Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Upgrading the antioxidant potential of cereals by their fungal fermentation under solid-state cultivation conditions T. Bhanja Dey and R.C. Kuhad Department of Microbiology, University of Delhi South Campus, New Delhi, India
Significance and Impact of the Study: Antioxidant-rich food products are getting popularity day by day. In this study, potential of solid-state fermentation (SSF) has been studied for the improvement of antioxidant potential of different cereals by GRAS micro-organisms. The comparative evaluation of the antioxidant potential of various fungal fermented products derived from whole grain cereals, such as wheat, brown rice, oat and maize, has been carried out. Among these, Rhizopus oryzae RCK2012-fermented wheat was observed as a potent source of natural antioxidants. A diet containing fermented cereals would be useful for the prevention of free radical-mediated diseases.
Keywords antioxidant, bioactive, cereals, fermentation, free radical, phenolics. Correspondence Ramesh Chander Kuhad, Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. E-mail: [email protected] 2014/0641: received 26 March 2014, revised 20 June 2014 and accepted 24 June 2014 doi:10.1111/lam.12300
Abstract Solid-state fermentation (SSF) at 30°C for 72 h with four generally recognized as safe (GRAS) filamentous fungi (Aspergillus oryzae NCIM 1212, Aspergillus awamori MTCC No. 548, Rhizopus oligosporus NCIM 1215 and Rhizopus oryzae RCK2012) showed high efficiency for the improvement of water-soluble total phenolic content (TPC) and antioxidant properties including ABTS●+ [2,20 azinobis (3-ethylbenzothiazoline-6-sulphonic acid)] and DPPH● (2,20 -diphenyl1-picrylhydrazyl) scavenging capacities of four whole grain cereals, namely wheat, brown rice, maize and oat. A maximum 14-fold improvement in TPC (1161 mg gallic acid equivalent g1 grain) was observed in A. oryzae fermented wheat, while extract of R. oryzae fermented wheat (ROFW) showed maximum of 66-fold and fivefold enhancement of DPPH● scavenging property (854 lmol Trolox equivalent g1 grain) and ABTS●+ scavenging activity (195 lmol Trolox equivalent g1 grain), respectively. The study demonstrates that SSF is an efficient method for the improvement of antioxidant potentials of cereals and R. oryzae RCK2012 fermented wheat can be a powerful source of natural antioxidants.
Introduction Antioxidant molecules with their reducing, free radical scavenging and metal-chelating properties can reduce oxidative stress maintaining equilibrium between oxidants and antioxidants in human body and decrease risk of many chronic diseases such as cardiovascular disease, obesity, type 2 diabetes and cancers (Bhanja et al. 2009). In recent times, cereals are considered beyond sources of energy and essential nutrients, as they contain phytochemicals or certain minor components with antioxidant properties, which are now recognized for their Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
health-promoting properties. Several reports also support that the in vitro antioxidant capacity of cereal grains is significantly correlated with their total phenolic content (Fardet et al. 2008). However, most of the cereal phenolic compounds (PCs) are bound to cell wall polysaccharides called as dietary fibre–phenolic compounds (DF-PCs). According to Adom and Liu (2002), the amount of bound PCs present in wheat, maize, rice and oats is 90, 87, 71 and 58%, respectively. The b-glucosidases and esterases produced by human microflora are unable to degrade the highly cross-linked water-insoluble DF. Therefore, predominately insoluble bound cereal PCs are 1
Development of antioxidant rich cereals
Results and discussion Identification of fungal isolate RCK2012 The fungal isolate RCK2012 was identified based on the sequence variation present in ITS region. A BLAST search in the NCBI database showed that fungal isolate RCK2012 had 98% identity with ITS sequence from Rhizopus oryzae, and based on this sequence homology, it was designated as R. oryzae RCK2012. The ITS sequence of RCK2012 was deposited in the GenBank database under Accession No. JQ906263. Total phenolic content (TPC) In most of the cases, different organic solvents such as ethanol, methanol or acetone are used for the extraction of PCs. In the present study, PCs were extracted in water because water extracts of cereal grain fractions are of great relevance to in vivo activity as they contain water-soluble antioxidants and thus more bioaccessible from food matrix in the digestive tract (Fardet et al. 2008). 2
As shown in Fig. 1, TPC of water extract of the fermented cereals ranged between 23 and 1161 mg GAE g1 grain and was higher than that of the unfermented cereal extracts (wheat: 081; brown rice: 079; maize: 081; oat: 206 mg GAE g1 grain). Exceptionally, TPC of Rhizopus oligosporus NCIM 1215 fermented maize (089 mg GAE g1 grain) was not found to be improved. Among the cereal samples, TPC was increased maximum in wheat (approx. 14-fold) fermented by Aspergillus oryzae NCIM 1212 (1161 mg GAE g1 grain) as compared to unfermented wheat. Similarly, about 22-fold improvement of TPC was observed in A. oryzae IFO 30103 fermented wheat grain (Bhanja et al. 2009). Hence, phenolics production may vary noticeably between species and even between strains of the same species. Moreover, it may vary according to the variety of the wheat grains and extracting solvent. There is no significant difference (P < 005) between TPC of A. oryzae NCIM 1212 and R. oryzae RCK2012 fermented oat. On the contrary, Cai et al. (2012b) showed that the R. oryzae fermented oats had the highest TPC in comparison with A. oryzae. In the present study, under SSF, water-soluble phenolic content of each of the cereals was increased, except R. oligosporus NCIM 1215 fermented maize. Hence, bioavailability of cereal phenolics can be enhanced through this process. Antioxidant status of fungal fermented cereals At present, more than 20 different indices of antioxidant activity assay with various mechanisms are in use. It is very difficult to compare the results obtained from different assay methods due to the diversity in substrates, probes, reaction conditions and quantification methods (Sreeramulu et al. 2013). In the present investigation,
14
TPC (mg GAE g–1 grain)
thought to have low bioavailability. Depending upon different epidemiological studies, prior to human consumption, it is advisable to convert insoluble cereal phenolics into soluble form to improve their bioavailability and hence maximize the possible health benefits of cereal PCs (Moore et al. 2007; Vitaglione et al. 2008). Solid-state fermentation (SSF) is a very common bioprocess frequently employed to produce bioactive compounds specially in Asian countries, and a variety of traditional foods are prepared employing this process. In this process, micro-organisms producing carbohydrate degrading enzymes such as cellulases, xylanases, amylase and esterases could be used to release the bound cereal PCs. The potential of SSF has been evaluated for the improvement of total phenolics content and antioxidant potential of wheat bran (Moore et al. 2007), maize (Daker et al. 2008), buckwheat, wheat germ, barley and rye (Dordevi c et al. 2010), rice (Bhanja et al. 2008), wheat grains (Bhanja et al. 2009) and oat (Cai et al. 2012a,b) employing several micro-organisms. During last few years, enrichment of phenolics by SSF is receiving great attention (Martins et al. 2011). Yet, an inadequate number of reports are available on antioxidant potential of fermented cereals as compared to unfermented whole grain cereals. For the first time, a study has been carried out to comparatively evaluate the antioxidant potential of various filamentous fungi-fermented products derived from different whole grain cereals, including wheat, brown rice, oat and maize.
T. Bhanja Dey and R.C. Kuhad
12 10 8 6 4 2 0 Wheat
Brown rice
Maize
Oat
Figure 1 TPC of unfermented and four filamentous fungi fermented cereals. ( Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; ( ) Rhizopus oryzae.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Development of antioxidant rich cereals
ABTS •+ scavenging property (µmol TE g–1grain)
ABTS●+ [2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt] and DPPH● (2,20 -diphenyl-1picrylhydrazyl) scavenging properties, the two most commonly used processes, were explored to determine antioxidant properties of cereal grains and their fermented products. Reaction between ABTS and potassium persulphate produces blue-coloured ABTS●+, and antioxidant compounds reduce this preformed cation radical. ABTS●+ scavenging antioxidant property of fermented and control cereals is depicted in Fig. 2. All the unfermented cereal grains tested in this study contained ABTS●+ scavenging antioxidant property of 385 (wheat), 430 (brown rice), 485 (maize) and 490 (oat) lmol TE g1 grain. Maximum improvement of ABTS●+ radical scavenging property was observed in case of wheat fermented by both R. oryzae RCK2012 (195 lmol TE g1 grain) and A. oryzae NCIM 1212 (194 lmol TE g1 grain) with fivefold increment. Through electron transfer or hydrogen atoms donation, antioxidant compounds neutralize the free radical character of DPPH and thus purple colour of the reaction mixture is changed to yellow (Bhanja et al. 2009). DPPH● scavenging property of unfermented cereals was 129 (wheat), 115 (brown rice), 184 (maize) and 298 (oat) in lmol TE g1 grain (Fig. 3). Among all the fermented cereals tested, maximum enhancement (66 times) of DPPH● scavenging property (854 lmol TE g1 grain) was observed in case of R. oryzae RCK2012-fermented wheat (ROFW) as compared to unfermented wheat. Moreover, R. oryzae RCK2012-fermented oat (96 in lmol TE g1 grain) showed highest DPPH● scavenging activity compared with other organisms. Rhizopus oryzae
25
20
15
10
5
0 Wheat
Brown rice
Maize
Oat
Figure 2 ABTS●+ scavenging property of unfermented and four filamentous fungi fermented cereals. ( ) Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; ( ) Rhizopus oryzae.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
DPPH• scavenging property (µmol TE g–1 grain)
T. Bhanja Dey and R.C. Kuhad
12 10 8 6 4 2 0 Wheat
Brown rice
Maize
Oat
Figure 3 DPPH● scavenging property of unfermented and four filamentous fungi fermented cereals. ( ) Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; (&) Rhizopus oryzae.
RCK2012 as well as Aspergillus awamori MTCC No. 548fermented maize showed equivalent amount of DPPH● scavenging property (571 and 548 lmol TE g1 grain, respectively). In the present study, both the ABTS●+ and DPPH● scavenging properties were enhanced after SSF of cereals by all the four micro-organisms (except R. oligosporus NCIM 1215-fermented maize) which might be related to the release of more soluble bioactive compounds. Among them, maximum improvement of both of the free radical scavenging properties was observed in case of ROFW. Several reports support that improvement of TPC and antioxidant potential depends on type of micro-organism involved in SSF (Moore et al. 2007; Dordevi c et al. 2010; Cai et al. 2012b). It is very difficult to compare our data with literature as different methods have been used for extraction and estimation of antioxidant property as well as TPC. Moreover, antioxidant property varies between species and varieties of grains (Zieli nski and Kozłowska 2000). Thamnidium elegans CCF 1456 and Cordyceps militaris have been observed to enhance TPC and antioxidant property of maize and wheat, respectively, yet with very less fold of increment compared with unfermented counter parts (Salar et al. 2012; Zhang et al. 2012). Basically production of antioxidant compounds during SSF is a complex and mysterious biochemical process. Several hydrolysing enzymes produced during SSF process are predicted to be associated with the release of PCs (Bhanja et al. 2008, 2009; Robledo et al. 2008; Cho et al. 2009). In the present investigation, the activities of various enzymes (a-amylase, xylanase, cellulase, pectinase and esterase) were higher in case of A. oryzae NCIM 1212fermented wheat (AOFW) compared with R. oryzae 3
Development of antioxidant rich cereals
T. Bhanja Dey and R.C. Kuhad
Table 1 Different enzyme activities, total protein, carbohydrate and reducing sugar content of AOFW and ROFW Enzyme activity/ protein/ carbohydrate/ reducing sugar
Phenolic acids AOFW
Enzymes activities (IU per gds) a-amylase 100964 2815 Xylanase 15657 3014 FPase 0 CMCase 538 025 b-glucosidase 054 004 Pectinase 1564103 266717 Esterase 386 069 Total protein 28 011 content (mg ml1) Total carbohydrate 179255 26 content (mg ml1) Reducing sugar 178 006 content (mg ml1)
ROFW
5024 2814 0 061 003 1357153 086 206
1025 57
002 .005 76831 038 015
402589 38 080 005
Values are expressed as means standard deviation.
RCK2012 (Table 1), which supported the maximum release of PCs but could not justify the elevated amount of each of the antioxidant property in ROFW. Following factors might be responsible for it: (i) Along with the enzymatic release of PCs, some other metabolic pathways might be involved in this process. (ii) Folin–Ciocalteu method, which is extensively used for TPC measurement, is a nonspecific process and it has numerous limitations especially in fermentation studies. (iii) In addition to PC, some other water-soluble compounds such as bioactive small peptides and oligosaccharides might be contributing in the antioxidant property (Veenashri and Muralikrishna 2011). Total protein, carbohydrate and reducing sugar content of AOFW and ROFW water extracts were shown in the Table 1. The % DPPH● scavenging property of polysaccharide fractions of AOFW and ROFW was 4364 and 3881%, respectively. Hence, polysaccharides might be contributing the antioxidant property of both AOFW and ROFW. (IV) The antioxidant activity of individual phenolic compounds can be different and depends on their donor proton capacity and electron delocalization capacity of their aromatic ring. Samples with same concentrations of TPC may vary remarkably in their antioxidant property (Bhanja et al. 2008; Dordevi c et al. 2010). HPLC analysis (Table 2) also supported that along with phenolic content, profiles of phenolic compounds were totally different for unfermented wheat (UFW), AOFW and ROFW. This study has demonstrated SSF as a convenient method to improve antioxidant potential of cereals. 4
Table 2 Phenolic acid composition of UFW, ROFW and AOFW
Gallic acid Protocatechuic acid 4-hydroxy benzoic acid 4-hydroxy 3-methoxy benzoic acid Caffeic acid Ferulic acid Trans-cinnamic acid
Retention time (min)
lg mg1 extract UFW
ROFW
AOFW
57 69 91 105
0237 0092 0007 0023
15219 0 0440 0049
0 1956 0 0439
112 171 266
0232 0087 0277
0372 0030 0220
0304 0651 0358
A diet containing these fermented cereals may therefore be useful for the prevention of free radical-mediated diseases. Hence, fermented cereals are suitable for the design of different functional foods and for the specific use as nutraceuticals. The study has clearly indicated that water extract of ROFW has strong antioxidant property against in vitro oxidative system compared with unfermented wheat. Moreover, these can be served as powerful sources of natural antioxidants over the synthetic antioxidant compounds used very often in food and pharmaceutical industry. Antioxidant properties of cereal grains can be further increased by optimization of different physicochemical parameters of SSF as well as optimizing the phenolics extraction conditions. Materials and methods Reagents DPPH, ABTS, Trolox and phenolic acid standards such as gallic, protocatechuic, caffeic, 4-hydroxy benzoic acid, 4-hydroxy 3-methoxy benzoic acid, trans-cinnamic acid and ferulic acid were procured from Sigma-Aldrich (Steinheim, Germany). All other reagents and chemicals were of analytical grade. Substrates for SSF Wheat, brown rice, maize and oat grains were purchased from local market, stored at room temperature, and whole grains without grinding were utilized as solid substrates in SSF to improve their antioxidant properties. Organisms, their maintenance and inoculum preparation Aspergillus oryzae NCIM 1212 and R. oligosporus NCIM 1215 were procured from NCIM, Pune, A. awamori (MTCC No. 548) was obtained from MTCC, Chandigarh, and a new fungus RCK2012 was isolated locally from Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
T. Bhanja Dey and R.C. Kuhad
Development of antioxidant rich cereals
rotten maize. Aspergillus awamori was cultured on Czapek Dox agar medium, while all other fungi were grown on potato dextrose agar (PDA). Inoculum was prepared from 5-day-old slant by suspending the fungal spores in sterile distilled water.
solution was added to it, mixed thoroughly and incubated for 15 min at room temperature. Thereafter, the mixture was diluted by 5 ml water and absorbance was measured at 725 nm. The amount of total phenolics was expressed as gallic acid equivalent (GAE) g1 grain.
Identification of the fungus
DPPH● scavenging assay
Fungal isolate RCK2012 was grown on potato dextrose medium at 30°C under static cultivation conditions for 3 days. The fungal mycelial mat was harvested by filtration through Whatman No. 1 filter paper, washed thoroughly with Milli Q water and ground in liquid nitrogen with a mortar and pestle. Genomic DNA was isolated following the method described by Kuhad et al. (2004). Sequence obtained was compared with ITS sequences available in GenBank.
The DPPH● scavenging activity of phenolic extract was determined according to the method of Brand-Williams et al. (1995), and %DPPH● scavenging activity was calculated according to the following equation:
SSF of cereals The whole grain cereal (10 g) taken in each 250-ml Erlenmeyer flask was mixed with 10 ml distilled water, autoclaved (121°C, 15 min) and subsequently cooled to ambient temperature. Fungal spore suspensions (1 9 106 spores ml1) were inoculated separately (at 10% inoculum, v/w) onto the surface of the autoclaved substrates, mixed properly and incubated for 3 days at 30°C. The flasks containing cereal grains without addition of fungal spore suspension served as controls (unfermented cereals). Extraction of phenolic compounds The fermented and unfermented cereal grains were taken out from the Erlenmeyer flask after 3 days, autoclaved and dried in an oven at 60°C for 24 h. The grains were ground in an electric grinder separately, and ground samples sieved through mesh 40 were defatted thrice by blending with hexane (1 : 5 w/v) for 5 min at ambient temperature. Defatted samples were air-dried for 24 h and stored at 20°C for further analysis. All samples were extracted with water (1 : 10 w/v) twice at 50°C for 60 min in water bath. After filtering through Whatman No.1 filter paper, the filtrate was used for comparative study of total phenolic contents (TPC), DPPH● and ABTS●+ scavenging properties. Determination of TPC TPC was determined following the modified method of Emmons and Peterson (2001). The properly diluted phenolic extract (05 ml) was mixed with 025 ml Folin–Ciocalteu reagent. 15 ml of 20% aqueous sodium carbonate Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
%DPPH scavenging activity ¼ ½ðAbCÞ ðAbSÞ=AbC100 where, AbC = absorbance of DPPH control and AbS = absorbance in presence of test sample. A standard curve was prepared using different concentrations of Trolox, and DPPH● scavenging property of the phenolic extracts was expressed as lmol Trolox equivalent (TE) g1 grain. ABTS radical cation scavenging assay It was determined following the improved ABTS decolourization assay method of Re et al. (1999). Similar to DPPH● scavenging activity, ABTS●+ scavenging property was expressed as lmol TE g1 grain. Enzyme activities Aspergillus oryzae NCIM 1212 and R. oryzae RCK2012-fermented wheat grains were suspended in water (1 : 10 w/ v) and agitated at 150 rev min1 for 1 h at ambient temperature. The extracts were squeezed through muslin cloth, centrifuged at 8000 g for 20 min at 4°C. The supernatants were assayed for different enzyme activities. Filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), a-amylase, pectinase and xylanase activities were estimated by determining the reducing sugars released from Whatman No. 1 filter paper, carboxymethyl cellulose, soluble starch, citrus pectin and birch wood xylan as substrates at 50°C and pH 50 following the method described by Ghose (1987), Bhanja et al. (2007), Gupta et al. (2008) and Kapoor et al. (2008), respectively, whereas b-glucosidase and esterase activities were assayed by estimating the p-nitrophenol content liberated from p-nitrophenyl glucopyranoside at 50°C and pH 50 and p-nitrophenyl acetate at 37°C and pH 70 employing the method of Wood and Bhat (1988) and Kordel et al. (1991), respectively. One unit of enzyme activity was defined as the amount of enzyme required to release 1 lmol of products from the respective substrates per min and expressed as IU per gram dry substrate (IU gds1). 5
Development of antioxidant rich cereals
Total protein, carbohydrate and reducing sugar content Total protein content, carbohydrate and reducing sugar content of water extracts of AOFW and ROFW were estimated according to the method described by Lowry et al. (1951), Dubois et al. (1956) and Miller (1959), respectively.
T. Bhanja Dey and R.C. Kuhad
(SA-II), for financial assistance. We would like to thank Dr. Rishi Gupta and Mr. Kavish Kumar Jain for their kind help during HPLC analysis and identification of the fungal isolate RCK2012. Conflict of Interest The authors declare no conflict of interest.
Isolation of the polysaccharide In addition to phenolic compounds, some water-soluble polysaccharides present in the water extract of A. oryzae NCIM 1212-fermented wheat (AOFW) and R. oryzae RCK2012 fermented wheat (ROFW) may have some antioxidant property. Polysaccharides were precipitated by ethanol (1 : 4 v/v) from water extract (5 ml) of AOFW and ROFW at 4°C, followed by centrifugation at 8000 g for 20 min. The precipitate was dissolved in 25 ml of water, and %DPPH● scavenging antioxidant property of 50 ll sample was estimated. HPLC analysis Identification of phenolic acids was performed by HPLC. Water extracts of unfermented wheat (UFW), AOFW and ROFW were freeze-dried, and 10 mg of each of the sample was mixed with 1 ml methanol and centrifuged at 8000 g for 10 min. The supernatant containing phenolic compounds was filtered through 045 lm Suporâ-450 membrane disc filter (Pall Corporation, Ann Arbor, MI) and injected in a Waters HPLC system connected to a Waters 2489 diode array detector (Waters, Milford, NY). The separation of phenolics was performed on a Symmetry C-18 RP column 250 9 46 mm with 5 mm particle size (Waters) with an appropriate guard column. Two mobile phases, 01% phosphoric acid (A) and acetonitrile (B), were used at a flow of 1 ml min1 with gradient profile of 20 min from 10 to 22% B, 20 min with a linear rise to 40% B and 5 min reverse to 10% B. Phenolic acids were identified from calibration curves obtained by injecting pure phenolic acids mixture as standards. Statistical analysis The mean values and the standard deviations were calculated from the data obtained from experiments carried out in triplicates. The data were analysed by one-way analysis of variance (ANOVA). Acknowledgement The authors gratefully acknowledge the University Grant Commission, Govt. of India, New Delhi (F. 15-83/2011
6
References Adom, K.K. and Liu, R.H. (2002) Antioxidant activity of grains. J Agric Food Chem 50, 6182–6187. Bhanja, T., Rout, S., Banerjee, R. and Bhattacharyya, B.C. (2007) Comparative profiles of a-amylase production in conventional tray reactor and GROWTEK bioreactor. Bioprocess Biosyst Eng 30, 369–376. Bhanja, T., Rout, S., Banerjee, R. and Bhattacharyya, B.C. (2008) Studies on the performance of a new bioreactor for improving antioxidant potential of rice. LWT-Food Sci Technol 41, 459–1465. Bhanja, T., Kumari, A. and Banerjee, R. (2009) Enrichment of phenolics and free radical scavenging property of wheat koji prepared with two filamentous fungi. Bioresour Technol 100, 2861–2866. Brand-Williams, W., Cuvelier, M.E. and Berset, C. (1995) Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 28, 25–30. Cai, S., Gao, F., Zhang, X., Wang, O., Wu, W., Zhu, S., Zhang, D., Zhou, F. et al. (2012a) Evaluation of c- aminobutyric acid, phytate and antioxidant activity of tempeh-like fermented oats (Avena sativa L.) prepared with different filamentous fungi. J Food Sci Technol. Doi: 10.1007/ s13197-012-0748-2. Cai, S., Wang, O., Wu, W., Zhu, S., Zhou, F., Ji, B., Gao, F., Zhang, D. et al. (2012b) Comparative study of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content (TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.). J Agric Food Chem 60, 507–513. Cho, K.M., Hong, S., Math, R.K., Lee, J.H., Kambiranda, D.M., Kim, J.M., Islam, S.M.A., Yun, M.G. et al. (2009) Biotransformation of phenolics (isoflavones, flavanols and phenolic acids) during the fermentation of cheonggukjang by Bacillus pumilus HY1. Food Chem 114, 413–419. Daker, M., Abdullah, N., Vikineswary, S., Goh, P.C. and Kuppusamy, U.R. (2008) Antioxidant from maize and maize fermented by Marasmiellus sp. as stabiliser of lipidrich foods. Food Chem 107, 1092–1098. Dor devi c, T.M., Siler-Marinkovic, S.S. and DimitrijevicBrankovi, S.I. (2010) Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chem 119, 957–963.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
T. Bhanja Dey and R.C. Kuhad
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350–356. Emmons, C.L. and Peterson, D.M. (2001) Antioxidant activity and phenolic content of Oat as affected by cultivar and location. Crop Sci 41, 1676–1681. Fardet, A., Rock, E. and Remesey, C. (2008) Is the in vitro antioxidant potential of whole-grain cereals and cereal products well reflected in vivo? J Cereal Sci 48, 258–276. Ghose, T.K. (1987) Measurements of cellulase activities. Pure Appl Chem 59, 257–268. Gupta, S., Kapoor, M., Sharma, K.K., Nair, L.M. and Kuhad, R.C. (2008) Production and recovery of an alkaline exopolygalacturonase from Bacillus subtilis RCK under solidstate fermentation using statistical approach. Bioresour Technol 99, 937–945. Kapoor, M., Nair, L.M. and Kuhad, R.C. (2008) Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora. Biochem Eng J 38, 88–97. Kordel, M., Hofmann, B., Schomburg, D. and Schmid, R.D. (1991) Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary x-ray diffraction data. J Bacteriol 173, 4836–4841. Kuhad, R.C., Kapoor, R.K. and Lal, R. (2004) Improving the yield and quality of DNA isolated from white-rot fungi. Folia Microbiol 49, 112–116. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. Martins, S., Mussatto, S.I., Martınez-Avila, G., Monta~ nezSaenz, J., Aguilar, C.N. and Teixeira, J.A. (2011) Bioactive phenolic compounds: Production and extraction by solidstate fermentation. A review. Biotechnol Adv 29, 365–373. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31, 426–429. Moore, J., Cheng, Z., Hao, J., Guo, G., Liu, J.G., Lin, C. and Yu, L.L. (2007) Effects of solid-state yeast treatment on the antioxidant properties and protein and fiber compositions
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Development of antioxidant rich cereals
of common hard wheat bran. J Agric Food Chem 55, 10173–10182. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26, 1231–1237. Robledo, A., Aguilera-Carb o, A., Rodrıguez, R., Martinez, J.L., Garza, Y. and Aguilar, C.N. (2008) Ellagic acid production by Aspergillus nigerin solid state fermentation of pomegranate residues. J Ind Microbiol Biotechnol 35, 507–513. Salar, R.K., Certik, M. and Brezova, V. (2012) Modulation of phenolic content and antioxidant activity of maize by solid state fermentation with Thamnidium elegans CCF 1456. Biotechnol Bioprocess Eng 17, 109–116. Sreeramulu, D., Reddy, C.V.K., Chauhan, A., Balakrishna, N. and Raghunath, M. (2013) Natural antioxidant activity of commonly consumed plant foods in India: effect of domestic processing. Oxid Med Cell Longev, 1–12. Veenashri, B.R. and Muralikrishna, G. (2011) In vitro antioxidant activity of xylo-oligosaccharides derived from cereal and millet brans - A comparative study. Food Chem 126, 1457–1481. Vitaglione, P., Aurora, N. and Fogliano, V. (2008) Cereal dietary fibre: a natural functional ingredient to deliver phenolic compounds into the gut. Trends Food Sci Technol 19, 451–463. Wood, T.M. and Bhat, M.K. (1988) Methods for measuring cellulase activities. In Methods in Enzymology (vol. 160) eds Wood, W.A. and Kellogg, S.T. pp. 87–112. London, UK: Academic Press Inc. Zhang, Z., Lv, G., Pan, H., Fan, L., Soccol, C.R. and Pandey, A. (2012) Production of powerful antioxidant supplements via solid-state fermentation of wheat (Triticum aestivum Linn.) by Cordyceps militaris. Food Technol Biotechnol 50, 32–39. Zieli nski, H. and Kozłowska, H. (2000) Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions. J Agric Food Chem 48, 2008–2016.
7
ORIGINAL ARTICLE
Upgrading the antioxidant potential of cereals by their fungal fermentation under solid-state cultivation conditions T. Bhanja Dey and R.C. Kuhad Department of Microbiology, University of Delhi South Campus, New Delhi, India
Significance and Impact of the Study: Antioxidant-rich food products are getting popularity day by day. In this study, potential of solid-state fermentation (SSF) has been studied for the improvement of antioxidant potential of different cereals by GRAS micro-organisms. The comparative evaluation of the antioxidant potential of various fungal fermented products derived from whole grain cereals, such as wheat, brown rice, oat and maize, has been carried out. Among these, Rhizopus oryzae RCK2012-fermented wheat was observed as a potent source of natural antioxidants. A diet containing fermented cereals would be useful for the prevention of free radical-mediated diseases.
Keywords antioxidant, bioactive, cereals, fermentation, free radical, phenolics. Correspondence Ramesh Chander Kuhad, Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India. E-mail: [email protected] 2014/0641: received 26 March 2014, revised 20 June 2014 and accepted 24 June 2014 doi:10.1111/lam.12300
Abstract Solid-state fermentation (SSF) at 30°C for 72 h with four generally recognized as safe (GRAS) filamentous fungi (Aspergillus oryzae NCIM 1212, Aspergillus awamori MTCC No. 548, Rhizopus oligosporus NCIM 1215 and Rhizopus oryzae RCK2012) showed high efficiency for the improvement of water-soluble total phenolic content (TPC) and antioxidant properties including ABTS●+ [2,20 azinobis (3-ethylbenzothiazoline-6-sulphonic acid)] and DPPH● (2,20 -diphenyl1-picrylhydrazyl) scavenging capacities of four whole grain cereals, namely wheat, brown rice, maize and oat. A maximum 14-fold improvement in TPC (1161 mg gallic acid equivalent g1 grain) was observed in A. oryzae fermented wheat, while extract of R. oryzae fermented wheat (ROFW) showed maximum of 66-fold and fivefold enhancement of DPPH● scavenging property (854 lmol Trolox equivalent g1 grain) and ABTS●+ scavenging activity (195 lmol Trolox equivalent g1 grain), respectively. The study demonstrates that SSF is an efficient method for the improvement of antioxidant potentials of cereals and R. oryzae RCK2012 fermented wheat can be a powerful source of natural antioxidants.
Introduction Antioxidant molecules with their reducing, free radical scavenging and metal-chelating properties can reduce oxidative stress maintaining equilibrium between oxidants and antioxidants in human body and decrease risk of many chronic diseases such as cardiovascular disease, obesity, type 2 diabetes and cancers (Bhanja et al. 2009). In recent times, cereals are considered beyond sources of energy and essential nutrients, as they contain phytochemicals or certain minor components with antioxidant properties, which are now recognized for their Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
health-promoting properties. Several reports also support that the in vitro antioxidant capacity of cereal grains is significantly correlated with their total phenolic content (Fardet et al. 2008). However, most of the cereal phenolic compounds (PCs) are bound to cell wall polysaccharides called as dietary fibre–phenolic compounds (DF-PCs). According to Adom and Liu (2002), the amount of bound PCs present in wheat, maize, rice and oats is 90, 87, 71 and 58%, respectively. The b-glucosidases and esterases produced by human microflora are unable to degrade the highly cross-linked water-insoluble DF. Therefore, predominately insoluble bound cereal PCs are 1
Development of antioxidant rich cereals
Results and discussion Identification of fungal isolate RCK2012 The fungal isolate RCK2012 was identified based on the sequence variation present in ITS region. A BLAST search in the NCBI database showed that fungal isolate RCK2012 had 98% identity with ITS sequence from Rhizopus oryzae, and based on this sequence homology, it was designated as R. oryzae RCK2012. The ITS sequence of RCK2012 was deposited in the GenBank database under Accession No. JQ906263. Total phenolic content (TPC) In most of the cases, different organic solvents such as ethanol, methanol or acetone are used for the extraction of PCs. In the present study, PCs were extracted in water because water extracts of cereal grain fractions are of great relevance to in vivo activity as they contain water-soluble antioxidants and thus more bioaccessible from food matrix in the digestive tract (Fardet et al. 2008). 2
As shown in Fig. 1, TPC of water extract of the fermented cereals ranged between 23 and 1161 mg GAE g1 grain and was higher than that of the unfermented cereal extracts (wheat: 081; brown rice: 079; maize: 081; oat: 206 mg GAE g1 grain). Exceptionally, TPC of Rhizopus oligosporus NCIM 1215 fermented maize (089 mg GAE g1 grain) was not found to be improved. Among the cereal samples, TPC was increased maximum in wheat (approx. 14-fold) fermented by Aspergillus oryzae NCIM 1212 (1161 mg GAE g1 grain) as compared to unfermented wheat. Similarly, about 22-fold improvement of TPC was observed in A. oryzae IFO 30103 fermented wheat grain (Bhanja et al. 2009). Hence, phenolics production may vary noticeably between species and even between strains of the same species. Moreover, it may vary according to the variety of the wheat grains and extracting solvent. There is no significant difference (P < 005) between TPC of A. oryzae NCIM 1212 and R. oryzae RCK2012 fermented oat. On the contrary, Cai et al. (2012b) showed that the R. oryzae fermented oats had the highest TPC in comparison with A. oryzae. In the present study, under SSF, water-soluble phenolic content of each of the cereals was increased, except R. oligosporus NCIM 1215 fermented maize. Hence, bioavailability of cereal phenolics can be enhanced through this process. Antioxidant status of fungal fermented cereals At present, more than 20 different indices of antioxidant activity assay with various mechanisms are in use. It is very difficult to compare the results obtained from different assay methods due to the diversity in substrates, probes, reaction conditions and quantification methods (Sreeramulu et al. 2013). In the present investigation,
14
TPC (mg GAE g–1 grain)
thought to have low bioavailability. Depending upon different epidemiological studies, prior to human consumption, it is advisable to convert insoluble cereal phenolics into soluble form to improve their bioavailability and hence maximize the possible health benefits of cereal PCs (Moore et al. 2007; Vitaglione et al. 2008). Solid-state fermentation (SSF) is a very common bioprocess frequently employed to produce bioactive compounds specially in Asian countries, and a variety of traditional foods are prepared employing this process. In this process, micro-organisms producing carbohydrate degrading enzymes such as cellulases, xylanases, amylase and esterases could be used to release the bound cereal PCs. The potential of SSF has been evaluated for the improvement of total phenolics content and antioxidant potential of wheat bran (Moore et al. 2007), maize (Daker et al. 2008), buckwheat, wheat germ, barley and rye (Dordevi c et al. 2010), rice (Bhanja et al. 2008), wheat grains (Bhanja et al. 2009) and oat (Cai et al. 2012a,b) employing several micro-organisms. During last few years, enrichment of phenolics by SSF is receiving great attention (Martins et al. 2011). Yet, an inadequate number of reports are available on antioxidant potential of fermented cereals as compared to unfermented whole grain cereals. For the first time, a study has been carried out to comparatively evaluate the antioxidant potential of various filamentous fungi-fermented products derived from different whole grain cereals, including wheat, brown rice, oat and maize.
T. Bhanja Dey and R.C. Kuhad
12 10 8 6 4 2 0 Wheat
Brown rice
Maize
Oat
Figure 1 TPC of unfermented and four filamentous fungi fermented cereals. ( Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; ( ) Rhizopus oryzae.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Development of antioxidant rich cereals
ABTS •+ scavenging property (µmol TE g–1grain)
ABTS●+ [2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt] and DPPH● (2,20 -diphenyl-1picrylhydrazyl) scavenging properties, the two most commonly used processes, were explored to determine antioxidant properties of cereal grains and their fermented products. Reaction between ABTS and potassium persulphate produces blue-coloured ABTS●+, and antioxidant compounds reduce this preformed cation radical. ABTS●+ scavenging antioxidant property of fermented and control cereals is depicted in Fig. 2. All the unfermented cereal grains tested in this study contained ABTS●+ scavenging antioxidant property of 385 (wheat), 430 (brown rice), 485 (maize) and 490 (oat) lmol TE g1 grain. Maximum improvement of ABTS●+ radical scavenging property was observed in case of wheat fermented by both R. oryzae RCK2012 (195 lmol TE g1 grain) and A. oryzae NCIM 1212 (194 lmol TE g1 grain) with fivefold increment. Through electron transfer or hydrogen atoms donation, antioxidant compounds neutralize the free radical character of DPPH and thus purple colour of the reaction mixture is changed to yellow (Bhanja et al. 2009). DPPH● scavenging property of unfermented cereals was 129 (wheat), 115 (brown rice), 184 (maize) and 298 (oat) in lmol TE g1 grain (Fig. 3). Among all the fermented cereals tested, maximum enhancement (66 times) of DPPH● scavenging property (854 lmol TE g1 grain) was observed in case of R. oryzae RCK2012-fermented wheat (ROFW) as compared to unfermented wheat. Moreover, R. oryzae RCK2012-fermented oat (96 in lmol TE g1 grain) showed highest DPPH● scavenging activity compared with other organisms. Rhizopus oryzae
25
20
15
10
5
0 Wheat
Brown rice
Maize
Oat
Figure 2 ABTS●+ scavenging property of unfermented and four filamentous fungi fermented cereals. ( ) Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; ( ) Rhizopus oryzae.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
DPPH• scavenging property (µmol TE g–1 grain)
T. Bhanja Dey and R.C. Kuhad
12 10 8 6 4 2 0 Wheat
Brown rice
Maize
Oat
Figure 3 DPPH● scavenging property of unfermented and four filamentous fungi fermented cereals. ( ) Control; ( ) Aspergillus oryzae; ( ) Aspergillus awamori; ( ) Rhizopus oligosporus; (&) Rhizopus oryzae.
RCK2012 as well as Aspergillus awamori MTCC No. 548fermented maize showed equivalent amount of DPPH● scavenging property (571 and 548 lmol TE g1 grain, respectively). In the present study, both the ABTS●+ and DPPH● scavenging properties were enhanced after SSF of cereals by all the four micro-organisms (except R. oligosporus NCIM 1215-fermented maize) which might be related to the release of more soluble bioactive compounds. Among them, maximum improvement of both of the free radical scavenging properties was observed in case of ROFW. Several reports support that improvement of TPC and antioxidant potential depends on type of micro-organism involved in SSF (Moore et al. 2007; Dordevi c et al. 2010; Cai et al. 2012b). It is very difficult to compare our data with literature as different methods have been used for extraction and estimation of antioxidant property as well as TPC. Moreover, antioxidant property varies between species and varieties of grains (Zieli nski and Kozłowska 2000). Thamnidium elegans CCF 1456 and Cordyceps militaris have been observed to enhance TPC and antioxidant property of maize and wheat, respectively, yet with very less fold of increment compared with unfermented counter parts (Salar et al. 2012; Zhang et al. 2012). Basically production of antioxidant compounds during SSF is a complex and mysterious biochemical process. Several hydrolysing enzymes produced during SSF process are predicted to be associated with the release of PCs (Bhanja et al. 2008, 2009; Robledo et al. 2008; Cho et al. 2009). In the present investigation, the activities of various enzymes (a-amylase, xylanase, cellulase, pectinase and esterase) were higher in case of A. oryzae NCIM 1212fermented wheat (AOFW) compared with R. oryzae 3
Development of antioxidant rich cereals
T. Bhanja Dey and R.C. Kuhad
Table 1 Different enzyme activities, total protein, carbohydrate and reducing sugar content of AOFW and ROFW Enzyme activity/ protein/ carbohydrate/ reducing sugar
Phenolic acids AOFW
Enzymes activities (IU per gds) a-amylase 100964 2815 Xylanase 15657 3014 FPase 0 CMCase 538 025 b-glucosidase 054 004 Pectinase 1564103 266717 Esterase 386 069 Total protein 28 011 content (mg ml1) Total carbohydrate 179255 26 content (mg ml1) Reducing sugar 178 006 content (mg ml1)
ROFW
5024 2814 0 061 003 1357153 086 206
1025 57
002 .005 76831 038 015
402589 38 080 005
Values are expressed as means standard deviation.
RCK2012 (Table 1), which supported the maximum release of PCs but could not justify the elevated amount of each of the antioxidant property in ROFW. Following factors might be responsible for it: (i) Along with the enzymatic release of PCs, some other metabolic pathways might be involved in this process. (ii) Folin–Ciocalteu method, which is extensively used for TPC measurement, is a nonspecific process and it has numerous limitations especially in fermentation studies. (iii) In addition to PC, some other water-soluble compounds such as bioactive small peptides and oligosaccharides might be contributing in the antioxidant property (Veenashri and Muralikrishna 2011). Total protein, carbohydrate and reducing sugar content of AOFW and ROFW water extracts were shown in the Table 1. The % DPPH● scavenging property of polysaccharide fractions of AOFW and ROFW was 4364 and 3881%, respectively. Hence, polysaccharides might be contributing the antioxidant property of both AOFW and ROFW. (IV) The antioxidant activity of individual phenolic compounds can be different and depends on their donor proton capacity and electron delocalization capacity of their aromatic ring. Samples with same concentrations of TPC may vary remarkably in their antioxidant property (Bhanja et al. 2008; Dordevi c et al. 2010). HPLC analysis (Table 2) also supported that along with phenolic content, profiles of phenolic compounds were totally different for unfermented wheat (UFW), AOFW and ROFW. This study has demonstrated SSF as a convenient method to improve antioxidant potential of cereals. 4
Table 2 Phenolic acid composition of UFW, ROFW and AOFW
Gallic acid Protocatechuic acid 4-hydroxy benzoic acid 4-hydroxy 3-methoxy benzoic acid Caffeic acid Ferulic acid Trans-cinnamic acid
Retention time (min)
lg mg1 extract UFW
ROFW
AOFW
57 69 91 105
0237 0092 0007 0023
15219 0 0440 0049
0 1956 0 0439
112 171 266
0232 0087 0277
0372 0030 0220
0304 0651 0358
A diet containing these fermented cereals may therefore be useful for the prevention of free radical-mediated diseases. Hence, fermented cereals are suitable for the design of different functional foods and for the specific use as nutraceuticals. The study has clearly indicated that water extract of ROFW has strong antioxidant property against in vitro oxidative system compared with unfermented wheat. Moreover, these can be served as powerful sources of natural antioxidants over the synthetic antioxidant compounds used very often in food and pharmaceutical industry. Antioxidant properties of cereal grains can be further increased by optimization of different physicochemical parameters of SSF as well as optimizing the phenolics extraction conditions. Materials and methods Reagents DPPH, ABTS, Trolox and phenolic acid standards such as gallic, protocatechuic, caffeic, 4-hydroxy benzoic acid, 4-hydroxy 3-methoxy benzoic acid, trans-cinnamic acid and ferulic acid were procured from Sigma-Aldrich (Steinheim, Germany). All other reagents and chemicals were of analytical grade. Substrates for SSF Wheat, brown rice, maize and oat grains were purchased from local market, stored at room temperature, and whole grains without grinding were utilized as solid substrates in SSF to improve their antioxidant properties. Organisms, their maintenance and inoculum preparation Aspergillus oryzae NCIM 1212 and R. oligosporus NCIM 1215 were procured from NCIM, Pune, A. awamori (MTCC No. 548) was obtained from MTCC, Chandigarh, and a new fungus RCK2012 was isolated locally from Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
T. Bhanja Dey and R.C. Kuhad
Development of antioxidant rich cereals
rotten maize. Aspergillus awamori was cultured on Czapek Dox agar medium, while all other fungi were grown on potato dextrose agar (PDA). Inoculum was prepared from 5-day-old slant by suspending the fungal spores in sterile distilled water.
solution was added to it, mixed thoroughly and incubated for 15 min at room temperature. Thereafter, the mixture was diluted by 5 ml water and absorbance was measured at 725 nm. The amount of total phenolics was expressed as gallic acid equivalent (GAE) g1 grain.
Identification of the fungus
DPPH● scavenging assay
Fungal isolate RCK2012 was grown on potato dextrose medium at 30°C under static cultivation conditions for 3 days. The fungal mycelial mat was harvested by filtration through Whatman No. 1 filter paper, washed thoroughly with Milli Q water and ground in liquid nitrogen with a mortar and pestle. Genomic DNA was isolated following the method described by Kuhad et al. (2004). Sequence obtained was compared with ITS sequences available in GenBank.
The DPPH● scavenging activity of phenolic extract was determined according to the method of Brand-Williams et al. (1995), and %DPPH● scavenging activity was calculated according to the following equation:
SSF of cereals The whole grain cereal (10 g) taken in each 250-ml Erlenmeyer flask was mixed with 10 ml distilled water, autoclaved (121°C, 15 min) and subsequently cooled to ambient temperature. Fungal spore suspensions (1 9 106 spores ml1) were inoculated separately (at 10% inoculum, v/w) onto the surface of the autoclaved substrates, mixed properly and incubated for 3 days at 30°C. The flasks containing cereal grains without addition of fungal spore suspension served as controls (unfermented cereals). Extraction of phenolic compounds The fermented and unfermented cereal grains were taken out from the Erlenmeyer flask after 3 days, autoclaved and dried in an oven at 60°C for 24 h. The grains were ground in an electric grinder separately, and ground samples sieved through mesh 40 were defatted thrice by blending with hexane (1 : 5 w/v) for 5 min at ambient temperature. Defatted samples were air-dried for 24 h and stored at 20°C for further analysis. All samples were extracted with water (1 : 10 w/v) twice at 50°C for 60 min in water bath. After filtering through Whatman No.1 filter paper, the filtrate was used for comparative study of total phenolic contents (TPC), DPPH● and ABTS●+ scavenging properties. Determination of TPC TPC was determined following the modified method of Emmons and Peterson (2001). The properly diluted phenolic extract (05 ml) was mixed with 025 ml Folin–Ciocalteu reagent. 15 ml of 20% aqueous sodium carbonate Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
%DPPH scavenging activity ¼ ½ðAbCÞ ðAbSÞ=AbC100 where, AbC = absorbance of DPPH control and AbS = absorbance in presence of test sample. A standard curve was prepared using different concentrations of Trolox, and DPPH● scavenging property of the phenolic extracts was expressed as lmol Trolox equivalent (TE) g1 grain. ABTS radical cation scavenging assay It was determined following the improved ABTS decolourization assay method of Re et al. (1999). Similar to DPPH● scavenging activity, ABTS●+ scavenging property was expressed as lmol TE g1 grain. Enzyme activities Aspergillus oryzae NCIM 1212 and R. oryzae RCK2012-fermented wheat grains were suspended in water (1 : 10 w/ v) and agitated at 150 rev min1 for 1 h at ambient temperature. The extracts were squeezed through muslin cloth, centrifuged at 8000 g for 20 min at 4°C. The supernatants were assayed for different enzyme activities. Filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), a-amylase, pectinase and xylanase activities were estimated by determining the reducing sugars released from Whatman No. 1 filter paper, carboxymethyl cellulose, soluble starch, citrus pectin and birch wood xylan as substrates at 50°C and pH 50 following the method described by Ghose (1987), Bhanja et al. (2007), Gupta et al. (2008) and Kapoor et al. (2008), respectively, whereas b-glucosidase and esterase activities were assayed by estimating the p-nitrophenol content liberated from p-nitrophenyl glucopyranoside at 50°C and pH 50 and p-nitrophenyl acetate at 37°C and pH 70 employing the method of Wood and Bhat (1988) and Kordel et al. (1991), respectively. One unit of enzyme activity was defined as the amount of enzyme required to release 1 lmol of products from the respective substrates per min and expressed as IU per gram dry substrate (IU gds1). 5
Development of antioxidant rich cereals
Total protein, carbohydrate and reducing sugar content Total protein content, carbohydrate and reducing sugar content of water extracts of AOFW and ROFW were estimated according to the method described by Lowry et al. (1951), Dubois et al. (1956) and Miller (1959), respectively.
T. Bhanja Dey and R.C. Kuhad
(SA-II), for financial assistance. We would like to thank Dr. Rishi Gupta and Mr. Kavish Kumar Jain for their kind help during HPLC analysis and identification of the fungal isolate RCK2012. Conflict of Interest The authors declare no conflict of interest.
Isolation of the polysaccharide In addition to phenolic compounds, some water-soluble polysaccharides present in the water extract of A. oryzae NCIM 1212-fermented wheat (AOFW) and R. oryzae RCK2012 fermented wheat (ROFW) may have some antioxidant property. Polysaccharides were precipitated by ethanol (1 : 4 v/v) from water extract (5 ml) of AOFW and ROFW at 4°C, followed by centrifugation at 8000 g for 20 min. The precipitate was dissolved in 25 ml of water, and %DPPH● scavenging antioxidant property of 50 ll sample was estimated. HPLC analysis Identification of phenolic acids was performed by HPLC. Water extracts of unfermented wheat (UFW), AOFW and ROFW were freeze-dried, and 10 mg of each of the sample was mixed with 1 ml methanol and centrifuged at 8000 g for 10 min. The supernatant containing phenolic compounds was filtered through 045 lm Suporâ-450 membrane disc filter (Pall Corporation, Ann Arbor, MI) and injected in a Waters HPLC system connected to a Waters 2489 diode array detector (Waters, Milford, NY). The separation of phenolics was performed on a Symmetry C-18 RP column 250 9 46 mm with 5 mm particle size (Waters) with an appropriate guard column. Two mobile phases, 01% phosphoric acid (A) and acetonitrile (B), were used at a flow of 1 ml min1 with gradient profile of 20 min from 10 to 22% B, 20 min with a linear rise to 40% B and 5 min reverse to 10% B. Phenolic acids were identified from calibration curves obtained by injecting pure phenolic acids mixture as standards. Statistical analysis The mean values and the standard deviations were calculated from the data obtained from experiments carried out in triplicates. The data were analysed by one-way analysis of variance (ANOVA). Acknowledgement The authors gratefully acknowledge the University Grant Commission, Govt. of India, New Delhi (F. 15-83/2011
6
References Adom, K.K. and Liu, R.H. (2002) Antioxidant activity of grains. J Agric Food Chem 50, 6182–6187. Bhanja, T., Rout, S., Banerjee, R. and Bhattacharyya, B.C. (2007) Comparative profiles of a-amylase production in conventional tray reactor and GROWTEK bioreactor. Bioprocess Biosyst Eng 30, 369–376. Bhanja, T., Rout, S., Banerjee, R. and Bhattacharyya, B.C. (2008) Studies on the performance of a new bioreactor for improving antioxidant potential of rice. LWT-Food Sci Technol 41, 459–1465. Bhanja, T., Kumari, A. and Banerjee, R. (2009) Enrichment of phenolics and free radical scavenging property of wheat koji prepared with two filamentous fungi. Bioresour Technol 100, 2861–2866. Brand-Williams, W., Cuvelier, M.E. and Berset, C. (1995) Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol 28, 25–30. Cai, S., Gao, F., Zhang, X., Wang, O., Wu, W., Zhu, S., Zhang, D., Zhou, F. et al. (2012a) Evaluation of c- aminobutyric acid, phytate and antioxidant activity of tempeh-like fermented oats (Avena sativa L.) prepared with different filamentous fungi. J Food Sci Technol. Doi: 10.1007/ s13197-012-0748-2. Cai, S., Wang, O., Wu, W., Zhu, S., Zhou, F., Ji, B., Gao, F., Zhang, D. et al. (2012b) Comparative study of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content (TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.). J Agric Food Chem 60, 507–513. Cho, K.M., Hong, S., Math, R.K., Lee, J.H., Kambiranda, D.M., Kim, J.M., Islam, S.M.A., Yun, M.G. et al. (2009) Biotransformation of phenolics (isoflavones, flavanols and phenolic acids) during the fermentation of cheonggukjang by Bacillus pumilus HY1. Food Chem 114, 413–419. Daker, M., Abdullah, N., Vikineswary, S., Goh, P.C. and Kuppusamy, U.R. (2008) Antioxidant from maize and maize fermented by Marasmiellus sp. as stabiliser of lipidrich foods. Food Chem 107, 1092–1098. Dor devi c, T.M., Siler-Marinkovic, S.S. and DimitrijevicBrankovi, S.I. (2010) Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chem 119, 957–963.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
T. Bhanja Dey and R.C. Kuhad
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350–356. Emmons, C.L. and Peterson, D.M. (2001) Antioxidant activity and phenolic content of Oat as affected by cultivar and location. Crop Sci 41, 1676–1681. Fardet, A., Rock, E. and Remesey, C. (2008) Is the in vitro antioxidant potential of whole-grain cereals and cereal products well reflected in vivo? J Cereal Sci 48, 258–276. Ghose, T.K. (1987) Measurements of cellulase activities. Pure Appl Chem 59, 257–268. Gupta, S., Kapoor, M., Sharma, K.K., Nair, L.M. and Kuhad, R.C. (2008) Production and recovery of an alkaline exopolygalacturonase from Bacillus subtilis RCK under solidstate fermentation using statistical approach. Bioresour Technol 99, 937–945. Kapoor, M., Nair, L.M. and Kuhad, R.C. (2008) Cost-effective xylanase production from free and immobilized Bacillus pumilus strain MK001 and its application in saccharification of Prosopis juliflora. Biochem Eng J 38, 88–97. Kordel, M., Hofmann, B., Schomburg, D. and Schmid, R.D. (1991) Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary x-ray diffraction data. J Bacteriol 173, 4836–4841. Kuhad, R.C., Kapoor, R.K. and Lal, R. (2004) Improving the yield and quality of DNA isolated from white-rot fungi. Folia Microbiol 49, 112–116. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. Martins, S., Mussatto, S.I., Martınez-Avila, G., Monta~ nezSaenz, J., Aguilar, C.N. and Teixeira, J.A. (2011) Bioactive phenolic compounds: Production and extraction by solidstate fermentation. A review. Biotechnol Adv 29, 365–373. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31, 426–429. Moore, J., Cheng, Z., Hao, J., Guo, G., Liu, J.G., Lin, C. and Yu, L.L. (2007) Effects of solid-state yeast treatment on the antioxidant properties and protein and fiber compositions
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Development of antioxidant rich cereals
of common hard wheat bran. J Agric Food Chem 55, 10173–10182. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26, 1231–1237. Robledo, A., Aguilera-Carb o, A., Rodrıguez, R., Martinez, J.L., Garza, Y. and Aguilar, C.N. (2008) Ellagic acid production by Aspergillus nigerin solid state fermentation of pomegranate residues. J Ind Microbiol Biotechnol 35, 507–513. Salar, R.K., Certik, M. and Brezova, V. (2012) Modulation of phenolic content and antioxidant activity of maize by solid state fermentation with Thamnidium elegans CCF 1456. Biotechnol Bioprocess Eng 17, 109–116. Sreeramulu, D., Reddy, C.V.K., Chauhan, A., Balakrishna, N. and Raghunath, M. (2013) Natural antioxidant activity of commonly consumed plant foods in India: effect of domestic processing. Oxid Med Cell Longev, 1–12. Veenashri, B.R. and Muralikrishna, G. (2011) In vitro antioxidant activity of xylo-oligosaccharides derived from cereal and millet brans - A comparative study. Food Chem 126, 1457–1481. Vitaglione, P., Aurora, N. and Fogliano, V. (2008) Cereal dietary fibre: a natural functional ingredient to deliver phenolic compounds into the gut. Trends Food Sci Technol 19, 451–463. Wood, T.M. and Bhat, M.K. (1988) Methods for measuring cellulase activities. In Methods in Enzymology (vol. 160) eds Wood, W.A. and Kellogg, S.T. pp. 87–112. London, UK: Academic Press Inc. Zhang, Z., Lv, G., Pan, H., Fan, L., Soccol, C.R. and Pandey, A. (2012) Production of powerful antioxidant supplements via solid-state fermentation of wheat (Triticum aestivum Linn.) by Cordyceps militaris. Food Technol Biotechnol 50, 32–39. Zieli nski, H. and Kozłowska, H. (2000) Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions. J Agric Food Chem 48, 2008–2016.
7
