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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