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Nutritional quality of rice bran protein in comparison to animal and vegetable protein
Accepted Manuscript Nutritional quality of rice bran protein in comparison to animal and vegetable protein Sung-Wook Han, Kyu-Man Chee, Seong-Jun Cho PII: DOI: Reference:
S0308-8146(14)01512-X http://dx.doi.org/10.1016/j.foodchem.2014.09.127 FOCH 16483
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
29 March 2014 17 August 2014 22 September 2014
Please cite this article as: Han, S-W., Chee, K-M., Cho, S-J., Nutritional quality of rice bran protein in comparison to animal and vegetable protein, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.09.127
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nutritional quality of rice bran protein in comparison to animal and vegetable protein
Sung-Wook Hana, Kyu-Man Cheeb, Seong-Jun Choa* a
Ingredients R&D Center, CJ Cheiljedang, 636 Guro-dong, Guro-gu, Seoul 152051, Korea
b
College of Life Sciences and Biotechnology, Korea University, Anam-dong 5-ga, Seongbuk-
gu, Seoul 136701, Korea
*Corresponding author Seong-Jun Cho Ingredients R&D Center CJ Cheiljedang, 636 Guro-dong Guro-gu Seoul 152051 Korea Tel: +82-2-2629-5258 Fax: +82-2-2629-5344 E-mail [email protected]
1
ABSTRACT Rice bran protein (RBP) was prepared by alkali extraction and isoelectric precipitation from defatted rice bran. The protein quality of RPB was evaluated and compared to two vegetable proteins[soy protein (ISP) and rice endosperm protein (RBP)] and two animal proteins[whey protein (WPI) and casein]. RPB contained 74.93% of protein and its pepsin digestibility and KOH solubility were 89.8% and 91.5%, respectively. In Sprague-Dawley rats, RBP showed protein efficiency ratio, net protein ratio, net protein utilization, and biological value of 2.39, 3.77, 70.7, and 72.6, which were comparable to those of animal proteins. The true digestibility of RBP (94.8%) was significantly higher than that of REP (90.8%), ISP (91.7%) and WPI (92.8%) and the same as that of casein. Protein digestibility corrected amino acid score (PDCAAS) of RBP is 0.90. These results suggest that rice bran protein appears to be a promising protein source with good biological values and digestibility.
Keywords Rice bran protein; protein quality; vegetable protein; Protein Digestibility Corrected Amino Acid Score
2
1. Introduction Proteins are considered to be the most important macronutrient for humans (Reeds, Schaafsma, Tome, & Young, 2000) (Otten, Hellwig, & Meyers, 2006), but protein-energy malnutrition is a worldwide problem, especially in developing countries. Interest in the potential utilization of protein from under-utilized biomass sources such as oil meal and legumes has increased in recent years (Enujiugha & Ayodele-Oni, 2003). The nutritional values of proteins differ substantially depending upon their amino acid composition and digestibility. Indeed, the nutritional value or quality of structurally different proteins is variable and is governed by amino acid composition, essential amino acid ratio, susceptibility to hydrolysis during digestion, purity, and processing effects such as heattreatment (Friedman, 1996) (Machado et al., 2008). The assessment of protein quality has relied on indirect approaches such as chemical analysis, which has limited use and sometimes does not match in vivo results, although it is easy and quick. Protein quality is a measure of bioavailability, and its evaluation is a means of determining the capacity of food proteins and diets to satisfy metabolic demands for amino acids and nitrogen.(Gilani, 2012) Rice bran is an underutilized by-product of rice milling, despite being a nutrient-dense product and a rich source of protein, fat, carbohydrate, and a number of micronutrients such as vitamins, minerals, antioxidants, and phytosterols (Schramm, Abadie, Hua, Xu, & Lima, 2007)(Iqbal, Bhanger, & Anwar, 2005)(Renuka Devi & Arumughan, 2007). Rice bran protein (RBP) has garnered interest in the food industry because of its unique nutritional value and nutraceutical properties (Saunders, 1990). In addition to its high nutritional value, rice protein is a hypoallergenic food ingredient and can be used in infant formulations (Helm & Burks, 1996). It has been reported to have anti-cancer properties (Shoji et al., 2001). The utility of this product in food applications depends on our 3
understanding of its nutritional and functional properties. Although rice bran protein could be a new protein source, few studies have been reported on its nutritional evaluation. Therefore, this study was conducted 1) to assess the effectiveness of rice bran protein and other protein sources in vivo and in vitro, 2) to investigate the comparative values of protein sources, and 3) to suggest rice bran protein as a new protein source.
2. Material and methods 2.1. Materials Rice bran (Oryza sativa L.) was supplied by Song-tan Rice Processing Center (Gyeonggi-do, Korea) and defatted with five volumes of n-hexane. Other protein sources were purchased commercially. Casein was purchased from LACTONATEN, Lactoprot, Germany; isolated soy protein (ISP), from Pro-FAM®, Decatur, USA; whey protein isolate (WPI), from IsoCilll™ 9010, Trega, USA; and rice endosperm protein (REP), from Remypro N 80+KB, REMY industries, Belgium.
2.2. Preparation of rice bran protein (RBP) Defatted rice bran was prepared by solvent extraction in a Soxhlet apparatus using hexane (rice bran: hexane ratio = 1:5w/v), followed by air-drying in a fume hood. The defatted rice bran was mixed thoroughly with five volumes of distilled water. The mixture was adjusted to pH 9 with 1 N NaOH, stirred for 1 h at 30°C, and centrifuged at 5,000 g for 20 min. Then, the supernatant was adjusted to pH 4 with 1 N HCl, maintained at 30°C for 30 min for protein precipitation, and centrifuged at 5,000 g for 20 min. The solid residues were washed with five volumes of distilled water and neutralized with 1 N NaOH. The neutralized protein solution was spray dried. 4
2.3. Proximate analysis Chemical compositions such as moisture content, crude protein, crude fat, crude ash and crude fibre were analyzed according to the Association of Official Agricultural Chemists (AOAC) method (2000). Amino acid composition was determined after acid hydrolysis with 6N HCl for 24 h and 110°C by high-performance liquid chromatography (Rayner, 1985). Analysis of samples was performed in triplicate.
2.4. Measurement of protein quality by in vitro methods Pepsin digestibility was analyzed by the AOAC method (2000). Pepsin was dissolved to a concentration of 0.002% in 0.1 N HCl and digested for 16 h. The KOH solubility was assessed by adding 0.2% KOH solution at 22°C for 20 min according to the methods described by Parsons et al. .(Parsons, Hashimoto, Wedekind, & Baker, 1991)
2.5. Animal test Sixty 5-week-old, male Sprague-Dawley rats (initial body weight: 111.8 ± 5.5) were used for measuring protein efficiency ratio (PER), net protein ratio (NPR), and net protein utilization (NPU). They were randomly divided into 6 treatment groups, each consisting of 10 rats, and allowed to adapt for 6 days on a conventional diet (AIN-93G, Dyets Inc. Bethlehem, PA). They were then fed the experimental diet for 14 days. Forty-eight 6-week-old, male Sprague-Dawley rats (initial body weight 171.2 ± 8.7) were used for measuring true digestibility, biological value, and PDCAAS. They were divided into 6 treatment groups, each consisting of 8 rats, and allowed to adapt for 7 days on a conventional diet (AIN-93G, Dyets Inc. Bethlehem, PA). They were then fed the 5
experimental diet for 5 days. Their faecal matter was collected for 5 days. They were housed individually in metabolic cages with adequate facilities for separate faecal and urinary collection, and kept in rooms lit for 12 h per day at a constant temperature of 23°C. Water and diets were provided ad libitum. All experiments and animal procedures conformed to protocols approved by the Institutional Animal Care and Use Committees of Korea University, Seoul, Korea.
2.6. Composition of experimental diet The experimental groups were provided feed that satisfied the requirements of rats for energy, fats, minerals, and vitamins space (National Research Council, 1995). The protein level was adjusted to around 10.0% (NPU standard) in all groups excluding the N-free diet group (Miller & Payne, 1961). The composition of experimental diets is shown in Table 1.
2.7. Protein quality measurements Body weight gain was determined by the difference in body weight between the start and end of the experiment. Empty body weight at the start of the experiment was measured after 12 h of fasting, and the empty body weight at the end of the experiment was measured by euthanasia of each animal without fasting, removing the intestinal contents, and measuring the weight of the animal. The feed/gain ratio of each animal was obtained by dividing the feed intake of the animal by its empty body weight gain. Protein efficiency ratio (PER) was determined by the following equation (Henry, 1965), which related to protein intake and empty body weight gain. PER =
.
Net Protein Ratio (NPR) was calculated from the body weight loss of the N-free diet group 6
according to the method of Bender and Doell (1975): NPR =
-
.
Net protein utilization (NPU) was measured by analyzing the carcass of each animal and measuring protein retention at the end of the experiment. Protein loss of the N-free diet group was calculated as follows: NPU =
True digestibility of each feed protein was determined by measuring the amount of metabolic fecal N from the N-free diet feed and performing the following equation according to the method of Schneider and Flatt (1975): TD!%# =
! $ % $ #
× 100.
The biological value (BV) of each feed was calculated from the relationship between NPU and true digestibility by Hackler (1977): BV!%# = NPU × True digestibility!%# PDCAAS was calculated according to the following equation. The AASs used to calculate PDCASS were based on the FAO/WHO/UNU reference (2007). 9 9 9 $ :
PDCAAS!%# = 9 9 9 $ : $ × true digestibility!%#.
2.8. Statistical analysis All results were subjected to analysis of variance by one-way ANOVA using SigmaStat(v.3.5. Systat Software Inc.). The significant difference between treatment means was determined at the P < 0.05 level by Duncan’s new multiple range test (Steel & Torrie, 1981).
3. Results and discussion 7
3.1. Proximate composition and amino acid contents Table 2 shows the proximate composition and essential amino acid content of 5 protein samples. Protein contents were highest in ISP (91.94%), followed by casein (90.02%), REP (86.89%), WPI (86.09%), and RBP (74.93%). Nine amino acids, including lysine, methionine, threonine, tryptophan, histidine, leucine, isoleucine, valine, and phenylalanine, are considered essential amino acids in human nutrition (Gropper, Smith, & Groff, 2009). Cysteine and tyrosine are non-essential amino acids, but are synthesized from methionine and phenylalanine, respectively, and can act as substitutes for some amino acids. Thus, the amounts of 2 amino acid combinations, namely, methionine + cysteine (TSAA, total sulfur-containing amino acids) and phenylalanine + tyrosine (AAA, aromatic amino acids), are used to determine protein nutritional value. RBP showed higher contents of threonine, valine, lysine, histidine, and tryptophan in comparison to REP. WPI had higher contents of threonine, isoleucine, leucine, lysine, TSAA, and tryptophan than the other protein sources, and casein had higher contents of valine, AAA, and histidine than the other protein sources. RBP had higher contents of valine, histidine, arginine, and TSAA, and lower contents of isoleucine, AAA, and lysine in comparison to ISP. In general, lysine is considered the first limiting amino acid in rice, as determined by the amino acid requirements of humans. The lysine content of rice protein derived from bran was about 37.4% higher than that of REP. In addition, RBP had the highest content of histidine. To compare the in vitro digestibility of each protein sample, the pepsin digestibility and KOH solubility were examined (Table 3). Pepsin digestibility was the highest for WPI (98.8%), followed by REP (94.6%), ISP (93.7%), RBP (89.8%), and casein (89.2%). KOH solubility was highest for casein and WPI, followed by RBP, ISP, and REP. The KOH solubility of REP was 44.5%, which was far lower than the 91.5% for RBP. Measurement of 8
protein solubility in KOH is frequently used to evaluate the quality of soybean meal (the degree of heat treatment). According to Parsons et al.(1991) , there is good correlation between the measured value of KOH solubility of soybean meal and the in vivo digestibility of soybean meal in chicken or pigs.
3.2. Protein quality measurement by animal experiments Body weight gain was highest in the WPI group, followed by casein, RBP, REP, and ISP. Body weight gain in the RBP group was significantly higher than that of the ISP group (P < 0.05, Table 4). There was no difference in feed intake between groups other than the N-free diet group. Since a lower feed/gain ratio indicates a better feed efficiency, the feed/gain ratio was best in the WPI and casein groups. The feed/gain ratio of the RBP group (4.21) improved by about 33% and 22% in comparison to the ISP (5.61) and REP groups (5.13; P < 0.05). The efficiency of protein utilization for each protein source is shown in Table 5. All measures of utilization efficiency were higher in the WPI group than the other protein groups. The PER and NPR of the RBP group were significantly higher than those of vegetable protein group (REP and ISP) (P < 0.05). The RBP shows the highest NPU value than ISP, REP, and casein. Thus, protein quality of RBP are better than those of REP and ISP, and RBP can replace ISP, which is widely used in human and animal nutrition as a vegetable protein source. The true digestibility, biological value, and PDCAAS of various protein sources are shown in Table 6. The true digestibility of RBP and casein were significantly better than those of ISP, WPI, and REP. The biological values were highest for WPI (78.8) and RBP (72.6), followed by REP (66.7), ISP (66.6), and casein (59.7). The PDCASS value of RBP is 0.90, which is lower than milk based protein, but comparable to ISP. Thus, the digestibility and retention of rice protein are not inferior to those of casein; WPI, 9
and ISP and may indeed be superior.
4. Conclusions In conclusion, RBP showed superior nutritional quality in comparison to vegetable proteins, but the biological availability of RBP is still lower than that of animal protein sources; however, there is a growing need to replace animal protein by vegetable protein, because of the increased cost and limited supply of animal proteins. Since rice bran is an economical nutritional protein source and is available in large quantities, its protein isolates could be used as new protein sources to provide nutritional and economical value.
10
References AOAC, (2000). Official Methods of Analysis (18th ed). Washington D.C.: Association of Official Analysis Chemists Bender, A. E., & Doell, B. H. (1957). Biological evaluation of proteins: a new aspect. British Journal of Nutrition, 11(02), 140–148. FAO/WHO/UNU. (2007). Protein and amino acid requirements in human nutrition: report of a joint WHO/FAO/UNU Expert Consultation. WHO Tech. Rep. Ser. N. 935. Geneva, Switzerland. Enujiugha, V. N., & Ayodele-Oni, O. (2003). Evaluation of nutrients and some anti-nutrients in lesser-known, underutilized oilseeds. International Journal of Food Science & Technology, 38(5), 525–528. Friedman, M. (1996). Nutritional Value of Proteins from Different Food Sources. A Review. Journal of Agricultural and Food Chemistry, 44(1), 6–29. Gilani, G. S. (2012). Background on international activities on protein quality assessment of foods. The British Journal of Nutrition, 108 Suppl (S2), S168–82. Gropper, S.S., Smith, J.L., & Groff, J.L. (2005). Advanced Nutrition and Human Metabolism (4th ed.). Belmont, CA : Wadsworth. Hackler, L. R. (1977). Methods of measuring protein quality: A review of bioassay procedure. Cereal Chemistry, 54, 984–995. Helm, R.M., & Burks, A.W. (1996). Hypoallerginicity of rice protein. Cereal Food World. 41, 836-842. Henry, K. M. (1965). A comparison of biological methods with rats for determining the nutritive value of proteins. The British Journal of Nutrition, 19, 125–35.
11
Iqbal, S., Bhanger, M. I., & Anwar, F. (2005). Antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Food Chemistry, 93(2), 265–272. Miller, D. S., & Payne, P. R. (1961). Problems in the prediction of protein values of diets. The influence of protein concentration. The British Journal of Nutrition, 15, 11–9. Machado, F. P. P., Queiróz, J. H., Oliveira, M. G. A., Piovesan, N. D., Peluzio, M. C. G., Costa, N. M. B., & Moreira, M. A. (2008). Effects of heating on protein quality of soybean flour devoid of Kunitz inhibitor and lectin. Food Chemistry, 107(2), 649–655. National Research Council. (1995). Nutrient Requirements of Laboratory Animals (Fourth Rev.). Washington, DC: The National Academies Press. Otten, J.J., Hellwig, J.P. & Meyers, L.D. (2006). Dietary Reference Intakes : The Essential Guide to Nutrient Requirements. Washigton, DC : The National Academics Press. Parsons, C. M., Hashimoto, K., Wedekind, K. J., & Baker, D. H. (1991). Soybean protein solubility in potassium hydroxide: an in vitro test of in vivo protein quality. Journal of Animal Science, 69, 2918–2924. Rayner, C. J. (1985). Protein hydrolysis of animal feeds for amino acid content. Journal of Agricultural and Food Chemistry, 33(4), 722–725. Reeds, P., Schaafsma, G., Tome, D., & Young, V. (2000). Criteria and significance of dietary protein sources in human; summary of the workshop with recommendations. Journal of Nutrition, 130(7), 1874S–1876. Renuka Devi, R., & Arumughan, C. (2007). Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment. Bioresource Technology, 98, 3037–3043.
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Saunders R.M. (1990). The properties of rice bran as a food studd. Cereal Food World. 35, 632-662. Schneider, B.H, & Flatt, W.P. (1975). The evaluation of feeds through digestibility experiments. Athens,GA : AthensUniversity of Georgia Press. Schramm, R., Abadie, A., Hua, N., Xu, Z., & Lima, M. (2007). Fractionation of the rice bran layer and quantification of vitamin E, oryzanol, protein, and rice bran saccharide. Journal of Biological Engineering, 1, 9. Shoji, Y., Mita, T., Isemura, M., Mega, T., Hase, S., Isemura, S., & Aoyagi, Y. (2001). A Fibronectin-binding Protein from Rice Bran with Cell Adhesion Activity for Animal Tumor Cells. Bioscience, Biotechnology, and Biochemistry, 65(5), 1181–1186. Steel, R.G.D., Torrie, J.H. (1981). Principles and procedures of statistics: a biomedical approach (2nd ed.), New York, NY : McGraw-Hill International Book Co.
13
Table 1 Ingredients of experimental diets Ingredient (g/100g of diet) Casein RBP ISP WPI REP Glucose Corn Starch Soy oil α-cellulose NaCl Choline bitartrate Vitamin Mix1 Mineral Mix1 Potassium phosphate
Casein
RBP
ISP
WPI
REP
N-Free
12.20 30.80 40.00 7.00 5.00 0.25 0.25 1.00 3.50 -
14.50 28.20 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
11.70 31.00 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
12.80 29.85 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.35
12.70 30.00 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
42.65 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.35
1 By AIN-93 vitamin and mineral mixture (Dyets, Inc., Bethlehem, PA) RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
14
Table 2 Chemical and essential amino acid composition of protein sources RBP Casein Chemical composition (g/100g dry matter) Crude protein 74.93 90.02 Crude fat 3.61 0.18 Crude fiber 0.00 0.26 Ash 14.28 5.08 Nitrogen free extract 7.18 4.46 Essential Amino acid (g /100 g protein) Threonine 3.68 4.30 Valine 5.53 5.88 Isoleucine 3.61 4.88 Leucine 7.69 9.73 Phenyalanine + tyrosine 8.24 10.73 Lysine 4.55 8.15 Histidine 4.48 2.97 Methionine + cystine 2.70 2.94 Tryptophan 1.17 1.05
ISP
WPI
REP
91.94 0.37 0.08 4.06 3.55
86.09 0.20 0.07 3.03 10.61
86.89 0.34 0.08 3.57 9.12
3.67 4.16 4.30 7.84 8.65 6.14 2.58 2.55 1.28
7.52 5.57 6.24 10.9 5.94 9.96 1.97 5.36 1.72
3.46 5.12 3.80 8.15 10.09 3.31 2.46 3.88 0.82
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein Nitrogen free extract = 100 – (moisture content + crude protein + crude fat + crude fibre + ash)
15
Table 3 Pepsin digestibility and KOH solubility of protein sources
In vitro digestibility
RBP
Casein
ISP
WPI
REP
Pepsin digestibility(%) KOH solubility(%)
89.8 91.5
89.2 94.0
93.7 85.0
98.8 93.3
94.6 44.5
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
16
Table 4 Effects of protein sources on the body weight gain, feed intake and feed/gain ratio of rats Protein sources RBP Casein ISP WPI REP N-free 1
Body weight gain1 (g/rat) 54.6±13.0b 55.7±9.1 b 40.1±10.0c 79.3±12.6a 47.1±10.0abc -30.9±3.6d
Feed intake1 (g/rat) 222.4±28.1a 214.7±21.3a 218.1±23.6a 235.2±18.9a 233.0±25.9a 138.0±7.4b
Feed/Gain ratio1 4.21±0.74b 3.91±0.44c 5.61±0.87a 3.04±0.57c 5.13±1.05a -
Values are for 10 animals in each group and different letters mean significant differences at p<0.05.
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
17
Table 5 Protein intake, protein efficiency ratio (PER), net protein retention (NPR), net protein utilization (NPU) value of each protein source. Protein sources RBP Casein ISP WPI REP
Protein intake (g/rat) 22.7±2.9 a 21.6±2.1 a 23.3±2.5 a 24.3±1.9 a 24.1±2.7 a
PER1
NPR1
NPU1
2.39±0.38 b 2.58±0.27 b 1.71±0.28c 3.26±0.43a 1.96±0.41c
3.77±0.36b 4.02±0.25a 3.04±0.23c 4.54±0.43a 3.26±0.45c
70.7±16.4ab 59.1±12.6 a 59.9±16.7 a 74.0±7.2b 61.4±8.9a
1 Values are for 10 animals in each group and different letters mean significant differences at p<0.05. RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
18
Table 6 Nitrogen intake, faecal nitrogen, true digestibility (TD), biological value (BV) and protein digestibility-corrected amino acid score (PDCASS) value of each protein source. Protein source RBP Casein ISP WPI REP
N intake1 (g/rat) 1.22 1.12 1.12 1.30 1.21
Fecal N1 (g/rat) 0.189 0.189 0.221 0.220 0.239
TD12
BV1
PDCAAS3
94.8±0.5a 94.8±0.7a 91.7±1.5c 92.8±0.7b 90.8±0.6c
72.6±0.4 c 59.7±0.4 a 66.6±1.1 b 78.8±0.6 d 66.7±0.4 b
0.90 1.00 0.95 1.00 0.63
1 Values are for 8 animals in each group and different letters mean significant differences at p<0.05 2 Endogenous faecal N loss was 0.126 g/rat in N-free diet group 3 The amino acid scores used to calculated PDCASS were based on the FAO/WHO/UNU reference RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
19
The first detailed in vitro and in vivo protein quality measurement of rice bran protein.
The rice bran protein (RBP) has better protein quality than vegetable protein.
RBP is comparable to casein and can replace it as non-animal protein source.
20
S0308-8146(14)01512-X http://dx.doi.org/10.1016/j.foodchem.2014.09.127 FOCH 16483
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
29 March 2014 17 August 2014 22 September 2014
Please cite this article as: Han, S-W., Chee, K-M., Cho, S-J., Nutritional quality of rice bran protein in comparison to animal and vegetable protein, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.09.127
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nutritional quality of rice bran protein in comparison to animal and vegetable protein
Sung-Wook Hana, Kyu-Man Cheeb, Seong-Jun Choa* a
Ingredients R&D Center, CJ Cheiljedang, 636 Guro-dong, Guro-gu, Seoul 152051, Korea
b
College of Life Sciences and Biotechnology, Korea University, Anam-dong 5-ga, Seongbuk-
gu, Seoul 136701, Korea
*Corresponding author Seong-Jun Cho Ingredients R&D Center CJ Cheiljedang, 636 Guro-dong Guro-gu Seoul 152051 Korea Tel: +82-2-2629-5258 Fax: +82-2-2629-5344 E-mail [email protected]
1
ABSTRACT Rice bran protein (RBP) was prepared by alkali extraction and isoelectric precipitation from defatted rice bran. The protein quality of RPB was evaluated and compared to two vegetable proteins[soy protein (ISP) and rice endosperm protein (RBP)] and two animal proteins[whey protein (WPI) and casein]. RPB contained 74.93% of protein and its pepsin digestibility and KOH solubility were 89.8% and 91.5%, respectively. In Sprague-Dawley rats, RBP showed protein efficiency ratio, net protein ratio, net protein utilization, and biological value of 2.39, 3.77, 70.7, and 72.6, which were comparable to those of animal proteins. The true digestibility of RBP (94.8%) was significantly higher than that of REP (90.8%), ISP (91.7%) and WPI (92.8%) and the same as that of casein. Protein digestibility corrected amino acid score (PDCAAS) of RBP is 0.90. These results suggest that rice bran protein appears to be a promising protein source with good biological values and digestibility.
Keywords Rice bran protein; protein quality; vegetable protein; Protein Digestibility Corrected Amino Acid Score
2
1. Introduction Proteins are considered to be the most important macronutrient for humans (Reeds, Schaafsma, Tome, & Young, 2000) (Otten, Hellwig, & Meyers, 2006), but protein-energy malnutrition is a worldwide problem, especially in developing countries. Interest in the potential utilization of protein from under-utilized biomass sources such as oil meal and legumes has increased in recent years (Enujiugha & Ayodele-Oni, 2003). The nutritional values of proteins differ substantially depending upon their amino acid composition and digestibility. Indeed, the nutritional value or quality of structurally different proteins is variable and is governed by amino acid composition, essential amino acid ratio, susceptibility to hydrolysis during digestion, purity, and processing effects such as heattreatment (Friedman, 1996) (Machado et al., 2008). The assessment of protein quality has relied on indirect approaches such as chemical analysis, which has limited use and sometimes does not match in vivo results, although it is easy and quick. Protein quality is a measure of bioavailability, and its evaluation is a means of determining the capacity of food proteins and diets to satisfy metabolic demands for amino acids and nitrogen.(Gilani, 2012) Rice bran is an underutilized by-product of rice milling, despite being a nutrient-dense product and a rich source of protein, fat, carbohydrate, and a number of micronutrients such as vitamins, minerals, antioxidants, and phytosterols (Schramm, Abadie, Hua, Xu, & Lima, 2007)(Iqbal, Bhanger, & Anwar, 2005)(Renuka Devi & Arumughan, 2007). Rice bran protein (RBP) has garnered interest in the food industry because of its unique nutritional value and nutraceutical properties (Saunders, 1990). In addition to its high nutritional value, rice protein is a hypoallergenic food ingredient and can be used in infant formulations (Helm & Burks, 1996). It has been reported to have anti-cancer properties (Shoji et al., 2001). The utility of this product in food applications depends on our 3
understanding of its nutritional and functional properties. Although rice bran protein could be a new protein source, few studies have been reported on its nutritional evaluation. Therefore, this study was conducted 1) to assess the effectiveness of rice bran protein and other protein sources in vivo and in vitro, 2) to investigate the comparative values of protein sources, and 3) to suggest rice bran protein as a new protein source.
2. Material and methods 2.1. Materials Rice bran (Oryza sativa L.) was supplied by Song-tan Rice Processing Center (Gyeonggi-do, Korea) and defatted with five volumes of n-hexane. Other protein sources were purchased commercially. Casein was purchased from LACTONATEN, Lactoprot, Germany; isolated soy protein (ISP), from Pro-FAM®, Decatur, USA; whey protein isolate (WPI), from IsoCilll™ 9010, Trega, USA; and rice endosperm protein (REP), from Remypro N 80+KB, REMY industries, Belgium.
2.2. Preparation of rice bran protein (RBP) Defatted rice bran was prepared by solvent extraction in a Soxhlet apparatus using hexane (rice bran: hexane ratio = 1:5w/v), followed by air-drying in a fume hood. The defatted rice bran was mixed thoroughly with five volumes of distilled water. The mixture was adjusted to pH 9 with 1 N NaOH, stirred for 1 h at 30°C, and centrifuged at 5,000 g for 20 min. Then, the supernatant was adjusted to pH 4 with 1 N HCl, maintained at 30°C for 30 min for protein precipitation, and centrifuged at 5,000 g for 20 min. The solid residues were washed with five volumes of distilled water and neutralized with 1 N NaOH. The neutralized protein solution was spray dried. 4
2.3. Proximate analysis Chemical compositions such as moisture content, crude protein, crude fat, crude ash and crude fibre were analyzed according to the Association of Official Agricultural Chemists (AOAC) method (2000). Amino acid composition was determined after acid hydrolysis with 6N HCl for 24 h and 110°C by high-performance liquid chromatography (Rayner, 1985). Analysis of samples was performed in triplicate.
2.4. Measurement of protein quality by in vitro methods Pepsin digestibility was analyzed by the AOAC method (2000). Pepsin was dissolved to a concentration of 0.002% in 0.1 N HCl and digested for 16 h. The KOH solubility was assessed by adding 0.2% KOH solution at 22°C for 20 min according to the methods described by Parsons et al. .(Parsons, Hashimoto, Wedekind, & Baker, 1991)
2.5. Animal test Sixty 5-week-old, male Sprague-Dawley rats (initial body weight: 111.8 ± 5.5) were used for measuring protein efficiency ratio (PER), net protein ratio (NPR), and net protein utilization (NPU). They were randomly divided into 6 treatment groups, each consisting of 10 rats, and allowed to adapt for 6 days on a conventional diet (AIN-93G, Dyets Inc. Bethlehem, PA). They were then fed the experimental diet for 14 days. Forty-eight 6-week-old, male Sprague-Dawley rats (initial body weight 171.2 ± 8.7) were used for measuring true digestibility, biological value, and PDCAAS. They were divided into 6 treatment groups, each consisting of 8 rats, and allowed to adapt for 7 days on a conventional diet (AIN-93G, Dyets Inc. Bethlehem, PA). They were then fed the 5
experimental diet for 5 days. Their faecal matter was collected for 5 days. They were housed individually in metabolic cages with adequate facilities for separate faecal and urinary collection, and kept in rooms lit for 12 h per day at a constant temperature of 23°C. Water and diets were provided ad libitum. All experiments and animal procedures conformed to protocols approved by the Institutional Animal Care and Use Committees of Korea University, Seoul, Korea.
2.6. Composition of experimental diet The experimental groups were provided feed that satisfied the requirements of rats for energy, fats, minerals, and vitamins space (National Research Council, 1995). The protein level was adjusted to around 10.0% (NPU standard) in all groups excluding the N-free diet group (Miller & Payne, 1961). The composition of experimental diets is shown in Table 1.
2.7. Protein quality measurements Body weight gain was determined by the difference in body weight between the start and end of the experiment. Empty body weight at the start of the experiment was measured after 12 h of fasting, and the empty body weight at the end of the experiment was measured by euthanasia of each animal without fasting, removing the intestinal contents, and measuring the weight of the animal. The feed/gain ratio of each animal was obtained by dividing the feed intake of the animal by its empty body weight gain. Protein efficiency ratio (PER) was determined by the following equation (Henry, 1965), which related to protein intake and empty body weight gain. PER =
.
Net Protein Ratio (NPR) was calculated from the body weight loss of the N-free diet group 6
according to the method of Bender and Doell (1975): NPR =
-
.
Net protein utilization (NPU) was measured by analyzing the carcass of each animal and measuring protein retention at the end of the experiment. Protein loss of the N-free diet group was calculated as follows: NPU =
True digestibility of each feed protein was determined by measuring the amount of metabolic fecal N from the N-free diet feed and performing the following equation according to the method of Schneider and Flatt (1975): TD!%# =
! $ % $ #
× 100.
The biological value (BV) of each feed was calculated from the relationship between NPU and true digestibility by Hackler (1977): BV!%# = NPU × True digestibility!%# PDCAAS was calculated according to the following equation. The AASs used to calculate PDCASS were based on the FAO/WHO/UNU reference (2007). 9 9 9 $ :
PDCAAS!%# = 9 9 9 $ : $ × true digestibility!%#.
2.8. Statistical analysis All results were subjected to analysis of variance by one-way ANOVA using SigmaStat(v.3.5. Systat Software Inc.). The significant difference between treatment means was determined at the P < 0.05 level by Duncan’s new multiple range test (Steel & Torrie, 1981).
3. Results and discussion 7
3.1. Proximate composition and amino acid contents Table 2 shows the proximate composition and essential amino acid content of 5 protein samples. Protein contents were highest in ISP (91.94%), followed by casein (90.02%), REP (86.89%), WPI (86.09%), and RBP (74.93%). Nine amino acids, including lysine, methionine, threonine, tryptophan, histidine, leucine, isoleucine, valine, and phenylalanine, are considered essential amino acids in human nutrition (Gropper, Smith, & Groff, 2009). Cysteine and tyrosine are non-essential amino acids, but are synthesized from methionine and phenylalanine, respectively, and can act as substitutes for some amino acids. Thus, the amounts of 2 amino acid combinations, namely, methionine + cysteine (TSAA, total sulfur-containing amino acids) and phenylalanine + tyrosine (AAA, aromatic amino acids), are used to determine protein nutritional value. RBP showed higher contents of threonine, valine, lysine, histidine, and tryptophan in comparison to REP. WPI had higher contents of threonine, isoleucine, leucine, lysine, TSAA, and tryptophan than the other protein sources, and casein had higher contents of valine, AAA, and histidine than the other protein sources. RBP had higher contents of valine, histidine, arginine, and TSAA, and lower contents of isoleucine, AAA, and lysine in comparison to ISP. In general, lysine is considered the first limiting amino acid in rice, as determined by the amino acid requirements of humans. The lysine content of rice protein derived from bran was about 37.4% higher than that of REP. In addition, RBP had the highest content of histidine. To compare the in vitro digestibility of each protein sample, the pepsin digestibility and KOH solubility were examined (Table 3). Pepsin digestibility was the highest for WPI (98.8%), followed by REP (94.6%), ISP (93.7%), RBP (89.8%), and casein (89.2%). KOH solubility was highest for casein and WPI, followed by RBP, ISP, and REP. The KOH solubility of REP was 44.5%, which was far lower than the 91.5% for RBP. Measurement of 8
protein solubility in KOH is frequently used to evaluate the quality of soybean meal (the degree of heat treatment). According to Parsons et al.(1991) , there is good correlation between the measured value of KOH solubility of soybean meal and the in vivo digestibility of soybean meal in chicken or pigs.
3.2. Protein quality measurement by animal experiments Body weight gain was highest in the WPI group, followed by casein, RBP, REP, and ISP. Body weight gain in the RBP group was significantly higher than that of the ISP group (P < 0.05, Table 4). There was no difference in feed intake between groups other than the N-free diet group. Since a lower feed/gain ratio indicates a better feed efficiency, the feed/gain ratio was best in the WPI and casein groups. The feed/gain ratio of the RBP group (4.21) improved by about 33% and 22% in comparison to the ISP (5.61) and REP groups (5.13; P < 0.05). The efficiency of protein utilization for each protein source is shown in Table 5. All measures of utilization efficiency were higher in the WPI group than the other protein groups. The PER and NPR of the RBP group were significantly higher than those of vegetable protein group (REP and ISP) (P < 0.05). The RBP shows the highest NPU value than ISP, REP, and casein. Thus, protein quality of RBP are better than those of REP and ISP, and RBP can replace ISP, which is widely used in human and animal nutrition as a vegetable protein source. The true digestibility, biological value, and PDCAAS of various protein sources are shown in Table 6. The true digestibility of RBP and casein were significantly better than those of ISP, WPI, and REP. The biological values were highest for WPI (78.8) and RBP (72.6), followed by REP (66.7), ISP (66.6), and casein (59.7). The PDCASS value of RBP is 0.90, which is lower than milk based protein, but comparable to ISP. Thus, the digestibility and retention of rice protein are not inferior to those of casein; WPI, 9
and ISP and may indeed be superior.
4. Conclusions In conclusion, RBP showed superior nutritional quality in comparison to vegetable proteins, but the biological availability of RBP is still lower than that of animal protein sources; however, there is a growing need to replace animal protein by vegetable protein, because of the increased cost and limited supply of animal proteins. Since rice bran is an economical nutritional protein source and is available in large quantities, its protein isolates could be used as new protein sources to provide nutritional and economical value.
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References AOAC, (2000). Official Methods of Analysis (18th ed). Washington D.C.: Association of Official Analysis Chemists Bender, A. E., & Doell, B. H. (1957). Biological evaluation of proteins: a new aspect. British Journal of Nutrition, 11(02), 140–148. FAO/WHO/UNU. (2007). Protein and amino acid requirements in human nutrition: report of a joint WHO/FAO/UNU Expert Consultation. WHO Tech. Rep. Ser. N. 935. Geneva, Switzerland. Enujiugha, V. N., & Ayodele-Oni, O. (2003). Evaluation of nutrients and some anti-nutrients in lesser-known, underutilized oilseeds. International Journal of Food Science & Technology, 38(5), 525–528. Friedman, M. (1996). Nutritional Value of Proteins from Different Food Sources. A Review. Journal of Agricultural and Food Chemistry, 44(1), 6–29. Gilani, G. S. (2012). Background on international activities on protein quality assessment of foods. The British Journal of Nutrition, 108 Suppl (S2), S168–82. Gropper, S.S., Smith, J.L., & Groff, J.L. (2005). Advanced Nutrition and Human Metabolism (4th ed.). Belmont, CA : Wadsworth. Hackler, L. R. (1977). Methods of measuring protein quality: A review of bioassay procedure. Cereal Chemistry, 54, 984–995. Helm, R.M., & Burks, A.W. (1996). Hypoallerginicity of rice protein. Cereal Food World. 41, 836-842. Henry, K. M. (1965). A comparison of biological methods with rats for determining the nutritive value of proteins. The British Journal of Nutrition, 19, 125–35.
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Iqbal, S., Bhanger, M. I., & Anwar, F. (2005). Antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Food Chemistry, 93(2), 265–272. Miller, D. S., & Payne, P. R. (1961). Problems in the prediction of protein values of diets. The influence of protein concentration. The British Journal of Nutrition, 15, 11–9. Machado, F. P. P., Queiróz, J. H., Oliveira, M. G. A., Piovesan, N. D., Peluzio, M. C. G., Costa, N. M. B., & Moreira, M. A. (2008). Effects of heating on protein quality of soybean flour devoid of Kunitz inhibitor and lectin. Food Chemistry, 107(2), 649–655. National Research Council. (1995). Nutrient Requirements of Laboratory Animals (Fourth Rev.). Washington, DC: The National Academies Press. Otten, J.J., Hellwig, J.P. & Meyers, L.D. (2006). Dietary Reference Intakes : The Essential Guide to Nutrient Requirements. Washigton, DC : The National Academics Press. Parsons, C. M., Hashimoto, K., Wedekind, K. J., & Baker, D. H. (1991). Soybean protein solubility in potassium hydroxide: an in vitro test of in vivo protein quality. Journal of Animal Science, 69, 2918–2924. Rayner, C. J. (1985). Protein hydrolysis of animal feeds for amino acid content. Journal of Agricultural and Food Chemistry, 33(4), 722–725. Reeds, P., Schaafsma, G., Tome, D., & Young, V. (2000). Criteria and significance of dietary protein sources in human; summary of the workshop with recommendations. Journal of Nutrition, 130(7), 1874S–1876. Renuka Devi, R., & Arumughan, C. (2007). Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment. Bioresource Technology, 98, 3037–3043.
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Saunders R.M. (1990). The properties of rice bran as a food studd. Cereal Food World. 35, 632-662. Schneider, B.H, & Flatt, W.P. (1975). The evaluation of feeds through digestibility experiments. Athens,GA : AthensUniversity of Georgia Press. Schramm, R., Abadie, A., Hua, N., Xu, Z., & Lima, M. (2007). Fractionation of the rice bran layer and quantification of vitamin E, oryzanol, protein, and rice bran saccharide. Journal of Biological Engineering, 1, 9. Shoji, Y., Mita, T., Isemura, M., Mega, T., Hase, S., Isemura, S., & Aoyagi, Y. (2001). A Fibronectin-binding Protein from Rice Bran with Cell Adhesion Activity for Animal Tumor Cells. Bioscience, Biotechnology, and Biochemistry, 65(5), 1181–1186. Steel, R.G.D., Torrie, J.H. (1981). Principles and procedures of statistics: a biomedical approach (2nd ed.), New York, NY : McGraw-Hill International Book Co.
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Table 1 Ingredients of experimental diets Ingredient (g/100g of diet) Casein RBP ISP WPI REP Glucose Corn Starch Soy oil α-cellulose NaCl Choline bitartrate Vitamin Mix1 Mineral Mix1 Potassium phosphate
Casein
RBP
ISP
WPI
REP
N-Free
12.20 30.80 40.00 7.00 5.00 0.25 0.25 1.00 3.50 -
14.50 28.20 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
11.70 31.00 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
12.80 29.85 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.35
12.70 30.00 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.30
42.65 40.00 7.00 5.00 0.25 0.25 1.00 3.50 0.35
1 By AIN-93 vitamin and mineral mixture (Dyets, Inc., Bethlehem, PA) RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
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Table 2 Chemical and essential amino acid composition of protein sources RBP Casein Chemical composition (g/100g dry matter) Crude protein 74.93 90.02 Crude fat 3.61 0.18 Crude fiber 0.00 0.26 Ash 14.28 5.08 Nitrogen free extract 7.18 4.46 Essential Amino acid (g /100 g protein) Threonine 3.68 4.30 Valine 5.53 5.88 Isoleucine 3.61 4.88 Leucine 7.69 9.73 Phenyalanine + tyrosine 8.24 10.73 Lysine 4.55 8.15 Histidine 4.48 2.97 Methionine + cystine 2.70 2.94 Tryptophan 1.17 1.05
ISP
WPI
REP
91.94 0.37 0.08 4.06 3.55
86.09 0.20 0.07 3.03 10.61
86.89 0.34 0.08 3.57 9.12
3.67 4.16 4.30 7.84 8.65 6.14 2.58 2.55 1.28
7.52 5.57 6.24 10.9 5.94 9.96 1.97 5.36 1.72
3.46 5.12 3.80 8.15 10.09 3.31 2.46 3.88 0.82
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein Nitrogen free extract = 100 – (moisture content + crude protein + crude fat + crude fibre + ash)
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Table 3 Pepsin digestibility and KOH solubility of protein sources
In vitro digestibility
RBP
Casein
ISP
WPI
REP
Pepsin digestibility(%) KOH solubility(%)
89.8 91.5
89.2 94.0
93.7 85.0
98.8 93.3
94.6 44.5
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
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Table 4 Effects of protein sources on the body weight gain, feed intake and feed/gain ratio of rats Protein sources RBP Casein ISP WPI REP N-free 1
Body weight gain1 (g/rat) 54.6±13.0b 55.7±9.1 b 40.1±10.0c 79.3±12.6a 47.1±10.0abc -30.9±3.6d
Feed intake1 (g/rat) 222.4±28.1a 214.7±21.3a 218.1±23.6a 235.2±18.9a 233.0±25.9a 138.0±7.4b
Feed/Gain ratio1 4.21±0.74b 3.91±0.44c 5.61±0.87a 3.04±0.57c 5.13±1.05a -
Values are for 10 animals in each group and different letters mean significant differences at p<0.05.
RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
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Table 5 Protein intake, protein efficiency ratio (PER), net protein retention (NPR), net protein utilization (NPU) value of each protein source. Protein sources RBP Casein ISP WPI REP
Protein intake (g/rat) 22.7±2.9 a 21.6±2.1 a 23.3±2.5 a 24.3±1.9 a 24.1±2.7 a
PER1
NPR1
NPU1
2.39±0.38 b 2.58±0.27 b 1.71±0.28c 3.26±0.43a 1.96±0.41c
3.77±0.36b 4.02±0.25a 3.04±0.23c 4.54±0.43a 3.26±0.45c
70.7±16.4ab 59.1±12.6 a 59.9±16.7 a 74.0±7.2b 61.4±8.9a
1 Values are for 10 animals in each group and different letters mean significant differences at p<0.05. RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
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Table 6 Nitrogen intake, faecal nitrogen, true digestibility (TD), biological value (BV) and protein digestibility-corrected amino acid score (PDCASS) value of each protein source. Protein source RBP Casein ISP WPI REP
N intake1 (g/rat) 1.22 1.12 1.12 1.30 1.21
Fecal N1 (g/rat) 0.189 0.189 0.221 0.220 0.239
TD12
BV1
PDCAAS3
94.8±0.5a 94.8±0.7a 91.7±1.5c 92.8±0.7b 90.8±0.6c
72.6±0.4 c 59.7±0.4 a 66.6±1.1 b 78.8±0.6 d 66.7±0.4 b
0.90 1.00 0.95 1.00 0.63
1 Values are for 8 animals in each group and different letters mean significant differences at p<0.05 2 Endogenous faecal N loss was 0.126 g/rat in N-free diet group 3 The amino acid scores used to calculated PDCASS were based on the FAO/WHO/UNU reference RBP: Rice bran protein, ISP: Isolated soy protein, WPI: Whey protein isolate, REP: Rice endosperm protein
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The first detailed in vitro and in vivo protein quality measurement of rice bran protein.
The rice bran protein (RBP) has better protein quality than vegetable protein.
RBP is comparable to casein and can replace it as non-animal protein source.
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