Micrograms per Day) of Microelements in the Consumption of 100 g of Biscuits with Okara Different Content with 60% okara

with 70% okara

genotype

Cr

Pb

Cr

Pb

Nena Krajina Novosadjanka Balkan ZPS-015 Lana

25.50 76.55 122.25 54.75 37.05 27.30

212.80 166.95 191.55 208.05 184.05 182.55

29.70 79.10 142.63 63.87 43.23 31.85

259.53 194.78 223.47 242.72 214.72 212.97

The investigated samples of okara bear no health risk regarding the contents of Cd and Co as these elements were below the limit of detection. Accumulation and translocation of Cd and Co as well as other toxic metals by plants depends not only of their content in soil but also on soil characteristics such as pH and its granulometric composition as well as organic 9021



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(5) Peñalvo, L. J.; Castilho, C. M.; Silveira, M. I. N.; Matallana, M. C.; Torija, M. E. Fatty acid profile of traditional soymilk. Eur. Food Res. Technol. 2004, 219, 251−253. (6) Waliszewski, K. N.; Pardio, V.; Carreon, E. Physicochemical and sensory properties of corn tortillas made from nixtamalized corn flour fortified with spent soymilk residue (okara). J. Food Sci. 2002, 67 (8), 3149−3197. (7) Stanojevic, S.; Barac, M.; Pesic, M.; Jankovic, V.; Vucelic-Radovic, B. Bioactive proteins and energy value of okara as a byproduct in hydrothermal processing of soymilk. J. Agric. Food Chem. 2013, 61, 9210−9219. (8) Xie, M.; Huff, H.; Hsieh, F.; Mustapha, A. Puffing of okara/rice blends using a rice cake machine. J. Food Sci. 2008, 73 (8), 341−348. (9) Sinha, L. K.; Khare, S. K.; Jha, K. Studies on okara fortified biscuits: preparation and nutritional evaluation. J. Dairying, Foods Home Sci. 1995, 14, 91−94. (10) Barać, M.; Stanojević, S.; Pesic, M. Biologically active components of soy bean − a review. Acta Periodica Technol. 2005, 36, 155−169. (11) Ž ilić, M. S.; Barać, B. M.; Pešić, B. M.; Mladenović-Drinić, D. S.; Ignjatović-Micić, D. D.; Srebrić, M. Characterization of proteins from kernel of different soybean varieties. J. Sci. Food Agric. 2010, 91, 60− 67. (12) Torres-Penaranda, V. A.; Reitmeier, A. C.; Wilson, A. L.; Fehr, R. W.; Narvel, M. J. Sensory characteristics of soymilk and tofu made from lipoxygenase-free and normal soybeans. J. Food Sci. 1998, 63 (6), 1084−1087. (13) Wolf, W. J. Physical and chemical properties of soybean proteins. J. Am. Oil Chem. Soc. 1977, 54, 112−117. (14) Finkel, T.; Holbrook, N. J. Oxidants, oxidative stress and the biology of ageing (review). Nature 2000, 408, 239−247. (15) Moure, A.; Cruz, J. M.; Franco, D.; Dominguez, J. M.; Sineiro, J.; Dominiguez, H.; Nunez, M. J.; Parajo, J. C. Natural antioxidants from residual sources (review). Food Chem. 2001, 72, 145−171. (16) Amin, I.; Mukhrizah, O. Antioxidant capacity of methanolic and water extracts prepared from food-processing by-products. J. Sci. Food Agric. 2006, 86 (5), 778−784. (17) Yokomizo, A.; Takenaka, Y.; Takenaka, T. Antioxidative activity of peptides prepared from okara protein. Food Sci. Technol. Res. 2002, 8 (4), 357−359. (18) Mateos-Aparicio, I.; Mateos-Peinado, C.; Jiméz-Escrig, A.; Rupéz, P. Multifunctional antioxidant activity of polysaccharide fractions from the soybean byproduct okara. Carbohydr. Polym. 2010, 82, 245−250. (19) Jiménez-Escrig, A.; Alaiz, M.; Vioque, J.; Rupérez, P. Healthpromoting activities of ultra-filtered okara protein hydrolysates released by in vitro gastrointestinal digestion: identification of active peptide from soybean lipoxygenase. Eur. Food Res. Technol. 2010, 230, 655−663. (20) Jiménez-Escrig, A.; Tenorio, M. D.; Espinosa-Martos, I.; Rupérez, P. Health-promoting effects of a dietary fiber concentrate from the soybean byproduct okara in rats. J. Agric. Food Chem. 2008, 56, 7495−7501. (21) Zhou, K.; Yu, L. Antioxidant properties of bran extracts from Trego wheat grown at different locations. J. Agric. Food Chem. 2004, 52, 1112−1117. (22) Martinez-Tome, M.; Murcia, M.; Frega, N.; Ruggieri, S.; Jimenez, A. M.; Roses, F. Evaluation of antioxidant capacity of cereal brans. J. Agric. Food Chem. 2004, 52, 4690−4699. (23) Zhu, P. Y.; Fan, F. J.; Cheng, Q.; Li, T. L. Improvement of the antioxidant activity of chinese traditional fermented okara (Meitauza) using Bacillus subtilis B2. Food Control 2008, 19, 654−661. (24) Tan, S. T.; Nagendra, P. K.; Amin, I. Antioxidant capacity, phenolics and isoflavones in soybean by-products. Food Chem. 2010, 123, 583−589. (25) Adom, K.; Sorrells, M.; Liu, R. Phytochemical profiles and antioxidant activity of wheat varieties. J. Agric. Food Chem. 2003, 51, 7825−7834.

matter content, etc., which all influence the availability of these metals to plants.51 The fact that Cd and Co were not detected points out favorable conditions that were present in the soil where soybeans were grown. In summary, the investigated soybean genotypes all produced okara containing very similar concentrations of Lox with Lox-I activity only in traces. Therefore, this okara would not produce undesirable effects on the sensory characteristics of foods. No significant correlation between the content of Lox in soybean and in okara was registered, thus indicating that the content of this enzyme in okara depended more on the technological process than on soybean genotypes. There was very strong correlation between okara Lox content and activity. Registered total antioxidant activity (soluble and insoluble components) certainly contributed to the nutritional value of raw okara. The minerals of the soybeans mainly found their way into the soy milk. The value of total mineral content could not indicate a trend change in the content of particular mineral elements. Potassium was the main macroelement in okara, approximately 2 times higher than phosphorus. Calcium also appeared in significant amounts. Iron was the predominant microelement, whereas the manganese contents were very similar in all samples. The content of zinc significantly depended on the soybean genotype. As the presence of some potentially harmful microelements was registered, their contents should be controlled in products intended for consumption. Finally, we can summarize that HTC treatment opens the way to a more effective use of okara as a plant source of nutritionally valuable minerals and antioxidants that could be used as feed or in food fortification.

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AUTHOR INFORMATION

Corresponding Author

*(S.P.S.) Phone: +381 11 2615 315, ext. 358. Fax: +381 11 2199 711. E-mail: [email protected]. Funding

We are grateful to the Serbian Ministry of Education, Science and Technological Development for financial support (Project TR 31022) and EU FP7 Project Grant Agreement 316004 (REGPOT-AREA). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are indebted to the Maize Research Institute, Zemun Polje, Serbia, and the Institute of Field and Vegetable Crops, Novi Sad, Serbia, for providing soybean genotypes.

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REFERENCES

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