Journal of Bioscience and Bioengineering VOL. 117 No. 4, 449e456, 2014 www.elsevier.com/locate/jbiosc

Antioxidant properties of certain cereals as affected by food-grade bacteria fermentation Chung-Yi Wang,1 Sz-Jie Wu,2 and Yuan-Tay Shyu3, * Food Industry Research and Development Institute, No. 569, Sec. 2, Bo-Ai Rd., Chiayi 600, Taiwan,1 Department of Food Science, China University of Science and Technology, No. 245, Sec. 3, Academia Rd., Taipei 115, Taiwan,2 and Department of Horticulture, National Taiwan University, No. 140, Sec. 4, Keelung Rd., Taipei 106, Taiwan3 Received 6 September 2013; accepted 7 October 2013 Available online 8 November 2013

The effects of fermentation by 2 food-grade bacteria (Bacillus subtilis and Lactobacillus plantarum) on antioxidant activities and the contents of phenolics and flavonoids in 4 cereals (specifically adlay, chestnut, lotus seed, and walnut) were determined and compared with those of their non-fermented counterparts. Results showed that antioxidant properties observed in the fermented and non-fermented cereals may vary with fermented starters. Fermentation was observed to increase the phenolic and flavonoid contents of the extracts. The effects on Bacillus-fermented cereals were stronger than on Lactobacillus-fermented cereals. In IC50 values (mg/mL) of extracts, the extracts of fermented cereal showed a stronger DPPH radical scavenging and ferric-reducing activities. Fermentation did not significantly alter the Fe2D-chelating activity in the extracts of chestnuts and lotus seeds. All cereals were shown significantly inhibited the production of LPS-induced intracellular reactive oxygen species (ROS) without creating obvious cytotoxic effects in the macrophage cells. These results suggest that the fermentation process enables cereal-based foods with enhanced antioxidant capacities to contribute to health and nutritional improvements in consumers. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cereals; Fermentation; Antioxidant activity; Bacillus; Lactobacillus]

Cereal grains constitute a major source of energy and nutrients in the world. The benefits of cereals to human health are the subject of extensive research and epidemiological studies, which have linked whole grain intake to the prevention of metabolic syndrome, obesity, and associated chronic diseases such as cardiovascular disease and type 2 diabetes. The health benefits of cereals are primarily caused by their phytochemicals including phenolic acids, flavonoids, vitamins, fiber and minerals, which act together to combat oxidative stress, inflammation, hyperglycemia and carcinogenesis (1). Recently, as part of a general trend, the cereal-processing industry has been challenged to produce new ingredients and foods with added values for consumer health. This includes revisiting dry milling and exploring wet enzyme-based fractionation processes, and fermentation to produce food ingredients or foods with increased levels of health-related components and structural features, which provide strong sensory properties (2). In particular, fermentation may be the simplest and most economical way of improving nutritional value, sensory properties, and functional qualities. These qualities include acid production suggested to retard starch digestibility, and adjustments of pH to a range that encourages the actions of certain endogenous enzymes, thus changing the bioavailability pattern of minerals and phytochemicals (3). Variety fermented cereal products, such as foods containing probiotic bacteria, rice vinegar, soy sauce, soybean-barley paste, natto and tempeh, are sold in food stores in Asia. These fermented * Corresponding author. Tel.: þ886 2 33664850; fax: þ886 2 23661441. E-mail addresses: [email protected], [email protected] (Y.-T. Shyu).

cereal-based foods are produced by traditional methods that exploit single or mixed cultures of various non-toxic microorganisms. Products produced from various cereal substrates fermented by lactic acid bacteria, yeast and/or fungi are included (4). Numerous traditional fermented foods have been studied and their health effects on metabolisms and immune systems have been demonstrated in vitro and in vivo models. Poutanen et al. (5) reported that new bioactive metabolites in cereals could be produced during fermentation from the starters present in the raw materials. Modification of the cereal matrix during fermentation could be tailored to increase the bioaccessibility of bioactive compounds. Nout (6) indicated that the nutritional properties of traditional cereal-fermented products can be enhanced by increasing their nutrient and energy densities, and by increasing their mineral status by combining mineral fortification and dephytinization. Currently, the use of Bacillus species in probiotic dietary supplements is rapidly expanding with an increasing number of studies demonstrating dietary supplementation in animals, growth promotion and competitive exclusion in animals and agents, and lastly demonstrating aquaculture in enhancing the growth and disease resistance of cultured shrimps (7). Bacillus products that have been historically consumed in Asia, such as Bacillus subtilis have been used extensively to ferment natto, a well-known traditional fermented food that is prepared with cooked soybeans. Natto contains various biological compounds, such as aglycone, vitamin K2, nattokinase and superoxide dismutase, which have been shown to suppress low-density lipoprotein oxidation, and also reduces the DNA injury caused by

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.002

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cyclophosphamide and exerts antioxidant and anti-mutagenic effects (8). Additionally, lactic acid bacteria fermented non-dairy foods have also be examined in the development of functional foods. Food and beverage probiotic additives may exert positive effects on the composition of gut microbiota and the subject of intensive research. Non-dairy probiotic foods are rich in plantbased functional components such as minerals, vitamins and various antioxidants, and are currently consumed frequently and consistently by a large percentage of the global population. Furthermore, they do not contain any dairy allergens and they lower fat content (9). To develop healthy foods and functional food ingredients, numerous studies have used bacteria and yeast to ferment various cereals, and to produce functional fermented foods. Juan and Chou (10) discovered that black soybean fermentation with B. subtilis enhanced antioxidant activity and raised total phenolic content (TPC) and total flavonoid content (TFC). Other studies showed that soybeans, rice, barley, emmer and oat fermentation with lactic acid bacteria resulted in higher antioxidant activities compared to unfermented samples (11,12). Therefore, our objective was to assay the influence of cereal fermentation with B. subtilis or Lactobacillus plantarum on antioxidant properties. MATERIALS AND METHODS Preparation of microbiological cultures The microorganisms of B. subtilis (BCRC 14718) were obtained from the Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. L. plantarum was isolated from pickled cabbages. Strains cultured at 37 C for 24 h were conducted in nutrient broth for B. subtilis and in De Man, Rogosa and. Sharpe (MRS) broth for L. plantarum. The bacterial cells were harvested in sterile distilled water, and after adjusting them to a concentration of 107e108 cells/mL served as the inoculum for cereal fermentation. Preparation of cereal extracts The cereal samples used in this study included adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), shelled chestnuts (Castanea crenata), lotus seeds (Nelumbo nucifera) and shelled walnuts (Juglans regia), which were purchased from a traditional market in Taipei, Taiwan. Approximately 100 g of each cereal grain was washed with distilled water and submerged in distillate water at room temperature for 12 h. The cereal grains were mixed in equal quantities and milled into flour. Cereal paste was prepared by adding 100 g of mixed cereal flour to 200 g distilled water and then mixed in a blender. Cereal paste was then steamed at 121 C for 1 h. Each steamed cereal paste (cooled to 40 C) was separated into 3 portions; the first samples were inoculated with 5 mL of B. subtilis fermentation at 37 C with shack at 180 rpm for 24 h. The second samples were inoculated with 5 mL of L. plantarum at 37 C for 24 h, and the third samples were the control and were not inoculated. At the end of fermentation, the samples were dried in a freeze dryer (Freeze-dryer Alpha 1-2/LD-2, Vacuum pump RZ-5, Christ, Germany). For extraction, lyophilized samples were subsequently milled using a commercial milling machine (Braun KSM2, Germany). Fine powders (10 g) were then extracted with 50 mL methanol and kept in a rotary shaker for 12 h and filtered through Whatman No. 4 filter paper. The methanolic extracts were then rotary-evaporated at 40 C to dryness. The dried extracts were sealed to prevent moisture absorption and stored at 4 C for future analyses of antioxidant properties. Assay of phenolics and total flavonoids The phenolic content was measured using the FolineCiocalteu method (13). An aliquot of 200 mL extract was mixed with 200 mL of FolineCiocalteu phenol reagent (Sigma, St. Louis, MO, USA) and allowed to react for 3 min. The 400 mL of 1 N Na2CO3 was then added and allowed to react for 90 min at room temperature. Absorbance was measured at 725 nm using a spectrophotometer (DU 800, Beckman Coulter). The results were expressed as milligram gallic acid equivalents (GAE) per gram of sample (mg GAE/ g extract). The flavonoid content of the samples was determined using the colorimetric method adopted from Jia et al. (14), and quercetin (QU) was used as a standard. Extracts or standard solutions (300 mL) were mixed with distilled water (1 mL) and 100 mL of 5% NaNO2 solution. The 100 mL of 10% AlCl3 solution was added to the mixture 5 min later. After 6 min, 0.65 mL of 1 M NaOH and 0.8 mL distilled water was added. The solutions were then mixed and absorbance was measured at 510 nm. The results were expressed as mg quercetin per g of sample (mg QU/ g extract). Free radical-scavenging assay The effect of cereal extracts on DPPH radicals was examined using the modified method by Shimada et al. (15). The 400 mM DPPH solution in methanol was prepared and 150 mL of this solution was added to 50 mL test samples in various concentrations. The reaction mixture was shaken well and incubated for 30 min at room temperature; the absorbance of the resulting

J. BIOSCI. BIOENG., solution was read at 517 nm against a blank. The inhibitory percentage of DPPH was calculated using Eq. 1 Scavenging effect ð%Þ ¼

  1  absorbancesample = absorbancecontrol  100%

(1)

Estimation of reducing activity The reducing power was measured by adopting the approach by Duh and Yen (16). A sample (0.2 mL) containing various amounts of extracts, phosphate buffer (0.2 mL, 0.2 M, pH 6.6), and a 1% (v/w) potassium ferric cyanide solution (0.2 mL) were mixed and heated at 50 C for 20 min. After the mixture had been cooled to room temperature, 0.2 mL 10% trichloroacetic acid was added. Following centrifugation at 700 g for 10 min at 4 C, a 0.5 mL aliquot of the supernatant was mixed with 0.5 mL of distilled water and 0.1 mL ferric chloride (0.1%). After 10 min of reaction, the absorbance at 700 nm was measured. Increased absorbance of the reaction mixture corresponds to higher reducing power. Evaluation of metal-chelating activity The Fe2þ-chelating ability of the samples was determined according to the method by Dinis et al. (17) with minor modifications. Briefly, 0.2 mL of the sample, methanol (0.74 mL), 2 mM ferrous chloride (0.02 mL), and 5 mM ferrozine (0.02 mL) were mixed. The mixture was shaken and left standing at room temperature for 10 min. The absorbance of the mixture was then determined at 562 nm. The ability to chelate the ferrous ion was calculated as follows: Chelating ability ð%Þ ¼

  1  absorbancesample = absorbancecontrol  100%

(2)

Intracellular ROS generation Murine RAW 264.7 macrophages (BCRC 60001) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillinestreptomycin. All cell cultures were incubated in a humidified chamber at 37 C with 5% CO2. Intracellular ROS levels were measured by detecting the fluorescence intensity of the oxidantsensitive probe 20 ,70 -dichlorfluorescein-diacetate (DCFH-DA). Dichlorofluorescein (DCFH) is converted from DCFH-DA by deacetylase within the cells, and is oxidized by various intracellular ROS to yield 20 ,70 -dichlorfluorescein (DCF), a highly fluorescent compound. The cells were incubated with 100 mg/mL of samples in the presence or absence of LPS (1 mg/mL) for 24 h. The cells were stained with 20 mM of DCFH-DA for 15 min at room temperature and subjected to determination of intracellular ROS production using a multimode microplate reader (Synergy H1, BioTek Instruments, Inc.). Statistical analysis The experimental results were the means from the triplicate experiments. The data were presented as mean  standard deviation (SD) and analyzed using a statistical analysis system (SAS Inc., NC, USA). One-way ANOVA analysis was conducted. Significant differences between the means were determined by Duncan’s multiple range tests. The results were regarded as statistically significant at p < 0.05.

RESULTS Contents of total phenolics and flavonoids Table 1 shows the content of phenolics and flavonoids in the unfermented and fermented cereal extracts expressed as mg of gallic acid and quercetin, respectively, per g of extract. A significantly (p < 0.05) higher content of total phenolics and total flavonoids was found in the fermented cereal extracts than in the unfermented cereals. Depended on the starter culture used for fermentation, the content of total phenolics and total flavonoids in Bacillus-fermented cereals was higher than those of Lactobacillus-fermented cereals. Total phenolic compound contents in the examined cereals were the highest in the Bacillus-fermented walnuts (33.8 mg GAE/g dried extract), and the lowest contents were 8.58 mg GAE/g dried extract in the adlay. However, the TPC of lotus seed fermented with B. subtilis had the best performance (increased w64%) than those of walnuts (49%). Similar to phenolics, the content of total flavonoids was also found to vary with the starter used for fermentation. Cereals fermented with B. subtilis showed a higher flavonoid content among the various extracts of the non-fermented and fermented with L. plantarum. The highest TFC were in chestnut 29.1 mg QU/g dried extract fermented with L. plantarum. In walnut extracts TPC increased from 22.8 mg GAE/g dried extract in the native unfermented sample to 33.8 mg GAE/g dried extract in the extract fermented with B. subtilis and 28.6 mg GAE/g dried extract in the extract fermented with L. plantarum. In chestnut, TFC varied from 23.1 mg QU/g dried extract to 27.8 and 29.1 mg QU/g dried extract, respectively in the 3 samples.

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TABLE 1. The contents of total phenolics and flavonoids and IC50 of the extracts of cereals. Cereals

Adlay

Chestnut

Lotus seed

Walnut

TPC (mg GAE/g dried extract) Native Fermented Fermented Native Fermented Fermented Native Fermented Fermented Native Fermented Fermented

with B. subtilis with L. plantarum with B. subtilis with L. plantarum with B. subtilis with L. plantarum with B. subtilis with L. plantarum

8.58 13.29 11.91 12.95 19.65 15.28 17.52 28.67 24.62 22.80 33.89 28.61

           

0.62b 1.67a 1.94a 0.57c 2.34a 1.85b 1.67c 2.95a 3.54b 4.23c 3.84a 4.24b

TFC (mg QU/g dried extract) 6.09 9.17 7.57 23.18 29.14 27.86 11.67 18.78 16.29 9.68 18.64 15.62

           

0.75b 1.54a 1.16ab 7.24b 2.60a 3.43a 1.57b 2.49a 2.51a 0.89c 2.37a 1.65b

IC50 (mg/mL) of extracts DPPH scavenging activity 3.12 1.19 2.24 2.43 1.89 2.31 2.93 1.67 1.83 1.92 0.80 0.89

           

0.12a 0.22c 0.19b 0.20a 0.14b 0.21a 0.25a 0.18b 0.21b 0.15a 0.13b 0.16b

Fe2þ-chelating ability 10.13 6.15 8.09 6.83 5.73 6.51 15.83 11.26 11.96 7.70 6.83 6.71

           

0.34a 0.26c 0.31b 0.30a 0.27b 0.22a 0.42a 0.38b 0.29b 0.22a 0.34b 0.27b

Reported values are the means  S.D. (n ¼ 3). Data bearing superscript in lowercase letters in the same column are significantly different (p < 0.05).

DPPH radical-scavenging effect The scavenging activity of extracts from the cereal against the DPPH radical has been evaluated at various concentrations, and the results are shown in Fig. 3. The extracts of non-fermented cereal and fermented cereal show the doseeresponse curve of the DPPH radical-scavenging activity, with BHA as the positive control. The extracts of Bacillusfermented cereal inhibited DPPH radical absorption at all concentrations and was superior to the Lactobacillus-fermented cereals and non-fermented cereals. Walnut extracts exhibited the highest DPPH radical-scavenging activity. At a concentration of 5 mg/mL, the scavenging activity of Bacillus-fermented walnut extracts reached a plateau of 88.6%, however, at the same

concentration the scavenging activities of Lactobacillus-fermented walnut extracts and non-fermented walnuts were 80.2% and 79.3%, respectively. In adlay extracts, at 5 mg/mL, the scavenging activity varied from 70.4% to 92.1% depending on the fermentation starter, and in lotus seed extracts, 72e85% scavenging ability was observed. In the chestnut extracts, a higher scavenging activity (71e87%) was observed at 10 mg/mL. However, the scavenging abilities of BHA were in the range of 94.3% at 0.5 mg/mL. In the half-inhibition concentration (IC50) value, which was obtained by interpolation by linear regression analysis of the data plotted in Fig. 1. Among the extracts examined, the Bacillus-fermented walnut extracts had the highest

FIG. 1. DPPH radical-scavenging effects of the various extracts of adlay (A), chestnut (B), lotus seed (C), and walnut (D). N, native; B, fermented with B. subtilis; L, fermented with L. plantarum. Each value represents mean  SD (n ¼ 3).

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scavenging activity, with the lowest IC50 of 0.80 mg/mL, and exhibited the strongest DPPH radical-scavenging effect. However, non-fermented adlay with the highest IC50, had the least scavenging activity. Fermentation with B. subtilis or L. plantarum had a positive influence on the DPPH inhibitory effect in the each cereal extracts (Table 1). For adlay, the scavenging effect of the DPPH radically increased from 70.4% in the unfermented sample to 92.1% and 91.5 % in the samples fermented with B. subtilis and L. plantarum, respectively, leading to IC50 values of 3.1, 1.1, and 2.2 mg/mL, respectively. Similar increase in IC50 of DPPH radical scavenging after fermentation with B. subtilis and L. plantarum were also noted in chestnuts (2.43, 1.89, and 2.31 mg/mL, respectively), lotus seeds (2.93, 1.67, and 1.83 mg/mL, respectively) and walnuts (1.92, 0.80 and 0.89 mg/mL, respectively). Reducing activity The doseeresponse curve for reducing activity of various extracts of cereals with or without fermentation is shown in Fig. 2. The reducing activity of the extracts of the fermented and non-fermented cereals showed a significant difference (p < 0.05). The Bacillus-fermented cereals exhibited a higher reducing activity than the extracts fermented with L. plantarum and non-fermented cereal. At 20 mg/mL, the reducing activity of adlay extracts was in the range of 0.18e064, it was in the range of 0.57e0.92 in chestnut extracts, 0.59e0.76 in lotus seed extracts, and 0.45e0.8 in walnut extracts. However, the reducing power of BHA was 1.2 at 0.1 mg/mL. Fe2D-chelating ability Fig. 3 shows the doseeresponse curve of the various extracts of cereals and fermented cereals for Fe2þ-

J. BIOSCI. BIOENG., chelating ability with EDTA as the positive control. The IC50 of the extracts for Fe2þ-chelating ability is presented in Table 1. As shown in Fig. 3, the Fe2þ-chelating ability of both non-fermented and fermented cereal extracts, regardless of the starters used to ferment the cereals, increased as the dosage of extract increased. The Fe2þ-chelating ability of cereals was enhanced after fermentation, and the results show that Bacillus-fermented cereals exhibited a significantly higher Fe2þ-chelating ability than did the respective extracts of Lactobacillus-fermented cereals and non-fermented cereals. At the same dosage level, the extracts of Bacillus-fermented adlay showed the highest Fe2þ-chelating ability followed by Lactobacillus-fermented chestnut extracts, Bacillus-fermented chestnut extracts, and Bacillus-fermented walnut extract in descending order. As shown in Table 1, the extracts of fermented cereal showed significantly (p < 0.05) lower IC50 in Fe2þ-chelating ability than those of non-fermented cereals, with the exception of Lactobacillus-fermented chestnut extracts. The highest IC50 of the Fe2þ-chelating ability was lotus seed extracts varying between 11.2 and 15.8 mg/mL, depending on the starters used, and depending on whether they were fermented. EDTA showed an excellent chelating agent for the Fe2þ-ion and a chelating ability of 99.2% at 0.1 mg/mL. Inhibition of intracellular ROS generation Fig. 4 shows the effects of various extracts of cereals and fermented cereals on intracellular ROS production in the LPS-induced RAW 264.7 cells. The cells stimulated with LPS showed an intracellular ROS elevation, as assessed by labeling the cells by using a fluorescent

FIG. 2. Reducing power of the various extracts of adlay (A), chestnut (B), lotus seed (C), and walnut (D). N, native; B, fermented with B. subtilis; L, fermented with L. plantarum. Each value represents mean  SD (n ¼ 3).

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FIG. 3. Fe2þ-chelating ability of the various extracts of adlay (A), chestnut (B), lotus seed (C), and walnut (D). N, native; B, fermented with B. subtilis; L, fermented with L. plantarum. Each value represents mean  SD (n ¼ 3).

probe DCFH-DA. During the LPS-stimulation, a large increase in oxygen uptake resulted in the massive release of intracellular ROS in the cells, as compared with the LPS treated cells. Treatment with 100 mg/mL of cereal extracts significantly inhibited LPSinduced intracellular ROS generation (approximately 17e35% inhibition for adlay, 14e28% inhibition for chestnut, 21e25% inhibition for lotus seed, and 10e21% inhibition for walnut). By contrast, a low dose of non-fermented cereals extracts (25 mg/mL) did not interfere with ROS production. DISCUSSION

FIG. 4. Effects of cereals on the LPS-induced ROS production in RAW 264.7 cells. Cells were incubated with adlay, chestnut, lotus seed, or walnut in the presence or absence of LPS (1 mg/mL) for 18 h. N, native; B, fermented with B. subtilis; L, fermented with L. plantarum. Data are expressed as mean  SD (n ¼ 3).

Cereals are traditional staples for Asians. The consumption of cereal-based food provides health benefits because they contain various phytochemicals and antioxidants. During the last decade, cereals alone or mixed with other ingredients have been used for the production of traditional fermented foods and for the development of new foods with enhanced health properties. Fermentation is one of the oldest and most economical methods of producing and preserving food. The nutritional properties of traditional cereal-fermented foods can be enhanced by increasing their nutrient and energy density, and by the health contribution that these foods promote in diets (6). It can be concluded that in tropical regions, mixed natural fermentations of cereals with

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probiotic microorganisms (lactic acid bacteria, Bacillus sp. and yeast) are widely practiced. These fermentations are important in providing healthy foods with attractive flavors and textures. In addition, recent research into functional foods is advancing the development of dietary supplementation including introducing the concept of probiotics, nutraceuticals, and prebiotics, which may affect gut microbial compositions and activities (18). Cereal-based foods are abundant in various non-digestible carbohydrates (inulin/ oligofructose and galacto-oligosaccharides), which act as microbial food supplements and may benefit people by selectively stimulating beneficial bacteria in the large intestine. Therefore, cereal grains are suitable for use as fermented material for food-grade bacteria to for produced healthy foods (19). Cereal substrates (adlay, chestnuts, lotus seeds, and walnuts) were fermented with B. subtilis or L. plantarum to study and compare the effects of starters on antioxidant properties. The results show that the antioxidant activities and bioactive compounds of these cereals could be considerably modified through changes in the inocula composition. The contents of phenolics and flavonoids of fermented cereals were significantly higher than in non-fermented cereals. ÐorCevi c et al. (20) showed that the contents of total phenolics in cereal grains were affected by the fermentation starter, and showed that the cereals fermented with Lactobacillus rhamnosus were higher than cereals fermented with Saccharomyces cerevisiae. These results can be explained by the levels of bioactive compounds that can be modified during fermentation through the metabolic activity of microbes. Fermentation-induced structural breakdown of cereal cell walls may also occur, leading to the liberation or synthesis of various bioactive compounds. During fermentation, enzymes such as amylases, xylanases and proteases derived from grain and microbes, contribute to the modification of grain composition; bound phenolics may be released through the enzymatic treatment of samples prior to extraction (21,22). Other scientists have also demonstrated that phenolic compounds in plants are typically seen in various forms, such as free or conjugated forms through hydroxyl groups with sugar as glycosides (23). The b-glucosidases of microbial origin could also be used to hydrolyze the phenolics and flavonoids, and improve their biological activity in the field of medicine, general biomedical research, and in the food industry (24). The B. subtilis and L. plantarum were documented as having strong glucosidase activity (25,26); therefore, increased active compounds might be converted from the enzymatic cleavage of corresponding glucosides. Pyo et al. (27) reported that the application of b-glucosidase-producing lactic acid bacteria as a functional starter in cultures derives bioactive isoflavones, genistein and daidzein in fermented soymilk. In another study, the phenolic compounds in cowpeas also increased during Lactobacillus-fermentation (25). Kuo et al. (26) reported that isoflavone aglycones are abundant in fermented soy products such as miso, natto, and tempeh, and they are produced by B. subtilis. A similar phenomenon was also observed in black soybeans fermented with B. subtilis, where the total phenolic and flavonoid contents were increased during fermentation (10). Therefore, these processes may be used to explain fermentation-induced increases in the total phenolic and flavonoid contents leading to higher antioxidant potentials in fermented cereal extracts. Traditional natto is fermented from soybeans; however, recent various natto-like foods fermented with B. subtilis were extensively developed to research their health benefits. Foods such as, adlay, red beans, black soybeans, and peanuts increased their antioxidant activity during fermentation (10,28e30). The B. subtilis is not only capable of enhancing the antioxidant activity of cereals, but can also positively enhance the contents of phenolics and flavonoids. During an antioxidant property assay, the colored DPPH radical is reduced to non-radical DPPH-H in the presence of an antioxidant or a hydrogen donor (31). The results show that significant DPPH radical inhibition

J. BIOSCI. BIOENG., ability in walnuts is consistent with data reported in other studies, except for differences in data interpretation. Akbari et al. (32) demonstrated that walnuts contained substantial amounts of phenolic antioxidants that effectively scavenged free radicals, particularly DPPH, superoxide anion, and nitric oxide radicals. Our results are in agreement with those reported by Tseng et al. (33). They reported an increase in DPPH radical-scavenging activity in soybeans after B. subtilis fermentation. Despite the variation in the DPPH radical-scavenging effects exerted by varied fermentation starters, this antioxidant activity was related to the content of the total phenolics in the extracts. For example, the Bacillus-fermented walnut extracts contained the highest content (33.89 mg GAE/ g dried extract) and exhibited the highest DPPH radical-scavenging effects among the various cereal extracts examined. Typically, the higher antioxidant activity of the extracts might be attributed to their contents of total phenolics and flavonoids. However, no marked correlation existed between the content of total flavonoids and DPPH radical-scavenging activity in cereal. The chestnut extracts with higher total flavonoid content values were not necessarily better in DPPH inhibition. Prior et al. (34) stated that numerous other compounds may contribute to the reaction, and may cause potential errors in bioactive content measurements. Meyers et al. (35) reported that free phenolic in strawberries was weakly correlated with their antioxidant activity, and flavonoid content did not correlate with antioxidant activity. Ghasemi et al. (36) also found no correlation between the total phenolic and flavonoid contents and antioxidant activity in tissues and/or peels of citrus. However, the synergism between antioxidants in the mixture caused the antioxidant activity to be not only dependent on antioxidant concentration but also on the structure and interactions among antioxidants. That is why samples with similar concentrations of total phenolics may vary significantly in their antioxidant activity (20). In the reducing power assay, the presence of reductants in cereal extracts results in reducing a Fe3þ/ferricyanide complex to form a Fe3þ ferrous complex. When antioxidants donate an electron or hydrogen, the Fe3þ/ferricyanide complex is reduced to Fe2þ, and the absorbance is increased at 700 nm. It is accepted that the higher the absorbance at 700 nm, the greater the reducing power (37). The doseeresponse curve for reducing activity of various extracts of cereals with or without fermentation is shown in Fig. 2. Fermented cereals were more effective in reducing activity than non-fermented cereals. Similar studies reported that Bacillus fermentation enhanced the reducing activity of soybeans and red beans (28,38). These results showed that the fermented cereal extracts are electron donors and can react with free radicals and convert them into more stable products, thus terminating the radical chain reactions. In addition, the contents of total phenolics and flavonoids in the fermented cereal extract were higher than in non-fermented cereals, and this may be attributed to the reducing power of the fermented cereal extracts. In addition to initiating lipid peroxidation, which leads to the deterioration of food, metal ions possessing catalytic ability have been correlated with the incidence of arthritis and cancer (39). In addition, ferrous ions, commonly found in the food system, are considered the most effective pro-oxidants (31). The chelating ability of the extracts of the cereals before and after fermentation toward ferrous ions was examined. The results implied that the fermented cereal extracts had the Fe2þ-chelating effect and could afford protection against oxidative damage. These data show that the fermented cereal extracts showing antioxidant activity may be attributed to their proton-donating ability, as evidenced through DPPH radicals scavenging results. In addition, the fermented cereal extracts had the Fe2þ-chelating effect and may afford protection against oxidative damage. Fermented cereal extracts can also be viewed as electron donors that could react with free radicals, convert them to more stable products, and terminate radical chain reactions.

VOL. 117, 2014

ANTIOXIDANT PROPERTIES OF CERTAIN CEREALS

The components of cereals, such as phenolic compounds, particularly phenolic acid and flavonoid derivatives, carotenoids, tocopherol and vitamin C, possess antioxidant activity. In addition, other ingredients in fermented cereal, such as garlic and carrots, have been reported to show antioxidant activity (1). They may contribute to the antioxidant activity observed in the cabbage mixture before and after fermentation. The fermented cereals containing B. subtilis or L. plantarum in addition to being capable of balancing intestinal microflora and stimulating the immune system, may further affect the antioxidant activity exhibited by fermented cereals. Fermentation may alter the composition, structure and polarity of antioxidant biofactors in fermented cereals, and therefore led to the variation in the antioxidant activity observed in the cereal substrates with and without fermentation. The phenolic compounds present in the extract, in addition to other chemical components, may suppress lipid peroxidation through different chemical mechanisms, including free radical quenching, electron transfer, radical addition, and radical recombination (40). Because the antioxidant activity of the extract cannot be predicted on the basis of its total phenolic content alone, a synergism of polyphenolic compounds, with one another, and other components present in an extract, may contribute to the overall observed antioxidant activity (41). Moreover, the antioxidant activities as attributed to the total phenolic content could be from properly oriented and substituted functional groups in phenolic compounds rather than by simply being present. That fermented cereals exhibited better antioxidant activity than non-fermented cereals could be because the phenolic compounds with high antioxidant activity might have been produced through fermentation. A high level of correlation between phenolics and antioxidant activities was shown in the studies of sorghum (30). Epidemiological studies associated with cereal grain consumption could contain important dietary antioxidants with benefits for human health and reduced risk of chronic diseases. This study showed that cereals fermented with B. subtilis or L. plantarum exhibit significant free radical-scavenging activities, ferric-reducing abilities, chelating Fe2þ-ion, and increased contents of phenolics and flavonoids. The starters of fermentation had a clear effect on potentially bioactive constituents of cereals, and improved their nutritional value. In addition, identification of both biological and food-processing conditions that impact the distribution, stability and activity of cereal antioxidants is necessary to be able to produce foods with maximum health benefits. Thus, the natural bioactivity of cereals can be further increased using fermentation bioprocesses to produce nutritionally superior healthy meals, which can be used as quality improvers in cereal-based foods. ACKNOWLEDGMENTS The authors would like to thank Chen Yung Memorial Foundation for their supporting on the funding of this research. References 1. Poutanen, K.: Past and future of cereal grains as food for health, Trends Food Sci. Technol., 25, 58e62 (2012). 2. Delcour, J. A., Rouau, X., Courtin, C. M., Poutanen, K., and Ranieri, R.: Technologies for enhanced exploitation of the health-promoting potential of cereals, Trends Food Sci. Technol., 25, 78e86 (2012). 3. Blandino, A., Al-Aseeri, M. E., Pandiella, S. S., Cantero, D., and Webb, C.: Cereal-based fermented foods and beverages, Food Res. Int., 36, 527e543 (2003). 4. Murooka, Y. and Yamshita, M.: Traditional healthful fermented products of Japan, J. Indian Microbiol. Biotechnol., 35, 791e798 (2008). 5. Poutanen, K., Flander, L., and Katina, K.: Sourdough and cereal fermentation in a nutritional perspective, Food Microbiol., 26, 693e699 (2009). 6. Nout, M. J. R.: Rich nutrition from the poorest e cereal fermentations in Africa and Asia, Food Microbiol., 26, 685e692 (2008).

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Antioxidant properties of certain cereals as affected by food-grade bacteria fermentation.

The effects of fermentation by 2 food-grade bacteria (Bacillus subtilis and Lactobacillus plantarum) on antioxidant activities and the contents of phe...
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