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Value-added subcritical water hydrolysate from rice bran and soybean meal
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Bioresource Technology 99 (2008) 6207–6213
Value-added subcritical water hydrolysate from rice bran and soybean meal Ketmanee Watchararuji a, Motonobu Goto b, Mitsuru Sasaki b, Artiwan Shotipruk a,* a
Department of Chemical Engineering, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand b Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 860-8555, Japan Received 28 August 2007; received in revised form 5 December 2007; accepted 6 December 2007 Available online 24 January 2008
Abstract New value-added product was derived from agricultural by-products: rice bran and soybean meal by means of subcritical water (SW) hydrolysis. The effect of temperature (200–220 °C), reaction time (10–30 min), raw material-to-water weight ratio (1:5 and 2:5), was determined on the yields of protein, total amino acids, and reducing sugars in the soluble products. The suitable hydrolysis time was 30 min and the proper weight ratio of the raw material-to-water was 1:5. The reaction temperature suitable for the production of protein and amino acids was 220 °C for raw and deoiled rice bran, 210 °C for raw soybean meal, and 200 °C for deoiled soybean meal. The products were also found to have antioxidant activity as tested by ABTS+ scavenging assay. In addition, sensory evaluation of milk added with the hydrolysis product of deoiled rice bran indicated the potential use of the product as a nutritious drink. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrothermal; Protein; Amino acids; Antioxidants; Hydrolysis
1. Introduction Rice and soybean are among the most important industrial food crops. Rice (Oryza sativa) is a cereal crop, which feeds more than half of the world’s population. About 617 million metric tons of rice is annually produced (IRRI, 2005) worldwide. In Thailand, 30.29 million tons of rice is produced annually (Office of Agricultural Economics, 2005), making Thailand the sixth largest rice producer in the world. Due to the consumers’ preference of white rice over brown rice, rice bran (approximately 8 wt% of milled rice) would be removed during the milling process (Shih et al., 1999). The removed rice bran is a rich source of proteins, oil, nutrients, and calories, and it is largely used as an animal feed. Recently, a number of studies were conducted on the utilization of rice bran as a nutrient source for fermentation processes (Gao et al., 2008; Sereewatthanawut et al., 2008); however, only a small proportion of rice bran *
Corresponding author. Tel.: +66 2 218 6868; fax: +66 2 218 6877. E-mail address: [email protected] (A. Shotipruk).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.12.021
is consumed by humans, and is usually in the form of rice bran oil. Similarly, soybean, Glycine max L. is considered an oil plant. It is a legume crop generally cultivated for oil production and protein source. For each ton of crude soybean oil, approximately 4.5 tons of soybean meals is produced, and 44 wt% of which is protein (Cheftel et al., 1985). In Thailand, approximately 0.22 million tons of soybean is produced annually (Office of Agricultural Economics, 2005). In addition, rice bran and soybean contain high amount of various antioxidant compounds that impart beneficial effects on human health. Those in rice bran include vitamin E (tocopherols and tocotrienols), vitamin C, anthocyanidins, isoflavones, beta-carotene, polyphenols and oryzanol. For soybean, the most powerful antioxidants are isoflavones, particulary daidzein and genistein, which have been reported for its ability to fight against breast cancer, prostate cancer, menopausal symptoms, and heart disease (Ruiz-Larrea et al., 1997). Several protein hydrolysates derived from rice bran and soybean meal which contain
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amino acids such as tyrosine, methionine, histidine, tryptophan, and proline have also been found to exhibit antioxidant activity (Saiga et al., 2003; Abdul-Hamid et al., 2007; Renuka Devi and Arumughan, 2007). It is, thus, the aim of this study to produce value-added products from rice bran and soybean meal. In our previous study, hydrolysis of deoiled rice bran by subcritical water was investigated for the production of protein and amino acids. At subcritical conditions (between 100 °C and its critical temperature (374.15 °C), water polarity decreases, thus making it a better solvent for the extraction of several organic bioactive compounds (Ibanez et al., 2003; Basile et al., 1998). In addition, at this condition, water ionization constant (KW) increases, causing water to readily ionize to hydrogen and hydroxide ions, thus increasing the degree of hydrolysis reaction. The results of the study show that the amounts of protein and amino acids produced by subcritical water hydrolysis were higher than those obtained by conventional alkali hydrolysis. In addition, the hydrolysis product was demonstrated to be suitable for use as nutrients for yeast growth (Sereewatthanawut et al., 2008). Another study reported on subcritical water hydrolysis of deoiled rice bran showed similar results on the protein yields and antioxidant activities of the soluble products, and the product emulsifying and emulsion-stabilizing activities were determined as a function of reaction temperature and time (Wiboonsirikul et al., 2007). In the present study, the application of subcritical water was extended to the production of valueadded products from other agricultural by-products including raw and deoiled rice bran as well as raw and deoiled soybean meal. The subcritical water hydrolysis of these agricultural by-products was first carried out to determine the suitable reaction conditions. Then, the antioxidant activity of the soluble product was analyzed and the possible application of the hydrolysis product as human food was evaluated. 2. Methods 2.1. Materials and chemicals Raw and deoiled rice bran was obtained from Thai Edible Oil Co., Ltd., Ayuthaya, Thailand. Raw and deoiled soybean meal (whose seed coat was removed) was obtained from Thanakorn Vegetable Oil Products Co., Ltd., Samutprakarn, Thailand. Bovine serum albumin (BSA) was purchased from Acros organics, USA. Dinitrosalicylic acid (DNS) from Fluka, Germany, and L-glutamic acid was purchased from Wako, Japan. D-Glucose and all other chemicals for Lowry’s and Ninhydrin’s assay were purchased from APS Fine Chem., NSW, Australia. 2.2. Subcritical water hydrolysis The raw material was suspended in distilled water at the raw material:water ratio of 1:5 or 2:5. This suspension was
charged into an 8.8-ml stainless steel (SUS-316) closed batch reactor (AKICO Co., Japan), which was then heated with an electric heater to the desired temperature (200– 220 °C). The pressure in the reactor was estimated to be between 101.35 kPa and 3.97 MPa based on saturated steam for the temperature range studied. After a desired reaction time (10–30 min), the reactor was immediately cooled to room temperature by immersion in a cool water bath (5 min). The liquid and solid contents in the reactor were collected and the remaining solid in the reactor was washed with 5 ml water. The residue bran was separated from the soluble product with a vacuum filter using a filter paper (Whatman No. 1) and weighed after drying in a vacuum oven at 65 °C. The soluble portion was assayed for the amount of protein, amino acids, reducing sugars, and antioxidant activity. All experiments were performed in duplicate. 2.3. Analysis of protein and amino acids The protein content of the soluble product was analyzed by Lowry’s assay (Lowry et al., 1951), using bovine serum albumin (BSA) as a standard. Amino acids content was analyzed by Ninhydrin assays using L-glutamic acid as a standard. Briefly, Ninhydrin reagent, containing 1 ml of 1% w/w ninhydrin solution, 2.4 ml of 55% v/v glycerol solution, 0.2 ml of 0.5 M citrate buffer, and 100 mg/ml manganese chloride, was added to 0.2 ml of the sample solution. The mixture was then shaken and boiled for 12 min, after which it was cooled in a water bath. Spectroscopic absorbance of the sample mixture was then measured at 570 nm. 2.4. Analysis of reducing sugar In addition, reducing sugars content was analyzed by dinitrosalicylic (DNS) colorimetric method (Miller, 1959), using D-glucose as a standard. For each of the 3 ml of the sample, 3 ml of DNS reagent was added. The mixture was then heated in boiling water for 5 min until the redbrown color developed. Then, 1 ml of 40% potassium sodium tartrate (Rochelle salt) solution was added to stabilize the color, after which the mixture was cooled to room temperature in a water bath. The absorbance was then measured with a spectrophotometer at 575 nm. 2.5. ABTS+ scavenging assay ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging assay was carried out following a modified method described by Re et al. (1999). The extract was diluted in series with water and each diluted solutions were added into ABTS+ solution (aqueous solution of 7 mM ABTS and 2.45 mM potassium persulfate having an absorbance of 0.70 ± 0.02 at 734 nm) with the volume ratio of 1:30 (sample solution:ABTS solution). The solutions were mixed using a vortex and the mix-
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PIð%Þ ¼ ½1 ðAt =Ar Þ 100 where At and Ar are the absorbance of test sample and that of the reference, respectively. 2.6. Color measurement The color of the product solution was measured using a Minolta Colorimeter CR-300 (Minolta Camera Co., Ltd. Japan) and the results were expressed in terms of 3-coordinate values (L, a*, and b* color space). L in the color space represents the luminance of the color (or value) on a numerical scale from 0 (black) to 100 (white). The color coordinates a* and b* represent the positions between red (+a*) and green (a*), and between yellow (+b*) and blue (b*). Prior to the color measurements, the instrument was calibrated against a standard white calibration plate to assure the reliability of the instrument. Two readings were taken from randomly selected locations and the mean values of L, a* and b* were recorded.
Table 1 Composition of raw materials Composition (%)
Raw rice bran
Deoiled rice bran
Raw soybean meal
Deoiled soybean meal
Moisture Oil Protein Fiber Non-protein
8.80 24.04 14.06 10.53 42.57
11.00 0.70 15.53 7.38 65.39
11.09 20.19 33.87 4.88 24.50
11.25 0.87 43.55 4.85 33.34
(42.57% vs. 24.50%). After deoiling, the protein content of both rice bran and soybean increased to 15.53% and 43.50%, respectively. 3.2. Color parameter The results of the color measurements are shown in Fig. 1 in which the soluble products gave the highest lightness values (L) at the hydrolysis time 10 min. The L values then decreased when the reaction time increased. Compared with the L values, the blueness (b*) values and redness values (+a*) were small, and were not significantly 10 min
20 min
30 min
40 30
L Value
tures were incubated in the dark at room temperature for 10 min, after which the absorbance was measured at 734 nm. For comparing the antioxidant activity of the extracts obtained at various conditions, concentration of sample producing 50% reduction of the radical absorbance (IC50) was used as an index. The IC50 values for various extracts were found from the plots of percent inhibition (PI) vs. the corresponding concentration of the sample. The values of PI were calculated using the following equation:
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20 10
2.7. Paired preference test
0 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
1.60
a* Value
Paired preference test is an initial consumer response test in which two sets of samples (a pair) are required for the comparison of the taste preference. In this study, two sets of paired preference tests were conducted. The first pair was to compare between milk control and the milk added with extraction product and the second pair was to compare between coffee control and the coffee added with extraction product. Forty testers were used and each tester was asked which of the pair of products they preferred and their responses were recorded and analysed.
1.20 0.80 0.40 0.00 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
3. Results and discussion 6.00
The compositions of rice bran and soybean both as raw and deoiled materials were analyzed by near infrared spectroscopy (NIR) and the results are shown in Table 1. The fat contents of the raw rice bran and raw soybean were high, and were approximately 24% and 20%, respectively. Protein content of the raw rice bran (14%) was much smaller than protein content of the soybean (34%), while the percentage of non-protein which includes ash and carbohydrates in the rice bran was higher than that of soybean
b* Value
3.1. Compositions of raw materials
4.00 2.00 0.00 -2.00 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
Fig. 1. The color parameter of solution products was measured using Colorimeter: (a) L value, (b) a* value and (c) b* value.
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different for hydrolysis samples obtained at various conditions. The dark color of the solution products was the result of Maillard reaction, a non-enzymatic browning process that depends on the reducing sugar content and amino acid contents, as well as the reaction temperature and reaction time. It is a chemical reaction between carbonyl group of a sugar and the nucleophilic amino group of an amino acid. This reaction usually requires the addition of heat and is highly time dependent. 3.3. Protein yields of soluble products The amounts of protein in the SW hydrolysis soluble products of raw and deoiled rice bran, and raw and deoiled soybean meal obtained at various reaction temperature and time were measured. The protein yields of raw rice bran and soybean meal were lower than those of deoiled materials possibly because the hydrophobic oil content within the materials made them less accessible to water. For rice bran, protein generally increased with increasing temperature, with the highest yields of 106.02 ± 5.01 mg/g raw rice bran and 130.17 ± 2.48 mg/g deoiled rice bran obtained from hydrolysis at 220 °C for 30 min. Based on the amount in the original bran reported by NIR in Table 1, these account for the protein recovery of 75% and 84%, respectively. These results suggested that the majority of protein in the original bran could be recovered in the soluble product. For soybean meal, the highest protein yield for raw soybean (165.72 ± 2.90 mg/g raw soybean) was obtained by hydrolysis at 210 °C for 30 min. The highest protein yield for deoiled soybean (204.64 ± 5.38 mg/g deoiled soybean) was obtained by hydrolysis at 200 °C for 30 min. The higher protein yield in soybean products compared with the rice bran products were due to the higher protein content in the raw materials as shown in Table 1. However, higher protein recovery was obtained for the rice bran product than the soybean products, in which only approximately 50% recovery was obtained. In general, at ambient temperature, protein normally has low solubility in water due to strong aggregation through hydrophobic interactions. At higher temperatures, however, the solubility in water increased. Moreover, the increase in protein yield at elevated temperature was due to the increased hydrolysis activity caused by the increase in water ionization constant (also called dissociation constant or ion product constant or Kw). Kw increases from 1 1014 at ambient temperature to 7 1012 at 220 °C, and thus the concentration of hydronium and hydroxide ions, which is equal to the square-root of the Kw, increases. In the presence of hydronium and hydroxide ions, peptide bonds are broken down into smaller molecules of soluble protein or amino acids, which could further degrade to low molecular weight carboxylic acids such as formic acids, acetic acids, propionic acids, etc. (Quitain et al., 2005). However, for both raw and deoiled soybean meal, the protein yield was found to decrease when the temperature increased from 200 °C to 220 °C for the reaction time of
20 min and 30 min, which was probably due to the decomposition of soluble protein into amino acids and other organic acids. Higher protein yield could also be obtained by increasing the hydrolysis time. Nevertheless, longer time did not always give the positive results. The effect of reaction time was less important when it was increased from 20 min to 30 min, for most of the samples tested. In particular, for the hydrolysis of deoiled soybean meal at hydrolysis temperature of 220 °C, increasing the hydrolysis time from 20 min to 30 min decreased the protein yields. This was possibly because the extent of protein decomposition to amino acids or organic acids was higher than the extent of protein production. 3.4. Total amino acid yields in soluble products The results for the amount of amino acids in soluble products indicated that the amino acid yields of the nondeoiled materials were lower than those from the deoiled materials, which was in agreement with the results of protein yields. The highest amino acids yield for raw rice bran (7.47 ± 0.14 mg/g raw rice bran) were obtained at 210 °C for 30 min, while the highest yield for deoiled rice bran was 9.74 ± 0.08 mg/g deoiled rice bran, which was obtained at 220 °C for 20 min. This relatively low yield indicated that the extent of amino acid decomposition to smaller molecules of organic acids or other products was higher than the amino acid production. For rice bran, the temperature and reaction time did not have significant effects on the amount of total amino acid yields. For soybean meals, the amino acid yields increased when the hydrolysis time increased for the hydrolysis at lower temperatures. At a higher temperature of 220 °C, the amount of amino acids in solution was not affected greatly by the reaction time. At this condition, the extent of amino acid production was comparable to the extent of amino acid decomposition into smaller organic acids. 3.5. Reducing sugars yields in soluble products When carbohydrate reacts with hydronium and hydroxide ions, reducing sugars are produced. The results in Fig. 2 show that, for raw and deoiled rice bran, the reducing sugar content in the soluble products increased with increasing temperature and reaction time, except for the hydrolysis product obtained at 220 °C and 30 min, whose reducing sugar content decreased. This result again indicated that at this condition, the decomposition of reducing sugar to other products was favored over its production. Similar decrease in reducing sugar as a result of increasing hydrolysis time was also observed for soybean meal. The reducing sugar contents of the soybean products were lower than those of the rice bran products with the maximum of 47 mg/g soybean meal obtained for hydrolysis at 200 °C for 20 min. This result agreed with the soybean composition shown in Table 1, which indicated lower
K. Watchararuji et al. / Bioresource Technology 99 (2008) 6207–6213 140 200 °C Raw
The weight of the rice bran hydrolyzed products that are not protein, amino acids and reducing sugar
210 °C Raw
The sum of protein, amino acids and reducing sugar yields
220 °C Raw
100
200 °C Deoiled
1000
210 °C Deoiled
80 60 40 20 0 0
200 °C
900
220 °C Deoiled
Extraction yield (mg/g deoiled rice bran)
Reducing sugar yield (mg/g rice bran)
120
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10
20
30
Time (min)
210 °C
220 °C
800 700 600 500 400 300 200 100
Fig. 2. Reducing sugar yields after hydrolysis of raw and deoiled rice bran at different temperature and time.
non-protein (mostly carbohydrate) content of soybean meal compared with rice bran. 3.6. Weight of solid residue Consideration of the weight of the residue after hydrolysis allowed the determination of the amount of the original materials that was converted into the extraction product. For all experiments, the residue was separated from the soluble product with a vacuum filter using a filter paper and weighed after drying at 65 °C in a vacuum oven. The weights of all sample residues generally decreased with reaction time and temperature. This trend agreed with the amount of protein, amino acid, and reducing sugar yields obtained in the soluble product which increased with reaction time, and to a certain extent, with temperature. The weight of the raw rice bran and soybean residue was higher than those of the residue of deoiled rice bran and soybean, indicating that smaller amount of raw rice bran and soybean could be converted into the soluble products, which also agreed with the results observed for extraction yield. It should be noted, however, that the original materials were not all converted into proteins, amino acids, and reducing sugars as the amount of the raw materials hydrolyzed (difference between the weight of original material and the weight of residue) was higher than the weight of protein, amino acids, and reducing sugar contents measured in the soluble products. Taking deoiled rice bran as an example, as shown in Fig. 3, the amount of rice bran hydrolyzed was 2–3 times as high as the sum of the amount of protein, amino acids, and reducing sugars in the soluble
0 10
20
30
10
20
30
10
20
30
min
Fig. 3. Extraction yield after subcritical water hydrolysis deoiled bran at different temperature and time.
product. Consider, for example, the experiment at 220 °C and 30 min, in which 33% of the starting rice bran remained (330 mg rice bran residue/g deoiled rice bran), which suggested that approximately 67% of deoiled rice bran (670 mg/g rice bran) was hydrolyzed. Since the measured contents (proteins, amino acids, and reducing sugars) of deoiled rice bran soluble product was only approximately 22% (220.125 mg/g deoiled bran), thus the remaining fraction of 45% (the difference between 67% and 22%) was the unmeasured product, which could be smaller organic acid hydrolysis products of non-protein components in rice bran. 3.7. Suitable conditions The yields of protein and amino acids were important factors for selecting suitable hydrolysis condition. The highest protein and amino acid yields were obtained mostly after 30 min hydrolysis time, the yields of protein and amino acids in the soluble products obtained after 30 min hydrolysis at various temperatures are summarized in Table 2. Note that when comparable yields were resulted from two different conditions, the lower temperature condition would be more desirable due to lower energy requirement. The bold-typed numbers in the table above represents the highest protein and amino acid yields for each raw material. Since temperature had a much smaller effect on the yield of amino acids, the most suitable temperature
Table 2 Protein and amino acid contents after hydrolysis of raw materials at different temperatures for 30 min Temperature(°C)
200 210 220
Raw rice bran
Deoiled rice bran
Raw soybean
Protein (mg)
Amino acid (mg)
Protein (mg)
Amino acid (mg)
Protein (mg)
Amino acid (mg)
Deoiled soybean Protein (mg)
Amino acid (mg)
97.14 100.50 106.02
7.20 7.47 7.09
128.60 128.79 130.17
9.59 9.27 9.14
162.00 165.72 151.37
17.96 18.62 17.23
204.64 188.22 142.96
19.65 20.67 18.47
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Table 3 Suitable hydrolysis conditions (the time was 30 min)
Raw rice bran Deoiled rice bran Raw soybean Deoiled soybean
Temperature (°C)
Protein yield (mg/g raw material)
220 220/210 210/200 200
0.74
˚ ˚
200 C 220 C
0.70
Amino acid yield (mg/g raw material)
106.02 130.17/128.79 165.72/162.00 204.64
7.09 9.14/9.27 18.62/17.96 19.65
0.60
IC50 (mg/ml)
Raw materials
0.80
0.49 0.43
0.50
0.30
0.40 0.22
0.30
0.23
0.19
0.20
20 min
30 min
3.8. Effect of the ratio of raw material-to-water on extraction yields The effect of the ratio of raw material-to-water on extraction yield was determined by comparing the product yields obtained from two different ratios of raw materialto-water: 1:5 and 2:5 and the results were found to be similar for all conditions. As an example, only the results from the hydrolysis experiment at 210 °C and 30 min are shown in Table 4, which shows that the yields of protein, amino acids, and reducing sugar decreased when the ratio raw of material-to-water was increased from 1:5 to 2:5. This was mainly because the too high amount of raw material caused an increase in the density and viscosity, resulting in poor mixing of raw material and water, thus mass transfer decreased and the accessibility of water to particles of raw materials became difficult. The ratio raw material-towater of 1:5 was therefore more suitable. 3.9. Antioxidant activity In this study, the antioxidant activity of the soluble products obtained by SW hydrolysis was evaluated with ABTS+ scavenging assay. Antioxidant activity was represented by IC50 index, which is the concentration of sample producing 50% reduction of the radical absorbance. This means that the higher IC50 of the product, the lower antioxidant activity. The antioxidant activity was measured for the hydrolysis temperature of 200 °C and 220 °C, and the hydrolysis time at 20 min and 30 min. These conditions were found to be the most suitable hydrolysis conditions and were therefore selected for the antioxidant test. Table 4 Effect of the ratio of raw material-to-water on extraction yields Material:water ratio
Rice bran Raw
Proteins AA Reducing sugars
Soybean Deoiled
Raw
1:5
2:5
1:5
2:5
1:5
2:5
1:5
Deoiled 2:5
101 7 62
73 6 61
129 9 111
95 5 89
166 19 35
123 15 21
188 21 38
136 17 24
0.28 0.24 0.20
0.20
0.19
0.19
0.20
0.19
0.10 0.00 Raw rice bran
for the production of each product could be drawn from the condition that gave the highest protein yield. From these results, the most suitable conditions are summarized in Table 3.
0.28
20 min
30 min
Deoiled rice bran
20 min
30 min
Raw soybean
20 min
30 min
Deoiled soybean
Raw material varieties
Fig. 4. Antioxidant activity (IC50) of the soluble products at hydrolysis times of 20 min and 30 min and temperatures of 200 °C and 220 °C.
The result in Fig. 4 indicated that with the hydrolysis time of 20 min, antioxidant activity increased as temperature increased from 200 °C to 220 °C for all materials. For the reaction time of 30 min, however, the antioxidant activity only increased slightly or stayed constant with the increase in temperature. The increase in water temperature does not only increase the ion product, which causes hydrolysis reaction, but it also causes the breakdown of hydrogen bonding between the water molecules. When the H-bonds break down, several antioxidative organic compounds within the rice bran and soybean samples become better able to dissolve in water, thus the soluble products exhibited higher antioxidant activity than those obtained at lower temperature. At long exposure time or at high temperature, however, some antioxidative compounds might be degraded, thus the activity might decrease (Clifford, 2000). 3.10. Paired preference test Paired preference test is an initial consumer response to the taste of the product in which a pair of samples is required to compare the taste preference. Here, only the product of deoiled rice bran was tested. In the samples in which the rice bran products were added, 2 g of dried products were added to 600 ml of the control samples. This is equivalent to the concentrations of 1.62 mg of protein/ml and 0.12 mg of amino acids/ml in the test samples. In this study, two pairs of samples were compared; the first pair was that of milk control and milk added with deoiled rice bran hydrolysis product and the second pair was that of coffee control and the coffee added with extraction product. Of the 40 testers, 14 and 26 testers preferred milk control and milk added with deoiled rice bran hydrolysis product, while 25 and 15 testers preferred coffee control and coffee added with extraction product, respectively. From the statistical analysis based on the 40 testers, the minimum correct answers necessary to establish significant differentiation at a 5% probability level was 26. From these results, it can be concluded that the preference for the milk added with hydrolysis product was significantly higher than that for the milk control (P 6 0.05), while the preferences for the coffee added with extraction product and the
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coffee control showed no significant differences (P > 0.05). It should be noted that the pair preference test is only a basic test conducted on testers with minimal training whose age range between 22 and 36, who were asked to select one of the two choices they preferred. The descriptive evaluation of the product was not the main result used in the analysis as these testers were inexperienced. Further detailed sensory evaluation such as descriptive analysis conducted on trained testers would be required. Furthermore, the product preference could be dependent on the age or gender of the testers, as well as on the serving size of the product, and thus further study is needed to determine the effect of these factors. Nevertheless, the results of the pair preference test conducted in this study proved the potential application of the SW hydrolysis products as functional food for humans.
4. Conclusions This study demonstrated that subcritical water could be used to potentially hydrolyze raw and deoiled rice bran, and raw and deoiled soybean meal into more valuable proteins, amino acids and reducing sugars. The suitable conditions for protein and amino acid production from rice bran (raw and deoiled) and soybean meal (raw and deoiled) by subcritical water hydrolysis were 1:5 raw material-to-water weight ratio at 30 min and hydrolysis temperature of 220 °C for raw and deoiled rice bran and 210 °C and 200 °C for raw and deoiled soybean meal, respectively. Apart from protein, amino acids and reducing sugars, active compounds that exhibit antioxidant activity were also extracted by subcritical water, and the products were proven to have potential application as human foods.
Acknowledgements Financial supports from Thailand Research Fund and Chulalongkorn University Graduate Program are appreciated. Mr. Pravit Santiwattanan from Thai Edible Oil Co., Ltd. is greatly acknowledged for the provision of raw and deoiled rice bran and Thanakorn Vegetable Oil Products Co., Ltd., Samuthprakarn, for the supplies of soybean meals.
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References Abdul-Hamid, A., Raja Sulaiman, R.R., Osman, A., Saari, N., 2007. Preliminary study of the chemical composition of rice milling fractions stabilized by microwave heating. J. Food. Compost. Anal. 20, 627– 637. Basile, A., Jimenez-Carmona, M.M., Clifford, A.A., 1998. Extraction of rosemary by superheated water. J. Agric. Food. Chem. 46, 5205–5209. Cheftel, J.C., Cuq, J.L., Lorient, D., 1985. Amino acids, peptides, and proteins. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekker, New York, pp. 245–369. Clifford, M.N., 2000. Anthocyanins-nature, occurrence and dietary burden. J. Sci. Food Agric. 80 (7), 1063–1072. Gao, M., Kaneko, M., Hirata, M., Toorisaka, E., Hano, T., 2008. Utilization of rice bran as nutrient source for fermentative lactic acid production. Bioresource Technol. 99, 3659–3664. Ibanez, E., Kubatova, A., Senorans, F.J., Cavero, S., Reglero, G., Hawthrone, S.B., 2003. Subcritical water extraction of antioxidant compounds from rosemary plants. J. Agric. Food. Chem. 51, 375–382. International Rice Research Institute (IRRI), 2005. World Rice Statistics.. Lowry, O.H., Rosebrough, N.J., Farr, A.W., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem 31, 426. Office of Agricultural Economics, 2005. Government of Thailand, Bangkok. . Quitain, A.T., Faisal, M., Kang, K., Daimon, H., 2005. Low-molecularweight carboxylic acids produced from hydrothermal treatment of organic wastes. J. Hazard. Mater. B93, 209–220. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice, E.C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 26, 1231–1237. Renuka Devi, R., Arumughan, C., 2007. Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment. Bioresource Technol. 98 (16), 3037–3043. Ruiz-Larrea, M., Mohan, A., Paganga, G., Miller, N., Bolwell, G., 1997. Antioxidant activity of phytoestrogenic isoflavones. Free Radical Res. 26, 63–70. Saiga, A., Tanabe, S., Nishimura, T., 2003. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J. Agric. Food. Chem. 51, 3661–3667. Sereewatthanawut, I., Prapintip, S., Watchiraruji, K., Goto, M., Sasaki, M., Shotipruk, A., 2008. Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis. J. Bioresource Technol. 99 (3), 555–561. Shih, F.F., Champagne, E.T., Daigle, K., Zarins, Z., 1999. Use of enzymes in the processing of protein products from rice bran and rice flour. Nahrung 43 (1), 14–18. Wiboonsirikul, J., Hata, S., Tsuno, T., Kimura, Y., Adchi, S., 2007. Production of functional substances from black rice bran by its treatment in subcritical water. LWT 40, 1732–1740.
Bioresource Technology 99 (2008) 6207–6213
Value-added subcritical water hydrolysate from rice bran and soybean meal Ketmanee Watchararuji a, Motonobu Goto b, Mitsuru Sasaki b, Artiwan Shotipruk a,* a
Department of Chemical Engineering, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand b Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto 860-8555, Japan Received 28 August 2007; received in revised form 5 December 2007; accepted 6 December 2007 Available online 24 January 2008
Abstract New value-added product was derived from agricultural by-products: rice bran and soybean meal by means of subcritical water (SW) hydrolysis. The effect of temperature (200–220 °C), reaction time (10–30 min), raw material-to-water weight ratio (1:5 and 2:5), was determined on the yields of protein, total amino acids, and reducing sugars in the soluble products. The suitable hydrolysis time was 30 min and the proper weight ratio of the raw material-to-water was 1:5. The reaction temperature suitable for the production of protein and amino acids was 220 °C for raw and deoiled rice bran, 210 °C for raw soybean meal, and 200 °C for deoiled soybean meal. The products were also found to have antioxidant activity as tested by ABTS+ scavenging assay. In addition, sensory evaluation of milk added with the hydrolysis product of deoiled rice bran indicated the potential use of the product as a nutritious drink. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Hydrothermal; Protein; Amino acids; Antioxidants; Hydrolysis
1. Introduction Rice and soybean are among the most important industrial food crops. Rice (Oryza sativa) is a cereal crop, which feeds more than half of the world’s population. About 617 million metric tons of rice is annually produced (IRRI, 2005) worldwide. In Thailand, 30.29 million tons of rice is produced annually (Office of Agricultural Economics, 2005), making Thailand the sixth largest rice producer in the world. Due to the consumers’ preference of white rice over brown rice, rice bran (approximately 8 wt% of milled rice) would be removed during the milling process (Shih et al., 1999). The removed rice bran is a rich source of proteins, oil, nutrients, and calories, and it is largely used as an animal feed. Recently, a number of studies were conducted on the utilization of rice bran as a nutrient source for fermentation processes (Gao et al., 2008; Sereewatthanawut et al., 2008); however, only a small proportion of rice bran *
Corresponding author. Tel.: +66 2 218 6868; fax: +66 2 218 6877. E-mail address: [email protected] (A. Shotipruk).
0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.12.021
is consumed by humans, and is usually in the form of rice bran oil. Similarly, soybean, Glycine max L. is considered an oil plant. It is a legume crop generally cultivated for oil production and protein source. For each ton of crude soybean oil, approximately 4.5 tons of soybean meals is produced, and 44 wt% of which is protein (Cheftel et al., 1985). In Thailand, approximately 0.22 million tons of soybean is produced annually (Office of Agricultural Economics, 2005). In addition, rice bran and soybean contain high amount of various antioxidant compounds that impart beneficial effects on human health. Those in rice bran include vitamin E (tocopherols and tocotrienols), vitamin C, anthocyanidins, isoflavones, beta-carotene, polyphenols and oryzanol. For soybean, the most powerful antioxidants are isoflavones, particulary daidzein and genistein, which have been reported for its ability to fight against breast cancer, prostate cancer, menopausal symptoms, and heart disease (Ruiz-Larrea et al., 1997). Several protein hydrolysates derived from rice bran and soybean meal which contain
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amino acids such as tyrosine, methionine, histidine, tryptophan, and proline have also been found to exhibit antioxidant activity (Saiga et al., 2003; Abdul-Hamid et al., 2007; Renuka Devi and Arumughan, 2007). It is, thus, the aim of this study to produce value-added products from rice bran and soybean meal. In our previous study, hydrolysis of deoiled rice bran by subcritical water was investigated for the production of protein and amino acids. At subcritical conditions (between 100 °C and its critical temperature (374.15 °C), water polarity decreases, thus making it a better solvent for the extraction of several organic bioactive compounds (Ibanez et al., 2003; Basile et al., 1998). In addition, at this condition, water ionization constant (KW) increases, causing water to readily ionize to hydrogen and hydroxide ions, thus increasing the degree of hydrolysis reaction. The results of the study show that the amounts of protein and amino acids produced by subcritical water hydrolysis were higher than those obtained by conventional alkali hydrolysis. In addition, the hydrolysis product was demonstrated to be suitable for use as nutrients for yeast growth (Sereewatthanawut et al., 2008). Another study reported on subcritical water hydrolysis of deoiled rice bran showed similar results on the protein yields and antioxidant activities of the soluble products, and the product emulsifying and emulsion-stabilizing activities were determined as a function of reaction temperature and time (Wiboonsirikul et al., 2007). In the present study, the application of subcritical water was extended to the production of valueadded products from other agricultural by-products including raw and deoiled rice bran as well as raw and deoiled soybean meal. The subcritical water hydrolysis of these agricultural by-products was first carried out to determine the suitable reaction conditions. Then, the antioxidant activity of the soluble product was analyzed and the possible application of the hydrolysis product as human food was evaluated. 2. Methods 2.1. Materials and chemicals Raw and deoiled rice bran was obtained from Thai Edible Oil Co., Ltd., Ayuthaya, Thailand. Raw and deoiled soybean meal (whose seed coat was removed) was obtained from Thanakorn Vegetable Oil Products Co., Ltd., Samutprakarn, Thailand. Bovine serum albumin (BSA) was purchased from Acros organics, USA. Dinitrosalicylic acid (DNS) from Fluka, Germany, and L-glutamic acid was purchased from Wako, Japan. D-Glucose and all other chemicals for Lowry’s and Ninhydrin’s assay were purchased from APS Fine Chem., NSW, Australia. 2.2. Subcritical water hydrolysis The raw material was suspended in distilled water at the raw material:water ratio of 1:5 or 2:5. This suspension was
charged into an 8.8-ml stainless steel (SUS-316) closed batch reactor (AKICO Co., Japan), which was then heated with an electric heater to the desired temperature (200– 220 °C). The pressure in the reactor was estimated to be between 101.35 kPa and 3.97 MPa based on saturated steam for the temperature range studied. After a desired reaction time (10–30 min), the reactor was immediately cooled to room temperature by immersion in a cool water bath (5 min). The liquid and solid contents in the reactor were collected and the remaining solid in the reactor was washed with 5 ml water. The residue bran was separated from the soluble product with a vacuum filter using a filter paper (Whatman No. 1) and weighed after drying in a vacuum oven at 65 °C. The soluble portion was assayed for the amount of protein, amino acids, reducing sugars, and antioxidant activity. All experiments were performed in duplicate. 2.3. Analysis of protein and amino acids The protein content of the soluble product was analyzed by Lowry’s assay (Lowry et al., 1951), using bovine serum albumin (BSA) as a standard. Amino acids content was analyzed by Ninhydrin assays using L-glutamic acid as a standard. Briefly, Ninhydrin reagent, containing 1 ml of 1% w/w ninhydrin solution, 2.4 ml of 55% v/v glycerol solution, 0.2 ml of 0.5 M citrate buffer, and 100 mg/ml manganese chloride, was added to 0.2 ml of the sample solution. The mixture was then shaken and boiled for 12 min, after which it was cooled in a water bath. Spectroscopic absorbance of the sample mixture was then measured at 570 nm. 2.4. Analysis of reducing sugar In addition, reducing sugars content was analyzed by dinitrosalicylic (DNS) colorimetric method (Miller, 1959), using D-glucose as a standard. For each of the 3 ml of the sample, 3 ml of DNS reagent was added. The mixture was then heated in boiling water for 5 min until the redbrown color developed. Then, 1 ml of 40% potassium sodium tartrate (Rochelle salt) solution was added to stabilize the color, after which the mixture was cooled to room temperature in a water bath. The absorbance was then measured with a spectrophotometer at 575 nm. 2.5. ABTS+ scavenging assay ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging assay was carried out following a modified method described by Re et al. (1999). The extract was diluted in series with water and each diluted solutions were added into ABTS+ solution (aqueous solution of 7 mM ABTS and 2.45 mM potassium persulfate having an absorbance of 0.70 ± 0.02 at 734 nm) with the volume ratio of 1:30 (sample solution:ABTS solution). The solutions were mixed using a vortex and the mix-
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PIð%Þ ¼ ½1 ðAt =Ar Þ 100 where At and Ar are the absorbance of test sample and that of the reference, respectively. 2.6. Color measurement The color of the product solution was measured using a Minolta Colorimeter CR-300 (Minolta Camera Co., Ltd. Japan) and the results were expressed in terms of 3-coordinate values (L, a*, and b* color space). L in the color space represents the luminance of the color (or value) on a numerical scale from 0 (black) to 100 (white). The color coordinates a* and b* represent the positions between red (+a*) and green (a*), and between yellow (+b*) and blue (b*). Prior to the color measurements, the instrument was calibrated against a standard white calibration plate to assure the reliability of the instrument. Two readings were taken from randomly selected locations and the mean values of L, a* and b* were recorded.
Table 1 Composition of raw materials Composition (%)
Raw rice bran
Deoiled rice bran
Raw soybean meal
Deoiled soybean meal
Moisture Oil Protein Fiber Non-protein
8.80 24.04 14.06 10.53 42.57
11.00 0.70 15.53 7.38 65.39
11.09 20.19 33.87 4.88 24.50
11.25 0.87 43.55 4.85 33.34
(42.57% vs. 24.50%). After deoiling, the protein content of both rice bran and soybean increased to 15.53% and 43.50%, respectively. 3.2. Color parameter The results of the color measurements are shown in Fig. 1 in which the soluble products gave the highest lightness values (L) at the hydrolysis time 10 min. The L values then decreased when the reaction time increased. Compared with the L values, the blueness (b*) values and redness values (+a*) were small, and were not significantly 10 min
20 min
30 min
40 30
L Value
tures were incubated in the dark at room temperature for 10 min, after which the absorbance was measured at 734 nm. For comparing the antioxidant activity of the extracts obtained at various conditions, concentration of sample producing 50% reduction of the radical absorbance (IC50) was used as an index. The IC50 values for various extracts were found from the plots of percent inhibition (PI) vs. the corresponding concentration of the sample. The values of PI were calculated using the following equation:
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20 10
2.7. Paired preference test
0 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
1.60
a* Value
Paired preference test is an initial consumer response test in which two sets of samples (a pair) are required for the comparison of the taste preference. In this study, two sets of paired preference tests were conducted. The first pair was to compare between milk control and the milk added with extraction product and the second pair was to compare between coffee control and the coffee added with extraction product. Forty testers were used and each tester was asked which of the pair of products they preferred and their responses were recorded and analysed.
1.20 0.80 0.40 0.00 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
3. Results and discussion 6.00
The compositions of rice bran and soybean both as raw and deoiled materials were analyzed by near infrared spectroscopy (NIR) and the results are shown in Table 1. The fat contents of the raw rice bran and raw soybean were high, and were approximately 24% and 20%, respectively. Protein content of the raw rice bran (14%) was much smaller than protein content of the soybean (34%), while the percentage of non-protein which includes ash and carbohydrates in the rice bran was higher than that of soybean
b* Value
3.1. Compositions of raw materials
4.00 2.00 0.00 -2.00 200
210
220
Raw rice bran
200
210
220
Deoiled rice bran
200
210
220
Raw soybean
200
210
220
(˚C)
Deoiled soybean
Fig. 1. The color parameter of solution products was measured using Colorimeter: (a) L value, (b) a* value and (c) b* value.
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different for hydrolysis samples obtained at various conditions. The dark color of the solution products was the result of Maillard reaction, a non-enzymatic browning process that depends on the reducing sugar content and amino acid contents, as well as the reaction temperature and reaction time. It is a chemical reaction between carbonyl group of a sugar and the nucleophilic amino group of an amino acid. This reaction usually requires the addition of heat and is highly time dependent. 3.3. Protein yields of soluble products The amounts of protein in the SW hydrolysis soluble products of raw and deoiled rice bran, and raw and deoiled soybean meal obtained at various reaction temperature and time were measured. The protein yields of raw rice bran and soybean meal were lower than those of deoiled materials possibly because the hydrophobic oil content within the materials made them less accessible to water. For rice bran, protein generally increased with increasing temperature, with the highest yields of 106.02 ± 5.01 mg/g raw rice bran and 130.17 ± 2.48 mg/g deoiled rice bran obtained from hydrolysis at 220 °C for 30 min. Based on the amount in the original bran reported by NIR in Table 1, these account for the protein recovery of 75% and 84%, respectively. These results suggested that the majority of protein in the original bran could be recovered in the soluble product. For soybean meal, the highest protein yield for raw soybean (165.72 ± 2.90 mg/g raw soybean) was obtained by hydrolysis at 210 °C for 30 min. The highest protein yield for deoiled soybean (204.64 ± 5.38 mg/g deoiled soybean) was obtained by hydrolysis at 200 °C for 30 min. The higher protein yield in soybean products compared with the rice bran products were due to the higher protein content in the raw materials as shown in Table 1. However, higher protein recovery was obtained for the rice bran product than the soybean products, in which only approximately 50% recovery was obtained. In general, at ambient temperature, protein normally has low solubility in water due to strong aggregation through hydrophobic interactions. At higher temperatures, however, the solubility in water increased. Moreover, the increase in protein yield at elevated temperature was due to the increased hydrolysis activity caused by the increase in water ionization constant (also called dissociation constant or ion product constant or Kw). Kw increases from 1 1014 at ambient temperature to 7 1012 at 220 °C, and thus the concentration of hydronium and hydroxide ions, which is equal to the square-root of the Kw, increases. In the presence of hydronium and hydroxide ions, peptide bonds are broken down into smaller molecules of soluble protein or amino acids, which could further degrade to low molecular weight carboxylic acids such as formic acids, acetic acids, propionic acids, etc. (Quitain et al., 2005). However, for both raw and deoiled soybean meal, the protein yield was found to decrease when the temperature increased from 200 °C to 220 °C for the reaction time of
20 min and 30 min, which was probably due to the decomposition of soluble protein into amino acids and other organic acids. Higher protein yield could also be obtained by increasing the hydrolysis time. Nevertheless, longer time did not always give the positive results. The effect of reaction time was less important when it was increased from 20 min to 30 min, for most of the samples tested. In particular, for the hydrolysis of deoiled soybean meal at hydrolysis temperature of 220 °C, increasing the hydrolysis time from 20 min to 30 min decreased the protein yields. This was possibly because the extent of protein decomposition to amino acids or organic acids was higher than the extent of protein production. 3.4. Total amino acid yields in soluble products The results for the amount of amino acids in soluble products indicated that the amino acid yields of the nondeoiled materials were lower than those from the deoiled materials, which was in agreement with the results of protein yields. The highest amino acids yield for raw rice bran (7.47 ± 0.14 mg/g raw rice bran) were obtained at 210 °C for 30 min, while the highest yield for deoiled rice bran was 9.74 ± 0.08 mg/g deoiled rice bran, which was obtained at 220 °C for 20 min. This relatively low yield indicated that the extent of amino acid decomposition to smaller molecules of organic acids or other products was higher than the amino acid production. For rice bran, the temperature and reaction time did not have significant effects on the amount of total amino acid yields. For soybean meals, the amino acid yields increased when the hydrolysis time increased for the hydrolysis at lower temperatures. At a higher temperature of 220 °C, the amount of amino acids in solution was not affected greatly by the reaction time. At this condition, the extent of amino acid production was comparable to the extent of amino acid decomposition into smaller organic acids. 3.5. Reducing sugars yields in soluble products When carbohydrate reacts with hydronium and hydroxide ions, reducing sugars are produced. The results in Fig. 2 show that, for raw and deoiled rice bran, the reducing sugar content in the soluble products increased with increasing temperature and reaction time, except for the hydrolysis product obtained at 220 °C and 30 min, whose reducing sugar content decreased. This result again indicated that at this condition, the decomposition of reducing sugar to other products was favored over its production. Similar decrease in reducing sugar as a result of increasing hydrolysis time was also observed for soybean meal. The reducing sugar contents of the soybean products were lower than those of the rice bran products with the maximum of 47 mg/g soybean meal obtained for hydrolysis at 200 °C for 20 min. This result agreed with the soybean composition shown in Table 1, which indicated lower
K. Watchararuji et al. / Bioresource Technology 99 (2008) 6207–6213 140 200 °C Raw
The weight of the rice bran hydrolyzed products that are not protein, amino acids and reducing sugar
210 °C Raw
The sum of protein, amino acids and reducing sugar yields
220 °C Raw
100
200 °C Deoiled
1000
210 °C Deoiled
80 60 40 20 0 0
200 °C
900
220 °C Deoiled
Extraction yield (mg/g deoiled rice bran)
Reducing sugar yield (mg/g rice bran)
120
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10
20
30
Time (min)
210 °C
220 °C
800 700 600 500 400 300 200 100
Fig. 2. Reducing sugar yields after hydrolysis of raw and deoiled rice bran at different temperature and time.
non-protein (mostly carbohydrate) content of soybean meal compared with rice bran. 3.6. Weight of solid residue Consideration of the weight of the residue after hydrolysis allowed the determination of the amount of the original materials that was converted into the extraction product. For all experiments, the residue was separated from the soluble product with a vacuum filter using a filter paper and weighed after drying at 65 °C in a vacuum oven. The weights of all sample residues generally decreased with reaction time and temperature. This trend agreed with the amount of protein, amino acid, and reducing sugar yields obtained in the soluble product which increased with reaction time, and to a certain extent, with temperature. The weight of the raw rice bran and soybean residue was higher than those of the residue of deoiled rice bran and soybean, indicating that smaller amount of raw rice bran and soybean could be converted into the soluble products, which also agreed with the results observed for extraction yield. It should be noted, however, that the original materials were not all converted into proteins, amino acids, and reducing sugars as the amount of the raw materials hydrolyzed (difference between the weight of original material and the weight of residue) was higher than the weight of protein, amino acids, and reducing sugar contents measured in the soluble products. Taking deoiled rice bran as an example, as shown in Fig. 3, the amount of rice bran hydrolyzed was 2–3 times as high as the sum of the amount of protein, amino acids, and reducing sugars in the soluble
0 10
20
30
10
20
30
10
20
30
min
Fig. 3. Extraction yield after subcritical water hydrolysis deoiled bran at different temperature and time.
product. Consider, for example, the experiment at 220 °C and 30 min, in which 33% of the starting rice bran remained (330 mg rice bran residue/g deoiled rice bran), which suggested that approximately 67% of deoiled rice bran (670 mg/g rice bran) was hydrolyzed. Since the measured contents (proteins, amino acids, and reducing sugars) of deoiled rice bran soluble product was only approximately 22% (220.125 mg/g deoiled bran), thus the remaining fraction of 45% (the difference between 67% and 22%) was the unmeasured product, which could be smaller organic acid hydrolysis products of non-protein components in rice bran. 3.7. Suitable conditions The yields of protein and amino acids were important factors for selecting suitable hydrolysis condition. The highest protein and amino acid yields were obtained mostly after 30 min hydrolysis time, the yields of protein and amino acids in the soluble products obtained after 30 min hydrolysis at various temperatures are summarized in Table 2. Note that when comparable yields were resulted from two different conditions, the lower temperature condition would be more desirable due to lower energy requirement. The bold-typed numbers in the table above represents the highest protein and amino acid yields for each raw material. Since temperature had a much smaller effect on the yield of amino acids, the most suitable temperature
Table 2 Protein and amino acid contents after hydrolysis of raw materials at different temperatures for 30 min Temperature(°C)
200 210 220
Raw rice bran
Deoiled rice bran
Raw soybean
Protein (mg)
Amino acid (mg)
Protein (mg)
Amino acid (mg)
Protein (mg)
Amino acid (mg)
Deoiled soybean Protein (mg)
Amino acid (mg)
97.14 100.50 106.02
7.20 7.47 7.09
128.60 128.79 130.17
9.59 9.27 9.14
162.00 165.72 151.37
17.96 18.62 17.23
204.64 188.22 142.96
19.65 20.67 18.47
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Table 3 Suitable hydrolysis conditions (the time was 30 min)
Raw rice bran Deoiled rice bran Raw soybean Deoiled soybean
Temperature (°C)
Protein yield (mg/g raw material)
220 220/210 210/200 200
0.74
˚ ˚
200 C 220 C
0.70
Amino acid yield (mg/g raw material)
106.02 130.17/128.79 165.72/162.00 204.64
7.09 9.14/9.27 18.62/17.96 19.65
0.60
IC50 (mg/ml)
Raw materials
0.80
0.49 0.43
0.50
0.30
0.40 0.22
0.30
0.23
0.19
0.20
20 min
30 min
3.8. Effect of the ratio of raw material-to-water on extraction yields The effect of the ratio of raw material-to-water on extraction yield was determined by comparing the product yields obtained from two different ratios of raw materialto-water: 1:5 and 2:5 and the results were found to be similar for all conditions. As an example, only the results from the hydrolysis experiment at 210 °C and 30 min are shown in Table 4, which shows that the yields of protein, amino acids, and reducing sugar decreased when the ratio raw of material-to-water was increased from 1:5 to 2:5. This was mainly because the too high amount of raw material caused an increase in the density and viscosity, resulting in poor mixing of raw material and water, thus mass transfer decreased and the accessibility of water to particles of raw materials became difficult. The ratio raw material-towater of 1:5 was therefore more suitable. 3.9. Antioxidant activity In this study, the antioxidant activity of the soluble products obtained by SW hydrolysis was evaluated with ABTS+ scavenging assay. Antioxidant activity was represented by IC50 index, which is the concentration of sample producing 50% reduction of the radical absorbance. This means that the higher IC50 of the product, the lower antioxidant activity. The antioxidant activity was measured for the hydrolysis temperature of 200 °C and 220 °C, and the hydrolysis time at 20 min and 30 min. These conditions were found to be the most suitable hydrolysis conditions and were therefore selected for the antioxidant test. Table 4 Effect of the ratio of raw material-to-water on extraction yields Material:water ratio
Rice bran Raw
Proteins AA Reducing sugars
Soybean Deoiled
Raw
1:5
2:5
1:5
2:5
1:5
2:5
1:5
Deoiled 2:5
101 7 62
73 6 61
129 9 111
95 5 89
166 19 35
123 15 21
188 21 38
136 17 24
0.28 0.24 0.20
0.20
0.19
0.19
0.20
0.19
0.10 0.00 Raw rice bran
for the production of each product could be drawn from the condition that gave the highest protein yield. From these results, the most suitable conditions are summarized in Table 3.
0.28
20 min
30 min
Deoiled rice bran
20 min
30 min
Raw soybean
20 min
30 min
Deoiled soybean
Raw material varieties
Fig. 4. Antioxidant activity (IC50) of the soluble products at hydrolysis times of 20 min and 30 min and temperatures of 200 °C and 220 °C.
The result in Fig. 4 indicated that with the hydrolysis time of 20 min, antioxidant activity increased as temperature increased from 200 °C to 220 °C for all materials. For the reaction time of 30 min, however, the antioxidant activity only increased slightly or stayed constant with the increase in temperature. The increase in water temperature does not only increase the ion product, which causes hydrolysis reaction, but it also causes the breakdown of hydrogen bonding between the water molecules. When the H-bonds break down, several antioxidative organic compounds within the rice bran and soybean samples become better able to dissolve in water, thus the soluble products exhibited higher antioxidant activity than those obtained at lower temperature. At long exposure time or at high temperature, however, some antioxidative compounds might be degraded, thus the activity might decrease (Clifford, 2000). 3.10. Paired preference test Paired preference test is an initial consumer response to the taste of the product in which a pair of samples is required to compare the taste preference. Here, only the product of deoiled rice bran was tested. In the samples in which the rice bran products were added, 2 g of dried products were added to 600 ml of the control samples. This is equivalent to the concentrations of 1.62 mg of protein/ml and 0.12 mg of amino acids/ml in the test samples. In this study, two pairs of samples were compared; the first pair was that of milk control and milk added with deoiled rice bran hydrolysis product and the second pair was that of coffee control and the coffee added with extraction product. Of the 40 testers, 14 and 26 testers preferred milk control and milk added with deoiled rice bran hydrolysis product, while 25 and 15 testers preferred coffee control and coffee added with extraction product, respectively. From the statistical analysis based on the 40 testers, the minimum correct answers necessary to establish significant differentiation at a 5% probability level was 26. From these results, it can be concluded that the preference for the milk added with hydrolysis product was significantly higher than that for the milk control (P 6 0.05), while the preferences for the coffee added with extraction product and the
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coffee control showed no significant differences (P > 0.05). It should be noted that the pair preference test is only a basic test conducted on testers with minimal training whose age range between 22 and 36, who were asked to select one of the two choices they preferred. The descriptive evaluation of the product was not the main result used in the analysis as these testers were inexperienced. Further detailed sensory evaluation such as descriptive analysis conducted on trained testers would be required. Furthermore, the product preference could be dependent on the age or gender of the testers, as well as on the serving size of the product, and thus further study is needed to determine the effect of these factors. Nevertheless, the results of the pair preference test conducted in this study proved the potential application of the SW hydrolysis products as functional food for humans.
4. Conclusions This study demonstrated that subcritical water could be used to potentially hydrolyze raw and deoiled rice bran, and raw and deoiled soybean meal into more valuable proteins, amino acids and reducing sugars. The suitable conditions for protein and amino acid production from rice bran (raw and deoiled) and soybean meal (raw and deoiled) by subcritical water hydrolysis were 1:5 raw material-to-water weight ratio at 30 min and hydrolysis temperature of 220 °C for raw and deoiled rice bran and 210 °C and 200 °C for raw and deoiled soybean meal, respectively. Apart from protein, amino acids and reducing sugars, active compounds that exhibit antioxidant activity were also extracted by subcritical water, and the products were proven to have potential application as human foods.
Acknowledgements Financial supports from Thailand Research Fund and Chulalongkorn University Graduate Program are appreciated. Mr. Pravit Santiwattanan from Thai Edible Oil Co., Ltd. is greatly acknowledged for the provision of raw and deoiled rice bran and Thanakorn Vegetable Oil Products Co., Ltd., Samuthprakarn, for the supplies of soybean meals.
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