Antioxidant activity of barley as affected by extrusion cooking

Food Chemistry 131 (2012) 1406–1413

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Antioxidant activity of barley as affected by extrusion cooking Paras Sharma a, Hardeep Singh Gujral a,⇑, Baljeet Singh b a b

Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, India Department of Food Science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India

a r t i c l e

i n f o

Article history: Received 8 February 2011 Received in revised form 17 July 2011 Accepted 5 October 2011 Available online 10 October 2011 Keywords: Barley Extrusion Total flavonoids content Antioxidant activity Metal chelating activity Reducing power

a b s t r a c t Grit from different hulled barley cultivars was subjected to extrusion cooking and the effect of extrusion moisture and temperature on the antioxidant properties was studied. A significant decrease in the total phenolic content (TPC) and total flavonoid content (TFC) was observed upon extrusion and a further decrease of 8–29% in TPC and 13–27% in TFC was observed when both the feed moisture and extrusion temperature were increased. The antioxidant activity (AOA) increased significantly upon extrusion and this increase was the highest (36–69%) at 150 °C and 20% feed moisture. The increase in feed moisture and temperature significantly increased the metal chelating activity. The reducing power decreased significantly upon extrusion as compared to their corresponding control samples. Extrusion lead to a greater increase in non-enzymatic browning (NEB) index however, increasing the moisture content of feed decreased the NEB index by 3–29% (at 180 °C) and 1–17% (150 °C), while increasing the temperature increased the NEB significantly. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Processed foods these days require the presence of bioactive ingredients to satisfy the demands of health conscious consumers. Barley (Hordeum vulgare L.) is considered as a nutraceutical grain because it contains bioactive compounds like b-glucan, phenolic compounds, B-complex vitamins, tocotrienols, tocopherols (Madhujith, Izydorczyk, & Shahidi, 2006; Sharma & Gujral, 2010a, 2010b). Among the cereal grains barley has higher antioxidant activity as compared to the more widely consumed cereals wheat and rice. The risk imposed by the consumption of free radicals and oxidation products towards various forms of cancer and cardiovascular disease could be lowered by the intake of dietary phenolics. Barley contains many phenolic compounds in the free and bound form; these compounds include benzoic and cinnamic acid derivatives, proanthocyanidins, quinines, flavonols, chalcones, flavones, flavanones, and amino phenolic compounds (Goupy, Hugues, Boivin, & Amiot, 1999; Shahidi, 2009). Extrusion is a rapid processing method involving high temperature and pressure and short time and is used to prepare a variety of processed foods like baby foods, snack foods, ready-to-eat breakfast cereals and pet foods. The consumer preference of extruded foods is mainly due to convenience, attractive appearance and texture and utilising barley in extruded foods would increase consumer acceptance as it contains bioactive functional

⇑ Corresponding author. Tel.: +91 183 2258802. E-mail address: [email protected] (H.S. Gujral). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.10.009

components. The effects of extrusion cooking on the polyphenol content and antioxidant activity in rye bran has been reported by Gumul and Korus (2006) and in a snack bar composed of chickpea, corn, oat carrot and hazelnut by Ozer, Herken, Guzel, Ainsworth, and Ibanoglu (2006). Korus, Gumul, and Czechowska (2006) studied the effects of extrusion on the phenolic composition and antioxidant activity of kidney beans. A maize bran/oat flour extruded breakfast cereal was developed by Holguin-Acuna et al. (2008) as a novel source of antioxidant and complex polysaccharides. An increase in the total phenols and DPPH radical scavenging activity in corn starch/common bean extrudates was reported by Anton, Fulcher, and Arntfield (2009). Shih, Kuo, and Chiang (2009) investigated the effects of drying and extrusion on the antioxidant activity of sweet potato and reported that the scavenging effect on DPPH radicals and total phenolic compounds increased. It is well documented that the minimal processed foods have more health benefits (Gujral, Sharma & Singh, 2011; Shahidi, 2009; Sharma, Gujral, & Rosell, 2011). Even though extrusion is a short time cooking process the temperatures encountered by the raw material in the barrel of the extruder is enough to bring about changes in the major and non-nutritive components. The most significant changes are brought about in the cereal starch and protein that contribute to form structure, texture, mouth feel and bulk density. The non-nutritive components mainly polyphenols having antioxidant properties may undergo various changes, thus altering their antioxidant activity. The objective of the present investigation was to study the effect of extrusion moisture and temperature on antioxidant activity of extrudates from different barley cultivars.

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2. Materials and methods

2.4. Total phenolic content (TPC)

2.1. Barley samples

The total phenolic content (TPC) was determined according the Folin–Ciocalteu specterophotometric method (Sharma & Gujral, 2010b). Extruded samples (200 mg) were extracted with 4 ml acidified methanol (HCl/methanol/water, 1:80:10, v/v/v) at room temperature (25 °C) for 2 h. An aliquot of extract (200 ll) was added to 1.5 ml freshly diluted (10-fold) Folin–Ciocalteu reagent. The mixture was allowed to equilibrate for 5 min and then mixed with 1.5 ml of sodium carbonate solution (60 g/l). After incubation at room temperature (25 °C) for 90 min, the absorbance of the mixture was read at 725 nm (Shimadzu, UV-1800, Japan). Acidified methanol was used as a blank. The results were expressed as lg of ferulic acid equivalents (FAE) per gram of sample.

Eight common hulled barley cultivars (PL-172, PL-426, RD2503, RD-2508, RD-2035, RD-2052, RD-2552 (six rowed) and DWR-28 (two rowed)) grown in different locations in the states of Punjab, Haryana, Uttar Pradesh and Rajasthan were collected from Central State Seed farm, Sriganganagar, Rajasthan, India. The grain was cleaned and stored for further evaluation. The barley cultivars varied significantly among themselves with the husk content ranging from 9.6% to 13.0% and bulk density ranging from 0.547 to 0.653 g/ml. The protein content and ash content ranged from 8.7% to 13.4% and 1.1% to 1.5%, respectively (Sharma & Gujral, 2010a). The total b-glucan ranged from 4.07% to 5.47% (Sharma et al., 2011a) while the amylose content ranged from 21.0% to 28.3% (Gujral, Sharma, Kaur & Singh, 2011). 2.2. Reagents Standard ferulic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrozine, protease (from Streptomyces griseus) and catechin were procured from Sigma–Aldrich (Steinheim, Germany). L-Ascorbic acid, potassium ferricyanide, ferric chloride, ferrous chloride, trichloroacetic acid, sodium carbonate and Folin Ciocalteu’s reagent were procured from Loba Chemie, Mumbai, India. All chemicals were of analytical grade. Each test was performed in triplicates on dry weight basis. The Milli Q water (Millipore, France) was used for all analytical tests.

2.5. Antioxidant activity (DPPH radical scavenging activity) Antioxidant activity (AOA) was measured using a modified version of the method described by Brand-Williams, Cuvelier, and Berset (1995). Extruded samples (100 mg) were extracted with 1 ml methanol for 2 h and centrifuged at 3000g for 10 min. The supernatant (100 ll) was reacted with 3.9 ml of a 6  105 mol/L of DPPH solution. The absorbance (A) at 515 nm was read at 0 and 30 min using a methanol blank. The antioxidant activity was calculated as % discoloration:

DPPH radical scavenging activityð%Þ ¼ ð1  ðA of samplet ¼ 30=A of controlt ¼ 0ÞÞ  100

2.3. Extrusion

2.6. Reducing power

Barley samples were dehusked as previously described by Sharma and Gujral (2010a). The grits (95% retained by 250 lm sieve) were prepared using a Super Mill 1500 (Newport Scientific, Australia). Further, the grits were conditioned to 15% and 20% moisture content and packed in polyethylene bags and allowed to equilibrate for 12 h. The extrusion was performed on a corotating twin screw extruder (Clextral, BC 21, Firminy, France). The screw diameter, (L/D) ratio and die diameter was 25 mm 16 and 6 mm, respectively. The feed rate (20 kg/h) and screw speed (400 rpm) were kept constant. The extrusion was carried out at 150 and 180 °C, the temperature of different barrel zones was 50, 100, 125 and 150 °C (for 150 °C extrusion) while it was 50, 100, 140 and 180 °C when the extrusion was carried out at 180 °C. The terminal section was heated by an induction heating belt and the feeding section of barrel was cooled with running water. The screw configuration in different sections of the extruder (from hopper to die) was as follows:

The reducing power was measured as described by Sharma and Gujral (2011). Extrudate samples (0.5 g) were extracted with 80% methanol (0.5 ml) on wrist action shaker for 2 h. The extract (1 ml) was mixed with phosphate buffer (2.5 ml) and 2.5 ml potassium ferricyanide were added followed by incubation at 50 °C. Trichloroacetic acid solution (10%) was added to mixture, which was then centrifuged at 10000g for 10 min. The upper layer of solution (2.5 ml) was mixed with 2.5 ml deionized water and 0.5 ml ferric chloride. The absorbance of the mixture was measured at 700 nm. A standard curve was prepared using various concentration of ascorbic acid and results were reported as ascorbic acid equivalents/g (AAE) of flour.

Screw section Screw element Length (mm) Pitch (mm)

1 BAGUE 20 –

2 C2F 50 50

3 C2F 50 33.33

4 C2F 50 25

The extrudates were classified as high temperature high moisture (HTHM, 180 °C temperature, and 20% moisture), high temperature low moisture (HTLM, 180 °C temperature, 15% moisture), low temperature high moisture (LTHM, 150 °C temperature, 20% moisture) and low temperature low moisture (LTLM, 150 °C temperature, 15% moisture). The extrudates were cooled to room temperature and packed in polyethylene bags. Further, all extrudates were ground in a grinder (Sujata, India) to particle size <250 lm and stored at 20 °C until further analysis.

2.7. Total flavonoids content (TFC) The total flavonoids content (TFC) was determined as previously described by Jia, Tang, and Wu (1998). The extract 5 C2F 50 25

6 C2F 50 16.66

7 INO 0 5 –

8 C1F 50 16.66

9 CF1C 25 12.5

10 C1F 50 12.5

(250 ll) was diluted with 1.25 ml distilled water. Sodium nitrite (75 ll) was added and the mixture was allowed to stand for 6 min. Further, 150 ll of aluminium chloride were added and the mixture was allowed to stand for 5 min. After that, 0.5 ml of sodium hydroxide (1 M) was added and the solution was mixed well. The absorbance was measured immediately at 510 nm using a spectrophotometer. Catechin was used as standard and the results were reported as microgram catechin equivalent (CE)/g of sample.

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2.8. Metal chelating (Fe+2) activity The metal chelating activity of 80% methanol extract was measured as reported by Sharma and Gujral (2011). The extract (0.5 ml) was mixed with 50 ll of ferrous chloride and 1.6 ml of 80% methanol was added. After 5 min, the reaction was initiated by the addition of 5 mM/l ferrozine and the mixture was shaken on a vortex. The mixture was incubated at room temperature (25 °C) for 10 min. The absorbance of solution was measured at 562 nm on a spectrophotometer. The chelating activity of the extract for Fe+2 was calculated as follows:

Iron ðFeþ2 Þ chelating activity ð%Þ ¼ f1  ðAbsorbance of sample at 562 nm= Absorbance of control at 562 nmÞg  100 2.9. Nonenzymatic browning (NEB) index Nonenzymatic browning index (NEB) of extrudates was carried out as previously reported by Sharma and Gujral (2011). The extrudate sample (100 mg) was mixed with 1 ml of deionized water. An aliquot (200 ll) of the mixture was transferred to a test tube which contained 200 ll of protease solution (in Tris buffer). The test tubes were incubated for 2 h at 45 °C, in a water bath (NSW-125, Narang Scientific Works, New Delhi, India). The test tubes were cooled and 300 ll trichloroacetic acid was added to each tube. Then, the tubes were centrifuged at 7000g for 20 min. The absorbance of supernatant was measured on a spectrophotometer. The browning index (DA) was calculated as follows:

DA ¼ Absorbance at 420 nm  Absorbance at 550 nm 2.10. Statistical analysis Analysis of variance (ANOVA) was carried out using Microsoft Excel software and Fishers least significant difference (LSD) test was used to describe means with 95% (p < 0.05) confidence. The Pearson correlation coefficients were calculated by SPSS statistical software (SPSS Inc., Chicago, Illinois, USA) at a probability level of p < 0.05. 3. Results and discussion 3.1. Effect of extrusion cooking on total phenolic content (TPC) The total phenolic content (TPC) in the eight barley cultivars ranged from 3070 to 4439 lg FAE/g (Sharma & Gujral, 2010b). Madhujith and Shahidi (2009) reported TPC value ranging from 2.63 to 4.51 mg of ferulic acid equivalents (FAE)/g in barley. Bonoli,

Verardo, Marconi, and Caboni (2004) reported a total phenolic content ranging from 0.18 to 0.68 mg gallic acid/g flour in barley flour. The differences in the total phenolic content can be attributed to differences in the solvent used in the extraction and the differences in the barley cultivars. The total phenolic content in all the cultivars decreased significantly upon extrusion as compared to their corresponding control (unextruded) samples (Table 1). These results are also consistent with previous study carried out by Delgado-Licon et al. (2009) on the extrusion of bean–corn mixture. The phenolic compounds are heat labile (Sharma & Gujral, 2011) and are less resistant to the heat, and heating over 80 °C may destroy or alter their nature (Zielinski, Kozlowska, & Lewczuk, 2001). The reduction in TPC may be attributed either to the decomposition of phenolic compounds due to the high extrusion temperature or alteration in molecular structure of phenolic compounds that may lead to reduction in the chemical reactivity of phenolic compounds or decrease their extractability due to certain degree of polymerisation (Altan, McCarthy, & Maskan, 2009). It has also been reported that the phenolic and flavonoids compounds may interacted with the proteins and may not exhibit their actual value (Arts, Haenen, & Wilms, 2002). The total phenolic content in HTHM extrudates, varied significantly (p < 0.05) among cultivars and ranged from 1374 to 1897 lg ferulic acid equivalents (FAE)/g with highest and lowest being for DWR-28 and RD-2552. Similarly, the TPC in HTLM extrudates, varied significantly (p < 0.05) among cultivars and ranged from 1721 to 2154 lg FAE/g. The highest and the lowest observed for DWR-28 and RD-2508. Stojceska, Ainsworth, Plunkett, and Ibanoglu (2009) reported total phenolic content ranging from 1.4 to 2.1 mM gallic acid equivalent/g in different extrudates prepared from barley–wheat flour and corn starch–barley flour blends. When the temperature was kept constant (180 °C) and the moisture of feed was increased from 15% to 20%, a significant (p < 0.05) decrease was observed in TPC for all cultivars and this decrease ranged from 8% to 29%, the highest and the lowest decrease was observed for RD-2552 and PL-426, respectively. This may be attributed to the higher moisture content of feed, as the moist heat is more destructive and produced a synergistic effect along with higher temperature. On the other hand, when the extrusion temperature was kept constant at 150 °C and the moisture content of feed increased from 15% to 20%, a significant (p < 0.05) increase was observed in TPC. In LTHM extrudates TPC varied significantly (p < 0.05) among cultivars and ranged from 1815 to 2195 lg FAE/g, the highest and the lowest being for RD2052 and RD-2508, respectively. This combination of extrusion variables had the highest TPC among all the extrudates. The TPC in the LTLM extrudates varied significantly and ranged from 1544 to 1995 lg FAE/g. The highest and the lowest were observed for DWR-28 and RD-2035, respectively. When the temperature was increased from 150 to 180 °C, keeping the moisture constant (15%) a significant (p < 0.05) increase was noticed for all the

Table 1 Total phenolic content of barley extrudates. Cultivars

DWR-28 RD-2503 RD-2508 RD-2035 RD-2052 RD-2552 PL-172 PL-426

Total phenolic content (lg FAE/g) Unextruded

HTLM (180 °C, 15%)

HTHM (180 °C, 20%)

LTLM (150 °C, 15%)

LTHM (150 °C, 20%)

3070as 3121as 3417bt 3485bt 3588ct 3441br 4439es 4180dt

2154cr (;30) 1782ar (;43) 1721ar (;50) 1733ar (;50) 1867br (;48) 1928bq (;44) 1949bq (;56) 1987br (;52)

1897dp (;38) 1515bp (;51) 1492bp (;56) 1449bp (;58) 1533bp (;57) 1374ap (;60) 1777cp (;60) 1823cp (;56)

1995dq (;35) 1705bq (;45) 1690bq (;51) 1544aq (;56) 1733bq (;52) 1923cq (;44) 1938cq (;56) 1895cq (;55)

2167dr (;29) 1815ar (;42) 1933bs (;43) 1915bs (;45) 2195ds (;39) 1959bq (;43) 2069cr (;53) 2164ds (;48)

a, b, c, d and e superscripts are significantly (p < 0.05) different within a column for different cultivars and p, q, r, s and t superscripts are significantly (p < 0.05) different within a row for each cultivar. Subscripts denote the percentage decrease (;) from control samples. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

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cultivars with RD-2035 showing the highest increase (12%). However, when the temperature was increased from 150 to 180 °C, keeping the moisture constant at 20%, the TPC decreased significantly (p < 0.05) by 12–30% with the highest and lowest being for RD-2052 and DWR-28, respectively. These results are also in agreement with those reported by Altan et al. (2009). 3.2. Effect of extrusion cooking on antioxidant activity (DPPH free radical scavenging activity) Estimation of the antioxidant activity (AOA) by scavenging of stable radicals, such as the chromogen radical DPPH in inorganic media has been extensively used for comparison of homogeneous series of antioxidants. This procedure measures the hydrogen donating capacity of the target substances in a methanolic media. The colour changes from purple to yellow by acceptance of a hydrogen radical from MRP and it becomes a stable diamagnetic molecule. Extrusion cooking led to a significant increase in DPPH free radical scavenging activity (antioxidant activity, AOA) in all the cultivars as compared to their corresponding control samples (Table 2). The earlier investigations have reported that dark colour pigments (brown colour) are produced during the thermal processing of foods (Sharma & Gujral, 2011; Xu & Chang, 2008) due to the Maillard browning. These pigments (particularly melanoidins) are extensively known to have antioxidant activity (Manzocco, Calligaris, Masrrocola, Nicoli, & Lerici, 2001). The increase in antioxidant activity could be explained by the formation of Maillard browning pigments which enhanced the antioxidant activity (Rufian-Henares & Delgado-Andrade, 2009) of extrudates as compared to their corresponding control samples. The thermal processing is also known to alter the antioxidant profile and generate more antioxidants that contribute in antioxidant activity. Increase in antioxidant activity due to thermal processing has been widely reported (Dewanto, Wu, Adom, & Liu, 2002; Nicoli, Anese, Parpinel, Franceschi, & Lerici, 1997) Antioxidant activity varied significantly among cultivars when the extrusion was carried out at 180 °C and 15% moisture (HTLM). The AOA ranged from 19.2% to 26.2% with highest and lowest being for PL-172 and RD-2503, respectively. Similarly, when the extrusion was carried at 180 °C and 20% moisture (HTHM), the AOA varied significantly (p < 0.05) among cultivars and ranged from 21.0% to 27.8%. The PL-172 showed the highest and DWR-28 showed the lowest AOA. The AOA in the extrudates LTLM did not vary significantly (p < 0.05) among the cultivars. Also, the AOA in the extrudates LTHM (150 °C, 15%) varied insignificantly among cultivars. Delgado-Licon et al. (2009) reported that the highest antioxidant activity was when they extruded the bean/corn mixture at 142 °C temperature and 16.5% feed moisture. Similarly, Shih et al. (2009) reported that the extrusion process significantly increased the DPPH radical scavenging activity in the different sweet potato extrudates.

Awika, Rooney, Wu, Prior, and Cisneros-Zevallos (2003) reported the highest antioxidant activity in the extruded sorghum as compared to baked and corresponding control sorghum samples. Furthermore, when the temperature of extrusion was kept constant (180 °C) and moisture content was increased from 15% to 20%, AOA was increased significantly (p < 0.05) in RD-2503, RD-2508 and PL-426 by 12%, 21% and 8%, respectively while the rest of the cultivars did not show significant (p < 0.05) increase. Keeping the temperature constant at 150 °C and increasing moisture from 15% to 20%; AOA showed a significant increase that ranged from 9% to 24%. The highest and the lowest increase were exhibited by RD-2052 and RD-2552, respectively. Interestingly, when the moisture was kept constant (20%) and temperature increased from 150 to 180 °C, a significant decrease in AOA was observed and this ranged from 18% to 33%. Similarly, increasing the temperature of extrusion from 150 to 180 °C and keeping the moisture constant at (15%), the AOA decreased significantly by 11% to 25%. A change in the extrusion temperature and moisture could have lead to the formation of different amounts of Maillard browning product (Manzocco et al., 2001). Maillard browning is influenced by many factors, including temperature, reactant concentration, reaction time and water activity (Stojceska et al., 2009). A significant (p < 0.05) positive correlation was observed (R = 0.82) between the AOA and TPC in control samples. The extrudates (LTHM and LTLM) also exhibited a positive correlation between the AOA and TPC (R = 0.65, p > 0.05) and (R = 0.78, p < 0.05), respectively. On the other hand, no correlation between TPC and AOA was observed (R = 0.22 and 0.28, p > 0.05) in extrudates, HTHM and HTLM. The antioxidant activity is not only affected by quantity but also the kind of free radical scavengers present in the material (Oomah, Cardador-Martinez, & Loarca-Pina, 2005). 3.3. Effect of extrusion cooking on metal chelating activity Numerous antioxidant methods have been reported to evaluate antioxidant activity of foods and to explain how antioxidants function. Of these, metal chelating activity, reducing power, DPPH assay and total antioxidant activity are most commonly used for the evaluation of antioxidant activities (Amarowicz, Naczk, & Shahidi, 2000; Sharma & Gujral, 2011). The interaction of Fe+2 with ferrozine produces a dark colour complex that is decreased by the action of metal chelator compounds present in the reaction mixture. The metal chelating activity was increased significantly upon extrusion cooking in all the cultivars tested as compared to their corresponding control samples (Table 3). The increase in the metal chelating activity may be as a consequence of the formation of novel compounds such as melanoidins during thermal processing (Sharma & Gujral, 2011; Shih et al., 2009). Maillard reaction products are found to have strong antioxidant properties comparable to

Table 2 DPPH radical scavenging activity of barley extrudates. Cultivars

DWR-28 RD-2503 RD-2508 RD-2035 RD-2052 RD-2552 PL-172 PL-426

Antioxidant activity (DPPH radical scavenging activity) (%) Unextruded (Control)

HTLM (180 °C, 15%)

HTHM (180 °C, 20%)

LTLM (150 °C, 15%)

LTHM (150 °C, 20%)

19.8bp 17.4ap 18.9bp 17.0ap 21.2bp 19.1bp 24.9cp 21.7bp

20.2bp ("2) 19.2aq ("10) 21.4bq ("13) 21.0bq ("23) 22.3bp ("5) 21.9bq ("15) 26.2dp ("5) 23.8cq ("10)

21.0ap ("6) 21.4ar ("23) 25.9dr ("37) 21.3aq ("25) 23.2cp ("10) 22.6bq ("19) 27.8ep ("38) 25.6dr ("38)

26.9aq ("36) 24.9as ("43) 27.0as ("43) 25.1ar ("47) 25.6aq ("21) 28.4ar ("48) 29.5ap ("18) 29.7as ("37)

31.3ar ("58) 29.1at ("67) 31.9at ("69) 30.1as ("77) 31.6ar ("49) 30.8as ("61) 34.0bq ("36) 34.3bt ("58)

a, b, c, d and e superscripts are significantly (p < 0.05) different within a column for different cultivars and p, q, r, s and t superscripts are significantly (p < 0.05) different within a row for each cultivar. Subscripts denote the percentage decrease (") from control sample. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

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Table 3 Metal chelating activity of barley extrudates. Cultivars

DWR-28 RD-2503 RD-2508 RD-2035 RD-2052 RD-2552 PL-172 PL-426

Metal chelating activity (%) Unextruded (Control)

HTLM (180 °C, 15%)

HTHM (180 °C, 20%)

LTLM (150 °C, 15%)

LTHM (150 °C, 20%)

37.5ap 57.3cp 42.2bp 61.5dp 37.8ap 37.9ap 58.2cp 55.8cp

66.6aq ("77) 68.8aq ("20) 60.0aq ("39) 66.3ap ("8) 65.9ar ("74) 61.2aq ("59) 63.8aq ("7) 65.9aq ("18)

71.4br ("90) 79.5dr ("39) 73.9bs ("75) 76.0cp ("23) 72.4bt ("92) 69.4as ("83) 73.7bs ("27) 81.6es ("46)

65.3aq ("74) 67.2bq ("17) 58.8aq ("42) 62.2ap ("1) 60.7aq ("61) 60.4aq ("61) 62.3aq ("10) 63.3aq ("13)

71.1ar ("89) 70.6aq ("23) 69.0ar ("64) 69.0ap ("12) 68.1as ("80) 65.5ar ("73) 67.2ar ("15) 68.7ar ("23)

a, b, c, d and e superscripts are significantly (p < 0.05) different within a column for different cultivars and p, q, r and s superscripts are significantly (p < 0.05) different within a row for each cultivar. Subscripts denote the percentage decrease (") from control samples. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

those of commonly used food antioxidants (Liu et al., 2010). The overall antioxidant properties of the food products may remain the same or even be enhanced by the development of Maillard reaction products, even though the concentration of natural antioxidants like phenolic compounds were significantly reduced as a consequence of the thermal treatments (Nicoli et al., 1997). When extrusion was carried out at 180 °C and the feed moisture was 15% the metal chelating activity varied insignificantly among the cultivars. On the other hand, the metal chelating activity varied significantly among the cultivars, when the extrusion was carried out at 180 °C and the feed moisture was 20%. The highest and the lowest metal chelating activity was exhibited by PL-426 (81.6%) and RD-2552 (69.4%) cultivars. Moreover, the metal chelating activity of the extrudates LTLM did not vary significantly among the cultivars except for RD-2503 that exhibited the highest metal chelating activity (67.2%) among all cultivars. Interestingly, when the temperature was kept constant (180 °C) and the feed moisture was increased from 15% to 20%, a significant increase in the metal chelating activity was observed. RD-2508 showed the highest increase (23%) in metal chelating activity. Furthermore, when the temperature was kept constant at 150 °C and the feed moisture was increased from 15% to 20% the metal chelating activity was increased significantly in all the cultivar. The increase in the metal chelating activity ranged from 5% to 17% with the highest being for RD-2508. On the other hand, when the moisture of the feed was kept constant (20%) and the temperature was increased from 150 to 180 °C, a significant increase in the metal chelating activity was observed. PL-426 showed the highest (18.7%) and DWR-28 showed the lowest (0.4%) increase in metal chelating activity. However, increasing the temperature from 150 to 180 °C and keeping the moisture content of feed constant (15%) an insignificant increase in metal chelating activity was observed, except for RD-2052 that showed the highest increase (9%). These results are in line and in the same order of magnitude

to those reported previously by other researchers (Dewanto et al., 2002; Nicoli et al., 1997). Similar results were also reported for sweet potato upon extrusion and for soybean upon cooking (Huang, Chang, & Shao 2006; Xu & Chang, 2008). Increase in metal chelating activity upon increase in the temperature of extrusion and moisture content of the feed, may be explained by the formations of different concentration of Maillard products. Furthermore, the Maillard products which are produced are high molecular weight (HMW) melanoidins and low molecular weight (LMW) heterocyclic compounds, which are thought to be mainly responsible for the antioxidant capacity and metal chelating activity (DelgadoAndrade & Morales, 2005). The soluble part of these compounds is known to have metal chelating activity (Rufian-Henares & Delgado-Andrade, 2009) and the formation of these compounds depends upon different factors, such as chemical composition of raw material (e.g., proteins, amino acids, reducing sugars, or carbohydrates and pH) process conditions (time, barrel temperature and screw speed) (Wang et al., 2010) and water activity (moisture content of feed). 3.4. Effect of extrusion cooking on reducing power The reducing power is also an indicator of antioxidant activity. The electron donor compounds are considered as a reducing agent and can reduce the oxidised intermediates of the lipid peroxidation reactions therefore there may be primary or secondary antioxidants (Sharma & Gujral, 2011). The reducing power of an antioxidant compound is associated with the presence of reductones. Further, the antioxidant capacity of reductones is based on the breaking of the free radical chain reaction by donating a hydrogen atom, and to prevent peroxide formation. The extrusion cooking showed a significant decrease in reducing power (Table 4). A similar decrease in reducing power has been reported by other authors upon thermal processing in different cereals (Xu & Chang, 2008).

Table 4 Reducing power of barley extrudates. Cultivars

DWR-28 RD-2503 RD-2508 RD-2035 RD-2052 RD-2552 PL-172 PL-426

Reducing power (lmol AAE/g) Unextruded (Control)

HTLM (180 °C, 15%)

HTHM (180 °C, 20%)

LTLM (150 °C, 15%)

LTHM (150 °C, 20%)

47.0as 53.1bs 61.2cr 56.4br 59.7cs 60.3cr 62.2cq 55.3bs

22.1ar (;53) 22.5ar (;58) 23.8bq (;61) 23.0aq (;59) 22.8ar (;62) 23.6bq (;61) 23.9bp (;60) 24.6cr (;56)

19.7bq (;58) 20.4bp (;62) 20.9bp (;66) 20.0bp (;64) 17.9ap (;70) 20.7bp (;66) 21.6cp (;64) 22.6dp (;59)

20.3ap (;57) 21.4bq (;60) 21.4bp (;65) 21.1bp (;63) 21.4bq (;64) 22.9cq (;62) 22.7cp (;62) 23.3dq (;58)

19.9ap (;58) 20.5ap (;61) 20.9ap (;66) 20.1ap (;64) 20.5aq (;66) 22.2bq (;63) 22.1bp (;63) 22.7bp (;59)

a, b, c and d superscripts are significantly (p < 0.05) different within a column for different cultivars and p, q, r and s superscripts are significantly (p < 0.05) different within a row for each cultivar. Subscripts denote the percentage decrease (;) from control samples. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

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3.5. Effect of extrusion cooking on total flavonoid content (TFC)

The reducing power of barley is mainly due to the phenolic compounds and flavonoids as the phenolic compounds and flavonoids have the ability to donate electrons and act as reductones (Omwamba & Hu, 2010) and play a major role in the reducing power of the extracts. Therefore, the decrease in total phenolic content and flavonoids may be a reason for the decrease in reducing power after extrusion cooking as compared to their corresponding control. The reducing power of HTLM extrudates varied significantly among the cultivars and ranged from 22.1 to 24.6 lmol ascorbic acid equivalents (AAE)/g. Similarly, the reducing power varied significantly among the cultivars in the HTHM extrudates and ranged from 17.9 to 22.6 lmol AAE/g. Furthermore, the reducing power of the LTLM extrudates varied significantly among cultivars and ranged from 20.3 to 23.3 lmol AAE/g. The highest and the lowest showed by PL-426 and DWR-28, respectively. Similarly, the reducing power of LTHM extrudates varied significantly among cultivars and ranged from 19.9 to 22.7 lmol AAE/g; PL-426 exhibited the highest, while DWR-28 showed the lowest reducing power. When the moisture of the feed increased from 15% to 20% keeping the temperature constant (180 °C), a significant decrease in the reducing power was observed that ranged from 8% to 22%. This decrease may be attributed to the greater decrease in phenolic compounds in the HTHM extrudates. An insignificant decrease in the reducing power was observed when the temperature of the extrusion was kept constant at 150 °C and the moisture content of feed increased from 15% to 20%. On the other hand, increasing the temperature of the extrusion cooking from 150 to 180 °C and keeping the feed moisture constant at 15%, increased the reducing power significantly. Similarly, the extrudates extruded at higher temperature (180 °C, 15% feed moisture) had a higher retention of phenolic compounds as compared to the extrudates extruded at 150 °C (15% feed moisture). It is widely accepted that the Maillard reaction products influence the antioxidant activity of foods but only the soluble fraction of the Maillard reaction products contribute to the reducing power (Rufian-Henares & Delgado-Andrade, 2009). Hence, it may be possible that the amount of soluble fraction of Maillard reaction products was also affected upon extrusion at different temperature and moisture combination. The reducing power showed positive correlations with antioxidant activity (HTLM, R = 0.75, p < 0.05; LTHM, R = 0.70, p > 0.05; LTHM, R = 0.80, p < 0.05) and the total flavonoids content (HTLM, R = 0.61, p > 0.05; LTHM, R = 0.79, p < 0.05; LTHM, R = 0.67, p > 0.05).

Flavonoids have generated interest because of their broad human health promoting effects, most of which are related to their antioxidant properties and to synergistic effects with other antioxidants. Another antioxidant mechanism of flavonoids may result from the interactions between flavonoids and metal ions especially iron and copper (Sharma & Gujral, 2011). A significant decrease in the total flavonoid content (TFC) was observed upon extrusion cooking (Fig. 1). The decrease in TFC may be attributed to the thermal destruction of flavonoids, as the flavonoids are reported to be heat sensitive (Sharma & Gujral, 2011; Xu & Chang, 2008), therefore the TFC decreased upon extrusion cooking. These results are in good agreement with those previously reported by Im, Huff, and Hsieh (2003) and Huang et al. (2006) for buckwheat and sweet potato, respectively, upon thermal processing. TFC varied significantly among cultivars in the HTLM extrudates and ranged from 857 to 1035 lg catechin equivalent (CE)/g. Similarly, TFC significantly varied among the cultivars in the HTHM extrudates and ranged from 800 to 991 lg CE equivalent/g. However, when the moisture of the feed increased from 15% to 20%, keeping the temperature constant at 180 °C a insignificant decrease was observed. When the moisture of the feed increased from 15% to 20%, keeping the temperature constant (150 °C), a significant decrease in TFC was observed that ranged from 13% to 27%. The increased moisture content of feed adversely affected the TFC at both temperatures (150 and 180 °C); this may be attributed to the higher moisture content of feed, as the moist heat is more destructive and produced the synergistic effect along with high temperature. The TFC significantly varied among cultivars in LTHM extrudates and ranged from 766 to 980 lg catechin equivalent (CE)/g. Furthermore, TFC varied significantly among cultivars in LTLM extrudates and ranged from 953 to 1329 lg catechin equivalent (CE)/g. However, when the temperature of extrusion increased from 150 to 180 °C keeping the moisture content constant at 15%, a significant decrease in TFC was observed and ranged from 6% to 24%. Furthermore, when the temperature of extrusion cooking increased from 150 to 180 °C keeping the moisture of feed constant at 20%, an insignificant increase in TFC was observed. The variation in temperature showed an unclear effect on the TFC. Although, the flavonoids showed a significant positive correlation with TPC in

Unextruded (Control)

HTLM

HTHM

LTLM

LTHM

2500 s Total flavonoid content (µg/g)

s t

2000

1500

r

s

s

r r

r 1000

s

t

qq

p

r q

s

qq p

r p

r

qp

qq

s q

r

p

p

qp

q p

pp

p

p

500

0 DWR-28

RD-2503

RD-2508

RD-2035

RD-2052

RD-2552

PL-172

PL-426

Fig. 1. Effect of extrusion cooking on the total flavonoid content (TFC). Superscripts p, q r, s and t show significant (p < 0.05) difference of extrusion cooking on the total flavonoid content within a cultivar. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

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Non enzymatic browning index (OD/0.1g flour)

Unextruded (Control)

HTLM

HTHM

LTLM

LTHM

0.350 0.300

t s

0.250 s 0.200

s r

0.150 0.100

s

s

q

q

p

r

q

r

r

qr q

p

p

RD-2503

RD-2508

t s

r qqq

qq

p

p

RD-2035

RD-2052

s r

qq

q

q

p

r r

p

p

0.050 0.000 DWR-28

RD-2552

PL-172

PL-426

Fig. 2. Effect of extrusion cooking on the non-enzymatic browning (NEB) index. Superscripts p, q r, s and t show significant (p < 0.05) difference of extrusion cooking on the NEB index within a cultivar. HTLM (high temperature low moisture; 180 °C, 15%), HTHM (high temperature high moisture; 180 °C, 20%), LTLM (low temperature low moisture; 150 °C, 15%) LTHM (low temperature high moisture; 150 °C, 20%).

the control samples (R = 0.71, p < 0.05), upon extrusion the correlation decreased in all types of extrudates (HTHM, R = 0.49, HTLM, R = 0.17, LTHM, R = 0.44, LTLM, R = 0.61, p > 0.05). A similar lack of correlation between TPC and TFC has been reported by Xu and Chang (2008) and Shih et al. (2009) upon thermal processing of soybeans and sweet potato, respectively. This lack of correlation may be due to the difference in nature of flavonoids and phenolic compounds and their sensitivity to thermal processing (Zielinska, Szawara-Nowak, & Zielinski, 2007). 3.6. Effect of extrusion cooking on non-enzymatic browning (NEB) index Extrusion cooking increased the non-enzymatic browning index (NEB) significantly in all cultivars tested (Fig. 2). Maillard products are produced during extrusion cooking that lead to higher NEB index as compared to their corresponding control samples (Sharma & Gujral, 2011). The NEB index varied significantly among the cultivars in HTHM extrudates and ranged from 0.148 to 0.201/0.1 g. In the HTLM extrudates the NEB index varied significantly among cultivars and ranged from 0.161 to 0.276/ 0.1 g. On the other hand, the NEB index for LTHM and LTLM extrudates ranged from 0.146 to 0.171 and 0.158 to 0.181/0.1 g. The highest and the lowest NEB index being for PL-426 and DWR-28 cultivars in the LTHM extrudates, while it was the highest and the lowest for PL-172 and RD-2508 cultivars, respectively, in the LTLM extrudates. When the moisture of the feed increased from 15% to 20%, keeping the temperature constant at 180 °C, a significant decrease in the NEB index was observed and it ranged from 3% to 29%. Similarly, when the moisture content of the feed increased from 15% to 20%, keeping the temperature of extrusion constant at 150 °C, a significant decrease in the NEB index was observed. This decrease ranged from 1% to 17%. Increasing the temperature from 150 to 180 °C and keeping the moisture content of feed constant at 20% increased the NEB index significantly by 0.9% to 22.5%. Similarly, when the temperature of extrusion increased from 150 to 180 °C, keeping the moisture of feed constant at 15%, the NEB index increased significantly and ranged from 1% to 52%. Similar results have been reported by Im et al. (2003) upon thermal processing of buckwheat. Sharma & Gujral (2011) have reported a significant increase in the NEB index for sand and microwave roasted barley. The different moisture of feed and temperature of extrusion may be the reason for variation in the NEB index of different extrudates. NEB exhibited a moderate positive correlation with AOA (HTHM, R = 0.49,

p > 0.05; HTLM, R = 0.83, p < 0.05; LTHM, R = 0.40, p > 0.05 and LTLM, R = 0.55, p > 0.05).

4. Conclusions Extrusion cooking exhibited a significant effect on the antioxidant properties of barley extrudates. Total phenolic content (TPC), total flavonoid content (TFC) and reducing power decreased upon extrusion while the NEB index, metal chelating activity and DPPH radical scavenging activity increased significantly. The feed moisture and extrusion temperature significantly affected the antioxidant properties of barley. Among the extrudates, the highest total phenolic content (TPC) and antioxidant activity was exhibited by extrudates prepared by the LTHM process. The extrudates from PL-426, PL-172 and DWR-28 cultivars exhibited the highest antioxidant properties. Acknowledgments Authors are highly thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing the Senior Research Fellowship (SRF) to Mr. Paras Sharma. References Altan, A., McCarthy, K. L., & Maskan, M. (2009). Effect of extrusion process on antioxidant activity, total phenolics and b-glucan content of extrudates developed from barley–fruit and vegetable by-products. International Journal of Food Science and Technology, 44, 1263–1271. Amarowicz, R., Naczk, M., & Shahidi, F. (2000). Antioxidant activity of crude tannins of canola and rapeseed hulls. Journal of American Oil Chemists Society, 77, 957–961. Anton, A. A., Fulcher, R. G., & Arntfield, S. D. (2009). Physical and nutritional impact of fortification of corn starch-based extruded snacks with common bean (Phaseolus vulgaris L.) flour: Effects of bean addition and extrusion. Food Chemistry, 113, 989–996. Arts, M. J. T. J., Haenen, G. R. M. M., & Wilms, L. C. (2002). Interactions between flavanoids and proteins: Effect on the total antioxidant capacity. Journal of Agricultural and Food Chemistry, 50, 1184–1187. Awika, J. M., Rooney, L. W., Wu, X., Prior, R. L., & Cisneros-Zevallos, L. (2003). Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. Journal of Agricultural and Food Chemistry, 51, 6657–6662. Bonoli, M., Verardo, V., Marconi, E., & Caboni, M. F. (2004). Antioxidant phenols in barley (Hordeum vulgare L.) flour: Comparative spectrophotometric study among extraction methods of free and bound phenolic compounds. Journal of Agricultural and Food Chemistry, 52, 5195–5200. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technology, 28, 245–251.

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