Food Chemistry 131 (2012) 1406–1413

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Food Chemistry

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Antioxidant activity of barley as affected by extrusion cooking

Paras Sharma a, Hardeep Singh Gujral a,⇑, Baljeet Singh b

a Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Indiab 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 2011Received in revised form 17 July 2011Accepted 5 October 2011Available online 10 October 2011

Keywords:BarleyExtrusionTotal flavonoids contentAntioxidant activityMetal chelating activityReducing power

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.10.009

⇑ Corresponding author. Tel.: +91 183 2258802.E-mail address: hsgujral7@yahoo.co.in (H.S. Gujral

a b s t r a c t

Grit from different hulled barley cultivars was subjected to extrusion cooking and the effect of extrusionmoisture and temperature on the antioxidant properties was studied. A significant decrease in the totalphenolic content (TPC) and total flavonoid content (TFC) was observed upon extrusion and a furtherdecrease of 8–29% in TPC and 13–27% in TFC was observed when both the feed moisture and extrusiontemperature were increased. The antioxidant activity (AOA) increased significantly upon extrusion andthis increase was the highest (36–69%) at 150 �C and 20% feed moisture. The increase in feed moistureand temperature significantly increased the metal chelating activity. The reducing power decreased sig-nificantly upon extrusion as compared to their corresponding control samples. Extrusion lead to a greaterincrease in non-enzymatic browning (NEB) index however, increasing the moisture content of feeddecreased the NEB index by 3–29% (at 180 �C) and 1–17% (150 �C), while increasing the temperatureincreased the NEB significantly.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Processed foods these days require the presence of bioactiveingredients to satisfy the demands of health conscious consumers.Barley (Hordeum vulgare L.) is considered as a nutraceutical grainbecause it contains bioactive compounds like b-glucan, phenoliccompounds, B-complex vitamins, tocotrienols, tocopherols(Madhujith, Izydorczyk, & Shahidi, 2006; Sharma & Gujral, 2010a,2010b). Among the cereal grains barley has higher antioxidantactivity as compared to the more widely consumed cereals wheatand rice. The risk imposed by the consumption of free radicals andoxidation products towards various forms of cancer and cardiovas-cular disease could be lowered by the intake of dietary phenolics.Barley contains many phenolic compounds in the free and boundform; these compounds include benzoic and cinnamic acid deriva-tives, 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 temper-ature and pressure and short time and is used to prepare a varietyof processed foods like baby foods, snack foods, ready-to-eatbreakfast cereals and pet foods. The consumer preference ofextruded foods is mainly due to convenience, attractive appear-ance and texture and utilising barley in extruded foods wouldincrease consumer acceptance as it contains bioactive functional

ll rights reserved.

).

components. The effects of extrusion cooking on the polyphenolcontent and antioxidant activity in rye bran has been reported byGumul 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) stud-ied the effects of extrusion on the phenolic composition and anti-oxidant activity of kidney beans. A maize bran/oat flour extrudedbreakfast cereal was developed by Holguin-Acuna et al. (2008) asa novel source of antioxidant and complex polysaccharides. Anincrease in the total phenols and DPPH radical scavenging activityin corn starch/common bean extrudates was reported by Anton,Fulcher, and Arntfield (2009). Shih, Kuo, and Chiang (2009) inves-tigated the effects of drying and extrusion on the antioxidant activ-ity of sweet potato and reported that the scavenging effect onDPPH radicals and total phenolic compounds increased.

It is well documented that the minimal processed foods havemore health benefits (Gujral, Sharma & Singh, 2011; Shahidi,2009; Sharma, Gujral, & Rosell, 2011). Even though extrusion is ashort time cooking process the temperatures encountered by theraw material in the barrel of the extruder is enough to bring aboutchanges in the major and non-nutritive components. The most sig-nificant changes are brought about in the cereal starch and proteinthat contribute to form structure, texture, mouth feel and bulkdensity. The non-nutritive components mainly polyphenols havingantioxidant properties may undergo various changes, thus alteringtheir antioxidant activity. The objective of the present investiga-tion was to study the effect of extrusion moisture and temperatureon antioxidant activity of extrudates from different barleycultivars.

P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413 1407

2. Materials and methods

2.1. Barley samples

Eight common hulled barley cultivars (PL-172, PL-426, RD-2503, RD-2508, RD-2035, RD-2052, RD-2552 (six rowed) andDWR-28 (two rowed)) grown in different locations in the statesof Punjab, Haryana, Uttar Pradesh and Rajasthan were collectedfrom Central State Seed farm, Sriganganagar, Rajasthan, India.The grain was cleaned and stored for further evaluation. The barleycultivars varied significantly among themselves with the husk con-tent ranging from 9.6% to 13.0% and bulk density ranging from0.547 to 0.653 g/ml. The protein content and ash content rangedfrom 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% (Sharmaet al., 2011a) while the amylose content ranged from 21.0% to28.3% (Gujral, Sharma, Kaur & Singh, 2011).

2.2. Reagents

Standard ferulic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH),ferrozine, protease (from Streptomyces griseus) and catechin wereprocured from Sigma–Aldrich (Steinheim, Germany). L-Ascorbicacid, potassium ferricyanide, ferric chloride, ferrous chloride, tri-chloroacetic acid, sodium carbonate and Folin Ciocalteu’s reagentwere procured from Loba Chemie, Mumbai, India. All chemicalswere of analytical grade. Each test was performed in triplicateson dry weight basis. The Milli Q water (Millipore, France) was usedfor all analytical tests.

2.3. Extrusion

Barley samples were dehusked as previously described bySharma and Gujral (2010a). The grits (95% retained by 250 lmsieve) 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 allowedto equilibrate for 12 h. The extrusion was performed on a co-rotating twin screw extruder (Clextral, BC 21, Firminy, France).The screw diameter, (L/D) ratio and die diameter was 25 mm 16and 6 mm, respectively. The feed rate (20 kg/h) and screw speed(400 rpm) were kept constant. The extrusion was carried out at150 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 beltand the feeding section of barrel was cooled with running water.The screw configuration in different sections of the extruder (fromhopper to die) was as follows:

Screw section 1 2 3 4 5 6 7 8 9 10Screw element BAGUE C2F C2F C2F C2F C2F INO 0 C1F CF1C C1FLength (mm) 20 50 50 50 50 50 5 50 25 50Pitch (mm) – 50 33.33 25 25 16.66 – 16.66 12.5 12.5

The extrudates were classified as high temperature high mois-ture (HTHM, 180 �C temperature, and 20% moisture), high temper-ature low moisture (HTLM, 180 �C temperature, 15% moisture), lowtemperature high moisture (LTHM, 150 �C temperature, 20% mois-ture) and low temperature low moisture (LTLM, 150 �C tempera-ture, 15% moisture). The extrudates were cooled to roomtemperature and packed in polyethylene bags. Further, all extru-dates were ground in a grinder (Sujata, India) to particle size<250 lm and stored at �20 �C until further analysis.

2.4. Total phenolic content (TPC)

The total phenolic content (TPC) was determined according theFolin–Ciocalteu specterophotometric method (Sharma & Gujral,2010b). Extruded samples (200 mg) were extracted with 4 ml acid-ified methanol (HCl/methanol/water, 1:80:10, v/v/v) at room tem-perature (25 �C) for 2 h. An aliquot of extract (200 ll) was added to1.5 ml freshly diluted (10-fold) Folin–Ciocalteu reagent. The mix-ture was allowed to equilibrate for 5 min and then mixed with1.5 ml of sodium carbonate solution (60 g/l). After incubation atroom temperature (25 �C) for 90 min, the absorbance of the mix-ture was read at 725 nm (Shimadzu, UV-1800, Japan). Acidifiedmethanol was used as a blank. The results were expressed as lgof ferulic acid equivalents (FAE) per gram of sample.

2.5. Antioxidant activity (DPPH radical scavenging activity)

Antioxidant activity (AOA) was measured using a modified ver-sion of the method described by Brand-Williams, Cuvelier, andBerset (1995). Extruded samples (100 mg) were extracted with1 ml methanol for 2 h and centrifuged at 3000g for 10 min. Thesupernatant (100 ll) was reacted with 3.9 ml of a 6 � 10�5 mol/Lof DPPH solution. The absorbance (A) at 515 nm was read at 0and 30 min using a methanol blank. The antioxidant activity wascalculated as % discoloration:

DPPH radical scavenging activityð%Þ¼ ð1� ðA of samplet ¼ 30=A of controlt ¼ 0ÞÞ � 100

2.6. Reducing power

The reducing power was measured as described by Sharma andGujral (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 potas-sium ferricyanide were added followed by incubation at 50 �C.Trichloroacetic acid solution (10%) was added to mixture, whichwas then centrifuged at 10000g for 10 min. The upper layer of solu-tion (2.5 ml) was mixed with 2.5 ml deionized water and 0.5 mlferric chloride. The absorbance of the mixture was measured at700 nm. A standard curve was prepared using various concentra-tion of ascorbic acid and results were reported as ascorbic acidequivalents/g (AAE) of flour.

2.7. Total flavonoids content (TFC)

The total flavonoids content (TFC) was determined as previ-ously described by Jia, Tang, and Wu (1998). The extract

(250 ll) was diluted with 1.25 ml distilled water. Sodium nitrite(75 ll) was added and the mixture was allowed to stand for6 min. Further, 150 ll of aluminium chloride were added andthe mixture was allowed to stand for 5 min. After that, 0.5 ml ofsodium hydroxide (1 M) was added and the solution was mixedwell. The absorbance was measured immediately at 510 nm usinga spectrophotometer. Catechin was used as standard and the re-sults were reported as microgram catechin equivalent (CE)/g ofsample.

1408 P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413

2.8. Metal chelating (Fe+2) activity

The metal chelating activity of 80% methanol extract was mea-sured as reported by Sharma and Gujral (2011). The extract(0.5 ml) was mixed with 50 ll of ferrous chloride and 1.6 ml of80% methanol was added. After 5 min, the reaction was initiatedby the addition of 5 mM/l ferrozine and the mixture was shakenon a vortex. The mixture was incubated at room temperature(25 �C) for 10 min. The absorbance of solution was measured at562 nm on a spectrophotometer. The chelating activity of theextract for Fe+2 was calculated as follows:

Iron ðFeþ2Þ chelating activity ð%Þ¼ f1� ðAbsorbance of sample at 562nm=

Absorbance of control at 562nmÞg � 100

2.9. Nonenzymatic browning (NEB) index

Nonenzymatic browning index (NEB) of extrudates was carriedout as previously reported by Sharma and Gujral (2011). The extru-date sample (100 mg) was mixed with 1 ml of deionized water. Analiquot (200 ll) of the mixture was transferred to a test tube whichcontained 200 ll of protease solution (in Tris buffer). The test tubeswere incubated for 2 h at 45 �C, in a water bath (NSW-125, NarangScientific Works, New Delhi, India). The test tubes were cooled and300 ll trichloroacetic acid was added to each tube. Then, the tubeswere centrifuged at 7000g for 20 min. The absorbance of superna-tant was measured on a spectrophotometer. The browning index(DA) was calculated as follows:

DA ¼ Absorbance at 420nm� Absorbance at 550nm

2.10. Statistical analysis

Analysis of variance (ANOVA) was carried out using MicrosoftExcel software and Fishers least significant difference (LSD) testwas used to describe means with 95% (p < 0.05) confidence. ThePearson correlation coefficients were calculated by SPSS statisticalsoftware (SPSS Inc., Chicago, Illinois, USA) at a probability level ofp < 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 cultivarsranged from 3070 to 4439 lg FAE/g (Sharma & Gujral, 2010b).Madhujith and Shahidi (2009) reported TPC value ranging from2.63 to 4.51 mg of ferulic acid equivalents (FAE)/g in barley. Bonoli,

Table 1Total phenolic content of barley extrudates.

Cultivars Total phenolic content (lg FAE/g)

Unextruded HTLM (180 �C, 15%) HTHM

DWR-28 3070as 2154cr(;30) 1897

RD-2503 3121as 1782ar(;43) 1515

RD-2508 3417bt 1721ar(;50) 1492

RD-2035 3485bt 1733ar(;50) 1449

RD-2052 3588ct 1867br(;48) 1533

RD-2552 3441br 1928bq(;44) 1374

PL-172 4439es 1949bq(;56) 1777

PL-426 4180dt 1987br(;52) 1823

a, b, c, d and e superscripts are significantly (p < 0.05) different within a column for difwithin a row for each cultivar. Subscripts denote the percentage decrease (;) from cotemperature high moisture; 180 �C, 20%), LTLM (low temperature low moisture; 150 �C

Verardo, Marconi, and Caboni (2004) reported a total phenolic con-tent 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 todifferences in the solvent used in the extraction and the differencesin the barley cultivars. The total phenolic content in all the culti-vars decreased significantly upon extrusion as compared to theircorresponding control (unextruded) samples (Table 1). Theseresults are also consistent with previous study carried out byDelgado-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 maydestroy or alter their nature (Zielinski, Kozlowska, & Lewczuk,2001). The reduction in TPC may be attributed either to the decom-position of phenolic compounds due to the high extrusion temper-ature or alteration in molecular structure of phenolic compoundsthat may lead to reduction in the chemical reactivity of phenoliccompounds or decrease their extractability due to certain degreeof polymerisation (Altan, McCarthy, & Maskan, 2009). It has alsobeen reported that the phenolic and flavonoids compounds mayinteracted with the proteins and may not exhibit their actual value(Arts, Haenen, & Wilms, 2002). The total phenolic content in HTHMextrudates, varied significantly (p < 0.05) among cultivars and ran-ged from 1374 to 1897 lg ferulic acid equivalents (FAE)/g withhighest and lowest being for DWR-28 and RD-2552. Similarly,the TPC in HTLM extrudates, varied significantly (p < 0.05) amongcultivars and ranged from 1721 to 2154 lg FAE/g. The highestand the lowest observed for DWR-28 and RD-2508. Stojceska,Ainsworth, Plunkett, and Ibanoglu (2009) reported total phenoliccontent ranging from 1.4 to 2.1 mM gallic acid equivalent/g in dif-ferent extrudates prepared from barley–wheat flour and cornstarch–barley flour blends. When the temperature was kept con-stant (180 �C) and the moisture of feed was increased from 15%to 20%, a significant (p < 0.05) decrease was observed in TPC forall cultivars and this decrease ranged from 8% to 29%, the highestand the lowest decrease was observed for RD-2552 and PL-426,respectively. This may be attributed to the higher moisture contentof feed, as the moist heat is more destructive and produced a syn-ergistic effect along with higher temperature. On the other hand,when the extrusion temperature was kept constant at 150 �C andthe moisture content of feed increased from 15% to 20%, a signifi-cant (p < 0.05) increase was observed in TPC. In LTHM extrudatesTPC varied significantly (p < 0.05) among cultivars and ranged from1815 to 2195 lg FAE/g, the highest and the lowest being for RD-2052 and RD-2508, respectively. This combination of extrusionvariables had the highest TPC among all the extrudates. The TPCin the LTLM extrudates varied significantly and ranged from1544 to 1995 lg FAE/g. The highest and the lowest were observedfor DWR-28 and RD-2035, respectively. When the temperaturewas increased from 150 to 180 �C, keeping the moisture constant(15%) a significant (p < 0.05) increase was noticed for all the

(180 �C, 20%) LTLM (150 �C, 15%) LTHM (150 �C, 20%)

dp(;38) 1995dq

(;35) 2167dr(;29)

bp(;51) 1705bq

(;45) 1815ar(;42)

bp(;56) 1690bq

(;51) 1933bs(;43)

bp(;58) 1544aq

(;56) 1915bs(;45)

bp(;57) 1733bq

(;52) 2195ds(;39)

ap(;60) 1923cq

(;44) 1959bq(;43)

cp(;60) 1938cq

(;56) 2069cr(;53)

cp(;56) 1895cq

(;55) 2164ds(;48)

ferent cultivars and p, q, r, s and t superscripts are significantly (p < 0.05) differentntrol samples. HTLM (high temperature low moisture; 180 �C, 15%), HTHM (high, 15%) LTHM (low temperature high moisture; 150 �C, 20%).

P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413 1409

cultivars with RD-2035 showing the highest increase (12%). How-ever, when the temperature was increased from 150 to 180 �C,keeping the moisture constant at 20%, the TPC decreased signifi-cantly (p < 0.05) by 12–30% with the highest and lowest being forRD-2052 and DWR-28, respectively. These results are also in agree-ment with those reported by Altan et al. (2009).

3.2. Effect of extrusion cooking on antioxidant activity (DPPH freeradical scavenging activity)

Estimation of the antioxidant activity (AOA) by scavenging ofstable radicals, such as the chromogen radical DPPH in inorganicmedia has been extensively used for comparison of homogeneousseries of antioxidants. This procedure measures the hydrogendonating capacity of the target substances in a methanolic media.The colour changes from purple to yellow by acceptance of ahydrogen radical from MRP and it becomes a stable diamagneticmolecule. Extrusion cooking led to a significant increase in DPPHfree radical scavenging activity (antioxidant activity, AOA) in allthe cultivars as compared to their corresponding control samples(Table 2). The earlier investigations have reported that dark colourpigments (brown colour) are produced during the thermal process-ing of foods (Sharma & Gujral, 2011; Xu & Chang, 2008) due to theMaillard browning. These pigments (particularly melanoidins) areextensively known to have antioxidant activity (Manzocco,Calligaris, Masrrocola, Nicoli, & Lerici, 2001). The increase in anti-oxidant activity could be explained by the formation of Maillardbrowning pigments which enhanced the antioxidant activity(Rufian-Henares & Delgado-Andrade, 2009) of extrudates ascompared to their corresponding control samples. The thermalprocessing is also known to alter the antioxidant profile andgenerate more antioxidants that contribute in antioxidant activity.Increase in antioxidant activity due to thermal processing has beenwidely reported (Dewanto, Wu, Adom, & Liu, 2002; Nicoli, Anese,Parpinel, Franceschi, & Lerici, 1997) Antioxidant activity varied sig-nificantly among cultivars when the extrusion was carried out at180 �C and 15% moisture (HTLM). The AOA ranged from 19.2% to26.2% with highest and lowest being for PL-172 and RD-2503,respectively. Similarly, when the extrusion was carried at 180 �Cand 20% moisture (HTHM), the AOA varied significantly (p < 0.05)among cultivars and ranged from 21.0% to 27.8%. The PL-172showed the highest and DWR-28 showed the lowest AOA. TheAOA 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-Liconet al. (2009) reported that the highest antioxidant activity waswhen they extruded the bean/corn mixture at 142 �C temperatureand 16.5% feed moisture. Similarly, Shih et al. (2009) reported thatthe extrusion process significantly increased the DPPH radicalscavenging activity in the different sweet potato extrudates.

Table 2DPPH radical scavenging activity of barley extrudates.

Cultivars Antioxidant activity (DPPH radical scavenging activity) (%)

Unextruded (Control) HTLM (180 �C, 15%) H

DWR-28 19.8bp 20.2bp("2) 2

RD-2503 17.4ap 19.2aq("10) 2

RD-2508 18.9bp 21.4bq("13) 2

RD-2035 17.0ap 21.0bq("23) 2

RD-2052 21.2bp 22.3bp("5) 2

RD-2552 19.1bp 21.9bq("15) 2

PL-172 24.9cp 26.2dp("5) 2

PL-426 21.7bp 23.8cq("10) 2

a, b, c, d and e superscripts are significantly (p < 0.05) different within a column for difwithin a row for each cultivar. Subscripts denote the percentage decrease (") from cotemperature high moisture; 180 �C, 20%), LTLM (low temperature low moisture; 150 �C

Awika, Rooney, Wu, Prior, and Cisneros-Zevallos (2003) reportedthe highest antioxidant activity in the extruded sorghum as com-pared to baked and corresponding control sorghum samples. Fur-thermore, 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-2508and PL-426 by 12%, 21% and 8%, respectively while the rest of thecultivars did not show significant (p < 0.05) increase. Keeping thetemperature constant at 150 �C and increasing moisture from15% to 20%; AOA showed a significant increase that ranged from9% to 24%. The highest and the lowest increase were exhibited byRD-2052 and RD-2552, respectively. Interestingly, when the mois-ture was kept constant (20%) and temperature increased from 150to 180 �C, a significant decrease in AOA was observed and this ran-ged from 18% to 33%. Similarly, increasing the temperature ofextrusion from 150 to 180 �C and keeping the moisture constantat (15%), the AOA decreased significantly by 11% to 25%. A changein the extrusion temperature and moisture could have lead to theformation of different amounts of Maillard browning product(Manzocco et al., 2001). Maillard browning is influenced by manyfactors, including temperature, reactant concentration, reactiontime and water activity (Stojceska et al., 2009). A significant(p < 0.05) positive correlation was observed (R = 0.82) betweenthe AOA and TPC in control samples. The extrudates (LTHM andLTLM) also exhibited a positive correlation between the AOA andTPC (R = 0.65, p > 0.05) and (R = 0.78, p < 0.05), respectively. Onthe other hand, no correlation between TPC and AOA was observed(R = 0.22 and 0.28, p > 0.05) in extrudates, HTHM and HTLM. Theantioxidant activity is not only affected by quantity but also thekind 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 evaluateantioxidant activity of foods and to explain how antioxidants func-tion. Of these, metal chelating activity, reducing power, DPPH as-say and total antioxidant activity are most commonly used forthe evaluation of antioxidant activities (Amarowicz, Naczk, &Shahidi, 2000; Sharma & Gujral, 2011). The interaction of Fe+2 withferrozine produces a dark colour complex that is decreased by theaction of metal chelator compounds present in the reactionmixture.

The metal chelating activity was increased significantly uponextrusion cooking in all the cultivars tested as compared to theircorresponding control samples (Table 3). The increase in the metalchelating activity may be as a consequence of the formation of no-vel compounds such as melanoidins during thermal processing(Sharma & Gujral, 2011; Shih et al., 2009). Maillard reaction prod-ucts are found to have strong antioxidant properties comparable to

THM (180 �C, 20%) LTLM (150 �C, 15%) LTHM (150 �C, 20%)

1.0ap("6) 26.9aq

("36) 31.3ar("58)

1.4ar("23) 24.9as

("43) 29.1at("67)

5.9dr("37) 27.0as

("43) 31.9at("69)

1.3aq("25) 25.1ar

("47) 30.1as("77)

3.2cp("10) 25.6aq

("21) 31.6ar("49)

2.6bq("19) 28.4ar

("48) 30.8as("61)

7.8ep("38) 29.5ap

("18) 34.0bq("36)

5.6dr("38) 29.7as

("37) 34.3bt("58)

ferent cultivars and p, q, r, s and t superscripts are significantly (p < 0.05) differentntrol sample. HTLM (high temperature low moisture; 180 �C, 15%), HTHM (high

, 15%) LTHM (low temperature high moisture; 150 �C, 20%).

Table 3Metal chelating activity of barley extrudates.

Cultivars Metal chelating activity (%)

Unextruded (Control) HTLM (180 �C, 15%) HTHM (180 �C, 20%) LTLM (150 �C, 15%) LTHM (150 �C, 20%)

DWR-28 37.5ap 66.6aq("77) 71.4br

("90) 65.3aq("74) 71.1ar

("89)

RD-2503 57.3cp 68.8aq("20) 79.5dr

("39) 67.2bq("17) 70.6aq

("23)

RD-2508 42.2bp 60.0aq("39) 73.9bs

("75) 58.8aq("42) 69.0ar

("64)

RD-2035 61.5dp 66.3ap("8) 76.0cp

("23) 62.2ap("1) 69.0ap

("12)

RD-2052 37.8ap 65.9ar("74) 72.4bt

("92) 60.7aq("61) 68.1as

("80)

RD-2552 37.9ap 61.2aq("59) 69.4as

("83) 60.4aq("61) 65.5ar

("73)

PL-172 58.2cp 63.8aq("7) 73.7bs

("27) 62.3aq("10) 67.2ar

("15)

PL-426 55.8cp 65.9aq("18) 81.6es

("46) 63.3aq("13) 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 withina row for each cultivar. Subscripts denote the percentage decrease (") from control samples. HTLM (high temperature low moisture; 180 �C, 15%), HTHM (high temperaturehigh moisture; 180 �C, 20%), LTLM (low temperature low moisture; 150 �C, 15%) LTHM (low temperature high moisture; 150 �C, 20%).

1410 P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413

those of commonly used food antioxidants (Liu et al., 2010). Theoverall antioxidant properties of the food products may remainthe same or even be enhanced by the development of Maillardreaction products, even though the concentration of natural anti-oxidants like phenolic compounds were significantly reduced asa consequence of the thermal treatments (Nicoli et al., 1997).When extrusion was carried out at 180 �C and the feed moisturewas 15% the metal chelating activity varied insignificantly amongthe cultivars. On the other hand, the metal chelating activity variedsignificantly among the cultivars, when the extrusion was carriedout at 180 �C and the feed moisture was 20%. The highest andthe lowest metal chelating activity was exhibited by PL-426(81.6%) and RD-2552 (69.4%) cultivars. Moreover, the metal chelat-ing activity of the extrudates LTLM did not vary significantlyamong the cultivars except for RD-2503 that exhibited the highestmetal 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 significantincrease in the metal chelating activity was observed. RD-2508showed the highest increase (23%) in metal chelating activity.Furthermore, when the temperature was kept constant at 150 �Cand the feed moisture was increased from 15% to 20% the metalchelating activity was increased significantly in all the cultivar.The increase in the metal chelating activity ranged from 5% to17% with the highest being for RD-2508. On the other hand, whenthe moisture of the feed was kept constant (20%) and the temper-ature was increased from 150 to 180 �C, a significant increase inthe metal chelating activity was observed. PL-426 showed thehighest (18.7%) and DWR-28 showed the lowest (0.4%) increasein metal chelating activity. However, increasing the temperaturefrom 150 to 180 �C and keeping the moisture content of feed con-stant (15%) an insignificant increase in metal chelating activity wasobserved, except for RD-2052 that showed the highest increase(9%). These results are in line and in the same order of magnitude

Table 4Reducing power of barley extrudates.

Cultivars Reducing power (lmol AAE/g)

Unextruded (Control) HTLM (180 �C, 15%) H

DWR-28 47.0as 22.1ar(;53) 1

RD-2503 53.1bs 22.5ar(;58) 2

RD-2508 61.2cr 23.8bq(;61) 2

RD-2035 56.4br 23.0aq(;59) 2

RD-2052 59.7cs 22.8ar(;62) 1

RD-2552 60.3cr 23.6bq(;61) 2

PL-172 62.2cq 23.9bp(;60) 2

PL-426 55.3bs 24.6cr(;56) 2

a, b, c and d superscripts are significantly (p < 0.05) different within a column for differenrow for each cultivar. Subscripts denote the percentage decrease (;) from control samphigh moisture; 180 �C, 20%), LTLM (low temperature low moisture; 150 �C, 15%) LTHM

to those reported previously by other researchers (Dewanto et al.,2002; Nicoli et al., 1997). Similar results were also reported forsweet potato upon extrusion and for soybean upon cooking(Huang, Chang, & Shao 2006; Xu & Chang, 2008). Increase in metalchelating activity upon increase in the temperature of extrusionand moisture content of the feed, may be explained by the forma-tions of different concentration of Maillard products. Furthermore,the Maillard products which are produced are high molecularweight (HMW) melanoidins and low molecular weight (LMW) het-erocyclic compounds, which are thought to be mainly responsiblefor the antioxidant capacity and metal chelating activity (Delgado-Andrade & Morales, 2005). The soluble part of these compounds isknown to have metal chelating activity (Rufian-Henares & Delgad-o-Andrade, 2009) and the formation of these compounds dependsupon different factors, such as chemical composition of raw mate-rial (e.g., proteins, amino acids, reducing sugars, or carbohydratesand pH) process conditions (time, barrel temperature and screwspeed) (Wang et al., 2010) and water activity (moisture contentof 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 agentand can reduce the oxidised intermediates of the lipid peroxidationreactions therefore there may be primary or secondary antioxi-dants (Sharma & Gujral, 2011). The reducing power of an antioxi-dant compound is associated with the presence of reductones.Further, the antioxidant capacity of reductones is based on thebreaking of the free radical chain reaction by donating a hydrogenatom, and to prevent peroxide formation. The extrusion cookingshowed a significant decrease in reducing power (Table 4). A sim-ilar decrease in reducing power has been reported by other authorsupon thermal processing in different cereals (Xu & Chang, 2008).

THM (180 �C, 20%) LTLM (150 �C, 15%) LTHM (150 �C, 20%)

9.7bq(;58) 20.3ap

(;57) 19.9ap(;58)

0.4bp(;62) 21.4bq

(;60) 20.5ap(;61)

0.9bp(;66) 21.4bp

(;65) 20.9ap(;66)

0.0bp(;64) 21.1bp

(;63) 20.1ap(;64)

7.9ap(;70) 21.4bq

(;64) 20.5aq(;66)

0.7bp(;66) 22.9cq

(;62) 22.2bq(;63)

1.6cp(;64) 22.7cp

(;62) 22.1bp(;63)

2.6dp(;59) 23.3dq

(;58) 22.7bp(;59)

t cultivars and p, q, r and s superscripts are significantly (p < 0.05) different within ales. HTLM (high temperature low moisture; 180 �C, 15%), HTHM (high temperature(low temperature high moisture; 150 �C, 20%).

P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413 1411

The reducing power of barley is mainly due to the phenolic com-pounds and flavonoids as the phenolic compounds and flavonoidshave the ability to donate electrons and act as reductones(Omwamba & Hu, 2010) and play a major role in the reducingpower of the extracts. Therefore, the decrease in total phenoliccontent and flavonoids may be a reason for the decrease inreducing power after extrusion cooking as compared to their corre-sponding control. The reducing power of HTLM extrudates variedsignificantly among the cultivars and ranged from 22.1 to24.6 lmol ascorbic acid equivalents (AAE)/g. Similarly, the reduc-ing power varied significantly among the cultivars in the HTHMextrudates and ranged from 17.9 to 22.6 lmol AAE/g. Furthermore,the reducing power of the LTLM extrudates varied significantlyamong cultivars and ranged from 20.3 to 23.3 lmol AAE/g. Thehighest and the lowest showed by PL-426 and DWR-28, respec-tively. Similarly, the reducing power of LTHM extrudates variedsignificantly among cultivars and ranged from 19.9 to 22.7 lmolAAE/g; PL-426 exhibited the highest, while DWR-28 showed thelowest reducing power. When the moisture of the feed increasedfrom 15% to 20% keeping the temperature constant (180 �C), a sig-nificant decrease in the reducing power was observed that rangedfrom 8% to 22%. This decrease may be attributed to the greater de-crease in phenolic compounds in the HTHM extrudates. An insig-nificant decrease in the reducing power was observed when thetemperature of the extrusion was kept constant at 150 �C and themoisture content of feed increased from 15% to 20%. On the otherhand, increasing the temperature of the extrusion cooking from150 to 180 �C and keeping the feed moisture constant at 15%, in-creased the reducing power significantly. Similarly, the extrudatesextruded at higher temperature (180 �C, 15% feed moisture) had ahigher retention of phenolic compounds as compared to the extru-dates extruded at 150 �C (15% feed moisture). It is widely acceptedthat the Maillard reaction products influence the antioxidant activ-ity of foods but only the soluble fraction of the Maillard reactionproducts contribute to the reducing power (Rufian-Henares &Delgado-Andrade, 2009). Hence, it may be possible that theamount of soluble fraction of Maillard reaction products was alsoaffected upon extrusion at different temperature and moisturecombination. The reducing power showed positive correlationswith 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 flavonoidscontent (HTLM, R = 0.61, p > 0.05; LTHM, R = 0.79, p < 0.05; LTHM,R = 0.67, p > 0.05).

s

ts

t

rqrqq

qqq

srsr

pppp

0

500

1000

1500

2000

2500

DWR-28 RD-2503 RD-2508 RD-2035

Tota

l fla

von

oid

co

nte

nt

(µg

/g)

Unextruded (Control) H

Fig. 1. Effect of extrusion cooking on the total flavonoid content (TFC). Superscripts p,flavonoid content within a cultivar. HTLM (high temperature low moisture; 180 �C, 15%)moisture; 150 �C, 15%) LTHM (low temperature high moisture; 150 �C, 20%).

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

Flavonoids have generated interest because of their broad hu-man health promoting effects, most of which are related to theirantioxidant properties and to synergistic effects with other anti-oxidants. Another antioxidant mechanism of flavonoids may re-sult from the interactions between flavonoids and metal ionsespecially iron and copper (Sharma & Gujral, 2011). A significantdecrease in the total flavonoid content (TFC) was observed uponextrusion cooking (Fig. 1). The decrease in TFC may be attributedto the thermal destruction of flavonoids, as the flavonoids are re-ported 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 re-ported by Im, Huff, and Hsieh (2003) and Huang et al. (2006)for buckwheat and sweet potato, respectively, upon thermalprocessing.

TFC varied significantly among cultivars in the HTLM extru-dates and ranged from 857 to 1035 lg catechin equivalent(CE)/g. Similarly, TFC significantly varied among the cultivars inthe HTHM extrudates and ranged from 800 to 991 lg CE equiva-lent/g. However, when the moisture of the feed increased from15% to 20%, keeping the temperature constant at 180 �C a insig-nificant decrease was observed. When the moisture of the feedincreased from 15% to 20%, keeping the temperature constant(150 �C), a significant decrease in TFC was observed that rangedfrom 13% to 27%. The increased moisture content of feed ad-versely 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 synergis-tic effect along with high temperature. The TFC significantly var-ied among cultivars in LTHM extrudates and ranged from 766 to980 lg catechin equivalent (CE)/g. Furthermore, TFC varied sig-nificantly among cultivars in LTLM extrudates and ranged from953 to 1329 lg catechin equivalent (CE)/g. However, when thetemperature of extrusion increased from 150 to 180 �C keepingthe moisture content constant at 15%, a significant decrease inTFC was observed and ranged from 6% to 24%. Furthermore,when the temperature of extrusion cooking increased from 150to 180 �C keeping the moisture of feed constant at 20%, an insig-nificant increase in TFC was observed. The variation in tempera-ture showed an unclear effect on the TFC. Although, theflavonoids showed a significant positive correlation with TPC in

s

s

s r

pqq

q pp

p

q

qr

rr

pp

pp

RD-2052 RD-2552 PL-172 PL-426

TLM HTHM LTLM LTHM

q r, s and t show significant (p < 0.05) difference of extrusion cooking on the total, HTHM (high temperature high moisture; 180 �C, 20%), LTLM (low temperature low

p p p pp

p

s sr

sr t

ts

r qq

r qs

s rs r

q rr q

qq

q

p p

r q q q q q q

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

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

enz

ymat

ic b

row

ning

inde

x (O

D/0

.1g

flour

)

Unextruded (Control) HTLM HTHM LTLM LTHM

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 theNEB index within a cultivar. HTLM (high temperature low moisture; 180 �C, 15%), HTHM (high temperature high moisture; 180 �C, 20%), LTLM (low temperature lowmoisture; 150 �C, 15%) LTHM (low temperature high moisture; 150 �C, 20%).

1412 P. Sharma et al. / Food Chemistry 131 (2012) 1406–1413

the control samples (R = 0.71, p < 0.05), upon extrusion the corre-lation 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 similarlack of correlation between TPC and TFC has been reported by Xuand Chang (2008) and Shih et al. (2009) upon thermal processingof soybeans and sweet potato, respectively. This lack of correla-tion may be due to the difference in nature of flavonoids andphenolic 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 in-dex (NEB) significantly in all cultivars tested (Fig. 2). Maillardproducts are produced during extrusion cooking that lead tohigher NEB index as compared to their corresponding controlsamples (Sharma & Gujral, 2011). The NEB index varied signifi-cantly among the cultivars in HTHM extrudates and ranged from0.148 to 0.201/0.1 g. In the HTLM extrudates the NEB index var-ied significantly among cultivars and ranged from 0.161 to 0.276/0.1 g. On the other hand, the NEB index for LTHM and LTLMextrudates 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 andDWR-28 cultivars in the LTHM extrudates, while it was the high-est and the lowest for PL-172 and RD-2508 cultivars, respectively,in the LTLM extrudates. When the moisture of the feed increasedfrom 15% to 20%, keeping the temperature constant at 180 �C, asignificant decrease in the NEB index was observed and it rangedfrom 3% to 29%. Similarly, when the moisture content of the feedincreased from 15% to 20%, keeping the temperature of extrusionconstant at 150 �C, a significant decrease in the NEB index wasobserved. This decrease ranged from 1% to 17%. Increasing thetemperature from 150 to 180 �C and keeping the moisture con-tent of feed constant at 20% increased the NEB index significantlyby 0.9% to 22.5%. Similarly, when the temperature of extrusionincreased from 150 to 180 �C, keeping the moisture of feed con-stant at 15%, the NEB index increased significantly and rangedfrom 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 forsand and microwave roasted barley. The different moisture offeed and temperature of extrusion may be the reason for varia-tion in the NEB index of different extrudates. NEB exhibited amoderate 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 andLTLM, R = 0.55, p > 0.05).

4. Conclusions

Extrusion cooking exhibited a significant effect on the antioxi-dant properties of barley extrudates. Total phenolic content(TPC), total flavonoid content (TFC) and reducing power decreasedupon extrusion while the NEB index, metal chelating activity andDPPH radical scavenging activity increased significantly. The feedmoisture and extrusion temperature significantly affected the anti-oxidant properties of barley. Among the extrudates, the highest to-tal phenolic content (TPC) and antioxidant activity was exhibitedby extrudates prepared by the LTHM process. The extrudates fromPL-426, PL-172 and DWR-28 cultivars exhibited the highest antiox-idant properties.

Acknowledgments

Authors are highly thankful to the Council of Scientific andIndustrial Research (CSIR), New Delhi, India, for providing the Se-nior Research Fellowship (SRF) to Mr. Paras Sharma.

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