Antioxidant activities of corn fiber and wheat bran and derived extracts

LWT - Food Science and Technology 50 (2013) 132e138

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Antioxidant activities of corn fiber and wheat bran and derived extracts Jonas Lewin Bauer*, Britta Harbaum-Piayda, Heiko Stöckmann, Karin Schwarz Department of Food Technology, Institute of Human Nutrition and Food Science, University of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 May 2011 Received in revised form 7 June 2012 Accepted 12 June 2012

The antioxidant activity of ground untreated corn fiber and wheat bran against lipid oxidation of rapeseed-fish oil-in-water emulsions was determined with respect to the formation of primary (lipid hydroperoxides) and secondary (propanal and hexanal) oxidation products. Corn fiber inhibited the formation of lipid hydroperoxides secondary products effectively at a concentration of 800 mg/kg emulsion, but wheat bran showed only low antioxidant effects. It was shown that the activity was based on the action of bound hydroxycinnamates like ferulic acid and p-coumaric acid, which was proven by the application of ferulated oligosaccharides (FOm) that showed higher activity than free ferulic acid on a molar basis. Enzymatic treatment with ferulic acid esterases did not increase the antioxidative activity. Extraction of corn fiber and wheat bran with methanol and isopropanol resulted in products which contained only low amounts of free soluble hydroxycinnamates and their marked activity against lipid oxidation and in antioxidant tests (TEAC, DPPH) gave reason for further fractionation. It has been shown that the polyaminconjugates diferuloylputrescine and p-coumaroyl-feruloylputrescine, which are major compounds of the corn fiber methanolic extract, exhibited high antioxidant activity and it can be assumed that they will take part in the antioxidant action of untreated fibers. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Corn fiber Wheat bran Ferulic acid Ferulated oligosaccharides Fish oil Emulsion

1. Introduction Corn fiber and wheat bran are agro-industrial derived byproducts, which arise during starch and flour production and are associated with high dietary fiber content. Cell walls of monocotyledons consist mainly of cellulose, hemicelluloses and pectin. It is well known that they contain numerous hydroxycinnamic acids mainly covalently bound to polysaccharides via ester linkages (Gallardo, Jimenez, & Garcia-Conesa, 2006). Ferulic acid is the most abundant hydroxycinnamic acid in cereals which is associated with the outer layers of the kernels (Andreasen, Christensen, Meyer, & Hansen, 2000; Barron, Surget, & Rouau, 2007). Other hydroxycinnamic acids were found in smaller quantities (p-coumaric acid, sinapic acid, caffeic acid) (Rhodes, Sadek, & Stone, 2002; Run Cang, Xiao Feng, & Shi Hong, 2001; Yadav, Moreau, & Hicks, 2007). Diferulic acids are products of the oxidative coupling of ferulic acid and they may form crosslinks in the arabinoxylan network and exert an important role in the stability of non-lignified cell walls

Abbreviations: Cf, corn fiber; CFP, p-coumaroyl-feruloylputrescine; DFP, diferuloylputrescine; FA, ferulic acid monomer; FOm, ferulated oligosaccharides; RFO, rapeseed fish oil mixture; Tx, trolox; Wb, wheat bran. * Corresponding author. Tel.: þ49 (0) 431 880 5034; fax: þ49 (0) 431 880 5544. E-mail addresses: [email protected] (J.L. Bauer), [email protected] (K. Schwarz). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.06.012

(Bunzel, Ralph, Marita, Hatfield, & Steinhart, 2001; Saulnier & Thibault, 1999). The industrial application of hydroxycinnamates had raised interest because they and their conjugates were shown to be bioactive compounds possessing beneficial antioxidant activities and health benefits (Thiyam, Stöckmann, & Schwarz, 2006; Faulds, 2010). Ferulic acid acts as a radical scavenger and displays antioxidant activity in lipid containing foods (Oehlke, Heins, Stöckmann, & Schwarz, 2010). The antioxidative capacity of ferulic acid is based on its ability to easily abstract a hydrogen atom to form a resonance stabilized phenoxyl radical which is unable to initiate or propagate a radical chain reaction (Graf, 1992). The increase of health beneficial polyunsaturated fatty acids in foods requires improved protection from lipid oxidation as the vulnerability increases with the degree of unsaturation. As a result, interest in finding natural sources of antioxidants has been raised, but commonly used natural antioxidants like herb extracts often possess strong flavors (Frankel, Huang, Aeschbach, & Prior, 1996). Wheat bran and corn fiber do not display strong tastes of their own and are therefore an interesting alternative source of natural antioxidants. There are two different procedures described to retrieve phenolic acids from fiber. On the one hand, alkaline hydrolysis of plant material is often applied to determine the content of cell wall bound phenolics. Further purification steps, such as solvent extraction, are necessary to obtain a product that could be used as

J.L. Bauer et al. / LWT - Food Science and Technology 50 (2013) 132e138

food additive (Tilay, Bule, Kishenkumar, & Annapure, 2008). On the other hand, another method to release cell wall bound phenolics is enzymatic hydrolysis of cell walls with cell wall degrading enzyme complexes. Ferulic acid and other hydroxycinnamates were successfully released from corn fiber and wheat bran by ferulic acid esterases from Aspergillus niger and Humicola insolens (Benoit et al., 2006; Faulds, Mandalari, LoCurto, Bisignano, & Waldron, 2004). Extracts derived from corn fiber and wheat bran containing free hydroxycinnamic acids, showed significant antioxidative properties in vitro such as scavenging of 2,20 -azinobis 3-ethylbenzothiaz oline-6-sulfonic acid (ABTS) cation, 2,2-diphenyl-1-picryhydrazyl (DPPH) or chelating activities against Fe2þ and Cu2þ, wherefore the contribution of bound phenolics to the predicted antioxidant activity of whole brans is often underestimated (Andjelkovic et al., 2006; LiyanaPathirana & Shahidi, 2006; Lopez-Martinez et al., 2009). The aim of the study is the investigation of the antioxidant effect of untreated and enzymatically treated corn fiber and wheat bran on lipid oxidation in systems containing fish oil. The action of untreated fibers, cell wall degrading enzymes and the influence of different extraction procedures with organic solvents were investigated to estimate the antioxidative action of bound or free ferulic acid in a lipid oxidation model. 2. Materials and methods 2.1. Materials and chemicals Samples of corn fiber were kindly provided by Habema GmbH, Hamburg, Germany. Wheat bran was purchased on the local market. Refined fish oil was provided by Cognis Deutschland GmbH & Co. KG, Illertissen, Germany and contained in sum approximately 267 mg/g eicosapentaenoic acid and docosahexaenoic acid as specified by the manufacturer. Refined rapeseed oil was purchased on the local market and contained 290 mg/g polyunsaturated fatty acids. The enzymes Depol 740 L and Depol 670 L were purchased from Biocatalysts Limited, Wales, UK. Aluminum oxide, Amberlite XAD-2, ammonium thiocyanate, a-amylase (TermamylÒ), Brij 58Ò, p-coumaric acid, ferulic acid, horseradish peroxidase, propyl gallate and Trolox were purchased from Sigma Aldrich, Munich, Germany. Acetic acid, acetone, barium chloride dihydrate, hydrochloric acid, ethyl acetate, n-heptane, n-hexane, iron (III) chloride dihydrate, iron (II) sulfate heptahydrate, isopropyl alcohol, methanol, sodium acetate, sodium hydroxide, sodium sulfate and trifluoracetic acid were purchased from Carl Roth GmbH & Co. KG, Karlsruhe, Germany. 2.2. Preparation of extracts All fibers were ground in a ball mill, sieved (<125 mm) and stored in a desiccator prior to analysis. Corn fiber (Cf) was treated with Depol 740 L which is a mono-active feruloyl esterase from H. insolens with a ferulic acid esterase activity of 36 U/g (manufacturers’ datasheet). Two grams of corn fiber were incubated for 24 h in an incubation shaker with 2.5 ml of enzyme in acetate buffer at pH 5 at a final volume of 100 ml. Wheat bran (Wb) was incubated under same conditions but with Depol 670 L, a cell wall degrading enzyme mixture with feruloyl esterase activity. Resulting hydrolysates were separated in three different extraction products which were further prepared. One aliquot was freeze dried and resulted in a powdered product containing the aqueous supernatant and nonhydrolyzed fibers (Cf-T; Wb-T). Another aliquot was centrifuged and freeze dried (Cf-S; Wb-S) containing only the supernatant. The third aliquot was acidified (HCl; pH <2) and the supernatant was extracted three times with EtOAc. The combined organic layers were dried over sodium sulfate and brought to dryness by a rotary evaporator (Cf-EtOAc, Wb-EtOAc).

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For preparation of non-enzymatic extracts two grams of fibers were weighted into centrifugation tubes and diluted with 30 ml of methanol or isopropyl alcohol and left to stand overnight at room temperature and further sonificated (cycle 9, 90% power, Bandelin sonopuls, sonication probe VS 70, Berlin, Germany) for 2 min. After centrifugation at 9000 rpm for 10 min the supernatant was decanted, 20 ml solvent was added again and extraction steps were repeated two times. The combined supernatants were dried on a rotary evaporator and stored at a freezer at 20  C until analysis (Cf-MeOH, Cf-P; Wb-MeOH, Wb-P). Ferulated oligosaccharides (FOm) from corn fibers were produced by mild acid hydrolysis according to the method of Saulnier, Vigouroux, and Thibault (1995) as described earlier (Bauer, Harbaum-Piayda, & Schwarz, 2012). Therefore destarched fibers were applied to acid hydrolysis with 50 mmol trifluoracetic acid and ferulated oligosaccharides were gained after column chromatography on Amberlite XAD-2. Two different ferulated fragments of the arabinoxylan network were identified tentatively as feruloyl arabinose (FA) and feruloyl-arabinose-xylose ester by HPLCeESI-MS (Bauer et al., 2012). 2.3. Fractionation of extracts The methanolic corn fiber extract was fractionated by preparative HPLC (Agilent 1100 series) using a 250 mm  21 mm, 5 mm RP C18ec Nucleodur column (Macherey-Nagel). Eluents were 100% water (A) and 100% methanol (B). The gradient was developed at initial conditions 30% B which rose up to 100% B within 9 min and kept on 100% B for another 7 min at a flow rate of 20 ml/min. Four fractions were separated as follows: fr. 1, 2.5e4.3 min; fr. 2, 4.3e7.7 min; fr. 3, 7.7e10.3 min; fr. 4, 10.1e14 min. 2.4. Alkali treatment The amounts of bound Hydroperoxides and their coupling products in untreated corn fiber and wheat bran were determined after alkaline hydrolysis as described by Kroon, Garcia-Conesa, Fillingham, Hazlewood, and Williamson (1999). 2 g of sample were hydrolyzed for 24 h with 60 ml of 2 mol/L NaOH under hydrogen atmosphere. The supernatant was obtained after centrifugation at 9000 rpm for 10 min. An aliquot of 5 ml was adjusted to pH <2 by the addition of hydrochloric acid and extracted three times with ethyl acetate. Combined organic layers were pooled and dried over sodium sulfate. The solvent was removed by rotary evaporation at 40  C and the residues were redissolved in 10 ml methanol and used for HPLC analysis. 2.5. HPLC analyses For quantitative analysis of phenolic acids a HP 1100 HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector was used as described by Bauer et al. (2012). Ferulic acid, p-coumaric acid, and diferulic acids were quantified by external standard calibration. Diferuloylputrescine was isolated by preparative HPLC from the methanolic corn fiber extract and used as reference compound for the quantification of diferuloylputrescine and p-coumaroyl-feruloylputrescine. The sum of these compounds was expressed as ferulic acid equivalents based on their molarities. 2.6. Emulsion preparation and storage Oxidation experiments were carried out with 10% oil in water emulsions with 1% Brij 58 as emulsifier. The aqueous phase consisted of 0.2 mol/L acetic acid/Na-acetate pH 5.0 buffer solution. An oil mixture of rapeseed oil and refined fish oil (RFO) 70:30 (w/w)

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was purified from antioxidant tocopherols, trace metals and other pro-oxidants by column chromatography as described by Lampi and Kamal-Eldin (1998) on aluminum oxide and was stored at 20  C under nitrogen. The absence of tocopherols was proved according to the DGF e FII 4a method. Emulsions were premixed using an Ultra-Turrax for one minute and finally homogenized in a high pressure homogenizer (Panda 2K, Niro Soavi, Lübeck, Germany) at 250/50 bar with two passes. Stock solutions of antioxidants and extracts were prepared in ethanol and an aliquot of 50 ml was added to the emulsions. Ground fibers were tested at different concentrations (Table 2) but during storage all fibers sedimented to the bottom of the vials and were redispersed by daily shaking. Emulsions (25 g) were stored in triplicate in 100 ml gas-tight glass bottles at 20  C in the dark and were analyzed several times until a final hydroperoxide content of 500 mmol kg1 oil was reached. Table 1 lists all extracts utilized in this study.

for 16 h. The resulting radical solution was diluted with ethanol to give an absorbance reading of 1.2 at 734 nm. 2.95 ml of ABTS_þ solution was used for an initial reading at 734 nm. A 50 ml aliquot of diluted sample was added and absorbance was measured again after a reaction time of 20 min. The Trolox equivalent antioxidant capacity of extracts was calculated with a standard curve prepared with known contents of Trolox. 2.10. DPPH assay Antioxidant activity of the obtained extracts was also tested using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) as previously described (Harbaum, Hubbermann, Zhu, & Schwarz, 2008). Calibration was done with Trolox and antioxidative activity was calculated as Trolox equivalents. 3. Results

2.7. Determination of hydroperoxide content The formation of hydroperoxides during storage of the emulsions was determined by the ferric thiocyanate method modified by Stöckmann, Schwarz, and Huynh-Ba (2000). Conjugated diene content of emulsions was determined on a spectric photometer at 234 nm (Stöckmann et al., 2000). 2.8. Determination of propanal and hexanal by static head space gas chromatography According to the method of Serfert, Drusch, and Schwarz (2009) one gram of emulsion was weighted into a head space vial (20 ml) which was equilibrated at 70  C for 15 min. An aliquot (1 ml) of the head space was injected into an Agilent 6890 series gas chromatograph running in split mode (4:1) equipped with a J&W DB1701 column (60 m  0.32 mm  3 mm). The injection port was operated at 220  C and detection was done with a flame ionization detector (FID) at 250  C. Initially, the oven temperature was set to 45  C where it was held for 2 min, then raised up to 85  C at 15  C min1 and maintained for 4 min and finally raised up to 220  C at 15  C min1 and held for 3 min. Peaks of propanal and hexanal were analyzed by their area under the curve.

3.1. Inhibition of lipid oxidation in RFO emulsions containing untreated fibers Untreated corn fiber tested at 8000, 4000 and 800 mg/kg (based on total emulsion weight) was most effective in inhibiting lipid oxidation in emulsions. In RFO containing emulsions untreated corn fiber showed a dose dependent inhibition of lipid oxidation products (Fig. 1). Dosage of 8000 mg/kg corn fiber resulted in a complete inhibition of hydroperoxide, propanal and hexanal formation within 25 days. Reducing the concentration to 4000 mg/ kg and 800 mg/kg resulted in a slight decrease of hydroperoxide inhibition. Ground wheat brans, tested at 8000, 4000 and 2000 mg/ kg, showed weaker inhibitory effects on hydroperoxide formation and secondary oxidation products and showed no or a slightly inverse concentration/activity performance in comparison to corn fiber (Fig. 1). A final hydroperoxide content of 500 mmol kg1 oil was reached within 6e7 days in control samples of emulsions. Therefore Table 2 lists the determined inhibition of hydroperoxide and propanal formation at day six and the ferulic acid contents for all tested samples added to the emulsions. 3.2. Inhibition of lipid oxidation in emulsions by enzymatically obtained extracts

2.9. TEAC assay The Trolox equivalent antioxidant capacity assay (TEAC) was performed according to the method of Re et al. (1999). The ABTS_þ cation was generated in the dark at room temperature by reacting a 8 mmol/L solution of ABTS with 3 mmol/L potassium persulfate

Table 1 Extracts tested in 10% oil in water emulsions to inhibit lipid oxidation. Sample code Corn fiber Cf-T Cf-S Cf-EtOAc Cf-U Cf-MeOH Cf-P FOm Wheat bran Wb-T Wb-S Wb-EtOAc Wb-U Wb-MeOH Wb-P

Enzyme

Extraction procedure

Depol 740 L Depol 740 L Depol 740 L Untreated Untreated Untreated Untreated

Total Supernatant EtOAc e MeOH Isopropyl alcohol Acid hydrolysis

Depol 670 L Depol 670 L Depol 670 L Untreated Untreated Untreated

Total Supernatant EtOAc e MeOH Isopropyl alcohol

When enzymatically treated corn fiber (Cf-T) and wheat bran (Wb-T) were tested at concentrations that correspond to 500 mmol ferulic acid kg1 oil (800 mg/kg for Cf, 4000 mg/kg for Wb) a lower inhibition in the tested RFO-emulsions was observed compared to untreated fibers (Fig. 1). The usage of the dried aqueous supernatant (Cf-S, Wb-S; 1000 mg/kg) led to moderate inhibition of lipid oxidation but the ferulic acid content was relatively low in comparison to untreated and enzymatically treated fibers (Table 2). While the dried ethyl acetate extract of treated corn fiber (CfEtOAc; 50 mg/kg) effectively inhibited the hydroperoxide formation, inhibition was decreased when Wb-EtOAc was tested and prooxidative effects have been shown with respect to the formation of volatile secondary lipid oxidation products. 3.3. Inhibition of lipid oxidation in emulsions containing extracts from corn fiber and wheat bran Solvent extracts of untreated corn fiber and wheat bran were tested at 1000 mg/kg in RFO-emulsions. The methanolic extract of untreated corn fiber (Cf-MeOH) effectively inhibited hydroperoxide formation (Table 2). Propanal and hexanal were not detected by head space GC after six days of storage. Wheat bran extracted with methanol protected the emulsions against lipid oxidation

J.L. Bauer et al. / LWT - Food Science and Technology 50 (2013) 132e138

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Table 2 Formation and inhibition of primary and secondary decomposition products in 10% oil in water emulsions containing rapeseed oil/fish oil 70:30 (w/w) at day six. Sample Control Cf-U Cf-U Cf-U Wb-U Wb-U Wb-U FA PG Tx Control Cf-T Cf-S Cf-EtOAc Wb-T Wb-S Wb-EtOAc FOm Control Cf-MeOH Cf-P Wb-MeOH Wb-P

FA contenta [mg/g dm]

Amount tested [mg dm/kg emulsion]

FA contentb [mmol/kg oil]

14.0  0.3 14.0  0.3 14.0  0.3 3.8  0.1 3.8  0.1 3.8  0.1

8000 4000 800 8000 4000 2000 9.7 10.6 12.4

5000 2500 500 1000 500 250 500 500 500

14.0  0.3 3.1  0.1 279.1  4.3 3.8  0.1 0.1  0.0 88.3  0.3 67.1  1.9

800 1000 50 4000 1000 50 145

500 174 962 500 4.7 227 500

0.9  0.0 0.1  0.0 n.d. n.d.

1000 1000 1000 1000

3138 547 11.3 6.6

Hydroperoxides [mmol kg1 oil] 453.2 4.9 9.9 18.7 187.1 133.3 99.9 119.5 34.0 0.4 467.6 72.3 79.1 98.8 175.6 141.1 315.6 22.8 384.9 7.3 73.2 7.1 21.4

 29.6  3.1  1.9  3.3  8.3  6.2  6.5  6.9  2.9  2.6  20.4  2.9  7.1  10.0  6.1  12.9  11.9  2.6  17.1  1.3  2.7  1.9  2.0

Inhibibitionc [%]

98.9 97.8 95.9 58.7 70.6 78.0 73.6 92.5 99.9 84.5 83.1 78.9 62.5 69.8 32.5 95.1 98.1 81.0 98.2 94.4

100 mmol kg1 oil [days]d

Propanal [area]

2 >25 >25 11 4 4,5 6 5.5 8.5 13 2 6.5 6.5 6 4 5 3 10 2 >25 7 >25 15

298.6  7.5 n.d. n.d. 7.1  1.5 99.4  8.6 70.0  7.3 71.3  4.6 115.0  8.8 44.6  7.1 3.0  0.8 141.2  10.3 19.8  1.4 21.8  2.7 64.2  4.1 58.2  6.2 50.5  4.7 177.9  6.2 8.6  1.88 104.0  6.6 n.d 16.7  3.2 n.d. 2.8  1.4

Inhibitionc [%]

100 100 97.6 66.7 76.5 76.1 61.5 85.1 99.0 86.0 84.6 54.5 58.8 64.2 26.0e 93.9 100 84.0 100 97.3

n.d., not detected. a Ferulic acid content in the additive (Bauer et al., 2012). b Calculated as ferulic acid equivalents. c Inhibition was calculated on basis of control values resulting from individual experiments. d Days determined to reach a hydroperoxide content of 100 mmol kg1 oil. e Negative values point to pro-oxidative effects.

effectively and no secondary lipid oxidation products were detected after six days of storage, however, only a low content of ferulic acid was found in this extract (Table 2). The inhibition of oxidation products by the isopropyl alcohol extracts of corn fiber (Cf-P) and wheat bran (Wb-P) was decreased in comparison to the methanolic extracts. The methanolic extract of corn fiber was fractionated by preparative HPLC and tested in rapeseed oil containing emulsions at a concentration of 50 mg/kg (Fig. 2). Fraction one and four showed no antioxidant effects while fraction two and three showed a 50% and 94% inhibition of hydroperoxide formation after nine days of storage respectively. Fraction three contained high amounts 500

3.4. Inhibition of lipid oxidation by ferulated oligosaccharides compared to reference compounds Ferulated oligosaccharides (FOm; 67 mg ferulic acid/g) gained from mild acid hydrolysis of corn fiber were tested at a concentration of 500 mmol FA kg1 oil and showed strong inhibition of lipid oxidation products compared to the ferulic acid monomer (FA) (Fig. 3). The same activity was found for Cf-U 800 mg/kg corresponding to 500 mmol FA kg1 oil. The hydroperoxide concentration was kept under 100 mmol kg1 oil after 10 days of storage. All

400 500 350 450 300

Hydroperoxides [mmol/kg oil]

Hydroperoxides [mmol/kg oil]

450

of the polyaminconjugates diferuloylputrescine (605 mg/g extract) and p-coumaroyl-feruloylputrescine (145 mg/g extract) but no free ferulic acid was detected. Fraction two contained free ferulic and pcoumaric acid and no phenolic compounds were detected in fraction one and four (data not shown).

250 200 150 100 50 0 0

5

10

15

20

Storage at 20 °C [days]

400 350 300 250 200 150 100 50 0 0

Fig. 1. Hydroperoxide formation (Means  SD, n ¼ 3) of oil in water emulsions (10% RFO) containing untreated and enzymatically treated corn fiber and wheat bran (-: control, C: Cf-U 8000 mg/kg, A: Cf-U 4000 mg/kg, :: Cf-U 800 mg/kg, : Cf-T 800 mg/kg, B: Wb-U 8000 mg/kg, >: Wb-U 4000 mg/kg, 6: Wb-U 2000 mg/ kg, : Wb-T 4000 mg/kg).

5

10

15

20

Storage at 20 °C [days]

Fig. 2. Hydroperoxide formation (Means  SD, n ¼ 3) of oil in water emulsions (10% RFO) containing four obtained fractions of the methanolic extract of corn fiber (-: control, C: Cf-MeOH fr.1, A: Cf-MeOH fr.2, :: Cf-MeOH fr.3, B: Cf-MeOH fr.4).

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J.L. Bauer et al. / LWT - Food Science and Technology 50 (2013) 132e138 Table 3 Antioxidant activities of obtained extracts from corn fiber and wheat bran.

500 450

Extract Hydroperoxides [mmol/kg oil]

400 350 300 250 200 150 100 50 0 0

5

10

15

20

Storage at 20 °C [days]

Fig. 3. Development of hydroperoxide content (Means  SD, n ¼ 3) in 10% oil (RFO) in water emulsions over time containing ferulated oligosaccharides, ferulic acid, and propyl gallate (-: control, B: FOm 145 mg/kg, C: FA 10 mg/kg, :: PG 11 mg/kg). All supplements containing 500 mmol ferulic acid kg1 oil.

reference antioxidants were tested at 500 mmol kg1 oil. Trolox (Tx) effectively inhibited hydroperoxide formation in RFO containing emulsions after six days of storage and kept under 100 mmol hydroperoxides kg1 oil after 13 days (Table 2). Propyl gallate (8.5 days) also showed stronger inhibition of lipid oxidation than FA but untreated corn fiber clearly showed a stronger inhibition. 3.5. Antioxidant capacity of the tested extracts and antioxidants The methanolic extracts of corn fiber (Cf-MeOH) and wheat bran (Wb-MeOH) showed a high activity in the DPPH and TEAC assays and the activity was higher than for the isopropyl alcohol extracts (Table 3). Since extracts from untreated fibers contained only low amounts of free hydroxycinnamic acids further testing was done with fractions of the methanolic extract. The highest activity was shown in fraction 3 followed by fraction 2 while fraction 1 and 4 exhibited lower activities. Ferulated oligosaccharides (FOm) obtained by acid hydrolysis of corn fiber showed strong antioxidant potential comparable to fraction 3 of the methanolic extract. On a molar basis FOm (3 mmol TE/mmol ferulic acid) showed a 1.5-fold higher activity compared to ferulic acid (1.9 mmol TE/mmol ferulic acid). Extracts from untreated wheat bran were less active than the corn fiber extracts (Table 2). The aqueous extracts of enzymatically treated corn fiber (Cf-S) and wheat bran (Wb-S) showed moderate activity but only low amounts of hydroxycinnamates were found in these extracts. Extracts from corn fiber indicated slightly higher potential. However, ethyl acetate extracts of the aqueous supernatant of enzymatic treatments which contained high amounts of ferulic acid (Table 2) acted as potent antioxidants in both assays. 4. Discussion 4.1. Activity of untreated corn fiber and wheat bran during storage of model RFO emulsions Ground untreated corn fiber and wheat bran were tested in different concentrations in 10% RFO-emulsions. Corn fiber inhibited the formation of hydroperoxides and volatile aldehydes in both systems effectively showing a dose dependent behavior. Best inhibition was observed at the highest concentration tested. In contrast to corn fiber, wheat bran caused only little antioxidant effects on the tested emulsions and showed a slight negative

DPPHa (mmol TE/g)

Untreated fibers Cf-MeOH 64.3  Cf-MeOH fr. 1 56.7  Cf-MeOH fr. 2 171.5  Cf-MeOH fr. 3 210.0  Cf-MeOH fr. 4 33.2  Cf-P 17.4  Wb-MeOH 18.9  Wb-P 14.4  FOm 249.8  Enzymatically treated fibers Cf-S 15.8  Cf-EtOAc 123.7  Wb-S 8.7  Wb-EtOAc 89.4  Reference compounds Ferulic acid 2018.9  Propyl gallate 4297.2  L-tryptophan e a

TEACa (mmol TE/g)

2.2 0.7 5.2 1.9 0.5 0.5 0.6 0.2 8.1

271.8 242.8 839.3 1047.4 103.4 254.6 190.3 93.1 1023.5

        

3.1 2.7 1.4 2.7 1.2 2.4 2.3 2.9 11.8

1.8 0.3 0.4 7.6

117.7 408.6 69.8 371.0

   

3.7 1.2 0.5 2.5

65.2 102.2

10000.9  102.9 20285.3  89.74 1258.4  31.6

Expressed as Trolox equivalents (mmol TE/g extract).

concentration/activity correlation. In addition, cereals also contain other compounds, such as carotenoids, flavonoids, or tocopherols (Adom, Sorrells, & Liu, 2003; Zhou & Yu, 2004) or other noncharacterized phenolics which significantly contribute to the final antioxidant capacity of cereal extracts. Gallardo et al. (2006) stated that the antioxidant activity is linked to the combined action of all available phytochemicals in cereals rather than to individual hydroxycinnamic acids. When untreated corn fiber was tested at a concentration of 1000 mg/kg (500 mmol ferulic acid kg1 oil) corn fibers showed stronger inhibition of lipid oxidation than monomeric ferulic acid, whereas inhibition decreased with ground wheat in comparison to monomeric ferulic acid. The ferulic acid content of corn fiber and wheat was shown previously to be 14.04 mg/g and 3.73 mg/g respectively (Bauer et al., 2012). Wheat bran may also contain prooxidants, like transition metals, which hinder the antioxidative action of wheat bran in the emulsion model, which could explain higher oxidation rates found at higher wheat bran concentrations. Corn fiber also contains high amounts of polyaminconjugates, which are associated with corn fiber oil and represent up to 10% of the oil with a reported ferulic acid like antioxidative capacity (Choi et al., 2007; Moreau & Hicks, 2005). Therefore free and cell wall bound ferulic acid appears not to be the only compound responsible for the antioxidative effects in the tested corn fiber. Ferulated oligosaccharides (FOm) were obtained after acid hydrolysis of corn fiber and separation with Amberlite XAD-2. These compounds were identified as ferulic acid esterified to one arabinose moiety and ferulic acid esterified to arabinose and xylose by HPLCeMS (Bauer et al., 2012). Free ferulic acid or other compounds were not detected in this fraction, and 67.09 mg ferulic acid/g was released by alkaline hydrolysis. Therefore the antioxidant effect of this fraction is likely to only be based on bound ferulic acid. FOm were tested in emulsions to estimate the antioxidative action of bound ferulic acid. In RFO containing emulsions added FOm (500 mmol ferulic acid kg1 oil) effectively inhibited the formation of hydroperoxides and volatile aldehydes. In comparison to untreated corn fiber and wheat bran, also tested with equal ferulic acid content, ferulated oligosaccharides inhibited the formation of lipid oxidation comparable to corn fiber. In addition, we found high antioxidant capacities in the TEAC and DPPH assay. The TEAC of ferulic acid was found to be 1.9 mmol TE/mmol ferulic acid, but FOm showed an activity of 3 mmol TE/mmol ferulic acid, i.e., the activity of FOm was 1.5-times higher as monomeric ferulic acid.

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This is in excellent accordance to the results reported by Kylli et al. (2008). They further reported that synthesized ferulic acid glycoside esters have shown improved antioxidative capacity compared to free ferulic acid in an emulsion model. Thereby the position of the ester bond to the sugar moiety seems to have a significant effect to the antioxidant activity, whereas 6-O-feruloyl glycosides showed higher activity than the 3-O- and 2-O-coupled esters. Rao and Muralikrishna (2006) have shown a seven fold higher activity in the DPPH assay when ferulated arabinoxylans were tested. They found a very strong positive correlation between the antioxidative activity and the molecular weight of ferulated arabinoxylans, and proposed that arabinoxylans with very high molecular weight show higher activity than small esters. The activity of ferulic acid esterified to arabinoxylans possesses free electron-donating groups on the benzene ring (3-methoxy, and more importantly, 4hydroxyl) and the ferulic acid moiety of the feruloyl oligosaccharides gave resonance structures of the resulting phenoxyl radical during lipid peroxidation (Ishii, 1997; Xiaoping, Jing, & Huiyuan, 2005). Adom et al. (2003) reported that bound phytochemicals contributed >82% of the total antioxidant activity and only 12e35% were attributed by free forms in wheat extracts. Katapodis et al. (2003) compared inhibitory effects of free ferulic acid and ferulated oligosaccharides in a human LDL oxidation and ferulated oligosaccharides inhibited LDL oxidation better than free ferulic acid. Ferulic acid sugar esters are more hydrophilic due to their sugar moiety than their free forms. More lipophilic antioxidants are generally favored because of their close positioning at the oilewater interface of emulsions. However emulsifiers are able to solubilize more hydrophilic compounds which allow them to get in contact with the oilewater interface where they can inhibit chain reactions (Stöckmann et al., 2000; Stöckmann & Schwarz, 1999). 4.2. Inhibition of lipid oxidation product formation during storage of emulsions by enzymatically treated fibers and derived extracts Compared to untreated corn fiber and wheat bran, tested at a concentration of 500 mmol ferulic acid kg1 oil, enzymatically treated fibers (Cf-T, Wb-T) showed weaker antioxidant effects on emulsions. Buffer salts and enzymes residues in the freeze dried powder may affect the antioxidant action of Cf-T. On the other hand, it has been shown that ferulic acid bound to arabinoxylan (FOm) exerted a high antioxidative potential which is considerably higher than the antioxidative potential of monomeric ferulic acid in emulsions. Therefore, it can be concluded that the release of bound ferulic acid into its free form will not increase the antioxidant activity. After treatment with Depol 740 L 43% of the bound ferulic acid was released into its free form in corn fiber and 36% of bound ferulic acid was released from Depol 670 L treated wheat bran (data not shown). Furthermore, corn fiber contains high amounts of the conjugated amines diferuloylputrescine and p-coumaroyl-feruloylputrescine, which are associated with corn fiber oil and it is likely that they take part in the antioxidant action of treated and untreated corn fibers (Bauer et al., 2012). These compounds are lost by enzymatic treatment when the aqueous supernatant is used as an extract due to their low water solubility. The amount of free ferulic acid in the enzymatic extracts is rather low (Cf-S: 3.1 mg/g extract; Wb-S: 0.09 mg/g extract) compared to the contents found in untreated ground fibers, thus lower inhibition was anticipated (data not shown). Cf-S and Wb-S also showed lower DPPH and TEAC values compared to other extracts tested in this study but their activity does not correlate with their content of released hydroxycinnamates. Moore, Zhihong, Lan, and Liangli (2006) showed that the enzymatic treatment of wheat bran improved the extractable and potentially bioaccessible antioxidant properties of wheat bran in the DPPH and TEAC assays.

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Other authors who described enzymatic treatments of brans with ferulic acid esterases only direct the attention to the extraction of free ferulic acid and no effects of crude hydrolysates on the antioxidative potential were published. The extraction of the aqueous supernatant of the enzymatic hydrolysis with ethyl acetate leads to extracts (CF-EtOAc and Wb-EtOAc) largely enriched with free ferulic acid (corn fiber, 279.1 mg/g extract, wheat bran, 88 mg/g extract) and p-coumaric acid (corn fiber, 22.7 mg/g extract; wheat bran, not detected) which can be added in smaller concentrations to the emulsions (data not shown). While Cf-EtOAc moderately inhibited the formation of lipid oxidation products in the emulsion model, the Wb-EtOAc extract exhibited pro-oxidative effects concerning the formation of secondary lipid oxidation products. Since wheat bran contains high amounts of phytic acid, a transfer of prooxidative metal ions into the ethyl acetate extracts may have occurred (Graf & Eaton, 1990). 4.3. Antioxidant action of extracts gained by extraction of untreated corn fiber and wheat bran on oil in water emulsions The corn fiber, as well as the wheat bran methanolic extract, showed strong antioxidant activity in the emulsion model which gave reason for further fractionation by preparative HPLC. Each fraction was tested in rapeseed oil containing emulsions. The polyaminconjugates diferuloylputrescine (DFP) and p-coumaroyl-feruloylputrescine (CFP) were tentatively identified as the main compounds in fraction 3 of the methanolic corn fiber extract (Bauer et al., 2012). Moreau, Nuñez, and Singh (2001) reported that corn fiber oil extracted with polar solvents contains up to 10% of polyamine conjugates. DFP and CFP were present in Cf-MeOH at levels of 57.4 mg/g and 9.9 mg/g respectively (Bauer et al., 2012). According to the lower activity conducted by isopropyl extracts from corn fiber (Cf-P) in the RFO emulsions the amount of ferulic acid (calculated as ferulic acid equivalents) was considerably lower than in the methanolic extracts (Bauer et al., 2012). The methanolic extract obtained in this study contained about 6 g/100 g of diferuloylputrescine which was found to be the major compound. Fraction 3 of the methanolic extract also showed a very high activity in the DPPH and TEAC assay, which was the highest activity among the tested extracts. However, on a molar basis, DFP and CFP are shown to be less active than ferulic acid. Choi et al. (2007) also showed that DFP and CFP exhibited considerable free DPPH radical and superoxide radical scavenging activities but slightly lower than reported for ferulic acid. However, the inhibitory effect on the formation of lipid oxidation products of Wb-MeOH cannot be explained by polyamine conjugates because they are only found in maize. Furthermore, no free hydroxycinnamic acids were found in this extract. Free L-tryptophan was found in high amounts in wheat bran (6.9 mg/g extract) and exerted moderate antioxidative activity (1258 mmol TE/g extract) in the TEAC assay (Bauer et al., 2012). It is possible that extraction of wheat bran with methanol and isopropyl alcohol excludes pro-oxidant compounds which causes prooxidant action in ethyl acetate extracts. 5. Conclusions It was shown that ground untreated corn fiber is an effective antioxidant comparable to propyl gallate which inhibited chain reaction in fish oil containing emulsions. This can be explained to a great extent by the antioxidant activity of bound ferulic acid and polyaminconjugates. Untreated wheat bran showed only low antioxidative activity in emulsions. Thereby ferulated oligosaccharides gained by acid hydrolysis of corn fiber were a more potent lipid oxidation inhibitor than free ferulic acid. Therefore the high activity of corn fiber in contrast to wheat bran can be attributed to

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