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Journal of Medicinal Plants Research Vol. 6(12), pp. 2396-2401, 30 March, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1190 ISSN 1996-0875 ©2012 <strong>Academic</strong> <strong>Journals</strong> Full Length Research Paper In vitro antioxidant capacities of rice residue hydrolysates from fermented broth of five mold strains Wei Tian 1 , Qinlu Lin 1,2* and Gao-Qiang Liu 2,3* 1 College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, P. R. China. 2 National Engineering Laboratory for Rice and By-product Further Processing, Central South University of Forestry and Technology, Changsha 410004, P. R. China. 3 College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004, P. R. China. Accepted 22 February, 2012 Rice residue was fermented by five different strains of molds, namely, Aspergillus oryzae, Mucor racemosus, Rhizopus oligosporrus, Aspergillium niger and Penicillium glaucum. Antioxidant activities of the fermented products, rice residue hydrolysates (RRHs) were evaluated using ferric reducing antioxidant power (FRAP) assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay, respectively. Among five types of RRHs, RRHs from fermented broth of A. niger (RRHsIV) exhibited higher antioxidant potential than those of other RRHs in the same concentration level, regardless of the applied assays. Moreover, there was no correlation between reducing power and total phenonic contents in the five RRHs. However, FRAP values were highly correlated with phenol contents. Key words: Rice residue, hydrolysates, antioxidant capacity, fermentation. INTRODUCTION Reactive oxygen species (ROS) including oxygencentered radicals and some non-radical derivatives of oxygen cause oxidative stress to cells. Oxidative stress can be defined as an imbalance between pro-oxidant/free radical production and opposing antioxidant defenses. It has been reported that acute and chronic oxidatives stress implicated in degenerative diseases, such as athrosclerosis, diabetes mellitus, ischemia/reperfusion injury, Alzheimer‟s disease, inflammatory diseases, carcinogenesis, neurodegenerative diseases, hypertension, ocular diseases, pulmonary diseases and hematological diseases (Maxwell, 1995; Opara, 2004).ROS can be scavenged by exogenously obtained antioxidants, which include synthetic antioxidants and natural antioxidants. The use of synthetic antioxidants, because of their potential health hazard and toxicity, is under strict regulation. Thus, there is recently an upsurge of interest *Corresponding author. E-mail: gaoliuedu@yahoo.com.cn. Tel/Fax: +86 731 8562 3498. in antioxidative compounds in many natural resources. Hydrolysates derived from dietary source have been demonstrated to possess potent antioxidant capacity. It is likely due to an array of antioxidative components (low molecular peptides, oligosaccharides and Maillard reaction products) formed during hydrolytic process (Mendis et al., 2005; Wang et al., 2010; Dittrich et al., 2003). Rice residue is the by-product of rice in the processing of starch sugar and monosodium glutamate, which contains more than 50% protein as well as 40% polysaccharide and dextrin, according to our previous determination. Whereas, it is usually used as an animal feed without efficient utilization in China. Microbial sources have been shown to be a potential means of producing natural antioxidants (Ishikawa, 1992). It has been reported that many molds produced antioxidants that can be extracted from broth culture filtrates by ethyl acetate (Yen and Lee, 1996). Yen et al. (2003) reported that antioxidant activity of ethyl acetate extracts from rice koji showed a marked antioxidants activity on radical scavenging effect and inhibitory effect on peroxidation of linoleic. Due to its abundant nitrogen and carbon sources,
ice residue is an ideal raw material for producing antioxidant hydrolysates by fermentation. To the best of our knowledge, so far, there is no report on antioxidant substances from rice residue hydrolysates (RRHs) fermented with molds. In this study, RRHs were produced during fermentation by five different strains of molds, namely, Aspergillus oryzae, Mucor racemosus, Rhizopus oligosporrus, Aspergillium niger and Penicillium glaucum. Furthermore, we investigated the total antioxidant capacity of fermented prepared RRHs via 1,1-diphenyl-2picrylhydrazyl (DPPH) assay, 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay and ferric reducing antioxidant power (FRAP) assay. In addition, we evaluated relationship between RRHs concentration and antioxidant potency. The objective of the present study was the in vitro antioxidant capacities of different RRHs fermented with different strains of molds. The obtained information will be helpful not only in optimization of fermentation process for further study, but also in providing a new way to improve the utilization value of rice residue. MATERIALS AND METHODS Rice residue is industrially produced by Hunan JinJian Cereals Industry Co., Ltd (Hunan, China). A. oryzae, M. racemosus, R. oligosporrus, A. niger and P. glaucum were purchased from Institute of Microbiology Chinese Academy of Science (Beijing, China). The 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox), ABTS, 2,4, 6-tripyridyls-triazine (TPTZ) and DPPH were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade. Preparation of RRHs The five different strains of molds were maintained in solid potato dextrose agar medium at 4°C, respectively. For primary fermentation, 14 ± 0.5 g rice residues were mixed with 14 ± 0.5 ml distilled water. After sterilization at 121°C for 30 min, the mixture was inoculated by A. oryzae, M. racemosus, R. oligosporrus, A. niger and P. glaucum as seed mediums, respectively. Fermentation was carried out at 28 ± 0.5°C for 7 days. For secondary fermentation, 150 ml sterile water was added into five sorts of seed mediums previously mentioned, respectively. Then, all the samples were incubated at 42 ± 0.5°C, in an electroheating standing-temperature cultivator for 7 days. After secondary fermentation, the fermented broth was centrifuged at 4000 g, 10 min and the supernatant was dried to powder by freeze dehydration, which was for usage of analysis. Determination of total phenolic compounds The content of total phenolic compounds of RRHs fermented by five different strains of molds was determined according to the method described by Singleton et al. (1999). Briefly, 0.5 ml of RRHs solution was transferred to a volumetric flask and the volume was adjusted to 46 ml by addition of distilled water. One milliliter (1 ml) Tian et al. 2397 of Folin-Ciocalteu reagent was then added to this mixture. After 3 min, 3 ml of sodium carbonate solution (20 g/l) was added to the volumetric flask. Subsequently, the mixture was shaken mechanically for 2 h at room temperature. The absorbance was measured at 760 nm using a ultraviolet (UV)-vis spectrophotometer. The content of total phenolic compounds of RRHs fermented by five different strains of molds was calculated using a standard curve prepared with gallic acid. Determination of degree of hydrolysis (DH) DH is operationally defined as the percentage of free N-terminal amino groups cleaved from proteins, which is calculated from the ratio of α–amino nitrogen to total nitrogen (You et al., 2010). According to the method of Nilsang et al. (2005), the amino nitrogen content was determined by a formaldehyde titration method. The total protein content was determined by Keldahl method (Chandi and Sogi 2007). The equation of DH is shown as follows: DH = [Amino nitrogen content (mg/ml) / total protein content (mg/ml)] × 100 Antioxidant activities of RRHs FRAP assay The FRAP assay was carried out according to the procedure of Benzie and Strain (1996). FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe(II) -tripyridyltriazine compound from colorless oxidized Fe(III) form by the action of electron donating antioxidants. Briefly, the FRAP reagent was prepared from acetate buffer (0.3 mol/L, pH 3.6), 10 mmol/L TPTZ solution in 40 mmol/L HCl and 20 mmol/L iron(III) chloride solution in proportions of 10: 1:1 (v/v), respectively. 3 ml of working FRAP reagent prepared daily was mixed with 100 ul of diluted sample; the reaction mixture was recorded at 593 nm, after 30 min incubation at 37°C. The standard curve was depicted using iron(II) sulfate solution. FRAP values were expressed as mmol of Fe(II) equivalents/kg. All the measurements were carried out in triplicate and the mean values were calculated. DPPH assay The DPPH radical scavenging activity of RRHs was determined using the method proposed by Von Gadow et al. (1997). Aliquot (100 ul) of the tested RRHs sample was placed in a cuvette, and 2 ml of 0.1 mmol/L methanolic solution of DPPH radical was added. Absorbance measurements commenced immediately. The decrease in absorbance at 517 nm was determined after 15 min for all samples. Methanol was used to zero spectrophotometer. The absorbance of the DPPH radical without antioxidant (control) was measured daily. Methanolic solutions of Trolox were tested. All determinations were performed in triplicate. The percentage inhibition of the DPPH radical by the samples was calculated according to the formula of Yen and Duh (1994): % inhibition = [(AC (0) )-AA (t) /AC (0) ] × 100 Where AC (0) is the absorbance of the control at t = 0 min, AA (t) is the absorbance of the antioxidant at t = 15 min ABTS assay The method is based on the ability of antioxidant molecules to
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Journal of Medicinal Plants Researc
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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Figure 1. Chuanxiong Rhizoma (http:
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Table 1. Identification of main pea
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Table 3. Pharmacokinetic parameters
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active component study, many invest
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and 1185 kg ha -1 in the first site
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Table 1. Recommendation for use fer
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Sadanandan AK, Hamza S (1996c). Stu
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and it has been traced to South Ame
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Table 3. Results of phytochemical a
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(a) (b) (c) Figure 1. (a) Normal ce
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emedies in the south of Brazil. J.
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known fatty acids and terpenoids we
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Table 1. Renal function tests of ra
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Al-Radahe et al. 2271 Figure 3.nnnn
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namely disruption of the vascular e
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Zayachkivska OS, Konturek SJ, Drozd
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β-carotene were purchased from Sig
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Figure 1. Effect of ethanol concent
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Table 3. Climatic and soil factors
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content of polysaccharides. Phospha
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Serge et al. 2285 Table 1. Relative
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Percentage inhibition % of inhibiti
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Table 1. Result of phytochemical sc
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Table 3. Result of thin layer chrom
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Table 2. MIC of a compound 1 from c
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Zirihi et al. 2301 Figure 2. Termin
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Figure 5. T. catappa Linn. (Combret
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Figure 8. A. fumigatus: Aspect of c
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Figure 12. Sensitivity of A. fumiga
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Noormi et al. 2311 Table 1. Callus
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Table 2. Contd. NoC=No callus. 4.0
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Figure 2. Contd. at 2.0 mg/L yellow
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Table 1. Biochemical parameters in
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Figure 3. Bcl-xL reactions in inter
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ACKNOWLEDGMENTS The authors would l
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Figure 1. The first page of the boo
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Figure 3. The most widely included
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Figure 4. Lithographic print “St.
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Table 1. List of plants included in
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Table 1. Contd. Nedelcheva 2333 Cin
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Table 1. Contd. Nedelcheva 2335 Rhe
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14 7 1 15 1 35 17 16 Imported plant
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Academy of Science Publishing House
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et al., 1996; van Wyk and Gericke,
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DPPH inhibition (%) DPPH % inhibiti
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2466 J. Med. Plants Res. AOAC (2000
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UPCOMING CONFERENCES 15th European
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