Biocatalysis and Agricultural Biotechnology 23 (2020) 101510

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Changes in rice bran bioactives, their bioactivity, bioaccessibility and

bioavailability with solid-state fermentation by Rhizopus oryzae
G. Janarny , K.D.P.P. Gunathilake *
Department of Food Science and Technology, Faculty of Livestock, Fisheries, and Nutrition, Wayamba University of Sri Lanka, Makandura, Gonawila, Sri Lanka

A R T I C L E I N F O A B S T R A C T

Keywords: Rice bran is a rich source of bioactives but an underutilized agro-industrial residue for human consumption.
Solid-state fermentation Solid-state fermentation (SSF) has been employed as a processing technique to increase the content of functional
Bioactives components from rice bran. This study has been carried out to examine the effect of SSF by Rhizopus oryzae on the
Bioaccessibility
bioactive content, bioactivity, bioaccessibility and bioavailability of 4 different varieties of rice bran. In vitro
Bioavailability
digestion with dialysis was carried out to assess the bioavailability and bioaccessibility of rice bran bioactives.
Methanolic extracts and digested fractions of fermented and unfermented rice brans were analyzed for total
phenolic (TPC), flavonoid (TF), carotenoid (TC), and total anthocyanin content (TAC). Additionally, antioxidant
activity, anti-inflammatory property and the anti-diabetic property was also evaluated. The results indicate that
TPC, TF, TAC, TC, total antioxidant capacity and ferric reducing power of Bg 352 and Bw 367 have increased
with SSF. Anti-inflammatory properties of all samples have increased with SSF whereas anti-diabetic properties
of all samples have decreased with SSF. Bg 406 and H4 have shown an increase in the bioaccessible and
bioavailable percentage of phenolics and flavonoids with SSF. Radical scavenging ability of digested fractions of
all varieties of rice bran has decreased with SSF. Among the digested fractions of fermented samples H 4
expressed the highest anti-inflammatory property and Bg 406 expressed the highest anti-diabetic property in all
phases of digestion. The results of this study indicate that SSF can be used to enhance bioactive content and
bioactivity of rice bran under appropriate conditions.

1. Introduction oxidative stress. Oxidative stress-induced damages in biological systems

Recently agro-industrial residues have gained attention for their
potential to provide valuable bioproducts. In line with the present ten­
dency for preventing disease through healthy food, the key feature that
consumers demand is food with health benefits. This demand lead to the
investigation of these residues and their beneficial effects on biological
systems. Such one abundantly available residue and a rich source of
bioactives that is of great interest at present is the rice bran. Rice is the
second most common agricultural commodity cultivated around the
world especially in developing countries. During paddy processing, rice
bran is obtained as a byproduct of milling. Milling of paddy yields 8%
rice bran (Chakraborthy et al., 2018). Though a huge amount of rice
bran is produced they remain to be an underutilized commodity for
human consumption, especially in Sri Lanka.
A higher prevalence of chronic disease at present has directed the
focus of food scientists and researchers on bioactive compounds due to
their ability to reduce the risk of chronic diseases by controlling
have been identified as one of the major causes of the higher prevalence
of chronic diseases like cardiovascular diseases, cancer, diabetes melli­
tus and neurodegenerative diseases (Dey and Kuhad, 2014). Epidemio­
logical studies reveal that bioactive compounds exert various
physiological effects on the human body including antioxidant activity,
prevention of chronic inflammation, platelet aggregation, cancer cell
invasion, anti-diabetic, anti-coagulant, anti-tumor and antimicrobial
properties (Seechamnanturakit et al., 2018)
As bioaccessibility and bioavailability of bioactives strongly influ­
ence the physiological function exerted by them, numerous processing
techniques are being employed to increase the bioaccessibility and
bioavailability of bioactive compounds from the plant matrix. Structural
and chemical modifications done to the bioactive compounds during
processing have a great impact on the release and absorption of these
compounds during the transition at digestion. An alternative efficient
and economically viable processing technique that is being adopted
recently to enhance bioactive content from agro-industrial residue is

* Corresponding author.
E-mail address: kdppgunathilake@yahoo.com (K.D.P.P. Gunathilake).

https://doi.org/10.1016/j.bcab.2020.101510
Received 8 September 2019; Received in revised form 31 December 2019; Accepted 21 January 2020
Available online 23 January 2020
1878-8181/© 2020 Elsevier Ltd. All rights reserved.
G. Janarny and K.D.P.P. Gunathilake
Solid State Fermentation. Bacteria and fungi are employed for this
purpose industrially, however mostly fungi are employed for this pur­
pose due to their ability to produce several extracellular enzymes and
tolerate and grow in any adverse conditions. This study aims to evaluate
the impact of SSF by Rhizopusoryzaeon the content of various bioactive
compounds of rice bran, and their bioactivity. Additionally, the influ­
ence of gastro-intestinal digestion on the bioactive content of rice bran
and their bioactivity also was studied.
2. Materials and methodology
2.1. Chemicals and reagents
Dichloran Rose Bengal Chloramphenicol Agar (DRBC agar), Lacto­
phenol cotton blue, Folin-Ciocalteu reagent, Gallic acid, Rutin, DPPH
solution, Ascorbic acid, Bovine serum albumin, Porcine pepsin, pig
pancreatin, bile salt, Phosphate buffered saline, alpha-amylase and 3,5-
Dinitrosalicylic acid were purchased from Sigma Aldrich. All other
chemicals used were of analytical grade.
2.2. Sample preparation
Four different varieties of rice bran samples were collected from Rice
Research Institute of Sri Lanka. (White rice varieties - Bg352, Bw367 and
Red rice varieties - Bg406, H 4). The collected bran samples were sta­
bilized by autoclaving at 115 ̊C for 5 min according to Moongngarm
et al. (2012) and defatted using hexane as described by Wang et al.
(1999). The defatted bran was air-dried overnight at room temperature
and ground using the laboratory grinder (Allianz industries, Noida -
India). The groundmass was sieved to get particles of size 0.35 mm–0.7
mm. Sieved bran samples were packed in separately labeled poly­
ethylene bags and stored at 4 ̊C until further analysis.
2.3. Isolation of Rhizopus oryzae
Indigenous strains of Rhizopus oryzae were isolated as described by
Kurniawati et al. (2014). Accordingly ripened Guava fruit was stored at
room temperature for 5 days. After a visible brown colony appeared on
the fruits, the infected parts were cut and surface disinfected by 0.4%
chlorine solution for 2 min and rinsed with distilled water for 2 min. The
disinfected part was then cut into small pieces and plated on DRBC agar
by a direct plating method. The plate was incubated at 40 ̊C for 5 days.
After incubation, a small number of fungal mycelia was scrapped using a
sterilized spatula and plated onto PDA agar, this medium was incubated
for 5 days at 40 ̊C to isolate the Rhizopusoryzae.
2.4. Preparation of spore suspension
A small number of mycelia was scrapped with a sterilized needle and
centrifuged (Hettich- Model D-78532, Germany) at a low speed to
separate the spores. After centrifugation, the solution was filtered using
Whatman # 1 filter paper and spores were collected. The spores were
suspended in distilled water and the spore solution was stored at -4 ̊C
until utilized.
2.5. Fermentation of rice bran
The procedure described by Kupski et al. (2012) was adopted to
ferment the rice bran. Fermentation of each variety was carried out
separately with 25 g of rice bran in a sterilized beaker. Initially, the rice
bran was homogenized with nutrient solution (KH2PO4 2 gL-1, MgSO4 1
gL-1, NH2CONH2 1.8 gL-1) at ratios of bran: nutrient solution 100: 45
(W/V). Then spore solution was added at a concentration of 4 � 106
spores/g bran. Distilled water was added to maintain 50% moisture. The
mixture was fermented for 96 h at 30 ̊C. At the end of fermentation, the
fermented mass was autoclaved at 105 ̊C for 5 min and then dried for 24
2
Biocatalysis and Agricultural Biotechnology 23 (2020) 101510
h. The dried fermented biomass was packed in separately labeled
polythene bags and stored at -18 ̊C until further analysis.
2.6. Preparation of methanolic extract
Sample extracts were obtained by simple solvent extraction accord­
ing to the method described by Wen (2015). Extract for analysis from
fermented and unfermented rice bran was prepared by mixing 5 g of rice
bran with 50 mL of methanol and stirring the mixture continuously for
30 min at room temperature. Then the mixture was centrifuged at 1000
rpm for 10 min. The supernatant was collected. The residual rice bran
was extracted again twice and all the supernatant was combined. The
solvent was evaporated at 35 ̊C using a rotary evaporator (Hahn Shin
Scientific, Korea), and the concentrated mixture was lyophilized for 24 h
and stored at -18 ̊C until further analysis.
2.7. In vitro gastrointestinal digestion
Rice bran was subjected to in vitro gastrointestinal digestion ac­
cording to the method described by Gunathilake et al. (2018,a,b). About
10 g of fermented and unfermented rice bran were ground using the
laboratory grinder for 1 min and the ground rice bran samples were
mixed with 50 mL, 0.9% NaCl solution and 4.0 mL pepsin solution (40
mg/mL in 0.1 M HCl). Then 0.1 M HCl was added to maintain the pH at
1. Using a shaking water bath (Daihen Lab tech, Korea) the mixture was
incubated at 37 ̊C for 1 h at 100 rpm. Aliquots of digested samples were
separated and filtered using Whatman # 42 filter paper and stored at -18
C̊ until further analysis.
The intestinal phase with dialysis was carried out using dialysis
tubing cellulose membrane (average flat width; 33 mm, MWCO 12,000
Da). Segments of 15 cm length were cut and both outer and inner sur­
faces of the membrane were rinsed using 0.9% NaCl solution. It was
made into a bag by fastening with clips at one end. Then it was filled
with 5.5 mL of 0.9% NaCl and 5.5 mL of 0.5 M NaHCO3. The bag was
entirely sealed and completely immersed into each gastric digesta. Using
shaking water bath the bag was incubated at37 ̊C for 45 min at 100 rpm.
Then using NaHCO3the pH of the mixture was brought to 6.5. The
pancreatin-bile mixture was prepared with 2 mg/mL pancreatin and 12
mg/mL bile extract dissolved in 0.1 M NaHCO3and was added to each
digesta. This mixture was incubated at 37 ̊C for 2 h. At the end of in­
cubation pH of each digesta was measured and aliquots from the in­
testinal digesta were separated for analysis, filtered using Whatman #
42 filter paper and stored at -18 ̊C until further analysis. The dialysis
bags immersed in digesta were separated from the beakers and carefully
rinsed with water and then dried using a paper cloth and weighed. The
mixture in the dialysis bag was transferred to a measuring cylinder and
the final volume was brought up to 14 mL by adding 0.9% NaCl, and
filtered using Whatman # 42 filter paper. The content was stored at -18
C̊ until further analysis.
2.8. Determination of total phenolic content
The total phenolic content of the methanolic extract as well as the
digested fractions were analyzed by the Folin-Ciocalteau method as
described in Gunathilake and Ranaweera (2016). Methanolic extrac­
ts/digested fractions of 10 μL were mixed with 50 μL of Folin-Ciocalteu
reagent and incubated at dark for 15 min at room temperature. Then 40
μL of 7.5% (W/V) Na2CO3 was added to the reaction mixture and
incubated at room temperature for 2 h in dark. The absorbance of the
mixture was measured at 765 nm using a UV/VIS spectrophotometer (JP
Selecta, Spain). Gallic acid was used for the standard curve preparation
and the total phenolic content was expressed as mg gallic acid equiva­
lents (GAE) per g of rice bran on a fresh weight basis (FW).
G. Janarny and K.D.P.P. Gunathilake
2.9. Determination of total flavonoid content
The total flavonoid content of the extract and digested samples was
determined using the aluminum chloride method of Zhishen et al.
(1999) with some modifications described in Gunathilake et al. (2018,a,
b). Accordingly, 1 mL of methanolic extracts/digested fractions were
mixed with 4 mL of distilled water and 0.3 mL of 5% NaNO2. After 5 min
3 mL of 10% AlCl3 was added to the mixture and mixed well and
allowed to stand for 6 min. Then 2 mL of 1 M NaOH was added and
mixture and volume up to 10 mL with distilled water and mixed well.
The absorbance was measured at 510 nm using a UV/VIS spectropho­
tometer. The total flavonoid content was expressed as mg rutin equiv­
alents (RE) per g of rice bran on a fresh weight basis (FW).
2.10. Determination of total anthocyanin content
Methanolic extract and digested fractions were analyzed for total
anthocyanin content by the pH differential method as described by
Giusti and Wrolstad (2001). Accordingly, 25 μL of the test sample will be
mixed with 175 μL of 0.024 M KCl buffer at pH 1 and incubated for 15
min at room temperature. The absorbance was measured at 510 nm and
700 nm. Another 25 μL of the test sample was mixed with 175 μL of
0.025 M sodium acetate at pH 4.5, the mixture was incubated at 15 min
and the absorbance was measured at 510 nm and 700 nm. The mono­
meric total anthocyanin content will be calculated and the results were
expressed as mg cyanidin-3-glucoside equivalents (cy 3-glc E).
2.11. Determination of total carotenoids
The total carotenoid content was analyzed according to the method
described by Sukran et al. (1998). Test samples of 0.5 mL were separated
and the absorbance was read at 470, 653, and 666 nm on UV/VIS
Spectrometer. Total carotenoid content was calculated according to the
formulas of Kichtenthaler and Wellburn (1983).
Chlorophyll aðCaÞ ¼ 11:75ðA662Þ 2:350ðA645Þ:
Chlorophyll bðCbÞ ¼ 18:61ðA645Þ 3:960ðA662Þ:
Carotenoids ¼ 1000ðA470Þ 2:270ðCaÞ 81:4ðCbÞ = 22
2.12. DPPH radical scavenging activity
DPPH radical scavenging activity of methanolic extracts and diges­
ted fractions were measured using the method in Gunathilake and
Ranaweera (2016). Accordingly, 3.9 mL of freshly prepared DPPH so­
lution was mixed with 0.1 mL of the sample and vortexed well. Then it
was incubated at room temperature in the dark for 30 min. DPPH so­
lution without the sample was used as the control. Then the absorbance
of the mixture was measured at 517 nm using UV/Visible spectropho­
tometer and the percentage inhibition of radical scavenging was
calculated.
2.13. Total antioxidant capacity
A method described by Prieto et al. (1999) mentioned in Gunathilake
and Ranaweera (2016) was used to estimate the total antioxidant ca­
pacity. Test samples of 0.3 mL were mixed with 3 mL of phosphomo­
lybdenumreagent and incubated at 90 ̊C for 90 min. Absorbance was
measured at 695 nm and ascorbic acid was used as the standard and total
antioxidant capacity was expressed as mg ascorbic acid equivalents
(AAE) per g of Fresh Weight.
2.14. Ferric reducing power
The method described byOyaizu (1986), mentioned in Gunathilake
3
Biocatalysis and Agricultural Biotechnology 23 (2020) 101510
and Ranaweera (2016) was used to determine the reducing power of
methanolic extracts and digested fractions. Accordingly, 1 mL of sample
was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of
a 1% (W/V) of potassium ferricyanide. The mixture was incubated in a
water bath at 50 ̊C for 20 min and then 2.5 mL of 10% (w/v) tri­
chloroacetic acid solution was added and the mixture was then centri­
fuged for 10 min at 3000 rpm. A 2.5 mL aliquot of the upper layer was
mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) ferric
chloride solution. The absorbance of the reaction mixture was read using
a UV/VIS spectrometer at 700 nm.
2.15. Anti-inflammatory properties
Protein denaturation assay described by Gambhire et al. (2009) with
some modifications described by Gunathilake et al. (2018,a,b) was fol­
lowed to evaluate the anti-inflammatory properties. Accordingly, 0.2 mL
of 1% bovine albumin, 4.78 mL of phosphate-buffered saline (PBS, pH
6.4) and 0.02 mL methanolic extract or digested fractions were mixed
and was incubated at 37 ̊ C for 15 min in a water bath. After incubation,
the reaction mixture was heated at 70 ̊ C for 5 min. After cooling the
absorbance of the solution was measured at 660 nm using a UV/Visible
spectrophotometer. PBS without sample was used as the control and the
percentage inhibition of protein denaturation was calculated.
2.16. Anti-diabetic properties
Alpha-amylase inhibition assay was performed based on the method
adopted from Bernfeld (1955) was used to evaluate the anti-diabetic
properties. Accordingly, 100 μL of the sample was mixed with 200 μL
of α-amylase enzyme and 100 μL of 2 mM of phosphate buffer (pH-6.9).
After 20 min of incubation, 100 μL of a 1% starch solution was added.
The same was performed for the controls where 200 μL of the enzyme
was replaced by the buffer. After incubation for 5 min, 500 μL of 3,
5-dinitrosalicylic acid reagent was added to both control and test.
They were kept in a boiling water bath for 5 min. The absorbance was
recorded at 540 nm using UV/Visible spectrophotometer and the per­
centage inhibition of the α-amylase enzyme was calculated.
2.17. Statistical analysis
All data were analyzed in triplicate with random sampling. Oneway
analysis of variance (ANOVA) test was used to analyze the data and
results are presented as the mean � standard deviation for all in vitro
assays. Oneway ANOVA was performed using SPSS 16. All the statistical
tests were conducted at a significance level of 5%.
3. Results& discussion
Rice bran has been identified as a rich source of several bioactive
compounds and this study evaluated total phenolic content, total
flavonoid content, total carotenoid content and total anthocyanin con­
tent of rice bran. This study has mainly focused on the changes in the
bioactive content and their bioactivity with solid-state fermentation by
Rhizopusoryzae. Additionally, the impact of gastro-intestinal digestion
on the bioactive content and bioactivity of unfermented and fermented
rice bran also has been evaluated.
The effect of SSF on the bioactive content of rice bran was evaluated
by comparing the results obtained for the unfermented and fermented
samples. Each sample was analyzed for the total phenolic content (TPC),
Total flavonoid content (TFC), Total anthocyanin content (TAC) and
Total carotenoid content (TC). The results obtained are summarized in
Table 1. According to the results, compared to the extract of white rice
bran varieties (Bw367 and Bg352) extract of red rice bran varieties
(Bg406 and H 4) had a higher TPC. With fermentation, there was a
significant increase (P < 0.05) in the TPC of white rice bran varieties.
TPC of Bw367 and Bg352 increased by 65.00% and 78.81% respectively.
G. Janarny and K.D.P.P. Gunathilake Biocatalysis and Agricultural Biotechnology 23 (2020) 101510

Table 1 be validated based on the studies of Sa

�nchez (2009). Accordingly, Fungi
Content of total phenolic, flavonoid, anthocyanin and carotenoids in unfer­ have two types of extracellular enzymatic system: the hydrolytic system,
mented and fermented rice bran. which produces hydrolases that are responsible for polysaccharide
Rice bran TPC (mg TFC (mg RE/ TAC (mg Cy 3- TC (mg/g degradation and a unique oxidative and extracellular ligninolytic sys­
GAE/gFW) gFW) glc/g FW) FW) tem, which degrades and opens phenyl rings. During fermentation,
Bw367 Rhizopusoryzaeproduces extracellular enzymes such as alpha-amylase,
Unfermented 5.33 � 1.14b 5.20 � 2.34c 0.10 � 0.03d 62.49 � beta-glycosidase, and xylanase which degrade lignocellulosic materials
4.32a and releases phenolic compounds. This can be explained as one of the
Fermented 8.81 � 1.24a 14.75 � 1.65 � 0.14a 71.82 �
major reasons for the increase in phenolic content of Bw367 and Bg352
5.76b 5.23a
Bg352 with fermentation. Schmidt et al. (2014) showed that in fermented rice
Unfermented 4.43 � 2.12b 2.11 � 0.15d 0.52 � 0.04c 1.03 � bran content of chlorogenic acids, p-hydroxybenzoic acids and vanillin
0.02d increased throughout the fermentation, whereas gallic acid and caffein
Fermented 7.89 � 3.12a 29.16 � 1.72 � 0.19a 47.61 � acid increased until 72 h, and syringic and ferulic acids increased up to
3.23a 3.12b
Bg406
120 h of fermentation. The changes produced by fermentation on the
Unfermented 26.88 � 4.23a 18.44 � 3.12 9.55 � 2.32 a 26.11 � profile of phenolic acids depend on the type of substrate, the fungus used
b
6.12b and the conditions of fermentation (Martins et al., 2011). Another
a c c
Fermented 4.13 � 1.21 3.09 � 0.88 0.01 � 0.00 3.26 � possible reason for the increment in phenolic content with fermentation
1.12d
is the synthesis of new phenolic compounds due to fugal metabolism and
H4
Unfermented 28.96 � 4.12a 41.11 � 2.97 � 0.76b 11.48 � bioconversion of metabolites into soluble forms. The decrease in
5.23a 1.34c bioactive content of Bg406 and H 4 with SSF is mainly due to the
Fermented 6.29 � 2.12 a
7.17 � 0.22b 0.21 � 0.09b
9.08 � extended period of fermentation which causes the breakdown of
2.34c bioactive compounds. Depending on the variety mostly after 72 h of
TPC ¼ Total Phenolic Content, TFC ¼ Total Flavanoid Content, TAC ¼ Total fermentation a decrease in the TPC contents can be observed for some
Anthocyanin Content, TC ¼ Total Carotenoid, GAE ¼ Gallic Acid Equivalent, RE varieties. This is mainly due to the degradation of phenolic compounds
¼ Rutin Equivalent, Cy 3-glc ¼ Cyanidin-3-glucoside, FW ¼ Fresh Weight. and due to the oxidation of bioactive compounds during SSF.
Values are expressed as mean � SD. Values followed by different letters for each Antioxidant activity of unfermented and fermented rice bran samples
condition (unfermented and fermented)in the same column are significantly was evaluated using DPPH radical scavenging activity, Ferric reducing
different (P < 0.05). power assay (FRAP) and total antioxidant capacity assays. The results
obtained are summarized in Table 2. From the results obtained for DPPH
However, the TPC of red rice bran varieties showed a significant (P < radical scavenging activity, among the unfermented samples the highest
0.05) decrease with fermentation. TFC of unfermented bran sample and the lowest radical scavenging ability was shown by Bg352 and
extracts ranged from 2.11 to 41.12 mg RE/g FW. Similar to TPC, red rice Bw367 respectively. However, the radical scavenging ability of all four
bran varieties contained higher flavonoids compared to white rice bran rice bran samples increased with fermentation. The reducing power of
varieties. With fermentation TFC of Bw367 and Bg352 increased by the extracts of unfermented samples ranged from 2.71 mg AAE/g FW to
183.66% and 92.74% respectively whereas TFC of Bg406 and H 4 6.53 mg AAE/g FW. Reducing the power of Bw367 and Bg352 has
decreased by 83.16% and 82.51%. Total carotenoid content of unfer­ increased with fermentation whereas reducing the power of Bg406 and
mented samples ranged from 1.03 to 62.49 mg/g FW with Bw367 having H4 has decreased with fermentation. The total antioxidant capacity of
the highest content and Bg352 having the lowest carotenoid content. unfermented samples ranged from 11.06 to 24.53 mg AAE/g FW with
Similar to TPC and TF, carotenoid content of white rice bran samples the highest capacity shown by Bg406. With fermentation, the total
increased significantly (P < 0.05) with fermentation whereas TC of red antioxidant capacity of Bw367 and Bg352 has increased by 38.78% and
rice bran samples decreased with fermentation. When considering the 44.32% whereas the total antioxidant capacity of Bg406 and H4 has
TAC, it was observed that Bg406 had the highest TAC with 9.55 mg cy-3- decreased by 69.09% and 21.07% respectively.
g E/g FW at the same time the lowest TAC after fermentation also was The antioxidative potential of rice bran extracts is not due to any
observed in Bg406. TAC of Bw367 and Bg352 increased by 155.32% and single compound but the combined effect of dietary fibers, phenolic
229.31% respectively, whereas the TAC of H 4 decreased by 92.3% with compounds and other bioactive compounds present in the bran fraction.
fermentation. The present study shows remarkable differences in
different bioactive compounds in different rice bran varieties. Higher
phenolic, flavonoid, anthocyanin, and carotenoid contents were shown Table 2
in red rice bran varieties (Bg406 and H 4) compared to white rice bran Antioxidant activities of unfermented and fermented rice bran samples.
varieties (Bw367 and Bg352). Studies by Prabhu and Jayadeep (2015) Rice bran DPPH (IC50) FRAP (mg Total antioxidant capacity
indicate that generally, non-pigmented rice bran (white rice bran) pro­ (mg/L) AAE/gFW) (mg AAE/g FW)
vide only phenolic acids whereas pigmented rice varieties like red or Bw367
black rice are rich in pro-anthocyanins and anthocyanin depending on Unfermented 137.39 � 2.11a 3.79 � 1.11a 11.06 � 2.35b
the variety. As a result, red rice bran varieties show higher content of Fermented 65.39 � 3.35a 6.00 � 2.15a 15.35 � 4.65b
bioactive compounds compared to white rice bran varieties. Bg352
Unfermented 35.10 � 3.3c 6.53 � 1.15a 17.08 � 4.78b
As described by Zhang et al. (2010), Phenolic compounds of rice
Fermented 23.54 � 6.5c 8.73 � 3.45a 24.65 � 5.12a
mainly include derivatives of hydroxycinnamic acids and benzoic acids. Bg406
These are generally present as chains or components of hydrolysable Unfermented 125.05 � 2.71 � 0.87a 24.53 � 2.56a
tannins and lignin or linked to the cell wall structural components such 11.23a
Fermented 20.16 � 3.45c 0.47 � 0.03c 7.58 � 1.56b
as cellulose and proteins by ester linkages. The soluble phenolics are
H4
located within the cell vacuoles, either in free or conjugated form, while Unfermented 55.47 � 6.8b 5.63 � 4.32a 14.38 � 6.7b
the insoluble phenolics are esterified with arabinose or galactose resi­ Fermented 37.98 � 4.23b 0.92 � 0.01b 11.35 � 2.34b
dues of hemicellulose or pectic components (Mira et al., 2008). The
IC ¼ Inhibition concentration, AAE ¼ Ascorbic Acid Equivalent, FW ¼ Fresh
increase shown in phenolic compounds and flavonoids in Bw367 and
Weight Values are expressed as mean � SD. Values followed by different letters
Bg352 is mainly due to the release of bound phenolics that are bound to in the same column for each condition (unfermented and fermented) are
cell wall components. An increase in total phenolic content with SSF can significantly different (P < 0.05).

4
G. Janarny and K.D.P.P. Gunathilake
Different compounds exhibit antioxidant potential by different mecha­
nisms thus each antioxidant assays assesses the different mechanisms of
antioxidant property of rice bran extract. This study has used DPPH
radical scavenging assay to evaluate the free radical scavenging ability
of the rice bran and ferric reducing power assay to assess the reducing
power of the extracts. The free radical of DPPH is generally quenched by
phenolic compounds by donating either electron or hydrogen atoms. A
higher percentage of DPPH radical scavenging activity of extracts in­
dicates higher antioxidant activity of samples in terms of hydrogen
donating capacity. (Amarowicz et al., 2004). The present study indicates
that DPPH radical scavenging activity of all rice bran increased with SSF,
and these results are similar to the result of Jung et al. (2017) obtained
for fermented Korean rice bran varieties. In the case of Bw367 and
Bg352 increase in phenolic content with SSF can be suggested as the
possible reason for the increased scavenging activity with SSF. Despite
the reduction of phenolic content with SSF, Bg406 and H4 showed
increasing radical scavenging and strong antioxidant activity due to the
presence of tocopherols and tocotrienols (Arab et al., 2011).
To determine the reducing capacity of the samples FRAP method was
used and it is based on the reduction of the Fe3þ complex to the intensely
blue-colored Fe2þ complex Fe (TPTZ)2þ by antioxidants in acidic me­
dium. The reducing power of Bw367 and Bg352 has increased with SSF
which relates to the increased content of total phenolic and flavonoids.
Also, studies by Cheng et al. (2016) suggests that small peptides and
some other secondary metabolites which are more active in their
reducing power and iron chelating activity are produced with SSF and it
may be one of the reason that reducing power is more notable after SSF.
The lower reducing power of BG 406 and H 4 after SSF can be attributed
to the reduced phenolic and flavonoid content of these varieties after
SSF. Total antioxidant capacity is related to the available phenolic
compounds which reduce Mo (VI) to Mo (V) by donating hydrogen. The
result indicates that increased content of phenolic after SSF has posi­
tively related to the increased total antioxidant capacity of Bw367 and
Bg352 whereas reduced phenolic content after SSF in Bg406 and H4 has
reduced the total antioxidant capacity of the samples.
The anti-inflammatory property of the samples was evaluated using
protein denaturation assay. Inflammation is a defense mechanism
initiated by the invasion of pathogens or tissue injury caused by bio­
logical, chemical or physical damage (Janeway et al., 2001). According
to the results obtained, when considering the IC50 values of each variety
it can be observed that IC50 values of fermented Bw367 (10.54 mg/L)
fermented Bg406 (8 mg/L) and fermented H 4 (12.3 mg/L) has
decreased compared to the unfermented samples (Bw367 21.2 mg/L,
Bg406 16.8 mg/L and H4 26.2 mg/L) indicating that anti-inflammatory
property has increased with fermentation. Interestingly both fermented
and unfermented Bg352 samples had the same IC50 values showing that
there was no effect of fermentation on their anti-inflammatory property.
Inflammation is a physiologic reaction to initiate healing and to act as a
barrier for injury propagation and infection. In this study
anti-inflammatory properties of fermented and unfermented rice bran
were evaluated using protein denaturation assay as denaturation of
tissue protein is one of the causes for inflammation and ability to reduce
protein denaturation indicate the ability of the sample to control
inflammation. Lower IC50 indicates higher anti-inflammatory proper­
ties. The IC50 values of unfermented samples were in the range of
8–23.81 mg/L. Previous studies have reported that bran phytosteryl
ferullates and isoprenoids exert anti-inflammatory effects by acting on
the immune cells. Also, Sakai et al. (2012) have reported that rice bran
gamma oryzanol has exerted anti-inflammatory activity in
LPS-stimulated vascular endothelial cells where it inhibited the
expression of adhesion molecules through inhibition of nuclear factor
kappa B (NF-jB). Studies in animal models also reveal that ferulic acid of
rice bran controlled inflammation and exerted neuroprotective effects.
Especially catechin and proanthocyanidin fractions of red rice varieties
contribute to the mechanism of controlling inflammation (Limtrakul
et al., 2016). The results of this study indicate that the anti-inflammatory
5
Biocatalysis and Agricultural Biotechnology 23 (2020) 101510
properties of all samples have increased with SSF. Since most of the
bioactive compounds responsible for anti-inflammation are released
from the cell walls and solubilized with SSF, it can be the possible reason
for the increase in the anti-inflammatory property with SSF.
Anti-diabetic property of unfermented and fermented samples was
evaluated using alpha-amylase inhibition assay. Alpha-amylase is one of
the key enzymes that is involved in starch digestion which breaks down
polysaccharides to monosaccharide and disaccharides and increases the
postprandial blood glucose. The α-amylase inhibition percentage of the
unfermented samples was in the range of 40.05%–70.05% with the
highest inhibition percentage shown by Bg406 and the lowest inhibition
percentage shown by Bw367. The inhibition percentage of fermented
samples was in the range of 11.47%–65.40%. This indicates that anti-
diabetic property of all 4 samples decreased with fermentation and the
percentage decrease of Bw367, Bg352, Bg406, H 4 are 71.36%, 15.40%,
6.64%, and 21.63% respectively. Inhibiting the activity of alpha-
amylase is a possible mechanism to reduce blood glucose and control
diabetic mellitus. The result of this study indicates that rice bran has a
good antidiabetic property compared to other potential sources of anti-
diabetic compounds. It is suggested that this inhibitory activity may be
due to the phenolic content (ferulic acid, ellagic acid, gallic acid, and
coumaric acid) and protein fraction of the rice bran (Sangeetha and
Vedasree, 2012). According to the results obtained from this study, it
can be observed that fermentation has reduced the anti-diabetic prop­
erties of all rice bran varieties. This reduction is due to the extended
period of fermentation of 96 h which has decreased the inhibitory ac­
tivity of alpha-amylase. Randhir and Shetty (2007) in their studies have
explained that higher inhibitory activity for alpha-amylase has been
observed at 48 h of SSF whereas at the latter stage of fermentation lower
inhibitory activity was observed. This low activity was due to the
breakdown of phenolic compounds by the fungi with the lack of nutri­
ents for fungal subsistence in an extended period of fermentation and
acute oxidative stress.
Impact of SSF in the total phenolic content and total flavonoid con­
tent of rice bran samples subjected to simulated in vitro gastrointestinal
digestion shown in Fig. 1 and Fig. 2 respectively. Accordingly, in un­
fermented samples, the percentage recovery of phenols after the gastric
digestion ranged from 23.65% to 84.92% with the highest and lowest
percentage of recovery shown by Bg352 and Bg406 respectively in terms
of the% recovery relative to the phenol content of the original fractions.
After the intestinal digestion highest recovery was observed in Bw367
(93.32%) whereas the lowest recovery was observed in Bg406 (15.66%).
More prominent dialyzable phenolic content in terms of the original
content was observed in Bg352 (1.03%) and the lowest dialyzable
Fig. 1. Percentage change with fermentation in total phenolic content of 4
different rice bran samples subjected to simulated in vitro gastric and intestinal
digestion and dialysis. The data presented in this figure consists of average
quantities � SD of three independent samples.
G. Janarny and K.D.P.P. Gunathilake
Fig. 2. Percentage change with fermentation in total flavanoid content of 4
different rice bran samples subjected to simulated in vitro gastric and intestinal
digestion and dialysis. The data presented in this figure consists of average
quantities � SD of three independent samples.
phenolic content was observed in Bg406 (0.78%). In fermented samples,
the percentage of phenolics recovered after gastric digestion in terms of
phenol content of the original fractions ranged from 63.93% to 82.84%
with the highest and lowest recovery shown by Bw367 and H4 respec­
tively. This indicates that SSF has increased the recovery of phenols after
gastric digestion. Phenolic content after intestinal digestion was high in
Bg352 and Bw367 compared to the phenolic content before intestinal
digestion. The dialyzable phenolic content of Bg406, Bg352, Bw367 and
H 4 showed recoveries of 9.41%, 3.66%, 3.61%, and 5.35% respectively.
This indicates that SSF has increased the dialyzable phenolic content in
all four rice bran samples. In unfermented samples, the bioaccessible
percentage of flavonoids of Bg406, Bg352, Bw367, and H4 was 6.67%,
26.82%, 24.45% and 4.09% respectively. A higher percentage of dia­
lyzable flavonoids was observed in Bg352 (2.95%) and Bw367 (1.65%)
whereas the comparatively lower percentage of dialyzable flavonoid
was observed in Bg406 (0.57%) and H 4 (0.40%). In fermented samples,
the bioaccessible percentage of flavonoids of Bg406, Bg352, Bw367, and
H4 was 40.81%, 4.99%, 9.22% and 49.34% indicating an increase in the
bioaccessibility of Bg406 and H 4. Changes in total anthocyanin content
with SSF of rice bran samples subjected to gastro-intestinal digestion are
shown in Fig. 3. Accordingly in unfermented samples, the percentage of
anthocyanin content that was available after gastric digestion in Bg406,
Bg352, Bw367, and H4 was 22.42%, 22.73%, 14.21%, and 27.64%
respectively. The percentage of anthocyanin available after pancreatic
Fig. 3. Percentage change with fermentation in total anthocyanin content of 4
different rice bran samples subjected to simulated in vitro gastric and intestinal
digestion and dialysis. The data presented in this figure consists of average
quantities � SD of three independent samples.
6
Biocatalysis and Agricultural Biotechnology 23 (2020) 101510
digestion ranged from 0.07% to 12.46% with the highest percentage
recovered in Bg352 and lowest percentage recovered in H 4. The dia­
lyzable percentage of anthocyanin in Bg406, Bg352, Bw367 and H 4
were 0.24%, 7.54%, 4.58% and 0.78% respectively. It indicates a high
bioavailable percentage of anthocyanin was present in white rice bran
varieties than red rice bran varieties. In fermented samples, the recov­
ered percentage of anthocyanin after gastric digestion ranged from
25.40% to 66.39% indicating an increase in the anthocyanin recovery
after gastric digestion with fermentation. The percentage available after
intestinal digestion ranged from 9.79% to 43.39% indicating a signifi­
cant (P < 0.05) increase in the anthocyanin content recovered after
intestinal digestion in fermented samples compared to that of unfer­
mented samples. The present study indicates that bioaccessibility and
bioavailability of phenolic, flavonoid and anthocyanin of Bg406 and H 4
have increased with SSF. A possible mechanism for this increase is the
extracellular enzymes produced by fungal metabolism which causes a
structural breakdown of the bran matrix and increases the bio­
accessibility of bound bioactive compounds (Đorđevi�c et al., 2010).
Also, an enzymatic transformation of bioactive compounds during
fermentation to more absorbable form at the intestine increases the
bioavailability of these compounds. However, changes in the trans­
formation of bioactive compounds associated with fermentation depend
on the fermentation conditions especially temperature, pH and time
along with the variety of rice. The effect of fermentation conditions may
be the possible reason for the reduced bioaccessibility and bioavail­
ability of bioactive compounds from Bg352 and Bw367.
Absorption and metabolism of polyphenolic compounds are deter­
mined by their molecular size, their basic structure, degree of poly­
merization or glycosylation, solubility, and conjugation with other
phenolics. In rice bran, most of the phenolic compounds are found as
glycosylated forms or as esters or polymers that must be hydrolyzed by
intestinal enzymes before they are released and their aglycones can be
absorbed. Gil-Izquierdo et al. (2002) observed that phenolic composi­
tion was not affected by pepsin digestion. However, anthocyanins can be
absorbed as glycosides and be available in the blood (D’Archivio, 2007).
In the case of the bioavailability of anthocyanin, a reduction in intestinal
phase observed may be due to the degradation of anthocyanin in alka­
line medium of anthocyanin however under in vivo conditions antho­
cyanin can be directly absorbed in the stomach (Manach et al., 2004).
Mostly, anthocyanin reaches the colon and is extensively metabolized
thereby bacteria, contributing to their bioavailability (Hidalgo et al.,
2012).
The impact of gastrointestinal digestion and SSF on the total carot­
enoid content of the rice bran sample is shown in Fig. 4. Accordingly, in
unfermented samples, the percentage recovery of carotenoids after the
Fig. 4. Percentage change with fermentation in total carotenoid content of 4
different rice bran samples subjected to simulated in vitro gastric and intestinal
digestion and dialysis. The data presented in this figure consists of average
quantities � SD of three independent samples.
G. Janarny and K.D.P.P. Gunathilake Biocatalysis and Agricultural Biotechnology 23 (2020) 101510

gastric digestion ranged from 9.69% to 58.84%, with the highest and Table 3
lowest percentage of recovery shown by H 4 and Bg352 respectively in Antioxidant activity of unfermented and fermented rice bran subjected to
terms of the percentage recovery relative to the carotenoid content of simulated in vitro gastric and intestinal digestion and dialysis.
the original fractions. The percentage of carotenoids recovered after Antioxidant assay Gastric Gastro intestinal Dialysis
gastric digestion in Bw367 and Bg406 was 46.43% and 21.06% digestion digestion
respectively. After the intestinal digestion highest recovery was DPPH (Inhibition % per gram of fresh weight)
observed in H 4 (70.85%) whereas the lowest recovery was observed in
Bw367
Bg352 (9.56%). Higher dialyzable carotenoid content was observed in Unfermented 5.50 � 0.11a 5.85 � 1.14a 3.37 � 0.43a
Bg352 (17.50) and H 4 (17.73%). In fermented samples the percentage Fermented 3.47 � 0.98b 1.12 � 0.17d 0.24 � 0.02c
of carotenoids recovered after gastric digestion ranged from 35.35% to Bg352
78.77% with the highest and lowest recovery shown by Bg352 and H 4 Unfermented 6.09 � 1.12a 6.21 � 0.92a 3.31 � 0.68a
Fermented 3.90 � 1.44b 2.24 � 0.23c 1.46 � 0.14a
respectively. This indicates that SSF has increased the recovery of ca­ Bg406
rotenoids after gastric digestion. The dialyzable carotenoid content of Unfermented 6.85 � 2.12a 5.32 � 0.87a 2.95 � 1.21a
Bg406, Bg352, Bw367 and H4 showed recoveries of 17.43%, 13.73%, Fermented 5.36 � 2.34ab 3.27 � 0.07b 0.78 � 0.05b
7.34% and 23.22% respectively. This indicates that SSF has increased H4
Unfermented 7.58 � 0.45a 6.35 � 0.14a 2.39 � 0.76a
the dialyzable carotenoid content in Bg406 and H4. Considering the
Fermented 6.95 � 0.37a 4.12 � 0.01a 1.59 � 0.33a
bioaccessibility of carotenoids only a very low proportion of carotenoids FRAP
has been reported to become bioaccessible (Courraud et al.; 2013). (mg AAE per g of FW)
Carotenoids are found in oil droplets in chromoplast and some mostly Bw367
esterified with fatty acids, being more easily extracted during digestion. Unfermented 2.85 � 0.35a 3.12 � 0.65a 0.56 � 0.02a
Fermented 4.00 � 0.23 a 2.11 � 0.17c 0.55 � 0.12a
Carotenoids’ bioavailability from foods varies greatly depending on Bg352
product-related factors and process related factors. With SSF the ester Unfermented 2.81 � 0.08a 3.04 � 0.09a 0.62 � 0.06a
linkages can be hydrolyzed releasing the bound carotenoid and Fermented 4.18 � 0.32a 3.18 � 0.56a 0.63 � 0.22a
increasing the bioaccessibility. Previous studies have identified that Bg406
Unfermented 3.49 � 1.12b 2.14 � 0.04b 0.51 � 0.01a
carboxyl ester lipase of fungal metabolism hydrolyzes and releases the
Fermented 3.42 � 0.16c 2.47 � 0.08b 0.51 � 0.08a
esterified carotenoids. Unfermented 3.11 � 0.78b 2.86 � 0.21a 0.62 � 0.11a
The antioxidant property, anti-inflammatory property and anti- Fermented 3.80 � 0.11b 1.44 � 0.05d 0.28 � 0.03b
diabetic property were evaluated for the samples subjected to in vitro Total antioxidant capacity
digestion. The radical scavenging ability of unfermented and fermented (mg AAE per g of FW)
Bw367
rice bran at different phases of digestion was systematically evaluated Unfermented 7.41 � 0.78a 9.59 � 0.31a 0.20 � 0.12a
using DPPH radical scavenging assay and the results are summarized in Fermented 8.87 � 0.83a 5.23 � 0.44a 0.27 � 0.06a
Table 3. The percentage of the unfermented sample of free radical in­ Bg352
hibition at the gastric phase ranged from 5.50% to 7.58%. In Bw367and Unfermented 8.76 � 1.23a 9.12 � 0.04a 0.22 � 0.08a
Fermented 8.61 � 1.64a 4.14 � 0.18b 0.25 � 0.01a
Bg352 radical scavenging ability has increased in the intestinal phase
Bg406
whereas in Bg406 and H4 there was a reduction in the radical scav­ Unfermented 8.36 � 0.98a 7.15 � 1.15b 0.19 � 0.01a
enging ability in the intestinal phase. All dialyzed samples showed a Fermented 9.55 � 2.43a 5.61 � 0.28a 0.28 � 0.01a
lower radical scavenging activity compared to the gastric and intestinal H4
phases. With fermentation, there was a reduction in the radical scav­ Unfermented 8.59 � 1.24a 5.73 � 0.23c 0.19 � 0.02a
Fermented 9.16 � 1.55a 6.23 � 0.41a 0.29 � 0.03a
enging ability of all samples in gastric, intestinal and dialyzable fractions
compared to the unfermented samples. Among the fermented samples, H Values are expressed as mean � SD Values followed by different letters in each
4 exhibited higher radical scavenging ability in all 3 phases of digestion. condition (unfermented and fermented) for each assay in the same column are
Reducing the power of the digested fractions of unfermented and significantly different (p < 0.05).
fermented samples was assessed using ferric reducing power assay and
the results are summarized in Table 3. The reducing power of unfer­ However, there were no significant differences among the total antiox­
mented samples in the gastric fraction was in the range of 2.81–3.89 mg idant capacity of the dialyzable fractions of the sample. With SSF total
AAE/g FW. In the intestinal fraction, there was an increase in the antioxidant capacity of Bw367, Bg406 and H 4 in gastric fractions have
reducing power of Bw367 and Bg352 whereas reducing the power of H 4 increased, whereas the total antioxidant capacity of Bg352, Bw367 and
and Bg406 has decreased in the intestinal fraction compared to the H 4 in the intestinal fractions have decreased. In all dialyzed samples SSF
gastric fraction. Reducing power of all samples has decreased in the has increased the total antioxidant capacity.
dialyzable fractions compared to the intestinal and gastric fractions The effect of gastrointestinal digestion on the inhibition percentage
however there was no significant difference in the reducing power of the of protein denaturation and α-amylase of fermented and unfermented
dialyzable fractions of all samples. With fermentation reducing the samples are shown in Table 4 below. Accordingly, in both fermented and
power of all samples except Bg406 has increased in gastric fractions. In unfermented samples, the inhibition percentage of intestinal and dia­
the intestinal fractions reducing the power of Bg352 and Bg406 has lyzable fractions was lower compared to the gastric fractions. The in­
increased with fermentation whereas reducing the power of Bw367 and hibition percentage of unfermented samples at the gastric fractions
H 4 has decreased with fermentation. There was no significant differ­ ranged from 1.66% to 6.00% whereas that of fermented samples ranged
ence (P > 0.05) in the reducing power of dialyzable fractions of the from 2.50% to 6.92% indicating an increase in the anti-inflammatory
fermented and unfermented samples of Bg352, Bw367, and Bg406, properties with SSF. The anti-inflammatory properties of Bg352,
however, a significant reduction was observed in the reducing power of Bw367 and Bg 406 in the intestinal phase increased with fermentation
H 4 at its dialyzable fraction. compared to the unfermented samples. However, in the dialyzable
The results indicate that the total antioxidant capacity in the gastric fractions, only Bw367 and Bg352 showed an increase in the anti-
fractions of unfermented samples was in the range of 7.41–8.49 mg inflammatory properties with fermentation.
AAE/g of fresh weight. Total antioxidant capacity of Bg352 and Bw367 The results indicated that the anti-diabetic properties of fermented
in the intestinal fractions has increased compared to that of gastric and unfermented samples have reduced in the intestinal fractions and
fractions whereas the total antioxidant capacity of Bg 406 and H 4 in the dialyzable fractions compared to the gastric fractions. Among the un­
intestinal fractions have decreased compared to that of gastric fractions. fermented samples, Bw367 exhibited a higher percentage of inhibition

7
G. Janarny and K.D.P.P. Gunathilake Biocatalysis and Agricultural Biotechnology 23 (2020) 101510

Table 4 compounds with bioactivity and these can be enhanced using SSF as a
Percentage inhibition of protein denaturation (anti-inflammatory property) and suitable processing technique. This area has great potential to expand
alpha amylase (anti-diabetic property) of unfermented and solid state fermented soon due to the increased consumer desire to improve health through
rice bran subjected to simulated in vitro gastric and intestinal digestion and food and solid-state fermented rice bran can be used as a functional
dialysis. ingredient for this purpose.
Assay Gastric digestion Gastro intestinal digestion Dialysis

Anti-inflammatory property (%Inhibition of protein denaturation per g of FW) CRediT authorship contribution statement
Bw367
Unfermented 4.00 � 1.21b 3.43 � 1.12a 2.00 � 1.23b G. Janarny: Data curation, Methodology, Writing - review & editing.
Fermented 6.92 � 2.31a 5.41 � 0.21a 4.26 � 2.31a K.D.P.P. Gunathilake: Conceptualization, Methodology.
Bg352
Unfermented 2.85 � 0.89c 1.71 � 0.24b 3.32 � 0.05a Acknowledgment
Fermented 6.36 � 2.11a 4.21 � 0.34b 3.14 � 3.18a
Bg406
Unfermented 1.66 � 0.45c 1.12 � 0.78b 1.25 � 3.12ab We greatly acknowledge the support provided by the Wayamba
Fermented 2.50 � 0.31b 1.98 � 0.05d 0.55 � 2.11b University of Sri Lanka. We also thank Mrs. LathaManamperiand Mr.
H4 Jeewananda Silva of the Wayamba University of Sri Lanka, Sri Lanka for
Unfermented 6.00 � 0.02a 4.69 � 0.31a 3.75 � 1.34ab
their technical support.
Fermented 4.28 � 1.12a 2.67 � 0.03c 2.00 � 1.22b
Anti-diabetic property (Inhibition % per g of FW)
Bw367 References
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