Effect of Dephytinization by Fermentation and Hydrothermal

Article pubs.acs.org/JAFC

Effect of Dephytinization by Fermentation and Hydrothermal Autoclaving Treatments on the Antioxidant Activity, Dietary Fiber, and Phenolic Content of Oat Bran H. Ö zkaya,† B. Ö zkaya,† B. Duman,† and S. Turksoy*,‡ †

Faculty of Engineering, Department of Food Engineering, Ankara University, Golbasi Campus, Golbasi, Ankara TR-06830, Turkey Faculty of Engineering, Department of Food Engineering, Hitit University, Ç orum TR-19030, Turkey

‡

ABSTRACT: Fermentation and hydrothermal methods were tested to reduce the phytic acid (PA) content of oat bran, and the effects of these methods on the dietary fiber (DF) and total phenolic (TP) contents as well as the antioxidant activity (AA) were also investigated. Fermentation with 6% yeast and for 6 h resulted in 88.2% reduction in PA content, while it only resulted in 32.5% reduction in the sample incubated for 6 h without yeast addition. The PA loss in autoclaved oat bran sample (1.5 h, pH 4.0) was 95.2% while it was 41.8% at most in the sample autoclaved without pH adjustment. In both methods, soluble, insoluble, and total DF contents of samples were remarkably higher than the control samples. Also for TP in the oat bran samples, both processes led to 17% and 39% increases, respectively, while AA values were 8% and 15%, respectively. Among all samples, the autoclaving process resulted in the lowest PA and the greatest amount of bioactive compounds. KEYWORDS: dephytinization, oat bran, hydrothermal process, fermentation process

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sclerosis agents.11 Similarly, it has been reported that antioxidant compounds from oats have a range of biological activities including cholesterol-lowering, antiatherosclerosis, scavenging reactive oxygen, and reducing oxidation levels of LDL.12 It has been also notified that consumption of oat products regularly is associated with reduced risk of coronary heart disease and of some types of cancer.13 However, oat bran may cause some problems due to its high phytic acid (PA, myo-inositol hexaphosphate) content, but even so it is a good source of DF, phenolic, and antioxidative compounds. These problems derive from the strong ability of PA to chelate multivalent metal ions, such as Ca2+, Zn2+, Fe2+, Mg2+, etc., resulting in insoluble complexes, changing their solubility and functionality, and also reducing their bioavailability in the gastrointestinal system. It is also determined that PA interacts with proteins and forms protein-phytate complexes which are stable against proteolytic enzymes. Due to the strong binding capacity of PA, oat bran can be considered an antinutrient. Thus, this fact limits the usage of oat bran as a functional ingredient in food formulations.14,15 A considerable amount of PA in oat bran remains in the processed foods even though some PA is biodegraded during processing methods such as fermentation and thermal treatments. According to the literature data, different treatments (i.e., soaking, malting, fermenting, baking, autoclaving, frying) attracted attention about reducing PA content during food processing.16−21 In our previous work, a decrease around 96% of PA was observed in dephytinizated wheat and rice brans by hydrothermal autoclaving treatment.22 Servi et al.23 noticed the

INTRODUCTION Particularly in countries where the majority of food consumption is from animal origin, a number of diseases associated with low dietary fiber (DF) intake have become widespread. Scientists studying food and nutrition have recommended enhancement of consumption of food products with DF compounds for the prevention of diseases. It has been stated that cereal brans are excellent DF sources, and their effects can vary depending on cereal variety, solubility of fiber, and chemical composition.1−3 Among different cereal brans, oat bran has been attracting a great deal of attention for its high DF (especially soluble dietary fiber (SDF)) content. Although there is no rigorously established explanation for mechanism of action, it has been reported that a meal containing oat bran lowers serum cholesterol levels dramatically, reduces the risk of cardiovascular diseases, decreases blood pressure, and also prevents chronic diseases such as cancer and obesity.4−6 Additionally, oat bran acts as an active ingredient for reducing symptoms of diabetes and also for balancing glucose and insulin metabolism.7 It has been reported that the daily insulin requirement of people following a high-oat bran diet for 10 days dropped from 20 units/day to 0 units/day.8 The hypocholesterolemic effect of oat bran is quite parallel with other plant fibers; however, there are many advantages of oat bran such as being appetizing, well metabolized, and suitable for many bakery product formulations.9 Besides their dietary fiber profiles, cereal brans contain significant amounts of bioactive functional components such as phenolics and antioxidants, which have crucial roles in preventing chronic diseases in human metabolism.10 Bran phenolics have diverse effects on the taste, flavor, appearance properties, and oxidative stability of food products. They can also serve in human body as antioxidants and antiapoptosis, antiaging, anticarcinogenic, anti-inflammation, and antiathero© 2017 American Chemical Society

Received: Revised: Accepted: Published: 5713

April 12, 2017 June 23, 2017 June 26, 2017 June 26, 2017 DOI: 10.1021/acs.jafc.7b01698 J. Agric. Food Chem. 2017, 65, 5713−5719

Article

Journal of Agricultural and Food Chemistry

mL DMSO and were kept at −20 °C under nitrogen gas in dark containers until further analysis. Analyses. The analyses of moisture, ash, and protein contents of bran samples were carried out according to the AACC approved methods 44-01, 08-01 and 46-12, respectively.24 The pH of the bran slurries was measured by a digital pH meter. Total dietary fiber content was determined according to the AOAC method 991.43.25 Phytic acid content was measured by using the colorimetric procedure of Haug and Lantzsch.26 After the samples were extracted by using the method of Adom and Liu,4 phenolic content and antioxidant capacity of samples were determined according to the methods described by Yu, Haley, Perret, Harris, Wilson, and Qian.10 The phenolic contents of each extract was determined using FolinCiocalteau reagent. Briefly, 100 μL of extracts were oxidized with Folin−Ciocalteau reagent, and the reaction was neutralized with sodium carbonate. The final volume was made up to 10 mL with pure water. After 2 h of incubation, the absorbance at 765 nm was measured and used to calculate the phenolic contents using gallic acid as standard. Results were expressed as milligram of gallic acid equivalent per kilogram of sample. The antioxidant activity of samples was determined using 2,2diphenyl-2-picryl-hydrazyl (DPPH) reagent. Trolox (6-hydroxy2,5,7,8-tetra-methyl-chroman-2-carboxylic acid) was used as an antioxidant standard. DMSO was used to prepare the solution of Trolox. The absorbance at 515 nm was measured, and the results were expressed as Trolox equivalents. Statistical Analyses. For each treatment, sampling was conducted according to a completely randomized experimental design with a factorial arrangement. For each treatment (fermentation and hydrothermal autoclaving), there are two factors (yeast level−fermentation time and pH−autoclaving time), and each factor has three levels. All statistical analyses were performed using SPSS software (V.11.0 for Windows, SPSS Inc., Chicago, IL). Results were analyzed by three-way analysis of variance (ANOVA) with the general linear model procedure, and the Duncan multiple test was used to determine whether significant differences exist among the different variable means. The level of significance applied to statistical tests was p< 0.05.

importance that the dephytinization process of bran samples should be performed before their incorporation into food matrix to keep the quality of end products. The determination of dephytinization in oat bran samples attracts great importance since all aforementioned dephytinization processes may cause changes in the nutritional properties of oat bran, which results in changes in the nutritional and quality properties of oat-bran-added food products, and there is insufficient literature about the dephytinization of oat bran and its effects on the functional compounds of food. The main aim of the study was to investigate the optimum conditions to achieve the maximum phytic acid degradation by using two different dephytinization methods. An additional aim was to evaluate its effect on the contents of dietary fibers, phenolic compounds, and antioxidants of oat bran under the dephytinization conditions mentioned above.

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MATERIALS AND METHODS

Sample Preparation. Oat bran sample was obtained from a commercial company in Adana, Turkey. Moisture, ash, and protein contents of oat bran were found as 10.8, 3.95, and 14.3%, respectively. Oat bran sample was passed through a laboratory type bran finisher (Buhler Type MLU-302), and later, bran slurries were prepared by mixing oat bran sample with distilled water (1:15, w/v). Bran slurries were divided into three groups and subjected to the following treatments. Yeast Fermentation of Oat Bran. The slurries were mixed with 3, 6, or 9% (w/w) of compressed baker’s yeast and fermented for 2, 4, or 6 h at 30 °C in a temperature controlled water-bath. Compressed baker’s yeasts were obtained in 500 g blocks wrapped in waxed paper from a commercial factory (Pakmaya Baker’s Yeast Company) and stored in a refrigerator at a temperature between 0 and 4 °C until usage. Hydrothermal Autoclaving Treatment of Oat Bran. The pH values of the slurries were adjusted to 4.5, 4.0, or 3.5, with acetic acid. The slurries were then held at 121 °C for 0.5, 1.0, or 1.5 h, in an autoclave. Control Experiments. The bran slurries without addition of yeast or without pH adjustment were used as comparison and left resting for the same periods as the related treatment. The slurries from the control and all of the three groups were further sieved (opening 250 μ). The sieved samples were rinsed five times with 500 mL of water each time and dried at 60 °C to moisture content of at most, 12%. Analyses of the bran samples were carried out in triplicate, and mean values on a dry basis were reported in the tables. Extraction of Free Phenolic Compounds. Free phenolic compounds of bran samples were extracted by blending 0.5 g of sample with 5 mL of acetone/water mixture (1:1, v/v) for 1 h in stirring shaker. After centrifugation at 2500g for 10 min, the supernatant was filtered through Whatman No. 42 filter paper, and extraction was repeated two times. Supernatants were pooled and evaporated at 40 °C using a rotary evaporator (Buchi, Rotavapor R210, Switzerland). The resulting solutions were kept at −20 °C under nitrogen gas in dark containers until further analysis, after the extracts were dissolved in 2 mL DMSO (dimethyl sulfoxide solution). Extraction of Bound Phenolic Compounds. The residues remained after extraction of free phenolics was digested with 2N sodium hydroxide (1:40, w/v) at room temperature for 4 h by stirring. Acidity of samples was adjusted to pH 2.0 with 6 M HCL solution. After neutralization, the mixture was extracted with 20 mL of hexane to remove lipids. After centrifugation at 2500g for 10 min, hexane was removed from the samples. This procedure was repeated one more time. The final solution was extracted five times with an appropriate amount of diethyl ether/ethyl acetate mixture (1:1, v/v). After centrifugation at 2500 g for 10 min the diethyl ether/ethyl acetate fraction was pooled and evaporated at 40 °C using a rotary evaporator (Buchi, Rotavapor R-210, Switzerland). Samples were dissolved in 2

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RESULTS AND DISCUSSION Phytic Acid Content. PA content of oat bran was found as 1890.2 mg/100 g. PA ratio in oat bran may change between 600 and 2406 mg/100g depending on the species, variety, growing conditions, and the method to obtain the bran.27,28 Changes in PA content of oat bran samples which were dephytinized by fermentation with different yeast levels or without yeast are given in Figure 1. As it can be observed, fermentation time and the amount of yeast had an important

Figure 1. Phytic acid content of oat bran dephytinized by fermentation for 2, 4, and 6 h with different yeast levels (3, 6, and 9%) and without yeast addition under the same fermentation conditions. 5714

DOI: 10.1021/acs.jafc.7b01698 J. Agric. Food Chem. 2017, 65, 5713−5719

Article


both wheat (maximum 95.2%, pH 4.0) and rice bran (maximum 95.6%, pH 4.0) samples. When autoclaving was performed without any pH adjustment, PA content of the samples decreased between 40.2% and 41.8%. This decrease can be partially attributed to soaking, washing, and filtering processes, but also the temperature contributed to the observed reduction. In the hydrothermal method, the greatest PA destruction was observed as 95.2%. This value is higher than that observed in the fermentation method. In the literature, there are several explanations about the effect of the autoclaving process on the PA loss. It has been reported that the thermal degradation of PA is controlled by high pressure and low pH throughout fermentation because of increased solubility of phytic acid−cation complexes, and also, it is indicated that the higher PA losses have been associated with discarding of the leached soluble PA salts with the cooking water.23,31−33 In the study of Majzoobi, Pashangeh, Farahnaky, Eskandari, and Jamalian,30 the combined effect of particle size reduction and hydrothermal and fermentation treatments on PA contents of wheat bran was investigated. They observed a PA reduction of 34% after hydrothermal treatment, while it was only 0.12% in the fermented sample. Dietary Fiber Content. IDF, SDF, and TDF contents of oat bran samples which were dephytinized by fermentation with different yeast levels at different times and without yeast under the same conditions and times are given in Table 1. In recent literature, insoluble dietary fiber (IDF) contents vary from 8.68−20.20%, soluble dietary fiber (SDF) from 3.45− 8.90%, and total dietary fiber (TDF) from 20.40−30.60%.1,2 The control samples (0% yeast level and no fermentation) in our study gave IDF, SDF, and TDF contents as 24.69, 3.91, and 28.60% respectively. Only the IDF value was found to be greater when compared with the literature data, which might be explained as the fiber content of oat bran may widely change depending on the variety, the growing conditions, and especially the dehulling procedure of the grain.1,2 This change also could be a result of the cleaning procedure in the machines which removes the bran and thus concentrates the fiber content. As observed, adding different levels of yeast resulted in a slight increase on the DF contents. However, for different fermentation durations at 0% yeast level, remarkable changes were obtained to indicate the clear effect of fermentation on the DF contents of the samples. Fermentation increased the IDF content (80%) more than SDF content (6%). This result might be dependent on the different interactions of soluble and insoluble fiber components during fermentation.34 This result might be due to some floury materials passing through the sieves during the washing and filtering processes and leaving material rich in dietary fiber above the filters. In hydrothermal process, the IDF, SDF, and TDF content of oat bran samples which were dephytinized by autoclaving 0.5, 1.0, and 1.5 h at 4.5, 4.0, and 3.5 pH values, adjusted by using acetic acid. The original pH values are given in Table 2. As found in the fermented samples, the fiber contents of hydrothermally processed oat bran samples were also found to be significantly higher than the control sample (p < 0.05). At the original pH level (6.4), autoclaving increased the IDF content (85%) more than the SDF content (7%). This difference might be related to the removal of some floury material, thus the soluble contents, by soaking, washing, and filtering procedures.

influence on the PA content of oat bran. Longer fermentation time lowered the PA content, and this decrease was found to be statistically significant (p < 0.05). Only one sample showed an effective change (p > 0.05) with the increase of the yeast level; however, increasing the yeast level from 6% to 9% did not result in a statistically significant alteration for the PA content. The highest reduction of PA content (88.2%) was observed in oat bran fermented for 6 h and with 6% yeast. Yeast-related dephytinization in oat bran samples is attributed to the phytase activity of the yeast throughout the hydrolysis of PA. Also, in previous studies, it has been claimed that the decrease of the pH with the release of carbon dioxide and organic acids during fermentation increases the PA solubility, thus the phytase activity.111113 In order to study the effect of soaking, washing, and filtering procedures on the PA amount in oat bran samples, the same process was followed without adding yeast, and PA loss was observed between 31.4% and 32.5% without any changing in pH and temperature. In some previous studies performed in cereal products, it was observed that some PA is dissolved after soaking.14,29 Accordingly, PA loss in our sample might be because of the removal of PA by being dissolved after soaking and washing. Servi, Ö zkaya, and Colakoglu23 reported similar PA losses in control samples, which can be attributed to the natural phytases present in cereal brans, were mainly responsible for the PA loss during the resting period. In the hydrothermal method, it was observed that the pH level affected the PA content of oat bran samples autoclaved at 120 °C with the pH levels of 4.5, 4.0, and 3.5 (Figure 2), and

Figure 2. Phytic acid content of oat bran dephytinized by autoclaving for 0.5, 1.0, and 1.5 h at different pH values (4.5, 4.0, 3.5) and its original pH (6.4) under the same autoclaving conditions.

the greater PA loss was found in the samples autoclaved at pH 4.0. Duration of autoclaving is not found to be effective on the samples, even though the long autoclaving time resulted in little change on the PA loss. Overall, the hydrothermal process led to an approximately 94% decrease in the PA content of oat bran samples. At pH 4.5, autoclaving time showed a more significant increase of PA loss, and it was found to be statistically significant (p < 0.05). Majzoobi et al.30 found similar PA reduction values in the hydrothermally treated wheat bran samples and explained this decrease as the effect of acetic acid (pH 4.5) enhancing the endogenous phytase activity of the bran resulting in the degradation of PA. In our previous study22 carried out with wheat and rice bran, all hydrothermal treatments caused significant decreases in the PA contents of 5715


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Table 1. Insoluble, Soluble, and Total Dietary Fiber Contents of Oat Bran Samples Fermented with and without Yeasta yeast level (%)

IDFb (%)

fermentation time (h) c

control sample 0

2 4 6 2 4 6 2 4 6 2 4 6

3

6

9

SDFb (%)

24.69 ± 0.18 44.25 ± 0.20 bA 44.42 ± 0.19 bA 44.48 ± 0.11 bA 45.16 ± 0.12 bB 45.59 ± 0.11 cB 45.92 ± 0.13 dB 45.33 ± 0.12 bBC 45.63 ± 0.09 cB 45.98 ± 0.17 dB 45.54 ± 0.13 bC 45.75 ± 0.18 bB 46.33 ± 0.14 cC aA

3.91 ± 0.02 4.18 ± 0.01 bA 4.19 ± 0.03 bC 4.15 ± 0.02 bA 4.28 ± 0.02 bB 4.29 ± 0.02 bD 4.25 ± 0.02 bB 4.19 ± 0.04 cA 4.09 ± 0.02 bB 4.29 ± 0.03 dB 4.21 ± 0.03 cA 4.01 ± 0.03 bA 4.17 ± 0.03 cA aA

TDFb (%)

IDF/SDF

28.60 ± 0.20 aA 48.43 ± 0.21 bA 48.61 ± 0.22 bA 48.63 ± 0.13 bA 49.44 ± 0.14 bB 49.88 ± 0.13 cB 50.17 ± 0.15 dB 49.52 ± 0.16 bBC 49.72 ± 0.11 bB 50.27 ± 0.20 cBC 49.75 ± 0.16 bC 49.76 ± 0.21 bB 50.50 ± 0.17 cC

6.3 10.6 10.6 10.7 10.6 10.6 10.8 10.8 11.2 10.7 10.8 11.4 11.1

IDF: insoluble dietary fiber, SDF: soluble dietary fiber, TDF: total dietary fiber. Lower case letters (a−c) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at same yeast level with different fermentation times. Upper case letters (A−C) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at different yeast levels under same fermentation times. Results are means ± SD. bOn a dry basis. cYeast level of 0% and no fermentation. a

This result might be due to the destruction or solubility of some fiber compounds at low pH values.30 When the effects of fermentation and autoclaving were compared, it can be stated that autoclaving gives slightly better results for the dietary fiber contents. Considering both oat bran samples being fermented or autoclaved, it can be stated that the increase in TDF content was derived from the increase in IDF content, which is substantially similar to our previous study carried out with wheat and rice bran. This fact must be due to the relative increase of IDF content after removing soluble substances.22 IDF/SDF fiber ratios for oat bran samples treated with both methods are important indicators in terms of the functional, dietary, structural, and sensorial properties of oat bran.22,35 Different parameters of autoclaving and fermentation processes almost doubled the IDF/SDF ratio of oat bran samples. Phenolic Compounds Content and Antioxidant Activity. Results of total, free, and bound phenolic compound contents of oat bran and total, free, and bound antioxidant activity are both presented in Table 3 and in Table 4. Total, free, and bound phenolic compounds content of oat bran were found as 2892.8, 588.7, and 2304.1 mg GAE/kg, respectively (Table 3). Total, free, and bound antioxidant values were also found as 583.6, 178.1, and 405.5 μmol TE/100 g, respectively (Table 4). It has been stated that phenolic compound content and antioxidant activity of cereal brans change in a wide range depending on the variety, growing conditions, dehulling procedure, and analysis method.36 Phenolic compounds and antioxidant activity results of oat bran samples are within the range of values in the literature.37−39 Phenolic compound contents and antioxidant activity of oat bran samples which were dephytinized by fermentation for 2, 4, and 6 h with 3, 6, and 9% compressed yeast and without yeast are shown in Table 3. In those samples prepared without adding yeast in order to study the effect of soaking, washing, and filtering processes on the levels of phenolic compounds and antioxidants, the free and total phenolic compound contents were found to be lower, and the bound phenolic compound content was found to be higher than the control sample. Decrease in free, bound, and total phenolic compounds contents depends on the fermentation time. Besides bound antioxidant activity, the same situation was observed for

Table 2. Insoluble, Soluble, and Total Dietary Fiber Contents of Oat Bran Samples Autoclaved at Different pH Values with Different Timesa pH level

holding time (h)

IDFb (%)

SDFb (%)

TDFb (%)

IDF/ SDF

control samplec

24.69 ± 0.18 aA

3.91 ±aA0.02

28.60 ± 0.20 aA

6.3

6.4

0.5

45.61 ± 0.06 bB

4.17 ±bA0.01

49.78 ± 0.07 bB

10.9

1.0

45.66 ± 0.21 bB

4.17 ±bA0.02

49.83 ± 0.23 bB

10.9

1.5

45.65 ± 0.17 bB

4.19 ±bA0.04

49.84 ± 0.21 bB

10.9

0.5

45.69 ± 0.07 bB

4.18 ±bA0.01

49.87 ± 0.08 bC

10.9

1.0

45.67 ± 0.16 bB

4.18 ±bA0.01

49.85 ± 0.17 bB

10.9

1.5

45.64 ± 0.12 bB

4.20 ±bA0.03

49.84 ± 0.15 bB

10.9

0.5

45.70 ± 0.09 bB

4.19 ±bA0.02

49.89 ± 0.11 bC

10.9

1.0

45.67 ± 0.07 bB

4.20 ±bA0.01

49.87 ± 0.08 bB

10.9

1.5

45.58 ± 0.11 bB

4.22 ±bA0.03

49.80 ± 0.14 bB

10.8

0.5

45.26 ± 0.12 bA

4.20 ±bA0.02

49.46 ± 0.14 bA

10.8

1.0

45.27 ± 0.10 bA

4.21 ±bA0.03

49.48 ± 0.13 bA

10.8

1.5

45.15 ± 0.11 bA

4.30 ±cB0.02

49.45 ± 0.13 bA

10.5

4.5

4

3.5

a IDF: insoluble dietary fiber, SDF: soluble dietary fiber, TDF: total dietary fiber. Lower case letters (a−c) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at same pH values with different holding times. Upper case letters (A−C) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at different pH values under same holding times. Results are means ± SD. bOn a dry basis. cAt original pH level of oat bran, 6.4, and no autoclaving.

After the autoclaving procedure, although a slight decrease in IDF and TDF content and a slight increase in SDF content were observed, changes were not statistically significant (p > 0.05) Decrease in pH did not have an important effect on fiber components, but the samples which were autoclaved at pH 3.5 gave less IDF and TDF in comparison with the other samples. 5716


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Table 3. Phenolic Compounds and Antioxidant Activity Values of Oat Bran Samples Fermented with or without Yeasta yeast level (%)

phenolic compoundsb (mg GAE/kg)

fermentation time (h) free

0

0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6

3

6

9

bound

588.7 ± 2.78 333.1 ± 2.89bA 330.2 ± 2.11bA 312.6 ± 1.17aA 588.7 ± 2.78dA 350.2 ± 2.66aB 371.3 ± 3.87bB 398.6 ± 2.72cB 588.7 ± 2.78cA 421.3 ± 1.78aC 424.7 ± 2.17aC 474.7 ± 3.48bC 588.7 ± 2.78dA 418.8 ± 3.12aC 428.5 ± 1.57bD 482.7 ± 2.41cD cA

antioxidant activityb (μmol TE/100g) total

2304.1 ± 7.13 2521.3 ± 7.26 dA 2470.7 ± 9.41 cA 2428.6 ± 4.26 bA 2304.1 ± 7.13 aA 2537.3 ± 8.23 bB 2615.0 ± 5.16 cB 2664.0 ± 9.16 dB 2304.1 ± 7.13 aA 2616.9 ± 8.14 bC 2764.8 ± 8.72 cC 2906.0 ± 11.61dC 2304.1 ± 7.13 aA 2635.5 ± 5.62 bD 2774.7 ± 8.81 cC 2907.3 ± 12.14dC aA

free

2892.8 ± 9.91 2854.4 ± 10.15cA 2800.9 ± 11.52bA 2741.2 ± 5.43 aA 2892.8 ± 9.91 aA 2887.5 ± 10.89aB 2986.3 ± 9.03 bB 3062.6 ± 11.88cB 2892.8 ± 9.91 aA 3038.2 ± 9.92 bC 3189.5 ± 10.89cC 3380.7 ± 15.09dC 2892.8 ± 9.91 aA 3054.3 ± 8.74 bD 3203.2 ± 10.38cC 3390.0 ± 14.55dC dA

bound

178.1 ± 0.97 172.2 ± 1.01cA 165.8 ± 1.11bA 161.1 ± 0.95aA 178.1 ± 0.97aA 179.1 ± 0.81aB 181.7 ± 1.08abB 183.2 ± 1.11bB 178.1 ± 0.97aA 186.0 ± 1.27bC 191.0 ± 1.31cC 197.8 ± 1.13dC 178.1 ± 0.97aA 192.1 ± 1.17bD 191.3 ± 0.93bC 198.1 ± 1.42cC dA

total

405.5 ± 1.13 404.2 ± 1.06aA 405.7 ± 1.14aA 409.8 ± 1.73bA 405.5 ± 1.13aA 408.0 ± 1.01bB 412.0 ± 1.53cB 419.5 ± 1.40dB 405.5 ± 1.13aA 419.6 ± 1.32bC 421.8 ± 1.27bC 421.5 ± 1.24bB 405.5 ± 1.13aA 422.5 ± 1.15bD 426.7 ± 1.27cD 429.9 ± 1.05dC aA

583.6 ± 2.10cA 576.4 ± 2.07bA 571.5 ± 2.25aA 570.9 ± 2.68aA 583.6 ± 2.10aA 587.1 ± 1.82bB 593.7 ± 2.61cB 602.7 ± 2.51dB 583.6 ± 2.10aA 605.6 ± 2.59bC 612.8 ± 2.58cC 619.3 ± 2.37dC 583.6 ± 2.10aA 614.6 ± 2.32bD 618.0 ± 2.20cD 628.0 ± 2.47dD

a GAE: gallic acid equivalent, TE: Trolox equivalent. Lower case letters (a−c) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at same yeast level with different fermenation times. Upper case letters (A−C) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at different yeast levels under same fermentation time. Results are means ± SD. bOn a dry basis.

Table 4. Phenolic Compounds and Antioxidant Activity of Oat Bran Samples Autoclaved at Different pH Valuesa pH level

holding time (h)

6.4c

0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5

4.5

4.0

3.5

phenolic compoundsb (mg GAE/kg)

antioxidant activityb (μmol TE/100g)

free

bound

total

free

bound

total

588.7 ± 2.78dA 446.1 ± 1.97aA 451.4 ± 2.11bA 457.6 ± 1.53cA 588.7 ± 2.78dA 456.0 ± 1.83aB 463.7 ± 2.21bB 477.2 ± 3.01cB 588.7 ± 2.78bA 480.0 ± 1.69aC 481.0 ± 2.13aC 479.2 ± 1.77aB 588.7 ± 2.78bA 478.5 ± 2.18aC 478.7 ± 2.73aC 479.3 ± 2.05aB

2304.1 ± 7.13 aA 2744.7 ± 9.14 bA 2874.7 ± 10.17cA 3225.8 ± 8.81 dA 2304.1 ± 7.13 aA 3301.3 ± 7.33 bB 3309.3 ± 7.65 bB 3311.3 ± 5.67 bB 2304.1 ± 7.13 aA 3357.3 ± 6.25 bD 3500.3 ± 8.14 cD 3528.2 ± 7.77 dD 2304.1 ± 7.13 aA 3337.2 ± 10.61bC 3474.6 ± 12.33cC 3480.1 ± 10.61cC

2892.8 ± 9.91 aA 3190.8 ± 11.11bA 3326.1 ± 12.28cA 3683.4 ± 10.34dA 2892.8 ± 9.91 aA 3757.3 ± 9.16 bB 3773.0 ± 9.86 cB 3788.5 ± 8.68 dB 2892.8 ± 9.91 aA 3837.3 ± 7.94 bD 3981.3 ± 10.27cD 4007.4 ± 9.54 dD 2892.8 ± 9.91 aA 3815.7 ± 12.79bC 3953.3 ± 15.06cC 3959.4 ± 12.66cC

178.1 ± 0.97bA 172.2 ± 1.03aA 177.8 ± 1.74bA 186.1 ± 1.26cA 178.1 ± 0.97aA 182.5 ± 1.52bB 189.5 ± 1.13cB 198.8 ± 1.42dB 178.1 ± 0.97aA 209.0 ± 1.38bC 217.7 ± 1.05cC 218.8 ± 1.48cD 178.1 ± 0.97aA 207.1 ± 1.27bC 216.1 ± 1.16dC 211.2 ± 1.47cC

405.5 ± 1.13aA 404.2 ± 1.33aA 405.7 ± 1.76aA 409.8 ± 2.11bA 405.5 ± 1.13aA 421.0 ± 1.66bB 428.0 ± 2.23cB 428.8 ± 1.96cB 405.5 ± 1.13aA 445.0 ± 1.03cD 441.5 ± 1.26bD 452.0 ± 1.99dD 405.5 ± 1.13aA 431.8 ± 2.11bC 435.0 ± 1.04cC 441.5 ± 1.88dC

583.6 ± 2.10bA 576.4 ± 2.36aA 583.5 ± 3.50bA 595.9 ± 3.37cA 583.6 ± 2.10aA 603.5 ± 3.18bB 617.5 ± 3.36cB 627.6 ± 3.38dB 583.6 ± 2.10aA 654.0 ± 2.41bD 659.2 ± 2.31cC 670.8 ± 3.47dD 583.6 ± 2.10aA 638.9 ± 3.38bC 651.1 ± 2.20cC 652.7 ± 3.35cC

a

GAE: gallic acid equivalent, TE: Trolox equivalent. Lower case letters (a−c) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at same pH value with different holding times. Upper case letters (A−C) indicate the differences of average values (which are significantly different at p < 0.05) for bran samples at different pH values under same holding time. Results are means ± SD. bOn a dry basis. cOriginal pH level of oat bran is 6.4.

antioxidant quantities of the samples. Decrease of the phenolic compound content most probably is because of the removal of some soluble phenolic compounds during the soaking and washing of the samples. During dephytinization by fermentation, longer fermentation time gave higher bound and total phenolic compounds content of oat brans (p < 0.05). Although a similar increase was observed in the free phenolic compounds of the samples, the dephytinized samples could not reach the original level of phenolic compounds content (Table 3). Yeast amount used during fermentation had an effect on the phenolic compounds, and the increase of the yeast amount usually resulted in an increase of the phenolic content of the free, bound, and total phenolic compounds contents of bran.

Even though the highest amount of yeast was used, the free phenolic compounds content of the dephytinized samples could not reach the free phenolic compound quantity of the control sample. To our knowledge, there are no studies performed on oat bran, but those performed in other cereals showed that the content of phenolic compounds increased after fermentation with two different microorganisms.40 It has been also reported that sourdough fermentation could increase the easily extracted phenolics and the ferulic acid ratio and fermentation could be useful in increasing the bioactive potential of cereals.41 Dephytinization process by fermentation increased the antioxidant activity of oat bran. Depending on the yeast 5717


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components of oat bran were found to be similar. However, the autoclaving process was found more effective than the fermentation for enriching both TP and AA of oat bran samples.

amount and fermentation time, free, bound, and total antioxidant values were substantially increased and gave statistically significant results (p < 0.05). Despite the free phenolics, the maximum yeast amount and the longest fermentation time yielded higher antioxidant values as compared to control sample. The effect of fermentation on the antioxidant activity of oat bran has not been investigated, but limited studies performed on wheat showed that this process increased its antioxidant capacity.34,40 The phenolic compounds and antioxidant activities of the samples dephytinized by autoclaving at different pH values and at their original pH values without any pH adjustment are given in Table 4. The pH value used during the dephytinization process by autoclaving affected the phenolics of oat bran, and the highest phenolic compounds content was observed at pH 4.0. Increasing duration of autoclaving increased the bound and total phenolic compounds, and the results were found to be statistically significant for most of the samples (p < 0.05). Time-related increase of the free phenolic compound content were not found to be statistically significant (p > 0.05), and the values of dephytinized samples were observed lower than those of the control sample. This result might be because of the loss of soluble compounds during soaking and washing processes. A similar case was observed on the phenolic content of the samples used at their original pH values. There are some findings showing an increase of phenolics in cereal products after autoclaving. Autoclaving of germinated cereals causes an increase in the phenolic compounds due to the thermal destruction which leads to the formation of simple phenolic compounds from conjugated tannin compounds and the release of phenolic compounds from the cell walls or vacuoles. Besides Maillard reactions, caramelization, maderization, or oxidation of phenols might have an effect on this issue.42,43 In autoclaving process, pH level and holding time have similar effects on both antioxidant activity and phenolic compounds content of oat bran. The effect of pH was also similar to that observed for phenolic compounds; significant effect was seen for the samples autoclaved at pH 4.0, but as contrary to the phenolics, free antioxidant activity was found higher than the ones in control sample. Previous studies showed that the thermal procedures applied to different materials increased the antioxidant capacity due to the release of antioxidant compounds after Maillard reactions or the change in the antioxidant profile of the products resulting in formation of new antioxidants. Better antioxidant features after the thermal procedure is due to the revealing of phenolics from the cell matrix, synergetic effect of phenolics and other phytochemicals, caramelization, and chemical oxidation of phenolics and maderization.43,44 To sum up briefly, oat bran is a good source of dietary fiber, phenolic compounds, and antioxidants, but phytic acid content might be a problem most of the time. In this study, fermentation and hydrothermal (autoclaving) procedures for dephytinization of oat bran decreased phytic acid content up to 90%. In both dephytinization methods, it was observed that dietary fiber, bound and total phenolic compounds, and antioxidant activity substantially increased in oat bran. Mostly washing and filtering processes were effective on the dietary fiber content of the samples compared to the controls because of removing some of the water-soluble materials from the samples. In this way, remaining material was enriched with dietary fiber. Positive effects of both methods on the functional

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AUTHOR INFORMATION

Corresponding Author

*S.T.: Tel.: +90 3642274533; E-mail: [email protected]; fax: +90 3642274535 ORCID

S. Turksoy: 0000-0001-5763-2744 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank The Scientific and Technological Research Council of Turkey (TUBITAKTOVAG-111O201) and Ankara University Scientific Research Projects (BAP, Project No: 12B4343004) for the financial support of this work.

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REFERENCES

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Effect of Dephytinization by Fermentation and Hydrothermal

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